Alpha-Synuclein Aggregation and Membrane

Transcription

Editor's Corner
Year Three: Growing Up
Michelle McKinzey '07
Faculty in Focus
The Translator Between Two Extremes
Shaun Davis '09
Studying Biology at a Liberal Arts Institution: Dr. Lynn Westley's Approach to Undergraduate
Science at Lake Forest College
Elizabeth Dean '09
Dr. Pliny Smith: New Kid on the Block
Lisa Jeziorny '07
The Science of Teaching
Tropical Ecology: The Glories of Experiential Learning
Benjamin Larsen '07
The Evolution of the Student
Beyond the Classroom
Clinical Shadowing: A Worthwhile Experience for all Premeds
Lokesh Kukreja '08
Series of Formal Talks Launched
Mithaq Vahedi '08
Lake Forest Students Present Their Research at Regional and National
Symposia
Michael White '07
Alumni in Focus
Life After Lake Forest College: Where are they now?
Michael Zorniak '07
News and Views
Bone-derived Microglia Clear Amyloid Plaques
Lokesh Kukreja '08
Alpha-Synuclein, and the Case of the Blocked ER-Golgi Pathway
Michael White '07
Book/Film/Fine Arts Review
Iris and Awakenings: Timeless Tear-Jerkers
Mohammed Ejaz Ali '10
Dissecting the Ethical Brain
Benjamin Bienia '10
Fact and Fantasy: The Beak of the Finch by Jonathan Weiner
Healthy Marketing: The Only Solution
Jason Prendergast '09
The Thin Line Between Madness and Sanity
Stephanie Valtierra '08
Ferocious Beauty: All Roar and Very Little Bite
Pete Wisnieff '10
Review Article
History Tend to Repeat: FMR-1 Silencing in Fragile X Syndrome
Joshua Haas '08
A Ride with Listeria monocytogenes: A Trojan Horse
Joshua Haas '08, Krista Kusinski '08, Shruti Pore '08, Solmaz Shadman '08,
Mithaq Vahedi '08
Nanotechnology May Replace Existing Treatments for Cancer
Ethan Helm '07
Coal Power: Providing Energy, Asthma, Cardiovascular Disease, and Free Abortions
Ethan Helm '07, Benjamin Larsen '07
Guts & Glory H. pylori: Cause of Peptic Ulcer
Ashley Johnson '07, Bryan Kratz '07, Lorraine Scanlon '08, Alina Spivak '07
Evolutionary Antibiotic Resistance as Documented in Multiple Strains of
Staphylococcus
Alpha-Synuclein Misfolding and Aggregation in Parkinson's Disease
Michael White '07
Mitochondrial Deficiencies and Oxidative Stress in Parkinson's Disease:
A Slippery Slope to Cell Death
Michael Zorniak '07
Grant Proposal
Characterization of Membrane Permeability Alterations in Plasmodium-infected Erythrocytes:
Insight into Novel Mechanisms for Malaria Chemotherapy
Chloe Wormser '06
Apical Membrane Antigen 1 (AMA-1): Role in Plasmodium yoelii Infectiviey
Michael Zorniak '07
Essay
Some Like it Hot: Astrobiology and Extremophile Life
Elizabeth Birnbaum '08
Agoutis and Seed Dispersal in Tropical Rainforests
Stephanne Levin '09
Senior Thesis
Reduced Sexual Attractiveness of Redundant Males in the Maintenance of
Guppy Color Polymorphism
Katherine Hampton '06
Calcium-stimulated Regulatory Volume Decrease in Salmo salar and Alligator
mississipiensis Erythrocytes
Chloe Wormser '06
Primary Article
Alpha-Synuclein Aggregation and Membrane Association in a Fission
Yeast Model: Implications for PD Pathogenesis
Lokesh Kukreja '08
Alpha-Synuclein Causes Non-specific Toxicity in vps34∆ Yeast
Mithaq Vahedi '08
Editor’s Corner
Eukaryon, Vol. 3, February 2007, Lake Forest College
Year Three: Growing Up
Michelle McKinzey
Department of Biology
Lake Forest College
Lake Forest, IL 60045
Dear Readers,
The goal of any journal is to put out a product better than the last, making each issue a more difficult job than
the one before. When you can actually step back and quantify that by just looking at the amount of interest there has
been in the work of your peers, there’s this great feeling like you’ve done something important and worthwhile. And
everyone wants to feel like their time is worth something to someone somewhere.
The authors in this year’s issue of Eukaryon are very excited to have their work showcased in an asset so
valuable to any college. But getting to the publication point is never easy. The volume and quality of submissions this
year was higher than previous years and the review board, working with new guidelines and greater scrutiny, did their
best to choose what will, hopefully, be viewed as the finest work. Sadly, you can never accept every submission and the
hardest part of any reviewer’s job is always rejecting papers. This year, we have gotten better about notifying authors of
reception and rejection but we are still not perfect. I hate rejection letters because I feel like the person who has to tell
someone their family pet just got run over by a car. It’s just important to remember that the article does have merit or no
one would have recommended it for submission in the first place.
With around fifty article submissions representing at least six professors and about 55 students, Eukaryon is
experiencing exponential growth. Our editorial board has dealt with this beautifully. Thanks to Michael Zorniak., the copy
editing board ran smoothly in spite of being a person short. Furthermore, while we were scrambling to make the
publication deadline last year, almost all of the formatting was finished in the beginning of January. More than a month
before our deadline giving us more time to plan for a bigger and better 2007 inaugural ceremony. Big thanks to Lokesh
for that - and to Chelsea for managing it so well.
Speaking of the 2007 Ceremony, this year we are so pleased the Professor Anne Houde agreed to be our
seminar speaker. Last year, we did not give a seminar before the ceremony and we are expecting a great crowd. So
rarely do our mentors share their own work within our academic community and they do such wonderful work. If it weren’t
for them and their support we would neither be the students nor the magazine we are today. Professor Houde has always
supported Eukaryon in spite of our discrepancies (which we are in the process of fixing) and we are grateful for her
patience and guidance.
Our publication, though growing quickly, has still only gone from infant to toddler. This year, in addition to new
review guidelines and restructuring the ceremony, we announced the creation of three new positions, amendments to the
constitution, and print issues. The new positions encompass Business Manager, Records Keeper, and Rolling Editor-inChief. Though all of these positions are important to us, the Rolling Editor is especially important. He/she will serve as
the understudy to the Editor-in-Chief and take over when the Editor steps down or is unable to serve.
The print issues mark the transformation of Eukaryon from a caterpillar to a butterfly. We are not only a web
magazine but we are a real journal that you can find various places throughout the campus. We hope you enjoy!
The board will lose half of its members to graduation come May and faces a great challenge next year. Many of
those who will be lost are founding members and we are sad to go. While there are some wonderful underclassmen who
show immense potential, they are going to have to step up and take control - training new members and keeping
everything organized. I have no doubt, however, that they will do a magnificent job with most likely more and better
submissions. They are not afraid of hard work and consumptive hours. With them, Eukaryon will continue to grow and be
a success. We know that we will never be Science or Nature, but maybe we can be teenagers to their parents one day.
Sincerely,
Michelle L. McKinzey
1
Faculty in Focus
The Translator between Two Extremes
Outside of academia, Ann Maine is an active
member of the Lake County Board. She is on the
committees for Public Works and Transportation,
Health and Human Services, and Forest Preserves. In
this position, she again takes the role of an intermediary
between the sciences and the local community. Using
her knowledge of the sciences, she is able to change
the scientific language into terms that an average
person would understand, while voicing the concerns of
the public to the scientists.
Over her years at Lake Forest College, she
has seen many changes. Faculty members have come
and gone and schedules change constantly. One of the
greatest improvements in the biology department is that,
“Students are better prepared than they were in the
past.” She accredits this to the fact that courses are
more rigorous and have higher standards set by an
excellent faculty. She did express some concern,
however, with the methods of scheduling courses.
When few people sign up for a course, the course gets
dropped, so her schedule is constantly changing.
Nonetheless, Professor Maine understands that this is
part of her job, so she prepares for it.
Year after year, Professor Ann Maine returns
to Lake Forest College in anticipation for the academic
year. “I get excited in August.” So while many students
may be saying their good bye’s to their families and
dreading going back to school, Professor Maine can be
found sitting in her office planning courses and
practicing lectures.
Shaun Davis
Lake Forest College
While teaching at Lake Forest College for 14 years,
Professor Ann Maine has certainly made a name for
herself. Offering courses for both science intensive and
non-science students, she has taken the position of an
intermediary, teaching the two extremes of an audience.
Being able to feel the passion she has for teaching
makes any biology course interesting. Her expertise in
a constantly changing environment allows her the
freedom to teach what she loves.
Professor Maine always liked a broad
curriculum. For her undergraduate work, she majored
in plant genetics and English. From there, she moved
on to cancer research before ending up at the
University of Rochester in New York to do her
postdoctoral research in molecular genetics. During
this time, she knew that she wanted to teach at a small,
liberal arts school. In 1991, she accepted a part-time
position in the biology department at Lake Forest
College, and has kept that position since.
At the completion of the 2006-2007 academic
year, Professor Maine will have taught a total of 15
different courses. She teaches both biology majors in
the Independent Research Colloquium course as well
as non-science students in numerous other courses. “I
end up with each end of the spectrum,” she explains.
She gets to work with current research projects, for
which she expressed great enthusiasm. Nevertheless,
she still enjoys working with non-science major
students.
“I understand where they come from,”
referring to their confusion towards the scientific
language. To help these students understand the
major biological processes, she teaches the specifics
about some things, like bacteria and viruses, but with
more of a broad concept, using analogies that people
are familiar with. For example, when explaining the
methods for cell signaling, she likes to uses the board
game Mouse Trap®. This way, students can relate how
one event can set off a whole set of chain reactions.
No matter whom she teaches, Professor
Maine always demands high standards. With her
background in English, she is able to help students with
their writing skills. “She wanted us to write a lot of
papers, but it was in preparation for more advanced
biology courses,” said junior Cory Querubin.
Note: Eukaryon is published by students at Lake Forest
College, who are solely responsible for its content. The
views expressed in Eukaryon do not necessarily reflect
those of the College. Articles published within Eukaryon
should not be cited in bibliographies. Material contained
herein should be treated as personal communication
and should be cited as such only with the consent of
the author.
3
Faculty in Focus
Studying Biology at a Liberal Arts Institution: Dr. Lynn Westley’s
Approach to Undergraduate Science at Lake Forest College
In addition to her roles as a lecturer and
internship coordinator, Dr. Westley works as the advisor
of a full set of students majoring in Biology. Her
connection with advisees and dedication as a professor
is evident in her desire to get to her students and to
help achieve their goals. “I enjoy talking to my students.
And if people are interested in things that I’m interested,
I know that I can help them succeed.”
“What really makes her great teacher,” says
Dr. Houde, “is the fact that she has an amazing feel for
what students are understanding from her. She gets
into students’ minds.”
Outside of teaching, Dr. Westley is known for
her work as co-author of a book on the ecological
relationships between animals and plants. The lack of
published work in the field of plant-animal interactions
inspired Dr. Westley to write on this subject matter. “I
was taking classes in graduate school,” she says, “and
nothing in the classes was relevant to what I was
interested. That’s what made me want to write the
book.”
Dr. Westley is also interested in the topic of
allocation to reproduction in plants, and she conducts
her research at a farm in central Wisconsin, where she
and her family often vacation. Regardless of whether
Dr. Westley is lecturing in a classroom, coordinating an
internship, or conducting research, she makes evident
her emphasis on the importance of experience within
the field of Biology.
Elizabeth Dean
Lake Forest College
As a graduate of Grinnell College, Dr. Lynn C. Westley,
Senior Lecturer of Biology at Lake Forest College, is no
stranger to the environment of a liberal arts institution.
The wide-ranging fields of study and close-knit
atmosphere of the liberal arts education initially drew
Dr. Westley to Lake Forest College, where she has
worked as a biology lecturer for nearly 15 years. Her
recent appointment as Internship Liaison for the Natural
Sciences brings with it even greater involvement with
the college; it enables her to connect undergraduate
science students with opportunities to study outside of
the classroom and gain a competitive edge in the areas
of research and further education.
Dr. Westley’s focus lies mainly in the
physiological ecology of plants. Of course, her favorite
class to teach is Plant Biology, but she also enjoys
teaching Ecology and Evolution “because sophomores
are exciting—they’re making important decisions and
are at the point at which they’re really learning how to
be biologists.”
Since the start of her teaching career, Dr.
Westley has seen undergraduate science become a
significantly more rigorous field of study. “When I
started teaching science to undergraduates, it was very
unusual for freshman and sophomores to be reading
primary research articles,” she says. “Now,
introductory-level courses require students to read this
type of literature.”
In terms of teaching her philosophy, Dr.
Westley emphasizes research and experience over
textbooks and memorization; she believes that such
methods
provide
progressive,
competitive
enhancement to the Biology curriculum. Students and
faculty alike take note of Dr. Westley’s emphasis on
experience-based learning. In fact, Dr. Anne E. Houde,
Professor of Biology at Lake Forest College, says, “if
you’ve ever been in one of Dr. Westley’s classes, you
know that the lectures are not the only thing that is
important.”
the author.
5
Faculty in Focus
Dr. Pliny Smith: New Kid on the Block
In fact, the Nobel Prize 2006 for biology was
given to a scientist for work done with C. elegans and
Dr. Smith could only smile. He is proud that the
spotlight is on his field of study and model organism.
While being a biology professor and scientist
takes up most of his time, do not be shocked to see him
out on a 30 mile bike ride or roaming around campus
with his wife, an immunologist, and two children, Jason,
6, and Gillian, 9.
Dr. Smith is eager to find out what Lake
Forest College has to offer in the way of
extracurriculars, so he can get involved. And because
of his late move to campus, don’t be surprised to find
his office and billboard undecorated. So, if you have
any posters lying around, you know where to donate
them.
Lisa Jeziorny
Lake Forest College
the author.
Dr. Pliny Smith is the “new kid on the block.” The
second floor of the Johnson block that is, taking over an
office previously occupied by a part-time faculty
member.
Dr. Smith relocated to Lake Forest College
for the start of the fall 2006 semester coming from Salt
Lake City, Utah. There he left two part time positions at
Huntsmen Cancer Institute and Westminster College.
As an undergraduate student, Dr. Smith attended
Grinnell College in Iowa, a school similar to Lake Forest
College. It was there he obtained his interest in Biology
and decided that becoming an undergraduate professor
was his calling. He reports that being able to do his own
research, interacting with faculty members from
different fields, and getting to know students are among
the many perks of being a professor. He notes that it is
rewarding to see students leave here with a new ideas
and that teaching is the best way for him to continue
learning.
What is he bringing to the table at Lake
Forest College? Dr. Smith, in addition to teaching
Organismal and Developmental Biology, hopes to one
day build a genetics course that can be offered to
biology students. Also, in spring 2007 he is teaching a
core seminar entitled The Biology of Aging. During this
course, although planning is still underway, Dr. Smith
hopes to start with the little concepts, like cellular based
aging, and work his way up to entire biological
explanations for population aging. This core class for
biology majors and minors will incorporate concepts
ranging from cell biology to evolution.
Dr. Smith, as a professor, reports he enjoys
teaching at the college level in hopes of turning
“undergraduate students into scholars” by teaching
them to think critically and providing them a way to
apply what they learn to the big picture.
In addition, he hopes to get to know a few
select students really well, by employing them in his
research lab. While the lab is not yet set up, Dr. Smith
is already recruiting interested students to help him with
his work on C. elegans and cell-fate. C. elegans have
been an important organism to Dr. Smith for years and
he reports it is because they are the smallest and best
specimens to do genetics research.
7
Eukaryon, Vol. 3 February 2007, Lake Forest College
Tropical Ecology: The Glories of Experiential Learning
college helps cover more than half the cost of the trip.
While the expenses may be difficult for some students
to cover, Dr. Gordon has never had a student express
financial concerns to him.
Benjamin Larsen
Lake Forest College
Spending your days in the lush tropical rainforest while
studying exotic birds, plants, and insects is not typically
what students expect to do during class. But then,
Tropical Ecology is anything but an ordinary class.
Dr. Caleb Gordon specializes in conservation
biology, entomology, and ornithology, and teaches the
course biannually. It includes a ten-day field trip over
spring break where students conduct research projects
on site.
Overall, the course provides an excellent
academic and personal experience. Students are able
to learn experientially in an amazing location, as well as
have fun bonding with both peers and professors.
In the course’s first year, Dr. Gordon planned
to take students to Bolivia. However, just weeks before
the trip, violence erupted and the political tensions
made the trip too dangerous. Scrambling to find
another location, Dr. Gordon consulted a longtime
colleague and friend who recommended the “best patch
of cloud forest” in Costa Rica. Dr. Gordon contacted
Savegre Lodge, located in the desired area, and hastily
made arrangements over the phone.
Luckily, this turned out to be “the perfect
spot” and there is no reason to find a new location;
transportation is easy, the Costa Rican government is
stable, and Savegre has a host of amenities that cater
to student needs. The lodge has a library, laboratory,
restaurant, dormitories, and laundry services. Still, this
class is no vacation.
Tropical Ecology students are required to
participate in two research projects, one independent
and one class-wide project. Prior to the trip, students
spend their time reading literature and preparing an
independent research project. Data is collected during
the spring break trip, which can be very strenuous.
Students should expect long hours in the field and must
be reasonably fit.
Field experience is crucial to the value of the
course, however. Dr. Gordon explains that hands-on
experience really makes people learn.
Students,
including Allison Toal ‘06, spend even more time in the
field than is required. She applauds Dr. Gordon for
making the experience both fun and rewarding. All
students from the course present their independent
research projects at the annual Student Symposium at
Lake Forest College. Both Lake Forest College and the
Biology Department strongly emphasize a lab based
and experimental curriculum. Because of this, the
those of the College
9
The Evolution of the Student
This is exactly the obstacle that Houde has to
tackle with her first-year students.
“I’m continually asking myself, at what point
are we going into logical details that [the non-science
majors] are going to dismiss,” she said.
With this in mind, Houde scheduled a diverse
set of trips. The second field trip was attended in
conjunction with the Medical Mysteries class and a
handful of upper level biology majors. A ballet entitled
“Ferocious Beauty: Genome” put on by the Liz Lerman
Dance Exchange at the Museum of Contemporary Art
combined dance with our understanding of human
genetics.
“They showed concepts of Evolution in ways
you wouldn’t think of,” said Campagna, an expected
biology major.
The final field trip of the semester tied into
what scientists still face in evolutionary science today:
disbelief. The class attended “Inherit the Wind” written
by Jerome Lawrence and Robert Edwin Lee based on
the 1925 Scopes Monkey Trial.
“I really didn’t want to go at first,” said Scott
Divine ’10. “But it ended up being really funny. I really
enjoyed it.”
Students expressed frustration at scenes
where the defense was not allowed to discuss the
theory of evolution and the fictional Scopes lawyer had
to make his case by disproving the bible.
What Scopes faced in 1925 is still being
faced today. Houde’s aim is to provide information
showing that Darwin’s ideas are more than just a theory
through science and thought. This includes principles,
simulations, and hard evidence like fossils and finches.
“I thought it would be more theoretical,” said
Clements. “But evolution isn’t a theory anymore, so, I
guess that’s okay.”
Over the semester, the students became
more and more convinced that evolution is not a theory.
One student used the idea of antibiotic resistance to
convince the elder lady next to her on a plane that
evolution is not just some quack idea.
The science students agree that this class
definitely helps them understand their other classes
better, but it is also helpful to the non-science students.
“It’s a good way to grasp concepts,” said
Clements. “I like the logic and that’s a good way to
think in college.”
Michelle McKinzey
Lake Forest College
Lake Forest, Illinois 60045
One the first day of class, 15 bright-eyed first-year
students wonder what is in store for them. The blonde,
curly-haired woman with the button nose at the front of
the room introduces herself as Anne Houde, professor
of biology at Lake Forest College, and welcomes them
to First-Year Studies 114: Origin, Adaptation, and
Evolution of Species.
“The driving principle is, as a first-year
studies class, to get students to think logically,” said
Houde, professor of biology at Lake Forest College for
13 years.
The First-Year Studies (FIYS) program at
Lake Forest College is designed to help incoming
students adjust to college classes and life. Its aim is to
promote critical thinking and expose students to
resources. Courses are offered in all disciplines. In the
biology department alone, there is Evolution, Medical
Mysteries and, previously, Biology of Sex and Gender.
“I think [Evolution] does a good job of
combining science and the implications of science,”
said Houde, who is also an editor for Behavioral
Ecology and reviewer for Nature magazine.
The goal of FIYS 114 is to familiarize all
students with the logic of Evolution or, what Darwin
called, descent with modification. This idea states that
all life forms on Earth are related to one another
through a common ancestor.
It isn’t just for biology students. One student
in the class, from Kansas, could not be taught Evolution
in High School. The class includes a number of
English, philosophy, art, and politics majors among
others.
“It mixes up my schedule for sure,” said
Kendall Clements ’10, an expected Spanish major.
The goal of any FIYS course is to work on
writing and teach students how to do their own learning
as well as let them experience Chicago. FIYS 114 did
the latter over trips to various events in the area.
The first of these was a viewing of the
Evolving Planet exhibit at the Field Museum of Natural
History. Joe Campagna ’10, said that the exhibit “broke
evolution down” and made it understandable through
visual displays.
the author.
11
Series of Formal Talks Launched
our bodies respond to a plethora of “stressors” like
temperature and lack of nutrients, using special
proteins called receptors. These receptors induce
different protective responses for the varied stressful
stimuli our cells experience. They can initiate
mechanisms enabling the cell to survive or mechanisms
to commit suicide through a systematic process known
as programmed cell-death, or apoptosis.
On average, our cells contain 1013 proteins!
Many of these proteins functions in multiple pathways.
Different proteins are also assembled into protein
machines, which help carry out cellular processes. Dr.
Morimoto explained that in order to make so many
proteins so rapidly and with diverse functions, the cell
has protein quality control machinery which makes sure
that proteins are folded correctly, have the right shape,
and are functioning well. Those that do not meet these
requirements are degraded. When proteins are
synthesized in our cells, about 10% have missense
mutations, which occurs when a protein building block
is misplaced in the sequence of building blocks. Thus,
the most important process in the cell is error prone.
Proteins with missense mutations, as a result, fold
differently and may have different functions. Lightening
up the mood in the auditorium, Dr. Morimoto compared
our cells to a Ford motor plant, rather than a Toyota
motor plant!
Proteins somehow know how to fold by
themselves and we are yet to discover how and why
this process happens. Misfolded proteins cause
“proteotoxic stress” due to their altered shape and
function. These misfolded proteins, if not degraded by
the cell, can be toxic, which is the major hypothesis for
the cause of some neurodegenerative diseases, like
Huntington’s, Alzheimer’s and Parkinson’s. To prevent
misfolding from occurring, cells have a special class of
proteins called chaperones, which help with correct
folding. Explaining how proteins can change shape
when thermally heated, Dr. Morimoto used an example
of making eggs where heating up the protein (or eggs),
changes its shape, form, and function. We certainly
don’t want that happening in our brains! Thankfully,
heat shock factors, or HSFs, help cells regulate
protein’s shape in cases of stress. As we age, these
HSFs do not function as efficiently, thus increasing
“proteotoxic stress” in our cells and subsequent toxicity.
Dr. Morimoto uses C. elegans to study HSFs.
He explained that this nematode is a good model
system to study diseases as each organism has only
959 somatic (non-gonadal) cells and 302 nerve cells.
Further, the worms are transparent and the fate of each
cell in the worm has been determined. C. elegans have
about 160 chaperones. He showed data from his lab,
where knocking out a heat shock factor, HSF-1 led to
the formation of many aggregates in the worm nervous
system. Overexpressing lots of HSF in the worms gave
them a longer life span!
This presentation tied in concepts from a
number of biology courses, including Organismal
Biology, Diseases around the Globe and Cell and
Molecular Biology. The importance of protein shape
and its relationship to the proteins’ function was a
review for students of Organismal Biology. Students
who had taken Diseases around the Globe had a clear
idea of the diseases Dr. Morimoto mentioned, while Cell
and Molecular Biology students could easily recall
protein formation in the cell!
Mithaq Vahedi
Lake Forest College
A series of exciting formal talks were held in the fall of
2006 on topics ranging from neurodegeneration to
intelligent design. These presentations, held for the first
time on such a regular basis throughout the semester,
were launched by Eukaryon, βββ, the Biology
Department, and the Center for Chicago Programs.
Students were exposed to the latest research of
distinguished
scientists
in
fields
such
as
neurodegeneration and psychology. Many students
majoring in biology, psychology and chemistry attended
these talks. The presentations also attracted students
from the social sciences and humanities.
The interactive nature of the presentations
added to the rich liberal arts education at Lake Forest
College and emphasized the importance of out-ofclassroom learning experiences. These presentations
highlighted the mission statement of the Biology
Department to help “students embark on hypothesisdriven journeys of discovery where answers are found
not in textbooks, but in the lab and the field”. All of the
speakers spoke in simple language, and welcomed
questions from the audience, which also included
professors and on occasion members of the public. A
brief summary of the six talks is given below:
Stress and Aging in Neurodegenerative Disease
Dr. Richard Morimoto, Bill and Gayle Cook Professor of
Biology, Northwestern University
Have you ever wondered if stress affects the
30 trillion cells of your body? What happens when you
are stressed? Can stress increase your chances of
getting neurodegenerative diseases, like Alzheimer’s
disease or Parkinson’s disease? Dr. Richard Morimoto,
professor of biochemistry and molecular and cell
biology at Northwestern University, addressed a packed
auditorium of students in the first of a series of six talks.
Specifically, the particular kind of stress being
spoken of was physiological stress, which includes a
number of factors like temperature, viruses, genetic
factors, and heavy metals to mention a few. Primarily,
stress affects a diverse class of molecules called
proteins, whose function depends on their natural
shape or conformation. At the molecular level, cells in
12
Junior Sina Vahedi, thought that the
presentation was very good but he would have wanted
to see more data and details about the experiments.
However, Sina understood that the talk’s lack of
experimental detail made it more accessible to the
many non-science majors in the audience.
Dr. Morimoto’s presentation was simple and
easy to understand. It explained the connection
between
stress,
protein
misfolding,
and
neurodegenerative diseases. Dr. Morimoto’s sense of
humor, calm disposition, and the tone and pace of his
voice made this presentation both educational and
enjoyable.
Increasing the amount of proteins in the cell
yields a greater chance of getting prions. However, the
fibers formed by these prions need to be broken in
order for it to be given to the daughter cell.
Dr. Liebman explained that a nonsense
mutation is one where there is an extra stop codon in
the DNA sequence. In her laboratory, when a mutation
was made in the sup35 gene, the protein was still
made, despite the stop mutation! Dr. Liebman’s lab also
discovered that a chaperone protein that dissolved
protein aggregates was required to propagate the prion.
The chaperone breaks the fiber and thus helps in
propagation of the protein. Inhibition of the chaperone
protein by hydrochloric acid leads to decreased prion
propagation.
This presentation touched on many topics
covered in Cell and Molecular Biology, as well as those
explored in Ecology and Evolution. Why does our
protein synthesis and degradation machinery differ only
slightly from that of yeast? Evolutionarily speaking, how
similar are we to yeast?
Dr. Liebman spoke in simple language and
explained cell biology terms throughout her talk. She
frequently asked the audience questions. This helped
almost everyone to understand the talk, and it also
made the presentation an unforgettable learning
experience.
Yeast as Small “Mad Cows” Demonstrate ProteinBased Inheritance
Dr. Susan Liebman, Distinguished University Professor,
University of Illinois-Chicago
How our Hands Help us Think
Dr. Susan Goldin-Meadow, Ruml Distinguished Service
Professor, University of Chicago
Did you know that we have a dogma in
biology? Yes, the central dogma of molecular biology
says that heredity is controlled by DNA, which spells
out protein formation. Dr. Liebman explained that in
Mad Cow disease, a pathogen, a prion (PrP), lacks
nucleic acids, yet can change a proteins original
formation. There are many cousins of PrP diseases,
like Creutzfeldt-Jakob disease, kuru, fatal familial
insomnia, scrapie of sheep, mad cow disease of cattle,
and chronic wasting syndrome of deer, all of which are
known as transmissible spongiform encephalopathy’s.
Dr Liebman’s talk was as exciting as it was
easy to follow. She explained that proteins can exist in
a normal or prion shape. Prions are infectious (selfperpetuating) proteins which form fibers that can be
seen under the microscope. Comparing the DNA
paradigm to the prion paradigm, Dr. Liebman pointed
out that in the case of a DNA mutation, a protein can
lose function or gain new function. However, in the case
of prions, a normal protein can change shape and
induce other molecules of that same protein to change
shape as well. There can also be mutations which
predispose proteins to change shape and act like a
prion.
Different strains of PrP cause different
disease pathologies in inbred animals. These prion
strain differences appear to be due to different heritable
prion conformations. Showing data from her lab, Dr.
Liebman pointed out that prion proteins in yeast are
infectious.
So why use yeast? Well, yeast contain
proteins that are highly conserved. In addition, many
cellular processes like DNA synthesis and repair, cellcycle progression, protein synthesis and processing,
and protein transport are also highly conserved. Yeast
grow by mitotic budding and propagate proteins that are
in the prion shape.
Dr. Susan Goldin-Meadow presented her talk
amid the excitement of the campus-wide Brain
Awareness week at Lake Forest College. Her
presentation was at the peak of this outreach campaign
organized by the first-year studies Medical Mysteries
class and Molecular Neuroscience students. She
shared exciting data from her research, which studies
the process of mismatch learning in children.
It was discovered that gestures change when
children or learners are “in transition.” Therefore
gestures are associated with learning. Dr. GoldinMeadow presented data to show that a gesture is not
only a reflection of human thought, but also a
mechanism of learning. Using data she collected, Dr.
Goldin-Meadow explained that in a child with gesturespeech match, the speech of the child about moving
and the gestures show the actual movement that
happened. However, in a gesture-speech mismatch,
the gesture of the child describing movement does not
correspond to the actual movement. Interestingly,
children with gesture-speech mismatch are more likely
to learn after training than children with gesture-speech
match.
13
Dr Goldin-Meadow found that while teaching,
one strategy in speech is a lot better than two. She also
discovered that gestures are powerful in their ability to
shape the way we think! If the children learned only the
gesture, they tended to learn much better than those
who repeated only the speech. In another experiment,
children were told to gesture everytime they were trying
to solve a problem. Interestingly, the number of new
strategies was much greater in those told to gesture.
Further, she found that children who are told to gesture
during a lesson remember what they learn. Also,
children remember more when they gesture, in addition
to coming up with new strategies to solve a problem.
She pointed out that making gestures encourages
experimentation and adding more ideas.
Dr. Goldin-Meadow mentioned that gesturing
lightens the cognitive load in the same way that writing
down a problem on paper does. Another benefit of
gestures is that they provide a second representational
formation. Further, notions in gesture can go
unchallenged.
This talk attracted a great number of
questions from the audience. One student requested
that Dr. Goldin-Meadow replay tapes of classroom
experiments involving children being taught by
gesturing and non-gesturing teachers.
to a new species. Last, the only force causing
evolutionary change is natural selection.
Dr. Coyne went on to present data supporting
the theory of evolution. He mentioned that the
Archaeopteryx which has a pelvis bone, indicating that
it evolved from dinosaurs. In embryology, scientists can
see that dolphins develop hindlimb buds, which then
regress. Further, humans develop a Lanugo (a coat of
hair), which we shed. Dr. Coyne pointed that vestigial
organs serve as “the senseless signs of evolutionary
history,” for example the kiwi is a flightless bird. Dr.
Coyne also cited the development of antibiotic
resistance in bacteria as evidence for natural selection.
Intelligent Design, or ID, claims that an
“intelligent agent” designed some of the features of
modern organisms. ID states that some features are
“irreducibly complex” and could not have evolved in a
stepwise fashion. They include such features as the
eye, the blood clotting system, the immune response
pathway and the bacterial flagellum. However, due to
new fossil evidence the vertebrate jaw can now be
explained. The problem with ID is that if we can’t think
of a way a feature evolved, then the intelligent designer
is credited with its creation. Another problem is that
nothing is known or can be known about the designer’s
goals and methods. Thus, claims by ID are not testable.
Dr. Coyne was very careful not to downplay
the important role of religion in society. He said that
Bible must not be taken literally and that we can
reconcile our beliefs with scientific evidence. Like a true
scientist, Dr. Coyne was very comfortable with
discussing evidence that would falsify or refute the
theory of evolution. He mentioned that a fossil in the
wrong place would be one. For example, a human fossil
that is older than 10 million years old!
Feeding and Gloating for More: Intelligent Design Vs
Evolution
Dr. Jerry Coyne, Professor of Ecology and Evolution,
University of Chicago
Alzheimer’s Disease: A Tangled Problem
Dr. Lester Binder, Abbott Professor of Biology,
Northwestern Feinberg School of Medicine
Do you accept evolution as a scientific theory
well supported by evidence or not? Well, only 1 in 5
Americans believes in evolution. And only 12 percent of
Americans think that evolution should be taught in
schools. Dr. Coyne pointed out that the theory of
evolution should be compared to the atomic theory of
matter, which is accepted by almost 100 percent of
Americans. This is because, like any other scientific
theories, it makes sense of wide-ranging data that were
previously unexplained, makes testable predictions and
is vulnerable to falsification. However, no evidence has
yet been found to falsify the theory of evolution.
Dr. Coyne’s talk was reminiscent of the
college’s Ecology and Evolution class! He explained
that there are four parts to the theory of evolution. First,
evolution occurred; that is, living species descended
from a common ancestor. Second, there were very
gradual changes in each descending generation . Third,
speciation occurred; that is, a single ancestor gives rise
Dr. Binder’s talk on Alzheimer’s disease (AD)
was the opening talk of an exciting one-day workshop
on neuroscience. Dr. Binder, who studied the control
elements of tau tangles found in AD patients,
enlightened the audience about the culprit thought to
cause the disease. Tau protein binds microtubules and
stabilizes them. Tau also aggregates to form filaments
that compose the neurofibrillary tangles found in brains
of AD victims. Phosphorylation of this protein controls
its binding to microtubules. Phosphorylated tau leads to
dynamic instability which allows for plastic changes to
the cell’s architecture. Hyperphosphorylation is a
hallmark of AD. In addition to the tangles, plaques
14
(amyloid) are also seen. The axons and dendrites of the
neurons are filled with tau tangles. The density of these
tangles correlates with the degree of dementia in the
AD patient. Tau mutations also cause certain forms of
familiar frontotemporal dementias (FTDs).
In an experiment involving neurons,
neurodegeneration is absent when tau is absent. Tau is
known to come off the microtubules. What is not known
is whether the disassociation or the aggregation of tau
is the problem. Thus, the role of tangles and other tau
aggregates in AD is still unknown.
In his laboratory, Dr. Binder designs and
conducts experiment using antibodies which recognize
tau conformations, modifications, and truncations. It
was found that one conformation of tau, ALZ50, was a
polymer. When tau is cleaved, the rate of assembly of
aggregates is increased. However, if the tail peptide is
added back, the rate of assembly is inhibited! Studies in
Dr. Binder’s laboratory indicated that making a tangle is
protective to the cell.
Other interesting data from Dr. Binder’s
laboratory indicated that the N terminus of tau facilitates
the assembly of full-length tau. And the deletion of a
region in the N terminus of the protein decreases the
rate of assembly. Findings from Dr. Binder’s laboratory
have made valuable contributions to AD research and
provided many targets for potential therapy.
Dr. Binder’s enlightening presentation was a
synthesis of concepts students had come across in Cell
and Molecular Biology. Students of Molecular
Neuroscience were able to appreciate Dr. Binder’s
research on AD to a greater extent than the others.
pattern of drug abuse characterized by overwhelming
involvement with the use of the drug (compulsive use),
the securing of its supply, and high tendency to relapse
after withdrawal. This pattern is thought to be “learned.”
In her laboratory, Dr. Napier used rats and
mice to study addiction. A drug was put at a certain
place so that the rats learned to associate
environmental cues with the drug. Drugs were given in
repeated, intermittent doses to induce addiction. This
led to the progressive enhancement of motor activity.
The animals were observed visiting this location even in
the absence of the drug.
Dr. Napier’s laboratory also carried out
research using amphetamines. Amphetamines have
common mechanisms in action. These bind to
receptors and are taken up, and subsequently displace
the transmitters. Thus there is a great increase in
transmitters. In other words, the brain is beefed up in a
very big way! It was found that rats could be weaned off
methamphetamine addiction by administration of the
drug mirtazapine!
Dr. Napier’s research and her promising
results with mirtazapine generated many intelligent
questions from the audience, who still seemed addicted
to neuroscience after a whole day workshop!
those of the College.
Neuroscience in Search for a cure for drug addiction
Dr. T. Celeste Napier, Professor of Pharmacology,
Loyola Stritch School of Medicine
Dr. T. Celeste Napier presented the last talk
in the fall series of formal seminars and the closing talk
of the day for the Neurofrontiers workshop. Her
presentation elicited many questions from students who
thoroughly enjoyed her talk. Dr. Napier mentioned that
an astonishing 9 percent of the population, or an
estimated 21.6 million people aged 12 or older, can be
classified with dependence or abuse on psychoactive
substances (alcohol or illicit addictive drugs). Recently,
there has been a large increase in ER visits for
methamphetamine related cases. Methamphetamine,
which is a most potent psychostimulant, is also called
meth, crystal, and crank.
Dr. Napier clarified that addiction refers to the
pattern of self-administration. Addiction is a behavioral
15
Clinical Shadowing: A Worthwhile Experience for all Premeds
“Sensing the Environment,” in which the curriculum
featured a three-week section on the visual pathway. It
turned out that topics like rods and cones, myopia,
refraction, and modern surgeries, like LASIK, were both
exciting and engaging to me.”
“I am currently interning for two of the best
pediatric optometrists in the Chicago land area, Dr.
Mary Lou French, and her partner, Dr. Amy HansenKwilose. Patients come from all over the United States
to see them, and they have been influential mentors,
thus far. After spending three months working for these
two doctors, I am able to say, with confidence, that this
profession is perfect for me. Everyday is different. My
main responsibility is using the auto-refractor to checkin new glasses, working hand-in-hand with the
dispensing department. During any free time, Dr.
Hansen teaches me how to view vessels and the optic
nerve. I am also in the training process as a pre-tester
and optician. Additionally, this internship has shed
some light on pediatric optometry as a potential area of
focus.”
Lokesh Kukreja
Lake Forest College
Introduction
Lokesh Kukreja ‘08
Besides good grades in classes and standardized exam
scores, medical schools look for students who are
deeply interested in medicine. In particular, these
schools are interested in students who have shadowed
physicians. Shadowing is an experience in which premed students get to observe patient-physician
interactions in a clinical setting. This experience should
not be taken flippantly. During this time, students
evaluate themselves and their desires to become
doctors. If shadowing cultivates and excites their
passion for medicine, the experience will definitely
encourage them to pursue medicine.
Shadowing is an indispensable activity for
pre-med students. There are many experiences that
students can lose if they dismiss the opportunity to jobshadow a physician. Medical careers, unlike other
careers, involve the direct contact with sick patients.
Life as a doctor is difficult to imagine unless students
have clinicians in the family. This is why shadowing is
essential; students gain a vicarious experience of the
day-to-day activities of doctors.
Careers in medicine are widespread.
Sometimes, shadowing helps narrow student’s interest
and also validates their pursuit of a medical career. But,
what makes one initially pursue a career in medicine? It
may be a clinical visit as a patient or sitting in a
classroom learning biology. These are few examples of
situations that inspire students to strive for a medical
degree.
Here is a compilation of experiences that
students at Lake Forest College had while shadowing
doctors. These students’ opinions are focused on how
their shadowing experiences influenced their decisions
of going into medical profession.
I am interested in going to medical school because I
would like to have a direct major impact on people’s
health. During my shadowing experiences, I wanted to
do shadowing where I can see the works of many
medical professionals. This is an opportunity that is
hard to find. I spent a summer between my sophomore
and junior year, following an anesthesiologist in the
operating rooms at Rush North Shore Medical Center in
Skokie, IL. Also, for a short period of time, I shadowed
a gynecological surgeon at Highland Park Hospital in
Highland Park, IL. So, I have been in many different
operating rooms in the two hospitals. Additionally,
during my shadowing experiences, I have been
fortunate to be able to see the most new and effective
surgeries by leading doctors.
I saw my shadowing experiences to be a
window of opportunity to learn more about various kinds
of medical professions in person. I was always overt
about my feelings of likes and dislikes on things I
observed in the hospitals. I observed many surgeries:
repairing abdominal aortic aneurysms endovascularly,
implanting a pacemaker, performing a quadruple
bypass by open-heart, laparscopic removing of a
cancerous kidney, laparscopic repair of an inguinal
hernia, and orthopedic surgeries of repairing rotator cuff
due to a shoulder RC tear and replacement of hips and
knees. When I was following the gynecologist, I
observed the doctor deliver a baby by C-section. The
next time I saw the same doctor, he removed unusual
fibroid formations in a woman’s uterus.
There are many virtues in shadowing. First of
all, I really found out that I am interested in medicine,
and along with my scientific research interests, I think I
want to become a clinical researcher. Second of all, I
have made important, close links, with doctors. This will
allow me to have expert guidance in the future. Third of
all, the shadowing experience has been an enriching
adventure. Through talking to doctors, nurses and
medical residents in the hospitals, I have learned the
qualities of hard work, responsibilities, and a sense of
humor, all of which are needed to become successful in
a medical profession.
Lisa Jeziorny ‘07
Lisa wants to become an optometrist. Her interests
generated by listening to biology class lectures about
vision, but when she shadowed an optometrist, her
passion for eye care became stronger.
“During the spring semester of my first year
at Lake Forest College, I became interested in a career
in vision. I enrolled in a biology course, entitled
13
Karina Nikogosian ‘07
compassionate, and collaborative relationship between
doctors and patients. Especially in pediatrics, the doctor
must attain the utmost trust of a child, so, the child
allows the doctor to perform a physical and touch the
child, in general. I simply followed the doctors as they
saw their patients. The majority of the cases were
monthly visits by children to get their immunizations,
physicals, and other general examinations. However,
there were also a lot of sick visits by both toddlers and
older children. I heard doctor's recommendations on
ear infections, flues, allergic reactions and other types
of infections. Since I got to shadow all five doctors
several times, I got an outstanding opportunity to see
how different doctors talk, treat, and even examine their
patients. I began to pick up on things that I liked how a
doctor does, or how I would do something a little bit
differently. One doctor told me, "The average time it
takes for a doctor to interrupt a patient in explaining
his/her symptoms is eight seconds". This doctor was
tremendously patient and gave his patients as much
time as necessary to explain what he/she felt as well as
made sure that his diagnosis/treatment made perfect
sense to the patient and the patient's parents. It was
great seeing children several times—I got to see how
they have grown and the way doctor's techniques
change as the child ages. I also learned a little bit about
the diet and proper care of children at different age
groups. A tremendously valuable experience all in all. I
definitely confirmed my desire to, not only become a
primary care doctor, but a pediatrician.”
Karina also wants to become an optometrist. A summer
of shadowing in an eye care clinic landed her a job in
the same clinic when next summer came around. The
shadowing experiences confirmed her interest in the
field of optometry. Though, she emphasizes that during
her shadowing experiences, she has made valuable
connections with optometrists, these doctors, Karina
believes, can help advise her so she becomes
successful in this field of medicine.
She explains, “two summers ago, I called
multiple optometry offices in the area near my house. A
doctor from one office, Johnson Eye Care, called me
back and I asked if she needed a volunteer. I told her
that I did not mind running errands or doing chores
around the office, as long as I received exposure to the
profession. One day a week for 5 hours, I sat in on eye
exams and kept a notebook for questions. After the
eye exams, I asked Dr. Schoepke about her choice of
treatment for the patient and other eye health related
issues. I also answered the phones, took out trash, and
dropped off mail. I was always friendly and helpful to
the patients, which is why I think Dr. Schoepke offered
me a job at her office. This past summer I worked at
Johnson Eye Care and got a complete exposure to
what the profession in optometry entails. My
experiences working at Johnson Eye Care helped me
refine my career goals. Shadowing or working in the
filed of interest is the best way to learn whether the
profession is right for the person. Working at Johnson
Eye Care made me realize that optometry best fits my
personality and now I have a mentor, Dr. Schoepke, to
give me advice about the application process and
further information about a career in optometry.”
Chloe Wormser ‘06
Chloe wants to become a veterinarian. After graduating
from Lake Forest College, she wanted to gain in-depth
experience in the veterinary field. One of the most
exciting things that a student like Chloe acquired during
her shadowing is lots of hands-on experience. During
many shadowing experiences, students solely observe.
There are liability issues that prevent pre-med students
to have hands-on experiences. However, the
opportunities to do hands-on activity will be valued by
students. This activity reflects on how students came
closer to experiencing physician-patient interactions.
Chloe explains, “I am interning at a small
animal veterinary clinic. I work with two small animal
veterinarians.
The experience has been very
beneficial. I have gotten a lot of hands-on experience
working with animals; I help during appointments by
holding animals for the veterinarians during
examinations as well as assist during dental cleanings
of cats and dogs. In addition, I have learned to use the
laboratory equipment essential for monitoring patients
and assessing animal health. For example, I have
learned how to run blood analyses and how to monitor
animals while they are under anesthesia using the
ECG, pulse/ox, and blood pressure readings. I am
planning to be a small animal veterinarian and will be
entering vet school next year. Therefore, getting a
head start and actually working alongside veterinarians
is very valuable. Not only have I become much more
comfortable working with animals, but I have really
learned a lot about how to communicate with owners.”
Shruti Pore ‘08
Shruti wants to be a dentist. Critically, her shadowing
experiences helped her explore and understand the
depth and breadth of knowledge in the field of dentistry.
“I am interested in dentistry. I have shadowed
a periodontist, an orthodontist, as well as a general
dentist. I spent the summer between my sophomore
and junior year shadowing. I thought that it would be
helpful in making up my mind. This is why I decided to
shadow some dentists. The people that I shadowed
were all very nice. By shadowing them I was able to
understand 'a day in the life of a dentist.' Also,
shadowing different kinds of dentists helped me see
how, within a single profession, there are many facets
to explore. I wanted to become a dentist before I ever
started shadowing. Shadowing confirmed my belief that
I will be happy in this particular profession.”
Alina Spivak ‘07
Alina is interested in both osteopathic (DO.) and
allopathic (M.D.) medicine. It is worth noting that prior to
shadowing, she already developed an interest in
specific area of medicine in primary care of pediatrics.
Shadowing helped her cultivate her interests in the
field.
Alina explains, “since I am interested in
pediatric medicine, I shadowed an office of five
pediatric physicians who were all also hospitalists (i.e.
making rounds in the hospital aside from working in
private practice). I shadowed for one to three hours
once a week for six months. This experience showed
me the importance of forming a sincere,
Michael Zorniak ‘07
How do students know that medicine is a right career
for them? When Michael shadowed a physician, he
wanted to use the experience to evaluate his future
career goals. He saw that more than half of the
14
shadowing experience is not only the observation of a
physician and patient interaction, but rather an
introspection that shapes your curiosity for medicine.
Michael explains, “I feel that shadowing a
physician was time well spent because I was given the
opportunity to objectively determine whether or not
being a doctor fits my personality. This objectivity did
not come with the experience, but it is something I
needed to bring with me. Before contacting a physician,
one must assess their own personality characteristics,
traits, and values. This can be done by writing a list.
Thus, when one finally contacts a physician, one can
objectively evaluate his/her personality fit with the
profession. Then one can ask themself, ‘Can I be a
doctor?’ It is important to shadow several doctors
because one does not want to limit their perspective on
the field of medicine and cut themselves short. I have
shadowed three different types of physicians (i.e.
ophthalmologist, internal medicine clinician, and a
family doctor). I have found that the scope of medicine
is very different and ophthalmology best suites my
interests and personality.”
“Shadowing an ophthalomologist was an
effective way for me to objectively evaluate my desire to
become a physician. Getting personal exposure to
medicine gave me an understanding of a clinician's role
on a health care team. This opportunity also gave me a
chance to place my feet in the shoes of a doctor. During
my experience, I was allowed to record patient
histories, administer basic ophthalmic examinations,
and research the pathology of different diseases of the
eye. Shadowing a doctor has helped me determine the
overlap of my interests with that of a physician's.”
“I have personally been treated by an
ophthalmologist who took immediate action to
functionally prevent the decline of my vision. Dr. Brian
Proctor, the ophthalmologist I have shadowed for over
6 months, immediately performed a procedure to repair
my condition and prevent the development of scar
tissue around the affected area. I would like to practice
medicine for the chance to aid individuals in the manner
that Dr. Proctor has aided me and many others. In my
life, I have had a taste of this and I am resolute in
gaining additional training. “
Conclusion
If students are considering careers in the medical
profession, shadowing is essential. The experiences
will make students think more seriously and attest to
their motivation for taking a path toward medicine. After
reading this compilation of shadowing experiences from
students at Lake Forest College, consider the great
benefits of shadowing if you’re interested in pursuing
medicine.
15
Life after Lake Forest College: Where are they now?
coordinator for the Health Literacy and Learning
Program. She and her group have published several
articles on health literacy and presented their findings in
conferences from Chicago to Basle, Switzerland.
Although Silvia has pursued various interests,
she reverts back to an experience she had in college
where she mentored freshman biology students, "I
really enjoy working with people in the teaching
environment, and whatever career I choose, it will have
to involve some kind of mentor-student relationship."
She misses being a student at Lake Forest where, "the
only thing I had to worry about was studying for
exams." No one could have predicted the experiences
Silvia has had after graduation. Despite this uncertainty,
she is still resolute in pursuing a doctoral degree, yet,
she maintains that, "nothing is set in stone."
Allison has traveled a similarly unpredictable
route. While writing a senior thesis in Dr. Kirk’s lab, she
applied to graduate programs in environmental
engineering and was accepted to Stanford University.
By pursuing a research project in college, Allison claims
she was able to, "gain admission to most of the
programs I applied to, even though I wanted to switch
fields." She also emphasizes that a research
experience is critical for any undergraduate. She then
switched gears, again, and received a master’s degree
in economics from the University of California, Santa
Barbara. Although her environmental engineering and
economics degree don’t seem to mix, she has
consolidated this education and finally pursued a
doctorate in environmental science and management at
UCSB. She expects to graduate in 2007. Allison
believes that the "secret to life is to find the job you like"
and that "sometimes it is worth taking the risks to
change directions."
With a plethora of academic experiences,
Allison is finally "at peace" with her decision to pursue a
career in environmental economics. After her unique
experiences, she has gained a wealth of advice for
undergraduates. She states that at a large research
university "you see how the envelope of knowledge is
being pushed." Yet, she cautions that "the graduate
school environment is not as nurturing as
undergraduate programs. There are a lot more hoops to
jump through and there are a lot of competitions. And
often times, you really need to expect more from
yourself and less from others."
Silvia and Allison have showed us how their
lives have been guided simply by their interests. Both
Silvia and Allison have emphasized the need to polish
writing and speaking skills in college. Silvia further
elaborated that these skills are the, "most necessary
[abilities] in any path of life you choose." Lake Forest
College has equipped these two students with skills that
they will value and use for the rest of their lives, "I feel
that Lake Forest College prepared me for the real
world," affirms Silvia.
Michael Zorniak
Lake Forest College
Top Left: Beth Ruedi ‘01, Top Right: Brandon Johnson ‘03,
Bottom Left: Allison Huang ‘98, Bottom Right: Silvia
Scripkauskas ‘04
While in college, many of you have probably wondered,
"What am I going to do with my life?" Unless you were
born knowing your fate, this question may be truly
disheartening. After interviewing four recent biology
alumni from Lake Forest College, I realized that
knowing your future is not as important as knowing
yourself.
Writing this article as a senior has forced me
to come to grips with my future. I understand that this
article is supposed to feature graduates from Lake
Forest College, but, being on the brink of
commencement, I feel that my soon-to-be alumnus
perspectives are simply an added bonus. Before talking
with the alumni, I felt that graduating from college
marked a fork in the road of life. I believed that by
choosing one path, one would have difficulty backtracking or switching paths. Upon receiving
corresponding with Allison Huang’s ’98, Silvia
Skripkauskas’ ’04, Allison Huang ’98, and Brandon
Johnson ’01, I realized that changing one’s mind is a
natural and sometimes necessary step in life.
Silvia and Allison
Let’s start with Silvia, who, at the moment, is pursuing a
master of arts degree in Public Policy and
Administration at Northwestern University. At Lake
Forest, she majored in biology and psychology while
managing to pick up a minor in chemistry with medical
school in mind for the future. After graduation, she
tacked from one job to another, only confirming her
desires for autonomy in a health-related field, "I need
more say in what I do, and that can be achieved only
after I get more training, whether it be the master’s
degree I am currently working towards, a Ph.D., or an
M.D." She first held a brief position as a research
technician at Abbott laboratories in the International
Pediatric Nutritional Regulatory Affairs Department.
Later, Silvia obtained another research position in the
Institute of Healthcare Studies at Northwestern
University, where she was appointed as a project
Beth and Brandon
Brandon Johnson ’03 and Beth Ruedi ’01 have
progressed through life in a more linear fashion when
compared to Silvia and Allison.
Beth, a biology and English double major,
found pleasure in studying genes and behavior when
she took Dr. Houde’s Ecology and Evolution class, for
which she was later a peer teacher. Following her
16
interests, she entered a doctoral program at the
University of Illinois in Champagne-Urbana. Beth’s goal
is to become a university-level professor, "I felt that I
needed to learn a great deal more about biology before
I could teach it." Taking steps to further her career,
Beth is employed as a teaching assistant and she is,
"reinforcing the fact that this was a good career path to
take." She says that although, "Lake Forest College
provides students with many opportunities for thesis
research and laboratory work, which is one of the key
elements that can help a student get into graduate
school," she was not prepared for the intense research
at a Level 1 institution, "due to Lake Forest College’s
comparatively relaxed atmosphere." All in all she
concludes that, “graduate school is challenging and
stressful. However, I can honestly say that the past five
years have been the best of my life.”
Brandon traveled a path no less direct than
Beth’s. While at Lake Forest, he majored in biology and
chemistry and wrote a thesis in Dr. DebBurman’s brand
new lab. As side-effect to this, he was a permanent
resident of Johnson Science Center during his junior
and senior year. He became so attached to Johnson
that whenever his experiments went awry he, “cursed
the entirety of Johnson building.” Despite his negative
sentiments toward the actual building, he says that, “the
Lake Forest College experience, as a whole, really
solidified my motivations towards graduate school…the
opportunity to write a thesis and defend it gave me the
confidence to conduct graduate research.” Now at
Stanford University, Brandon is pursuing a doctorate in
cell biology, which may lead him into a career in
teaching where he has, “a passion for science and
maintaining a high level of scientific achievement in the
US.” Brandon may even want to, “reenergize scientific
awareness and research,” possibly by teaching science
at the high school level. He thinks that, “high school
students are not receiving adequate scientific training.
Right now, we are seeing reduced scientific funding in
the United States and a general apathy towards basic,
non-clinical scientific discovery.”
Like Allison, Brandon cautions undergraduates,
“graduate school is all about learning how to conduct a
long term, independent, primary research project.”
Unlike Lake Forest College he says, “Classes are
secondary to conducting primary research.” He faces
the same challenges at Stanford as he did in Johnson,
“there are many times when my project doesn’t move
forward for months at a time, and brief periods where
the science advances rapidly.” Even though there are
ups and downs in science, Brandon looks to other
successful scientists, with similar experiences, for
inspiration, “I take comfort in knowing that I am on the
same rollercoaster.” Additionally, Brandon is no
stranger to the distress and adventure he experiences
in science. He avidly surfs and hikes in California’s
wilderness and has recently competed in an 11 mile
relay swim.
Silvia, Allison, Beth, and Brandon have all found value
in immersing themselves in the academic climate at
Lake Forest College. By looking inside themselves and
pursuing their interests, they have become successful
scholars in their respective fields. One thing they all
miss is the, sometimes, daily interactions with faculty,
which have strengthened their understanding of biology
as well as themselves.
17
Lake Forest Students Present Their Research at Regional and
National Symposia
wandered around the San Diego Convention Center
checking out all the latest biotechnology.
Many famous scientists, such as Susan Lindquist,
presented their research or participated in panel discussions
involving the audience. Surprisingly, the NIH Director was in
attendance and answered passionate questions on the
current state of research funding. In addition to all the
serious research being presented, there was an event called
“Cell Slam” that was one of the most memorable parts of the
trip. Participants were given several minutes to do anything
they wanted in regards to cells. One scientist sang a parody
of “Let it Snow” that went, “The funding situation is frightful
but my grant is so insightful…Let it go, let it go, let it go”.
The crowd loved it. This demonstrated that scientists loved
taking a humorous attitude toward science.
One afternoon during the symposium, Lokesh,
Michael, and I decided to take a break from the science and
travel to Tijuana, Mexico. It was a great opportunity for us to
take advantage of the community surround San Diego. At
the U.S.-Mexico border we took a cab into downtown Tijuana
and had a drink at a local restaurant. Surprisingly, there
were pharmacies on every corner. To get back into the U.S.
we waited over an hour and wondered if we would ever get
back to the symposium. Several hours later, we returned
and had numerous stories to tell, such as the donkey painted
as a zebra.
Michael White*
Lake Forest College
During the fall of 2006, undergraduates from Lake Forest
College (LFC), including myself, organized their research
into posters and PowerPoint presentations to exhibit at
regional and national academic symposia. Specifically,
participants conducted laboratory or field research on either
Parkinson’s disease, cell volume regulation, RNAi, addiction,
or avian biology. Fourteen LFC students presented this
great diversity of biological study at the Argonne National
Laboratories Undergraduate Symposium in lecture form.
Lokesh Kukreja, Michael Zorniak, and myself went a step
further and traveled to San Diego, California to present two
posters on our fission and budding yeast models of
Parkinson’s disease at the American Society for Cell
Biology. Whether presenting on the regional or national
scale, young scientists from LFC demonstrated their
individual research in biology and enjoyed the fellowship of
other undergraduate and graduate students.
The
Argonne
symposium
consisted
of
undergraduate research, organized into mini-symposia
consisting of several twenty-minute individual talks.
Hundreds of students from the states surrounding Illinois
attended. There were many presentations to choose from,
and it was interesting to learn about a vast array of topics. I
noticed that the structure of each talk differed significantly
between colleges, indicating differences in teaching styles.
Michael Zorniak said that Argonne, “strengthened my
communication skills by challenging me with a diverse
audience.” In the morning, a local high school physics
teacher conducted a series of insightful demonstrations on
the characteristics of shadows. The evening lecture was
given by a chemist and, like the first, used a series of colorful
chemical reactions to demonstrate the digestive functions of
the stomach. Humorously, his chemists’ view of acid
digesting food neglected most of the biology behind the
process.
Caption: Tijuana, Mexico 2006. Michael Zorniak (left) and
Michael White (right).
On the final day, Michael and I presented our posted on
budding yeast and Lokesh on fission yeast. Initially, I felt
that the graduate students would be extremely critical of our
work but once several visited and discovered we were
undergraduates and our research was respectable, their
comments were insightful and full of praise. A young
scientist from Mayo Graduate School also worked with yeast
and gave great suggestions for future experiments, one of
which I am currently performing. In addition, all three of us
discussed our research with others in similar fields and
found these conversations very beneficial. This experience
increased our confidence and demonstrated that
undergraduate research can be presented alongside more
advanced studies. When Michael Zorniak was asked what
he liked most about attending the ASCB he said, “I was able
to interact with scientists from as far as Tokyo, Japan that
were interested in the same research questions. Even as an
In December, Lokesh Kukreja, Michael Zorniak,
and myself traveled with our P.I Shubhik DebBurman to the
American Society for Cell Biology symposium in San Diego,
California for what would be one of my most memorable
experiences. There were nearly 15,000 attendees, the
majority of which were graduate and post-doctoral
researchers. Fewer than 1,000 undergraduates attended.
We presented two posters on our study of α-synuclein, the
protein involved in Parkinson’s disease, on the fifth and final
day of the symposium. With several days of free time, we
visited a variety of lectures, poster presentations, and
21
Caption: American Society for Cell Biology San Diego,
California 2006. Michael White (left), Michael Zorniak
(center), and Lokesh Kukreja (right).
undergraduate, I was able to propose experiments to
graduate students in the same field.”
Together, presenting undergraduate research on
both regional and international levels, provided others and
myself with an indispensable asset to our intellectual growth
as well as peer fellowship. Furthermore, it allows students to
gather perspective on the broader research community and
realize that they are a significant part of it. And of course, a
trip to Tijuana adds a little more excitement to an already
intriguing experience.
herein should be treated as personal communication and
should be cited as such only with the consent of the author.
22
News and Views
Bone-derived microglia clear amyloid plaques
Nevertheless, studying the role of microglia in AD
animal model is more relevant than any study in the test
tube. In AD mouse model, the outcome of clearing Aβ
deposits by microglia has been questionable because
Aβ deposits are abundant in the brain and form faster
than they can be cleared by the microglia (Wegiel et al.,
2004). Prior to the research done by Simard et al.
(2006), the microglia have been shown to be inefficient
at degrading Aβ deposits.
Now, in the study of AD using transgenic mice
model, Simard et al. (2006) show that there are other
efficient microglia of blood origin which specifically
phagocytose amyloid plaques. Simard and colleagues
demonstrate that the monocytes pass through the blood
brain barrier of CNS and differentiate into microglia.
These blood-derived microglia are shown to closely
associate with Aβ deposits. In the AD affected region of
the hippocampus, Simard et al. found bone marrowderived miroglia to colocalize with the β-amyloid 40/42.
Following the first evidence, Simard et al. (2006)
test a very important question of whether blood-derived
microglia are beneficial in slowing down the build up of
amyloid plaques. They treat the undifferentiated blood
derived cells with ganciclovir drug that impedes the
cells differentiation into microglia. The scientists closely
observe the changes in Aβ formation strictly when no
blood-derive microglia are created. They discover that
the size and the number of amyloid plaques increase
with ganciclovir treatment.
In addition, they see a second type of non-blood
derived (resident) microglia associate with Aβ deposits
but unlike the blood microglia, they are not able to clear
amyloid plaques. This observation seriously suggests
that the blood-derived microglia are specific species of
the brain immune cells better capable of removing the
amyloid plaques and possibly further preventing the
plaque formations.
Simard et al. (2006) observe that as Aβ deposits
associate with bone marrow-derived microglia, an
immune response is elicited. Astonishingly, this
response happens to be concurrent with the decrease
in the size and the number of amyloid plaques. As
such, blood-derived microglia draws a beneficial
mechanism. It is a converse of the response that
resident microglia produce in which dangerous
proinflammatory cytokines secrete.
Past in vivo experiments have shown that resident
and blood-derived microglial cells are not distinguished
in their function. Usually, these studies comment on
the idea that microglia are incapable of phagocytosing
amyloid plaques. However, Simard et al. (2006) make
an attempt to clear up the confusion that exists in
explaining the role of microglia. There are two types of
microglia: resident microglia and blood-derived
microglia. The latter perform a beneficial mechanism for
the cell by carrying out phagocytosis and protecting the
central nervous system from a neurodegenerative
disease.
In the last decade, therapeutic methods of
preventing and curing AD have failed. In the Simard et
al. (2006) study, the bone marrow stem cells shine on a
novel strategy of eliminating amyloid plaques to
possibly treat AD patients (Figure 1). There is a strong
belief now that the prospect of treating Alzheimer’s
Disease will come from learning more about how the
immune response plays a role in the degenerative
process (Monsonego and Weiner, 2003).
Lokesh Kukreja*
Lake Forest College
Alzheimer’s
disease
(AD)
is
an
incurable
neurodegenerative disease and is the most common
cause of dementia that affects elderly people (Izenberg,
2000). Today as citizens are living longer, AD is
reaching epidemic proportions with no cure available
(Tanzi and Bertram, 2005). In America, 4 million people
are affected by this disease and it is estimated that the
epidemic will jump 44 percent by the year of 2025
(Medline Plus, 2006). AD patients live debilitating lives
of faulty memory, judgment, and reasoning (Tanzi and
Bertram, 2005). However, a promising study published
in Neuron by Simard et al. (2006) suggests a future
therapeutic strategy. They employ stem cells that
specifically target the amyloid deposits, the toxic culprit
in this disease pathology.
When Alzheimer’s disease strikes the brain, it
makes three main aberrant structural changes. One
change is the extensive loss of neurons in the
hippocampus and neocortex. The second change is the
accumulation of intracellular protein deposits called
neurofibrillary tangles. The third change is the
accumulation of extracellular protein deposits termed
amyloid (Αβ, also called senile plaques, surrounded by
damaged neurites (George-Hyslop and Westaway,
1999).
The build up of Aβ in the brain is considered to be
a major cause toward AD pathogenesis. Aβ i s naturally
produced by the breakdown of a bigger protein called βamyloid precursor protein (APP). However, in AD
patients, the problem occurs when the APP is mutant.
The mutation progresses the production of too much Aβ
(Hardy and Selkoe, 2002).
Thus far, the research on amyloid plaques has
shown that they influence significant immunological
changes in their cellular environment. When Aβ
deposits build up, they elicit an innate immune
response on the central nervous system (CNS)
(Monsonego and Weiner, 2003). Specifically, they
activate the microglia, which are the immune cells of
brain. The inflammatory response triggers the microglia
to surround the amyloid deposits. This behavior of
microglia immune cells has been observed in the rodent
transgenic model of AD (Malm et al., 2005).
Interestingly, the scientific community is in a
debate over the role of microglia in Alzheimer’s
Disease. Since there is a large amount of microglia in
the diseased brain, they must, undisputably, play an
important pathological role. (Rogers et al., 2002).
Studies show activated brain microglia to have the
capacity to be either potentially neurotoxic or beneficial
to the brain. In test tube studies, when cultured
microglial cells encounter Aβ peptides they trigger an
immune response and secrete high levels of
proinflammatory cytokines. However, the secretion of
cytokines in the brain would be fatal. (Rogers et al.,
2002). On the other hand, test tube studies also show
that the microglial cells play a positive role. They clear
up cellular debris and certainly are capable of clearing
Aβ deposits by phagocytosis (Wegiel et al., 2004).
*This paper was written for BIOL346, taught by Dr. Shubhik DebBurman.
25
Figure 1: Blood-derived microglia specifically target amyloid plaques for elimination by phagocytosis.
Monsonego, A. and Weiner, H. L. (2003). Immunotherapeutic
Approaches to Alzheimer’s Disease. Science 302: 834-838.
Since the activation of blood-derived microglial
cells create an immune response which reduces the
size and the number of amyloid plaques, these
microglial cells may be the key for AD therapy.
Rogers et al. (2002). Microglia and InflammatoryMechanisms
in the Clearance of Amyloid -PeptideGlia 40:260–269.
the author.
Simard et al. (2006). Bone Marrow-Derived Microglia Play a
Critical Role in Restricting Senile Plaque Formation in
Alzheimer’s DiseaseNeuron 49, 489–502.
St. George-Hyslop, P. H. and Westaway D. A. (1999). Antibody
clears senile plaques. Nature 400: 116-117.
Tanzi, R. E. and Bertram, L. (2005). Twenty Years of the
Alzheimer’s Review Disease Amyloid Hypothesis:A Genetic
Perspective. Cell 120: 545–555
References
Wegiel, J., Imaki, H., Wang, K.C., and Rubenstein, R. (2004).
Cells of monocyte/microglial lineage are involved in both
microvessel amyloidosis and fibrillar plaque formation in APPsw
tg mice. Brain Res. 1022, 19–29.
Alzheimer's Disease. (2006). MedlinePlus. U.S. Library of
Medicine and NIH
Accessed October 23rd , 2006.
http://www.nlm.nih.gov/medlineplus/alzheimersdisease.html#ov
erviews
Hardy, J. and Selkoe, D. J. (2002). The Amyloid Hypothesis of
Alzheimer’s Disease: Progress and Problems on the Road to
Therapeutics. Science Review: Medicine 297: 353-356.
Izenberg, N. (2000). Human Diseases and Conditions. Volume
1 A-D. Charles Scribner’s Sons, New York.
Malm et al. (2005). Bone-marrow-derived cells contribute to the
recruitment of microglial cells in response to beta-amyloid
deposition in APP/PS1 double transgenic Alzheimer mice.
Neurobiol. Dis. 18, 134–142.
Kim, S. U. and Vellis J. de. (2005). Microglia in Health and
Disease Journal of Neuroscience Research 81:302–313.
26
26
Dissecting the Ethical Brain
biomedical world have been debating. Gazzaniga
initially embarks upon the notion that consciousness is
the pièces de résistance of human life itself; without a
brain you are unable to sustain a conscious life and
therefore, undeserving of the moral status of a human
(23). Through a detailed synopsis of the path to a
conscious life, Gazzaniga is able to genuinely convey
his belief that embryo research has validation on the
basis of good intention and only during the preembryonic stage.
Gazzaniga elucidates the immense
apprehension that commonly follows scientific
progress,especially in gene and brain enhancement,
rationalizing that the notion of hyperagency is
misplaced and that the extremes such as the
humanzee are often something of science fiction.
Gazzaniga acknowledges the possibility of negative
side effects, but reminds us that “ in the end, we
humans are good at adapting to what works, what is
good and beneficial, and in the end, jettisoning the
unwise, the intemperate, the silly and self-aggrandizing
behaviors that will always be present in certain
proportions in our species” (53). The Ethical Brain
provides an insightful testimony for the enhancement of
the human brain using precedents as well as
substantiating evidence in a fluid argument that carries
itself.
The complex judicial system, which is based
on recollection and testimony, may be forever changed
from recent understanding of how the brain works.
Gazzaniga relays the flaws associated with memory
and suggests innovative brain scanning and brain
fingerprinting as a possible alternative to incriminate or
acquit a defendant. Gazzaniga makes the stunning
revelation that each person is responsible for his or her
actions, indicating that the insanity plea holds no value
in a courtroom. He explains, “brains are automatic,
rule-governed, determined devices, while people are
personally responsible agents, free to make their own
decisions” (90). The author exemplifies the possibility
that soon the fate of a defendant may not lie in the
deliberation of the courtroom but at a click of a button.
Gazzaniga enthralls the reader with riveting accounts of
endless possibilities the field of neuroscience has
brought upon the judicial system at the turn of the
twenty-first century. Distinguishing from whether or not
a defendant was associated with a terrorist group or
crime scene, by means of brain fingerprinting, has the
reader drooling for more.
The Ethical Brain controversially reassesses
our position on moral beliefs, particularly on our
religious beliefs. The author claims that humans react
to an event, interpret it, and from their interpretation
beliefs emerge about rules to live by (146). Startling
evidence has shown that religious visions or “religiosity”
could have an organic basis within the normally
functioning brain. Gazzaniga is quick to point out that
the temporal lobes are active during the perception of
intense religious experience and during auditory
hallucinations. He adds that disruption of this region by
electrical stimulation, epilepsy, or
overexcitement,
might cause such out-of-body experiences (161). As a
result, this new evidence introduces a gripping reality
for society and possibly the way we may view religious
beliefs.
The Ethical Brain is a lively confrontational
and thought-provoking book about the world of
Benjamin Bienia*
Lake Forest College
By Michael S. Gazzaniga
Dana P (2005)
On September 12, 2006, Edmund D. Pellegrino,
Chairman of The President’s Council on Bioethics said,
“To advance human good and avoid harm,
biotechnology must be used within ethical constraints.
It is the task of bioethics to help society develop those
constraints and bioethics, therefore, must be a concern
to all of us.”
Evolution through natural selection has
endowed our species with the innate capacity to
process information by the use of our brains. Through
natural gene selection, our ability to process this
information varies from individual to individual. What if
the genome dictating the variation of an individual’s
intellect, athletic ability, or even personality, can be
enhanced by pharmaceuticals or brain therapy? How
can we differentiate between an embryo and human
life? When do powerful brain imaging technologies, that
can literally “read” you brain, cross the abstract line of
an individual’s privacy and right to self? How do we
diffuse the gray cloud that surrounds ethics today?
Michael S. Gazzaniga, an outspoken member of the
President’s Council on Bioethics, may not have all the
answers, but he provides much insight in his critically
acclaimed book, The Ethical Brain. This thrilling eyeopener helps us debunk many medical ethical
dilemmas our society has come to face in recent years
with insightful developments in the field of
neuroscience.
Gazzaniga, a world-renowned neuroscientist,
argues that the field of neuroethics alleviates much
uncertainty about the arbitrary limitations imposed on
life.
He explains that through a scrupulous
understanding of how the brain and its underlying
mechanisms work, humans will be able to pursue a true
set of universal ethics. According to Gazzaniga, “ it is
the job of neuroethics to use what we know about how
the brain works to help better define what it is to be a
human and how we can and should interact socially”.
The Ethical Brain helps define the intangibles that
encompass ethical dilemmas through his exceptional
understanding of the brain mechanisms in an easily
digestible manner for the reader.
What marks the beginning of human life?
This has been the million dollar question stem cell
researches, policy makers, and the rest of the
* This paper was written for FIYS 106 Medical Mysteries, taught by Dr.
Shubhik DebBurman.
26
neuroethics and its solutions to numerous social
problems. Gazzaniga illuminates scientific findings in
this enjoyable read in hopes that it will write a new page
in the understanding of bioethics. After reading the
book, one walks away with not only academic merit but
with a greater sense of self. This father of cognitive
science will have you basking in his fruit of enjoyable
scientific discovery and understanding.
References:
Gazzinaga, Michael. The Ethical Brain. New York: Dana P, 2005.
"The President's Council on Bioethics." Bioethics. 12 Sept. 2006
<http://www.bioethics.gov/topics/neuro_index.html.>
27
News and Views
α-Synuclein, and the Case of the Blocked ER-Golgi Pathway
pathway. In order to accomplish this task, they took
two approaches; one genetic and the other cellular.
Together these different pathways would converge to
implicate αSyn in the blocking of ER-Golgi traffic and
cell death.
αSyn was expressed in yeast and regulated
with a galactose inducible promoter.
After αSyn
expression, ER stress was measured and found to be
increased for cells expressing αSyn-WT and further
increased for the familial mutant αSyn-A53T. Lindquist
et al. (2006) hypothesized that αSyn was causing ER
stress by blocking the function of endoplasmic reticulum
associated degradation (ERAD). As misfolded proteins
accumulate in the ER, the ERAD process functions by
retrotranslocating them back into the cytoplasm for
proteasomal degradation (McCracken and Bdodsky
2006). They found that out of two commonly misfolded
proteins in the ER, CPY and Sec61-2p (both ERAD
substrates), the rate of CPY degradation decreased
even though proteasomal function was unaltered.
Interestingly, Caldwell et al. (2001) demonstrated that
ERAD degradation of CPY required transport through
the Golgi.
Because the failure of the ERAD
translocation through the ER to the Golgi during αSyn
expression may be an indicator of general pathway
blockage, Lindquist et al. (2006) hypothesized that
αSyn may be blocking ER-Golgi traffic. To determine if
this was the case, they followed two proteins, CPY and
ALP, through the ER-Golgi circuit when αSyn was
expressed. Within three hours, ER-Golgi traffic was
greatly reduced and at four hours nearly nonexistent.
Simultaneously, cell growth inhibition also occurred.
Thus, αSyn blocks ER-Golgi traffic (2006).
Following their cellular approach, Lindquist et
al. (2006) initiated a genetics approach aimed at
determining if genes that enhance ER-Golgi transport
could reduce αSyn’s ability to block the pathway. They
identified the yeast protein Ypt1p as a promoter of
traffic, and Gyp8P as a suppresser of traffic. This
finding led Lindquist et al. (2006) to hypothesize that
over-expression of the Ypt1p (yeast) or Rab1
(mammalian) in a variety of models would rescue them
from αSyn toxicity.
This final study yielded profound results that
provided the strongest evidence, yet, that αSyn’s
impairment of ER-Golgi traffic was the source for
toxicity. They overexpressed Rab1 along with αSyn in
Drosophila melanogaster (fruit fly), C. elegans (worm),
and mammalian dopaminergic neurons to determine if
Rab1 would prevent αSyn toxicity by enhancing ERGolgi traffic. In all three models, the cells were rescued
from death when overexpressing Rab1.
As a result of Ypt1p/Rab1 re-establishing ERGolgi traffic, it was hypothesized that αSyn interacted at
the ER-Golgi junction. This was based on two lines of
evidence; 1) CSP requires transport into the Golgi to be
degraded and 2) Ypt1p/Rab1 functions within the ERGolgi vesicular binding pathway. Therefore, when
Ypt1P/Rab1 is over-expressed, vesicular binding
efficiency increases.
Returning to the Gosavi et al. (2002) and Lee
et al. (2005) manuscripts, the Lindquist et al. (2006)
data provides two established lines of evidence (i.e.
detailed previously) supporting the Gosavi et al. (2002)
Michael White*
Lake Forest College
Summary
Parkinson’s disease has long been associated with
Lewy Bodies composed of the protein α-synuclein.
A groundbreaking new study has demonstrated the
pathological function of α-synuclein may be
impairment of ER-Golgi traffic.
Introduction
Parkinson’s disease (PD) is a fatal neurodegenerative
disorder of the brain. It affects 1 in 100 individuals over
the age of 60 of which 5-10% of cases occur in
individuals under 40, and another ~5-10% are familial
(NPF, 2006). PD is the result of neuronal atrophy
within the substantia nigra located in the brain stem.
The substantia nigra is part of a complex circuit called
the basal ganglia. It is responsible for the initiation of
movement (Purves et al., 2004). The hallmark feature
of PD is neurofibrillary inclusions, Lewy Bodies,
composed primarily of the protein α-synuclein (αSyn;
Spillantini et al., 1998). Familial forms of PD have been
linked to the αSyn mutations A30P (Krueger et al.,
1998), A53T (Polymeropoulos et al., 1997), and
recently E46K (Zarranz et al., 2004). However, the
reason these cells are dying in PD patients remains
unknown even after more than a decade of heavily
funded research!
αSyn’s pathological component has often
been associated with its role in Lewy Bodies. One
widely accepted hypothesis is that αSyn is pathological
when in a protofibrillar form that occurs between
monomeric αSyn disappearance and Lewy Body
appearance (Lansbury et al., 2003). However, a
remarkable new manuscript, “α-Synuclein Blocks ERGolgi Traffic and Rab1 Rescues Neuron Loss in
Parkinson’s Models”, by Lindquist et al. (2006) has
demonstrated that the pathogenicity of αSyn may be
due to the impairment of ER-Golgi traffic, resulting in a
halt of critical cellular secretory processes.
Prior to their research, little was known about
α-Syn’s relationship with the ER-Golgi pathway.
However, αSyn expression led to the fragmentation of
the Golgi apparatus (Fujita et al., 2006 and Gosavi et
al., 2002). Notably, Gosavi et al. (2002) found Golgi
fragmentation to occur before Lewy Body formation but
after the disappearance of monomeric αSyn. Contrary
to the αSyn-Golgi interaction, Lee et al. (2005) revealed
αSyn to be excreted from the cell via a vesicular, ERGolgi independent, exocytotic pathway. Thus, debate
exists over which pathway αSyn is involved in.
The Case of the Blocked ER-Golgi Pathway
In the recent Lindquist et al. (2006) study, they wanted
to determine the effect of αSyn on the ER-Golgi
*This paper was written for BIOL 493 Independent Study taught by Dr.
Shubhik K. DebBurman.
27
Endoplasmic Reticulum (ER)
(-)α
αSyn
(+)α
αSyn/(+Ypt1p/Rab1)
(+)α
αSyn
Cell Death
Traffic
Blocked
Golgi Apparatus
Figure 1: Rab1 enhances ER-Golgi vesicular binding affinity. This diagram portrays the ER-Golgi junction and the vesicular
transport that occurs between the two organelles. (+) indicates the presence of the indicated protein and (-) indicates its absence.
The black circles represent a vesicle full of cargo (ex. CPY), the red circles αSyn, and the blue cross Ypt1p/Rab1. αSyn blocks ERGolgi traffic and leads to cell death (far right). Overexpression of Ypt1p/Rab1 rescues cells from atrophy by increasing the affinity of
the vesicle for the Golgi (middle). Vesicular transport without αSyn is shown on the left.
References
conclusion that αSyn interacts with the ER-Golgi to
yield toxicity. Though αSyn is continuously being
secreted through a Golgi-ER independent pathway (Lee
et al., 2005), it is plausible that a defect in this excretory
system may function to exacerbate toxicity, but not
produce it.
Caldwell, Sabrina R., Hill, Kathryn J., and Cooper, Antony A.,
Degradation of Endoplasmic Reticulum (ER) Quality Control
Substrates Requires Transport between the ER and Golgi,
Journal of Biological Chemistry, volume 276, no. 26, pages
23296-23303, 2001.
Fujita, Tukio et al., Fragmentation of Golgi apparatus of nigral
neurons with α-synuclein-positive inclusions in patients with
Parkinson’s disease, Acta Neuropathol, volume 112, pages
261-265, 2006.
Future Research
The Lindquist et al. (2006) manuscript has provided the
Parkinson’s disease community with what appears to
be an opened door, leading to a whole new frontier in
PD research and understanding. As with the relentless
pursuit of the protofibrillar discovery by Dr. Lansbury, all
methods of research must be exhausted on finding the
mechanism by which αSyn is able to turn off the ERGolgi pathway. It is feasible that the same lentivirus
used by Lindquist et al. (2006) to carry the Rab1 gene
into mammalian neurons in their experiments could be
re-configured to enter the cells of PD patients and reestablish traffic between the ER and Golgi. If this is, in
fact, the reason these cells are dying, one of the most
prevalent and debilitating neurodegenerative diseases
could be cured.
Gosavi, Nirmal et al., Golgi Fragmentation Occurs in the Cells
with Prefibrillar α-Synuclein Aggregates and Precedes the
Formation of Fibrillar Inclusion, Journal of Biological Chemistry,
volume 277, no. 50, pages 48984-48992, 2002.
Lansbury, Peter Jr., and Volles, Michael J., Zeroing in on
the Pathogenic Form of α-Synuclein and Its Mechanism
of Neurotoxicity in Parkinson’s Disease, Biochemistry,
volume 42, no. 26, pages 7871-7878, 2003.
Lee, He-Jin et al., Intravesicular Localization and Exocytosis of
α-Synuclein and its Aggregates, Journal of Neuroscience,
volume 25, issue 25, pages 6016-6024, 2005.
Lindquist, Susan et al., α-Synuclein Blocks ER-Golgi Traffic and
Rab1 Rescues Neuron Loss in Parkinson’s Models, Science,
volume 313, issue 5785, pages 324-328, Epub 2006.
the author.
McCracken, A., and Brodsky, J., Recognition and Delivery of
ERAD Substrates to the Proteasome and Alternative Paths for
Cell Survival, CTMI, volume 300, pages 17-40, 2006.
PD Statistics Provided by the National Parkinson Foundation.
Retrieved
on
2
September
2006
from
http://www.parkinson.org/site/pp.asp?c=9dJFJLPwB&b=71354.
Purves, Dale et al., Neuroscience 3rd Edition, Sinauer
Associates Inc., Maryland, 2004.
28
Smith, Wanli et al., Endoplasmic reticulum stress and
mitochondrial cell death pathways mediate A53T mutant alphasynuclein-induced toxicity, Human Molecular Genetics, volume
14, no. 24, pages 3801-3811, 2005.
Spillantini, Maria G. et al., α-Synuclein in filamentous inclusions
of Lewy Bodies from Parkinson’s disease and dementia with
Lewy Bodies, PNAS, volume 95, pages 6469-6473, 1998
28
Iris and Awakenings: Timeless Tear-Jerkers
her plan for the future. Bygone events, such as the death
of her girlfriend, don’t weigh her down. Instead, Iris, with
an untarnished outlook towards life, spends every moment
fulfilling one impulse after another. She has not only been
unchained from the shackles that memory creates, she
has been liberated from the pressures of working towards
a goal. This is evident in the scene when Iris lifts the rocks
she had placed on her papers, which contained the rough
beginning to her novel, and lets them blow away.
Activities aimed at achieving future gain forestall us from
living in the present. Planners, time tables, and calendars
rob us of flexibility of time that could be spent in
enjoyment. Life is simply too short to waste.
Iris and Awakenings playfully tackle the concept
of age-appropriate behavior and suggest that life is too
short to stick to formulated rules of what suits somebody at
what age.
Like inquisitive children, Iris repeatedly
questions John Bayley to the point of frustration and
Leonard, at the age of forty, also bombards Dr. Malcolm
Sayer (Robin Williams) with simple questions. Leonard
realizes that he has been given a second chance and he
takes full advantage of it without worrying if his actions
correspond with his age or not. At one point, even Iris is
shown watching the Teletubbies. These scenes challenge
the old notion of age-appropriate behavior and suggest
that age need not restrict one to a set pattern of behavior.
For example, Leonard is attracted to a girl considerably
younger than him, while Iris dates men way over her age.
In Awakenings, other patients, who also reawaken due to
L-Dopa, challenge the concept of age-appropriate
behavior as well. An old woman, after looking at herself in
the mirror, cries out for hair dye. These scenes tie
perception to age and suggest that you are, after all, as
young as you feel. Indeed, life is too short to follow
society’s rules of what is appropriate at what age.
Along with the importance of time, Iris and
Awakenings emphasize the significance of love, friendship
and family. Dr. Malcolm Sayer, initially in the movie
Awakenings, is a lonely guy with little to no social skills.
He is engrossed in science to a level that leaves no room
for company. For example, Dr. Sayer refuses Julie’s
dinner invitation because he has to attend to an
experiment that he is conducting on plants. However, after
he gets to know Leonard, he comes to the conclusion:
That the human spirit is more powerful than any drug
- and THAT is what needs to be nourished: with work,
play, friendship, family. THESE are the things that
matter. This is what we've forgotten - the simplest
things. (“Memorable” 2)
This socially inept and shy doctor, even though hesitantly,
asks Julie out. Iris too, subtly advocates that love is the
language that ought to be spoken by all.
However, unlike Awakenings, Iris suggests that
the physical aspect of life is just as important. We need to
find a balance between lust and love. When Iris and John
are kissing each other’s hand, John says that as a married
couple, they could be doing this all the time. Iris corrects
him and says that perhaps not all but nearly all the time.
The theme of balance is introduced.
Throughout the film, Iris is in touch with her inner
animal while John Bayley is simply a product of society.
Iris has unshaved armpits and wrinkled skin. Yet she does
not resort to cosmetics because she is comfortable within
her own body. She is open to lesbianism and promiscuity
– attitudes that show her instinctual nature. However,
John Bayley is a figure of formality. Together these
Mohammed Ejaz Ali*
Lake Forest College
Iris (2001 film) Directed By Richard Eyre
Awakenings (1990 film) Directed By Penny Marshall
To sum up my classmates’ reactions to Iris and
Awakenings: that was so cool. However, these movies
provide more than just sheer visual pleasure. They supply
food for thought. Provocative in several arenas, Iris and
Awakenings are like potent cannonballs that shake you to
your core. Moreover, the acting is simply impeccable. In
fact, several actors were nominated and given Academy
Awards for their heart-rending performances. Iris and
Awakenings are two of those rare films that effectively
balance comedy and drama. Truly, as films, they are
simply successful.
Iris vividly narrates the tale of the enduring love
between noted British philosopher and author Iris Murdoch
(Kate Winslet and Judie Dench) and college professor
John Bayley (Jim Broadbent), love that surpasses even
the hurdles created by Alzheimer’s. Awakenings portrays
the miraculous return of Leonard Lowe (Robert De Niro)
from a state of paralysis brought about by the
neurodegenerative encephalitis. Despite their differences,
these movies are remarkably similar and parallel.
Iris and Awakenings both advocate that one
should seize the day (carpe diem) while it lasts. This is
explicitly conveyed by the frequent introduction of the
concept of time. These movies highlight the point that one
should not let one’s future plans or past rule him or her,
but instead should always live in the present.
As
Alzheimer’s progresses, Iris Murdoch forgets her past and
*
This paper was written for FIYS 106 Medical Mysteries: Neuroscience in
Chicago, taught by Dr. Shubhik K. DebBurman.
29
contrasting characters emphasize the importance of
establishing balance.
At one point in the movie, a cat is shown hissing
at a fox and to Iris’s utter disappointment, the fox leaves.
This scene also symbolically points out the conflict
between formality and informality.
Cats are tamed
creatures while foxes are simply pure beasts.
Metaphorically it basically suggests that society unjustly
dominates us. Even in the subtle scene when John is
reading a passage to Iris from Pride and Prejudice, this
conflict is highlighted because the novel Pride and
Prejudice by Jane Austen is a Victorian classic that
exposes the sexual repression ensued by the prevalent,
overbearing, straitlaced, societal etiquettes and deals with
the conception of a balance between love and lust. It
prompts the audience to imitate the fashion in which the
characters in the book challenge, cross, and abide various
dictatorial societal customs and establish a baseline for
themselves. The nudity in the movie also renders the
point that while taking into considerations the pressures
created by the puritanical society, one must not stifle his or
her wild, untamed, corporeal, earthly instincts. Iris makes
one revaluate his or her priorities and ultimately settle the
conflict between society and individuality.
Thematically powerful, Iris and Awakenings stir
one at various levels.
They educate the social
consequences of having a member of one’s family suffer a
fatal neurodegenerative disease. Although the biological
aspect of the diseases – Alzheimer’s and encephalitis –
are not stressed enough, Iris and Awakenings do a fair job
of highlighting the neurological and behavioral changes
that accompany the diseases. Moreover, the insufficiency
of biological substance is substituted by the presence of
ample social consequences of the diseases. Iris runs at a
much slower pace than Awakenings and the presence of
elements such as homosexuality, nudity, and promiscuity
make it all the more controversial while Awakenings is a
simple, tender, love story with emotional ups and downs.
Hence, I would recommend Awakenings to all but
cautiously recommend Iris to all above the age of
seventeen.
References
Memorable Quotes from Awakenings. 2 Oct. 2006.
<http://www.imdb.com/title/tt0099077/quotes>
30
Ferocious Beauty: All Roar and Very Little Bite
grace that is sufficient to catch the attention of even the
most skeptical observers. With impressive diversity,
the cast performs a wide variety of dance routines,
placing their dancing prowess on display for the general
public to see. In the eyes of some, the fluidity of the
dancer’s movements may more than make up for the
performance’s lack of educational value.
On the whole, I believe that the creators of
the show achieved the finished product that they had
aimed to achieve when in the process of producing their
gift to the sophisticated world. With that said, any
viewer who should happen to attend a future
performance with the hopes of gaining a meaningful
educational experience should think twice before
dropping twenty dollars at the door. However, for the
prospective viewer who aspires to lay eyes upon an
impressive dance performance, the ticket is well worth
its cost. When the performance had ended on the
evening of Thursday, September 28, this viewer felt that
there was definitely no need for an encore.
Pete Wisnieff*
Lake Forest College
Liz Lerman Dance Exchange’s interpretive dance
performance Ferocious Beauty: Genome has come and
gone from Chicago’s Museum Of Contemporary Art.
The performance was given nightly from September 28
through September 30 and opened before a large
audience on its first night. A cast of dancers as diverse
as the dances they perform place the role of the human
genome on display from the very beginning. A unique
production, the experimental piece attempts to educate
its audiences about the nature of the human genome
and the ethical dilemmas that loom in the near future as
imminent advances in the study of genetics come with
ever increasing ethical implications. In an attempt to
help the average audience member attain a better
knowledge of a complicated subject matter, the group
decided to present the material through both short
video clips and long sequences of dance.
After sitting through the entire performance, I
was left with the impression that the performance fell
short in one of two ways. Either the piece failed to
delve deep enough into the subject to make the
performance worthwhile, or interpretive dance is simply
too vague to successfully educate the masses about
the nature of such a concrete scientific phenomenon.
Despite the fact that dance was not a viable
way to illuminate the nature of the genome, the short
video clips were to the point and proved to be very
informative. Unfortunately, they were short video clips.
These abbreviated video information sessions made up
only a small portion of the performance and left the
viewer desiring more. When all was said and done,
Ferocious Beauty: Genome provides only a very
shallow view of the nature of the genome and continues
to present a decidedly one-dimensional view of the
ethical issues associated with it.
There is a chance however, that I am wrong
to jump all over this apparent shortcoming. Perhaps
the presentation’s lack of depth is intentional; perhaps it
is intended to leave the audience wanting more, and
thus promoting continued awareness of genetic
research. If this is the case, then they have succeeded
and the video clips serve their purpose admirably.
Either way, the amount and quality of the information
provided by the video clips is sufficient and cannot be
blamed for the feeling of emptiness I felt as I headed for
the exit following the performance’s welcome
conclusion.
One thing is for certain, however, interpretive dance is
not a successful vehicle for educating the public on the
mysteries of the human genome. As a rule, interpretive
dance is intriguing and thought provoking; but, by
definition, it cannot provide concrete answers. For
instance, in almost all sciences, the study of genetics is
extremely concrete and requires definitive answers.
This is a need that interpretive dance just cannot fulfill.
The one saving grace of the whole
performance is that the dancing is advertised.
Throughout the show the dancers on stage move with a
*
This paper was written for FIYS 106 Medical Mysteries: Neuroscience
in Chicago, taught by Dr. Shubhik K. DebBurman.
31
Fact and Fantasy: The Beak of the Finch by Jonathan Weiner
were to be repeated over many generations, we may
see a new species of birds with larger beaks.
This leads the reader to question the idea of
speciation, or how new species arise. Why are there so
many species of finches and what did they evolve
from? Weiner quotes Thomas Henry Huxley saying
that, “A race does not attract our attention until it has, in
all probability, existed for a considerable time, and then
it is too late to inquire into the conditions of its origin.”
Thankfully, the Grants noticed Darwin’s finches in time
to learn so much.
Many may argue that Darwin never
addresses this in his Origin of Species. However,
Darwin does describe the idea of speciation. He never
claims that natural selection is the only mechanism
working in evolution to create new species. Over time,
as natural selection tinkers with the variations within a
population, adaptations accumulate. Eventually, after
so many adaptations have built-up, whether between
two separated populations or within a single population,
a new species is observed.
These ideas apply to all species of organisms,
including plants, as well as us. Weiner uses the story
of Cleopatra to demonstrate this. “If Cleopatra’s nose
had been shaped a little less like the Grecian ideal, and
a little more like Cleopatra’s Needle, there would have
been no Alexandrine War, no sea-fight at Actium. The
whole arc of the Roman Empire would have been
reshaped by Cleopatra’s beak.” One day, Homo
sapiens may be obsolete and some species of Homo
somethingorother may be the dominant species on the
earth. It is important to study both the process and its
relation to our lives.
Analogies like these allow a reader, who may
not be adept in the sciences, to clearly comprehend the
process of evolution. Weiner’s method of alternating
the Grant’s story and Darwin’s principles of evolution,
with non-science philosophies, succeed in driving his
ideas home. While some readers may find certain
ideas familiar, they will find them juxtaposed with an
exceptionally engaging story.
Time and time again, Weiner revisits Daphne
Major with the Grants and their colleagues. Every time,
slightly more of the island and the puzzle are revealed.
One drought, one rainy season, hybrids, beak
variations, adaptations, the list continues today as more
people study Darwin’s finches. Each occasion the
Grants visit the island, Weiner makes it seem as though
instead of being on scientific study, they are there for
pure adventure.
We forget that the finches are the exemplar
of Darwin’s theories and begin to think of them as a
child thinks of the peacock at the zoo. They are not a
spectacle of science, just a spectacle. Weiner is quick
to remind us, though, that they are indeed spectacles of
science. And extraordinary ones at that; the finches
show us that evolution is not unidirectional as
previously thought, but bi-directional and capable of
reversing itself.
An adaptation that is beneficial to one
generation may be a hindrance to the next. In turn, the
variation that led to the most survivors in the previously
generation, such as large beak size, may be the
downfall of the next generation. This shows that life
itself is more evolutionary than we thought. Weiner
says, “The closer you look at life, the more rapid and
Michelle McKinzey*
Lake Forest College
By Jonathan Weiner
Vintage (May 30, 1995)
The sun sets on a small island off the coast of South
America. Thirteen species of Galapagos finches settle
in their various homes for the evening. Peter and
Rosemary Grant along with their crew settle in, as well,
and you feel like you are right there with them thanks to
Jonathan Weiner.
Weiner presents the story of
Darwin’s finches and the Grants as if it were just that: a
fabulous story. It’s no wonder, however, that Weiner’s
book is a Pulitzer Prize winner.
Along with the Grants’ tale, Weiner ties in
Darwin’s theories of Evolution and Natural Selection,
examples outside of the finches, and even philosophy
making the book both a key source of public
understanding and an entertaining read.
Weiner reconstructs the research of the
Grants’ twenty years after they first discover their
amazing finches and follows them back and forth
through time in his interviews. While the Grants and
their colleagues struggle to discern exactly what the
finches mean, we struggle along with them. Bit by bit,
Darwin’s finches reveal to the Grants that evolution is
not a slow process, in so doing; other common
misconceptions about evolution are cleared up.
Weiner presents the argument that perhaps
the finches, because they can still interbreed and
produce viable offspring, are in a sort of transitional
phase. They are separate species but are recently
diverged from a common ancestor and are constantly
evolving. (This was first seen by Darwin, which is why
they are termed Darwin’s finches.)
Had it not been for the periods of drought and
excessive rain, we may not have realized that evolution,
the process of change in organisms, need not be slow.
The gene pool of the finches varies from generation to
generation depending upon their environment. Each
successive generation is consequently better suited for
their environment.
For instance, in periods of drought, birds with
larger beaks are better able to survive and reproduce,
thus the next generation has, on average, a larger beak
size and a better chance of survival. If this
* This paper was written for an independent study with Dr. Anne Houde.
33
intense the rate of evolutionary change. The further
back in time you stand, the less you see.”
It’s easy to see that Weiner researched this
subject very well. Nonetheless, the book can feel a
little drawn out. Sometimes, the reader may wonder at
the connections between a sub-topic and Darwin, but
with a little further reading and brainpower, the
haziness soon vanishes. The timeline can seem a little
muddled at times, but may be for clarity and aesthetic
purposes.
Despite these few minor qualms, Weiner
does prove, in essence, his statement that, “The beak
of the finch is an icon of evolution the way the Bohr
atom is an icon of modern physics, and the study of
either one shows us more primal energy and internal
change than our minds are built to take in.”
It’s clear that Weiner’s aim is to further
impress the knowledge of evolution as well as entertain
the reader. He succeeds. For those of us who already
know the outcome, we are immersed in a wonderful
story.
For those concerned with understanding
evolution, there is much to be learned from this book.
the author.
34
Healthy Marketing: The Only Solution
he felt well by any stretch of the imagination. Morgan’s
cholesterol went from 165 mg/dL at his first weigh-in to
225 mg/dL after the third weigh-in; one of the doctors
said that anyone would say that Morgan is sick.
The doctors concurred that Morgan was at
risk of liver failure, which usually is a product of
alcoholism, not a high-fat diet. By day 21, Morgan woke
up in a hot sweat with difficulty in breathing. One of the
physicians stated that Morgan needed to stop, because
his liver resembled that of an alcoholic. Morgan
continued despite pleas from his girlfriend and
physicians to stop the, “McDiet.” In his final
assessments, Morgan weighed 210 lbs–24.5 lbs
heavier–and increased his body fat from 11% to 18%.
When Morgan returned to a normal diet and exercise it
took him 5 months to lose 20 lbs and 9 months to lose
4.5 lbs. This one-month test of a McDonald’s diet
proved that McDonald’s food definitely has adverse
effects on health.
In the American society, we seem to be
having a difficult time digesting the fact that our own
family members are overly obese. We look for escape
routes when we are pinned in the corner of being
labeled overweight from proclaiming that the Body
Mass Index is a faulty measuring tool, to trying every fat
burning pill on the market. In an interview in Super Size
Me, Dr. David Sattch stated, “We live in an environment
that makes us sick, a toxic environment” (Sattch 2004).
The environment that Dr. Sattch is referring to is the
trend of replacing home cooked meals to on-the-go fast
food that is pumping our arteries full of saturated fat.
The marketing teams of fast food and junk food
manufacturers are undoubtedly the cause of this drastic
transgression.
There are over 10,000 advertisements a year
that are targeted at the youth of America. Since the
early days of Michael Jordan endorsing Big Macs, the
healthy food industry has been matched to an unfair
fight with the fast food and junk food industries. To
show the considerable variation in available capitol
between marketing of healthy foods and fast food,
consider that McDonalds spent 1.4 billion dollars on
advertising in 2005, while healthy marketers were able
to dig into their pockets for a meager 2 million dollars.
Such disparity is evident in the way that fast food faces
like Ronald McDonald, the playpens in fast food
establishments, and Happy Meal toys are all unfair
ways in which the fast food industry gains the upper
hand in parental food choices and overcomes the small
healthy food market. These marketing tactics have
allowed being overweight a socially acceptable idea.
The fact is that pressuring someone to quit smoking is
socially permissible, but telling someone to lose weight
is completely out of the question. Maybe it should be
considered impolite to demand another to lose weight,
because according to nationwide statistics, obesity is
about to take over smoking as the number one cause of
preventable deaths in the United States.
The obesity epidemic is unquestionably an
intense dilemma for the youth of the nation, and it all
starts with healthy eating habits at home and school.
Rarely are school lunches made from fresh ingredients,
rather they are reheated packages with disturbing
amounts of preservatives. Food for the majority of
schools is government-issued, which poses an up-hill
challenge in the attempt to offer children affordable,
healthy lunches. This government task of providing
Jason Prendergast*
Lake Forest College
Hart Sharp Video (2004)
Little Jonathon scurries down the aisle in search of his
usual supermarket purchase. As he impatiently tugs at
his mother’s arm, she drops boring food items like
oatmeal and fresh fruit into the cart. After quite some
time, Jonathon’s mother gives him permission to go to
the “fun food aisle.” There, Jonathon drops a cereal box
into the cart with Sponge Bob on the front, and a bag of
chips with his favorite basketball players’ (i.e. LeBron
James) picture on it. These unsafe advertising
strategies are raking in millions of dollars for cereal and
junk food manufacturers. As Morgan Spurlock points
out in his documentary Super Size Me, when young
children are raised to idealize cartoons and athletes
that market unhealthy food, it should be no shock that
the United States is the fattest country in the world.
Morgan’s
unprecedented
documentary
shines light on the unknown effects of regular diets
based solely on the American fast food industry. In his
first-hand analysis, Morgan attempts to tackle an
unimaginable task: eat only McDonald’s for three meals
a day, eat everything that McDonald’s offers on their
menu, and super-size his meal whenever offered the
option to upgrade. In order to affirm that his high fat
“Mac diet” was not going to cause any serious health
concerns, he consulted cardiologists and nutritionists to
evaluate his health and to predict any health risks or
side effects of the diet. The main predictions that the
team of doctors decided was that Morgan would
increase his blood pressure based on high cholesterol
and sodium intakes, gain overall weight, and feel
sluggish and miserable.
Such forecasts by the team of doctors
seemed to be relatively accurate when analyzing
Morgan’s weekly results. After just five days of eating
nothing but McDonald’s food, Morgan gained 10 lbs, he
started feeling pressure on his chest, he felt depressed,
and he could not seem to quell the intense hunger
attacks that he experienced shortly after eating a meal.
Morgan’s second weigh-in put him at 203 lbs with no
apparent side effects. The doctors hypothesized
that his body was beginning to adapt to the intense
increase in fat-intake. Although his health seemed to be
adequate after the second weigh-in, the same could not
be said for the time between the second and third
weigh-ins. When Morgan showed-up for his third health
update, he weighed 202 lbs, but that did not mean that
* This paper was written for BIOL106 Food , taught by Dr. Nicole Sleiter.
35
healthy lunches is a long-term predicament that will
take years to sort out. For that reason, America needs
to achieve short-term success in the obesity epidemic
by turning our attention to snack food marketing. Shortterm incentives for marketing healthy foods seems to
be a step towards slowly changing the nutritional habits
of our youth. Frito Lays has a healthy marketing
scheme that marks snacks that are low in calories and
fat, known as “Smart Choices Made Easy.” Such a
conversion between product concept and healthy
marketing is the only way in which healthy foods have a
chance to compete with the evil empires of the junk
food and fast food industries. While cartoon characters
and sports heroes continue to be brandished on junk
food packages, moms like Jonathan’s will have to
struggle to quickly pass by the unfair marketing
strategies of junk and fast food companies, whether
pushing a cart or driving a car.
the author.
36
The Thin Line Between Madness and Sanity
loss of countless relationships. As a patient, Jamison’s
story is one of a personal struggle to carry on, a
personal struggle to love and to live.
As the doctor, part of Jamison’s struggle was
to keep her illness a secret from those whom she was
supposed to treat, those who just like her, were ill. With
this, several questions arise. Was she capable of
helping others who were ill when she had been, up to
that point, unable to help herself? Dr. Jamison faced
struggles every day of her life, in many dimensions. As
a woman in the field of psychiatry, she had to prove
herself to others. As a manic-depressive she had to
prove to herself that a life without invigorating highs
was better than no life at all. Dr. Jamison had to
convince herself to take the medications. She had to
prove to herself that just as she was capable of helping
others, she could also help herself.
Would others be understanding? Dr. Jamison
was very conscious of the fact that this illness could
have meant the revocation of her clinical license and
the end of her career. Her illness represented a threat
to everything she had accomplished until then. This
meant that she would have to fight to stay at the top of
her field and she was not willing to lose this fight.
Dr. Jamison had a daily battle just to live
normally. She found her sense of normalcy
professionally, by treating others with the disease that
she dealt with, but also found her sense of normalcy in
love. A strong emphasis is made on the need for
acceptance and love. Love from her family, her
husband and later her partners allowed Jamison to be
able to value her life and gave her a reason to want to
get better. Through her stories of love we get the
message of acceptance and understanding that is
needed by those ill.
Jamison describes to the reader her struggle
with a disease which eventually affected her personal
life, her relationships, her family, friends, and her
professional life with coworkers and patients. This book
gives us an insight as to how it feels to be on both sides
of the desk, the healer and the healed. As the disease
and its consequences are described in detail, one feels
almost as if she is speaking directly to you. Jamison’s
story allows us to see the sick person as that, a human
being. This captivating book is written in a way that
anybody, even those with no scientific background, can
pick it up, enjoy it and most importantly, be educated by
it. Along with learning about the ups and downs of
manic depression and becoming informed individuals,
we begin to partially understand what manic depression
is like. We begin to understand where the line between
madness and sanity lies and why in some cases it gets
blurred. With Dr. Jamison’s story, our image of the
mentally ill is changed from one of pity and fear to one
of understanding.
Stephanie Valtierra*
Lake Forest College
By K. R. Jamison
Vintage Books (1996)
As we go about our daily lives we experience many
emotions. We are annoyed by traffic, saddened by bad
news and angered by delays. While most of us quickly
get over what has angered us or saddened us and
continue to go about our day, many others live a very
different life. What would it feel like to go through rapid
mood swings, where one can go from a debilitating
depression to a time filled with invigorating highs?
What would it feel like knowing that for you, there is a
thin line between madness and sanity? Dr. Kay
Redfield Jamison has had a life full of these highs and
lows and was able to tell us her first-hand experience
with disorder that affects millions.
Dr. Jamison tells us the story of her struggle
with manic-depressive disorder, also known as bipolar
disorder, in her memoir, An Unquiet Mind: A Memoir of
the Moods and Madness. Jamison gives us the
perspectives both of the patient and the healer. The
author was born into a good, middle class American
family and she was an intelligent, bright young woman
with aspiration of going to medical school. Mood swings
started plaguing her life as a teen. Jamison did not see
these mood changes or the persistent depressions as
serious problems. She believed that these mood
changes were just another part of her, something by
which she could be defined. The mood swings not only
began to define her, but also connected her to her
father and sister, who also suffered from these mood
swings. The symptoms soon turned into advanced
manias, depressions and suicide attempts. Medication
was an option and could make her life “normal,” but this
meant that she would not experience the invigorating
highs and the accompanying devastating lows. This
highly educated doctor became just another patient.
The many highs and lows of manic
depression did not only affect Jamison, but also
affected those around her. A strong emphasis is placed
of the affects that this disease has on those who live
around and with the ill. The disease lead to the
destruction of her marriage, loss of friendships, and the
the author.
* This paper was written for BIOL346, taught by Dr. Shubhik K.
DebBurman.
37
Review Article
History Tends to Repeat: FMR-1 Silencing in Fragile X Syndrome
Joshua G. Haas*
Lake Forest College
of the FMR1 gene with binding sites and the CpG island
is depicted in Figure 1.
Studies have revealed a significant decrease
in the expression of FMR1 mRNA in fragile X cases as
a result of the hypermethylation of the CpG island (35).
An important feature of repressing of this particular
gene is the presence of FMR1in fetal tissue. The
varying levels of expression between normal and fragile
X fetal tissue reflects a very important role in
development. This data also suggests early acquisition
of the methylation, probably during embryogenesis
(38,40).
Early acquisition of methylation points out the
importance of the loss of FMR1 expression.
The
human FMR1 gene, being highly conserved among
species (16), has been shown to display alternative
splicing properties suggesting the presence of many
isoforms (4). Mouse models have been developed to
study fragile X syndrome due to the allelic similarities
(16). Three different levels of repeat within the gene
have been reported: normal (7-50 repeats), permutation
(50-200 repeats), and full mutation (> 200 repeats).
The class of permutation is interesting because it only
becomes affective in successive generations when the
repeat region has a chance to expand leading to
Shermans’s paradox.
The permutation allele is
unstable when transmitted from generation to
generation.
All males with the full mutation display the
fragile X phenotype where only approximately 50% of
females with the mutation display the fragile X
phenotype (4, 40). Taken together with data from our
more recent studies this proves that the germ line is
susceptible to full expansion (32). There seem to be no
new mutations appearing to cause fragile X syndrome.
The only known mutation of resulting in
fragile X syndrome interestingly shows increases in the
transcription of FMR1 have been proportionally linked
to the CGG tri-nucleotide repeat length and the repeat
number approaches 200 in permutations. As the
number of repeats increases the number of FMR1
mRNA levels increase.
Unexpectedly, the FMR1
protein products decrease in relation to the CGG repeat
length in permutation (25). This finding shows the
development of the disease at various points of repeat
length during the permutation stage of fragile X
syndrome.
Upon extending from the permutation to the
full mutation, the FMR1 gene is silenced. The silencing
of the FMR1 gene is interesting because it remains
unclear as to how the tri-nucleotide repeat expansion
occurs in an organism. Moreover, questions still remain
as to how the loss of the FMR1 protein, FMRP can
have such broad effects as those observed in fragile X
syndrome (21, 40). Studies have started to reveal the
role and importance of FMRP in patient and mouse
models uncovering mRNA binding properties (5,29,17)
Within the past few years we have uncovered
information linking FMRP to protein synthesis (30) and
a micro-RNA dependent translational suppression
pathway (23). In this review we will focus on explaining
the neuronal functions and importance of FMRP in
model systems. We will also propose a mechanism for
both translational and transcriptional repression through
a micro-RNA pathway.
[Role Playing: Steven Warren
Howard Hughes Medical Institute and Department of
Biochemistry and Pediatrics, Emory University School
of Medicine, Atlanta GA 30322]
Abstract
Fragile X syndrome is the most common form of
inherited mental retardation, and afflicts 1 in 1250
males and 1 in 2500 females. The symptoms
include connective tissue displasia, mental
retardation, and macroorchidism (enlarged testis).
My lab discovered that the most common forms of
this disorder are caused by the expansion of CGG
tri-nucleotide repeats on the X chromosome at the
FMR-1 gene locus; an excess of 200 repeats in
diseased individuals suppresses the translation of
FMR-1. The CGG repeat expansion leads to hypermethylation of a CpG island distal to the repeat,
leading to transcriptional repression of FMR-1. This
gene silencing is also aided by RNA interference
and de-acetylation of histones H3 and H4. The FMR1 gene codes for fragile X mental retardation
protein (FMRP), which plays a functional role in
protein translation in neurons. FMRP selectively
binds to specific mRNAs essential to development
of the brain and other parts of the body, and plays a
major role in shuttling its ligands from the
nucleoplasm to the dendritic cytoplasm. FMRP
knockout mice models demonstrate abnormal
dendritic spine growth, suggesting altered synaptic
plasticity, which may be responsible for the fragile
X phenotype. Elucidating the fragile X mechanism
of pathogenesis can aid the development of
possible treatments to the world’s leading cause of
mental retardation.
Introduction
Fragile X syndrome is one of the most prevalent forms
of mental retardation affecting approximately 1 in 4,000
males and 1 in 8,000 females. FMR1 has been
identified as the gene associated with fragile X
syndrome (1, 2, 6). This gene was mapped revealing a
CGG tri-nucleotide repeat in the 5’ non-coding region of
the gene. This region was noted to expand in repeat
length in fragile X syndrome (1). The expansions of the
CGG repeat to levels of 200 repeats or greater is
responsible for the instability of the fragile site on the X
chromosome. Normal cases posses an average of 3050 repeats of the CGG region (27). This instability
results in the hypermethylation of a CpG island distal to
the tri-nucleotide repeat (6,1,26,35). Examination of
fragile X patients has consistently shown that the
hypermethylation of the CpG island is the primary factor
implementing the fragile X phenotype being that
methylation is responsible for approximately 99% of
known fragile X phenotypes (35, 41). A representation
*This paper was written for BIOL 346 Molecular Neuroscience taught by
Dr. Shubhick DebBurman.
41
Figure 1. Molecular Basis of Fragile X Syndrome: A model of the normal FMR1 gene (top) and the fragile X FMR1 gene (bottom)
with the defining difference in CCG repeat number between the two labeled. In the case of repeats >200 (fragile X syndrome), the
CpG island is hypermethylated, histone 3 and 4 experience changes in acetylation, or RNA interference can all inhibit transcription of
the FRM1 gene which codes for FMRP.
Functions of FMRP
X syndrome, a functional role of FMRP was
investigated (38). FMRP was found to be associated
with ribosomes in the dendritic structures of neurons
elucidating a possible role of the proteins related to
dendritic structure and neuronal plasticity (18). The
association of FRMP with polyribosomes was
eliminated in I304N mutations of extreme fragile X
syndrome (19). The tissue specific expression of FMR1
connects the protein to important developmental
structures in the brain such as the hippocampus (21).
A set of FMR1 knockout mouse models were used to
understand the relationship between drendritic
formation and FMRP. Knock out mice were found to
posses irregular dendritic spines (14). The importance
of altered neuronal formation is discussed later.
The next important link connecting neuronal
alterations and the function of FMRP is the evidence
pointing to negative regulation of mRNA translation. By
removing the binding site of FMRP we found a lack of
translational inhibition (30, 31). Specifically, FMRP has
been shown to regulate the production of MAP1B, a
protein. In Futsch mutant mice show inverse regulation
of MAP1B and altered synaptic growth (31, 45). This
shows that FMRP possibly plays a major role in
development of neuronal structure throughout the body.
In fetal tissue, FMRP is found to be most abundantly
and universally expressed in the testis and the brain
(21). The differentiation of neuronal stems cells related
to the fragile X phenotype has shown alteration in
fragile X patients linking the disease to development
(10). FMRP was discovered to assert properties of
RNA-binding proteins in areas of expression (40). The
protein itself encompasses three RNA binding domains,
two K domains and an RGG box (40, 15). Importantly,
the FMRP protein maintains both a nuclear location
signal (NLS) at the amino terminus end of the FMRP
and a nuclear export signal (NES) encoded by exon 14
of the FMRP. These signals help to confirm RNA
binding properties of FMRP by hinting at the possible
translocation of various mRNAs via the FMRP pathway
(17).
In order to bind mRNAs, FMRP forms an
RNP complex containing FMRP, FRX1P, FRX2P,
nucleolin, and three other proteins. The particles
making up human RNPs are conserved in mice as well.
(11). This RNP binds mRNA selectively by associating
the G-quartet structure of the mRNA to the RGG box of
the FMRP (5, 15). The G-quartet structure acts as a
target for FMRP explaining the selective binding
properties and the importance of transcriptional
regulation via FMRP of the targeted mRNAs (15).
FMRP binds directly to mRNAs via this interaction (9).
This direct interaction with mRNA allows
FMRP to act as a translational control factor for many of
these targeted mRNAs. Evidence for this was found
because a large majority of FMRP is associated with
ribosomes, translational machinery (38). Because the
absence of the FMR1 gene was associated with fragile
mRNA ligands associated with FMRP
Because FMRP is shown to play significant roles in
both mRNA transportation and translation, the
characteristics of the specific mRNAs associated with
FMRP have been explored. A variety of mRNAs are
associated with FRMP. One of our recent studies
examined 13 potential candidate mRNAs associated
with FMRP and found that at least ten encode proteins
42
involved in synaptic plasticity or neuronal development.
This data helped to identify some of the specific mRNA
translation that is affected in mental retardation (15,
33). One specific mRNA was the mRNA coding for
MAP1B.
MAP1B is negatively regulated in the
Drosophila nervous system. In the absence of FMRP,
MAP1B experiences no inhibition and altered dendrite
and axon development is noted (45).
We also interestingly found that FMRP
associates with the non-translatable BC1 RNA. In
addition to binding directly to FMRP, BC1 can associate
with the FMRP target mRNAs in the absence of FMRP
(43, 44, 45). This data suggests that BC1 is involved in
specificity of FMRP to mRNAs and BC1 helps to inhibit
translation of some mRNAs by blocking the initiation
codon (43, 44). Large numbers of mRNAs have been
recently associated with FMRP. Of these mRNAs,
many have been found to differ in expression and
distribution among wild type and FMR1 knockout mice
(31). These recent studies identifying specific mRNAs
associated with FMRP and their coded proteins have
shown the importance of FMRP in neuronal
development.
Altered neuronal
Syndrome
development
in
Fragile
phosphorylated where the FMRP associated with
actively translating polyribosomes was consistently
dephorphorylated in both brian cells and cultured cells
(12). This is possibly the mechanism by which FMRP
releases its inhibitory effect on targeted mRNAs.
Recent models have associated the release
of translational inhibition with the presence of mRNA
granules and mGluR-induced translation (3). FMRP
normally inhibits mRNA translation, but induction of
translation has been associated with glutamate
receptors.
In fragile X syndrome mGluR-induced
translation is heightened due to the lack of inhibition
normally provided by FMRP. The decrease in mRNA
granules in knockout mice supports this finding.
Furthermore, upon excitation with an mGluR5 agonist
increased granule levels were observed (3). The lack
of rapid protein translation in sites mediated by
neurotransmitters such as glutamate has been
proposed to cause several abnormalities observed in
fragile X syndrome (41).
The micro-RNA silencing of FMR1
The FMR1 gene is silenced by methylation induced by
the CGG tri-nucleotide repeat expansion alone, but also
by interaction with micro-RNAs (23, 24).
FMRP
interacts with Argonaute (AGO; 33) and components of
the micro-RNA pathway such as Dicer (33, 24). In
Drosophila melanogaster models the AGO ortholog was
found to be suppressed in the presence of FMRP.
When FMRP was removed the models experienced a
loss of AGO suppression leading to a rough eye
phenotype. Upon induced suppression of AGO, the
rough eye phenotype was significantly reduced to levels
of almost normal (24). AGO was found to be important
to the biological functions of synapses, but not totally
dependent on FMRP.
FMRP interacts with many molecules like
AGO to influence translation. FMRP also has recently
been associated with translational regulation through a
micro-RNA pathway. Transcripts are produced from
the expanded fragile X allele at some point early in
development before complete methylation of the CpG
island (23, 24, 34). These transcripts form structures
referred to as hairpins that are cleaved by the enzyme
Dicer (23) resulting in small mRNAs approximately 20
nucleotides in length.
These small mRNAs
communicate with the RITS complex, a transcriptional
silencer of genes. The small RNAs direct the RITS
complex
to
homologous
mRNAs
through
complementary base pairing.
The RITS complex
recruits methylation machinery eventually leading to the
suppression of the FMR1 gene as observed in fragile X
syndrome (24). The RITS complex also may change
the acetylation of specific histones (7, 24).
The FMR1 promoting region has shown to
possess qualities of varying chromatin conformations
linked to altered histones (20). When FMR1 is inactive
as in fragile X cells the chromatin was displayed
uniform conformation. In normal cells the interactions
are far lesser than in fragile X cells (20). Histones
acetylation is associated with normally expressed
FMR1, but not the absence of FMR1 as in fragile X
syndrome (7, 8, 20). Recent studies have treated
fragile X cells with 5-aza-2-deoxycytidine and observed
re-acetylation of histones 3 and 4. The re-acetlyation of
these histones resulted in FMR1 transcriptional
reactivation (8). This finding presents an alley of
treatment investigation for fragile X patients in the
future.
X
FMRP regulates the translation of many mRNAs which
code for proteins involved in neuronal development.
FMRP has been specifically found to be highly
expressed in neurons. Areas controlling cognition are
commonly affected in fragile X syndrome. A mouse
model study shows deficient amygdale and
hippocampal functions in FMR1 knockout mice during
fear and conditioning tests (36). Specifically, FMRP is
highly expressed in the dendritic formations of nonfragile X organisms (18). The dendritic functions
encoded by the target mRNAs of FMRP are deficient in
fragile X patients thus leading to altered synaptic
function as observed in the fragile X phenotype (18).
Further proving the neuronal effects of FMR1
silencing mouse models have elucidated interesting
findings in the area of altered synaptic plasticity related
to fragile X mental retardation (22).
Long term
depression (LTD) dependent on glutamate receptors
was found to be significantly altered in the
hippocampus cells of knockout mice. By using DHPG
to induce the glutamate dependent LTD, enhanced
results were found in the knockout mice (22). This
supports the earlier hypothesis that FMRP is important
in regulating protein production in the synapse.
In mice lacking the expression of the FMR1
gene, irregular dendritic spines were observed (26)
connecting the FMRP protein with synaptic growth.
Dendritic spines in the visual cortex were compared
among FMR1 knock out mice and wild type mice to
explore the specific differences in dendritic growth. The
dendrites of the knockout mice show a high incidence
of long thin dendritic spines as well as dendritic spines
of higher density (13). This data combined with the
mRNAs targeted by FMRP that regulate neuronal
structure connect fragile X syndrome with altered
synaptic plasticity.
FMRP influences synaptic growth by normally
inhibiting translation of functional mRNAs in
synaptosomes (18). Two methods of the release of
inhibition in normal models have been discovered: the
first is dephosphorylation (12) and the second is
excitation by glutamate receptors (3, 42). FMRP
associated with stalled polyribosomes was consistently
43
2 proposes an over all view of the FMRP interactions in
a normal neuron.
The alterations observed in fragile X
syndrome are due to the loss of translational regulation
via the various pathways that lead to transcriptional
silencing of FMR1 (methylation of the CpG-island, miroRNA interactions, and histone deacetlyation). Altered
synaptic plasticity leads to dysfunctional communication
between neurons. This altered communication leads
to the fragile X phenotype observed in humans.
The new insights into the AGO (in fly models,
Drosophila melangaster) and the micro-RNA pathway
provide insight into a more specific cause of FMR1
silencing with possible drug treatment options. The
mGluR induced translation provides the same exciting
knowledge about translational regulation and drug
treatment. Knowledge pertaining to further functions of
FMRP still remains elusive along with the cause for the
CGG tri-nucleotide repeat expansion which is thought
to be the central cause behind fragile X syndrome.
Interesting studies have also shown the
influence of environmental factors on fragile X
phenotypes. When knockout mouse models were
raised in enriched environments there were found to
show increased dendrite branching, length, and
dendrite spine density to levels near normal (39). This
Discussion
FMRP has shown tissue specific expression in areas of
the brain and other parts of the body associated with
the observed phenotype of fragile X syndrome such as
the hippocampus and the testis. Specific binding of
FMRP to various mRNAs has been elucidated also
linking FMRP to an inhibitory role in the translation of
respective mRNAs. Translation inhibition and mRNA
targeting may be influence by non-translatable mRNAs
such as BC1. The silencing of the FMR1 gene is the
central link in fragile X syndrome, however, recent
studies have shown gene silencing by the binding of
transcription factors to the promoter regions of FMR1
(28). This is data another example of the mechanism
by which methylation silences the FMR1 gene.
The specific mRNAs associated with FMRP
have proven to reveal a wealth of information explaining
the gap of knowledge between the silencing of the
FMR1 gene and the fragile X phenotype. MAP1B is an
example of one such mRNA. MAP1B encodes for
microtubule structural functions, thereby influencing
physical features in the development of neurons. The
absence of FMRPs regulatory influence leads to
severely altered neurons in fragile X syndrome. Figure
44
8. Bradford, Coffee, Fuping Zhang, Stephen T. Warren, and
Daniel Reines. "Acetylated Histones are Associated with the
FMR1 in Normal But Not Fragile X-Syndrome Cells." Nature
Genetics 22 (1999): 98-101.
provides non-invasive treatment options to counter the
silencing of the fragile X gene, FMR1.
Transgenic genes have also been explored
as a possible means of treatment. Because mouse
models display some human-like symptoms in
knockouts, a transgenic line of mice with yeast artificial
chromosomes were generated.
The study found
behavioral and morphological changes. Also, overexpressing the gene did not have the opposite effect
being a important consideration in treatment (37).
9. Brown, Victoria, Kersten Small, Lisa Lakkis, Yue Feng, Chris
Gunter, Keith D. Wilkinson, and Stephen T. Warren. "Purified
10. Recombinant Fmrp Exhibits Selective RNA Binding as an
Intrinsic Property of the Fragile X Mental Retardation Protein."
The Journal of Biological Chemistry 273 (1998): 1521-15527.
10. Castren, Maija, Topi Tervonen, Virve Karkkainen, Seppo
Heinonen, Ero Castren, Kim Larsson, Cathy E. Bakker, Ben
Oostra, and Karl Akerman. "Altered Differentiation of Neuronal
Stem Cells in Fragile X Syndrome." PNAS 102 (2005): 1783417839.
Acknowledgements
I would like to thank Jenny Riddle for all of the time and
dedication as a mentor through out the process of
developing my project. I would also like to thank my
peers for their support. Lastly, I would like to thank Dr.
DebBurman for his constant advice, aid, and faith.
11. Ceman, Stehpanie, Victoria Brown, and Stephen T.
Warren. "Isolation of an FMRP-Associated Messenger
Ribonucleoprotein Particle and Identification of Nucleolin and
the Fragile X-Related Proteins as Components of the
Complex." Molecular and Cellular Biology 19 (1999): 79257932.
the author.
12.
Ceman, Stephen, William T. O'donnell, Matt Reed,
Stephana Patton, Jan Pohl, and Stephen T. Warren.
"Phosphorylation Influences the Translation State of FMRPAssociated Polyribosomes." Human Molecular Genetics 12
(2003): 3295-3305.
13. Comery, Thomas A., Jennifer B. Harris, Patrick J Willems,
Ben A. Oostra, Scott A. Irwin, Ivan Jeanne Weiler, and William
T. Greenough. “Abnormal dedritic spines in fragile X knockout
mice: Maturation and pruning deficits,” PNAS 94 (1997):
5401-5404.
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46
Review Article
A Ride with Listeria monocytogenes: A Trojan Horse
Joshua Haas*, Krista Kusinski*, Shruti Pore*, Solmaz
Shadman* and Mithaq Vahedi*
Lake Forest College
extensively in murine macrophages in the past 40-50
years. The study of the life cycle of LM in this
experimental model has contributed significantly to the
understanding of the immune response to intracellular
pathogens (Portnoy et al., 2002).
Listeriosis manifests itself through flu-like
symptoms and can lead to diarrhea, meningitis,
encephalitis, meningoencephalitis and stillbirths. In
humans, it primarily infects immunocompromised
individuals like pregnant women, neonates, and the
elderly. (Portnoy et al., 2002, Vázquez-Boland et al.,
2001, and Dyer et al., 2002). Listeriosis is more
dominant in females because of differential production
of the immunosuppressive cytokine IL-10 (Pasche et
al., 2005). Immunocompetent individuals usually
survive the infection, whereas those with debilitating
diseases often die (mean mortality rate of 30-40%)
(Vázquez-Boland et al., 2001).
LM can cross the placental and blood brain
barriers; however, in order to do so, it needs to pass the
intestinal barrier and survive the harsh environment of
the stomach. The primary method of entry into
endothelial cells is believed to be via a zipper-like
mechanism (Alberts et. al., 2003) Invasion proteins on
the surface of the bacteria, like Internalin A, and
Internalin B, and P60, help the bacterium bind to host
surface receptors (Drevets et al., 2004). This binding
induces phagocytosis of the bacteria into the host cell.
Another mechanism by which LM is internalized is
phagocytosis by macrophages. Once inside the host
cells, the bacteria secrete a pore forming hemolysin,
known as Listeriolysin O (LLO) and two distinct
phospholipases, PI-PLC and PC-PLC. These
hemolysins, along with the phospholipases mediate the
degradation of the phagolysosome and the escape of
the bacteria from the vacuole into the intracytoplasmic
environment of the host cell. Once LM is in the cytosol,
the ActA protein recruits host cell Arp2/3 complexes to
enable efficient actin-based motility. Actin-based
motility enables the bacteria to form filopods
(pseudopod-like extensions) and propels it into
neighboring cells, resulting in spread of the infection
(Portnoy et al., 2002 and Vázquez-Boland et al., 2001).
The immune response to L. monocytogenes
is entirely cell mediated. CD8+ T-cells recognize and
lyse infected cells. The NF-κB pathway is used to
activate immune response genes and in the
subsequent production of interleukins (IL’s) (Portnoy et
al., 2002 and Vázquez-Boland et al., 2001).
Current treatments of Listeriosis are
intravenous administration of ampicillin and gentamicin.
Treatment can last for 10 days, but depends on the
body’s ability to fight the infection (www.kidshealth.org).
This paper will review the current understanding of
mechanisms underlying the internalization of Listeria
monocytogenes, its subsequent escape from the
phagolysosome, replication in the host cytosol and its
actin-based motility (Fig 1). Currently available
treatments will be discussed along with future
experiments which could lead to more effective
therapies.
Summary
Listeriosis, a disease caused by Listeria
monocytogenesa
facultative,
intracellular
bacterium, spreads through contaminated food. It
affects epithelial cells and macrophages and has a
mortality rate of about 30%. The bacterium can
cross the blood brain barrier, causing meningitis,
and the placental barrier, causing abortion. Some
mechanisms for entry into cells include the InlA- Ecadherin adhesion and InlB-Met pathway. hly, one
of the many genes activated during infection, leads
to the production of Listeriolysin O (LLO). LLO and
two distinct phospholipases are indispensable to
the spread of Listeria. Phosphatidylinositol-specific
phospholipase C (PI-PLC) activates a host protein
kinase C (PKC), which facilitates the escape of the
bacterium from the primary vacuole, along with
LLO. Once inside the cell’s cytoplasm, Listeria
replicates. At this point, both the original bacterium
and the daughter cells use Act A protein to exploit
the cell’s machinery to polymerize actin. Actinbased motility propels the Listeria throughout the
cell and facilitates its intercellular spread. Current
curative methods include ampicillin, gentamicin,
and chloramphenicol, reserved for life threatening
infections. Treatment via plant extracts of Pluchea
quitoc is in the experimental stage. This review
focuses on tracking the progression of the L.
monocytogenes bacterium from its entry to spread.
The story of the Trojan horse: Introduction
Listeria monocytogenes (LM) is a ubiquitous, facultative
intracellular bacterium which can thrive in a variety of
environments and hosts. It is a Gram-positive bacterium
and spreads primarily through contaminated foods
(Portnoy et al., 2002). Foods that are most commonly
contaminated by Listeria are meats, milk, soft cheese,
dairy products and industrially-produced refrigerated
food products (Vázquez-Boland et al., 2001). LM can
survive and proliferate in acidic environments, high salt
concentrations and even at very low temperatures
(www.textbookofbacteriology.net, Vázquez-Boland et
al., 2001).
Listeria microorganisms were first discovered
in 1924 by E.G.D. Murray, R.A. Webb, and M. B.R.
Swann, as the microorganisms causing a septicemic
disease in rabbits and guinea pigs in their laboratory in
England. However, the first case of the disease was
reported in humans in Denmark in 1929 (VázquezBoland et al., 2001). An outbreak of Listeriosis, the
infection caused by L. monocytogenes, in California, in
1985, claimed the lives of 18 adults and 30 fetuses,
outlining the high mortality of this disease
(www.textbookofbacteriology.net). LM has been studied
The horse looks like a present, but…: Listeria
monocytogenes entry mechanisms
* This paper was written in BIO221 Cellular and Molecular Biology, taught
by Dr. Shubhick DebBurman
LM being an intracellular bacterium, it is very important
for this pathogen to gain entry into a cell in order to
47
Figure 1: Model showing the entry and spread of Listeria monocytogenes
The bacterium is phagocytosed by the host cell, using cell adhesion proteins. It escapes the vacuole by secreting a pore-forming toxin,
Listeriolysin, and the action of phospholipases. Subsequent to its escape from the vacuole, LM acquires actin based motility, which enables
it to form filopods and spread from cell to cell (Reproduced from http://textbookofbacteriology.net/Listeria.html).
the “N-terminal cap” and at the C terminus there is a
conserved sequence known as the IR or Inter repeat
region (Marrino et al., 2000). The LRR regions are
structurally and functionally important to the
internalization of LM (Dussurget et al., 2004).
replicate and thus cause Listeriosis. LM has many
mechanisms by which it can gain entry into a cell. A
widely studied pathway involves proteins of the
Internalin family. Internalin (InlA) and B (InlB) are
involved in two distinct pathways by which they allow
the bacterium to enter the cell.
Bacterial Internalin (InlA) is a ligand for E-Cadherin
receptors
Internalin (InlA) and Internalin B (InlB) dependent
internalization of LM
InlA is involved primarily in the infection of epithelial
cells. InlA, specifically, is anchored to the cell wall of the
bacteria through a LPXTG motif in its carboxyl terminal
region (Lecuit et al., 1997). The receptor for InlA is ECadherin. E-cadherin is a part of the cadherin superfamily of transmembrane glycoproteins that act as
adhesion molecules. It is located at the adherens’
junctions and allows for the Ca2+ dependent adhesion
of two cells (Dussurget et al., 2004). In addition, not all
E-cadherins are receptors for InlA. Rat and mouse Ecadherins cannot bind InlA. This may be due to the
absence of a proline at position 16 of the rat and mouse
E-cadherins that allows for such specificity (Lecuit et
al., 2001). In species that do present a favorable Ecadherin receptor, InlA interacts with the first two
ectodomains(protein domains outside the cell) of Ecadherin. Specifically, the amino terminal region of InlA
that contains the LRR and IR regions interacts with Ecadherin and is necessary and sufficient to promote the
internalization of LM. Furthermore, it is the LRR region
that directly interacts with the E-cadherin ectodomains,
whereas the IR region is important in the folding of the
LRR region (Lecuit et al., 1997).
The carboxyl terminal of E-cadherin directly
interacts with the intracellular β-catenin. α-catenin, in
The Internalin family of Listeria proteins is very large. It
is composed of at least seven members including InlC,
InlC2, InlD, InlE, InlF, InlG, and InlH (Marrino et al.,
2000). The two members of this family that are relevant
to entry of LM into the host cell’s are InlA and InlB.
These proteins are found on the cell surface of LM and
are involved in its internalization. Internalins bind
specific receptors on the host cells surface and thus
trigger phagocytosis of LM. Through these
mechanisms, LM can induce phagocytosis in nonphagocytic cells in vitro. This phagocytosis is attributed
to the reorganization of the actin cytoskeleton, which, in
turn, leads to membrane folding (Marrino et al., 2000).
These two proteins as well as other proteins that belong
to this family have some structural features in common.
Both proteins have a Leucine Rich Repeat region (LRR)
and a β repeat region, as well as an Inter repeat region
between the two (IR) that is extremely conserved
(Lecuit et al., 1997). The LRR motif is involved in
protein-protein interaction and is repeated in a highly
regular fashion (Marrino et al., 2000). The LRR regions
are flanked by highly-conserved sequences on either
side that may play a role in the stability of the protein.
At the N terminus there is a hydrophilic cap known as
48
phagocytosis and membrane ruffling (Bierne et al.,
2002).
Gene Expression in the Intracellular Life Cycle of
Listeria monocytogenes
After the bacterium has been internalized, its gene
expression changes with respect to its new
environment. The virulence genes (prfA, plcA, hly ,mpl,
actA, and plcB) located in the major virulence gene
cluster are strongly regulated during intracellular growth
(Chatterjee, et al., 2006). The PrfA gene is an
autoregulatory positive regulatory factor required for the
regulation of the virulence genes, in addition to
expression of other genes elsewhere on the
chromosome, like inlA. PrfA is regulated by the general
stress-response alternative sigma factor σB, which
plays a crucial role in the invasion of cells, but not the
systemic spread (Garner et al., 2004).
Genes important for the escape of bacteria
from the phagolysosome to the intracytoplasmic
environment are hly and plcA. hly encodes for the poreforming toxin listeriolysin O (LLO), while the plcA gene
is responsible for the production of phosphatidylinositol
phospholipase C. Both of these proteins are essential
for the escape of the pathogen from the primary
vacuole (phagolysosome) into the cytoplasm of the host
cell. In a study conducted using murine macrophages, it
was shown that ∆hly and ∆plcA double-deletion mutant
Listeria are rapidly killed (Chatterjee, et al., 2006). It
was also shown that these genes are up-regulated
during the intracellular phase of growth.
In an intracellular milieu, LM changes its
normal sugar metabolism. Genes encoding enzymes in
the second part of glycolysis were reduced during
infection. Further, it was found that an operon encoding
glycerol kinase and the glycerol uptake facilitator (lmo
1538 to lmo 1539) and glycerol-3-phosphate
dehydrogenase (lmo 1293) were upregulated, indicating
that glycerol was being used as the additional carbon
source for intracellular growth. This could be a
mechanism whereby the bacteria do not affect the host
cell’s energy source and are able to spread more
rapidly, rather than killing the host cell. Glucose also
inhibits the expression of prfA, and hence the
expression of the major virulence gene cluster
(Chatterjee et al., 2006).
Genes required for the normal replication of
LM were downregulated. The ftsZ and ftsA genes,
which are the major bacterial cell division determinants,
were downregulated, suggesting lowered cell division.
This lowered cell division activity is probably a result of
the host cell’s defense mechanism to keep bacterial
multiplication in check.
Another important gene that is altered during the
intracellular phase of LM’s growth is lmo0593, which is
a nitrite transporter gene. The increased transport of
nitrite by the bacteria suggests that nitrite is used in
place of oxygen as the final electron acceptor in the
electron transport chain. This mechanism allows the
bacteria to survive under oxygen deprived conditions in
the host cell (Chatterjee et al., 2006).
Fig 2: Model for InlA-dependent entry of LM into epithelial
cells.
Proteins known to play a role in entry are indicated, including Ecadherin, α and ß-catenins, vezatin, myosin VIIA, and actin.
This model highlights how myosin VIIA could help the
membrane rearrangements during LM entry. (Modified from
Sousa et al., 2003)
turn, binds to β-catenin and interacts with actin (Lecuit
et al., 2000) This interaction leads to the formation of a
fusion molecule consisting of the ectodomains of the Ecadherin and the actin binding site of the α-catenin
which eventually leads to LM entry (Dussurget et al.,
2004). Furthermore, myosin VIIA and its ligand vezatin
together function as the molecular motor in the
internalization of Listeria. When myosin VIIA binds
vezatin, coupled with an actin polymerization process, it
provides the tension necessary for bacterial
internalization (Fig. 2) (Sousa et al., 2003).
InlB as a virulence factor for hepatocytes and other
non-epithelial cells during Listeriosis
InlB is a bacterial protein that is anchored to the cell
wall of LM through a series of GW repeats (Lecuit et al.,
1997). InlB has an elongated structure and its main
receptor for LM invasion is the hepatocyte growth factor
receptor, also known as Met (Dussurget et al., 2004). It
is interesting to note that InlB does not strictly mimic the
hepatocyte growth factor (HGF), in that HGF and InlB
do not share sequence similarities. Once Met has been
activated by InlB, it autophosphorylates two tyrosine
kinase residues and recruits Gab 1, Sch and Cbl as
well as PI 3-kinase. PI 3- kinase, which is known to be
involved in control of the actin cytoskeleton, activates
PLC-γ1 (Vazquez-Boland et al., 2001). The activation of
Met
is
enhanced
in
the
presence
of
glycosaminoglycans (GAG’s) (Bierne et al., 2002).
These are normally involved in the oligomerization as
well as storage and protection from extracellular
proteases (Dussurget et al., 2004).
Another receptor for InlB is some form of the
surface associated gC1q-R. However, the specific
mechanism by which InlB binds and interacts with
gC1q-R remains to be determined. Since this protein
lacks a transmembrane domain, it may act as a
signaling co-receptor (Vazquez-Boland et al., 2001). In
addition, the partial inhibition of InlB-mediated signaling
pathway due to gC1q-R antibodies supports this
hypothesis. InlB-Met signaling leads to both
There was no door, so how did they get out? :
Escaping into the cell cytoplasm
Once inside the host cell, the bacterium is enclosed by
the phagosomal membrane. LM must have a way to
escape the vacuole because this is where the bacteria
replicate. It is here that LM uses the host cell
49
(A)
(B)
Figure 3: Transmission Electron Micrographs of Listeria monocytogenes
(A) Wild type Listeria monocytogenes free in the cytosol of a macrophage (size bar = 2µm). (B) A secondary macrophage with LM in a
double membrane vacuole (size bar = 0.5µm) (Gedde et al, 1999).
In a study by Lety et al, it was shown that a
PEST-like motif in LLO is required for the escape of LM
from the vacuole and for causing virulence. The
removal of this PEST-like sequence results in a strain
that is extremely toxic to host cells and is 10,000 times
less virulent in mice macrophages (Portnoy et al.,
2002).
In contrast to LLO and PFO, streptolysin O
(SLO) was found to have a 10-fold lower activity. B.
subtilis expressing SLO could not grow in the cytoplasm
of host cells efficiently, presumably because they were
unable to escape the phagolysosome. It is possible that
SLO is less stable in an acidic environment and is not
able to lyse the vacuole (Portnoy et al., 1992). Gedde et
al used a genetic approach to investigate the role of
LLO in intracellular growth and cell-to-cell spread. SixHis-tagged LLO (HisLLO), noncovalently bonded to the
surface of nickel-treated LLO lacking LM, enabled some
of these cells to escape the host vacuole and replicate
in the cytoplasm. Both LLO lacking LM and wild type
LM were able to replicate in the cytosol of the host cell
(Fig 3A). LLO lacking LM could also spread to adjacent
cells, however, these LM were trapped in doublemembrane
vacuoles
(Fig.
3B).
Surprisingly,
phospholipase C (PC-PLC) and PI-PLC were also not
required in the spread of LLO negative LM into
secondary cells.
machinery. In order for LM to enter the cytosol it first
needs to escape the phagosome. Both LLO and two
types of PLCs play a key role in mediating this escape.
Virulence Factors:
Listeriolysin O
LM is one of the many bacteria that produce
hemolysins. Listeria secretes Listeriolysin O (LLO),
which, along with a phospholipase, PlcA, plays a very
important role in the escape of the pathogen from a
vacuolar compartment (Portnoy et al., 1992).
Listeriolysin is a member of the thiol-activated
cytolysins, which include perfringolysin O (PFO),
streptolysin O (SLO), and pneumolysin, among others.
These hemolysins are inhibited by free cholesterol and
cysteine; free cholesterol is the common hemolysin
receptor, and cysteine oxidation causes reversible
protein inactivation (Portnoy et al., 1992). It has been
shown that the expression of listeriolysin in an
extracellular non-pathogenic soil bacterium, Bacillus
subtilis, enables the bacterium to grow in the cytoplasm
of mammalian cells. In a subsequent study, it was
shown that LLO is not the only cytolysin that allows
bacteria to proliferate in the cytoplasm of host cells.
When B. subtilis expresses PFO, it is also able to
escape the phagolysosome and replicate in the
cytoplasm. However, unlike LLO, PFO causes damage
to host cells (Portnoy et al.,1992,1994). It has also been
shown that LM expressing PFO instead of LLO is much
less virulent (Portnoy et al., 1994). This result is
consistent with the observation that LLO works best in
an acidic pH, whereas PFO functions in both acidic and
neutral environments. The increased efficiency of LLO
at a pH of 5.5 is a mechanism by which LM
compartmentalizes the activity of LLO to escape the
vacuole and subsequently replicates in the cytoplasm
without causing damage to the host cell (Portnoy et al.,
1992).
Phospholipases
LM also uses phospholipases C to aid in the
escape from the vacuole. Two specific phospholipases
(PLCS) are used. One is the phosphatidylinostiolspecific PLC (PI-PLC), and the other is more general,
phosphatidylinostiol-specific (PC-PLC) (Portony, et al.,
2002). LLO has been studied in great detail as the
major factor for the permeation which leads to
phagosomal escape. Studies have also attributed PCPLC to the escape even in the absence of LLO
(Sibelius et al., 1992).
50
The role of the PI-PLC secreted by LM is to
catalyze the production of inositol phosphate and
diacylglycerol (DAG) through cleavage of the
membrane lipid PI. DAG then has the ability to activate
protein kinase C (PKC). There are four types of PKCs,
but the PKC β of the host is shown to be linked with the
PI-PLC signaling cascade. The PKC β has been shown
to facilitate the permeation of the phagosomal
membrane before the bacteria escape (Poussin et al.,
2005).
PI-PLC has another component which allows
for the escape of the bacterium into the cytosol. It is
known that LM has a weaker effect on the GPIanchored proteins than most bacteria do. This has been
found to be the result of the PI-PLCs of LM differing
from those of other bacteria. There is a Vb β-strand
which is found in other bacterial PI-PLCs which is
absent from L. monocytogenes. The Vb β-strand is
known to give a contact for the glycan linker of GPIanchored protein. This contact enhances the ability of
the cell to cleave the GPI anchors. When Vb β-strand
was absent in LM, the cell’s ability for the bacteria to
escape the phagosome and be released into the
cytosol of the host cell was increased. It is through
these observations, as well previous knowledge of Vb
β-strand, and PI-PLCs that it can be hypothesized that
LM have evolved this absence or loss in order to
promote growth inside the host cell (Wei et al., 2005).
PI-PLC and PC-PLC have also been shown
to have an activating effect on immune response during
LM infection. NF-κB is a transcription factor which can
be used by a number of genes. Some of these genes
are activated during infection. One which is activated
and is specific to LM is Listeriosis biphasic NF-κB
activation. This phase of NF-κB is regulated by IκBβ.
When IκBβ is degraded NF-κB becomes active. This
activation can be seen as a result of the degradation of
IκBβ by lipoteichoic acid (LTA), but also through the
listerial PI-PLCs and PC-PLCs which is in correlation
with the IκBβ degradation. The NF-κB transcription
factor can then be used by LM to enter the host cell. LM
can then use the host cell machinery for its own
replication. The nuclear NF-κB complexes which are
formed can therefore be attributed to the effect of the
Listerial PLCs on the host cell (Hauf et al., 1997).
In summary, the secretion of PLCs during
listerial infection has several effects on the host cell.
One of these effects has been shown to increase the
permeation of phagosomal membrane. The activation
of PKC β through PI-PLC facilitates the escape of the
bacterium. The decreased affinity of L. monocytogenes
for the glycan linker of the GPI-anchored protein due to
the lack or absence of the Vb β-strand also increases
the ability for the bacterium to escape during infection.
The degradation of IκBβ by listerial phospholipases PIPLC and PC-PLC leads to the activation of NF-κB,
which allows the bacteria to exploit the host cell
machinery.
(Portnoy et al., 2002). The ActA protein activates the
Arp2/3 complex by mimicking proteins of the WASP
family. Research is underway to understand the nature
of the binding between ActA and the Arp2/3 complex.
In order to determine what is most important
in actin polymerization, many different proteins and
their functions have been examined in past studies. The
Apr2/3 complex has been found to organize signals of
actin cytoskeletons and initiate actin assembly because
of its specific function. The Arp2/3 complex is
comprised of the actin related proteins Arp2 and Arp3,
along with p41-Arc, p34-Arc, p21-Arc, and p16-Arc.
Arp2 and Arp3 both have insertions in loops that are
exposed to the cytosol, and Arp2 contains a profiling
binding site. However, neither Arp2 nor Arp3 are
capable of the polymerization of actin alone. The Arp2/3
complex nucleates actin filaments, elongating the
barbed ends. Also, the complex binds to other actin
filaments and produces a branching formation when
present at a filament pointed end. Once the polar actin
tails become long enough, LM is propelled inter/intracellularly with actin-based motility (Fig 4).
Why Only Act A Is Needed
WASP family proteins become activated by
interactions with Cdc42 and PIP2 region and other
proteins. This interaction opens the given protein
exposing the C-terminal region. The Arp2/3 complex
and the C-terminal ends of these proteins interact.
Arp2/3 binds to the CA-like region which all of the
WASP family proteins share. Equivalent regions of the
Act protein are also capable of activating the Arp2/3
complex in this way. Because of this unique ability to
activate the complex, LM can by-pass the machinery for
polymerization and directly interact with the complex.
The complex attracts G-actin or F-actin to further actin
polymerization.
In addition to the Arp2/3 complex, other host
cell proteins are involved in actin polymerization.
Research has found that VASP proteins bind to the
proline-rich region of ActA at the EVH1 domain. This
was proven by mutating VASP proteins, which resulted
in aslower rate bacterial locomotion. Many different
ligands exist for the ActA protein. Therefore, ActA has
more than one mechanism for polymerizing actin and
eventually for locomotion.
Listeria monocytogenes Infects Neighboring Cells
After polymerizing actin, Listeria combines
other mechanisms in order to spread to neighboring
cells. In the areas of newly polymerized actin, two
molecules have been found to concentrate.
Phosphatidylinositol 3,4,5-biphosphate (PtdIns(4,5)P3)
and
phosphatidylinositol3,4,5-biphosphate
(PtdIns(4,5)P2) play essential roles in the actin-based
motility of LM. Recent studies have found that reducing
the amount of PtdIns(4,5)P3 and PtdIns(4,5)P2 with AktPH-GFP and PCLδ-PH-GFP, respectively, significantly
slows the movement of actin within a cell. As expected,
this also inhibits the filopod formation process.
When a PI 3-Kinase was used to allow the
concentration of PtdIns(4,5)P3 by degrading Akt-PH
GFP, but not PCLδ-PH-GFP, actin based motility was
completely inhibited. When this PI 3-kinase was
removed, full recovery of actin based motility and
filopod formation was observed. The specific kinase
used was LY294002. These results imply that
Charge! The Main Act: ActA Protein Fills the Role
Quickly after Listeria enters the host cell’s
cytosol, the protein ActA interacts with the cell’s
proteins to mediate actin-based motility. ActA is a 610amino-acid protein containing a charged N-terminal
end, proline rich repeats, and a C-terminal end to
anchor the protein to the bacteria surface (Cossart et
al., 2000). Specifically, ActA directly activates the
Arp2/3 complex, followed by other proteins that exploit
the host cell’s machinery for actin polymerization
51
Figure 4: Activation of the Arp2/3 complex by the ActA protein
Recruitment of the Arp2/3 complex by the ActA protein followed by nucleation and elongation of the actin tail. Activation of the Arp2/3
complex results in propulsion of LM.
PtdIns(4,5)P2 is the substrate for formation of
PtdIns(4,5)P3. Thus, the newly formed actin polymers
(formed through the recruitment and activation of the
Arp2/3 complex by Act A proteins) dissociate when
PtdIns(4,5)P2 and PtdIns(4,5)P3 are not present.
This charged actin filament polymerization
allows for the directional force, actin-based motility,
through the cells’ cytoplasm. Eventually the bacteria are
propelled to the peripheral membrane of the host cell.
Enough force is present to actually push the membrane
outward, forming distinct filopods on the host cell using
polymerized actin as support (Fig 5). The filopods are
then ingested by adjacent cells, and the cell cycle
continues to repeat. Filopod formation and the survival
of newly polymerized actin is directly correlated with the
presence of PtdIns(4,5)P2 and PtdIns(4,5)P3 (Vingjevic
et al., 2003). Also, the PI 3-Kinase activity can play a
major inhibitory role in the actin-based motility of LM
and formation of filopods (Sidhu et al., 2005). The
formation of filopods, as well as the specific interactions
of the proteins involved, is still being researched.
bacteriostatic, for L. monocytogenes (Taege et al.,
1999). Often ampicillin is combined with gentamicin for
synergy (Kamath et al., 2002 and Taege et al., 1999).
With the interaction of these two agents, the overall
effect will be greater than the sum of their individual
effects.While ampicillin targets the cell wall; gentamicin
hampers metabolic actives in the bacteria. Gentamicin
operates by binding to a site located on the bacterial
ribosome, which results in the misreading of the genetic
code.
For patients who are allergic to ampicillin, an
alternative treatment option including Trimethoprim and
sulfamethoxazole may be used to treat Listeriosis.
Trimethoprim interferes with the action of bacterial
dihydrofolate reductase to prevent the synthesis of folic
acid, which is an essential precursor in the new
synthesis of the DNA nucleosides thymidine and
uridine. When the bacterium is unable to take up folic
acid from the environment, enzyme inhibition starves
the bacteria of two bases necessary for DNA replication
and transcription (Taege et al., 1999). These two
antibiotics are used in a combination known as cotrimoxazole. Co-trimoxazole works by inhibiting the
successive
steps
in
folate
synthesis
(http://www.pubmedcentral.nih.gov).
Other treatments include chloramphenicol,
which is reserved for life-threatening infections (Wei et
al., 2005). This antibiotic has acute side-effects,
including damage to bone marrow in humans.
Chloramphenicol is used by the World Health
Organization (WHO) in third world countries in the
absence of cheaper alternatives. Chloramphenicol
operates by stopping bacterial growth by hindering the
ribosomal enzyme peptidyl transerase which assists in
the formation of peptide links between amino acids
during the translation process of protein biosynthesis
(Wei et al., 2005).
Figure 5: Formation of Filopods and cell to cell spread.
Filopod formation and entry into adjacent host cells is mediated
by actin-based motility.
Current Experimental Treatments via Plants
Attacking the
Listeriosis
Trojan
horse:
Treatments
Treatment via plant extracts from Pluchea
quitoc is in the experimental stage, studying the effects
it has on Listeria infection. Pluchea quitoc extract is a
well known remedy used in South American traditional
medicine for the treatment of digestive diseases. In
research, it has demonstrated strong anti-inflammatory
and antioxidant activities. An experiment to study the
effects of the P. quitoc extracts determined that it is
helpful in defending the host against L. moncytogenes
infection by increasing the number of leukocytes in the
model mice (Queiroz et al., 2000).
Results indicated that the administration of
P. quitoc increased hematopoietic recovery in the mice
infected with LM. The increase in the number of.
for
To
diagnose
Listeriosis,
blood
or
cerebrospinal fluid cultures are used to establish
bacteterium growth, and characterization of the
bacterium (Taege et al., 1999).The most common and
current curative method of treating Listeriosis includes
the use of ampicillin. Ampicillin works by targeting the
cell wall of the bacterium, inhibiting the third and final
stage of bacterial cell wall synthesis, which ultimately
causes the cell to lyse. However, ampicillin is only
52
Figure 6: Model showing current understanding of Listeria monocytogenes infection in host cells.
Bacterial entry into the cell is facilitated via the internalin adhesion proteins. Listeriolysin O (LLO) and PI-PLC’s mediate lysis of vacuoles
and escape of the bacteria into the cytoplasm. Recruitment of actin filaments aids in bacterial movement and spread (Reproduced with
modifications from Vaquez-Boland et al, 2001).
leukocytes enhances the host’s ability to defend itself
against the infection. It is still unclear what mechanism
improves the survival of mice that are treated with P.
quitoc, however, when this mechanism is discovered,
further studies could make these extracts a useful
remedy for humans.
the author.
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54
Review Article
Nanotechnology may replace existing treatments for cancer
The cell cycle can be perpetuated through
two types of genetic mutations: oncogenes and tumor
suppressor genes (Kruh et al., 2000).
Tumor
suppressor genes normally are involved with the repair
of damaged DNA. Thus, whenever these genes are
inactivated, damaged DNA is not properly repaired
(Moossa et al., 1990). According to Ames and Gold
(1991), every cell in the body experiences 105 DNA
damaging events daily. Thus, the regulatory process of
repairing DNA is an active and important process.
Tumor suppressor genes can be broken down into two
categories: caretakers and gatekeepers (Kruh et al.,
2000). Gatekeepers have a direct roll in controlling
cellular proliferation, while caretakers help preserve the
integrity of the genome by preventing mutations from
occurring. An inactivated caretaker does not lead
directly to tumor initiation, but instead it causes genetic
instability, which causes subsequent mutations. In
contrast, inactivated gatekeepers play a more direct
role in the tumorigenesis process (Kruh et al., 2000).
While tumor suppressor genes are
dangerous when inactivated, oncogenes are only
hazardous when active, at which point they are capable
of inducing cancer in normal cells (McKinnel et al.,
1998). Due to this, oncogenes are highly regulated in
the body. Additionally, oncogenes have a wide variety
of functions. For instance, some encode for growth
factors that increase the proliferation of cells, others
bind to DNA and regulate transcription, and yet others
code for receptors or ligands involved in the cell cycle
(Kruh et al., 2000). If over expressed, however, all of
them can contribute to the development of cancer by
promoting cell division (McKinnel et al., 1998).
Tumorigenesis, or tumor formation, is a
multistep process requiring more than one active
oncogene or inactive tumor suppressor gene. If a
group of cells has a small number of these mutations, a
benign tumor may form. These tumors lack the ability
to metastasize or spread to other parts of the body.
However, if the benign tumor has more mutations, it is
possible for it to become malignant (McKinnel et al.,
1998).
The process of carcinogenesis involves four
steps. The first step is initiation, in which a carcinogen
reacts with DNA causing a strand break or altering a
nucleotide to form an adduct (McKinnel et al., 1998).
Normally, a DNA polymerase repairs this problem,
however, if the DNA replicates before the repair, the
error can be permanently fixed into the genome (Kruh
et al., 2000). Most errors of this type have no real
effect on the body, but if a tumor suppressor is
inactivated or an oncogene activated, the cell has a
significant growth advantage, and the next step,
promotion, may begin. During promotion, a molecule
called a promoter causes selective proliferation, which
may lead to the formation of multiple benign tumors
(Alberts et al., 2003). Through one or more additional
genetic alterations, the third step, known as
progression, may occur. In this step, the tumor cells
develop a significant growth advantage, which is so
strong that they are able to break through the blood
vessel membrane and travel to other areas through the
process of metastasis. This actual conversion is the
last step, and is referred to as malignant conversion
(McKinnel et al., 1998).
This further establishes the importance of
multiple mutated tumor suppressor genes and
Ethan Helm*
Lake Forest College
In 2002, 23% of all deaths in the United States were
caused by cancer making it the second biggest killer,
only ranking behind heart disease (Jemal et al., 2005).
Every year, over a half million Americans die of cancer
and more than a million are diagnosed with the disease.
It is also the second biggest killer among children, with
nearly 12% of all childhood deaths coming from the
disease (Jemal et al., 2005).
Cancer is a disease in which cells proliferate
uncontrollably (Campbell et al., 2002). Unlike most
cells, cancerous cells do not display density dependent
growth, meaning they divide with little spatial regulation
(Moossa et al., 1990). Moreover, these cells have the
ability to spread by breaking into blood vessels and
moving to other systems (Moossa et al., 1990).
Cancer can be fatal due to a combination of
its properties. For instance, cancerous cells lose their
ability to function normally.
That is, they stop
responding normally to cellular signals and therefore no
longer perform their job (McKinnel et al., 1998). Not
only do cancer cells cease working, they also affect
neighboring cells because cell division and metabolism
require nutrients and energy; eventually the cells
require more nutrients than the body can provide and
slowly organ systems begin to fail, a process known as
cachexia
(American
Cancer
Society,
2000).
Additionally, the growths themselves can cause
immense pain or death in hollow organs (such as the
colon) by blocking the lumen and preventing proper
function. Moreover, tumors can cause pressure on the
brain which can lead to brain failure, seizures, or partial
lack of function depending on the location of the tumor
(McKinnel et al., 1998).
The formation of cancer requires several
genes to be altered through mutations, which can be
caused by spontaneous errors in replication or by
exposure to carcinogens that alter nucleotides or break
the DNA strand. In order for a mutation to lead to
cancer, it has to perpetuate the cell cycle (Kruh et al.,
2000).
The cell cycle is a highly regulated process
that ultimately results in the division of one cell into two
(Campbell et al., 2002). In somatic cells, this cycle
includes four phases: G1, S, G2, and mitosis (M).
During G1 phase, the cell grows as it prepares for DNA
synthesis, S phase. Then in G2 phase, the cell grows
in preparation for mitosis, in which the replicated DNA
is equally divided into two newly formed daughter cells
(Campbell et al., 2002).
Errors in the cell cycle are normally corrected
during specific checkpoints at G1 to S, intra-S phase,
and S to M. At these points, the cell cycle is
temporarily arrested while regulatory enzymes ensure
that there are no errors in the DNA sequence. If an
error is found, the DNA damage is either repaired or the
cell is tagged by a marker protein to commit suicide
through apoptosis (Alberts et al., 2003). If inhibited, the
cell cannot properly identify damage, and the cell cycle
continues without the appropriate regulation (Kruh et
al., 2000).
*This paper was as part of an independent study on Oncology.
55
oncogenes in cancer development. In other words, the
growth advantage brought about by one mutation is not
significant enough to overcome the natural immunity of
the body. Tumors of this nature are contained because
they are unable to break into the blood vessels
(McKinnel et al., 1998). However, through multiple
mutations, the growth advantage may be increased
sufficiently to break through blood vessel membranes
(McKinnel et al., 1998). For many years, scientists had
no clue how to deal with this growth advantage. As a
result, cancer was virtually untreatable, and even today,
many types have no specific treatment.
Chemotherapy’s potential to treat cancer was
discovered during December of 1943, when an Allied
warship holding mustard gas exploded (Williams,
2000). As a response to this, the army performed
autopsies on the soldiers, which showed that their bone
marrow had been destroyed by the gas, thereby
inhibiting the production of red blood cells, white blood
cells,
and
platelets.
Accordingly,
scientists
hypothesized that the chemical may be used to fight
cancer. To test this hypothesis, a chemical derived from
mustard gas, known as mustine, was given to
Hodgkin’s disease patients and, even in some patients
with late-stage Hodgkin’s, the disease responded to the
drug (Williams, 2000). In fact, this drug is still a key
component of the MOPP (mustine, vincristine,
procarbazine, and predinisone) regimen (Rüffer et al.,
1998), which is one of the two primary treatments for
Hodgkin’s disease, the other being ABVD (Adriamycin,
bleomycin, vinblastine, and dacarbazine) (Kennedy et
al., 2003; Murphy et al., 1997).
Unfortunately, the treatments commonly used
for cancer (radiation and chemotherapy) are both
deleterious to the health of patients, and can actually
cause death themselves by weakening the immune
system and making patients more susceptible to other
diseases (Schnell et al., 2003). The problem with these
treatments is that they are not selective. That is, they
act on all rapidly dividing cells causing the most
recognizable symptom of cancer treatment: loss of
hair. These treatments also inhibit the production of
erythrocytes and white blood cells, causing patients to
become anemic and neutropenic (Schnell, 2003).
Anemia, a state of insufficient O2 delivery to tissues,
can cause problems with blood clotting, as well as lead
to dizziness and lethargy. Neutropenia refers to a
decrease in the number of neutrophils in the blood
signifying a weakened immune system.
When
neutropenic, patients are more susceptible to
secondary infections; even a common cold can be fatal.
Furthermore, chemotherapy triggers neuroreceptors,
such as those that bind dopamine and serotonin, which
stimulate nausea and cause vomiting (Schnell, 2003).
Not only are chemotherapy and radiation
dangerous, they also are not completely effective.
According to Dr. Frank Balis, “We attribute our inability
to cure many adults with more common forms of solid
tumors to the ineffectiveness of chemotherapy to these
diseases” (1998). In fact, the average five year survival
rate among all cancers in the United States is only 63%
(Jemal et al., 2005). Thus, newer and more effective
treatments are being sought by scientists and
pharmaceutical companies alike.
In the last few years, the field of
nanotechnology has exploded as some scientists
believe tiny objects known as nanoparticles may be
able to help treat a variety of diseases, including
cancer. By definition, nanoparticles can range in size
from 1 to 100 nanometers (Cervellino et al., 2005). The
nanoparticles being studied have a variety of
compositions, shapes, and sizes. The most common
composition includes either a carbon backbone or the
presence of an inorganic metal, such as a gold (Zharov
et al., 2003).
Recently, scientists have discovered that
nanoparticles can easily enter cells. However, it is
uncertain how this occurs. Dai et al. (2005) claims the
influx of nanoparticles occurs by endocytosis. In
contrast, Bianco et al. (2005) suggest the process
happens through insertion and diffusion of particles
through the lipid bilayer of the cell membrane.
Furthermore and surprisingly, these particles can be
linked to proteins, such as antibodies, and still enter
cells (Dai et al., 2005). Fortunately, cancer cells
express certain receptors that are not expressed by
normal cells.
Thus, nanoparticles attached to
antibodies for these receptors can be directed to
cancerous cells exclusively (Dai et al., 2005).
The ability of nanoparticles to selectively
enter cancer cells has duel significance.
Firstly,
nanoparticles can work as drug deliverers.
For
instance, by linking certain proteins, such as tumor
necrosis factor (TNF), a protein with known antitumor
activity, to the particles a new mechanism for fighting
cancer can be utilized (Paciotti et al., 2004). Secondly,
nanoparticles have been shown to absorb different
wavelengths of light than the body, and when exposed
to appropriate wavelengths nanoparticles heat up, but
the body does not.
This method, known as
hyperthermia, can be used to selectively kill cancer
cells by heating nanoparticles that are linked to
antibodies (Ito et al., 2003a).
The specificity of these techniques is key,
because unlike the deleterious effects of chemotherapy
and radiation, treatment with nanoparticles should
result in no major side effects.
Furthermore, in
preliminary studies, hyperthermia and drug delivery
have both been successful, and currently, both
hyperthermia and drug delivery are being heavily
investigated as treatments for cancer (Dai et al., 2005;
Onishi et al., 2003). The purpose of this review is to
discuss the nanoparticle techniques of hyperthermia
and drug delivery and determine whether they may one
day replace the current techniques of chemotherapy
and radiation as a treatment for cancer.
Imaging to Detect Cancer Cells
Beyond having the power to treat cancer, nanoparticles
may also be used to detect the disease. Moreover,
some therapies hope to utilize hyperthermia in such a
way that diagnosis and treatment can occur together.
There are several techniques scientists are
investigating to improve cancer detection and couple it
with hyperthermia (Loo et al., 2004).
One popular technique involves attaching
bioconjugates, such as antibodies, to the nanoparticles.
Loo et al. (2005) attempted to analyze this technique by
utilizing the tendency of breast carcinoma cells to
overexpress the HER2 biomarker.
Thus, by
conjugating an antibody of HER2 to a PEG linker
complex, which enhances biocompatibility and blood
flow, and then attaching the complex to a gold
nanoshell, the particle is linked exclusively to breast
cancer cells (Loo et al., 2005).
Using this, Loo et al. (2005) cultured three
types of cells: cells with the anti-HER2/PEG/nanoshell
complex, cells with a non-cancer specific antibody, and
cells without nanoshells. These cells were viewed with
56
Figure 1. Imaging and hyperthermia using nanoparticles. Imaging and therapy of SKbr3 breast cancer cells using HER2 linked
nanoshells. Top row: darkfield imaging of of HER2 expression based on light scattering. Bottom row: cell viability assessed through
calcein staining with exposure to ~820 nm near infrared (NIR). Cell death was observed only in cells treated with anti-HER2 nanoshell
take from (Loo et al., 2005).
a darkfield microscope sensitive to scattered light, and
only the Anti-HER2 cells showed much light scattering
(Figure 1). In contrast, the cells with the non-specific
antibody showed some light scattering, but it was not as
dense. This illustrates that the Anti-HER2 treated cells
attached exclusively to cancer cells, and exposure of
light identified cancer cells. Furthermore, when treated
with near-infrared (NIR) light of around 800 nm,
cytotoxicity was observed only in the presence of the
cells treated with Anti-HER2 nanoshells (Figure 1) (Loo
et al., 2005). Thus, the hyperthermia treatment was
successful, but only with the Anti-HER2 treated cells.
protein 70 (HSP70), in conjunction with hyperthermia
with MCLs. Expression of this protein protects cells
from heat-induced apoptosis (Mosser et al., 2000), but
recently, it has also been shown to be a key component
in immune reactions (Srivastava et al., 1998).
To analyze HSP70 gene therapy combined
with hyperthermia, Ito et al (2003a) analyzed how mice
with malignant melanoma reacted to tumors that had
been given a plasmid containing human-inducible
hsp70 complimentary DNA. The primary finding was
that hsp70 gene transfer successfully boosted the
immune system during hyperthermia (Ito et al., 2003a).
They determined this by comparing tumor size after
exposure to hsp70 containing plasmid, hyperthermia,
and the combined treatment. Both treatments alone
showed improvement, but in each case, additional
treatments would be required because the tumors
began to grow again at around the tenth day. The
combined therapy, however, completely eradicated
cancer in 3 of the 10 mice with only one treatment.
Because hyperthermia can be used multiple times
without any negative effects, it is believed that the
cancer could have been eradicated in the other mice
with subsequent treatments. Moreover, tumors with the
combined therapy were 16 times smaller than the
hyperthermia only treated tumors after thirty days, and
24 times smaller than the tumors given hsp70 (Ito et al.,
2003a).
Hyperthermia to Kill Cancer Cells
As mentioned earlier, hyperthermia is the killing of cells
through the heating of nanoparticles. One of the
problems of hyperthermia is containing the heat in such
a way that it does not affect other cells. To combat this,
scientists use specific types of nanoparticles for
hyperthermia, such as magnetite cationic liposomes
(MCLs) (Kobayashi et al., 2005). These spherical
particles contain a positively charged phospholipid
exterior that interacts with the negatively charged cell
surface, easily entering cells. The inside of the MCLs is
a 10 nm magnetite nanoparticle (Kobayashi et al.,
2005). Additionally, these particles have maintained
the ability to bind to antibodies and can provide tumorspecific contrast enhancement.
Hyperthermia with Dendritic Cell Addition
The use of immune triggering proteins is not the only
way to boost anti-tumor activity. For instance, mature
dendritic cells (DC) are an integral part of a normal
immune response, which stimulate the growth of CD4+
T cells, CD8+ cytotoxic T lympocytes, and natural killer
cells (Palucka et al., 1999). Unfortunately, mature DCs
cannot take up antigen, and thus addition of these cells
would not result in the proper immune response.
Injection of immature DCs, however, has been reported
to cause antitumor activity (Celluzzi et al., 1998).
Tanaka et al. (2005) decided to go straight to
the source by actually adding additional dendritic cells
(DC) after mouse EL4 T- lymphoma tumors were
treated with hyperthermia. While only 1 in 8 of the mice
Gene Therapy/Hyperthermia Combination
Hyperthermia appears to be effective in some cases by
itself, however, in advanced stages of several types of
cancer, such as melanoma, it may not be sufficient (Ito
et al., 2003a).
Furthermore, to treat cancer,
hyperthermia requires many treatments. However, in
conjunction with other processes, scientists hope to find
a way to use one round of hyperthermia to eradicate
the disease.
The combination therapies revolved
around the use of substances to boost anti-tumor
immunity. Thus, in addition to hyperthermia, the cancer
cells will be assaulted by a revamped immune system
(Ito et al., 2003b). Ito et al. (2003a) have been
analyzing the use of one such protein, heat shock
57
treated with hyperthermia alone had complete tumor
regression, 6 in 8 of the mice treated with hyperthermia
and immature DCs had complete tumor regression.
Based on this, it appears the tumor cells killed by
hyperthermia release antigen proteins which the
immature DCs take up and are then presented to T
cells via MHC class I and/or II antigens (Tanaka et al.,
2005).
the drugs not only have increased cytotoxic activity, but
also adverse side effects are limited (Alberts et al.,
1985).
Doxorubicin
Doxorubicin hydrochloride (Dox), also known as
adriamycin, is a cytotoxic anthracycline that is an
essential component of chemotherapeutic regimens
used to treat acute lymphoblastic leukemia, breast
carcinoma, Hodgkin’s and Non-Hodgkin’s lymphoma
(Murphy et al., 1997). The drug works by halting DNA
replication, and thereby preventing further proliferation
of the disease (Reddy et al., 2004a).
Fortunately, Dox’s anti-tumor activity has
been widely documented, and there is no reason to
think it would behave differently if attached to a
nanoparticle. At the same time, intravenous treatment
of Dox causes systemic toxicity that can cause severe
diarrhea, neutropenia, anemia, hair loss, and heart
damage. Thus, scientists are investigating the use of
different types of nanoparticles that can be used to
deliver Dox directly to cancer cells, ultimately
preventing systemic toxicity (Wilkes et al., 2000).
Reddy and Murthy (2004a) investigated this
by analyzing two different polymerization techniques for
making polybutyl cyanoacrylate (PRC) nanoparticles:
dispersion polymerization (DP) and emulsion
polymerization (EP). The result of each polymerization
technique produced structurally similar molecules. The
difference, however, was that the EP nanoparticles
were smaller. Therefore, Reddy and Murthy (2004a)
sought to find out whether the size difference of the
PRCs affected the nanoparticles’ ability to deliver Dox.
They found that EP particles provided a longer half-life
of Dox in the blood and a lower tissue distribution,
which is consistent with their previous finding that EP
nanoparticles have enhanced permeability and
retention effects (Murthy and Harivardhan, 2003).
Conversely, DP nanoparticles were quickly cleared into
the RES. Both techniques demonstrated a significant
increase in bioavailability of Dox compared to
intravenous injection of Dox solution (Reddy and
Murthy., 2004a). Together, the experiment identified
the EP nanoparticles as a potential method of
improving Dox therapy by reducing systemic toxicity
(Reddy and Murthy, 2004a).
Following the polymerization study, Reddy et
al. (2004b) examined the affect of Doxorubican loaded
poly(butyl cyanoacrylate) (DPBC) nanoparticles on
Dalton’s lymphoma.
They found that the DPBC
nanoparticles sequestered in the tumor after
subcutaneous injection much better than did free Dox.
Additionally, they noted that there was a low amount of
Dox found in the heart from the DPBC nanoparticles,
and confirmed that Dox delivered by DPBC
nanoparticles has an increased retention time within
tumors. This confirms the results of the previous
experiment, and also shows that cardiac toxicity may be
limited through this technique.
Ma et al. (2004) developed another type of
nanoparticle to be used for Dox delivery to tumor cells.
The particles, known as carbon magnetic nanoparticles
(CMNP), were created using a new technology known
as dense medium plasma (DMP) technology. The
particles consist of a carbon-based host structure with
iron and iron oxide particles evenly dispersed (Ma et al.,
2004). The CMNP-Dox and intravenous free Dox were
applied to osteosarcoma cells to test antiproliferative
activity. The results showed that at the highest dose,
Drug Delivery Using Nanoparticles
Drug delivery is the carrying of drugs using
nanoparticles specifically to the cells causing the
disorder. In the case of cancer, these drugs are
frequently
known
chemotherapeutic
agents.
Intravenously, these drugs cause a variety of side
effects. However, by linking them to nanoparticles the
drugs go directly to the source and do not affect healthy
cells (Paciotti et al., 2004). As is the case with
hyperthermia, certain types of nanoparticles are better
adapted for drug delivery than others. For instance,
nanoparticles composed of colloidal gold easily attach
various drugs. Colloidal gold is a dispersed solution of
nanoparticles of Au0 (Paciotti et al., 2004). Additionally,
polybutyl cyanoacrylate (PCB) nanoparticles attach
drugs, protect them against enzymatic degradation,
reduce their toxic effects, and limit distribution of the
drug outside the target area (Reddy et al., 2004a).
Tumor Necrosis Factor and Colloidal Gold
Tumor necrosis factor (TNF) is a cytokine that affects
coagulation, lipid metabolism, insulin resistance, and
proper function of endothelial cells (Paciotti et al.,
2004).
It is produced during immune response
primarily by monocytes and macrophages and has the
ability to induce death in tumor cells (Elliott et al., 1994).
Unfortunately, TNF causes systemic toxicities that have
prevented it from being used as an anti-cancer drug
(Furman et al., 1993). This toxicity can be attributed to
rapid uptake of TNF by the reticuloendothelial system
(RES) (Paciotti et al., 2004). Through the use of
colloidal gold nanoparticles, Paciotti et al. (2004) were
able to construct a vector which can avoid detection
and clearance by the RES. Thus, the nanoparticles
(PT-cAu) delivered TNF specifically to tumor cells,
eliminating the associated systemic toxicity.
Next, Paciotti et al. (2004) compared
treatment using native TNF and PT-cAu-TNF which
showed both reduced tumor size in a concentration
dependent manner. However, mice given 12 µg native
TNF suffered 25% fatality and all given 24 µg native
TNF died whereas none of the mice treated with PTcAu-TNF perished. Furthermore, Figure 2b illustrates
that while 15µg of Native TNF has approximately the
same affect on tumor size as PT-cAu-TNF through 16
days, the survival rate using the native form is 40%
lower. Thus, without the colloidal gold nanoparticles,
TNF is extremely toxic. These nanoparticles help TNF
circumvent the RES and enter selectively into cancer
cells, which ultimately causes tumor cells to die
(Paciotti et al., 2004).
Localized Chemotherapy
As mentioned earlier, the main problem with
chemotherapy is that it is not tumor specific. Thus,
chemotherapy drugs tend to act on all rapidly dividing
cells. Through the use of nanoparticles, however, the
same drugs can be linked specifically to cancer cells at
higher concentrations for longer periods of time. Thus,
58
Figure 2: TNF effect on tumor volume in mice MC-38 colon carcinoma tumors.
a.) Antitumor efficacy of native TNF and the cAu-TNF vector. Mice with MC-38 colon carcinoma tumors were intravenously injected with
increasing concentrations of native TNF of cAu-TNF vector (n=4/group/dose). Tumors were measured 10 days after treatment using three
dimensional measurements (L x W x H). b.) Antitumor efficacy of native TNF and PT-cAu-TNF vector using one group as a control. Two
groups with either 7.5 or 15 g of intravenously injected PT-cAu-TNF. Another two groups were intravenously injected with 7.5 or 15 µg of
native TNF. The size of tumors were then measured on various days (Paciotti et al., 2004).
free Dox had no significant effect on the tumor cells
compared to CMNP-Dox, which completely stopped
proliferation at 120 µg/ml Dox. Interestingly, at 240
µg/ml, CMNP-Dox had a reduced effect, believed to be
because of steric hindrance caused by excess
nanoparticles (Ma et al., 2004). One of the chief
advantages of this system, however, is that it can be
made in one step under atmospheric pressure using
inexpensive chemicals, such as benzene and
acetonitrile, making it both effective and cost efficient
(Ma et al., 2004).
urothelium utilized gelatin nanoparticles loaded with the
drug. These nanoparticles are hydrophilic and thus
uptake fluid rapidly allowing for paclitaxel to be released
easily. This is important because the quicker the drug
is released, the longer its exposure to cancer cells
before urination. The concentration of paclitaxel in the
urine, which was collected during treatment, was 2.6x
that of the cremophor/EtOH formula. Additionally, 87%
of the drug was released in two hours (Wientjes, et al.,
2004), compared to only 45% after 3 days for
paclitaxel-loaded
poly(ethylene
oxide)poly
(lactide/glycolide) nanospheres used to regulate
smooth muscle cell regulation (Suh et al., 1998). In
summary, paclitaxel loaded gelatin nanoparticles were
able to penetrate the urothelium of the bladder and
rapidly release the drug, making them a promising
treatment for bladder cancer (Wientjes et al., 2004).
Paclitaxel
Paclitaxel is a chemotherapy drug that can be used to
treat Kaposi’s sarcoma and metastatic breast, ovarian,
and bladder cancer (Wilkes et al., 2000). It is an antimicrotubule compound that prevents continuation of the
cell cycle and thus proliferation (Wientjes et al., 2004).
In the case of bladder cancer, doxorubicin and
mitomycin C are ineffective treatment options due to
their inability to pass through the transitional epithelium
in the wall of the bladder known as the urothelium.
Since paclitaxel is lipophilic, however, it can freely pass
through the urothelium (Wientjes et al., 2004). The
FDA approved formulation for paclitaxel includes the
solvent Cremophor. Cremaphor causes paclitaxel to
become entrapped in the micelles of the bladder, which
lowers the drugs ability to penetrate the urothelium
(Knemeyer et al., 1999). To combat this, Wientjes
(2003) used DMSO as a surface-active agent that
disrupted Cremaphore micelles and enabled paclitaxel
to be delivered to the tumors; however, this technique
caused increased urine production and associated drug
removal. Consequently, with less time in contact with
the cancerous cells, paclitaxel was less effective.
Wientjes’s et al. (2004) second attempt to
facilitate the transfer of paclitaxel through the
Gene Delivery using Nanoparticles
Nanoparticles can deliver proteins with anti-tumor
activity into tumor cells and additionally, they can be
used to deliver chemotherapeutic drugs directly to
tumors, avoiding systematic toxicity. The versatility of
these small particles also allows them to transport
plasmid DNA with tumor suppressor genes to tumor
cells. This causes a tumor suppressing protein to be
produced which induces tumor cell apoptosis,
effectively fighting the cancer (Ramesh et al., 2004).
MDA-7
First identified in human melanoma cells
(Jiang et al., 1995), the human melanoma
differentiation associated gene 7 (mda-7 or IL-24) is a
tumor suppressor gene.
In late stage human
melanoma, MDA-7 protein is absent, whereas in early
stage melanoma it is present. Accordingly, this gene
59
product is likely involved with progression of the
disease (Ellerhorst et al., 2002). Furthermore, the
protein is absent in a variety of human tumors including
lung, breast, and colorectal carcinomas and sarcomas,
and thus, it is believed to be involved in both the
development and progression of these human cancers
(Chada, et al., 2003).
Previous studies have shown that through
using adenoviral vectors, expression of MDA-7/IL-24
triggers cytotoxic related cell death and growth
suppression in several human cancer cells (Ramesh et
al., 2004). Moreover, normal cells are not affected by
exposure to mda-7gene, making it a potentially strong
anti-tumor therapy. In 2003, Chada et al. used an
adenoviral receptor to deliver mda-7 to tumors in the
lungs. The results were promising, because this
procedure caused expression of MDA-7 induced
apoptosis in the tumors. Unfortunately, the adenovirus
vector can cause an immune response and liver toxicity
(Vlachaki et al., 2002). Therefore, a new vector for
mda-7 delivery to disseminated cancers is needed.
Ito et al. (2003c) demonstrated that DOTAP:
cholesterol nanoparticles can transport tumor
suppressor genes to tumors in the lungs and increase
the transgrene expression of these genes. Based on
this, Ramesh et al. (2004) tested the use of cationic
DOTAP: cholesterol (Chol) nanoparticles as a vector for
delivery of mda-7 gene. They found that cells treated
with the DOTAP/mda-7 gene showed significantly fewer
tumors (Figure 3).
Additionally, they found no
resistance to multiple treatments with this therapy, as
well as no systematic toxicity.
Furthermore, the
treatment was still successful in immunodeficient and
immunocompetent organisms. Thus, using DOTAP:
Chol nanoparticles as a vector for the mda-7 gene is a
novel approach for cancer therapy that shows much
promise (Ramesh et al., 2004).
Firstly, neither of these treatments causes systematic
toxicity. In fact, both hyperthermia and drug delivery
can be directed specifically to cancer cells. Ultimately,
this is advantageous because it greatly reduces the
physically and psychologically demanding side effects
of chemotherapy and radiation, which include, but are
not limited to anemia, neutropenia, hair loss, diarrhea,
sterility, and nausea.
These side effects are thought to be
worthwhile because of chemotherapy’s effect on
cancer, but all cancer cells are not responsive to
chemotherapy. Furthermore, some cancers develop
resistance to chemotherapeutic drugs (Gottesman,
2002).
There are several reasons for this. As
mentioned in the beginning, tumor cells have a variety
of mutations, and all tumor cells do not have the same
mutation. Some mutations allow cells to randomly
develop resistance to drugs because they no longer
express the protein receptors to which the drug
interacts. Thus, the cells without the receptor have a
growth advantage, and if another drug is not used,
these cells will proliferate rapidly (Gottesman, 2002).
Additionally, tumor cells may produce more target
proteins than the drugs can bind.
Since
chemotherapeutic agents are not specific, the
concentration of the drugs cannot be raised, as other
systems of the body would be effected as well
(Gottesman,
2004).
Furthermore,
enhanced
amplification of the MDR1 (Multiple Drug Resistance)
gene results in the encoding of a large transmembrane
protein which can stop certain drugs from entering a
cell and also eject drugs already in it (Bredel et al.,
2002).
With chemotherapy, any form of resistance
requires another type of drug; however, nanoparticles
may hold the key to circumventing such resistance.
Early trials with hyperthermia and gene delivery show
that each technique may be used multiple times.
Hyperthemia, for instance, does not work on hindering
processes inside the cell, but instead, it heats the cell
up to such high temperatures that it denatures proteins
and DNA (Dai et al., 2005). Heat shock proteins that
stabilize proteins to prevent denaturing are themselves
denatured when exposed to heat of this magnitude (Ito
et al., 2003).
Thus, hyperthermia can be done
Discussion
In this paper, I have chronicled three promising
techniques for treatment of cancer using nanoparticles:
hyperthermia, drug delivery, and gene therapy. These
techniques each have several advantages over the
current treatments of radiation and chemotherapy.
Figure 3- Mice treated with mda-7 exhibit a lower number of tumors.
Mice with A549 and UV2237m lung tumors were treated daily for a total of six doses (50 g/dose) with phosphate-buffered saline
(PBS), DOTAP:Chol-chloramphenicol acetyl transferace (CAT) nanoparticles, or DOTAP: Chol-mda-7 nanoparticles. Tumor growth
was only inhibited by DOTAP:Chol-mda-7 nanoparticles (P<0.05; Ramesh et al., 2004).
60
Bredel, M. and J. Zentner. "Brain-tumour drug resistance: the
bare essentials." Lancet Oncology, 2002.
repeatedly without detrimental effects to other systems
or the threat of tumor cells becoming resistant to it (Dai
et al., 2005).
Moreover, through gene delivery,
expression of tumor suppressor genes inside tumors
can be controlled, so in essence, tumors are forced to
fight themselves. Early experiments suggest, that using
the mda-7 gene in this manner can be performed
repeatedly and cells develop no resistance (Ramesh et
al., 2004).
While hypothermia and gene therapy appear
to circumvent resistance, through the use of higher drug
concentrations and exposure time, localized drug
delivery provides another option.
The use of
nanoparticles allows for higher concentrations of drugs,
such as native tumor necrosis factor and doxorubicin, to
be used. This is possible because the nanoparticles
specifically target cancer cells, and thus, there will be
no associated systemic toxicity. Because a higher
amount of the drugs can be used, the initial treatment
has a larger effect, as more of the drug is able to
interact with the tumor cells. Additionally, a second
treatment can be administered much more rapidly
afterward, since the rest of the body does not have to
recover. Together, there is a much smaller chance that
the tumor would develop resistance, because treatment
can take a shorter period of time. However, it is
possible that some of the tumor cells have innate
resistance, in which case, another drug would have to
be used.
In summary, the techniques of drug delivery
and hyperthermia using nanoparticles have the
potential to decrease side effects while increasing the
cure rate of cancer patients. These techniques promise
a substantial improvement over chemotherapy and
radiation. Over the next few years, if the research
conducted on nanoparticles continues to find promising
results, the treatment of cancer all over the world may
be substantially altered. The cure for cancer may in
fact be close at hand.
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“Impact of preimmunization on adenoviral vector
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Williams, Penelope. New Cancer Therapies: The Patients
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Wientjes, M. G., D. Chen, D. Song., J. L. Au. “Effect of
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Onishi, H., Yoshiaki Machida, and Yoshiharu Machida.
“Antitumor properties of irinotecan-containing nanoparticles
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“Paclitaxel-Loaded Gelatin Nanoparticles for
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62
Review Article
Coal Power: Providing Energy, Asthma, Cardiovascular
Disease, and Free Abortions
Ethan Helm* and Benjamin N. Larsen*
Lake Forest College
Pulmonary Effects of Coal Power Pollution
Nearly eighty years ago, a dense mist settled to the
east of Belgium’s third largest metropolitan area, Liege
(Roholm, 1937). Soon after the emergence of the mist,
doctors in the area began to realize something was
wrong. Several thousand acute pulmonary attacks had
been caused in the area and there were sixty confirmed
deaths (Firket, 1931). In the small town of Meuse,
people did not know what hit them. They would later
find out that a poison in the form of sulfur dioxide was
being produced by a local factory (Roholm, 1937). This
discovery was met with much skepticism, and sulfur
dioxide emission standards were not increased until the
1970s. Since then, the scientific world has investigated
the role of the pollutants released through industrialized
processes, and as a result, we know that sulfur dioxide,
nitrogen oxides, particulate matter, and ozone all have
deleterious pulmonary effects (Pope et al., 2002). A
recent study conducted by Kan and Chen in Shanghai
found that mortality caused by pulmonary problems was
associated pollution from coal power plants; these
findings lead to the conclusion that current pollution
levels are a threat to the general population’s health in
Shanghai (2003).
Over 23,600 people die from it yearly. It causes more
than 554,000 asthma attacks and 38,200 nonfatal heart
attacks. (Schneider, 2004). It has left twenty-five
percent of New York’s Adirondack Lakes uninhabitable
(Wu, 2003). It is a large contributor the global warming
trend which is melting the artic and glaciers world wide
and pushing many species to the brink of extinction
(Schneider, 2004). Coal power plants are not only
affecting our cities by cloaking them with ominous
clouds of pollution, but also our children, us, and the
world we live in.
Most of this is preventable. Over half of the
electricity in the United States comes from coal power
and 548 power plants have not been modified to reduce
emissions in the last thirty years (Schneider, 2004). In
this time period, huge technological advances have
been made that could significantly reduce pollution.
Unfortunately, such technology has mainly been
implemented in newly constructed power plants and
current legislation does not require old power plants to
be modified (Corrigan, 2005). As such, over 70% of the
sulfur dioxide and 62% of nitrogen oxides released from
these plants could have been prevented (Wu, 2003).
Additionally, large amounts of ozone, particulate matter,
and carbon dioxide could also be reduced if older plants
implemented extant technology.
Part of the problem with coal power is that it
does not produce just one pollutant. Carbon dioxide,
sulfur dioxide, nitrogen oxides, ozone, and particulate
matter are all released through burning coal. Nitrogen
oxides, ozone, sulfur dioxides, and particulate matter
have all been linked with respiratory problems.
Additionally, particulate matter has been associated
with cardiac disease, and all of the pollutants have
been shown to be harmful and potentially fatal to
children (Schneider, 2004).
While there are many similarities, pollutants
work in a variety of different ways. For example,
particulate matter, nitrogen oxides, and sulfur dioxides
form acids with other compounds, which cause scarring
in the lungs (Schneider, 2004).
Also, ozone is
damaging because it catalyzes oxidation inside the
body. Through oxidation, it can produce free radicals,
which cause muscle pain, inhibit hormone function, and
disrupt neural impulses (Wu, 2003).
While coal power pollution affects people in a
variety of ways, we are focusing on the direct
physiological effects of pollution on humans. As such,
we are not researching the heat trapping capabilities of
carbon dioxide. Additionally, we will not be focusing on
the environmental impacts on biodiversity due to
nitrogen oxides. Instead, we concentrated on the
cardiac, pulmonary, and developmental problems
associated with pollution from coal power.
General Effects
Pollution does not even have to affect people in the
form of disease.
In fact, exposure to air borne
pollutants can reduce lung function (AckermannLiebrich, 1997). This should be of concern to those
whom enjoy the outdoors or breathing in general.
Reduced lung function may also be a concern for
athletes during competitions in which the body needs
oxygen to produce ATP that fuels muscle contractions.
An increase in particulate matter of just 10 µg/m3
corresponded to a 3.4% decrease in FEV (forced
expiratory volume, or the amount of air your lungs
exhale after a deep breath). Not to be left out, nitrogen
dioxide and sulfur dioxide were also correlated with
decreased pulmonary function (Ackermann-Liebrich,
1997).
Asthma
Particulate matter has been linked to both cardiac and
pulmonary dysfunction. In Barcelona, for example,
there was a strong correlation between levels of
particulate matter and the number of asthma related
hospital visits over a three year period (Llorca et al.,
2005). Research by Castellsague et al. also describes
a correlation between particulate matter exposure and
emergency room hospitalizations for asthma (1995).
Some of particulate matter’s health impacts may be
actually be underestimated due to “masking” by
nitrogen dioxide (Llorca et al., 2005).
Also, Nitrogen dioxide has been linked to
asthma related symptoms. It was shown to decrease
tolerance to allergens among allergic asthmatic
individuals. Subjects exposed to NO2 for short periods
of time experienced allergic symptoms when
subsequently exposed to allergen levels that had
previously left them asymptomatic. Both early and late
phase airway responses were more severe after
nitrogen dioxide exposure and the histamine response
doubled. The increase in symptom intensity may be
*This article was written for BIOL 375, Conservation Biology taught
by Dr. Caleb Gordon.
63
even more pronounced in individuals with more severe
asthma or other health problems (Strand, 1998). The
findings of Castellsague et al. support this trend; they
found nitrogen dioxide increased symptoms of brachial
asthma among adults (1995) and Llorca et al. noticed
more emergency room treatments for asthma when
ambient levels of nitrogen dioxide were higher. The
number of emergency room visits increased by about
70% for each additional 100 µg/m3 of nitrogen dioxide
in the air (Llorca et al., 2005).
People who do not have preexisting
conditions can also be affected by coal power plant
pollution.
Chronic obstructive pulmonary disease
makes breathing difficult for those inflicted. Generally,
the disease is associated with smokers suffering from
emphysema and or chronic bronchitis.
However,
Tolbert et al. found a correlation between chronic
obstructive pulmonary disease and exposure to
ambient nitrogen dioxide, ozone, and particulate matter
(2000). Death due to chronic disease has been linked
to sulfur dioxide (Kan, 2003), although the affected
individuals are probably sickened by the effect of
multiple pollutants.
al. went a step farther by linking particulate matter to
infant mortality due to respiratory illness (1997).
Research on the affect of nitrogen oxides on
lung development has not been as consistent. In 1990,
Dijkstra et al. did a study on children in Holland and
found that nitrogen oxides do not prevent lung
development. Conversely, Ware et al. found that
nitrogen oxides do affect large air pathways (1984).
The same lab later refuted these claims (Berkey, 1986).
The involvement of nitrogen oxides by Gauderman and
his coworkers regarding lung development in children
represents a debate in the scientific community that has
not been resolved (2000).
While Gauderman et al. did not find that
ozone had a statistically significant effect on lung
development in children; other scientists had conflicting
results (2005). For example, Bates (1995) found that
long-term exposure to ozone caused developmental
problems associated with the lungs (1995). Frischer et
al. confirmed this by analyzing the affect of ambient
ozone on children in Austria (1999). The entire debate
on ozone’s effect was questioned by Tager whom
stated that scientists were not adequately addressing
co-pollutants of ozone (1999). This critique can be
broadly applied to much of pollution research, and
many researchers, such as Gauderman, choose to
acknowledge the limitations of their studies (2000).
Pollution Hinders Development
In early December of 1952, a stationary front moved
through London thereby reducing wind. At the same
time, a thermal inversion trapped coal smoke in the
Thames Valley (Schwartz, 2004). The combination of
these two events caused a prolific build up of pollution,
which resulted in the deaths of around 4,000 people
over a four-day period, and many others in the following
weeks (Anderson, 1999). Of those who died, the
mortality rate of infants was twice that of adults
(Schwartz, 1994). In fact, from the time of birth to 4
years of age, the number of alveoli in the human lungs
increases by over ten fold (Schwartz, 2004). Thus,
infants are not able to obtain oxygen as readily as
adults. This demonstrates that development extends
past the amniotic sac, and as such, children are even
more susceptible to the effects of pollution (Schwartz,
2004).
Preterm Delivery and Associated Mortality
Xu et al. (1994) linked the use of coal stoves to low
birth rate and premature birth. Low birth weight has
been established to be the most important factor for
predicting neonatal mortality (McCormick, 1985). How
pollution affects premature delivery, however, is not
fully understood. It is known, however, that infections
can be passed from mother to child causing premature
birth (Xu et al., 1995). If disease can be passed
through the amniotic sac, pollution can affect the fetus
as well. In 1995, Xu et al. noticed that there were
significant seasonal changes in concentrations of sulfur
dioxide and particulate matter around Beijing, China.
Based on this, they looked at medical data to determine
whether gestation periods were lower during the
periods of higher pollution. Using 24,370 pregnant
women from four parts of Beijing, Xu et al. were able to
determine that there is a statistically significant
correlation between concentration of SO2 and
particulate matter and average gestational age of new
born children (1995).
This result was confirmed by Wang et al.
Using the same area over a longer period of time, they
analyzed the gestational age of 74,621 births (1997).
The data showed that for every 100 µg/m3 of sulfur
dioxide there was an 11% greater chance of premature
birth with an estimated reduction of birth weight of 7.3g
for each 100 µg/m3 increase. Likewise, for every 100
µg/m3 of particulate matter preterm delivery was 10%
more likely to result in a reduction of birth weight by
approximately 6.3g (Wang et al., 1997).
Additionally, Bobak and Leon found mortality
increased from the lowest to highest measured
amounts of particulate matter in the Czech Republic.
However, no correlation between infant mortality and
nitrogen oxides and sulfur dioxides was found (1992).
In Brazil, particulate matter was more indirectly related
to infant mortality through pneumonia (Penna and
Duchaide, 1991).
While these studies do not
demonstrate that pollution from coal power directly
causes preterm mortality, there is undoubtedly a
Lung Development in Children
Children spend more time outdoors, are more active,
and breathe more rapidly than adults (Gauderman et
al., 2000). Thus, children are more susceptible to
inhaling pollutants, all while their bodies are still
developing. Gauderman et al. investigated this by
monitoring the large and small airways of 3035 fourth
graders around Los Angeles, California over four years
(2000). By comparing the development of the lungs
and the amount of ozone, particulate matter, and
nitrogen oxides, they found that each pollutant except
ozone decreased lung development. Nitrogen oxides
and particulate matter reduced the maximum volume of
large air pathways by 0.77% and 0.90% annually
(2000). Both values are larger than the 0.2% annual
decrease believed to be caused by secondary smoking
(Berkey et al., 1986). This demonstrates that the effect
of coal power pollution on lung development in children
could be physiologically significant (Gauderman et. al.,
2000) and cause as much as a 16.7% decrease in lung
capacity over a ten year period if both pollutants are
independent.
Other studies have confirmed that particulate
matter obstructs proper lung development.
For
example, Jedrychowski et al. found similar results to
Gauderman and his associates (1999). Woodward et
64
correlation between preterm mortality and increased
sulfur dioxide and particulate matter.
closely associated with ischemic strokes in Taiwan
(2003). The preponderance of the evidence suggests
that coal power pollution is linked to ischemic strokes,
but more research must be done to determine which
pollutants have the most effect.
Coal Powered Pollution Hurts the Heart
Plasma Viscosity
An increase in mortality has been associated with Air
pollution (Bobak, 1992; Kan, 2003; Penna and
Duchaide, 1991; Peters and Doring, 1997). Much of
the mortality is related to cardiac dysfunction rather
than pulmonary trouble (Pope et al., 2004). Peters and
Doring investigated hospitalization and mortality during
pollution episodes due to cardiac disease. A large
cross-sectional survey revealed that during pollution
episodes, plasma viscosity increased dramatically. In
fact, there was nearly a 25% chance of plasma
viscosity exceeding the 95th percentile among men.
Such considerable thickening may be part of the
physiological chain reaction linking ambient air pollution
to hospitalization and mortality from cardiovascular
illness (Peters and Doring, 1997) also observed by
Tolbert et al. (2000) and Pope et al. (2004). Another
link in this chain may be vaso and arterial constriction
caused by exposure to particulate matter and ozone
(Brooke et al., 2004). The effects of such constriction
would likely be compounded by quickened
atherosclerosis (Pope et al., 2004).
Discussion
There is a large body of research linking pollutants
released by coal power plants to pernicious health
effects. Exposure to pollutants like nitrogen dioxide,
sulfur dioxide, particulate matter, and ozone has been
correlated to increasing emergency room visits for
asthmatics (Castellsague et al., 1995; Llorca et al.,
2005), heightened allergic response (Strand, 1998),
decreased lung function in healthy individuals
(Ackermann-Liebrich, 1997), chronic obstructive
pulmonary disorder (Tolbert et al., 2000), and even
death from chronic disease (Kan, 2003).
These
pollutants have also been tied to pulmonary
developmental retardation in children (Gauderman,
2000; Jedrychowski et al., 1999). Further studies have
linked some pollutants to lowered birth weight and or
premature birth (Wang et al., 1997; Xu et al., 1994)
while others link particulate matter to infant mortality
(Bobak and Leon, 1992; Penna and Duchaide, 1991).
In addition to pulmonary problems, coal
power pollution can be dangerous to the circulatory
system. Hospitalization due to cardiac dysfunction also
increased with ambient air pollution (Pope et al., 2004;
Tolbert et al., 2000). Such hospitalization is likely
related to high blood plasma viscosity correlated to
increasing ambient particulate matter (Peters and
Doring, 1997).
While not all of these studies were directly
linked to point source pollution from coal power plants,
the fact that many pollutants released when coal is
combusted in power plants are associated with
negative health impacts is alarming. The potential
damage caused by coal power pollution should
reinforce pleas to maintain or increase existing
emissions standards as declared in the Clean Air Act.
Moreover, attempts to loosen regulations might cause
an increase in chronic health problems, interference
with children’s development, and premature death.
Cardiovascular Disease
Other cardiac problems have also been associated with
air pollution. In Atlanta, a survey of over two million
emergency room visits revealed an association
between cardiovascular disease and ambient air
pollution, especially particulate matter (Tolbert et al.,
2000). Dysrythmia, cardiac arrest, and heart failure
were all associated with exposure to particulate matter
(Tolbert et al., 2000; Pope, 2004). As suggested by
Peters and Doring, symptoms of cardiovascular disease
are probably related to thickening of the blood (1997).
Smokers are at particular risk of cardiac disease and
exposure to air borne pollutants may increase the risk
of disease synergistically (Pope et al., 2004).
Ischemic Strokes
Scientists have demonstrated that pollution can also
cause death through strokes (Tolbert et al., 2000).
There are two types of strokes:
ischemic and
hemorrhagic. Hemorrhagic strokes occur because of a
burst blood vessel in the brain, while ischemic strokes
are a result blockage of blood flow to the brain.
Moreover, an increase in plasma viscosity and heart
rate variability cause an increased risk for ischemic
strokes (Hong et al., 2002a; Peters and Doring, 1997;
Peters et al., 1999).
Unfortunately, the affect of pollution on
strokes has not been widely studied. In South Korea,
strokes are much more common than in the United
States. Additionally, rapid industrialization in the form
of cars and coal power plants has marked a huge
increase in pollution in South Korea, Taiwan, India, and
China (Hong et al., 2002a). By comparing hospital
records in relation to pollution levels over time, Hong et
al. found that particulate matter, nitrogen oxides, sulfur
dioxide, and ozone are all correlated with an increase in
ischemic stroke mortality (2002a). Another study in
Hong Kong found similar results (Wong et al., 2002).
Further studies in South Korea illustrated that ozone
and particulate matter have a stronger correlation
(Hong et al., 2002b). Conversely, Tsai et al. postulated
that particulate matter and nitrogen oxides are more
the author.
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66
Review Article
Eukaryon, Vol 3, February 2007, Lake Forest College
Guts & Glory H. pylori: Cause of Peptic Ulcer
Ashley Johnson*, Bryan Kratz*, Lorraine
Scanlon*, and Alina Spivak*
Lake Forest College
Bicarbonate secretions, and reduce blood circulation
which aids in cell renewal and repair. With the host’s
defenses down, stomach acid can irritate the sensitive
lining, thus causing ulcers (5, 6).
Summary
Peptic Ulcers
Peptic ulcers form on the epithelial cells of the stomach
lining. An ulcer consists of two major structures: a
distinct ulcer margin and granulation tissue at the ulcer
base. A distinct ulcer margin is formed by the adjacent
non-necrotic mucosa - the epithelial component. The
granulation tissue consists of fibroblasts, macrophages,
and proliferating endothelial cells,
which form
microvessels. On the molecular level, the pathogenesis
of ulcer disease is believed to reflect an imbalance
between increased corrosive stomach byproducts and
decreased protective factors. As a result of stimulation
arising from the sight, smell, taste, or thought of food,
acetylcholine, a neurotransmitter, and gastrin, a
hormone, are released and act on the parietal cells to
produce acid. The mast cells in turn release histamine,
which also stimulates gastric acid secretion. In patients
infected with H. pylori, the parietal cells have increased
sensitivity to gastrin and possibly to histamine. The
increased sensitivity causes corrosion of the stomach
lining, leading to the formation of an ulcer (2).
Gut Wrenching Diseases
Due to the 1983 discovery of H. pylori bacteria as
the leading cause of peptic ulcers, the
understanding of the disease dramatically changed.
We now know that stress and spicy foods are not
the leading causes of peptic ulcers. Symptoms
including acute abdominal pain, vomiting of blood,
and weight loss are characteristic of peptic ulcers.
Ulcers form because of the inflammation caused by
H. pylori leading to sensitivity of gastric cells to the
acid secreted by the infected patient’s stomach.
Although more than half of the world’s population
is infected with H. pylori, most people remain
asymptomatic. Current research suggests that
several bacterial virulence genes such as CagA and
VacA, as well as the individual host’s genetic
predisposition,
are
factors
that
influence
progression of disease. The mechanism of H. pylori
infection has been recently examined in detail
clarifying the morphological changes of the host
cell and how this promotes the formation of a
peptic ulcer. Present studies
to explain the
persistence of H. pylori and propose how this
bacterium evolved key mechanisms to evade the
host’s immune response. Due to the advances in
the understanding of peptic ulcers, effective
treatments have been proposed to treat and
eliminate this disease.
Gastric Cancer
H. pylori trigger the host's immune system to release
immune response mediators. These molecules, such as
reactive oxygen species and nitrogen made by
neutrophils, are released in the stomach and undergo
lysis due to low pH levels. These molecules can often
damage DNA. Patients with gastric cancer often have
constantly activated oncogenes, such as c-met, c-erbB2, K-sam, or inactivated tumor-suppressor genes, such
as p53, p16, and APC. Those affected also show
abnormal alterations of genes implicated in cell
proliferation and apoptosis, such as cyclin D1, bcl-2,
E2F-1, and SC-1(7).
History
In 1983, Australian scientists Robin Warren and Barry
Marshall showed that the leading cause of peptic ulcers
is the infection of the stomach lining with a helical
(spiral) shaped gram negative bacterium Helicobacter
pylori (H. pylori). It was previously believed that peptic
ulcers were caused by stress and consumption of spicy
foods (1).
Not-So-Glorious Symptoms
One of the major symptoms of gastric ulcers is
abdominal pain, which usually occurs during mealtimes
as more acid is secreted into the stomach.
Hematemesis (vomiting of blood) is often seen in
patients with gastric ulcers, which leads to a noticeable
reduction in the patient’s weight. Melena (i.e. foul
smelling feces) is another symptom of gastric ulcers
that is often caused by the presence of oxidized iron
from hemoglobin (2).
Characteristics
H. pylori infection leads to inflammation of the gastric
mucosa in 80% of peptic ulcer cases. H. pylori cause
elevated acid secretion in people who develop
duodenal ulcers, and decreased acid secretion in those
who develop gastric ulcers and gastric cancer (3, 5).
Duodenal ulcers form due to acid hypersecretion in
response to antral inflammation. In patients with gastric
ulcers, H. pylori cause corpus inflammation which leads
to decreased acid secretion and gastric atrophy (Figure
1). Peptic ulcers are 0.3-0.4 cm in diameter in the
affected area of the stomach. The remaining 20% of
peptic ulcer cases are caused by nonsteroidal antiinflammatory drugs (NSAIDS) like aspirin, which irritate
the stomach lining (3). NSAIDS hinder the protective
mechanisms of the stomach including mucus and
Environmental Pitfalls
A diet high in salt and lacking antioxidant vitamins might
promote low acid secretion and cause gastritis, which
leads to gastric ulcers and gastric cancer. Salt may
change acid secretion by suppressing parietal cells,
causing gastric atrophy. Also, the antioxidant vitamins
in fresh fruit might protect specialized gastric cells from
reactive oxygen species released by inflammatory cells.
Diet confirms why there is a high prevalence of ulcers in
China and Japan. These countries not only have a high
prevalence of H. pylori but also a traditionally salty diet.
* This paper was written in BIO221 Cellular and Molecular Biology, taught
by Dr. Shubhick DebBurman
67
Normal
Stomach
Acid
Production
Gastrin
Food
Normal digestion
functioning
Infected with H.pylori
Food
antral
Increased Stomach Acid
Gastric
ation
Increased Inflamm
Production
Gastrin
Production Gastric
Decreased Stomach Acid
Inflam corpus Production
mation
Duodenal Ulcers
Gastric Atrophy
Gastric Cancer
Gastric Ulcer
Figure 1. H. pylori induced changes in stomach functioning lead to several gastrointestinal diseases Infection with H. pylori leads to
hypergastrinemia which may lead to inflammation depending on bacterial virulence, environmental factors, and host genetic differences.
Patients who develop inflammation of the gastric corpus exhibit decreased acid production which may lead to gastric cancer or gastric
ulcer. Those who maintain a healthy gastric corpus but have gastric antral inflammation exhibit increased acid production, which may lead
to duodenal ulcers.
Cigarette smoking also strongly predisposes to both
duodenal ulcer and gastric cancer (3).
of developing gastric carcinoma as a result of chronic
infection (4, 5, 6).
You’ve “Gut” to Take These
VacA: Evacuate my body
The most effective treatment for peptic ulcers is a three
drug regimen consisting of a proton pump inhibitor
(PPI) and two antibiotics. Proton pump inhibitors work
to expose H. pylori to the drug treatment. The most
common antibiotic used is amoxicillin and the most
prevalent PPI is Omeprazole. Once the bacteria are
eradicated by the drug regimen, the normal immune
response has the full potential to regenerate the
stomach lining and heal the ulcer.
What’s Left to Stomach
Although a lot is already known about H.
pylori and the diseases it causes, there are four major
areas that are currently being expanded on. The fact
that the bacterium resides in so many people yet
symptoms of disease only appear in a few people is
intriguing. This question led researchers to investigate
whether bacterial virulence factors and differences in
the host attribute to this discrepancy. In addition, the
persistence of the bacterium in the infected individual
suggests the possibility, addressed by current research,
that H. pylori evolved key mechanisms to evade the
host's immune responses. Studies of the molecular
mechanism of the invasion of the gastric cells with H.
pylori have been elaborated upon in recent years. Also,
new therapeutic agents and methods of treatment of
gastrointestinal diseases have been proposed in
response to new findings.
The H. pylori vacuolating cytotoxin gene, vacA, is
naturally polymorphic. The two most diverse regions
being are the signal region (which can be type s1 or s2)
and the mid region (m1 or m2). The type s1/m1 and
s1/m2 strains of vacA are associated with peptic ulcer
and gastric cancer, whereas while the type s2/m2
strains are non-toxic and associated with lower risk of
peptic ulcer and gastric cancer. The features of vacA
that determine the nontoxicity of these strains were
determined by Letley et. al. (2003). They did this by
deleting parts of vacA and constructing isogenic hybrid
strains in which regions of vacA were exchanged
between toxigenic and non-toxigenic strains. They
showed that a naturally-occurring 12-amino acid
hydrophilic N-terminal extension found on
s2 VacA blocks vacuolating activity as while its removal
(making the strain s1-like) confers activity. They did
chromosomal replacement of vaca in a nontoxigenic
strain with vacA from a toxigenic strain and found full
activating activity, proving that the vacoulation is
controlled entirely by elements within vacA. This
research defined why determined that H. pylori strains
with different vacA allelic structures have differing
toxicity (10).
Virulent Strain Carries CagA Gene
Another bacterial virulence factor is the polymorphism
of the CagA protein. All H. pylori strains have the
cagPAI DNA segment, but only some strains have the
cagA gene that encodes the 145 -kDA CagA protein.
These strains are called cagA+ strains, and while the
strains lacking the cagA gene are called cagA-. The
cagA+ strains are more virulent than the cagA- strains
and are associated with gastric carcinoma. The CagA
is injected by the bacterium and subsequently
Disease or No Disease…That is the question
Current research suggests that several bacterial
virulence factors such as CagA and VacA genes, as
well as the individual host’s genetic predisposition,
influence progression of H. pylori-related diseases. The
World Health Organization recently has classified H.
pylori as a class I carcinogen because of the risk factor
68
undergoes
tyrosine
phosphorylation.
The
phosphorylated CagA specifically binds SHP-2
phophatase, activates the phophatase activity, and
thereby induces morphological transformation of cells.
SHP-2 plays an important role in both cell growth and
cell motility. This morphological change is referred to
as the hummingbird phenotype because the cell
undergoes dramatic elongation by means of the
attachment of cagA+. Higashi et. al. (2002) found that
Western and East Asian CagA both contain tyrosine
phosphorylation sites but they differ in structure.
Western strains can have repeating tyrosine
phosphorylation sites. The larger the number of binding
sites, the greater the amount of tyrosine
phosphorylation, which leads to increased SHP-2
binding and greater morphological changes.
In
contrast, the East Asian strains have a different tyrosine
phosphorylation sequence at the region corresponding
to the Western sequence that binds SHP-2 stronger
and induces greater morphological changes to the cell
than the Western sequence, causing East Asian CagA
proteins to be more potent and leading to high gastric
cancer incidence rates (9).
H. pylori Persistence
The primary response of the body to infection of H.
pylori is inflammation. This is caused by the infiltration
of the gastric mucosa with neutrophils, macrophages, B
cells, and T cells following release of interleukins. T
lymphocyte responses in acute H. pylori infection are
predominantly of the CD4+ Th1 (mainly cell-mediated)
cell phenotype (3, 5). Although a seemingly large
immune response is initiated, it is mostly ineffective,
because H. pylori bacteria are rarely completely
eradicated from an infected individual. The persistence
of H. pylori and the high reinfection rate suggest that
the host has significant anergy and is unable to build
protective immunity.
Interleukin-1 β  What alleles do you have?
VacA Does What?
Host genetic factors that affect interleukin-1-beta may
determine why some individuals affected with H. pylori
develop gastric cancer while others do not.
Polymorphisms in human cytokine genes affect the
level of cytokine production by cells after contact with
H. pylori. Specific polymorphisms in the IL-1b gene and
the IL-1 receptor-antagonist gene (IR-1RN) lead to
increased gastric mucosal levels of IL-1b in individuals
infected with H. pylori. IL-1b (interleukin-1-beta) is an
important pro-inflammatory cytokine and a powerful
inhibitor of gastric acid secretion (5).
The three
reported diallelic polymorphisms in IL1B which have
been reported all represent C-to-T base transitions at
positions –511, -31, and +3954 basepairs from the
transcriptional start site. El-Omar et al., demonstrated
that individuals who were carriers for of the interleukin-1
beta- 31T allele had low acid secretion. The
polymorphisms also increase the risk of gastric atrophy,
hypochlorhydria, intestinal metaplasia, and gastric
cancer (5, 8). Using electrophoretic mobility shift
analysis to assess DNA binding in vitro, the interleukin1 beta -31T allele was associated with a five-fold
increase in DNA-binding after lipopolsaccharide
stimulation. Individuals carrying the interleukin-1 beta 31 T allele are more susceptible to developing
hypochlorhydria, and subsequently gastric cancer, in
the case of an infection by the bacterium H. pylori.
Thus, the interleukin-1 beta gene is a crucial factor in
determining if a person will develop gastric cancer (8).
Previous studies showed that VacA inhibits release of
IL-2 in Jurkat cells (human T-cell leukemia cells). This
inhibition is linked to the ability of VacA to inactivate the
Nuclear Factor of Activated T-cells (NFAT). This
transcription factor is critical to the transcription of IL-2;
therefore, if VacA inactivates NFAT, IL-2 secretion is
inhibited, and Jurkat T cell proliferation is therefore
decreased (11).
However, Sundrud et al. (2004) propose that
VacA has a different effect on primary human Th cells.
Similar testing with the human Th cells suggested that
VacA has only a modest effect on IL-2 secretion. VacA
did not cause a reduction in IL-2 levels in either naïve
or memory Th cells. Therefore, it is now predicted that
VacA inhibits IL-2-driven proliferation of primary human
Th cells by a non-NFAT mechanism. Further studies
suggested that VacA suppresses cell cycle progression
in Th cells, similar to drugs such as rapamycin which
induce G1 arrest. Therefore, instead of blocking IL-2
secretion, and by that inhibiting Th cell proliferation in
human Th cells, VacA might be blocking normal cell
cycle progression of these cells. Sundrud et al. found
that VacA must have an intact hydrophobic domain
within its N-terminal region. This component of VacA
structure is necessary for both inhibition of IL-2
secretion in Jurkat cells and inhibition of IL-2-driven
proliferation of human primary Th cells. This region is
attributed to making VacA anion-selective channels,
which may cause depolarization of the Th cell plasma
membrane and lead to inhibition of IL-2- dependent Tcell proliferation. Interestingly, a mutant VacA (VacA(6-27) that completely lacks this entire hydrophobic
region actually has a dominant negative effect and fully
blocks the wildtype VacA mediated inhibition of T cell
proliferation both in Jurkat and primary human Th cells.
Thus, these scientists concluded that VacA has
immunosuppressive properties that help H.pylori evade
the host’s immune response (11).
associated binder 1 or growth factor receptor–bound
protein 2. The H. pylori–induced motogenic response is
suppressed and blocked by the inhibition of PLCγ and
of MAPK, respectively. Thus, upon translocation, CagA
modulates cellular functions by deregulating c-Met
receptor signaling. The activation of the motogenic
response in H. pylori–infected epithelial cells suggests
that CagA could be involved in tumor progression (8).
Gastric Cancer
Infection with the human microbial pathogen
Helicobacter pylori is assumed to lead to invasive
gastric cancer. H. pylori activate the hepatocyte growth
factor/scatter factor receptor c-Met (oncogene), which is
involved in invasive growth of tumor cells. The H. pylori
effector protein CagA intracellularly targets the c-Met
receptor and acts as an oncoprotein, promoting cellular
processes that lead to changes in cell polarity, motility
and differentiation. These changes may be related to
the development of gastric cancer. CagA could
represent a bacterial adaptor protein that associates
with phospholipase Cγ (PCγ), but not with Grb2-
Treg Cells don’t regulate but promote disease
Recent studies show that the host’s immune response
often leads to immunopathology in an infected person
(3, 5, 7). This conclusion stems from the fact that Th
69
H. pylori Learned to Avoid TLR
cells have a poor responsiveness to H. pylori antigens.
CD4+ T cells proliferate more during H. pylori infection
in comparison to CD8+ T cells. Also, Lundgren et al.
(2003) suggest that memory cells in infected individuals
proliferate a lot less in comparison to the memory cells
of healthy individuals, and naïve cells barely proliferate
in either case. In fact, this difference in proliferation
rates of memory cells was nonexistent when both
individuals were treated with another toxin (Tetanustoxin). This implies that the reduced responsiveness of
memory T cell proliferation in infected individuals was
limited to H. pylori specific cells. This finding led to the
assumption that regulatory CD4+CD25high T cells (Treg
cells) suppress proliferation of memory T-cells. Treg
cells are vital for controlling the immune response to
foreign
antigens
and
preventing
autoimmune
responses. Therefore, it is currently suggested that
repetitive stimulation of T cells with H. pylori antigen
may lead to activation of Treg cells that actively
suppress the response of memory cells. Therefore,
these authors showed that with prolonged infection, the
host’s own immunity activates H. pylori specific Treg
cells, which suppress memory cell proliferation
promoting pathogenesis (12).
It is widely known that eukaryotic organisms have
evolved many mechanisms to recognize bacterial
agents so that a proper immune response can be
activated to eradicate the bacterium. One such
immunity are Toll-like receptors (TLRs), which
recognize components of bacterial membrane LPS and
a bacterial protein flagellin that are released by many
gram-negative bacteria. Gewirtz et al. (2004) suggested
that although H. pylori contain both LPS and flagella,
they are still able to evade this immune response. The
scientists found that H. pylori releases much smaller
amounts of flagellin than other gram-negative bacteria
and the flagellin that they do release is barely potent.
The flagellin that is released does not play a large role
in mediating proinflammatory gene expression in the
host. The usual effect of gram-negative bacteria is the
activation of TLR, which induces IL-8 secretion of a
proinflammatory cytokine. However, H. pylori are able
to evade TLR mediated immunity by producing
impotent flagellin and preventing the release of this
potentially immunogenic, proinflammatory protein (15).
CagA vs. Mucus
(COX) 4 Lowers Immune Response
CagA plays a major role in morphological changes
induced by the Helicobacter pylori bacterium upon entry
of the gastric epithelial cells. Al-Mahroon et al. (2004)
preformed an experiment to test the effect of CagA (+)
or CagA (-) strains of H. pylori on the mean gastric
mucus thickness in humans when compared to an
uninfected individual. Biopsies taken from each of the
patients were submitted to PCR to determine the
presence of CagA (+). After staining and treating the
biopsies, the mucus layer thickness was determined
using an integration of light microscopy, CCD camera,
and specific computer software. The results showed
that, on average, the mucus layer thickness was not
affected in a manner that was statistically significant
(20).
Meyer et al. (2003) found that H. pylori induce
production of cyclooxygenase (COX) 4-2. COX is an
enzyme that is attributed to inhibition of epithelial
apoptosis,
increased
cell
proliferation,
and
angiogenesis. Studies have shown that a byproduct of
H. pylori, urease, allows the bacteria to survive the
acidic pH of the stomach and also induces (COX) 4-2
expression (5, 13). (COX) 4-2 then produces
prostaglandins such as prostaglandin E2 (PGE2) which
mediate inflammation. Therefore, the induction of
(COX) 4-2 by the host is a defense strategy that works
by making PGE2 that reduces inflammation. Also,
Meyer et al. (2003) found that a decrease of
inflammation has been attributed to increased bacterial
colonization. Therefore, H. pylori inhibit the
effectiveness of the host’s immune response leading to
increased pathology (13).
I SAID Drop Your Apical Junctions Now!
Another side effect of H. pylori infection is faulty apical
junctions and loss of cell-to-cell adhesion. Scientists
wondered if CagA plays a role in the mediation of this
effect and how it causes this abnormal morphological
change. Bagnoli (2005) preformed an experiment in
which CagA and ZO-1(a known tight junction
scaffolding protein) were tagged with antibodies so that
they could be easily seen under the microscope. The
results showed that in CagA expressing cells, the ZO-1
protein was mislocated to the basolateral membrane
(Figure 2). It was also found that the apical junction
perimeter and the surface area of the apical membrane
had become substantially reduced. As a result and
consistent with their hypothesis, CagA expressing cells
acquired an elongated, spindle-shaped morphology,
and lost their connections with the apical junctions of
neighboring cells (19).
Le+ H. pylori have an advantage
Horizontal gene transfer and translational frame shifts
contribute to the large genetic diversity of this bacterium
(5). Bergman et al (2005) showed that H. pylori
express Lewis blood group Antigen (Le) in their
lipopolysaccharide (LPS) that is phase variable,
resulting in Le+ and Le- population of H. pylori within a
single strain. Similar to HIV, Le+ antigen of H. pylori
variants can bind to the C-type lectin DC-SIGN and
present on gastric dendritic cells (DCs). This interaction
induces inhibition of Th1 cell differentiation as
compared to nonbinding variants. Le+ antigen alter the
host’s T cell ability to differentiate by reducing the
amount of IL-6 produced and blocks Th 1 cell
polarization. Similar to the Treg suppression of the
immune response, the binding of Le+ antigen to DCSIGN reduces IL-6 levels which may lead to increased
T cell sensitivity to suppression. Therefore, H. pylori
targets DC-SIGN to block a polarized Th1 cell response
by phase-variable expression of Le antigens. Once
again, decreased proliferation of Th1 cells lead to a
decrease in the host’s immune response (14).
Hey SHP-2 Wanna Bind Tonight?
A study conducted by Shiho Yamazaki et al. (2003)
suggests that the CagA protein then may bind, undergo
tyrosine phosphorylation, and form an activatedcomplex with SRC homology 2 Domain (SHP-2). The
phosphorylation of CagA and activation of SHP-2 are
thought to induce the hummingbird phenotype: a
70
CagA (+)
Immune
Response:
Inflammation
Infected Host
VacA
(Induces Vacuolation)
CagA Disrupts
ZO-1
(Paracellular leakage)
SHP-2
IL-1β
Decreased Acid
Secretion
Cytoskeletal change
c-Met
Erk
Increased
proliferation
Drug Regimen (PPI and Antibiotics)
Genome based drugs
?
Increased motility
Ulcer and
Cancer
Treatment
Mastic Gum ?
Vaccines?
Figure 2. Pathogen-Host Interactions in the Pathogenesis of H. pylori Infection
Bacterial virulence factors CagA and VacA cause damage to the host cell. In response to bacterial colonization, a host mounts an immune
response which often leads to disease. Several host proteins: ZO-1, SHP-2, c-Met, IL-1b, and Erk have been noted to be affected by CagA
leading to formation of duodenal and gastric ulcers as well as gastric cancer. However, there are other effects of CagA that remain
unknown and must be addressed in future research. Several treatments are used as a means to eradicate H. pylori infection such as
antibiotics. Recent research proposes several alternative methods of treatment such as: mastic gum, genome-based drugs, and vaccines.
Further research is still needed to clearly understand the pathology caused by H. pylori and optimal treatments.
morphological change characterized by elongation and
contraction of the cell and increased cell motility.
Normal SHP-2 is actively involved in regulation
adhesion, spreading, and migration of cells. The
scientists took biopsies at eight different parts of the
stomach lining from fifteen patients who had either
gastritis or early gastric cancer. The biopsies were
submitted to immunoblotting and immunoprecipitation in
conjunction with antibodies to detect CagA,
phosphotyrosine, and SHP-2. The results detected the
presence of tyrosine phosphorylated CagA protein and
CagA-coimmunoprecipitated endogenous SHP-2. This
suggests that deregulation of SHP-2 by translocated
CagA can cause abnormal morphology and movement
of gastric epithelial cells (16).
cells. In a study conducted by Yuri Churin et al. (2003),
the interaction of CagA with this receptor was tested.
Small interference RNA was used to block the
expression of c-met. The blocking of c-Met expression
inhibited scattering in AGS cells infected with CagA (+)
H. pylori (18).
What’s a Gut to do?
Current treatment of Helicobacter pylori infection, which
ultimately leads to the development of peptic ulcers, is
based on multiple drug therapies (22). Currently, the
most effective therapy consists of a proton-pump
inhibitor and a series of three antibiotics chosen from
macrolide antibiotics, ß-Lactam antibiotics, or
metronidazole antibiotics (22). Other therapies,
including two drugs (proton pump inhibitor and an
antibiotic) and four drugs (proton pump inhibitor, three
antibiotics), have also proven to eradicate H. pylori
infection in humans (21).
Please Don’t Phosphorylate When Erk is Home
Hideaki Higashi et. al (2004) investigated cellular
proteins that bind to phosphorylated tyrosine but not
non-phosphorylated CagA and form complexes SHP-2
and subsequently with extracellular signal-regulated
kinase (Erk), a MAP kinase signaling molecule that is
thought to effect cell proliferation and motility. To test
the effect on the humming bird phenotype, they created
a knock-out SHP-2 and transfected it into AGS cells.
They found that only phosphorylated CagA complex
with SHP-2 binds to and abnormally prolongs the
activation of Erk (17).
The Basic PPIs
Proton pump inhibitors (PPIs) play an essential role in
the eradication of H. pylori. PPIs act within the parietal
cells of the stomach to inhibit H+, K+-ATPase activity.
This enzyme maintains the balance of H+ and K+ ions
within the cell so that pH is maintained inside and
outside of the cell. PPIs bind to the H+, K+-ATPase on
the outer luminal membrane and inhibit phosphorylation
of ATP molecules. This in turn prevents the exchange
of H+ and K+ ions. With the enzyme blocked, the acidic
pH of the stomach is made more basic so that
CagA Sticks like C-Met
C-Met is a hepatocyte growth factor/scatter factor
receptor that is involved in invasive growth of tumor
71
Just Say No to Drugs
antibiotics, which are taken along with PPIs, may reach
the H. pylori living within the epithelial cells of the
stomach (23).
The reason that there are so many choices in antibiotic
combinations when considering treatment of H. pylori is
antibiotic resistance. A patient’s level of resistance to
an antibiotic can cause a drug regimen to fail in
erradicating infection. Ecclissato et. al. (2002) studied
the effects of antibiotic resistance in two common
regimens used to treat infection by H. pylori. In both a
three drug regimen and a two drug regimen, it was
shown that when a patient was resistant to just one
antibiotic, the overall eradication rate of the regimen
was decreased by half (28). This has serious
implications for the treatment of patients for H. pylori
infection.
Currently, doctors do not test patients for
antibacterial resistance before they are prescribed a
regimen to treat H. pylori infection (28). If these patients
are resistant to the bacteria, the regimen is likely to fail.
In countries such as the United States, where drugs are
readily available ,regimen failure is not as serious as in
countries where drugs are not easily obtained (28).
Bacteria form resistance to antibiotics in ways
unique to each antibiotic. H. pylori resistance to ßLactam antibiotics is due to alteration in the Penicillin
Binding Protein (PBP) (26). Studies have shown that
the replacement of the the wild-type HP0597 (PBP1A)
gene by the Hardenberg PBP1A resulted in a huge
increase in the minumum inhibitory concentration (MIC)
of amoxicillin (a ß-Lactam antibiotic) (26). Antibacterial
resistance is usually due to the bacteria evolving ways
to produce ß-Lactamase even in the presence of anti βlactam antibiotics. Structural alterations in a PBP or
changes in other proteins that are involved in cell wall
synthesis are also involved in antibacterial resistance.
Macrolide antibiotics face two main modes of
resistance. There is target site modification, during
which the bacterium makes an enzyme that methylates
the rRNA, thus inhibiting the binding of erythromycin (or
other macrolides) (24). The second mode of resistance
is alteration in transport of the antibiotic. This mode of
resistance involves two macrolide efflux pumps: A and
E. The pumps pump macrolides out of the cell;
however, this mode of resistance only works on
fourteen or fifteen membered macrolides (24).
Metronidazole resistance has been accredited to
mutations in the rdxA gene that make the gene
inoperative (29). This gene coded for an oxygeninsensitive NADPH nitroreductase (29). Without the
expression of this gene, the Metronidazole cannot
energize its anabolic functions.
All PPIs are NOT Created Equal
There are several PPIs that may be used in
combination with antibiotics to eradicate H. pylori. The
most common PPIs used are Omeprazole,
Pantoprazole, Lansoprazole, and Rabeprazole. PPI
differences depend on the H+, K+-ATPase binding
location and their pharmacokinetic properties.
In comparison, Hellstrom and Sigurd (2004) found that
Rabeprazole was very quick to inhibit acid production
compared to the others; however, Omeprazole offered
the most potent acid inhibition. Pantoprazole and
Lansoprazole are not far behind Omeprazole and
Rabeprazole in speed and potency, indicating that all
four of these PPIs are effective ways to inhibit the
function of the H+, K+-ATPase enzyme (23).
Dealing the Drugs
Current therapies used to eradicate H. pylori in the
stomach all include at least one antibiotic in
combination with a PPI. The main categories of
antibiotics used are: macrolide antibiotics, ß-Lactam
antibiotics, and Metronidazole antibiotics (21).
Holy Macrolide
Macrolide antibiotics accumulate in the epithelial
tissues of the stomach. Here, they are able to inhibit
RNA- dependent protein synthesis by binding to the
23S ribosomal RNA in the 50S subunit of prokaryotic
ribosomes.When the macrolides bind to the ribosomes,
they inhibit peptidyl transferase reactions and cause
incomplete peptide chains to be detatched from the
ribosome. Proteins are essential for a cell to funciton,
so without properly formed proteins, the bacteria die
quickly (24).
And The Walls Came Tumbling Down
ß-Lactam antibiotics are analogues of D-alanyl-Dalanine, which is an amino acid that makes up
peptidoglycan. This close relationship allows ß-Lactam
antibiotics to bind to the active site of penicillin binding
protiens (PBPs) within bacteria. PBPs facilitate the
transpeptidation of the cell walls of bacteria. When ßLactam
antibiotics bind to the active site of PBPs and inhibit
transpeptidation of peptidoglycan, they prevent cell wall
synthesis within the bacteria (25). Without cell walls,
parent cells are not able to undergo mitosis to generate
a new generation of bacterial cells. Therefore, the
bacteria are soon eradicated (26).
Glimpses of Future Glory
Hit Them Where it Hurts
Currently, Genome-based drugs and vaccines are
being worked on. Genome-based drugs are drugs that
attack a specific target, which is essential to cell
function (21). Researchers are trying to find proteins
involved in cell envelope synthesis and integrity, cell
division, protein synthesis, nucleic acid biosynthesis,
gene expression and regulation, cell metabolism and
other protein essential to H. pylori function that may be
easily and safely targeted (21).
Where’s the Air
Metronidazole antibiotics only work on anaerobic
bacteria like H. pylori (27). When a metronadizole
antibiotic enocunters an anaerobic bacterium, the nitro
group of the metronidazole is reduced, thus interfering
with DNA synthesis and making it possible for the
antibiotic to interact with intracellular macromolecules
and ultimately kill the bacterium (28).
Just Give it a Shot
An important topic of research that many scientists are
72
Conclusion
very interested in is the possibility of a vaccination for
H. pylori infection. It is believed that a vaccination is
possible due to the immune response generated by the
host at the onset of H. pylori infection (29). It has been
found that H. pylori actually benefit from this response
when first colonizing a new host. The antigens formed
in this process may be used to treat established
infections (30).
Most research concerning vaccines has been
carried out in animal models with promising results. It
was found by Ghiara et. al. (1997) that mice that had
chronic H. pylori infection were able to receive
therapeutic vaccinations of recombinant VacA and
CagA together with a genetically detoxified mutant of
the heat-liable enterotoxin LTK63, intragastrically, to
eradicate H. pylori infection (31). Furthermore, the
vaccination protected the mice from re-infection for 12
weeks after eradication (31).
Using animal models, scientists are currently
testing different possible vaccines for efficacy and
safety, as well as considering the best mode of delivery
(32). A big challenge for scientists to overcome in
eradicating H .pylori is antibiotic resistance.
Since its discovery in 1983, research has shown
Helicobacter pylori to be the cause of peptic ulcers and
a contributor to gastric cancer. Further studies on the
bacterium have given scientists insight into how the
bacterium functions in the human body and how it may
be eradicated. Advances in the knowledge of H. pylori
will help scientists and physicians effectively treat
gastric and duodenal ulcers as well as gastric cancer.
Acknowledgements
We would also like to thank Michael Zorniak, Jenny
Riddle, Katie Hampton and Michael Wollar for their
guidance and expertise. We would like to thank Dr.
DebBurman for his time and patience.
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74
Review Article
Evolutionary Antibiotic Resistance as Documented in Multiple
Strains of Staphylococcus
Michelle McKinzey*
Lake Forest College
threatening Toxic Shock Syndrome (Bauman). Some
strains can be resistant to antibiotics causing
complications in treating the associated illnesses.
Summary
Studies have suggested that many species of
Staphylococcus have only recently diverged from a
common ancestor. Comparative analyses have shown,
for example, that S. intermedius closely resembles S.
aureus and S. epidermidis genetically (Cookson et. al.,
2004). Thus, the term intermedius. (Fig. 1)
Genes thought to be responsible for the
resistance of staphylococci species may have
originated in the species S. scuiri, possibly an ancestral
species, and later transferred to other species (aureus,
intermedius, and epidermidis) or vice-versa (Stepanovic
et. al., 2005). Transfer of resistance genes is possible
due to the close evolutionary relationship of one
species of staph to another.
Genetic similarities
interfere with a bacterium’s ability to distinguish its own
species from another species, which facilitates gene
transfer.
Evolutionary Tree of Staph Species
Staphylococcus (staph) is a genus of bacteria
found almost everywhere including in the soil and
on the skin of many animal species. Staph species
are responsible for a considerable number of
diseases ranging from carbuncles, bacteremia, and
endocarditis.
Treatment of these illnesses is
becoming increasingly difficult due to developing
resistance. Genes thought to be responsible for
the resistance of Staphylococcus species may have
originated in the species S. scuiri, possibly an
ancestral species, and later transferred to other
species. Horizontal transfer of resistance genes is
possible due to the close evolutionary relationship
of one species of staph to another. The emergence
of antibiotic resistant bacteria, such as some
strains of staph, can be attributed to the increase in
use of antibiotics. Recently, resistance to
medicines such as vancomycin has manifested.
Antibiotic resistance of staph can be attributed to
the transfer of the genes mecA, pls, and more by
transduction. This creates large problems for the
medical community as new treatments against
antibiotic resistant strains must be engineered.
Mechanisms of Antimicrobial Activity
There are several mechanisms by which
antimicrobials can function against bacteria. Each
mechanism interferes with some vital process or
structure of the cell. These include interference of
protein synthesis, nucleic acid synthesis, metabolic
activity, cell membrane function, or cell wall synthesis
(Krasner). The first three mechanisms require the
antimicrobial agent to enter the cell for success.
Examples of these drugs include sulfa drugs,
erythromycin, and polymyxin B. These types of drugs
work on mostly on gram-negative bacteria that do not
have a thick layer of peptidoglycan.
Staphylococci, though, are gram-positive and
are coated with a very thick layer of peptidogylcan.
Antibiotics, such as penicillin and cephalosporin-based
medicines, target this type of bacteria by interfering with
synthesis of the cell wall. Their molecular structures
contain beta-lactam rings that interfere with the
enzymes responsible for cell wall construction
(Krasner).
Resistance to these antimircobials can exist
naturally within a population or random variants may
arise through genetic mutation.
Prevalence of Staphylococcus
Staphylococcus (staph) is a genus of bacteria
found almost everywhere including in the soil, and on
the skin of many animal species. Staphylococcus
aureus is commonly found on skin and in nasal
passages of humans (Darini et. al., 2004). It is
widespread throughout the human community but
strains acquired by individuals in hospitals are often
highly pathogenic (Ewald 1994). Recent studies have
shown that the overall isolation rate of Staphylococcus
sciuri in hospital environments is 10.5% (Stepanovic et.
al., 2005).
Invasive procedures in hospitals are
generally accompanied with special precautions to
prevent the transmission of this bacterium from one
patient to another, however post-procedure infection
cannot be avoided.
Other species, such as S. intermedius, are
commonly found in horses, pigeons, dogs and other
animals (Cookson et. al., 2004).
In dogs, S.
intermedius is recognized as common skin flora that
can also cause invasive disease in humans. The
isolation rate of S. intermedius is 18.5% from canine
inflicted wounds (Cookson et. al., 2004).
Staphylococcus species are responsible for a variety of
diseases ranging from carbuncles and food poisoning
to bacteremia and endocarditis (Parkhill et. al., 2004).
Furthermore, it can be responsible for infections of the
breast, in new mothers, and impetigo (Ewald 1994).
Staphylococcus can even be the source of life-
Antibiotic Resistance
The emergence of antibiotic resistant
bacteria, such as some strains of staph, can be
attributed to the increase in humans’ use of antibiotics.
For example, bacteria isolated from patients 65 years
ago, before the introduction of antimicrobial agents,
show almost no resistance to antibiotics (O’Brien 2002).
As the use of antimicrobial agents becomes more
frequent, the appearance of bacterial resistance
becomes more common and more rapid.
Resistance to an antibiotic can be attributed
to differences in gene products on or in a cell to
interfere with the mode of action of the antibiotic. Such
differences in the genome can arise through mutation
*This paper was written as part of an independent study with Dr. Anne
Houde
75
al., 2004) and of MRSA coexisting with vancomycin
resistant enterococci in 2005 (Samore et. al., 2005).
The emergence of these resistant strains is
exemplar to the theory of evolutionary antibiotic
resistance in infectious diseases.
Source of Resistance in Staph
The administration of antimicrobials for
nontheraputic purposes is one proposed source of
resistance in bacteria. It has been shown to select for
resistance in multiple strains of pathogenic bacteria
coexisting in concentrated animal feeding operations
(Schwab et. al., 2005) making food products a source
of resistant bacteria for humans.
Strains
of
Staphylococcus
exhibiting
resistance to beta-lactam antibiotics have a common
resistance gene. This gene has been identified in an
evolutionary precursor to S. scuiri, and as a homolog of
a resistance gene, vanA, found in other bacterial
species (Parkhill et. al., 2004). This gene, denoted as
mecA, is involved in the normal process of cell wall
synthesis and does not contribute to resistance in the
wild. However, overexpression of the gene is shown to
increase antibiotic resistance (Cookson et. al., 2004).
Isolates of resistant strains acquired in
hospitals have much larger regions of this gene and
tend to show more resistance to the antibiotics (Samore
et. al., 2005).
MecA, which codes for penicillin-binding
protein 2A, works in conjunction with immune evasion
genes, such as pls, to give Staph strains resistance.
Figure 1. Evolutionary Tree of Staphylococcus species
discussed in this paper. Question marks indicate that this is not
a complete tree and does not contain other species.
or by gene transfer from other bacteria of the same or
different species.
Gene swapping is fairly regular in bacteria
and genomic similarities allow for highly facilitated gene
transfer since some species of bacteria can partake in
gene transfer only within their own species (Bauman).
Genomic similarities allow for this barrier to be
circumvented. This can be highly detrimental for the
medical community as resistance to one antibiotic can
develop in one species and be transferred to another.
This is then perpetuated by the introduction of a new
antibiotic for the first species, which develops new
resistance and passing it to the next creating a
revolving door for the engineering of antimicrobial
agents.
The mecA Gene
A study conducted by Parkhill et al. (2004)
showed that a genomic stretch in several strains of
MRSA was absent in methicillin susceptible strains of
Staph. This stretch contains the mecA gene as well as
other genes that have not been identified thus far.
(Fig. 2)
MecA, when active, increases resistance to
beta-lactam antibiotics (Tomasz, 2005). Tomasz and
colleagues demonstrated that a mecA homolog from
MR S. scuiri introduced into methicillin susceptible S.
aureus generated MRSA. They confirmed this result by
documenting that elimination of the plasmid containing
mecA, resulting in methicillin susceptible strains. This
shows that the transfer of resistance genes between
species confers resistance. This also supports the idea
that a close evolutionary relationship between staph
species leads to increased ability to transfer genes.
Emergence of Staph Resistance
A study conducted in 1989 by Talan et al.
showed that only 79% of S. intermedius isolates were
susceptible to penicillin, one of the first known
antibiotics. Since then, many strains of staph have
been identified as resistant to penicillin derivatives such
as oxacillin and methicillin. (Cookson et. al., 2004).
Resistance level of S. intermedius to oxacillin-based
drugs is documented as 60 to 85%, meaning that
higher dose levels of the drug are needed to eradicate
the bacteria. S. intermedius is also beginning to show
signs of methicillin resistance.
Methicillin resistant S. aureus (MRSA) were
first noted in 1961 in Europe only two years after the
introduction of the drug (Dohar et. al., 2005). At first,
only 3% of isolates were MR. This number soon
increased to 38%. As it stands now, MR strains are still
less common than methicillin susceptible (MS) strains.
Thus, resistance to antibiotics is more costly in
competition between strains. Furthermore, patients
who developed MR strains have had increased
exposure to broad-spectrum antibiotics.
Of Staphylococcus scuiri isolates obtained
from hospital sampling in 2002, 73% showed resistance
to one or more types of antimicrobial agents (Stapnovic
et. al., 2005; 62.5% were resistant to penicillin, 64.3%
were resistant to oxacillin, 3.5% were resistant to
tetracycline, and 4.3% were multiresistant, among
others).
Reports
of
vancomycin
resistant
staphylococci from Brazil surfaced in 2004 (Darini et.
Figure 2. Genetic Comparison between MRSA (left) and MSSA
taken from Parkhill et al. 2004. (mecA is located on the outer
ring at the top)
76
corresponding genes.
Additionally,
when
MR
strains
of
staphylococci are stored for long periods of time, they
lose the mecA gene and show signs of susceptibility to
beta-lactam antibiotics (Kluytmans et. al., 2005).
Tomasz et. al. (2001), also saw that, in
conjunction with the mecA, staphylococci strains
showed greater resistance to antimicrobials when
penicillin-binding protein 2A (PBP2A) was expressed.
He suggests that mecA codes for the expression the
protein and overexpression of mecA leads to an
increased production of PBP2A.
another species also transferred natural or mutated pls
genes.
Different Resistance, Different Genes
Resistance to other antibiotics, such as
tetracycline and vancomycin, has been documented in
various species of Staphylococcus (Schwarz et. al.,
2006). S. lentus, for example, has some strains which
exhibit tetracycline resistance.
This resistance is
caused, at least in part, by the plasmid pSTE2 (Table
1). Sequencing of the plasmid has shown that it has
similarities to other known resistance plasmids. Given
what is already known about evolutionary antibiotic
resistance, this plasmid, along with others, can be
transferred easily to other species of bacteria leading to
an allover increased resistance level.
Penicillin-Binding Protein 2A
PBP2A is implicated in the production of the
peptidoglycan layer (Tomasz et. al., 2001).
The
strategy of normal beta-lactam antibiotics is to acetylate
the transpeptidase domain of normal PBPs thereby
inhibiting cell wall synthesis.
MecA expression,
nonetheless, leads to the assembly of PBP2A as well
as normal PBP2. PBP2A is an extra subunit of
transpeptidase. PBP2 only is composed of both a
transpeptidase and transglycolase subunit.
The
production of PBP2A allows for the transpeptidation of
the peptidoglycan layer, in the presence of antibiotics,
to regain its normal funcion (Tomazs et. al., 2001). The
function of PBP2A in non-antimicrobial conditions is
unknown.
Acquisition of Resistance
A study conducted by van Strijp and
colleagues (2005) describes two newly discovered
immune modulators: chemotaxis inhibitory protein of
Staphylococcus aureus (CHIPS) and staphylococcal
complement inhibitor (SCIN). These genes also carry
genes for immune evasion molecules. They showed
that 90% of S. aureus carry these genes, or variants of
them, in the same stretches of DNA in the genome. All
forms of these genes have some effect on the human
immune system.
Furthermore, van Strijp demonstrated that βhemolysin converting bacteriophages transfer these
genes from one microbe to another.
Given that these genes are easily transferred
via bacteriophage, it is possible that transduction is the
method of resistance gene transfer among
staphylococcal species. Whether or not the virus
responsible for resistance in staph is able to infect other
species, it may play a role in the number of resistance
genes transferred and which bacteria they come from.
The pls Gene
Resistance
in
Staphylococcus
is
characterized not only by the abnormal function of cell
processes, but an abnormally low adherence rate to a
host’s extracellular proteins (Kuusela et. al., 2001).
This often gives a negative result when testing for
MRSA. By introducing a point mutation into the gene
coding for pls, Kuusela et. al. (2001), illustrated that pls
is responsible for negative test results. The idea that
pls prevents adhesion of resistant staph to cell is highly
favored. Previoius studies showed that susceptible
staph strains do not code or express the pls gene or its
protein.
Pls is found in the same regional DNA
fragment as mecA. Homologs of the pls gene are
found in other species of antibiotic resistant bacteria,
including multiple species of resistant staph. This
indicates that the species of bacteria that first
developed novel mecA genes and swapped them with
Precautionary Steps
Common Sense
One way to combat the spread of antibiotic
resistant bacteria is simply the exercise of common
knowledge such as washing your hands before you
touch a patient in the hospital or hand washing between
patients by staff. For example, of the staph isolates in
Stepanovic’s study (2005), none were found on the
hands of medical personnel. The reason was that all
doctors and nurses at all medical institutions are
required to wash their hands between patients; a
practice started in 1848 by Ignaz Semmelweis who
eventually got fired for implementing the routine even
though the rate of post-procedure infection declined
dramatically (Bauman).
A second exercise of common knowledge is
one that most people have been hearing for years:
elimination of the overuse of antibiotics. The hog
farming industry uses 10.3 million pounds of antibiotics
regularly and have been found to produce high-level
drug resistance in staph species as well as other
bacteria species (Schwab et. a., 2005). Without the
presence of antimicrobials in the environment, there
would be no selection for antibiotic resistance. Lacking
Table 1. Species exhibiting resistance to antibiotics and the
Species
Genes
Antibiotic
aureus
mecA, pls
Penicillins,
vancomycin
lentus
pSTE2
Tetracycline
sciuri
mecA
Penicillins,
tetracylines
haemolytic
blaZ
Vancomycin
werneri
ermC
Eyrthromyci
77
this pressure for selection, on staph strains harboring
resistance genes, the bacteria are likely to kick out the
plasmid (Kluytmans).
which will lower the exposure of the bacteria and
decrease the probability of resistance acquisition.
Scientists have recently begun studying the
use of bacteriophages in the reduction of the spread of
resistant Staphylococcus strains (O’Flaherty et. al.,
2005). Phage K, one such virus, affects nine different
species of Staphylococcus including S. aureus and S.
epidermidis. In the case of newly emerged resistant
strains, 39 of the 53 strains tested showed sensitivity to
unmodified Phage K and the other 14 were sensitive to
modified Phage K. Phage K infects the bacteria and
ultimately causes death. It is important to note that a
Phage K wash reduces staphylococcal cells on the skin
but does not completely eliminate the bacteria, which
leads one to believe that it may be possible to develop
some resistance. The effects of Phage K in vitro have
not been established.
Natural Remedies
Due to the rapid development of resistance in
bacteria to all antibiotics, synthetic or not, other
alternatives must be found. A study conducted by
Molan et al. (2005) examines the antibacterial activity of
honey against Staphylococcus. They found that a
concentration of 2.7-5% inhibited the growth of 18 staph
isolates with no significant differences between honey
type, antibiotic resistant vs. antibiotic susceptible, or
species. Furthermore, Molan showed that the inhibitory
effect of natural honey as opposed to simulated honey
was 5.5-11.7 times greater and that it could be diluted
20 fold without losing its ability to behave as an
antimicrobial. They speculate that this activity is due to
the enzymic production of hydrogen peroxide or some
other phytochemical element.
A second option is to use a compound not
based on existing antibiotics. Plant extracts can be one
source of these. S. aureus shows susceptibility to
plants used in Columbian folkloric medicinal practices
(Munoz et. al., 2006). These plants include Bixa
orellana, Gliricidia sepium, Jacaranda mimosifolia, and
Piper pulchrum.
Such plants are used to treat a
number of illnesses from gingivitis to bronchitis to
infected wounds. Extracts from this flora taken using
distilled water, ethanol, and hexane all exhibited
antimicrobial activity against S. aureus as well as other
species of bacteria. By creating new treatments from
these plants, the likelihood of previously acquired
resistance is lower.
Conclusion
Emerging antibiotic resistance can be the
product of close evolutionary relationships between
bacterial species and the administration of antimicrobial
agents. Isolates collected before the use of antibiotics
harbor no resistance genes (O’brien 2002). Many
strains of Staphylococcus show not only resistance but
also mulit-drug resistance. Evolutionary ties between
species add complication to this task. Many bacterial
species can transfer genes within a species; close ties
between two species may stop them from recognizing
their differences and allow transfer. In some cases,
such as that of staph, the host range of a bacteriophage
may play a part in gene transfer between closely tied
species.
Close monitoring of the use of antibiotics
through diagnostics can aid in combating resistance.
This will aid in reducing unnecessary exposure of
bacteria to antimicrobials. Reduced exposure can
decrease the acquisition of resistance genes. Newly
discovered natural treatments, such as the use of plant
extracts and honey, in conjunction with the use of
engineered bacteriophages, Phage K, can also
discourage these developments.
“Artificial” Fixes
One quick fix used to surmount the antibiotic
resistance in bacterial species is to develop new
antibiotics. For example, new cephalosporin antibiotics
interfere with the actions of PBP2A, consequently
inhibiting cell wall synthesis in MRSA (Mobashery et.
al., 2006). Cephalosporins facilitate a conformational
change in the active site of PBP2A. This change
decreases the affinity for the protein to help piece
together segments of the cell wall. As a result, osmotic
pressure on the cell causes it to collapse and die.
However, this is not the best method of
controlling antibiotic resistance in light of the ability of
bacteria to rapidly develop resistance. Often, new
antibiotics are based off of existing antimicrobials for
which bacteria may already exhibit resistance. It is
likely that many new developments would not be viable
methods of treatment for a considerable amount of
time.
Nevertheless, this process can be deterred
with the advancement of detection methods. Given that
many strains of staph have developed resistance, it is
suggested that all patient samples should be
immediately tested for penicillin, oxacillin, and
vancomycin resistance. The problem comes with
results from phenotypic assays, such as disk diffusion,
which take at least 24-48 hours to incubate (Appelbaum
et. al., 2006). Microassays, such as PCR and gene
probing, have been shown to give accurate detection
rates in S. aureus and S. epidermidis (Unai et. al.,
2005) for the mecA gene. Rapid detection can reduce
administration of unnecessary or incorrect antibiotics,
the author.
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79
Review Article
α-Synuclein Misfolding and Aggregation in Parkinson’s Disease
indicates degeneration. At the cellular level, substantianigral
neurons contain large intraneuronal aggregates, Lewy
Bodies, which consist primarily of the protein α-synuclein
(Spillantini et al., 1998). Interestingly, aggregates are found
only in these neurons even though α-synuclein is abundantly
expressed throughout the human brain.
In comparison of PD with the neurodegenerative
disorders Alzheimer’s disease (AD), and Amyotrophic
Lateral Sclerosis (ALS, Lou Gehrig’s), a common pathology
is observed (Lansbury et al., 2003). Upon autopsy, the AD
brain is characterized by neuronal atrophy localized within
the medial temporal lobe. Like PD patients, the medial
temporal neurons in AD contain large intraneuronal
aggregates composed of the protein tau, as well as extracellular β-amyloid plaques (Lansbury et al., 2003). ALS
patients have degeneration of α-motor neurons which also
exhibit large intraneuronal aggregates consisting of
superoxide dismutase-1 (SOD1) (Ray et al., 2004). Overall,
each of these diseases has a common pathology of protein
misfolding and aggregation.
This review will focus on the characteristics of α-synuclein,
primarily misfolding and aggregation. However, many of the
characteristics of α-synuclein provide valuable insight into
the proteins involved in AD and ALS.
Significant
commonalities will be referred to within the text.
Michael White*
Lake Forest College
Summary
disorder that affects 1.5 millions Americans and 1 in 100
individuals over the age of 60. It results from neuronal
atrophy localized within the substantia nigra pars
compacta.
Upon autopsy, PD patients have large
intraneuronal fibrils, Lewy Bodies, composed of αsynuclein. Familial forms of PD result from the A30P
and A53T mutations within α-synuclein. Wild-type (WT),
A30P, and A53T-α
α-synuclein aggregate from monomers
into protofibrils before forming fibrils. Previously, fibrils
were thought to be the PD causative agent however,
recent evidence suggests that the protofibril may be the
true toxic conformation of α-synuclein. In 2004, Zarranz
et al. discovered a novel mutation, α-synuclein-E46K, in
a Spanish family. Little is known about this mutation,
except that it increases the rate and quantity of fibril
formation as well as the lipid binding affinity of αsynuclein. The fibril formation pathway for E46K is
unknown, though it likely includes a protofibrillar
intermediate. Future research is needed to characterize
E46K and compare it to the other familial mutants.
Treatment approaches aimed at reducing the
concentration of protofibrils could be accomplished
through accelerated fibril formation, decreased αsynuclein
expression,
increased
α-synuclein
degradation, or reduction of intracellular dopamine
which binds and stabilizes protofibril.
Biological Basis
The actual biological basis of PD remains to be discovered,
however, mutations within several proteins have been linked
to familial forms of PD. PD results from the mutations A30P
(Krueger et al., 1998), A53T (Polymeropoulos et al., 1997),
or E46K (Zarranz et al., 2004) within the protein α-synuclein.
It can also result from mutations within parkin (Kitada et al.,
1998), UCH-L1 (Leroy et al., 1998), PINK1 (Valente et al.,
2004), DJ-1 (Bonifati et al., 2003), and LRRK2 (Funayama et
al., 2002). Because A30P, A53T, and E46K mutations
cause PD and α-synuclein is the primary component of Lewy
Bodies, it has remained the main target of research.
Introduction
disorder of the central nervous system. It is characterized by
motor initiation deficits, rigidity, bradykinesia, and resting
tremor. It affects 1 in 100 individuals over the age of 60 and
90 to 95% of all PD cases are sporadic (NPD Foundation,
2006).
Heritable forms of the disease constitute the
remaining 5-10% of cases (NPD Foundation, 2006). Within
the whole of PD cases, another 5 to 10% occur in individuals
under the age of 40 (NPD Foundation 2006). For the past
decade, millions of dollars in federal and private funding
along with numerous researchers have provided us with a
large body of knowledge on PD. However, there is still no
cure and all patients are destined to die as a result.
Systems for Modeling PD
Several different systems are used for the study of PD
including mice, drosophila, yeast, primary neuron cultures,
and various in vitro techniques. A line of transgenic mice
overexpressing human α-synuclein has been established,
and found to exhibit substantia nigral atrophy and motor
deficits similar to those in PD (Masliah et al., 2000).
Interestingly, these mice have Lewy Body like structures that
differ from those found in human neurons because they are
non-fibrillar (unorganized) aggregates (Goldberg and
Lansbury, 2000). In contrast, the α-synuclein aggregates in
humans are highly organized fibrils (Lansbury et al., 2003).
Mice expressing both human α-synuclein and its homologue,
β-synuclein, have fewer aggregates and show no symptoms
of PD (Hashimoto et al., 2001). A drosophila model
expressing human wild-type (WT), A30P, and A53T αsynuclein has also been synthesized and found to exhibit
aggregation, dopaminergic neuronal atrophy, and motor
deficits (Feany and Bender, 2000). Finally, several yeast
models expressing human α-synuclein-WT, A30P, and A53T
have been established (Outeiro et al., 2003; Dixon et al.,
2005; Zabrocki et al., 2005; Sharma et al., 2006; Brandis et
al., 2006).
Pathology
As Dr. James Parkinson first observed in 1817, the PD brain
is characterized by neuronal atrophy localized within the
pars compacta region of the substantia nigra. Most PD
patients do not display any symptoms until 60-80% of
substantia nigral neurons are dead (Purves et al., 2004).
These neurons have a black coloration because they contain
the molecule melanin (Purves et al., 2004). Upon autopsy,
little pigmentation is observed in the substantia nigra, which
* This paper was written for BIO 493 Independent Study, taught by Dr. Shubhik
K. DebBurman.
81
α-Synuclein
toxic form of α-synuclein, it would account for the lack of
A30P propensity for aggregation yet still being capable of
causing PD.
Following the discovery of protofibrils, a study
conducted by Rochet et al. compared the in vitro fibrillization
properties of human α-synuclein-WT, A30P, and A53T to
mouse α-synuclein (2000). Like human α-synuclein, mouse
α-synuclein exists in a natively unfolded structure, and has a
threonine amino acid at the 53rd codon (Rochet et al., 2000).
Mouse α-synuclein aggregates even faster than human
A53T (Rochet et al., 2000). However, mouse aggregates
are similar because they are β-sheet rich (Rochet et al.,
2000). Interestingly, mixtures of mouse α-synuclein with
human WT or A53T led to the slowing of fibrillization but an
increase in protofibril formation (Rochet et al., 2000). These
findings further support the hypothesis of a toxic protofibrillar
intermediate because transgenic mice can express PD
symptoms without organized fibril formation (Goldberg and
Lansbury, 2000).
As previously mentioned, α-synuclein is the principal
component of Lewy Bodies and causes PD when A30P,
A53T, or E46K mutations are present. It is a small, 140
amino acid, 14 kDa, natively unfolded protein, but highly αhelical in the presence of fluorinated alcohols, detergents, or
vesicles (Weinreb et al., 1996; Davidson et al., 1998). αsynuclein is divided into three distinctive regions. The Nterminus is amphipathic and consists of amino acids 1-60,
which contain seven highly conserved 11-amino acid slightly
variable repeats, XKTKEGVXXXX (Kessler et al., 2003;
Uversky et al., 2002). The central portion of α-synuclein is
amyloidagenic and consists of amino acids 61-95. It
contains two of the above repeats above (Uversky et al.,
2002). Finally, the C-terminus consists of amino acids 96140 and contains many acidic residues as well as numerous
prolines which are characteristic of unfolded proteins
(Uversky et al., 2002). α-synuclein is expressed throughout
human brains primarily in pre-synaptic terminals. There it
may be involved in vesicular trafficking (Maroteaux et al.,
1988), regulation of neurotransmitter (Jensen et al., 1998;
Murphy et al., 2000) as well as several other possible
functions not listed. The actual function of α-synuclein
remains unknown.
α-Synuclein Membrane Affinity
In addition to the folding, misfolding, and aggregative
properties of α-synuclein, it also closely associates with
lipids (Davidson et al., 1998; Jensen et al., 1998; Bussell, et
al., 2004; Sharma et al., 2006). Lipid affinity may be the
result of α-synuclein’s numerous 11-amino acid repeats
which are characteristic of many apolipoproteins (Davidson
et al., 1998).
Detailed analysis of α-synuclein phospholipid
interaction by Davidson et al. demonstrated that the αsynuclein secondary structure stabilized into an α-helical
conformation following binding to phospholipid bilayers
(1998). Furthermore, this experiment was conducted on a
variety of vesicular sizes which revealed α-synuclein to
preferentially bind to those 20-25 nm in diameter. Similar
studies using the familial mutants A30P and A53T found αsynuclein membrane affinity to differ between the familial
mutants (Jensen et al., 1998, Perrin et al., 2000). The A30P
mutation decreases α-synuclein’s ability to bind to
membranes whereas A53T affinity is similar to WT (Jensen
et al., 1998, Perrin et al., 2000). These observations are
consistent with those found in a budding yeast model
(Sharma et al., 2006). In budding yeast, WT and A53T are
both found to membrane localize whereas A30P is observed
to remain diffuse (Sharma et al., 2006).
In depth analyses of the α-synuclein secondary
structure have revealed the N-terminus to bind membranes
while the C-terminus remains unbound and unfolded (Eliezer
et al., 2001). Specifically, amino acids 1-102 confer αsynuclein membrane binding capabilities (Perrin et al.,
2000). Because both A30P and A53T reside within this
domain, it is not surprising that A30P diminishes membrane
binding capability. High resolution NMR spectroscopy of αsynuclein confirmed that the N-terminus binds to lipid
membranes and consequently acquires a helical
conformation (Eliezer et al., 2001). Interestingly, the Cterminus remains unbound and unfolded suggesting it may
be involved in binding to other molecules or vesicles (Eliezer
et al., 2001).
Thus, it is plausible that α-synuclein is involved in
vesicular traffic at the pre-synaptic terminal based on two
lines of evidence; 1) α-synuclein preferentially binds to
vesicles 20-25 nm in diameter, which corresponds to the
size of those carrying neurotransmitter (Davidson et al.,
1998) and 2) the C-terminus remains unfolded and free to
associate with other vesicles or molecules (Eliezer et al.,
2001).
A30P, A53T and Fibrillization
Lewy Bodies are the end products of monomeric α-synuclein
aggregation and are present in every PD brain (Purves et al.,
2004).
Initial studies of α-synuclein were directed at
determining the mechanism by which it transformed from
monomers into highly organized fibrils. In order to begin this
enormous task, Conway et al. conducted an in vitro analysis
of A30P, and A53T to determine their rates of aggregation
(1998). At low concentrations of monomer, they found both
isomers to be natively unfolded.
However, as their
concentrations increased fibrils began to form at different
rates depending on the isoform, with A30P fibrillizing the
slowest and A53T the fastest (Conway et al., 1998). In
addition to increases in monomer concentration, increased
molecular crowding increases the rate of fibrillization
(Shtilerman et al., 2002). The Conway et al. study led to the
hypothesis that familial PD resulted from increased
aggregation of α-synuclein (1998).
Discovery and Properties of Protofibrils
This “old” model quickly changed when Conway et al.
conducted a subsequent experiment that specifically
quantified the rate of monomeric α-synuclein disappearance
and rate of fibril formation (2000).
At equimolar
concentrations of A30P, A53T, WT, the rates of monomeric
A30P and A53T consumption were increased compared to
WT with A53T being the fastest. In addition, a mixture of
A30P and WT was consumed slower than A30P alone and
A53T mixed with WT. Interestingly, the rates of fibril
formation for both A53T and A30P did not account for their
rates of monomeric disappearance. Thus, Conway et al.
hypothesized that a protofibrillar intermediate existed (2000).
The existence of the protofibril was confirmed using atomic
force microscopy (AFM) and gel filtration chromatography on
solutions of A30P, and A53T (Conway et al., 2000). Gel
filtration isolated oligomeric intermediates between monomer
and fibril forms. AFM of these protofibrils revealed them to
exist in either a spherical conformation, chain of spheres, or
rings of spheres. Thus, Conway et al. revised their previous
hypothesis by proposing that a protofibrillar intermediate was
the causative agent in PD (2000). If protofibrils were the
82
Dopaminergic Neurons
Stable Protofibril
L-dopa
Oxidation
Toxic Buildup
Dopamine
A53T
A30P
Lashuel et al., 2002
PD
Substantia Nigra Courtesy of
http://alzheimer.wustl.edu/adrc2/Research/Neuropath
ology/images/cerad_sn.bw.gif,
Figure 1: This figure illustrates the possible relationship between PD and the substantia nigra. L-dopa is an oxidized form of dopamine that
has been binds and stabilizes protofibrils (Lashuel et al., 2002). In this model, L-dopa (red) binds and stabilizes protofibrils. The stabilized protofibrils
have a high affinity for membranes and form pores similar to those of bacterial toxins which are depicted as AFM images from Lashuel et al., 2002.
Those pores may alter the permeability of substantial nigral neurons consequently, causing PD.
pathogenic agent has not. Of all three (monomer, protofibril,
and fibril) conformations α-synuclein is known to exist in,
emerging evidence has indicated that the protofibril is the
toxic agent. Critical to this hypothesis was an experiment by
Volles et al. using circular dichromism (2001). They found αsynuclein protofibrils to bind in a β-sheet rich structure to
membranes isolated from rat brains. Significantly, AFM
revealed protofibrils to bind to membranes and permeabilize
them (Volles et al., 2001). Another study conducted by Ding
et al. compared the binding affinity of protofibril and
monomeric α-synuclein to synthetic vesicles, rat-brain
derived membranes, and mitochondria (2002). Though both
monomeric and protofibril α-synuclein bind membranes,
protofibils bound to each membrane more tightly than the
monomers (Ding et al., 2002). Interestingly, protofibrils
formed pores resembling those of bacterial toxins (Volles et
al., 2001).
In addition, both A30P and A53T
permeabilization was greater per mole of protofibrils
compared to WT (Volles et al., 2001). Unlike protofibrils and
monomers, fibrils do not bind membranes (Volles et al.,
2001; Ding et al., 2002). Together, these studies provide
evidence supporting the toxic protofibril hypothesis (Figure
1).
α-Synuclein Post-Translational Modification
In a transgenic mouse model for PD, it was shown that αsynuclein is post-translationally modified into an N-terminally
truncated variant found only in regions of neurodegeneration
(Lee et al., 2002; Kessler et al., 2003). Based on this
observation and that the N-terminus confers several of αsynculein’s characteristics (previously described), Kessler et
al. evaluated the significance of seven N-terminus repeat
sequences (2003).
Two α-synuclein variations were
synthesized; one with the addition of two additional repeats,
and the other with two fewer repeats (both in the N-terminus)
(Kessler et al., 2003). Interestingly, their results indicated
that the plus two mutants prefer a non-amyloid, α-helical
conformation while the minus two acquired a β-sheet rich
conformation. This finding is significant because potentially
pathogenic protofibrils and fibrils are β-sheet rich (Lansbury
et al., 2003). Therefore, the repeat sequences may have
been highly conserved in order to protect against
aggregation (Kessler et al., 2003).
In AD, the APP is post-translationally modified into
two fragments, Aβ40 and Aβ42, which are linked to familial
forms of the disease (Lansbury et al., 2003). It is feasible
that a mechanism similar to this one may occur in PD
patients, producing toxic truncated variations of α-synuclein
(Kessler et al., 2003).
Recently Discovered E46K
In 2004, Zarranz et al. discovered the autosomal dominant
familial PD mutation α-synuclein-E46K in a Spanish family
(2004). Since its discovery, there have only been five
published manuscripts on E46K out of the thousands of PD
papers. The first in vitro study examined the rate of E46K
Protofibril Membrane Affinity
The pathway toward Lewy Body formation has been
elucidated for WT, A30P, and A53T; however, the
83
fibrillization, and lipid binding affinity (Choi et al., 2004).
E46K formed aggregates at a faster rate than WT but similar
to A53T (Choi et al., 2004). The E46K fibrils were also
conformationally different than A30P and A53T (Choi et al.,
2004). In addition, E46K increased α-synuclein vesicular
binding affinity whereas A30P decreased it significantly, and
A53T slightly (Choi et al., 2004).
Greenbaum et al. demonstrated that E46K increases αsynuclein’s rate of fibrillization as well (2005). Time-course
circular dichromism
analysis of the E46K fibrillization
process revealed α-synuclein to transition from primarily αhelical to β-sheet as fibrils formed (Greenbaum et al., 2005).
This transition occurred more rapidly for E46K compared to
WT (Greenbaum et al., 2005).
A single in vivo analysis of E46K aggregation was
performed by Pandey et al. using human catecholaminergic
neuroblastoma cells (2006). They demonstrated that 18% of
E46K, 12% of A53T, 2% of A30P, and 6% of WT expressing
cells contained aggregates. Electron microscopy identified
two types of aggregates within these neurons (Pandey et al.,
2006). Furthermore, western analysis showed that E46K
formed greater quantities of aggregates compared to A53T
thus, indicating the higher propensity for E46K to aggregate
(Pandey et al., 2006).
The β-sheet nature of E46K aggregates is similar
to those of A30P and A53T (Conway et al., 2000;
Greenbaum et al., 2005). Therefore, it is probable that E46K
forms β-sheet rich protofibrils before fibrils as well (Figure 2).
However, detailed analysis of E46K fibrillization still remains
to be conducted.
2003). A relationship has been established between α and
β-synuclein co-expression and α-synuclein aggregation
(Hashimoto et al., 2001; Park et al., 2003). Doubletransgenic mice expressing both human α and β-synucleins
do not exhibit neuronal atrophy, aggregates, or motor deficits
(Hashimoto et al., 2001). In vitro, β-synuclein prevents αsynuclein-A53T from forming protofibrils and fibrils (Park et
al., 2003). Analysis of β-synuclein’s relationship with the
other familial mutants has yet to be performed.
PD Specificity for the Substantia Nigra
Though α-synuclein is abundantly expressed in the human
brain, neurodegeneration is localized to the substantia nigra
(Purves et al., 2004). Conway et al. performed a wide
screen of pharmaceuticals to find molecules that prevent
fibril formation (2001). Interestingly, catecholamines related
to dopamine act as inhibitors of fibrillization (Conway et al.,
2001). Furthermore, an oxidized form of dopamine, L-dopa,
bound and stabilized protofibrils (Conway et al., 2001).
Under oxidative conditions within substantia nigral neurons it
is feasible that toxic protofibrils accumulate and are
stabilized
by
L-dopa,
consequently
linking
neurodegeneration to the substantia nigra (Conway et al.,
2001)
Possibilities for Treatment
After reviewing the characteristics of α-synuclein and its
relationship to PD, several possibilities for treatment arise.
Currently, PD is widely treated by replenishing the substantia
nigra synaptic clefts with dopamine (Purves et al., 2004).
However, this neither cures nor prevents neurodegeneration.
In order to prevent or inhibit neurodegeneration, the
concentration of dopamine in the cytoplasm could be
reduced (Conway et al., 2001). This would decrease the
concentration of potentially toxic protofibrils and
α and β Synucleins
The synuclein family consists of α, β, and γ homologues, all
of which are expressed in humans (Park et al., 2003). α and
β synucleins are primarily expressed in the brain whereas γsynuclein is in the peripheral nervous system (Park et al.,
A53T
A30P
PD?
Old
New
http://alzheimer.wustl.edu/adrc2/Research/Neur
opathology/images/cerad_sn.bw.gif,
?
E46K
PD?
Monomer
Protofibril
Fibril
Figure 2: Comparison of the toxic protofibrillar hypothesis (“New”) to the toxic fibril hypothesis (“Old”). The upper portion of the scheme
depicts the two previously discovered familial mutants A30P and A53T, and their pathway toward fibril formation ending in possible neurodegeneration.
The lower part of the diagram shows E46K and what is known of it’s aggregation pathway from monomer to fibril and possibly neurodegeneration. The
E46K protofibril has yet to be discovered. The image on the right is of the substantia nigra from a PD brain upon autopsy. Adapted from White, 2006.
84
consequently prevent neuronal atrophy (Conway et al.,
2001). Because protofibrils are not necessarily the PD
causative agent, therapeutics aimed at increasing the
degradation of α-synuclein or reducing its expression may
prevent buildup of the toxic species as well.
Conclusion
PD affects millions of individuals world wide and is always
fatal. Familial forms of PD are known to result from the
mutations A30P, A53T, and recently discovered E46K in αsynuclein (Krueger et al., 1998; Polymeropoulos et al., 1997;
Zarranz et al., 2004). The transition from monomeric WT,
A30P, and A53T to fibrillar α-synuclein includes a
protofibrillar intermediate (Conway et al., 2000 and 2001).
However, the pathway to fibril formation has not been
determined for E46K. If E46K were to fibrillize without
forming protofibrils, this would refute the toxic protofibril
hypothesis. This possibility is unlikely because WT, A30P,
and A53T all form protofibrils. Future research is needed to
determine the characteristics of E46K and how they
compare to those of A30P and A53T. Finally, the study of
misfolding and aggregation of α-synuclein provides many
valuable insights into PD as well as numerous other
neurodegenerative disorders. Currently, protofibrils have
been identified in AD and ALS patients as well (Harper et al.,
1997; Lansbury et al. 2000; Ray et al., 2004).
8.
Ding, Tomas T. et al., Annular α-Synuclein Protofibrils Are
Produced When Spherical Protofibrils Are Incubated in Solution
or Bound to Brain-Derived Membranes, Biochemistry, volume
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Eliezer, David et al., Conformational Properties of α-Synuclein
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Parkinson’s disease, Nature, volume 303, pages 394-398,
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(PARK8) maps to chromosome 12p11.2-q13.1, Ann. Neurol,
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Is Abolished by Familial Parkinson’s Disease Mutation, Journal
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recessive juvenile parkinsonism, Nature, volume 392, pages
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Biochemistry, volume 42, pages 672-678, 2003.
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Lansbury, Peter Jr., and Goldberg, Matthew., Is there a causeand-effect relationship between α-synuclein fibrillization and
Parkinson’s disease?, Nature Cell Biology, volume 2, pages
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Acknowledgements
I would like to acknowledge Dr. Shubhik DebBurman for
oversight and review of this manuscript.
herein should be treated as personal communication and
should be cited as such only with the consent of the author.
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86
Review Article
Mitochondrial Deficiencies and Oxidative Stress in Parkinson’s
Disease: A Slippery Slope to Cell Death
Parkinson’s Disease Etiology
Michael Zorniak*
*
Lake Forest College
PD is a movement disorder characterized by a triad of
symptoms: bradykinesia, postural rigidity, and resting
tremors (Dauer and Przedborski, 2003). Voluntary
movement is disrupted by the loss of dopamine in the
intact striatum. Specific death of dopamine-producing
neurons in the substantia nigra pars compacta (SNpc)
leads to this loss-of-function phenotype (Dauer and
Przedborski, 2003). α-Synuclein-dominant protein
inclusions, or Lewy bodies (LB), are suspect in causing
neuronal death in PD (Spillantini et. al., 1997).
Additionally, post-mortem PD reveals oxidative damage
partially due to redox-sensitive dopaminergic neurons
(Beal, 2003). Yet, an exact mechanism that engages αsynuclein-toxicity with oxidative stress has not been
determined.
Sporadic mutations in α-synuclein constitute
95% of PD cases. The other 5% of occurrences are
attributed to a mixture of dominant and recessive
genes. Dominant forms of PD have familial mutations in
α-synuclein which increase its tendency to aggregate
and form LB. Yet, recessive forms of PD are LB
negative where the onset of symptoms occurs much
earlier than in familial dominant forms. Two of these
recessive genes, DJ-1 (Bonifati et. al., 2003) and
PINK1 (Valente et. al., 2004), are involved in
mitochondrial protection. Perturbations in these genes
can impair mitochondrial function and lead to apoptosis
and rapid generation of reactive oxygen species (ROS;
Dauer and Przedborski, 2003). Whether or not αsynuclein is involved in the mitochondrial disease
pathway is unresolved. Dominant and recessive forms
of PD may involve two different mechanisms that
achieve the same symptoms. Both mechanisms,
however, will have oxidative stress as a key player in
PD pathogenesis.
Summary
Parkinson’s disease (PD) affects over 500,000
Americans. Most cases of PD are idiopathic, or
occurring without a known cause. Two pathological
features of PD, α-synuclein-rich Lewy bodies (LB)
and oxidative damage, hint at the cause of the
disease. Yet, disparities in recessive forms of PD
increase the complexity of the disease mechanism.
These recessive forms occur earlier in life and are
devoid of LB. One common feature among these
forms is the extensive presence of reactive oxygen
species (ROS). Studies with the toxin MPTP
produced similar pathologies to recessive PD but
intriguingly showed inhibition of complex I in the
mitochondria. These and other studies chased the
mitochondria as the progenitor of oxidative stress.
These investigations also uncovered several
disparate mitochondrial proteins, one of which is a
Kreb’s
cycle
enzyme,
α-ketoglutarate
dehydrogenase (α-KGDH). Interestingly, α-KGDH
activity is reduced in both Alzheimer’s disease (AD)
and PD. Links to both diseases may be due to its
role in the inactivation of complex I. This review will
focus on how mitochondrial impairments enhance
neuronal toxicity in PD.
Introduction
Neurodegenerative diseases (NDD) are incurable,
progressive, and fatal disorders of the central nervous
system (CNS; Muchowski, 2002). Alongside this
commonality, a culprit protein is frequently found
tangled in symptomatic patients. Protein accumulation
and subsequent aggregation is correlated with cell
death in brains of the afflicted. Alzheimer’s disease
(AD) and Parkinson’s disease (PD) are both NDD that
have key similarities and differences. Investigations in
both diseases have elucidated complementary
mechanisms involving different genes.
In both AD and PD brains, insoluble protein
deposits of tau and α-synuclein are, respectively, found
(Caughey et. al., 2003; Dauer and Przedborski, 2003).
In conjunction with protein aggregation, the
accumulation of toxic oxidants, like superoxide and
hydrogen peroxide, is another hallmark of both
diseases (Mizuno et. al., 1995). Yet, the specificity of
degeneration is unique to each disorder; AD has global
neuronal death in the CNS, whereas PD has localized
death (Caughey et. al., 2003). These similarities and
differences have led scientists on a search to find the
genes implicated in both diseases. This review will span
the discoveries made in PD that point to dysfunctions in
the mitochondria and its respiratory chain, some of
which are also found in AD.
Oxidative Stress: The Main Cause of PD?
The question remains: does the accumulation of ROS
initially cause specific nigrostriatal death in PD? Studies
performed with the toxin MPTP (1-methyl-4-phenyl1,2,3,6-tetrahydropyridine) have yielded support for this
hypothesis (Langston et. al., 2003). The elucidation of
PD pathogenesis has been largely dependent on the
introduction of MPTP.
MPTP gains toxicity when converted to MPP+
(1-methyl-4-phenylpyridinium ion) by monoamine
oxidase B (Chiba et. al., 1984). MPP+ is transported in
neurons through a dopamine transporter (DAT; Chiba
et. al., 1985). This is the only way MPP+ can enter a
cell; thus, specific dopaminergic cell death can be
ascribed by this quality. Complex I of the electron
transport chain is inhibited by MPP+ (Nicklas et. al.,
1985). The inhibition of oxidative phosphorylation has
many harmful effects including: decreased ATP
production and increased oxidant production. This toxic
state is further insulted by increased intracellular
calcium, which enhances the release of dopamine to
further promote oxidative damage (Fiskum et. al.,
2003). It is uncertain whether idiopathic PD is causally
related to dopaminergic loss by complex I inhibition
(Abou-Sleiman et. al., 2006). Regardless of the
* This paper was written for BIOL493, taught by Dr. Shubhik K.
DebBurman.
87
three, dopamine-quinone, like MPP+, may inhibit
mitochondrial complex I activity. Cumulatively,
dopamine metabolism primes SNpc neurons for selfdestruction. Thus, programmed cell-death, induced by
ROS and mitochondrial dysfunction, may lead to
specific death of SNpc neurons.
In mitochondrial respiratory dysfunction, ATP
production is decreased. The lack of energy supports
the necrotic cell death hypothesis of PD neurons
(Mizuno et. al., 2005). However, since PD is a
progressive disease, occurring over a long period of
time, cellular necrosis cannot be the only theory for
SNpc atrophy in PD. The decrease of ATP levels
perturbs calcium homeostasis, which activates
apoptotic pathways. Low levels of ATP decrease
sodium ion transport to the outside of the cell. Thus,
sodium must be expelled by the exchange of
extracellular calcium (Reeves et. al., 1992). As stated
above, high levels of intracellular calcium create an
excitotoxic environment. Calcium is a second
messenger that activates many cellular signaling
pathways. Most notably, degenerate proteases are
activated which induce apoptosis (Mizuno et. al., 2005).
Furthermore, disruption in calcium homeostasis
increases ROS production by the mitochondria.
Imbalances in calcium homeostasis from mitochondrial
respiratory failure feedback into the mitochondria and
further degrade its normal activity (Mizuno et. al., 2005).
To further support the apoptosis based celldeath scheme in PD, Hartmann et. al. (2001), describe
the proapoptotic mitochondrial mediator, Bax, a
member of the Bcl-2 family of proteins. Its primary role
in cell death is to release cytochrome c from the inner
mitochondrial membrane, in effect, purging its
potentiation. This facilitates the activation of caspases,
a group of cysteine proteases, which cleave numerous
cellular proteins. They found that Bax levels were
significantly higher in dopaminergic neurons containing
LB than in overall melanized areas. These and other
studies have strongly suggested dopamine’s role in
SNpc specific cell death. These data support that
dopamine metabolism enhances toxic ROS levels.
Collectively, mitochondrial deficiencies and
dopamine metabolism leave PD neurons in a
compromised state. We are still uncertain as to the
exact role of the mitochondria in PD. The next section
will explore the biochemical aspects of mitochondrial
dysfunction.
pathway, oxidative modification plays an intimate role in
the PD pathogenesis.
Oxidative stress may play a role in SNpc
specific atrophy. Neuromelanin, and its high iron
content which pigments the SNpc, may provide a
necessary oxidative stress mechanism to specifically
destroy the SNpc (Zecca et al., 2006). Iron may
aggressively catalyze ROS generation from oxidized
substrates by the Fenton reaction (Mizuno et. al.,
1995). The increase of reactive oxidants can be
measured by cellular responses. One such response is
superoxide dismutase activity (SOD). This enzyme’s
activity is elevated in the SNpc in PD (Saggu et. al.,
1989). Saggu and colleagues (1989) reported that Mn
SOD activity was elevated over Cu-Zn SOD levels. This
finding is significant because Mn SOD is localized in the
mitochondria. Another indication of increased oxidant
presence is the reduction of antioxidants. Glutathione is
found reduced in PD (Perry and Yong, 1986). These
data suggest that increased ROS are present in the
mitochondria in PD. MPP+, SOD, and glutathione all
point to the mitochondria as a putative producer of
ROS.
Respiratory failure and increased oxidative
stress both characterize PD. Yet, which event comes
first? Jenner and colleagues (1992) studied an
analogous system, Lewy body disease, to answer this
question. They found loss of both glutathione and
complex I substrates, yet glutathione was slightly less
than complex I. They concluded that oxidative stress
precedes respiratory failure in a PD. Other groups,
however, disagree with Jenner and colleagues’
conclusions.
PD has a twofold loss of respiratory activity,
which may exceed glutathione loss. The Kreb’s cycle
enzyme, α-ketoglutarate dehydrogenase (α-KGDH), is
also found deficient along with inhibition of complex I
(Mizuno et. al., 1994). In fact, reduction of respiratory
activity may be even more deleterious than oxidative
stress alone. Chance et. al., (1979) suggest that αKGDH and complex I inactivity triggers enhanced
generation of oxidative stress. When the ETC slows
down due to inadequate activities of complex I and αKGDH, the leaky mitochondrial membrane generates
increased free radicals. Thus, oxidative stress alone is
not enough to explain PD pathogenesis. A pathway
including mitochondrial respiratory failures must be
engendered to understand how radicals are produced
and sustained. In the next section, radical production
from dopamine metabolism further explains SNpc
specific degeneration
The Science behind Mitochondrial Dysfunction
The mitochondrion is the gate keeper for ROS
production. Significant rises in oxidative damage can
only occur through deficiencies in respiratory
metabolism (Cookson, 2005). Since oxidant levels are
raised in the SNpc due to dopamine metabolism, SNpc
neurons are predisposed to increased oxidant damage.
When neurons are incapable of reducing this oxidantrich environment, the mitochondria is deleteriously
impacted. The mitochondrion fuels its own destruction
by rapidly producing even more oxidants. Nevertheless,
what structures and pathways engender this response?
Normally during oxidative phosphorylation,
electrons travel along the respiratory chain to complex
I, II, III, and IV, along with cytochrome c and
ubiquinone. The chain is commonly characterized as
“leaky”, which promotes the reduction of substrates,
such as oxygen, thereby producing a superoxide
molecule (O2•–). Iron-sulfur clusters within the
complexes provide the donation of one electron to
The SNpc is Sensitive to ROS: Dopamine AutoOxidation
Dopamine auto-oxidation has long been an attractive
hypothesis for SNpc selective death due to its
endogenous metabolism in the SNpc (Graham, 1978).
Normal metabolism of dopamine produces hydrogen
peroxide and superoxide radicals, which oxidize
dopamine to form dopamine-quinone (Dauer et. al.,
2003). Dopamine-quinone then proceeds to disrupt all
proteins with disulfide linkages (Dauer et. al., 2003).
Auto-oxidation has thus had a three-fold effect on the
cell. One, the disassembly of proteins with disulfide
linkages places an unnecessary stress and load on the
ubiquitin-proteasome protein degradation system (UPS;
Dauer et. al., 2003). Two, ROS generated by dopamine
metabolism increases general protein misfolding in the
cell, thus further increasing the load onto the UPS. And
88
Figure 1. Effects of MPP+ Inhibition on Complex I and α-KGDH. A. Normal function of electron transport chain. B. MPP+ introduction into
the mitochondria arrests ubiquinone (Q), thus electrons are not continuously transferred to complex III and IV. Oxygen is not reduced to
water, so it is available for superoxide formation (O2•–). This inactivity disrupts the proton gradient needed to make ATP in complex V (not
shown).
make the toxic superoxide molecule. This donation
increases in probability as the ETC becomes more and
more inhibited. The inhibition of the respiratory chain
leaves upstream components reduced for relatively
long periods of time. The escape of electrons is most
favorable during this time. Consequently, since the
redox potential decreases, electrons are not shuttled to
the next carrier. The release of electrons in this window
of time produces ROS (Adam-Vizi, 2005).
Alternatively, superoxide production by
complex I requires a pH gradient across the inner
membrane space (Lambert et. al., 2004), which can be
achieved with ubiquinone inhibitors. Ubiquinone is
produced at two places in the ETC, complex I and
complex II. ROS production requires inhibition of both
sites so that ubiquinone is unable to carry electrons to
complex III and continue to complex IV. If electrons are
not shuttled to complex IV, oxygen is not reduced to
water and remains saturated in the cell. Oxygen
saturation is another prerequisite of ROS production
(Boveris and Chance, 1973).
Thus, inhibition of
complex I only is not adequate to produce toxic
oxidants.
Nonetheless, in the MPTP model of PD
described above, MPP+ inhibits complex I. According to
Lambert and colleagues (2004), complex I inhibition is
not enough to produce superoxide radicals. The MPTP
pathway must be developed further. Strikingly, MPP+
inhibits α-KGDH activity as well (McNaught et. al.
1995). Reduction of α-KGDH thereby reduces
succinate concentrations, the substrate for complex II.
Succinate is then unable to carry electrons to complex
II, in so doing inhibiting its activity. Thus, MPTP does, in
effect, inhibit complex I and II (Figure 1).
Until recently, the respiratory chain, or
electron transport chain, has been assumed as the
89
Figure 2. α-KGDH Regulation of Complex I Activity. A. ROS inhibits α-KGDH which decreases the production of NADH in the
Kreb’s cycle. The lack of NADH lowers complex I activity, thus ATP is not synthesized. B. A high concentration of NADH increases
the activity of both α-KGDH and complex I. Yet, this dual activation is counterintuitive. α-KGDH produces ROS when NADH levels
are high. This may, in turn, inhibit previously activated complex I.
principal generator or ROS (Starkov et. al., 2004).
Previous studies (Chance et. al., 1979) have suggested
the duality of α-KGDH and complex I as sources of
ROS. Yet, complex I-dependent ROS production was
always thought to surpass α-KGDH-related ROS
production. New evidence suggests that α-KGDH
regulates complex I ROS production. In the next
section, the relationship between α-KGDH and complex
I activity will be discussed further.
Non-Electron
Transport
Ketoglutarate Dehydrogenase
Deficiencies:
radicals (Tretter and Adam-Vizi, 2004). These results
were found to hold true in situ as well (Starkov et. al.,
2004). This generation of ROS was dependent on the
NADH/NAD+ ratio, where increasing NADH levels
account for this phenomenon. Increasing ROS levels
actually inhibit α-KGDH (Tretter and Adam-Vizi, 1999).
The inhibition of α-KGDH reduces NADH for the ETC,
thus decreasing ATP production. Consequently, by
these inverse pathways, α-KGDH is both a target and
generator of oxidative stress
Markedly, α-KGDH may regulate complex I
activity by these inverse pathways (Adam-Vizi, 2005).
As stated before, inhibition of α-KGDH reduces NADH
production which kinetically decreases complex I
(NADH ubiquinone oxidoreductase) activity. The other
inhibitory pathway is set in motion by a high NADH
concentration. Thus, complex I is kinetically active
under these conditions. The generation of ROS by αKGDH in the mitochondrial matrix may disrupt complex
I activity (Figure 2). Also, the ROS generated by αKGDH would increase lipid peroxidation thereby
disrupting calcium homeostasis. Lipid disruption would
furthermore perturb the pH gradient across the inner
membrane space. This loss of membrane potentiation
is a step towards apoptosis. Loss of α-KGDH activity
and inhibition of complex I is a similar to the MPTPbased model of PD.
α-
The toxin MPTP serves as a convenient model for PD,
but most cases are idiopathic. MPTP fails to replicate
two other qualities of classical PD: Lewy body
inclusions and progressive onset. MPTP-induced
Parkinsonism occurs rapidly after exposure thus
making it, at most, a model of PD. This begs the
question, can complex I be inhibited by any other
means? Extensive studies with the α-KGDH have
revealed that possibility and more.
α-KGDH is regulated by the NADH/NAD+
ratio, Ca2+, and ADP (Adam-Vizi, 2005). Its loss-offunction would thus eliminate a key modulating location
in glucose metabolism. Interestingly, the isolated
enzyme produces hydrogen peroxide and superoxide
90
Loss of α-KGDH also occurs in AD. Gibson
and colleagues (1988) have demonstrated that αKGDH activity is reduced by 40-75% in AD brains.
Reduction in α-KGDH levels is negatively correlated
with neurofibrillary tau tangle counts, suggesting this
enzyme is involved in the neurodegenerative cascade.
Even 10 to 15% reduction in available glucose or
oxygen can reduce brain function, including decline in
memory (Gibson et. al., 2005). A current study shows
how brain regions with low metabolic activity
accumulate more plaques in conjunction with dementia
(Shoghi-Jadid et. al., 2002). The return of glucose to
these brain regions reverses some behavioral deficits
found in AD. This suggests that symptoms in AD are
not always attributed to neurodegeneration. The
question remains, how does α-KGDH activity decrease
in AD and PD?
Genetic studies have attempted to answer
this question. Two different groups have attempted to
track the allele responsible for α-KGDH deficiencies in
AD and PD. Of the three subunits of α-KGDH, the
second has received most attention because of its
noted ROS producing activity (Starkov et. al., 2004).
The other two subunits do not produce ROS.
Polymorphisms on the second subunit have been
paired with the apolipoprotein E4 gene in AD to cause
dementia. Apolipoprotein E4 is not a factor on its own
until it is paired with the α-KGDH polymorphism (Sheu
et. al., 1998). Similarly, a bi-allelic intragenic
polymorphism of α-KGDH was found to constitute a
genetic risk factor for PD (Kobayashi et. al., 1998). The
dysfunction of α-KGDH caused by the polymorphism
suggests a causal genetic link to NDD. Whether αKGDH activity is inhibited by a genetic link or ROS
remains to be determined.
Since the identification of α-KGDH as a
critical enzyme in AD and PD pathogenesis, several
studies have sought to reverse the phenotypes in these
NDD. In PD, acute lipoic acid, a key cofactor for αKGDH, administration increases cerebral metabolism,
thus reversing the α-KGDH deficit (Seaton et. al.,
1996). This was performed under the assumption that
α-KGDH activity is decreased in PD brains. This study
indirectly supports the α-KGDH-centric hypothesis of
PD. Likewise, in AD, thiamine supplementation proved
to have beneficial effects in patients (Mimori et. al.,
1996). Thiamine is necessary for α-KGDH activity, as
well. Both of these studies support the hypothesis that
α-KGDH is involved in AD and PD pathogenesis.
There are other mitochondrial connections to
PD pathogenesis. Two recently discovered genes,
when mutated, were found to cause LB negative
Parkinsonism (Bonifati et. al., 2003; Valente et. al.,
2004), similar to MPTP models. The genes DJ-1 and
PINK1 will be discussed next.
DJ-1 is customarily oxidized in non cell death
conditions. Accordingly, it’s localization to the outer
membrane of mitochondria may suppress apoptosis
(Canet-Aviles et al., 2004).
PINK1 is a serine/threonine kinase with an Nterminal mitochondrial localization signal (Valente et.
al., 2004). Valente and collegues (2004) also observed
that PINK1 protects cells against apoptosis induced by
proteasome inhibitors. The PINK1 substrate is still
unknown as well as its protection specificity. One
feature is clear, however, both DJ-1 and PINK1 protect
against loss of mitochondrial function.
Conclusion
The redox status of a cell is delicately modulated by
several mechanisms. Mitochondria play an integral part
in this regulation. The loss-of-function of mitochondrial
proteins like complex I, α-KGDH, and PINK1 have
implications for the viability of cells in NDD. In PD,
dopaminergic neurons respond to stress in a unique
way. Dopamine metabolism decreases the oxidative
stress threshold required for apoptosis. Thus,
mitochondrial
impairments
selectively
target
dopaminergic neurons. These impairments, in turn,
further devastate mitochondrial function. The slippery
slope of cell death in PD is characterized by the
additively detrimental interactions between oxidative
stress and mitochondrial dysfunction.
Acknowledgments
The author would like to thank Dr. Shubhik K.
DebBurman for inspiration and support to write this
article.
the author.
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93
Grant Proposal
Characterization of Membrane Permeability Alterations in
Plasmodium-Infected Erythrocytes:
Insight into Novel
Mechanisms for Malaria Chemotherapy
Chloe Wormser*
Lake Forest College
osmolyte influx pathways typically inactive in
erythrocytes while simultaneously inhibiting efflux
pathways normally stimulated during apoptosis. To this
end, the individual goals of this study are 1) to discern
the origin of the new permeability pathways induced by
Plasmodium on human erythrocytes, and 2) to identify
whether deficient apoptosis in Plasmodium-infected
cells results from inhibition of endogenous efflux
pathways. The origin of the new permeability pathways
expressed in infected erythrocytes will be assessed
through electrophysiological studies comparing plasma
membrane permeability of infected erythrocytes with
that of uninfected erythrocytes. To identify whether
Plasmodium infection results in inhibition of
endogenous osmolyte efflux pathways, cell volume
studies will be performed using electronic sizing to
characterize the responses of uninfected and infected
erythrocytes to varying extracellular media.
Introduction
Current malaria research is geared toward identifying
novel targets for malaria chemotherapy because of the
growing resistance of Plasmodium falciparum to
existing drug options (Trager et. al., 1997). Potential
targets not yet explored are the new permeability
pathways induced by Plasmodium on host erythrocytes.
These pathways confer increased permeability to
inorganic ions including chloride, sodium, and calcium
(Adovelande et al. 1993, Brand et al. 2003, Garcia et al.
1996, Lang et al. 2003), as well as organic solutes such
as sorbitol (Tanneur et al. 2005), lactic acid, and
hemoglobin-derived amino acids (Duranton et al. 2004).
Evidence suggests that the primary function of these
pathways is to allow for abundant access to nutrients
and vitamins essential for parasite growth (Brand et al.
2003, Duranton et al. 2004), while facilitating
elimination of metabolic waste products (Duranton et al.
2004).
Due to the apparent dependence of
Plasmodium survival on these transport pathways
(Brand et al. 2003), targeting the pathways could be a
potent method for inhibiting the blood stage life cycle of
Plasmodium and, in turn, arresting disease progression.
However, the precise nature of the new
permeability pathways induced by Plasmodium on host
erythrocytes is ill-defined. That is, it is not clear
whether the pathways are endogenous membrane
proteins activated by Plasmodium or, alternatively, if
they are xenoproteins encoded by Plasmodium and
shuttled to the host cell membrane. Classification of
these membrane channels is essential before
pharmacological antagonists can be developed.
Additionally, it is unclear how Plasmodium prevents
premature cell death of erythrocytes, which is one of
the expected consequences of parasite-induced new
permeability pathways. Specifically, osmolyte influx via
these pathways leads to a breakdown of plasma
membrane asymmetry (Brand et al. 2003), which in
normal erythrocytes triggers apoptosis (Lang et al.
2003). Obviously, it is crucial to understand the
mechanisms by which Plasmodium avoids programmed
cell death if we are to formulate ways to initiate parasite
destruction. One possibility is that Plasmodium
activates osmolyte pathways that allow for nutrient
influx, while
simultaneously inhibiting efflux pathways normally
stimulated during apoptosis that allow for a decrease in
erythrocyte volume. Research aimed at assessing this
possibility is severely lacking.
Experimental Proposal
Discerning
the
Origin
of
Parasite-Induced
Permeability Pathways: Electrophysiology Studies
The results of previous studies suggest that
erythrocytes incur oxidative stress as a result of
Plasmodium infection (Brand et al. 2003, Tanneur et al.
2005). One possibility not yet fully examined is that
oxidative stress, in turn, activates endogenous
channels in the host cell membrane, and that these
channels are the new permeability pathways previously
observed in infected cells. To test this hypothesis, the
effects of oxidation on uninfected erythrocytes will be
determined using electrophysiology. Presumably, if the
new permeability pathways present in infected
erythrocytes are indeed endogenous channels
activated by oxidation, then treating uninfected
erythrocytes with oxidizing agents should result in
activation of permeability pathways identical to those
observed in Plasmodium-infected cells, even in the
absence of parasite infection. In contrast, if activation
of new permeability pathways is not the result of
oxidation, or if the ion channels activated by
Plasmodium are xenoproteins, oxidation of uninfected
cells should not result in activation of permeability
pathways similar to those observed in infected cells.
To determine which of the above scenarios is
correct, comparative studies between uninfected,
oxidized
erythrocytes
and
Plasmodium-infected
erythrocytes will be performed by monitoring ion
channel activity in the cell membranes of each cell type.
This involves using a glass pipette tip attached to the
cell membrane; a microelectrode present within the
pipette detects current (ions) flowing through the
membrane protein channels (Peterson et. al., 1986).
By exposing cells to varying extracellular solutions, it is
possible to discern the selectivity of the ion channels
(i.e., what ions they allow to traverse the membrane)
and, in turn, characterize permeability pathways active
in cells.
These patch-clamp experiments will be
performed on control cells (uninfected human
erythrocytes), Plasmodium-infected cells, and oxidized,
Specific Aims
The purpose of this research effort is to find evidence to
support the hypothesis that Plasmodium activates
*This paper was written for BIOL 320 Microbiology and Immunology,
taught by Dr. Karen Kirk
93
uninfected cells. Infected cells will be prepared by
growing Plasmodium falciparum in complete liquid
medium, and then the culture will be used to infect
human erythrocytes (the technique used will be
adapted from Trager et. al., 1997).
Oxidized,
uninfected cells will be prepared by treating human
erythrocytes
with
the
oxidizing
agent
tertbutylhydroperoxide (tBHP), as described by Brand et al.
(2003). Patch clamp recordings of each cell type will
then be obtained by bathing cells in a sodium chloride
solution (control solution that mimics blood plasma), a
medium in which chloride is replaced by the
impermeable anion gluconate (to determine the degree
to which each cell type is permeable to chloride), a
medium in which sodium is replaced by the
impermeable cation NMDG (to determine whether any
component of the current carried across the membrane
is carried by sodium), and a medium in which calcium is
chelated by EGTA (to determine the degree in which
each cell type allows for calcium influx).
It is expected that current recordings in all
test solutions will be low for control cells, as uninfected
erythrocytes have a low resting ion conductance (Huber
et al. 2004). In contrast, it has previously been shown
that Plasmodium-infected erythrocytes have a very high
chloride conductance and modest cation conductance
compared to uninfected cells (Brand et al. 2003).
Therefore, high current recordings should be seen in
the sodium chloride solution, whereas reduced currents
are expected to be seen in the chloride-free, sodiumfree, and low-calcium media. In all solutions, however,
currents from infected cells should be greater than
those observed in control cells. Lastly, it is expected
that current recordings from oxidized, uninfected cells
should match those of infected cells, if the hypothesis
that oxidation results in activation of endogenous
permeability pathways in the host cell membrane is
indeed correct. If unexpected results are obtained and
parallels are not observed, this would imply that the
method used by Plasmodium to stimulate new ion
conductance is something other than oxidation, or that
the ion channels activated by Plasmodium are foreign,
not endogenous. In order to verify such a result,
however, alternate concentrations of the oxidizing agent
tBHP will be tested and other oxidizing agents will be
used to rule out the possibility that non-specific effects
are occurring. That is, a small degree of oxidation
might activate ion channel activity, whereas a
pharmacological increase in oxidation might have an
inhibitory effect.
It should be noted that there are limitations to
what information can be drawn from the above
experiment. One cannot conclude definitively that if
oxidation of uninfected cells does not induce ion
channel activity identical to infected cells that the
mechanism responsible for activating new permeability
pathways is not Plasmodium-induced oxidation. To
explain, the oxidative events characteristic of
Plasmodium infection might be different than those
triggered by pharmacological agents in vitro.
Additionally, it is possible that although oxidation might
have a role in activating new permeability pathways,
other factors, such as parasite-derived enzymes, might
also be involved. If this is the case, then the absence
of these enzymes in uninfected cells would prevent
expression of novel permeability pathways, even in the
presence of oxidation.
Determining if Plasmodium Inhibits
Volume Decrease: Cell Volume Studies
Apoptotic
It is well known that apoptosis, or programmed cell
death, occurs by activation of osmolyte efflux pathways
that result in volume decrease (Lang et al. 2003, Okada
et. al., 2001). This, in turn, reduces cells to a size that
is easily engulfable by phagocytic cells. One cellular
signal known to trigger apoptosis is a breakdown of
membrane asymmetry, which occurs following
translocation of phosphatidyl serine from the inner
leaflet of the plasma membrane, where it is
predominantly if not exclusively localized under normal
conditions, to the outer leaflet of the membrane (Lang
et al. 2003). This translocation event has been shown
to occur in Plasmodium-infected erythrocytes.
However, unlike in uninfected cells, it does not trigger
apoptosis (Brand et al. 2003). A possible explanation
for this observation is that Plasmodium inhibits
membrane channels crucial for osmolyte efflux and
subsequent apoptosis.
To test this possibility, control cells and
Plasmodium-infected cells (prepared as described
above) will be exposed to an extracellular solution that
should stimulate volume regulatory efflux pathways
similar to those activated during programmed cell
death. Specifically, each cell type will be exposed to an
isosmotic (control) solution and a hypotonic solution,
and the effects of this exposure on cell volume will be
monitored using a Coulter Counter. This machine
electronically sizes and counts cells based on the
change in resistance that occurs as cells pass through
an aperture opening.
Cell volume is directly
proportional
to
this
change
in
resistance
(www.beckman.com). By monitoring the reduction in
cell size following exposure to varying experimental
media, it will be possible to discern whether osmolyte
efflux pathways involved in volume decrease are active
in Plasmodium-infected cells.
It is expected that exposure to a dilute
medium will result in cell swelling of both control and
Plasmodium-infected cells due to the unavoidable influx
of water. Control cells, which are able to counteract cell
swelling by activating efflux pathways (Okada et. al.,
2001), should gradually recover from swelling and
approach steady-state cell size. However, if the efflux
pathways necessary for this recovery are inhibited by
Plasmodium as hypothesized above, then infected cells
should lack the compensatory mechanism that offsets
cell swelling and should remain swollen. It is also
possible that Plasmodium cells lyse in hypotonic
solution due to dramatic cell swelling. Again, this would
indicate that the efflux pathways required for volume
regulation are inactive. Alternatively, if Plasmodium
infection does not correspond with inhibited efflux
pathways, then the response of infected cells to
hypotonic challenge should match that of uninfected
cells (i.e., cell volume recovery should proceed). If this
turns out to be the case, then one could conclude that
Plasmodium does not bypass apoptosis by inactivating
efflux pathways.
Therefore, other potential
mechanisms by which Plasmodium-infected cells avoid
premature destruction will be examined. For example,
phosphatidyl serine translocation might actually assist
in Plasmodium infection if it allows Plasmodiuminfected erythrocytes to cytoadhere more effectively to
endothelial cells and thus evade the host immune
system, in particularly the spleen. This could be
assessed using cytoadhesion, flow-based assays such
94
References
as those described by Cooke et. al. (1995) and Cooke
et al. (1995).
Although the cell volume study described
above will provide insight into how Plasmodium
prevents the erythrocyte death that typically
accompanies phosphatidyl serine translocation, it is
limited in some respects. Specifically, apoptotic events
can be simulated by activating volume regulatory
mechanisms because the efflux pathways involved in
apoptosis are believed to be identical to those involved
in regulatory volume decrease. However, the exact
mechanics of apoptotic volume decrease are not
completely understood and may vary slightly from other
cell volume regulatory processes. Additionally, it is
possible that Plasmodium inactivates efflux pathways
normally active in erythrocytes, while activating other
efflux pathways (such as those that would allow for
waste elimination).
Clearly, if these Plasmodiuminduced efflux pathways could somehow be recruited
during volume recovery, this would confound the results
of cell volume studies. Therefore, although the findings
of this experiment can be applied to apoptotic events,
such applications must be done with some degree of
skepticism.
Adovelande, J., Bastide, B., Deleze, J., and Schrevel, J. 1993. Cytosolic
free calcium in Plasmodium falciparum-infected erythrocytes and the
effect of verapamil: a cytofluorimetric study. Exp Parasitol 76: 247-258.
Brand, V.B., Sandu, C.D., Duranton, C., Tanneur V., Lang K.S., Huber
S.M., and Lang, F. 2003. Dependence of Plasmodium falciparum in vitro
growth on the cation permeability of the human host erythrocyte. Cell
Physiol Biochem 13: 347-356.
Cooke, B.M., and Coppel, R.L. 1995. Cytoadhesion and falciparum
malaria: going with the flow. Parisitol Today 11(8): 282-287.
Cooke, B.M., Morris-Jones, S., Greenwood, B.M., and Nash, G.B. 1995.
Mechanisms of cytoadhesion of flowing, parasitized red blood cells from
Gambian children with falciparum malaria. Am J Trop Med Hyg 53(1): 2935.
Duranton, C., Huber, S.M., Tanneur, V., Brand, V.B., Akkaya, C.,
Shumilina, E.V., Sandu, C.D., and Lang, F. 2004. Organic osmolyte
permeabilities of the malaria-induced anion conductances in human
erythrocytes. J Gen Physiol 123: 417-426.
Garcia, C.R.S., Dluzewski, A.R., Catalani, L.H., Burting, R., Hoyland, J.,
and Mason, W.T. 1996. Calcium homeostasis in intraerythrocytic malaria
parasites. Eur J Cell Biol 71: 409-413.
Huber, S.M., Duranton, C., Henke, G., Van de Sand, C., Heussler, V.,
Shumilina, E., Sandu, C.D., Tanneur, V., Brand, V., Kasinathan, R.S.,
Lang, K.S., Kremsner, P.G., Hubner, C.A., Rust, M.B., Dedek, K., Jentsch,
T.J., and Lang, F. 2004. Plasmodium induces swelling-activated ClC-2
anion channels in the host erythrocyte. J Biol Chem 279(40): 4144441452.
Conclusion
Lang, K.S., Duranton, C., Poehlmann, H., Myssina, S., Bauer, C., Lang,
F., Wieder, T., and Huber, S.M. 2003. Cation channels trigger apoptotic
death of erythrocytes. Cell Death Differ 10: 249-256.
Studies aimed at characterizing the alterations in
erythrocyte membrane properties
induced by
Plasmodium infection and the mechanisms by which
Plasmodium
compensates
for
the
adverse
consequences of these alterations are beneficial to the
field of malaria research. They will assist in elucidating
the underlying processes involved in growth and
survival of Plasmodium during blood stage infection,
which are a crucial aspect of malaria pathophysiology.
Further, more complete knowledge of how Plasmodium
evades the host immune response and apoptosis will
facilitate our ability to recognize, track, and prevent
malaria. And, most importantly, this information could
be applied when developing novel mechanisms for
malaria treatment that bypass current limitations in the
field.
Okada, Y., and Maeno, E. 2001. Apoptosis, cell volume regulation, and
volume-regulatory chloride channels. Comp Biochem Physiol A Mol Integr
Physiol 130(3): 377-383.
Peterson, O.H., and Peterson, C.C.H. 1986. The patch-clamp technique:
recording ionic currents through single pores in the cell membrane. Int
Union Physiol Sci/Am Physiol Soc 1: 5-8.
Tanneur, V., Duranton, C., Brand, V.B., Sandu, C.D., Akkaya, C.,
Kasinathan, R.S., Gachet, C., Sluyter, R., Barden, J.A., Siley, J.S., Lang,
F., and Huber, S.M. 2005. Purinoceptors are involved in the induction of
an osmolyte permeability in malaria-infected and oxidized human
erythrocytes. FASEB J 20(1): 133-135.
Trager, W., and Jensen, J.B. 1997. Continuous culture of Plasmodium
falciparum: its impact on malaria research. Int J Parasitol 27(9): 989-1006.
Articles published within
Eukaryon should not be cited in bibliographies.
Material contained herein should be treated as personal
communication and should be cited as such only with
the consent of the author.
95
Grant Proposal
Apical Membrane Antigen 1 (AMA-1): Role in Plasmodium yoelii
Infectivity
Michael Zorniak*
Lake Forest College
mAb against AMA-1. I propose to perform a similar
experiment using a primary hepatocyte cell culture from
mice with Plasmodium yoelii. Further understanding of
AMA-1 role in parasite entry will allow us to develop a
potential synergistic vaccine with a CSP inoculum in a
murine model, which cannot be immediately done in
HepG2 cell cultures (Gantt et. al., 2000).
Introduction
Plasmodium enters the blood stream of a
mammalian host via a bite by an infected Anopheles
mosquito. Translocation to the liver and introduction
into a hepatocyte is a critical step for infectivity of the
malaria parasite. Entry of the parasite follows two
distinct pathways: rupturing of the hepatocyte
membrane by migration or by the adhesion,
internalization, and formation of a vacuole within the
hepatocyte (Silvie et. al., 2004b). Only the latter
pathway is necessary for the differentiation and
proliferation of the blood-stage pathogen.
My focus will be primarily on the interaction of
cell-surface proteins between the hepatocyte and the
parasite. There are two well-studied proteins secreted
by apical micronemes (i.e. vesicles at the anterior tip of
the protozoan which secrete enzymes for parasite
entry): the circumsporozoite protein (CSP) and the
thrombospodin-related adhesive protein (TRAP; Silvie
et. al., 2004b). The exocytosis of CSP and TRAP from
micronemes within the parasite is dependent on the
transient increase of intracellular calcium. Once the
micronemes are excreted, CSP and TRAP localize to
the membrane of Plasmodium. This process exposes
CSP and TRAP to interact with hepatocyte cell-surface
proteins, thus allowing the internalization and infection
of the parasite by an unknown mechanism. A
parasitophorous vacuole (PV) is formed after
internalization, which is required for the differentiation of
the exoerythrocytic form (EEF; Silvie et. al., 2004b).
CSP has many roles in the life of
Plasmodium including sporozoite formation in oocysts,
gliding locomotion, hepatocyte invasion, as well as
inhibition of ribosomes (Menard, 2000). During gliding
motility, CSP is shed from the cell surface. Previous
studies have used monoclonal antibodies (mAb)
against CSP and prevented locomotion of Plasmodium,
thereby preventing infection (Mota et. al., 2002b).
CSP’s array of functions present many therapeutic
targets. Plasmodium falciparum CSP vaccines have
had some triumph in human trials, yet effective blood
titer of antibody were not sustained (Gantt et. al., 2000).
Yet, success has not been observed with TRAP
because its exposure is limited to the intimate contact
with unknown hepatocyte cell-surface proteins. CSP, on
the other hand, is evenly distributed on Plasmodium,
allowing for more binding sites on hepatocytes for
antibody neutralization.
Much success has been achieved with the study
of TRAP and CSP. These accomplishments give basis
for further study of other parasite-surface proteins.
Apical Membrane Antigen 1 (AMA-1) is an understudied
protein also found within micronemes and is released
onto the cell-surface with the advent of intracellular
calcium. Silvie and colleagues (2004a) have inhibited
HepG2 cell Plasmodium falciparum infectivity with a
Aims
Based on evidence seen in HepG2 cells, I will
test the efficacy of anti-AMA-1 mAb in mice to
neutralize the infectivity of Plasmodium yoelii in primary
hepatocyte cultures from wild-type mice. Previously,
HepG2 cells were protected against Plasmodium
infectivity with increasing concentrations of anti-AMA-1
mAb (Silvie et. al., 2004a).
Secondly, I will test the infectivity of P. yoelli
on primary hepatocytes from mice preincubated with
anti-AMA-1 mAb and treated 3 hours post sporozoite
introduction. In the past, HepG2 cells were found
susceptible to Plasmodium infection after this
procedure (Silvie et. al., 2004a). . This study will further
verify the necessity of the AMA-1 protein for sporozoite
entry into hepatocytes and subsequent infection.
Experimental Procedure
Firstly, I hypothesize that the anti-AMA-1
mAb will prevent Plasmodium yoelii entry and infection
of wild-type mouse hepatocytes as noted in HepG2
cells (Silvie et. al., 2004). The anti-AMA-1 mAb for P.
yoelii will be obtained as described in Silvie and
colleagues (2004a). Note, the ectodomain of AMA-1 is
conserved across many Plasmodium species including
parasites that infect rats. Primary hepatocyte cultures
from wild-type mice will be treated with anti-AMA-1 mAb
and inculated with P. yoelii sporozoites in the presence
of rhodamine-labeled dextran (Silvie et. al., 2003c).
Dextran-positve cells will indicate the migration of
sporozoites without parasitophorous vacuole (PV)
formation. Dextran-negative cells will indicate PV
formation without disruption of the hepatocyte
membrane.
I believe that AMA-1 is necessary for
Plasmodium invasion and proliferation in the
hepatocyte, thus treated cells should be Dextranpositive (i.e. noting the migration of parasites thru
hepatocytes without PV formation due to mAb
competitive inhibition). One pitfall may be the presence
of calcium in this procedure. Calcium has not been
added to induce exposure of the AMA-1 protein from
the microneme. A calcium ionophore may be necessary
to trigger the apical exocytosis upon invasion (Mota et.
al, 2002a). A green phycoerythrin labeled monoclonal
antibody will be used to stain for the presence of the
PV, or circumsporozoite proteins (CSP) as described in
Silvie and colleagues (2003c) thus noting infectious
entry. A culture of P. yoelii infected cells not treated
with the anti-AMA-1 mAb will serve as the control for
this experiment. The control cells should be both
Dextran-negative and Dextran-positive because of the
two invasion pathways observed by Plasmodium:
migration and infectious entry.
*This paper was written for BIOL 320 Microbiology and Immunology,
taught by Dr. Karen Kirk
97
Secondly, I will further verify the role of AMA-1
in Plasmodium infectivity by preincubating a primary
wild-type mouse culture with anti-AMA-1 mAb and P.
yoelii. Microscopy as well as immunofluorescence
decribed above will be used to assess the percentage
of exoerythrocytic forms (i.e. blood stage pathogens)
within hepatocyte tissue. This experiment is significant
because it will assess the level of toxicity induced by
the antibody treatment on P. yoelli. I hypothesize no
significant induced toxicity of P. yoelii. This will serve as
the control for the following experiment. Primary culture,
wild-type mouse, and hepatocytes will be inoculated
with P. yoeli,i which will be incubated for 3 hours. After
this time period, anti-AMA-1 mAb will be used to treat
the infected hepatocytes. The percent of EEFs will be
monitored for a 48 hour period.
I hypothesize a significant increase of EEFs in
comparison to the control. If the result is positive, this
experiment will confirm the necessity of the AMA-1
protein for invasion and formation of a PV for
Plasmodium proliferation. If negative, the AMA-1
protein may be operating in an undetermined
mechanism. This mechanism may be similar to the
TRAP protein, in that, exposure of AMA-1 from the
apical complex occurs only after the influx of calcium
and close proximity to the hepatocyte. Thus, the
antibody treatment may not present a satisfactory way
to bind AMA-1 because of the discrete affinity of AMA-1
to unknown cell-surface proteins on the hepatocyte.
This proposed model contrasts the uniform
arrangement of CSP around Plasmodium.
to TRAP and CSP, AMA-1 may serve as a putative
receptor for sporozoite invasion and be used to
construct a vaccine similar to the human CSP vaccine.
A combined CSP/AMA-1 immunization may prove to
have synergistic effects on Plasmodium protection
(Gantt et. al., 2000).
Articles published within
Eukaryon should not be cited in bibliographies.
Material contained herein should be treated as personal
communication and should be cited as such only with
the consent of the author.
References
Gantt, S., Persson, C., Rose, K., Birkett, A. J., Abagayan, R., and
Nussenzweig, V. (2000). Antibodies against thrombospondin-related
anonymous protein do not inhibit Plasmodium sporozoite infectivity in
vitro. Infect. Immun. 68, 3667–3673.
Menard, R. (2000). The journey of the malaria sporozoite through its
hosts: two parasite proteins lead the way. Microbes Infec. 2, 633-642.
Mota, M.M., Hafala, J.C.R., and Rodriguez, A. (2002a). Migration through
host cells activates Plasmodium sporozoites for infection. Nat. Med. 8,
1318-1322.
Mota, M. M., and Rodriguez, A. (2002b). Invasion of mammalian host cells
by Plasmodium sporozoites. BioEssays 24, 149-156.
Silvie, O., Franetich, J. F., Charrin, S., Mueller, M. S., Siau, A., Bodescot,
M., Rubinstein, E., Hannoun, L., Charoenvit, Y., Kocken, C. H., Thomas,
A. W., Van Gemert, G. J., Sauerwein, R. W., Blackman, M. J., Anders, R.
F., Pluschke, G., and Mazier, D. (2004a). A role for apical membrane
antigen 1 during invasion of hepatocytes by Plasmodium falciparum
sporozoites. J. Biol. Chem. 279, 9490-9496.
Conclusion
The above experiments have been used
previously
to
elucidate
the
significance
of
parasitophorous vacuole formation for infectivity of
Plasmodium in Silvie and colleagues (2003c). With the
conclusion of the above research, we will have further
characterized the function of an additional microneme
protein in context with sporozoite infectivity. In addition
Silvie, O., Franetich, J.F., Renia, L., and Mazier, D. (2004b). Malaria
sporozoite: migration for a living. Trends Molec. Med. 10, 97-100.
Silvie, O., Rubinstein, E., Franetich, J.F., Prenant, M., Belnoue, E., Renia,
L., Hannoun, L., Eling, W., Levy, S., Boucheix, C., Mazier, D. (2003c).
Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium
yoelii sporozoite infectivity. Nat. Med. 9,93-96.
98
Essays
Some Like it Hot: Astrobiology and Extremophilic Life
at which they live,” (Bennett et al 2002) but this is
notably not a necessary characteristic of all
extremophilic life because of its variety.
Almost 20 years ago, everyone seemed to
accept that nature could only harbor life on the thin
covering of Earth’s surface because of the necessity of
sunlight, but since then this view has changed. In the
late 1980s, researchers found microbes living in rock
500 meters below the surface in South Carolina
(Monastersky, 1997). We have since pushed the
known threshold much deeper, and “microbes have
apparently remained prisoners of the deep for millions
of years, making such colonies veritable living
fossils.”(Monastersky, 1997) This discovery certainly
pushed the envelope of life deeper, but it also sets the
stage for inquiry.
The heated debate over the origin of life
produces various models that either revise or
completely disagree with other theories. One current
theory is that life originated deep beneath the surface of
the ocean in hydrothermal vents. Since the discovery of
hyperthermophilic life in hydrothermal fluids recovered
from "smoker" vents on the East Pacific Rise, Lilley
Baross and Jody Deming have studied the widely
accepted upper temperature limit for life. They have
revealed that the temperature at which the
hyperthermophilic organisms thrive is approximately
300 degrees Farenheit and possibly beyond (Frontiers
1997). Many microbiologists are even willing to
speculate that the maximum may above this limit.
These boiling volcanic vents on the ocean floor may
have provided the nutrients and conditions required for
life to begin, but under truly intense pressure and heat.
The supporters of the thermal vent theory will argue
that the deep oceans of the early earth would have
sheltered early microbial life. The microorganisms may
also have adapted to the heat from the period of heavy
bombardment about 65.5 million years ago, the K-T
event. Even so, this thermal vent theory is among a
myriad of other beliefs that try to distinguish the most
plausible scientific explanation of the origin of life.
Whatever the origin of life was, we need to examine
where it may have begun to understand the amazing
variety as we see today.
Theories for the evolution of life were not
invented by Charles Darwin, but rather were solidified
by his voyage to the Galapagos on the HMS Beagle,
and his book The Origin of Species. He proposed the
mechanism of natural selection to explain his
observations of the finches and other species on the
islands. Darwinian states, “if it could be demonstrated
that any complex organ existed, which could not have
been formed by numerous, successive, slight
modifications, my theory would absolutely break down”
(Origin of Species, 1859). Following this logic, it is
plausible that certain bacteria have adapted to extreme
environments.
Every environment produces challenges that
an organism must meet in order to survive. In 1996,
Enrique Querol and his colleagues reviewed the protein
structural modifications for life at temperature extremes.
They demonstrated that the amino acid sequence of a
protein in thermostable and mesostable isoproteins
relate to changes in structure, stability, and function.
With close examination of previous research, they
questioned “simplistic” explanations and that “eight of
the replacements in β-strands would accomplish
Elizabeth Birnbaum*
Lake Forest College
Though there were a multitude of intervals of time
before us, we can barely imagine the immensity of our
very own Earth’s history because the presence of
humans only occupies a fraction of that time. Fossil
records aid in understanding what came before us,
including dinosaurs, trilobites, and ancient microbial life,
and what we learn from these applies to the
commencement of life on Earth. The pattern of life’s
origin on Earth may subsequently apply to other planets
in other galaxies and star systems in profoundly
beneficial ways. Thus, understanding the vast range of
environments within our own planet is a wonderful
place to start exploring.
Though there are a variety of environments
where life can survive, humans are limited to the
troposphere—the lowest atmospheric layer—and have
adapted to varied climates and elevations within this
expanse. Oxygen, relative temperatures, and pressure
are among the requirements for humans to survive. In
stark contrast to these requirements for human life,
consider the extremophiles, organisms that thrive in
extreme conditions. They are typically unicellular
prokaryotes—either bacteria or archaea. In relation to
what we know of the vast majority of life on our planet,
they are the rule breakers. Some of these organisms
do not need carbon beyond carbon dioxide, and can
survive without oxygen or even the relatively mild
temperatures of Earth as we experience them. Still,
from the perspective of the extremophilic organism,
their environment is completely normal. This paper will
explore their extreme character, as it may lead to clues
about how we can conceptualize life beyond our
present realm.
Understanding extremophiles is something
that is pertinent not only to realizing life’s variety and
splendor within the territory of our planet, but their
abilities are imperative to the search for life beyond
Earth—and even beyond our own star system. This
range includes the unbearably hot temperatures (i.e. to
human standards) of the inner terrestrial planets of the
solar system, to the much colder outer reaches in the
moons of Saturn and Jupiter. To commence our
understanding, we must know where these organisms
have thrived and lived; thermophilic, heat-loving, life is
naturally found in deep-sea thermal vent environments
(Frontiers, 1997) and in the Hot Springs of Yellowstone
National Park (Stahl et al, 1985; Barns and Burggraf,
1997; Spear, 2005).
The properties of extremophiles are
interestingly not the same among all in this extremeloving class—they have adapted to their environment. It
has become increasingly clear that life has modified to
and thrived in amazing places: nutritionally limited
environments, under high pressure, and astoundingly
high temperatures. Still, there are strong indices for life
in places we have not even discovered yet. We have
learned that some extremophiles die when brought to
so-called normal temperatures “because their enzymes
have evolved to function only at the high temperatures
* This paper was written in BIO 111 Astrobiology: In Search of Life
Beyond Planet Earth, taught by Dr. Ken Weik.
99
enhanced thermal stability upon stabilizing their strand
dipoles.”(Querol et al, 1996). As thermophilic
microorganisms are not able to shield their cellular
components from the environment, they have adapted
to maintain their structural integrity. The study by
Querol is one attempt to explain such adaptations.
Extremophiles living near smoking and sulfur-rich
ocean floor vents are not easily sampled. Yet, an article
reviewing the work of Biochemist Mike Adams
described his extensive work with organisms in this
territory (Hivley, 1993). These organisms have a love
for heat, and “that sets them apart from all other life. At
212 degrees Fahrenheit, the molecules that we’re made
of—that all life as we know it are made of—fall apart.
DNA comes unglued, and proteins collaps[e] in a
tangled heap, usually within seconds” (Hively, 1993).
So, it is staggering that some extremophiles thrive at
such unsympathetic temperatures. Even 16 years ago,
it was accepted that as we grow in our understanding of
these organisms, they had the promise of
revolutionizing ideas on the very chemistry and origins
of life in profound ways.
Specifically, Adams studied the bacterial
enzyme hydrogenase, which can strip water molecules
of hydrogen. Adams read the reports of German
Microbiologist Karl Stetter who “in 1982 discovered the
first organisms that thrive above 212 degrees, in
shallow hot springs off the coast of Sicily. Later, he and
other researchers began finding them in vents up to
three miles deep at the bottom of the ocean.” (Hively,
1993). From these reports, Adams ordered cultures
from both thermophilic organisms, and found that they
had a “superhot” version of the enzyme hydrogenase.
Through this finding and a subsequent series of tests,
Adams found metals in the hyperthermophilic
organisms when attempting to isolate the enzyme.
Tungsten, an especially rare element, was discovered
in this isolation process. The organisms thrived on this
element, but most importantly, tungsten induced minor
changes in protein structure which gave “dramatic
changes in stability” because of allowances in
enhanced enzyme flexibility over evolutionary history
(Hively, 1993).
The volcanic hot springs of Yellowstone
National Park are studied at present, and resaerch by
Spear, Walker, McCollom and Pace (2005) gave
special attention to this geothermal microbial
ecosystem as a whole. The brilliant colors of the hot
springs and geysers at Yellowstone range from brilliant
orange, blue, red, and yellow, to green. As we marvel at
these hues, we may also imagine how such amazingly
adapted creatures would live inside pools like these—
on another hot planet. We are discovering new news
about exoplanets and characteristics of planetary
bodies on a nearly daily basis as science progresses.
What we are learning about our galaxy and planetary
neighbors can be utilized with the knowledge
accumulating about extreme microorganisms.
The accumulating discoveries are answering
many questions, but also raising more. Are there or
have there ever been extremophiles on Mars? Or on
Jupiter’s moon, Europa? Recently discovered was a
plume of icy water from Saturn’s moon Enceladus:
“Detected last year by the Cassini probe orbiting
Saturn, the plume opens up the possibility that icy
moons considered uninhabitable may actually harbor
water, and life” (Figure 1; Vergano, 2006). A current
project is “Icepick: the Europa Ocean Explorer,” which
is an effort to create a plan for a future mission; the
Figure 1. Plumes of icy material extends above
the southern polar region of Saturn's moon
Enceladus. Courtesy of NASA, JPL, Space
Science Institute via AP.
spacecraft “would explore the liquid water oceans that
may exist beneath Europa's surface” (Figure 2; Icepick,
2006). To think of such grand-scale projects may seem
far-fetched, but with the accumulating literature on
extremophiles, the prospects look all but grim.
Figure 2. Icepick: the Europa Ocean Explorer
project
It is important that we, as humans, are
reminded that our world contains spectacularly diverse
forms of life. Lessons gleaned from studying the
extremophilic life on Earth are applicable elsewhere in
the solar system. The collaborative efforts of science
will enhance our understanding of life here on Earth,
and subsequent theories may be generated from this
knowledge for life’s existence elsewhere. We may look
forward to journeys managed quite different from, but
with the same spirit of over a century ago in Jules
Verne’s novel: A Journey to the Center of the Earth.
The ways which humans are exploring the depths and
high temperatures of the Earth today will apply to the
missions beyond it, tomorrow.
the author.
References
Barns, S. and Burggraf, S. (1997). Crenarchaeota. Retrieved May 2, 2006
from http://tolweb.org/Crenarchaeota/9/1997.01.01 in The Tree of Life
Web Project, http://tolweb.org.
100
Bennett, J., Shostak, S. & Jakosky, B. (2002). Life in the Universe.
Boston: Addison Wesley.
Bizarre Life Forms Thrive Beneath Earth’s Surface. Frontiers. [Electronic
Version] (July 1997).
Darwin, Charles. (1859). On the Origin of Species by Means of Natural
Selection, or the Preservation of Favoured Races in the Struggle for Life.
Hivley, W. (1993). Life beyond boiling. Discover. 14, 86+. Icepick: The
europa
ocean
explorer.
Retrieved
May
2,
2006
from
http://www.klx.com/europa/.
Monastersky, Richard. (1997). Deep Dwellers: Microbes thrive far below
ground.
Retrieved
April
8,
2006
from
http://www.sciencenews.org/pages/sn_arc97/3_29_97/bob1.htm.
Querol, E., Perez-Pons, J. & Mozo-Villarias, A. (1996). Analysis of protein
conformational characteristics related to thermostability. Protein
Engineering. 9, 265-271.
Spear, J.R., Walker, J.J., McCollom, T.M., & Pace, N. R. (2005).
Hydrogen and bioenergetics in the Yellowstone geothermal ecosystem.
PNAS. 102, 2555-2560.
Vergano, D. (2006, March 9). Saturn moon spurts icy plume. USA Today.
Retrieved
May
2,
2006
from
http://www.usatoday.com/tech/science/space/2006-03-09-enceladuswater_x.htm.
101
Essays
Agoutis and Seed Dispersal in Tropical Rainforest
Stephanne Levin*
Lake Forest College
Seed dispersal is a vital component of all tropical
rainforests; it is the means by which plants can spread
their seeds and successfully reproduce. Without the
use of dispersal agents, seeds must compete with
parent plants and with one another in order to survive.
Many of the seeds within neotropical forests are
dispersed by animals. These plant-animal interactions
have considerable effects on plant distribution and
diversity and the structure of rainforest communities
(Howe & Smallwood 1982). In some cases, animals act
as both seed predators and seed dispersers, ingesting
and damaging some seeds, while dispersing others
(Theimer 2005). The species of plants and animals
involved in these intricate relationships are thus able to
rely on one another for their own survival.
A number of the seeds in these forests are
adapted for dispersal by either species that live within
the forest canopy, such as primates, or species, such
as birds and bats, that are capable of flight. They are
generally dispersed after being swallowed and passing,
intact, through the animals’ digestive system. These
seeds are sometimes dropped after being partially
eaten, or they simply fall to the ground if they have not
been picked for consumption (Smythe 1986).
A
significant proportion of the trees in neotropical forests,
however, produce fleshy fruits with relatively large
seeds that are often encased in a tough pod or seed
coat. These seeds are generally too large to be
swallowed by birds, bats, and primates, which suggests
that seeds of this type evolved in such a way that they
would be dispersed by terrestrial mammals (Smythe
1986). In neotropical forests, the dispersal of these
seeds is most often facilitated by various species of
rodents.
Agoutis (Dasyprocta spp.) are among the
terrestrial mammals that act as seed dispersers in
neotropical forests. Agoutis are relatively large (3.0-5.9
kg) caviomorph rodents (Peres et. al. 1997) that are
primarily frugivorous, which suggests that the majority
of their diet is comprised of fruit and/or seeds (Smythe
1986). Dasyprocta have incisors that that allow them to
open hard fruit pits and gnaw through the hard outer
layer of some seeds. Agoutis play a critical role in the
dispersal of the seeds of many large-seeded plant
species found within tropical rainforests (Silvius &
Fragoso 2003). They scatterhoard seeds, collecting
and burying seeds within their home ranges for future
use, thus providing security for times when food may be
scarce. Agoutis inhabit a wide range of tropical
rainforests, from areas in southern Mexico to northern
Argentina (Nowak 1991, as cited in Jorge & Peres
2005).
Their relatively widespread distribution in
neotropical forests is indicative of their importance as a
member of their ecological communities.
Furthermore, during the fruiting season,
agoutis are thought to survive primarily on ripe fruit,
which is abundant and easily accessible on the forest
* This paper was written in BIO 133: Tropical Rain Forests, taught by
Lynn Westley.
103
floor. During this time, they eat until they are full and
then search for surplus food to bury in caches, which
they rely on heavily during the rainforest’s dry season,
when fruit is no longer readily available (Henry 1999).
Additionally, when fruit is in limited supply, agoutis use
visual and olfactory cues to find their caches,
depending on these resources to help fulfill their energy
requirements (Henry 1999). Agoutis have also been
known to eat animal material, leaves, and fiber,
particularly during the dry season. The consumption of
these materials is thought to compensate for the extra
time and energy that agoutis spend when they must
forage and search for caches of stored food when few
resources are available on the forest floor (Henry
1999). It is unlikely that other species will deplete the
agoutis’ stores of food by hunting for the buried seeds
because doing so would be energetically inefficient; as
a result, it is more likely that these species would
depend on sources of food that are more readily
available (Smythe 1986). Thus, agoutis are able to
maintain an adequate diet during both tropical seasons
by consuming buried seeds and relying on alternative
food sources when fruit is not available.
Theimer (2005) argues that the relationship
between plants and the rodents, such as agoutis, that
scatterhoard their seeds is a conditional mutualism.
This is based on his observations that the interaction is
often, but not always, beneficial to both species. In
addition to providing food during the dry season,
scatterhoarding seeds for later consumption makes it
possible for agoutis to reproduce year round, which can
prove to be an ecological advantage (Henry 1999).
Moreover, scatterhoarding provides protection for the
seeds from pathogens and other predators and gives
them the opportunity to germinate if they are not
consumed (Smythe 1986; Theimer 2005). Hence, the
complex interaction between agoutis and large-seeded
plants is essential for the success of both species.
The importance of this relationship is
demonstrated by one species of agouti, Dasyprocta
leporina or the red-rumped agouti, and Brazil nut trees
(Bertholletia excelsa) found in Amazonian rainforests
(Jorge & Peres 2005; Peres & Baider 1997; Peres et al.
1997). In fact, agoutis are thought to be the principal
predators and dispersers of this species (Peres et al.
1997). Dasyporcta is one of the only species of rodent
that is known to consistently bury large seeds intact
after removing the hard seed capsule, or pxyidium
(Dubost 1988; Forget 1990, 1991; Smythe 1978; as
cited in Peres and Baider 1997). Bertholletia seeds that
remained inside mature pyxidia were found to
encounter mortality nearly 100% of the time due to
attacks by fungal pathogens and rotting (Peres et al.
1997). As a result, the regeneration of Brazil nut trees
is largely dependent on the dispersal of seeds by
agoutis following their removal from inside of the hard
seed capsule (Peres & Baider 1997 & Peres et al.
1997), which has significant implications for the survival
of this species of tree.
The effect of agoutis on the distribution of
Brazil nut trees has been relatively well established, but
little is known about the trees’ influence on agouti
populations. Jorge & Peres (2005) conducted a study
in southeastern Amazonia to determine whether local
agouti density and home range size are dependent on
the presence of Bertholletia trees. The study indicated
that the prevalence of Brazil nut trees, in an area, does
in fact affect agouti populations. There were nearly
twice as many agoutis in a large Brazil nut stand than in
an area where Brazil nut trees were absent.
Furthermore, the mean home range size for agoutis
living in the Brazil nut stand was approximately half that
for agoutis who lived in areas devoid of Bertholletia
excelsa. This implies that when there is a rich supply of
Brazil nuts, agouti densities increase and agouti home
range sizes decrease. In an attempt to explain their
findings, Jorge & Peres (2005) conjectured that agoutis
might prefer some species of large seeds to others,
which could result in larger agouti populations where
these large-seeded plants are abundant. Thus, one
can conclude that, while agoutis play a significant role
in the distribution of many large-seeded plants, some of
these plants are similarly influential on the distribution
of agouti populations in neotropical forests, as well.
Understanding
the
complex
interdependences between agoutis and the plant
species, such as the Brazil nut tree, that they disperse
has caused concern among a number of conservation
biologists. It is likely that in the absence of Dasyprocta,
tree species that depend exclusively on agoutis for
seed dispersal would become locally extinct (Asquith, et
al., 1999). A reduced agouti population has also been
associated with a reduction in overall forest diversity
(Asquith, et. al., 1999). Furthermore, in areas where
large mammalian predators have been hunted to
nonexistence, agouti populations have increased
substantially; this has resulted in increased seed
predation and decreased seed dispersal by agoutis,
which has had a negative effect on the recruitment of
large-seeded tree species within these forests
(Redford, 1992). Therefore, future conservation efforts
should focus on preserving tropical rainforest
ecosystems through the maintenance of important
plant-animal interactions.
the author.
References
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size of red-rumped agoutis (Dasyprocta leporina) within and outside a
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37 (2), 317-321.
Peres, C.A., & Baider, C. (1997, July). Seed dispersal, spatial distribution
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southeastern Amazonia. Journal of Tropical Ecology, 13(4), 595-616.
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excelsa,
Lecythidaceae), an agouti-dispersed Amazonian seed crop: a test of the
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March 29, 2006 from JSTOR database.
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leporina) home range use in an Amazonian forest: Implications for the
aggregated distribution of forest trees. Biotropica, 35(1), 74-83. Retrieved
March 25 2006 from Ovid database.
Smythe, N. (1986). Competition and resource partitioning in the guild of
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Theimer, T.C. (2005). Rodent scatterhoarders as conditional mutualists.
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283-295).
CAB
International.
104
Senior Thesis
Reduced Sexual Attractiveness of Redundant Males in the
Maintenance of Guppy Color Polymorphism
behavior of guppies (Poecilia reticulata) in order to
further the understanding of evolution of sex-based
coloration in the males.
Wild guppies display a wide range of color
pattern variants among the males within a natural
population; they exhibit color patterns on their bodies,
caudal fins, or dorsal fins that greatly vary in size,
shape, position, and color (blue, green, purple, yellow,
orange, red, and black). In fact, guppies are one of the
most polymorphic species in existence, with few males
sharing the same color pattern (Farr 1997; Lank et al.
1995). Inheritance of male color patterns has a genetic
basis and is predominantly sex-linked (Winge 1922,
1927). It remains a mystery as to why such extreme
polymorphism is seen in guppies.
In many populations, female guppies
generally choose to mate with males that display a
large amount of orange coloration (Houde 1997). In
contrast, Endler (1980) discovered that predators tend
to favor less conspicuous male morphs.
Later,
Olendorf et al. (2006) and colleagues found predators
form a search image for the common male morph and
therefore indirectly affect selection. Although male
color patterns are directionally affected by sexual
selection, somehow variation is still powerfully
sustained. One of the predominant theories for the
maintenance of polymorphism is frequency-dependent
selection (Crow & Kimura 1970; Cressman 1988; Roff
1992; Judson 1995).
The theory of frequency-dependent selection
involves rare phenotypes being favored over common
ones through survival, reproductive or mating success.
Since the rare phenotypes are being favored, they will
increase in frequency until their advantage disappears.
For this reason, many morphs can coexist in a
population, and no one phenotype will predominate. In
many species, this mechanism has been proven to
uphold high levels of polymorphism.
Negative frequency-dependent selection has
been studied in various species as a mechanism for the
maintenance of polymorphism in a population. This
event has been shown to occur in Drosophila (Petit
1958; Petit & Ehrman 1969; Ehrman & Spiess 1969),
Tribolium castaneum (Sinnock 1970), Nasonia
vitripennis (Grant et al 1974), Philomachus pugnax
(Lank et al. 1995), and Escherichia coli (Elena & Lenski
1997).
In the guppy, extensive research into this
mechanism has been conducted. Most of these studies
have examined mating success of males with color
patterns that are unfamiliar to females. Some studies
suggest that females may be likely to choose mates
that have a rare color pattern (Farr 1977, 1980), while
others suggest that novel color patterns are attractive to
females (Hughes et al. 1999; Eakley & Houde 2004).
Potentially, choosing novel males could increase the
fitness of a female’s offspring. Mating with a novel
male would introduce genetic diversity into the
population and decrease inbreeding depression. This
mechanism could explain the maintenance of color
polymorphism in guppies.
Although
frequency-dependent
sexual
selection is hypothesized to be a major factor
contributing to polymorphism in guppies, non-sexual
selection may also play a role. In a study by Olendorf
Katherine J. Hampton*
Lake Forest College
Summary
The Trinidad guppy, Poecilia reticulata, is one of
the most polymorphic species in existence.
Guppies exhibit predominantly Y-linked inheritance
of male color patterns, which appear on the body,
caudal fin, or dorsal fin and are highly variable
between individuals. Little investigation behind the
mechanism maintaining polymorphism in guppies
has been done. One hypothesis is that guppy
polymorphism results from frequency-dependent
selection. My study sought to investigate whether
reduced sexual attractiveness of “redundant”
males, with similar color patterns, may contribute
to polymorphism. Experimental groups consisted
of two males with similar color patterns (redundant)
and two males with distinctly different color
patterns (unique). The four males were placed in a
tank with four virgin females. I compared sexual
responsiveness of female guppies to the redundant
vs. the unique males. Females were significantly
less likely to show sexual response to the
redundant males than they were to the unique
males.
This suggests that female guppies’
preference for unique males contributes to the
maintenance of polymorphism in this population.
Introduction
Coloration in animals has evolved for reasons related to
defense against predators, sex, and thermoregulation
(Guilford 1988). For example, many insects evolved
their coloration for defensive purposes.
O.
nicaraguensis displays cryptic coloration, making the
beetle inconspicuous to predators as they blend in with
the forest floor. Other insects’ defense coloration has
evolved to mimic species with known hazards to
predators. For instance, the Pseudoxycheila species
mimics the stinging wasp in coloration and therefore
protects itself from predators in this way. Additionally,
the conspicuous color pattern of P. tarsalis serves as
an aposematic signal to predators after eating them, as
they contain distasteful compounds (Schultz 2001). In
birds, the males often display conspicuous coloration in
order to attract mates, as a form of sex-based
coloration.
Male Quetzals have evolved long,
exaggerated tails, bright plummage coloration, and
facial ornamentation to attract females (Wingquist &
Lemon 1994). Some animals use coloration as a
thermoregulatory device, like the butterfly Colias
eurytheme. In cold weather, the butterfly has darkwings in order to form heat faster in sunlight, which is
required for flight. However, it turns a lighter color in
warm seasons to minimize overheating (Watt 1969). It
is important to study the evolutionary mechanisms
which underlie the origins and maintenance of such
coloration. In this study, we examined the sexual
*This article was written as a Senior thesis under the direction of Dr. Anne
Houde
105
Polymorphism in Guppies
et al. (2006), predation was examined as a possible
mechanism for the maintenance of polymorphism.
Their study manipulated frequencies of males with
different color patterns in three natural populations in
Trinidad and estimated the survival of rare and common
phenotypes.
The results concluded that rare
phenotypes survive possibly because guppy predators
develop a search image for common male morphs.
The first studies on frequency-dependent
sexual selection in guppies were conducted by Farr
(1977, 1980). He examined female responses to “rare”
male courtship displays and their reproductive success.
Farr noted that female guppies rarely respond to male
displays, in general. But when Farr placed a new male
into the tank, females readily responded and showed
preference for the male. Farr also showed that rare
morphs had a higher mating success.
Hughes et al. (1999) also examined
frequency-dependent selection. In this study, Hughes
and colleagues exposed females to a particular male
color pattern and then introduced a new male type in
equal numbers. As in the Farr study, Hughes et al.
concluded that females familiar with a certain group of
males (having similar phenotype to one another) had a
significantly lower probability of mating with those
males after exposure to novel males. Researchers
found that familiar males also produced significantly
fewer offspring. These findings suggest a frequencydependent mechanism of selection, in that females
prefer to mate with unfamiliar males. This relates to the
rare male mating advantage, in that rare males are
likely to be unfamiliar.
Eakley and Houde (2004) later studied how a
previous mate affects the choice of a subsequent mate.
They found that females avoid males they mated with
previously and males similar to them. Our study
expanded on these studies since we showed females
discriminate against males with similar color patterns
(redundant) relative to males with unique color patterns.
This is the first study of frequency-dependence to look
at female sexual responses to males that vary in
frequency of color pattern types.
Two redundant males and two males with
unique color patterns were observed interacting with
four virgin females, during which time female sexual
response scores were taken for each male. Unlike
other frequency-dependent studies, where virgin
females are first familiarized with a particular male
phenotype and subsequently introduced to a new male
phenotype, this study presents all male morphs at the
same time. An advantage of our study is that we
observe a variety of morphs.
Other frequencydependent studies used a limited number of male
morphs, however, we observed three different morphs
in each trial. Every trial presents a new selection of
morphs as well, and in this way we are able to study the
effects of a wide-range of phenotypes. This enables us
to investigate frequency-dependent sexual selection as
it may occur in the wild. That is, in natural populations,
females are presented with many different male
phenotypes from which to choose. We predict that
unique males will have higher female response scores
because females will discriminate against redundant
males.
One reason for possible discrimination
against redundant males may be females’ lack of
interest in re-mating with a male similar to their previous
mate as Eakley and Houde showed. Alternatively, it
may be that females will simply have a preference for
less-common phenotypes per se, as in Farr’s studies.
Guppies are extremely polymorphic organisms in
respect to male color patterns; they exhibit a wide
range of colors in multifarious combinations and
patterns. The species is highly sexually dimorphic, with
females being nearly twice the size of males and
displaying a uniform grayish-brown color. Although
females may contain the genes for male color pattern,
they are not expressed visibly as in the case of males
(Winge & Ditlevsen 1947). Normally, guppies have
typical X-Y sex determination, however, XX males and
XY females have been observed (Winge & Ditlevsen
1947).
These findings indicate the X and Y
chromosomes are not sole determinants of sex but
rather that the accumulation of male/femaledetermining
autosomes
may
contribute more
significantly towards sex determination (Winge &
Ditlevsen 1947). At the beginning of this century,
Ojvind Winge affirmed the concept of “one-sided
masculine inheritance,” making the guppy the first
organism in which a Y-linked inheritance was
demonstrated (Winge 1922). In the beginnings of his
research, Winge believed the female did not influence
the pigmentation of her sons.
A year later, he
discovered a case of “crossing over” between the X and
Y chromosome and determined the X chromosome
contained a gene responsible for an elongated caudal
fin characteristic (Winge 1923). This led to Winge’s
thorough investigation of the frequency and types of
“cross-over” events in guppies.
Winge found the X and Y chromosomes in
guppies are so similar that in fact they are completely
homologous, with the exception of a single gene—the
male-determining gene (Winge 1927). Because of the
homology of the X and Y chromosomes, “crossing-over”
is a frequent occurrence. In his research, Winge (1947)
discovered 20 color genes, eighteen being X or Ylinked and 2 being autosomal. He states that the Y
chromosome always contains at least one of the
absolute Y-linked alleles—Ma, Pa, Ir, or Ar, which code
for whole color patterns (Fig 1). However, Y-linked
genes not located near the male-determining gene can
cross over onto the X chromosome, and X-linked genes
can, alternatively, cross over onto the half of the Y
chromosome that is not male-determining. Winge
(1947) found the maximum cross over percentage to be
ten percent. A more recent review lists 16 Y-linked
traits, 24 that recombine between the X and Y
chromosome, two that are X-linked, and two that are
autosomal (Lindholm & Breden 2002).
Winge’s and other studies are based on
laboratory varieties of guppies descended from
individual males from the wild, and may not represent
the full range of color pattern variation. In order to
evaluate the full representation of male color patterns,
we set up a breeding experiment.
In conjugation
with my behavioral study, I conducted a genetics study
to further investigate sex linked traits in a laboratory
population derived from wild guppies. Twenty-three
males in the study were bred with three females each,
and offspring of each female were examined. Orange
body color patterns, spots, and tail patterns of the dads
and sons were analyzed. This was accomplished by
first identifying each of these traits in the entire
population of parents and offspring in the study. Then
the traits were numbered, and every male fish was
analyzed by marking all of their individual traits.
Looking at all the traits represented in this study, I have
106
possible confounding effects, redundant males in the
last 16 trials were more orange than those in the
previous trials (Table 1), and these redundant males
were significantly more orange than unique males in the
last 16 trials. Overall, redundant males were slightly
more orange than unique males.
In order to control for possible effects of
males’ area of orange and variation between groups on
female response rates and male display rates, general
linear models were used to test for an effect of unique
vs. redundant males. These analyses confirmed that
females rate of response was significantly higher for
unique males than for redundant males (Table 2). Due
to the discrepancy in orange between the two data sets,
analyses were performed separately, representing the
first 12 trials for set 1 and the last 16 trials for set 2.
Unique males elicited a much higher response rate in
both sets. Display rates for unique and redundant
males were very similar in set 1, and in set 2 redundant
males displayed more. These analyses demonstrated
that the area of orange coloration did not affect the
female response rate or the male display rate (Table 3).
Paired t tests with corrections for orange area also had
similar results, showing that response rates were
significantly higher for unique than for redundant males
(set 1: t=3.3, d.f.=11, P=0.007; set 2: t=3.4, d.f.=15,
P=0.004). Paired tests for male display rates showed
significant difference between unique and redundant
males (set 1: t=0.07, d.f.=11, P=0.95; set 2: t=1.54,
d.f.=15, P=0.14).
(Winge 1947)
been able to identify traits inherited directly by the
fathers and ones influenced by the mothers.
Preliminary data also shows, as in Winge’s studies, that
fathers, sons, and brothers tend to look strikingly similar.
Some families were more similar in color patterns than
others, suggesting that certain traits are more likely
directly inherited from the father.
Discussion
Our results showed female guppies were significantly
more responsive to unique males than to the redundant
males. This result is consistent with the idea that
patterns of mate choice may lead to frequency
dependent selection. Previous studies of guppies have
Results
In initial analyses, the mean female response in the 28
trials was significantly higher for unique males than
redundant males (fig 2, t=-7.25, df=27, P<0.001). That
is, females were much more likely to respond to a
unique male and continue in the courtship sequence.
Although few actual copulations were observed, unique
males, on average, scored higher on the scale than did
redundant males. The fraction of responses with
scores of 2 and higher was significantly greater in
unique males than for redundant males (Fig 3, t=7.22,
df=27, P<0.001). Redundant males tended to display
slightly more frequently than unique males. However,
this difference was not significant (Fig 4, t=1.73, df=27,
P=0.10).
For each individual male, area of orange was
measured. Males in the first 12 trials were selected
differently than those in the last 16 trials. Due to
0.9
Fraction of Responses
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Redundant
Unique
Figure 3. Fraction of female responses to redundant and
unique males with scores of two and higher. Error bars
represent standard errors.
70
Mean Number of Displays
Within 20 Minutes
Mean Female Response
on 0-5 Scale
2.5
2.0
1.5
1.0
0.5
60
50
40
30
20
10
0
0.0
Redundant
Redundant
Unique
Unique
Figure 4. Mean number of displays between redundant and
unique males with a 20- minute observation session. Error bars
represent standard error.
Figure 2. Mean female response to redundant and unique
males. Female response was rated on a 0-5 scale. Error bars
represent standard errors.
107
Table 1. Fraction of Orange (Orange/Body) in Redundant and
Unique Males for two sets of trials.
Set
1
2
Redundant
0.12 (0.01)
0.21 (0.01)
Unique
0.10 (0.01)
0.14 (0.01)
T
1.77
-5.36
DF
23
31
note that when a novel male was placed into a group of
males and females, the novel male’s presence elicited
sexual responses from otherwise unresponsive
females. In order to assess the reproductive success of
the “rare” males, Farr formed groups consisting of
males with two different color patterns in a 9:1 ratio.
The rare males sired a disproportionate number of
offspring (> 30%). One limitation in Farr’s study, which
was corrected for in our research, was the fact that Farr
used a limited number of male morphs.
Our
observations were conducted with a different set of
male morphs for each of the 28 trials, representing the
full range of variation in a natural population.
Other studies on the effects of morph
frequency on male mating success have been
conducted with Drosophila. Ehrman (1966) found
females mated equally with males of different
genotypes when present in equal ratios. However,
when introduced at different frequencies, rare males
had higher mating success. Experiments done on the
“rare-male effect” have posed problems because their
experimental designs contained biases (Partridge & Hill
1984; Partridge 1988).
Some biases could have
existed because individual females had fixed
preferences for different male types and male-male
competition occurred between males of similar color
patterns.
Hughes et al. (1999) overcame these biases
by using a “familiarization” period.
They tested the
hypothesis that females may prefer to mate with
unfamiliar (or novel) males. In their study, they sought
to determine whether female preference for males with
a particular color pattern would be affected by prior
experience with those males. The results showed
females were less likely to mate with familiar than
unfamiliar males and this could provide a mating
success advantage to rare types that are more likely to
be unfamiliar.
Eakley and Houde (2004) also found
evidence that male guppies discriminate in favor of
novel males.
Their study examined the sexual
responses of female guppies one day after mating with
an initial male. They found that females were more
responsive sexually to a novel male than to either the
original mate or to a male with a color pattern similar to
the original mate’s. Eakley and Houde examined
females’ preference for a second mate and concluded
that females greatly preferred what they termed ‘novel’
males.
Since female guppies generally prefer males
with a larger area of orange coloration, we measured
the area of orange on every fish. Our results showed
that the redundant males, on average, actually had a
slightly higher area of orange coloration than did the
unique males. This may be because very similar males
with higher amounts of orange were more readily
available for choice as redundant male sets. Also, half
of the trial groups came from a genetics study in which
fish may have been more orange and attractive, due to
chooser bias. The redundant males from the second
P
0.09
<0.001
Set 1 represents the first 12 trials and set 2 the last 16 trials.
Standard errors are given for redundant and unique groups.
suggested that “rare” males may have increased mating
success (Farr 1977, 1980) and that females prefer
unfamiliar over familiar males (Hughes et al. 1999,
Eakley & Houde 2004). However, no previous study
has examined the sexual response behavior of female
guppies when presented with a range of naturally
occurring male color pattern morphs that differ in
frequency. Our study shows that females prefer unique
males and discriminate against redundant males when
presented simultaneously to females. This behavior
could potentially lead to a mating success advantage to
unique males, which in turn could help maintain color
pattern variation in the population through negative
frequency dependent selection.
The fraction of male displays eliciting female
responses was significantly higher for unique males
than redundant males. There are a number of possible
behavioral mechanisms that could account for this
result. For instance, there were two redundant males
with a single color pattern phenotype but only one of
each of the unique male phenotypes. Therefore,
females were more likely to meet by chance a
redundant male than they were one of the unique
males. Hypothetically, if a female was courted by one
of the redundant males and rejected him, and then was
later courted by the other redundant male, she could
extend her rejection to a similar male. Additionally, she
could discriminate against the first male as well.
Females may thus become less responsive to
redundant males. A second possibility is that female
guppies may discriminate against males they have
already mated with, given the females are more likely to
have mated previously with a redundant male than
either unique male (Eakley & Houde 2004). Finally, in
nature, it could be possible that females can calculate
morph frequencies and discriminate against common
male morphs, favoring rare morphs within the
population.
While our study suggests that females may
discriminate against redundant males, a number of
other studies have also suggested that mate choice
may result in frequency dependence. These include
studies suggesting a “rare male mating advantage,” in
which females may favor uncommon morphs (Farr
1977, 1980), and studies of familiarity or novelty
(Hughes 1999; Eakley & Houde 2004).
As in our study, Farr (1977) introduced
females to two male phenotypes which were presented
in different frequencies. However, Farr did not conduct
behavioral observations in his study; instead, he simply
recorded the reproductive success of the males. He did
Table 2. Least-square means (standard errors), on the untransformed scale, and significance tests for redundancy calculated on
transformed variables.
Trait
Response
Response
Display
Display
Set
1
2
1
2
Unique
0.71 (0.05)
0.65 (0.04)
20.4 (1.65)
26.3 (2.18)
Redundant
0.40 (0.06)
0.38 (0.05)
20.6 (3.72)
33.8 (3.22)
Set 1 represents the first 12 trials and set 2 the last 16 trials.
108
F
11.1
14.3
0.01
2.44
DF
1, 11
1, 15
1, 11
1, 15
P
0.007
0.002
0.99
0.14
Table 3. Intercepts and slopes (standard errors) and significance tests for effects of orange area on male response and display rates.
Trait
Response
Response
Display
Display
Set
1
2
1
2
O Int (se)
0.70 (0.22)
0.24 (0.15)
19.3 (9.06)
34.0 (10.1)
O Slope (se)
-0.90 (0.58)
0.34 (0.31)
4.02 (23.3)
-0.43 (20.6)
F
3.79
1.07
0.13
0.03
DF
1, 34
1, 46
1, 34
1, 46
P
0.06
0.31
0.72
0.87
Set 1 represents the first 12 trials and set 2 the last 16 trials. O is amount of orange coloration.
mature age. Unique males in this set of data were collected
from the same 40 liter tanks as in the previous trials. Two 40
liter aquaria containing 4 compartments each were used to
house the groups of males for two to eight days, until needed
for observations. Four nonvirgin, sexually mature females were
added to each compartment with the males.
Fish were placed in an observation tank the day
before observations were conducted, allowing sufficient
interaction time between all individual fish.
Experimental
groups consisted of one group of males (2 redundant males
and 2 unique males) and 4 virgin females (matched for size).
Observations were conducted in a dark room at 8:30 am, 30
minutes after the fish were fed flakes and the aquarium light
came on. The 40 L observation tank was illuminated by a
fluorescent light on a 12-h light: 12-h dark cycle and contained
bottom gravel and filtered water only.
We observed the sexual behavior of virgin females
the morning after they were introduced to a group of males.
The virgin females were placed in the tank with the males a day
before actually conducting observations, in order to allow them
to complete initial matings. Virgin females reared apart from
males mate, possibly indiscriminately, within minutes of first
encountering males and then enter a refractory period during
which little sexual behavior occurs for an hour or more (Houde
1997). The next morning, females can still be reliably observed
showing sexual responses to males.
Each male was observed for 10 minutes at a time
and then observed again for another 10 minutes, at least 10
minutes after the first observation ended. The observation
order of the males was selected at random by using cards
marked with the males’ identification numbers. A total of 28
groups of males were observed.
Female response to the males’ courtship displays
was marked on a 0-5 scale described by Houde (1997). On
this scale, a score of zero indicates no female response to the
male’s sigmoid display, and a score of 1 is recorded when the
female simply turns her head toward the displaying male but
subsequently either turns away or does not progress in the
courtship sequence. A response score of 2 and above was
considered to be a sexual response. This was recorded when
a receptive female glided towards the displaying male (Liley
1966; Houde 1997). Scores of 3, 4, and 5 represented
advanced stages in the courtship sequence after stage 2 had
already occurred. The fraction of displays eliciting a sexual
response was calculated using Microsoft Excel as a measure of
each male’s attractiveness.
After an 80 minute observation session, the males
were removed from the tank and photographed on both sides.
Later, body area and orange coloration measurements were
made for each male in the study using NIH Image J Software
(http://rsb.info.nih.gov/ij). Measurements were done by tracing
each orange spot on an individual fish’s body and adding the
total areas of orange. The body of the fish, excluding the tail,
was also traced to measure the fish’s body area. Calculations
were then made to assess the fraction of orange relative to
body area.
An initial set of statistical analyses was
performed using paired t-tests on the response scores, with
fraction of responses and display rates averaged for the unique
and redundant males within each group. Averaged data for
redundant and unique males were used because the possibility
of non-independence within groups required that this analysis
be done with trial (group) as the experimental unit. A more
complex analysis, taking into account orange coloration and
possible between-group variation, was performed by Kimberly
Hughes (University of Illinois, Champaign-Urbana).
She
created a general linear model for each set of data separately;
set 1 was the first 12 trials and set 2 was the last 16 trials. See
Appendix 1 for full details of statistical analyses provided by Dr.
Hughes. She looked to see how the dependent variables
half of the trials were more orange than in the first half
of the trials. Since the unique males did not have a
larger area of orange, the difference in female
preference could not have been due to a preference for
more orange males.
Further analysis of the data
showed no effect of area of orange on attractiveness to
females. Females’ response to redundant vs. unique
males appears to have significantly overridden the
effect of orange found in previous studies.
Male guppies tend to inherit most of their
color patterns from their fathers and often look identical
or at least similar to their fathers and brothers (Winge
1922, 1927; Houde 1992; Hampton unpublished data),
making the maintenance of color pattern polymorphism
especially difficult to explain. Our study presents a
possible mechanism for sexual frequency-dependent
selection, in which female guppies discriminate against
males that are similar to other males in the group. Our
data strongly support female preference for unique
males, however, it will be important to obtain data on
mating success of redundant and unique males.
Genetic variation may be maintained if females mate
with rare males. Mating preference for rare males may
benefit females, because males introduced to a
population from outside the local pool may provide
heterozygosity and mating with these males could lead
to higher fitness of offspring and a reduction of
inbreeding effects. Furthermore, females who mate
with a second different male would likely have offspring
with greater variation, and this would reduce the risk of
a single incompatible mating.
Our study provides a possible mechanism for
frequency-dependent sexual selection. However, the
frequency-dependent survival found by Olendorf et al.
(2006) may also promote polymorphism in guppy
populations.
In conclusion, the behavior of both
predators and females may be responsible for
maintaining polymophism, but it will be important to
show that both lead to direct fitness effects.
Materials and Methods
Fish used in this study were descendents of wild guppies from
the Paria River population in Trinidad. They were housed in 40
liter aquaria, which contained filtered and conditioned tap water,
gravel, and moss. Each aquarium was exposed to a 12-h light:
12-h dark illumination cycle by fluorescent lights, and a
temperature of about 22-27ºC was constantly maintained.
Virgin females used in each trial were raised in isolation from
males in 40 liter aquaria separated into 4 compartments with 6
females in each; they were used in experiments after reaching
sexual maturity at about 12 weeks of age. All fish were fed
twice daily with Tetramin flake food in the morning and brine
shrimp in the afternoon.
Experimental groups were set up consisting of two
“redundant” males with similar color patterns and two unique
males (selected at random), all reared in the same community
aquarium and all of sexually mature age. Redundant males
from the first 12 trials were collected from an aquarium by
examining all the males caught and selecting 2 males whose
color patterns were similar (redundant) and 2 whose were not
similar (unique). In the second set of 16 trials, redundant males
were collected from a breeding experiment in which full sibs
were housed in the same 4 liter tanks; twins were all of sexually
109
(fraction responses or displays) were predicted by category
(redundant or unique), amount of orange, group, and group-bycategory interaction. The interaction of category and orange
coloration was not significant in either set for either trait, so it
was not included in the final model. In addition, Dr. Hughes
conducted paired t-tests to compare the response rates and
display rates between the unique and redundant males, in
which the dependent variables were corrected for the effect of
amount of orange coloration.
significant (set 1: t=0.07, d.f.=11, P=0.95; set 2: t=1.54,
d.f.=15, P=0.14).
Appendix
I would like to thank Dr. Anne Houde for advising me
throughout the extent of my research experience. She
has been a great mentor who has devoted huge
amounts of time revising my thesis and encouraging me.
Additionally, great thanks to Dr. Douglas Light and Dr.
Bernice Gallagher for participating in my thesis
committee and for their noteworthy advice. I would also
like to thank Estefania Luna for feeding my fish twice a
day and The National Science Foundation for providing
the funding for this research.
There was no significant effect of orange coloration on
either female response rate or male display rate (Table
2).
Acknowledgments
Details of statistical methods and results of analyses
carried out by Dr. Kim Hughes
(This text written by Dr. Hughes)
The two variables that were measured as proportions
(response rate and the amount of orange per male)
were transformed by taking arc sine square root, after
which both the variables themselves and the residuals
from all analyses were normally distributed. For the
count variable (displays) we used a square root
transformation, after which the variable and the
residuals were normally distributed. Because of the
differences in sampling procedures between the two
sets, we conducted separate statistical analyses within
sets. We tested for differences between unique and
redundant males within sets using general linear
models of the form y = µ+R+O+G+G*R+e, where y is
the dependent variable (fraction responses or displays),
R is the category (redundant or unique), O is the
amount of orange, G is the group, and G*R is the
group-by-category interaction.
The interaction of
category and orange coloration was not significant in
either set for either trait, so we did not include it in the
final model. We treated R and O as fixed effects, G and
G*R as random effects, and assumed that the errors
within groups were correlated (using the SUBJECT and
GROUP options within SAS Proc Mixed). This model
accounts for the non-independence of males tested at
the same time and for additional non-independence of
males in the same color category within group, and it
adjusts the degrees of freedom accordingly. We also
conducted paired t tests within sets on the difference
between unique and redundant males, by first
calculating the mean trait value for the redundant and
unique males within each group and then treating mean
scores as paired observations within groups. In this
analysis, we first removed effects of orange coloration
by taking the residuals from a regression of the
dependent variable on the amount orange.
the author.
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111
Senior Thesis
Calcium-Stimulated Regulatory Volume Decrease in Salmo salar
and Alligator mississippiensis Erythrocytes
Chloe Wormser*
Lake Forest College
common function: maintaining a homeostatic balance,
both within the intracellular environment and between
the intracellular and extracellular environments (Lang &
Waldegger 1997).
Securing this steady-state
equilibrium is not an easy task, as there are
unavoidable instances over the course of a cell’s life in
which homeostasis may be challenged (O’Neill 1999).
Fortunately, cells have acquired distinctive features that
aid in preventing cellular imbalance and recovering
from inevitable challenges.
One such feature is the plasma membrane,
which is responsible for compartmentalizing the cell and
allowing metabolic processes crucial for cell survival to
take place in isolation from the external environment
(Schultz 1989). The basic structure of the plasma
membrane is a phospholipid bilayer embedded with
proteins (Goodman 2002). The phospholipids that
compose the membrane form a stable bilayer as a
result of their amphipathic nature; they possess both
hydrophobic phosphate moieties and hydrophilic
hydrocarbon tails. As a result of this composition, the
plasma membrane permits the selective movement of
fat-soluble solutes, both in and out of the cell, in
accordance with the concentration gradient established
between the extracellular and intracellular environments
(Goodman 2002). For ions or molecules that cannot
readily traverse the membrane, either due to their size
or charge, carriers and channels allow for their
transport (Goodman 2002). This transport may be
active, if the molecule or ion is moving against its
concentration gradient, or passive, if the molecule or
ion is moving with its concentration gradient (McCarty &
O’Neil 1992). Both types of transport, active and
passive, play a role in daily cellular function and are
necessary for proper communication between the cell
and its surroundings.
Active transport of molecules across the
plasma membrane requires both energy and the
assistance of a pump (O’Neill 1999). Protein pumps
function to recognize and bind a specific substrate
molecule and transfer that molecule across the
membrane in the direction that opposes its
concentration gradient (Singer & Nicolson 1972). This
process requires energy input, often in the form of ATP
(Singer & Nicolson 1972). In contrast, other carriers
transport molecules with their concentration gradient;
this passive process is known as “facilitated diffusion”
(Singer & Nicolson 1972).
Protein channels function as pores that shield
water-soluble ions from direct contact with the lipid
bilayer, thus allowing the ions to travel passively into or
out of the cell (Lewis & Donaldson 1990). These
channels are often highly regulated, with this regulation
being dependent on both the type of channel and the
specific membrane in which the channel is embedded.
For example, some channels are ligand gated, in which
the binding of a signal molecule to the channel leads to
its opening or closing (Singer & Nicolson 1972). In
contrast, other channels are voltage gated, where a
change in membrane potential either activates or
deactivates the channel (Lewis & Donaldson 1990). Ion
channels may also be mechanically gated such that
mechanical stress or distortion to the plasma
membrane controls the channel’s activity (O’Neill 1999).
Summary
The mechanisms by which cells compensate for
volume fluctuations are not clearly understood and
vary among species. Research efforts in our lab
have focused on elucidating the pathways involved
in regulatory volume decrease (RVD), the process
activated in response to cell swelling that allows for
volume recovery.
Previously, fluorescence
microscopy studies performed by Light et al. (2005)
revealed that in salmon red blood cells, cell
swelling elicits a rise in intracellular Ca2+
concentration (visualized using fluorescence
microscopy and the Ca2+ indicator fluo-4-AM). This
was most likely due Ca2+ influx from the
extracellular environment, because it was not
observed in cells bathed in a hypotonic, low Ca2+
medium. The goal of this study, therefore, was to
confirm a role for extracellular Ca2+ in the RVD
response, using both Salmo salar (Atlantic salmon)
and Alligator mississippiensis (American alligator)
red blood cells. This was done by exposing cells to
different
extracellular
environments
and
pharmacological agents that block Ca2+ influx
2+
pathways or Ca -mediated intracellular signaling
cascades.
To asses the effects of these
manipulations on RVD, median cell volume
changes over a 90 minute time course were
determined by electronic sizing using a Coulter
counter. Salmon cells exposed to a low Ca2+
environment failed to recover from cell swelling,
indicating that extracellular Ca2+ was needed for a
successful RVD response.
Similarly, volume
regulation of alligator red blood cells occurred by a
2+
Ca -dependent mechanism. Additionally, RVD in
alligator cells appeared to occur through an
intracellular signaling cascade involving Ca2+
activation of phospholipase A2 and the subsequent
formation of arachidonic acid. Arachidonic acid
itself, as opposed to one of its potential breakdown
products, aided in volume recovery by stimulating
K+ efflux. In conclusion, the results from this study
indicate that Ca2+ plays a pivotal role in the RVD
response of both salmon and alligator red blood
cells.
Introduction
Cells are the basic building blocks of all life forms, no
matter how simple or complex. In fact, despite the
enormous diversity that exists among organisms, all
have the same basic level of organization in that they
are composed of one or more cells. Although cell
morphology and physiology may vary, both within an
organism and among species, all cells conform to a
* The report of the investigation undertaken as a Senior Thesis, to carry
one course of credit in the Department of Biology.
113
Having numerous membrane channels and multiple
gating mechanisms at their disposal allow cells to
detect different types of perturbations and respond
accordingly.
Carriers, pumps, and ion channels are
distinct from one another in their affinity and specificity
for a given solute. Despite these differences, they all
function similarly to assist the cell in responding to
physiological conditions by altering plasma membrane
permeability to certain molecules and ions (O’Neill
1999). This change in permeability, in turn, adjusts
intracellular solute concentration and affects the
osmotic gradient between a cell and its surrounding
medium (O’Neill 1999). As a result, the movement of
osmotically obligated water occurs in coordination with
solute transport (Lang & Waldegger 1997). This water
and solute flux across the plasma membrane is linked
to a major cellular threat: a change in cell volume
(McCarty & O’Neil 1992).
The difference in osmolality (i.e., the number
of dissolved particles per kg H2O) existing between two
media separated by a semi-permeable membrane
determines the direction of water flow between them.
Specifically, water will travel passively across the
membrane from a medium of lower osmolality (more
dilute) to higher osmolality (more concentrated); this
process is termed osmosis (Lang & Waldegger 1997).
Because cells are bathed in an extracellular medium,
an osmotic gradient might exist between the
intracellular
and
extracellular
environments.
Consequently, if the osmolality inside the cell is greater
than the osmolality outside the cell, water will travel
passively into the cell, resulting in cell swelling. In
contrast, if the solute concentration within the cell is
lower than the solute concentration of the extracellular
environment, water will diffuse out of the cell, and the
cell will shrink.
Such cell volume changes are
characteristic of organisms composed of cells that are
faced with variable extracellular solute concentrations
(O’Neill 1999). For example, many intertidal animals
are exposed to an external medium that dramatically
and continually changes salinity due to environmental
factors, such as evaporation, rainfall, and tidal
movements of water (Vidolin et al. 2002). Similarly, the
gill epithelia of many teleosts, particularly euryhaline
species, are bathed in external environments of
fluctuating salinity (Kultz 2002). Such fluctuations may
lead to volume changes at the cellular level.
Even cells bathed in a fairly invariable
extracellular medium experience shifts in cell volume as
a result of changes in intracellular osmolality, which can
occur from a variety of cellular processes (O’Neill
1999). For example, the cortical proximal tubule cells
of the kidney are challenged with significant fluctuations
in intracellular osmolality as a consequence of their role
in water and nutrient resorption (Linshaw 1991).
Similarly, the crucial role that intestinal cells play in
solute absorption leads to changes in the number of
osmotically active particles within them and, in turn,
changes in their volume (O’Neill 1999). Further, the
protein synthesis and degradation that occur in
nucleated cells lead to osmotic perturbations, as the
synthesis of proteins from amino acids reduces
intracellular osmolality, whereas the degradation of
proteins into amino acid monomers increases
intracellular osmolality (Lang & Waldegger 1997).
Clearly, some cell volume fluctuations are unavoidable
events that occur as a result of common cellular
activities and metabolic functions.
Extreme volume changes, changes that a cell
would not typically face under normal physiological
conditions, can be induced in vitro. The direct effects of
such perturbations on cell homeostasis have been
determined experimentally by placing cells in either
dilute or concentrated extracellular media. These
experiments have shown that drastic shifts in
extracellular osmolality result in rapid cell swelling or
shrinking and are accompanied by altered cellular
functioning (Schultz 1989).
For example, the
expression of certain genes is osmosensitive, and the
activating or deactivating effects that changes in cell
volume have on these genes alter the expression of the
proteins for which they encode (Lang & Waldegger
1997). Additionally, some genes cannot tolerate abrupt
changes in cell volume, so such perturbations result in
deleterious effects on the metabolic and enzymatic
activities they control (Kultz 2002). In the most basic
sense, the mechanical strain imposed on a cell from
volume fluctuations affects cell morphology and may
even threaten cellular existence altogether; a cell can
only expand to a given point before bursting or shrink to
a given point before shriveling up in atrophy (McCarty &
O’Neil 1992). Thus, to prevent the potential loss of cell
integrity resulting from cell swelling or shrinkage, it is
crucial that a cell be able to employ specific recovery
mechanisms.
These mechanisms do exist in a wide
number of cell types. Specifically, certain cells are able
to respond to and recover from hypotonic or hypertonic
shock by activating the compensatory processes known
as regulatory volume decrease (RVD) or regulatory
volume increase (RVI) (Chamberlin & Strange 1989).
Regulatory volume increase is the process by which a
cell counteracts cell shrinkage by activating intracellular
processes that increase cell volume through solute and
water uptake. In contrast, RVD is the compensatory
response by which cells decrease their volume through
the loss of solutes and water to recover from exposure
to a dilute extracellular medium and subsequent
swelling (McCarty & O’Neil 1992). The fact that RVI
and RVD allow for cell volume restoration following
instances of severe anisosmotic stress make them
critical parts of the cellular machinery. This explains
why they are such highly conserved mechanisms;
indeed, rudimentary cell volume regulatory processes
occur in most prokaryotic cells (Schultz 1989).
The processes involved in RVD have been
studied extensively in many cell types. In those studied
to date, such as human ciliary epithelial cells (Adorante
& Cala 1995), Necturus red blood cells (Bergeron et al.
1996), Erlich ascites tumor cells (Hoffman et al. 1986),
and Madin-Darby canine kidney cells (Rothstein &
Mack 1992), the RVD mechanism activated by cell
swelling ultimately leads to net K+ and Cl- efflux. This
efflux is essential for volume recovery, as it reverses
the driving force for water flux across the membrane.
The intracellular signaling cascades that result in K+
efflux have also been examined, and some of these
cascades involve protein kinase C (PKC) (McCarty &
O’Neil 1992).
For instance, in Necturus red blood
cells, RVD occurs through a PKC-dependent cascade
that eventually results in the loss of K+ and Cl- from
cells (Light et al. 1998). Similarly, changes in cell
volume and cell volume recovery mechanisms in
astrocytes are PKC-sensitive (Bender et al. 1992).
Additional intracellular messengers have
been shown to play a role in activation of K+-Cl- loss,
such as calmodulin and arachidonic acid. Calmodulin
is a Ca2+ binding protein involved in many biological
114
pathways, including cell volume regulation in certain
cell types (Huang et al. 2001). Specifically, activation of
this cellular messenger is crucial for RVD in astrocytes
(Quesada et al. 1999, Bender et al., 1992), and
Necturus red blood cells depend on calmodulin for a
successful RVD response (Bergeron et al. 1996).
Arachidonic acid metabolites, called eicosanoids, are
also involved in volume recovery mechanisms. For
example, eicosanoids mediate RVD in trout proximal
renal tubules (Kanli & Norderhus 1998). Similarly, a 5lipoxygenase metabolite of arachidonic acid is involved
in regulation of Necturus cell volume (Light et al. 1997).
Clearly, the intracellular processes involved in volume
decrease are often rather complex, with numerous cell
signaling agents working in coordination to activate and
carry out the RVD response.
Despite the necessity of K+-Cl- efflux for cell
volume recovery, large shifts in the intracellular
concentrations of such ions can have destabilizing
effects on cells (Lang & Waldegger 1997). Therefore,
the loss of organic solutes, such as sugars and amino
acids, is also commonly involved in cell volume
regulation (Chamberlin & Strange 1989). For example,
loss of the amino acid taurine has been implicated in
the RVD processes that take place in trout erythrocytes
(Garcia-Romeu et al. 1991, Huang et al. 2001), and in
Ehrlich ascites tumor cells, both taurine and glycine
efflux occur following cell swelling (Hoffman & Lambert
1983). A cell’s ability to use organic osmolytes to
adjust
intracellular
solute
concentration
is
advantageous, as the loss of many of these molecules
does not cause deleterious effects on cell functioning
(Chamberlin & Strange 1989).
This is of key
importance because preventing cell damage from
volume fluctuations must not occur at the cost of ionic
imbalance, as this could also be detrimental to cell
integrity.
Despite what has been learned regarding cell
volume regulatory mechanisms, much is still unknown
concerning the initial steps that lead to activation of
RVD. In particular, the role that Ca2+ plays in RVD
initiation and signal transduction is widely debated.
Calcium is known for the essential role it plays in many
cellular processes, such as muscle contraction
(Martonosi 2000) and neurotransmission (DeLorenzo
1981), but its utility extends much further. For example,
Ca2+ is involved in activities that range from inducing
apoptosis (programmed cell death) (Orrenius et al.
2003) to stimulating sperm motility in carp (Krasznai et
al. 2000).
A role for this signal molecule has also been
identified in the volume regulatory processes of
numerous cell types, including human ciliary epithelial
cells (Adorante & Cala 1995), choroids plexus epithelial
cells (Christensen 1987), Necturus red cells (Light et al.
1999), Madin-Darby canine kidney cells (Rothstein &
Mack 1992), proximal tubules (McCarty & O’Neil 1990),
and rat lacrimal acinar cells (Speake et al. 1998). In
these cell types, Ca2+ plays an important role in
activating intracellular events that culminate in solute
efflux. However, in other biological systems, Ca2+ is not
essential for volume recovery, although it does assist in
achieving an optimal RVD response. This is the case
for rat liver cells (Bear 1990) and cultured astrocytes
(Quesada et al. 1999). In contrast, findings from other
research indicate either an RVD scheme completely
independent of Ca2+ or a mechanism by which a rise in
Ca2+ during cell swelling is a non-crucial
epiphenomenon.
For example, rat cerebellar
astrocytes employ a volume recovery response that
A
B
C
D
2+
Figure 1. A rise in intracellular Ca accompanies hypotonic
shock in salmon red blood cells.
The amount of fluorescence visible in red blood cells loaded
M) and exposed to UV light
with fluorescent dye (fluo-4, 10
µ
was dependent on the extracellular medium the cells were
bathed in. (A) Cells bathed in isosmotic solution failed to
fluoresce. (B) Cells treated with the Ca2+ ionophore A23187
(positive control) in an isosmotic solution displayed
fluorescence. (C) Exposure to hypotonic shock increased the
degree of fluorescence in the red blood cells. (D) When Ca2+
was buffered to 10 nM with EGTA in a hypotonic medium, no
fluorescence was observed. The above fluorescent microscopy
studies were conducted by Light et al. (2005).
lacks Ca2+ involvement (Morales-Mulia et al. 1998), as
do trout proximal renal tubules (Kanli & Norderhus
1998) and trout erythrocytes (Garcia-Romeu et al.
1991). Clearly, whether a dependency on Ca2+ for RVD
exists is controversial and appears to vary among cell
types. Furthermore, for those cell types in which Ca2+
does appear to play a role in volume restoration, the
exact pathway by which it operates has yet to be
determined.
The goal of my research was to help clarify
the inconclusive relationship between Ca2+ and
regulatory volume decrease. To do so, I studied the
effects of Ca2+ manipulation on RVD in both Atlantic
salmon (Salmo salar) and American alligator (Alligator
mississippiensis) erythrocytes, in an effort to determine
whether the signal transduction pathways stimulated by
cell swelling are Ca2+-dependent or Ca2+-independent.
Previous research in our laboratory provides evidence
for potential involvement of Ca2+ in the RVD response
of salmon red blood cells (Light et al. 2005). Using
fluorescent microscopy, Light et al. (2005) tracked
changes in the intracellular Ca2+ of the cells (Figure 1).
They observed that cells bathed in an isosmotic
medium displayed virtually no fluorescence, which was
indicative of low Ca2+ levels under basal conditions.
However, exposure to a dilute extracellular environment
elicited a dramatic increase in fluorescence, which
suggests that salmon cells experience a rise in
intracellular Ca2+ in response to hypotonic shock.
Based on the findings of Light et al. (2005),
my aim was to characterize the role of Ca2+ in RVD
further. Specifically, my study rested on the following
three hypotheses: 1) Cell swelling is accompanied by a
rise in intracellular Ca2+ necessary to stimulate RVD, 2)
the source of this Ca2+ is extracellular, and 3) Ca2+
activates an intracellular event essential for volume
recovery. If RVD does indeed depend on extracellular
Ca2+, then I expected removing this Ca2+ source would
inhibit the volume recovery response. In contrast, if
RVD is Ca2+-independent, then Ca2+ removal should
115
have no effect on the ability of cells to regulate their
volume following hypotonic challenge. Additionally,
blocking Ca2+ entry into cells using Ca2+ transport
antagonists should inhibit RVD if Ca2+ influx is required
to facilitate the volume recovery process. However, if
Ca2+ influx is not an essential step, then no effect
should be observed following inhibition of Ca2+
permeability pathways. Lastly, if the role of Ca2+ is to
stimulate an intracellular signaling cascade crucial for
volume recovery, then blocking the signaling events
occurring downstream of Ca2+ activation should also
result in the failure of cells to respond appropriately to
anisosmotic challenges. But if these Ca2+-activated
signaling events are not involved in RVD, antagonizing
them should not inhibit volume regulation.
I chose to focus on salmon and alligator cells
because the lifestyles of these organisms suggest that
they would be good models for studies on cell volume
recovery. In particular, salmon are euryhaline marine
teleosts, meaning they can tolerate both freshwater and
saltwater environments (Handeland et al. 1996). They
are also anadromous, making multiple migratory trips
between freshwater and saltwater throughout the
course of their lives (Handeland et al. 1996). The fact
that they are able to adapt to such varying degrees of
salinity suggests that their cells, which may come in
contact with the extracellular environment during
transport through the gills, have a finely-tuned
mechanism for coping with cell volume deviations.
Likewise, although alligators are primarily
found in freshwater, they can tolerate abbreviated
episodes in brackish environments (Richards et al.
2004). Additionally, they hibernate during the winter
and can also endure freezing conditions for short
periods of time (Lance & Elsey 1999). Hibernation
results in reduced blood flow to the kidneys and
consequent reduced renal activity (Zancanaro et al.
1999). Kidney cells play a crucial role in adjusting
extracellular fluid osmolarity.
Therefore, abrupt
changes in kidney cell activity as a result of hibernation
could result in cell volume changes. In addition,
exposure of alligators to freezing conditions has a fairly
rapid stimulatory effect on stress hormone production,
which initiates a change in plasma ion concentration
(Lance & Elsey 1999). A more long-term effect of such
exposure includes retardation of protein synthesis
(Lance & Elsey 1999). As stated earlier, such changes
in osmolyte concentrations correspond with a shift in
the osmotic gradient between intracellular and
extracellular environments and, consequently, lead to
either cell swelling or shrinkage (McCarty & O’Neil
1992). Therefore, the ability of alligators to cope with
such challenges is presumably allowed for, at least in
part, by a well-developed cell volume regulatory
mechanism.
In addition, unlike mammalian erythrocytes,
red blood cells from salmon and alligators are
nucleated.
This allows for protein synthesis and
degradation to take place within them (Lang &
Waldegger 1997), activities that alter intracellular
osmolality. This suggests that the red blood cells of
both species may be adapted to deal with shifts in
solute concentration (Lang & Waldegger 1997).
Furthermore, these cells are free floating, so they do
not require a substrate on which to grow. Additionally,
the characteristic pigmentation of these cells makes
osmotic fragility studies possible.
The continuation of research in this field
promotes a better understanding of cell physiology, and
uncovering the underlying processes involved in RVD
has practical applications, as well, including the study of
certain pathophysiological conditions. For example,
apoptosis is the process by which cells marked for
destruction lose water and solutes in a feed-forward
mechanism until they are reduced to a size that can be
easily engulfed by phagocytic cells (Okada & Maeno
2001).
Overactive or dysfunctional apoptosis are
characteristic of diseases including neurodegeneration
and cancer (Okada & Maeno 2001). The obvious
parallels between apoptosis and RVD, mainly that both
proceed in a controlled manner that allows for volume
reduction (Okada & Maeno 2001), suggest that
clarification of the RVD pathway may be useful when
developing methods for either inducing apoptosis in
apoptotically deficient cells or inhibiting it in cells
displaying overactive apoptosis.
Another disease state related to RVD in its
mechanics is ischemia, which is characterized by a lack
of oxygenated blood flow to cells and often
accompanies a blood clot or stroke (Pasantes-Morales
et al. 2000). One of the hallmark events that occurs
during ischemia is
significant cell swelling.
Subsequently, ischemic cells are able to recover from
this swelling by the loss of K+ and organic osmolytes
(Pasantes-Morales et al. 2000). However, the recovery
rarely occurs quickly enough to prevent some amount
of cell damage (Diaz et al. 2003). It has been proposed
that enhancing the rate of cell volume recovery may be
the key to preventing irreversible damage to these cells
(Diaz et al. 2003). To develop a method for quickening
this rate, further research on cell volume recovery
processes is needed.
Finally, while similarities can be recognized
between RVD, apoptosis, and ischemia, other disorders
are largely the direct consequence of insufficient or
faulty cell volume regulation. These include renal
disease, diabetes mellitus, dehydration, and cerebral
edema (McManus et al. 1995). The potential for
developing better techniques for managing these
disease states lies in the ability to elucidate the
pathways involved in the underlying cell volume
regulatory mechanisms.
Experimental Design
The first hypothesis I set out to test was whether
extracellular Ca2+ is necessary for RVD. Presumably, if
extracellular Ca2+ is required for a successful RVD
response, then in its absence cells should fail to
regulate their volume effectively.
However, this
manipulation should have no effect if RVD is Ca2+independent.
To distinguish between these two
possibilities, I carried out calcium influx studies in which
Ca2+ was removed from the extracellular environment
using the extracellular Ca2+ chelator ethylene glycolbis(2-aminoethylether)-N,N,N′,N′-tetraacetic
acid
(EGTA). Additionally, I performed experiments where
plasma membrane permeability to Ca2+ was increased
using A23198, an ionophore that facilitates Ca2+ entry
into cells (Light et al. 2003). I expected this to have the
opposite effect of removing Ca2+. That is, increasing
Ca2+ influx should enhance RVD if Ca2+ plays a role in
stimulating the volume regulatory response.
If the results of the above studies supported
my first hypothesis (i.e., Ca2+ is necessary for RVD and
is extracellular in origin), I next wanted to characterize
the Ca2+ influx pathways activated subsequent to cell
swelling to identify how Ca2+ was entering cells. There
116
are three main entry sites that could facilitate Ca2+
influx:
stretch-activated
channels,
ligand-gated
channels, and voltage-sensitive channels (McCarty &
O’Neil 1992). Stretch-activated channels have been
shown to be a major site for Ca2+ entry in numerous cell
types (McCarty & O’Neil 1992).
For example,
Christensen (1987) concluded that hypotonic shock
stimulates the opening of stretch-activated channels
that are permeable to Ca2+ in the plasma membrane of
choroids plexus epithelia. Also, Hoyer et al. (1994)
named stretch-activated ion channels as the site of
Ca2+ entry following hypotonic exposure of porcine
endocardial endothelia. Ligand-gated cation channels,
such as P2 receptors, also allow significant amounts of
Ca2+ to cross the plasma membrane of many cells,
including red blood cells (Ralevic & Burnstock 1998).
Light et al. (2003), for example, showed P2 receptors as
a likely site for Ca2+ entry into Necturus red blood cells
following hypotonic shock. In contrast to stretchactivated and ligand-gated channels, voltage-sensitive
channels are typically restricted to neurons and muscle
cells (Jones 1998). Accordingly, I decided to first
examine those entry pathways that have been strongly
implicated in solute influx during RVD.
To address the role of stretch-activated
channels in RVD, I used the pharmacological agents
lanthanum and gadolinium. Lanthanum is a broadspectrum Ca2+ channel inhibitor, but it shows some
selectivity toward stretch-activated channels, which
tend to be permeable to Ca2+ (Yang & Sachs 1989).
Gadolinium primarily blocks stretch-activated channels,
but there is some evidence suggesting that this agent
also antagonizes P2 receptors (Nakazawa et al. 1997).
These agents have proven to be potent inhibitors in
many cell volume studies. For example, Adorante &
Cala (1995) and Rothstein & Mack (1992) found that
treating cells with lanthanum has an inhibitory effect on
cell volume recovery following hypotonic shock of
nonpigmented human ciliary epithelial cells and MadinDarby canine kidney cells, respectively. Light et al.
(2003) discovered that Ca2+ entry into Necturus red
blood cells after hypotonic shock occurs through a
gadolinium-sensitive channel, as use of this agent
inhibits volume recovery. Similar RVD inhibition in
response to gadolinium treatment was observed in rat
lacrimal acinar cells by Speake et al. (1998).
Accordingly, I expected volume recovery to be
weakened in the presence of these Ca2+ transport
antagonists if their sites of action correlate with the
calcium entry pathways active during RVD of salmon
and alligator cells, whereas they should have no effect
on volume recovery if they act on influx pathways not
involved in the volume regulatory responses of these
cell types.
To further determine the entry site for Ca2+, I
examined the effects of targeting P2 receptors,
specifically the P2X class, which .are ATP-gated, Ca2+
permeable channels (Nakazawa et al. 1997). To
address whether they are involved in the RVD process,
I utilized the ATP scavenger hexokinase, as
extracellular ATP has been shown to stimulate RVD in
several cell types by activating P2 receptors (Dezaki et
al. 2000, Light et al. 2001, Schwiebert et al. 1995).
Hexokinase can be used to remove this ATP source in
the presence of glucose, thus preventing P2 receptor
activation (Schwiebert et al. 1995). In addition, I
directly inhibited P2 receptors using suramin, an agent
that binds to and inactivates this receptor type (Dezaki
et al. 2000). If P2 receptors play a role in Ca2+ entry
following hypotonic shock, then these agents should
have an attenuating effect on RVD. I also attempted to
potentiate volume recovery by adding ATP to the
extracellular medium. I predicted that if P2 receptors
are indeed involved in volume regulation, then this
addition should have the opposite effect of hexokinase
and suramin, and thus stimulate a decrease in cell
volume.
After studying the processes by which Ca2+
entered cells, I wanted to address my hypothesis that
Ca2+ functions as an intracellular messenger to activate
an event necessary for volume recovery.
I
accomplished this by first determining whether K+ efflux
is required for a decrease in cell volume, and whether
this efflux is Ca2+-dependent, as K+ loss from swollen
cells is a necessary step in RVD of virtually all cell
types that have been studied (Adorante & Cala 1995,
Bergeron et al. 1996, Hoffman et al. 1986, Rothstein &
Mack 1992). To do so, I observed the effects of
This agent
enhancing K+ efflux with gramicidin.
increases plasma membrane permeability to K+ by
forming pores in the cell membrane specific for
monovalent cations, thereby allowing for the movement
of cations either in or out of the cell as dictated by their
electrochemical gradients (Light et al. 1998). In my
studies, I was able to make these pores essentially K+
specific by removing Na+ from the extracellular
environment and replacing it with choline (a large,
impermeant cation that does not readily traverse the
plasma membrane, nor alter RVD). In doing so, I could
assess whether RVD is dependent on K+ efflux, and
whether K+ efflux, in turn, is dependent on Ca2+. If this
is indeed the case, then bypassing the Ca2+-dependent
step (with gramicidin) should reverse the inhibitory
effects of placing cells in a hypotonic, Ca2+-free
solution.
Then, I sought to establish whether Ca2+ acts
as a direct modulator of K+ efflux or, alternatively, if it
plays an indirect role by acting as a second messenger.
To this end, I inhibited Ca2+-activated K+ channels using
quinine, a selective blocker of this channel type
(Hoffman et al. 1986), expecting to observe a reduction
in RVD upon doing so if K+ loss from cells occurred by
this route. I also examined the potential role Ca2+ might
play as an intracellular messenger by targeting different
Ca2+-activated signal transduction pathways that could
ultimately lead to K+ efflux. One pathway I thought
could be involved was calmodulin, which plays a role in
the RVD signaling cascade of the astrocytes studied by
both Quesada et al. (1999) and Bender et al. (1992),
Necturus erythrocytes studied by Bergeron et al.
(1996), and the human erythroleukemia cells studied by
Huang et al. (2001). In these cells, inhibition of
calmodulin resulted in a reduced ability of cells to
regulate their volume while under hypotonic challenge.
I assessed a potential role for calmodulin in volume
recovery using pimozide, an antagonist of calmodulin
activity (Bergeron et al. 1996, Hoffman et al. 1986,
Huang et al. 2001). Hypothetically, if calmodulindependent signaling occurs downstream of Ca2+ and is
required to stimulate volume decrease, then inhibiting
calmodulin should correlate with a reduced RVD
response.
In addition, I targeted the enzyme
phospholipase A2, whose activation results in the
formation of arachidonic acid.
Metabolites of
arachidonic acid, in particular, leukotrienes, have been
implicated in the RVD response in a diverse number of
cell types (Kanli & Norderhus 1998, Light et al. 1998,
Pasantes-Morales et al. 2000). The first step in
determining whether arachidonic acid or its metabolites
117
Agent
Function
Predicted Effect on RVD
EGTA
Extracellular calcium chelator
Inhibition if Ca2+ is necessary for volume recovery
A23187
Calcium ionophore
Enhancement if Ca2+ influx stimulates RVD
Gadolinium
Stretch-activated channel blocker
Inhibition if Ca2+ influx occurs through a SA channel
Lanthanum
Stretch-activated channel blocker
Inhibition if Ca2+ entry occurs through a SA channel
Hexokinase
ATP scavenger
Inhibition if Ca2+ entry occurs through a P2 receptor
Suramin
P2 receptor antagonist
ATP
Natural ligand for P2 receptors
Enhancement if P2 receptor activity facilitates RVD
Gramicidin
Ionophore for monovalent cations
Enhancement of K+ efflux is required for volume recovery
Quinine
Ca2+-activated K+ channel blocker
Inhibition if K+ efflux occurs via a Ca2+-activated K+ channel
Pimozide
Calmodulin antagonist
Inhibition if RVD requires calmodulin activation
ONO-RS-082
Phospholipase A2 inhibitor
Inhibition if volume recovery is PLA2-dependent
2,3 DBAP
Phospholipase A2 inhibitor
ETYA
Antagonist of eicosanoid synthesis
Inhibition if an eicosanoid stimulates RVD
NDGA
Antagonist of leukotriene production
Inhibition if a leukotriene is needed for volume decrease
Table 1. Pharmacological agents used in cell volume studies, their functions, and my predictions as to their effects on regulatory
volume decrease.
are involved in RVD of the cells I studied was to identify
whether RVD is PLA2 dependent, because PLA2 (a
Ca2+ activated enzyme) is responsible for arachidonic
acid formation (Pasantes-Morales et al. 2000). I used
the PLA2 antagonists ONO-RS-082 and 2,4dibromoacetophenone to do this, as these agents have
been shown to be useful PLA2 inhibitors in other RVD
studies (Balsinde et al. 1999, Nakashima et al. 1989).
If arachidonic acid or its metabolites are essential for
volume recovery following hypotonic shock, then I
expected to observe an inhibited volume recovery in
response to preventing arachidonic acid formation.
If my PLA2 inhibition studies implicated
arachidonic acid formation as a necessary event in the
RVD response, my next step would be to determine
whether arachidonic acid itself modulates volume
recovery, or, alternatively, if one of its metabolites
(known as eicosanoids) does. To distinguish between
these two possibilities, I treated cells with the general
eicosanoid antagonist 5,8,11,14-eicosatetraynoic acid
(ETYA).
Additionally, I targeted the lipoxygenase
pathway of arachidonic acid metabolism using
nordihydroguaiaretic acid (NDGA), as this pathway has
been shown to be activated during RVD of several cell
types (Lambert et al. 1987, Light et al. 1997). I
predicted that if arachidonic acid, but not an eicosanoid,
is required for RVD, then inhibiting eicosanoid formation
should not affect the volume recovery response.
However, if an eicosanoid is involved in volume
regulation, then antagonizing the pathways responsible
for forming the metabolite should inhibit the ability of
cells to recover from cell swelling.
Table 1 summarizes the experimental
approach I used to test my hypotheses. Listed are the
various pharmacological agents I used and their modes
of action. Additionally, my predictions as to the effects
of these agents are also given.
Animals
A common pool (3-4 fish) of salmon blood was
purchased from Bioreclamation (Hicksville, NY).
Alligator whole blood was purchased from Carolina
Biological Supply (Burlington, NC). The blood was
stored at 4oC for no longer than one month and was
inverted daily. Visualization of salmon erythrocytes
(Figure 2) was achieved using a Nikon phase contrast
microscope (Fryer Co. Scientific Instruments,
Carpentersville, IL) along with a mounted camera (Spot
Insight Color and Spot software). For visualizing
alligator erythrocytes (Figure 2), a Nikon Eclipse
TE2000-U DIC microscope (Fryer Co. Scientific
Instruments, Carpentersville, IL) was used.
Cell Volume
A Z2™ COULTER COUNTER®
used to electronically size and
(Figure 3, Coulter Electronics,
aperture orifice was 100 µ m
with Channelyzer was
count red blood cells
Fullerton, CA). The
in diameter, and the
metered volume was 0.5 ml. The Coulter counter was
calibrated using latex beads of known size. Median cell
volumes (fl) were obtained from distribution curves
between 50 fl and 1200 fl. The lower limit (50 fl) was
chosen to prevent the counting of thrombocytes, which
are smaller than red blood cells and comprise a
significant portion of total blood volume (Histology of
Blood,
Creighton
University
Health
Sciences
Laboratory, www.hsl.creighton.edu). Although white
blood cells were counted, as they are approximately the
118
A
Figure 3. Z2™ COULTER COUNTER® and aperture.
The Coulter counter electronically sizes and counts cells based
on a change in resistance that occurs as the cells pass through
a narrow aperture opening. Cell volume is proportional to this
change in resistance (figure is courtesy of Beckman Coulter,
Inc., www.beckmancoulter.com).
B
Solutions and Reagents
Isosmotic and hypotonic Ringer solutions were used to
mimic blood plasma environments. The compositions
of the Ringers used in this study were based on those
provided by the Marine Biology Laboratory in Woods
Hole, MA (www.mbl.edu). The osmolality and pH of all
solutions were determined using a Vapor Pressure
Osmometer (Wescor 5500) and an Accumet Basic
AB15 pH meter (Fisher Scientific), respectively.
For salmon, isosmotic high Na+ Ringer
contained (in mM) 178 NaCl, 2.5 KCl, 2.0 CaCl2, 1.8
MgCl2, 8.75 Hepes hemisodium salt, 1.25 Hepes, 5.5
Glucose, and 1.0 NaHCO3. Hypotonic high Na+ Ringer
had the same composition, except the NaCl content
was reduced to 67 mM. Hypotonic low Ca2+ (10 nM
free Ca2+) contained (in mM) 67 NaCl, 2.5 KCl, 0.5
CaCl2, 4 EGTA, 1.5 MgCl2, 10 Hepes, 8 Trizma base,
5.5 Glucose, and 1.0 NaHCO3. The MAXC downloads
website
(http://www.stanford.edu/%7ECpatton/downloads.htm)
was used to calculate the appropriate free Ca2+
concentration.
For alligator, isosmotic high Na+ Ringer
contained (in mM) 140 NaCl, 6 KCl, 6 CaCl2, 1.5 MgCl2,
5.45 Hepes hemisodium salt, 4.55 Hepes, 5.5 Glucose,
and 1.0 NaHCO3. For the hypotonic high Na+ Ringer,
NaCl content was reduced to 70 mM, and for the NaClfree hypotonic Ringer, NaCl was replaced with 70 mM
choline chloride. Hypotonic low Ca2+ (10 nM) Ringer
contained (in mM) 62.5 NaCl, 6 KCl, 0.5 CaCl2, 1.5
MgCl2, 3.8 EGTA, 5.45 Hepes hemisodium salt, 4.55
Hepes, 9.16 Trizma base, 5.5 Glucose, and 1.0
NaHCO3.
All chemicals and pharmacological agents
were acquired from Sigma Chemical Co. (St. Louis,
MO), Axxora LLC (San Diego, CA), and ICN
Biomedicals (Aurora, OH). Aqueous stock solutions
were made at 100X their final concentration and were
then diluted 100X when used to achieve the final
working concentration desired. Nonaqueous stock
solutions (using methanol, ethanol, or DMSO as
vehicles) were made at 1000X their final concentration
and diluted 1000X to their final working concentration
(Light et al. 2001). All experiments were carried out at
room temperature (21-23oC).
Figure 2. Salmon and alligator erythrocytes.
(A) Salmon erythrocytes photographed at 400X. The cells are
approximately 15
m long and 11
m wide (Nash &
µ
µ
Egginton 1993); as a comparison, this is about twice the size of
human red blood cells. They are oval in shape, free-floating,
and, unlike mammalian red cells, they are nucleated. (B)
Alligator erythrocytes photographed at 1000X (taken by Spivak,
Pore, & Silverman, Light Lab). These oval-shaped cells
measure approximately 21
m in length and 11
m in width
µ
µ
(Hartman & Lessler 1964).
nucleated.
They are free-floating and
same size as red blood cells, the proportion of white
cell
to
red
cell
is
typically
1/1000
(www.hsl.creighton.edu); therefore, their contribution
was assumed to be insignificant. Readings were taken
from cell suspensions containing between 20,00040,000 cells over a time course of 90 min. Specifically,
median cell volumes were recorded at 30 sec intervals
from 0 to 3 min, at 5 min, and at 10 min intervals from
10 to 90 min.
Using the volume measurements obtained
from electronic sizing, two different assessments were
made. First, the final relative volume of experimental
cells was compared to control cells, with relative
volume being the absolute cell volume divided by the
volume of untreated cells equilibrated in isosmotic
solution. This was done to determine differences in the
overall degree by which cells reduced in size following
hypotonic challenge. Additionally, the rate of volume
recovery was used to compare the RVD responses of
experimental and control cells. This was done by
comparing the relative volume of experimental and
control cells at both 40 and 90 min to the maximum
relative volume of control cells. To do so, the following
formulas were used (Light et al. 1999): percent volume
recovery at time x is equal to
[ (V
max –
Vx)/(Vmax – V0)
]x
100%. In this equation, Vmax represents maximum
relative control cell volume, Vx represents the relative
cell volume measured at time x, and V0 represents
relative cell volume at time 0 min. Overall percent
volume
decrease
was
determined
recoveryexperimental)/percent recoverycontrol)
as
[ (percent
Statistics
] x 100%.
Cell volume changes were graphically represented
using KaleidaGraph 4.0 (Synergy Software; Reading,
119
recovery by increasing intracellular Ca2+ concentration
using the Ca2+ ionophore A23187 (0.5 µ M, n=6).
PA) and Microsoft Excel (Microsoft Corporation;
Redmond, WA). The statistical significance of the
experimental results (i.e., differences in final cell
volume and percent volume decrease of control cells
compared to experimental cells) was assessed by
performing either paired t-tests or analysis of variance
(ANOVA) and multivariate analysis of variance
(MANOVA). Statistics were computed using Data Desk
Software (Ithaca, NY).
Each experiment was replicated at least five
times. Each maneuver was compared to its own
control, allowing for paired comparisons. A separate
blood sample was used for each replicate experiment.
All samples were taken from several common pools.
Unexpectedly, the ionophore had a pronounced
inhibitory effect on the ability of alligator red blood cells
to regulate their volume following hypotonic shock
(Figure 8A, P<0.001 at 90 min compared to control
cells). Not only did A23187-treated cells fail to recover
their volume, but they continued to swell throughout the
90 min time course (Figure 8B, P<0.001 at 40 min and
P<0.001 at 90 min).
To examine the mechanism for Ca2+ influx
into alligator cells, I used gadolinium (50 µ M, n=7).
Unlike in salmon (Figure 6), this agent had a distinct
inhibitory effect on cell volume recovery (Figure 9A,
P<0.01 at 90 min compared to control cells). That is,
unlike control cells, percent volume recovery of
gadolinium-treated cells was completely inhibited
(Figure 9B, P<0.001 at 40 min and P<0.01 at 90 min).
After observing an inhibitory effect following
gadolinium treatment, I wanted to pinpoint the
gadolinium-sensitive transport pathway involved in Ca2+
influx in response to cell swelling. To this end, I
examined whether P2X receptors were involved in the
alligator RVD process. To accomplish this, I utilized the
ATP scavenger hexokinase (2.5 units/mL H2O, n=6), as
extracellular ATP is known to enhance RVD by
stimulating P2 receptors (Dezaki et al. 2000, Light et al.
2001, Schwiebert et al. 1995). Consistent with my
prediction, this agent had a significant inhibitory effect
on cell volume recovery, as hexokinase-treated cells
were more swollen than control cells at 90 min (Figure
10A, P<0.01). Furthermore, upon hexokinase addition,
percent volume recovery was reduced (Figure 10B,
P<0.01 at 40 min and P<0.001 at 90 min).
To further verify a role for P2 receptors, I
treated cells with suramin (100 µ M, n=5), which
Results
Salmon
Challenging salmon cells with a hypotonic (~0.5x)
extracellular environment caused them to swell rapidly.
Following this initial increase in volume, the cells
gradually and spontaneously recovered toward their
normal size over a 90 minute time course (Figure 4A). I
then bathed cells in a low Ca2+ medium to test whether
There was a
RVD requires extracellular Ca2+.
significant difference in final cell volume of cells bathed
in the low Ca2+ hypotonic Ringer (buffered to 10 nM
with EGTA) compared to that of control cells (Figure
4A, n=5, P<0.001 at 90 min). Additionally, in the low
Ca2+ environment, percent volume recovery was
inhibited both at 40 min and at 90 min (Figure 4B, n=5,
P<0.001).
The next series of experiments were
designed to inhibit Ca2+ influx. Surprisingly, cells
treated with lanthanum (10 µ M, n=5), an inorganic
inhibitor of Ca2+ channels (Rothstein & Mack 1992),
displayed an enhanced volume recovery (Figure 5A,
P<0.001 at 90 min compared to control). The percent
volume recovery was dramatically potentiated at both
40 min and 90 min (Figure 5B, P<0.001 for both 40 and
90 min), and the total percent volume decrease of
lanthanum-treated cells compared to control cells was
188%.
directly inhibits this receptor type (Dezaki et al. 2000,
Light et al. 2001). Cells bathed in a hypotonic solution
containing this agent showed a decreased ability to
regulate their volume compared to control cells (Figure
11A, p<0.05 at 90 min). Furthermore, percent volume
recovery of experimental cells was also limited in
comparison to control cell percent volume recovery
(Figure 11B, P<0.01 at both 40 and 90 min).
After successfully inhibiting the RVD
response of alligator cells using antagonists of P2
receptor activation, I attempted to potentiate volume
recovery using an agonist of this receptor type. I
provided cells with an exogenous source of ATP
(100 µ M, n=6), as this has been shown to stimulate P2
Alligator
Alligator erythrocytes swelled rapidly in response to
hypotonic challenge (Figure 7A). The initial swelling
was followed by a subtle volume recovery. However, in
a hypotonic, low-Ca2+ extracellular medium, volume
decrease was completely inhibited (Figure 7A, n=7,
P<0.001 at 90 min compared to control cells).
Specifically, in the absence of extracellular Ca2+,
percent volume recovery was abolished (Figure 7B,
n=7, P<0.01 at 40 and 90 min). Upon examining
whether the cationophore gramicidin would influence
RVD by enhancing K+ efflux, I found that addition of
this agent (0.5 µ M added at peak cell volume, n=5) to
receptor-dependent RVD responses (Dezaki et al.
2000, Schwiebert et al. 1995). Although I predicted this
would enhance RVD, the degree by which cells were
able to regulate their volume was unaffected by
increasing their exogenous ATP source. Specifically,
volume recovery of ATP-treated cells matched that of
control cells (Figure 12A), as did the percent volume
recovery at both 40 and 90 min (Figure 12B).
One potential method by which Ca2+ could
stimulate volume recovery would be to directly activate
K+ efflux. To assess this possibility, I used quinine (1
mM, n=5), a Ca2+-activated K+ channel antagonist
(Bergeron et al. 1996, Hoffman et al. 1986). Quinine
had no significant effect on the RVD response of
alligator red blood cells (Figure 13A). Specifically, there
was no difference in the overall volume recovery of
a low Ca2+ choline Ringer reversed EGTA-mediated
RVD inhibition.
In fact, despite the lack of an
extracellular Ca2+ source, in the presence of gramicidin,
final cell volume recovery exceeded that of control cells
(Figure 7A, n=5, P<0.05 at 90 min), as did percent
volume recovery at 90 min (Figure 7B, n=5, P<0.05).
This I attempted to address my hypothesis that
facilitating Ca2+ influx should potentiate volume
120
A
B
3
50
Percent Volume Recovery
Relative Volume
C o n t ro l
EG TA
40
2 .5
2
1 .5
C o ntrol
EGTA
1
30
20
10
0
-1 0
0 .5
0
20
40
60
80
10 0
40
T im e ( m in )
90
T im e ( m in )
2+
Figure 4. Cells failed to regulate their volume in the absence of extracellular Ca .
(A) At time 0, cells were exposed to hypotonic shock, which resulted in a rapid rise in cell volume. Following this initial swelling, control
2+
cells recovered toward their normal size over a 90 min time course. In contrast, cells in a low Ca medium remained swollen (n=5,
P<0.001 at 90 min compared to control). (B) Percent volume decrease was significantly inhibited in the absence of extracellular Ca2+, both
at 40 min (n=5, P<0.001 compared to control at 40 min) and at 90 min (n=5, P<0.001 compared to control at 90 min). Cells swelled in both
instances, as indicated by the negative percent volume recoveries of -5% (40 min) and -8% (90 min) compared to the control cells’ percent
volume recoveries of 31% (40 min) and 50% (90 min). Values represent mean + standard error.
B
3
100
2 .5
80
Relative Volume
A
2
1 .5
1
Control
Lanthanum
60
40
20
C o n tro l
L a n th a n u m
0
0 .5
0
20
40
60
80
40
100
90
Time (min)
T im e (m in )
Figure 5. Lanthanum treatment potentiated cell volume recovery.
(A) At time 0, cells were exposed to hypotonic shock. Lanthanum-treated cells expressed a substantially enhanced cell volume recovery
compared to control cells, both in terms of rate and final cell volume (n=5, P<0.001 at 90 min). (B) Control cells decreased in volume by
30% and 49% at 40 min and 90 min, respectively. However, cells treated with lanthanum had remarkably recovered by 87% and 92% at 40
min and 90 min, respectively (n=5, P<0.001 for both 40 and 90 minutes). Values represent mean + standard error.
A
B
3
100
C o n tro l
G a d o lin iu m
Relative Volume
2 .5
2
1 .5
1
80
60
40
20
C o n tr o l
G a d o lin iu m
0 .5
0
0
20
40
60
80
10 0
40
T im e (m in )
T im e ( m i n )
90
Figure 6. Treating cells with gadolinium enhanced cell volume recovery after exposure to a hypotonic medium.
(A) Gadolinium-treated cells had more successfully regulated their volume after 90 minutes when compared to control cells (n=5, P<0.05
at 90 min). (B) The rate of volume recovery of gadolinium-treated cells was elevated to 73% at 40 min and 83% at 90 min compared to
control values of 32% at 40 min and 47% at 90 min (n=5, P<0.001 at both 40 and 90 min). Values represent mean + standard error.
121
A
B
1 .8
C o n tr o l
EG TA
E G T A + G ra m ic id in
40
Relative Volume
1 .6
1 .4
1 .2
C o n tr o l
EGTA
E G T A + G r a m ic id in
1
30
20
10
0
-10
-20
0
20
40
60
80
10 0
40
90
T im e (m in )
T im e ( m in )
Figure 7. Removing extracellular Ca2+ inhibited volume recovery, and gramicidin reversed this inhibition.
(A) At time 0, cells were challenged with a hypotonic medium. RVD was inhibited by the removal of Ca2+ from the extracellular medium
(n=7, P<0.001 at 90 min compared to control). However, with the addition of gramicidin (added at 30 sec, n=5), volume recovery exceeded
that of control cells (P<0.01 at 90 min compared to control), even in the absence of extracellular Ca2+ (B) Percent volume recovery was 13% at 40 min and -16% at 90 min in EGTA-treated cells compared to control cell percent volume recovery of 18% at 40 and 90 min (n=5,
P<0.01 at 40 and 90 min). Percent volume recovery of gramicidin-treated cells (n=5) was enhanced to 36% compared to control cell
percent volume recovery of 18% at 90 min (P<0.05 at 90 min). Values are mean + standard error.
A
B
1 .7
1 .4
1 .3
1 .2
1 .1
C o n tro l
A 23187
1
10
1 .5
0
-10
-20
-30
-40
-50
0 .9
0
20
40
60
80
100
40
T im e (m in )
90
Time (min)
Figure 8. A23187 inhibited recovery following hypotonic shock in alligator cells.
(A) Control cells gradually recovered from cell swelling, whereas A23187-treated cells continued to increase in volume over the 90 min time
course studied (n=6, P<0.001 at 90 min compared to control). (B) Following hypotonic shock, control cells recovered their volume by 13%
and 18% at 40 and 90 min, respectively. In contrast, A23187-treated cells displayed a negative percent volume recovery of -42% at 40 min
(n=6, P<0.001 compared to control) and -48% at 90 min (n=6, P<0.001 compared to control). Values are mean + standard error.
A
B
25
C o n tr o l
G a d o lin iu m
1 .6
1 .5
1 .4
1 .3
1 .2
1 .1
C o n tro l
G a d o l in i u m
1
20
Relative Volume
Relative Volume
Control
A23187
20
1 .6
15
10
5
0
-5
-10
0 .9
-15
0
20
40
60
80
100
40
90
T im e ( m in )
T im e ( m in )
Figure 9. Gadolinium inhibited regulatory volume decrease of alligator red blood cells.
(A) Unlike control cells, which slowly recovered toward steady state cell volume following hypotonic shock, recovery was abolished in
gadolinium-treated cells (n=7, P<0.01 at 90 min compared to control cells). (B) Alligator red cells exposed to gadolinium showed a percent
volume recovery of -3% and -9% at 40 and 90 min respectively. This was in comparison to control cell percent volume recovery of 18% at
the corresponding times (n=7, P<0.001 at 40 min and P<0.01 at 90 min). Values are means + standard error.
122
A
B
20
1.5
Control
Hexokinase
Relative Volume
1.4
1.3
1.2
1.1
Control
Hexokinase
1
15
10
0.9
0
20
40
60
80
5
0
100
40
90
Time (min)
Time (min)
Figure 10. Cell volume recovery was attenuated in the presence of hexokinase.
(A) Hexokinase-treated cells remained more swollen throughout the 90 min time course than control cells (n=6, P<0.01 at 90 min
compared to control). (B) Percent volume recovery of hexokinase-treated cells was limited to 2% at both 40 and 90 min. This was in
contrast to control cells, which showed a percent volume recovery of 13% and 15% at 40 and 90 min, respectively (n=6, P<0.01 at 40 min
and P<0.001 at 90 min compared to control). Values are mean + standard error.
A
B
25
1.6
Control
Suramin
1.5
Relative Volume
1.4
1.3
1.2
1.1
Control
Suramin
1
20
15
10
5
0
0.9
0
20
40
60
80
40
100
Time (min)
90
Time (min)
Figure 11. Suramin reduced alligator cell volume recovery.
(A) Addition of suramin to the extracellular medium limited the ability of cells to reduce in volume over the 90 min time course (n=5, P<0.05
at 90 min compared to control). (B) In the presence of suramin, percent volume recovery was reduced to 6% at 40 min and 9% at 90 min,
in comparison to control cell percent volume recovery of 16% and 19% at the corresponding times (n=6, P<0.01 at 40 min and 90 min).
Values are mean + standard error.
A
B
1.6
20
Control
ATP
Relative Volume
1.5
1.4
1.3
1.2
1.1
Control
ATP
1
0.9
0
20
40
60
80
15
10
5
0
100
40
Time (min)
90
Time (min)
Figure 12. Addition of ATP to the extracellular medium had no effect on regulatory volume decrease.
(A) Cells bathed in a hypotonic medium containing ATP showed a volume recovery response that paralleled the response of control cells
(n=6). (B) Percent volume recovery of ATP-treated cells was equivalent to control cell percent volume recovery at both 40 and 90 min
(n=6). Values are mean + standard error.
123
A
B
1.5
25
C o ntrol
Q uinine
Relative Volume
1.4
1.3
1.2
1.1
C o ntrol
Q u in ine
1
20
15
10
5
0.9
0
0
20
40
60
80
10 0
40
90
T im e (m in )
T im e (m in)
Figure 13. Quinine failed to inhibit volume recovery of alligator cells.
(A) After exposure to hypotonic shock, there was no significant difference in the overall volume recovery of quinine-treated cells when
compared to control cells (n=5). (B) Quinine-treated cells and control cells displayed similar percent volume recoveries, both at 40 and 90
min (n=5). Values are means + standard error.
A
B
25
1 .5
C o n tro l
P im o z id e
1 .3
1 .2
1 .1
C o n tro l
P im oz id e
1
20
Relative Volume
1 .4
15
10
5
0 .9
0
20
40
60
80
0
100
40
90
T im e (m in)
T im e (m in)
Figure 14. Pimozide-treated cells and control cells responded similarly to hypotonic challenge. (A) There was no significant
difference between control cell volume recovery and cell volume recovery of pimozide-treated cells (n=5). (B) There was no difference
between the percent volume recovery of control and pimozide-treated cells at either 40 min or 90 min (n=5). Values are means + standard
error.
A
B
1 .6
25
20
Relative Volume
1 .5
1 .4
1 .3
1 .2
1 .1
C o n tro l
1
C o n tro l
ONO
15
10
5
0
-5
-1 0
ONO
0 .9
-1 5
0
20
40
60
80
100
40
90
T im e (m in )
T im e (m in)
Figure 15. ONO-RS-082 abolished the RVD response of alligator cells.
(A) Control cells gradually recovered toward resting cell volume following hypotonic shock, but cells treated with ONO showed no volume
recovery response and instead continued to swell over the 90 min time course (n=5, P<0.001 at 90 min compared to control cells). (B)
Upon ONO treatment, a reduction in alligator cell percent volume recovery to -2% and -8% was observed at 40 and 90 min, respectively.
This was in contrast to control cell percent volume recovery of 16% and 17% at the corresponding times (n=5, P<0.01 at 40 min and
P<0.001 at 90 min). Values are mean + standard error.
124
A
B
1.6
25
Control
DBAP
Relative Volume
1.5
1.4
1.3
1.2
1.1
Control
DBAP
20
15
10
5
1
0.9
0
0
20
40
60
80
100
40
90
Time (min)
Time (min)
Figure 16. DHAP attenuated percent volume recovery of alligator cells. (A) There was no significant difference in the final volume of
control cells and DHAP-treated cells. (B) Addition of DHAP reduced percent volume recovery to 11% and 12% at 40 and 90 min,
respectively. This was in contrast to control cells, which recovered by 16% and 19% at the corresponding times (n=6, P<0.05 at 40 min
and 90 min). Values are mean + standard error.
A
B
1.5
1.4
Relative Volume
Control
ETYA
25
1.3
1.2
1.1
Control
ETYA
1
0.9
0
20
40
60
80
20
15
10
5
0
100
40
Time (min)
90
Time (min)
Figure 17. ETYA had no effect on cell volume recovery. (A) There was no difference in the volume of ETYA-treated cells and control
cells at 90 min (n=6). (B) The percent volume recovery of ETYA-treated cell matched that of control cells (n=6). Values are mean +
standard error.
B
35
1.4
30
Relative Volume
A
1.5
1.3
1.2
1.1
Control
NDGA
1
20
40
60
80
25
20
15
10
5
0.9
0
Control
NDGA
0
100
40
Time (min)
Time (min)
90
Figure 18. NDGA-treated cells displayed a potentiated volume recovery response following hypotonic challenge.
(A) Cells exposed to NDGA were slightly more successful in regulating their volume than control cells, as reflected in their smaller final
volume (n=6, P<0.05 at 90 min compared to control cells). (B) The percent volume recovery of control cells and NDGA-treated cells was
similar at 40 min (approximately 16%). However, the percent volume recovery of 30% experienced by NDGA-treated cells at 90 min
surpassed the 17% volume recovery of control cells at this time (n=6, P<0.01). Values represent mean + standard error.
125
Agent
Prediction
Observed Effect on RVD
EGTA
Inhibition if Ca2+ is necessary for volume recovery
Inhibition in salmon
Inhibition in alligator
A23187
Enhancement if Ca2+ influx stimulates RVD
Gadolinium
Inhibition if Ca2+ influx occurs through a SA channel
Enhancement in salmon
Lanthanum
Inhibition if Ca2+ entry occurs through a SA channel
Enhancement in salmon
Hexokinase
2+
Suramin
Inhibition if Ca entry occurs through a P2 receptor
ATP
Enhancement if P2 receptor activity facilitates RVD
No effect in alligator
+
Gramicidin
Enhancement of K efflux is required for volume recovery
Reversed inhibition in alligator
Quinine
Inhibition if K+ efflux occurs via a Ca2+-activated K+ channel
Pimozide
Inhibition if RVD requires calmodulin activation
ONO-RS-082
2,3 DBAP
ETYA
Inhibition if an eicosanoid stimulates RVD
NDGA
Inhibition if a leukotriene is needed for volume decrease
Enhancement in alligator
Table 2. Pharmacological agents used in cell volume studies, my predictions as to their effects, and the experimental outcomes
of their usage.
quinine-treated cells in comparison to control cells
(Figure 13A). Likewise, the percent volume recovery of
cells was unchanged by the addition of quinine (Figure
13B).
I then turned my attention to the various Ca2+mediated intracellular signaling cascades that might be
initiated during RVD. I used pimozide (10 µ M, n=5), a
n=6), a PLA2 inhibitor (Balsinde et al. 1999). Although
this agent’s effect on the relative volume of alligator
cells at 90 min fell shy of statistical significance (Figure
16A), it did significantly reduce the percent volume
recovery at both 40 and 90 min (Figure 16B, P<0.05
compared to control cells).
Arachidonic acid could act directly on ion
channels or could be metabolized further into
eicosanoids, which in turn can activate K+ channels. To
determine whether it was arachidonic acid or an
eicosanoid stimulating K+ efflux in alligator cells, I used
5,8,11,14-eicosatetraynoic acid (10 µ M, n=6), an
blocker of calmodulin activity (Bergeron et al. 1996,
Hoffman et al. 1986, Huang et al. 2001), to address the
possibility of the involvement in this Ca2+ binding protein
in alligator RVD. Pimozide had no significant effect on
alligator cell volume regulation, as the RVD response of
pimozide-treated cells paralleled that of control cells
(Figure 14A). There was no difference in percent
volume recovery of pimozide-treated cells compared to
unmanipulated control cells, either (Figure 14B).
Having ruled out calmodulin, I next targeted
arachidonic acid metabolism because this also is a
2+
Ca -activated system. I blocked arachidonic acid
production using ONO-RS-082 (10 µ M, n=5). This
agent that blocks the production of all eicosanoids (Lehr
& Griessbach 2000). This antagonist had no effect on
RVD. That is, the final cell volume of ETYA-treated
cells was not significantly different to that of control
cells (Figure 17A). Likewise, no difference in percent
volume recovery was apparent at either 40 or 90 min
(Figure 17B), suggesting eicosanoid formation is not
necessary for volume recovery.
Nonetheless, I still examined the role of the
lipoxygenase pathway of arachidonic acid metabolism
to ensure that the lack of effect following ETYA
treatment was not due to this agent’s relatively low
potency. I treated cells with nordihydroguaiaretic acid
(10 µ M, n=6), an effective inhibitor of the lipoxygenase
agent is a potent inhibitor of phospholipase A2 (Light et
al. 1998, Nakashima et al. 1989), a Ca2+-activated
enzyme that functions to release arachidonic acid from
the phospholipid bilayer (Balsinde et al. 1999). Treating
cells with ONO abolished their ability to regulate their
volume (Figure 15A, P<0.001 at 90 min compared to
control cells). In fact, ONO-treated cells continued to
swell over the time course studied (Figure 15B, P<0.01
at 40 min and 90 min).
The results of my ONO studies implicated
PLA2 involvement in alligator RVD, as inhibiting this
enzyme resulted in the failed ability of cells to reduce in
volume following cell swelling. To further confirm a role
for PLA2, I used 2,4-dibromoacetophenone (5 µ M,
pathway (Lambert et al. 1987). This agent had a
modest potentiating effect on alligator RVD.
Specifically, cells treated with NDGA showed a more
extensive volume recovery, indicated by their reduced
final cell volume compared to control cells (Figure 18A,
P<0.05 at 90 min compared to control). Additionally,
the rate of volume recovery was potentiated in NDGAtreated cells, which showed a final percent volume
126
Na+ ??
Ca2+
RVD
Hypotonic shock
Cell at resting
volume
Swollen cell
Recovered cell
Figure 19. Proposed pathway for volume recovery of salmon red blood cells.
Hypotonic challenge leads to a rise in intracellular Ca2+ as a result of influx from the extracellular
2+
medium by way of a gadolinium- and lanthanum-insensitive transport pathway. The rise in Ca
activates the RVD response, allowing for volume recovery. Additionally, Na+ influx at the onset of
RVD might attenuate volume decrease.
recovery exceeding that of control cells (Figure 18B,
P<0.01 at 90 min).
The results of my cell volume studies are
summarized in Table 2. Listed are the various agents I
used in my experiments, the predictions I made as to
the outcomes of my studies, and my actual
experimental findings.
kidney cells is due to influx from the extracellular
environment as opposed to release from intracellular
stores. They found that use of EGTA to form a low
Ca2+ hypotonic medium inhibits this increase. The
results of McCarty & O’Neil (1990) further corroborate
this finding; they failed to observe a rise in cytosolic
Ca2+ upon removal of Ca2+ from the hypotonic medium
in proximal tubules. However, in other cell types, the
rise in Ca2+ concentration appears to be the result of
both influx from the extracellular medium as well as
release from the intracellular medium. This is the case,
for example, in rat cultured suspended cerebellar
astrocytes (Morales-Mulia et al. 1998). Additionally, it
should be noted that hypotonic shock does not
invariably cause a rise in intracellular Ca2+. For
example, Kanli & Norderhus (1998), who tracked
intracellular Ca2+ levels using epifluorescence,
observed no change in the degree of fluorescence upon
hypotonic stimulation of trout proximal renal tubules.
Clearly, the specific events that occur as a result of
changing extracellular osmolality vary as a function of
cell type.
Although my results suggest that extracellular
Ca2+ is the primary activator of the RVD response, more
complex mechanisms than the one I proposed above
could be responsible for the volume recovery
mechanism employed by salmon cells. For example,
extracellular Ca2+ might function to stimulate release of
Ca2+ from intracellular storages, and the release of this
Ca2+ source, in turn, might then be responsible for
activation of downstream RVD processes (McCarty &
O’Neil 1992). If such a sequence of events was
responsible for volume recovery, then removal of
extracellular Ca2+ would indeed inhibit this process, as
the extracellular Ca2+-dependent event occurs
upstream from that of intracellular Ca2+ release. Such a
cascade has been demonstrated by Tinel et al. (2002)
using rabbit TALH cells. In this cell type, Ca2+ release
from intracellular stores is dependent on Ca2+ influx
from the extracellular medium. When Ca2+ influx and, in
turn, Ca2+ release, was blocked using a low Ca2+
Ringer, cells were unable to recover from hypotonic
challenge. Future studies could address this possibility
in salmon red blood cells by exposing the cells to
pharmacological agents that deplete intracellular Ca2+
stores, such as thapsigargin, ryanodine, or caffeine
(Morales-Mulia et al. 1998, Quesada et al. 1999, Tinel
et al. 2002). If the primary role of extracellular Ca2+ is to
stimulate Ca2+ release, then use of these agents prior to
hypotonic exposure should inhibit RVD, even in the
presence of extracellular Ca2+.
Discussion
A role for Ca2+ in salmon RVD
Cell volume recovery
extracellular Ca2+
was
dependent
on
The findings of my study are consistent with a role for
Ca2+ in regulatory volume decrease. That is, in a low
Ca2+ environment, salmon red blood cells failed to
recover from the rapid swelling that accompanied
hypotonic shock (Figure 4). Furthermore, the Ca2+
source needed for the RVD response appears to be
extracellular in origin, because if intracellular Ca2+ was
the main source involved, then removal of extracellular
Ca2+ should not have inhibited volume recovery. And if
both intracellular and extracellular Ca2+ played a role in
RVD, then even in the absence of an extracellular Ca2+
source, cells might have experienced a partial volume
recovery.
Therefore, my results indicate that
extracellular Ca2+ plays the critical role in activating the
RVD transduction pathway, thus supporting my original
hypothesis.
This finding is in agreement with the
microscopy studies of Light et al. (2005). Specifically, if
the conclusion that extracellular Ca2+ is needed for
volume regulation is correct, then one would expect a
rise in intracellular Ca2+ levels to follow hypotonic
exposure in order to activate the RVD response. Light
et al. (2005) showed that an elevation in Ca2+
concentration does indeed occur (Figure 1).
Additionally, they assessed the source of this Ca2+ by
bathing salmon cells in a hypotonic, low Ca2+-EGTA
Ringer. In this environment, fluorescence was virtually
abolished.
This reinforces my suggestion that
hypotonic shock is followed by Ca2+ influx from the
extracellular medium as opposed to Ca2+ release from
intracellular storages.
The conclusion that Ca2+ influx plays the
predominant role in elevating intracellular Ca2+ levels is
shared by Rothstein & Mack (1992), who determined
that the increasing cytosolic Ca2+ concentration
accompanying hypotonic shock of Madin-Darby canine
127
Extracellular Ca2+ influx did not occur through
lanthanum- or gadolinium-sensitive transport
pathways
observed in the gadolinium and lanthanum
experiments.
Finally, future studies are needed to
determine what pathways are responsible for the Ca2+
influx essential for cell volume regulation by salmon red
blood cells. The findings of this study do not support
Ca2+ entry through stretch-activated channels, as
gadolinium and lanthanum failed to inhibit volume
recovery. Because gadolinium has also been shown to
block P2 receptors (Nakazawa et al. 1997), this entry
site does not seem likely, either. This is consistent with
the findings of Light et al. (2005), who examined the
potential role for P2 receptors in salmon RVD. They
performed experiments using the ATP-scavenger,
hexokinase, which inhibits P2 receptor activation. They
found this agent to have no significant effect on volume
recovery, which indicates a P2 receptor-independent
RVD response. Considering that the specific Ca2+ entry
site has yet to be determined for this cell type, future
experiments using additional Ca2+ transport antagonists
are required.
Both lanthanum and gadolinium had similar, and
unexpected, effects on the RVD response of salmon
erythrocytes. Specifically, these agents dramatically
enhanced the rate of cell volume recovery (Figures 5 &
6), which was contrary to my original prediction and the
findings of other researchers (Adorante & Cala 1995,
Rothstein & Mack 1992, Light et al. 2003).
Furthermore, there is no clear explanation as to why
these agents would potentiate volume decrease. One
possibility, however, is that gadolinium and lanthanum
had non-specific effects that caused the blockage of
Na+ influx. To explain, both the fluorescent studies
(Light et al. 2005) and EGTA studies show
unequivocally that RVD in salmon cells is Ca2+dependent. Therefore, it is unlikely that lanthanum and
gadolinium blocked Ca2+ entry pathways, for if they did,
cells would not have been able to respond appropriately
to hypotonic challenge. However, if these agents
instead prevented Na+ entry into the cells, the result
could be an enhanced volume recovery. For instance,
Garcia-Romeu et al. (1991) showed that Na+ influx at
the onset of RVD attenuates volume recovery in
rainbow trout erythrocytes.
Although few studies
identify a weakening effect of Na+ on the RVD response
as proposed here, the fact that rainbow trout is a close
relative to Atlantic salmon suggests that RVD in salmon
might share similar characteristics to the RVD process
observed by Garcia-Romeu at al. (1991). Furthermore,
there is evidence that lanthanides can be somewhat
non-selective and block the influx of other cations
besides Ca2+, including Na+ (Caldwell et al. 1998).
Taking the limitations of these inhibitors’ specificities
into account and my observation, the suggestion that
they might block Na+ influx as opposed to Ca2+ influx in
this cell type is not unreasonable.
Furthermore, Na+ influx might play an
important role in buffering the cell volume recovery
response of salmon erythrocytes, because if lanthanum
and gadolinium did inhibit Na+ influx, the result was a
strikingly rapid RVD response compared to control
cells. In this sense, Na+ entry might serve to ensure
that cells do not decrease in volume too quickly or too
much; just as cell swelling can have devastating effects,
a reduction in cell volume beyond a critical point is
equally threatening to cell integrity. Another possibility
is that Na+ influx is a concomitant occurrence. That is,
it might serve no purpose in the overall RVD scheme,
but at the same time, it might not be preventable. For
example, if cell swelling initiates the opening of nonselective stretch activated cation channels, then these
permeability pathways would inevitably allow for Na+
entry.
Due to the unavailability of blood, I was
unable to further study the salmon RVD response.
Accordingly, future studies are needed to better
pinpoint the events activated subsequent to cell
swelling. To assess more directly whether Na+ entry at
the onset of RVD was indeed responsible for the
observed effects of lanthanum and gadolinium, studies
could be performed to examine the outcome of
removing Na+ from the extracellular environment and
replacing it with an impermeant cation such as Nmethyl-D-glucamine (NMDG). If the proposed scenario
outlined above is indeed what is occurring in this cell
type, then inhibiting Na+ entry directly should also have
an enhancing effect on RVD that mimics the effect
Summary
Taken collectively, my findings suggest that swelling of
salmon erythrocytes triggers an influx of Ca2+ and the
subsequent rise in intracellular Ca2+ levels. This, in
turn, plays a role in activating the volume regulatory
response (Figure 19).
RVD in alligator erythrocytes is Ca2+-dependent
Extracellular Ca2+ was necessary for volume
recovery of alligator red blood cells
Similar to my results from salmon (Figure 4), alligator
cells placed in a hypotonic medium lacking Ca2+ failed
to recover toward steady state cell volume (Figure 7).
This finding is consistent with Ca2+ playing a primary
role in alligator volume recovery, which is not
surprising; as mentioned previously, such a Ca2+dependent RVD response is common among numerous
cell types (Christensen 1987, Hoffman et al. 1986, Light
et al. 1998, Rothstein & Mack 1992). This most likely
reflects the ubiquitous nature of this signal molecule.
Taking into account the Ca2+-dependent
nature of alligator RVD, I hypothesized that increasing
plasma membrane permeability to Ca2+, which allows
for increased Ca2+ influx, should enhance the RVD
process. However, the results of my studies using the
calcium ionophore A23187 contradicted this prediction.
Unexpectedly, this agent dramatically inhibited volume
recovery following hypotonic shock (Figure 8).
Therefore, it appears that in this cell type, a subtle rise
in intracellular Ca2+ might be required to activate RVD,
whereas a dramatic rise might have the opposite effect.
The lack of RVD rate enhancement upon large
increases in intracellular Ca2+ is not restricted to
alligator erythrocytes (McCarty and O’Neil 1992). For
instance, Montrose-Rafizadeh & Guggino (1991) found
that in rabbit medullary thick ascending limb cells, the
rate of RVD is proportional to intracellular Ca2+
concentration only when this concentration is lower
than basal Ca2+ levels. They concluded that the resting
Ca2+ concentration in this cell type is sufficient to fully
maximize the RVD response, which explains why an
increase in Ca2+ cannot further enhance the rate of
volume recovery. Beck et al. (1991) also found that no
rise in intracellular Ca2+ is necessary for RVD in rabbit
proximal convoluted tubules. Although Ca2+ appears to
128
Ca2+ influx occurred through gadolinium-sensitive
ion channels
be involved in the RVD process in this cell type (as
removing Ca2+ attenuates volume recovery), the
observed increase in intracellular Ca2+ following
addition of A23187 did not result in RVD rate
enhancement.
Similarly, it might be that alligator cells have a
low Ca2+ threshold such that only a slight increase in
2+
Ca is needed to fully saturate the Ca2+-stimulated
volume recovery response. However, many cell types,
including Necturus erythrocytes (Light et al. 2003),
dissociated epithelial cells (Rothstein & Mack 1990),
and proximal tubules (McCarty & O’Neil 1990) do not
display such a low Ca2+ threshold. In these cell types,
fairly large increases in cytosolic Ca2+ (achieved by
A23187 addition) do, indeed, potentiate volume
recovery. The dissimilarity in Ca2+ sensitivity among
cell types might be due to differences in the cellular
machinery involved in their RVD processes or
differences in the specific role played by Ca2+ in their
volume recovery mechanisms.
The fact that alligator cells responded in an
inhibitory fashion to A23187, as opposed to showing no
response, was rather puzzling.
This observation is
best explained as a concentration effect—small
increases in Ca2+ levels stimulate RVD, whereas
pharmacological increases inhibit the volume recovery
process. Additionally, A23187 might have a permissive
effect on both Ca2+ and Na+ entry into cells (Escobales
& Canessa 1985). And sodium influx, in turn, could
result in additional cell swelling (Garcia-Romeu at al.
1991). Because some researchers suggest that cell
swelling itself plays a role in inactivating certain aspects
of the RVD process (McCarty & O’Neil 1990), excessive
Na+ entry might prematurely arrest the volume recovery
process in alligator cells, accounting for the dramatic
inhibition of RVD following A23187 treatment.
However, experimental evidence must be obtained to
draw more definitive conclusions. Specifically, studies
in which the concentration of A23187 used or the
concentration of Ca2+ present in the extracellular
medium is adjusted would be useful. By observing the
effects of such manipulations on volume recovery,
insight into the effects of A23187 on alligator RVD
might be gained.
Consistent with my original hypothesis, a gadoliniumsensitive channel appears to account for the Ca2+ influx
needed to activate the RVD response in alligator cells,
because gadolinium-treated cells showed a complete
inability to regulate their volume (figure 9).
The
response of alligator cells to this antagonist is not
unique, for similar results have been obtained from
studies on rat lacrimal acinar cells (Speake et al. 1998)
as well as on Necturus erythrocytes (Light et al. 2003).
These similarities suggest that some continuity exists in
terms of calcium entry pathways.
However,
interestingly, the membrane transport pathway active in
alligator RVD appears to be fundamentally different to
that stimulated in response to salmon cell swelling
(figure 6). That is, salmon cells displayed the complete
opposite response to gadolinium treatment.
In addition, it seems that the gadoliniumsensitive transport pathway involved in Ca2+ entry into
alligator cells is, at least in part, a P2 receptor in
contrast to a stretch-activated channel. Evidence for
this can be seen in figures 10 and 11, which reveal that
inhibition of alligator RVD occurred following addition of
the ATP-scavenger hexokinase and the P2 receptor
antagonist suramin. Thus, a role for P2 receptors in the
volume recovery response of alligator cells is supported
by this study. Similarly, in Necturus red blood cells,
RVD is dependent on P2 receptor activation, which is
followed by the increase in cytosolic Ca2+ required to
stimulate RVD (Light et al. 1999). Dezaki et al. (2000)
also showed that in a human epithelial cell line,
hypotonic shock triggers the release of ATP into the
extracellular medium, the subsequent activation of P2
receptors, and an increase in intracellular Ca2+. The
fact that the findings of Light et al. (1999) and Dezaki et
al. (2000) are consistent with this study strengthens the
notion of a P2 receptor-mediated RVD mechanism.
However, unlike Necturus erythrocytes (Light
et al.1999) and human epithelial cells (Dezaki et al.
2000), it appears that alligator cells release sufficient
ATP at the onset of the RVD response to fully saturate
their P2 receptors. Whereas ATP addition to the
extracellular medium enhanced volume recovery in
Necturus and human epithelial cells, no effect was
observed upon ATP addition to alligator cells (Figure
12). Therefore, the most logical conclusion, based on
the fact that P2 receptors do appear to be necessary in
alligator RVD, is that they are maximally active under
normal conditions. In future studies, ATP γ S, a non-
Extracellular Ca2+ functioned to activate K+ efflux
It appears that Ca2+ is involved in stimulating K+ efflux
from alligator cells. Support for this conclusion comes
from the finding that the cationophore gramicidin
reversed the inhibitory effect of extracellular Ca2+
removal (Figure 7), which was the expected result if
Ca2+ has a permissive effect on K+ efflux. The finding
that K+ efflux is a crucial component of alligator RVD is
consistent with virtually all cell types studied to date
(Chamberlin & Strange 1989, McCarty & O’Neil 1992,
Pasantes-Morales et al. 2000). Furthermore, other
researchers have similarly concluded that this efflux is
Ca2+ dependent. For example, Adorante & Cala (1995)
found that Ca2+ stimulates K+ efflux in nonpigmented
human ciliary epithelial cells, as the inhibition of RVD by
Ca2+ chelation was removed with the addition of
gramicidin. Likewise, Light et al. (2003) found that
gramicidin reverses the RVD inhibition that
accompanies
EGTA
treatment
of
Necturus
erythrocytes. These parallel findings lend support for
the results of this study.
hydrolyzable form of ATP (Light et al. 2001) could be
used. This might discern whether the lack of effect
upon ATP addition observed in this study was indeed
the result of receptor saturation, or, alternatively, if
these cells have very active exo-ATPases (Gordon
1986) that prevent the rise in extracellular ATP
concentration necessary to observe an enhanced
volume recovery.
It should be noted that the inhibition of RVD
evoked by hexokinase and suramin was not as robust
as that of gadolinium. This suggests that P2 receptors
might not be entirely responsible for Ca2+ entry during
RVD. In addition to a P2 receptor, another gadoliniumsensitive transport pathway (such as a stretch-activated
channel) could also be active during the volume
recovery process. In future studies, using additional P2
receptor antagonists, in particular those specific for P2X
receptors, would be beneficial for further elucidation of
129
the specific Ca2+ transport pathways functioning in this
cell type.
Additionally, Schliess et al. (1996) determined that
calmodulin is not involved in the cell signaling events
stimulated by cell swelling in rat cerebral astrocytes.
The fact that both calmodulin dependent and
independent signaling processes have been observed
implies that there are variations in the steps ultimately
leading to volume restoration.
K+ efflux does not occur through Ca2+-activated K+
channels
The effect of gramicidin on RVD in alligator erythrocytes
suggested that the rate limiting step in volume recovery
was K+ efflux, as facilitating K+ loss from cells with
exogenous pores resulted in a more efficient RVD
response (Figure 7). Furthermore, it appears that Ca2+
had a permissive effect on K+ efflux, as I was able to
reverse the inhibitory effect of a low-Ca2+ medium by
artificially permitting K+ loss from alligator cells. As
mentioned earlier, this was the expected result if K+
efflux occurs downstream of Ca2+ entry into cells.
However, my results suggest that Ca2+ does not
modulate K+ permeability directly, because quinine, a
Ca2+-gated K+ channel blocker (Hoffman et al. 1986),
had no significant effect on volume recovery (Figure
13). That is, it is unlikely that Ca2+ stimulates K+ efflux
by binding to and activating a K+ efflux site, as such a
channel does not appear to be operating in alligator
erythrocytes during the RVD response. Similar results
were found by Arrazola et al. (1993) in rat thymocytes.
In this cell type, RVD was unaffected by the addition of
quinine.
Similarly, K+ efflux during cultured lens
epithelial cell RVD occurs via a quinine-insensitive
transport pathway (Diecke & Beyer-Mears 1997).
However, in the case of alligator erythrocyte RVD, it is
not possible to rule out the involvement of a Ca2+activated K+ channel entirely, as a quinine insensitive
Ca2+-dependent K+ channel might be present and
operating during volume recovery. In order to account
for this possibility, experiments using other Ca2+activated K+ channel antagonists would have to be
performed.
The lack of an effect following quinine
addition to alligator red blood cells is in contrast to the
studies performed by Hoffman et al. (1986) on Erlich
ascites tumor cells, Adorante & Cala (1995) on
nonpigmented human ciliary epithelial cells, and
Bergeron et al. (1996) on Necturus red cells. In these
cell types, quinine did have an inhibitory effect on RVD,
which lends support to Ca2+ activated K+ channels
being present in these cell types. Therefore, although
K+ efflux is a universal step in the volume recovery
process, the pathway for this efflux seems to vary
among cell types.
RVD depends on Phospholipase A2 activation and
arachidonic acid production
The results of my PLA2 inhibition studies were in
agreement with my early predictions. Specifically, as
was expected if PLA2 activation is a crucial component
to the RVD response, the PLA2 antagonist ONO had a
robust inhibitory effect on the ability of alligator cells to
regulate their volume (Figure 15). Additionally, the PLA2
inhibitor 2,4-dibromoacetophenone attenuated percent
volume recovery in alligator cells (Figure 16). In
combination, these findings imply that arachidonic acid
and/or its metabolites are efficacious in alligator volume
recovery. As stated previously, a role for arachidonic
acid in volume recovery following hypotonic challenge
is not unique to this cell type; RVD in neuroblastoma
(Pasantes-Morales et al. 2000), Necturus erythrocytes
(Light et al. 1998), and trout proximal renal tubules
(Kanli & Norderhus 1998) all involve arachidonic acid
metabolism.
It appears that in alligator erythrocytes,
arachidonic acid itself, as opposed to one of its
metabolites, potentiates the RVD signaling cascade.
Support for this conclusion comes from the observation
that suppressing the formation of all eicosanoids (the
further breakdown products of arachidonic acid) using
ETYA failed to inhibit volume regulation (Figure 17).
Interestingly, the more specific antagonist NDGA (an
inhibitor of the lipoxygenase pathway thereby blocking
production of leukotrienes) slightly potentiated volume
recovery (Figure 18). If anything, this result further
supports the direct role I propose for arachidonic acid in
alligator cell volume regulation. That is, by blocking
arachidonic acid breakdown, NDGA might enhance
RVD by increasing the concentration of arachidonic
acid available to participate in volume restoration.
A primary role for arachidonic acid itself in
volume regulation is consistent with the findings of
other researchers. Basavappa et al. (1998) found that
inhibiting arachidonic acid formation in human
neuroblastoma cells results in a reduced RVD.
However, similar to this study, RVD inhibition was not
observed upon selective blockade of arachidonic acid
breakdown. Sanchez-Olea et al. (1995) also showed
that arachidonic acid stimulates RVD in astrocytes
directly; RVD was significantly affected in the absence
of the fatty acid itself, whereas antagonizing its further
metabolism did not alter RVD. In contrast, if an
arachidonic acid metabolite was required to stimulate
RVD, then blocking its formation would attenuate
volume recovery. This has been shown to occur in
kidney cells (Tinel et al. 2000), where addition of ETYA
results in a weakened RVD mechanism. Additional
support for arachidonic acid metabolites as volume
recovery agents comes from studies performed on
Necturus erythrocytes (Light et al. 1997). In this cell
type, both ETYA and NDGA had inhibitory effects on
RVD, indicating a volume regulatory response
dependent on a lipoxygenase metabolite.
Obviously, there is a clear continuity with
respect to the RVD mechanisms among cell types, as
many of them involve a signal transduction pathway
RVD does not involve a Ca2+/calmodulin signaling
pathway
Since Ca2+ did not appear to directly activate K+ efflux
during alligator RVD, I assessed whether a signal
transduction pathway involving calmodulin is at play in
alligator erythrocytes. Although calmodulin has been
implicated in the RVD process in many cell types
(McCarty & O’Neil 1992), it does not seem to play a role
in alligator volume recovery. That is, I failed to observe
an inhibitory effect in response to preventing calmodulin
activation with pimozide (Figure 14). Hence, it seems
unlikely that RVD in alligator cells is modulated by a
calmodulin system.
Calmodulin-independent
transduction
pathways similar to the one found in alligator cells have
been identified in other cell types. For example,
Fincham et al. (1987) showed that the response of
erythrocytes from euryhaline fish species to hypotonic
shock is independent of calmodulin activation.
130
dependent on arachidonic acid. At the same time,
however, obvious differences are apparent, particularly
when it comes to the degree of arachidonic acid
metabolism required to stimulate the RVD response.
This may reflect differences in the specific role played
by arachidonic acid in the overall RVD scheme, which
has yet to be elucidated in many cell types.
Future studies should be performed to
confirm a role for arachidonic acid in alligator volume
2+
Ca
regulation. This might include utilizing additional PLA2
inhibitors. Also, using PLA2 inhibitors in coordination
with the cationophore gramicidin would discern what
role arachidonic acid plays in the RVD cascade. If
arachidonic acid mediates K+ efflux, then gramicidin
should reverse the inhibitory effect of PLA2 inactivation.
Additionally, experiments could be carried out in which
respond appropriately to hypotonic challenge in the
This might reflect
absence of this Ca2+ source.
similarities in their RVD pathways, if Ca2+ is operating in
a similar fashion in salmon and alligator cells.
However, Ca2+ serves as an activator of a wide variety
of cellular processes. Thus, although RVD in both cell
types clearly relies on Ca2+, it might be serving a
different function in each species. In alligator cells,
Ca2+ appears to stimulate arachidonic acid formation,
which then leads to K+ efflux. I was unable to study the
signaling cascade downstream of Ca2+ influx in salmon
cells, but future research could examine this topic. It
would be interesting to determine whether RVD in
salmon similarly occurs through a pathway mediated by
arachidonic acid.
The most obvious difference between salmon
and alligator RVD is the rate and extent by which the
cell types are able to recover from cell swelling.
Salmon cells displayed a robust RVD response,
whereas volume decrease by alligator cells was much
less pronounced. This difference might indicate that
salmon erythrocytes possess a more highly tuned and
efficient RVD mechanism.
This might be
physiologically relevant, as severe hypotonic shock is
probably a much greater threat to the cells of marine
teleosts, especially those cells that travel through the
gills. In contrast, alligator cells are not directly exposed
to the external environment. Instead, it is likely that
their RVD mechanism is adapted to counteract milder
and more gradual osmotic fluctuations, such as those
that accompany altered metabolic activity and kidney
function during hibernation. In other words, the differing
lifestyles of these two organisms should be taken into
account when comparing their RVD responses.
cell swelling H2O
P2 receptor
PLA2
AA
PLB
K
+
Figure 20. Proposed RVD signal transduction pathway in
alligator red blood cells.
Cell swelling leads to Ca2+ influx, possibly by activating a P2
receptor. The rise in intracellular Ca2+ stimulates PLA2, which
catalyzes the breakdown of membrane phospholipids to form
arachidonic acid. Finally, the actions of arachidonic acid result
in K+ efflux, thereby allowing for cell volume recovery.
arachidonic acid is added to the extracellular medium. I
would expect this to enhance volume recovery, if RVD
is indeed stimulated by arachidonic acid.
Summary
The results from my studies on alligator RVD are
consistent with cell swelling being followed by the influx
of Ca2+ through a gadolinium-sensitive entry site,
possibly a P2 receptor. In turn, calcium stimulates the
volume regulatory response through the activation of
PLA2 and the subsequent formation of arachidonic acid.
Arachidonic acid leads to volume restoration by
activating K+ efflux (Figure 20).
the author.
Conclusion
Ca2+ as a signaling agent in regulatory volume
decrease
Cell swelling appears to correlate with a rise in
intracellular Ca2+ in both salmon and alligator red blood
cells following an influx from the extracellular medium,
as opposed to release from intracellular stores. The
increase in Ca2+ levels is not an epiphenomenon, as
Ca2+ is needed to activate the RVD response.
Therefore, it seems that Ca2+ is a pivotal signaling
agent in the intracellular processes that allow for
volume decrease following hypotonic challenge.
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133
Primary Article
α-Synuclein Aggregation and Membrane Association in a
Fission Yeast Model: Implications for PD Pathogenesis
2001). Numerous model systems in mice (Dauer and
Przedborski, 2003), worms (Lasko et al., 2003), flies
(Feany and Bender, 2000) and yeast (Outeiro and
Muchowski, 2004) have been designed to elucidate αsynuclein’s biology and the molecular mechanism of
misfolding, aggregation and cytotoxicity.
Fission yeast (Schizosaccharomyces pombe)
is the model organism that our lab (Brandis et al. 2006)
recently developed to study α-synuclein’s misfolding,
aggregation, and cytotoxic properties in vivo. The
advantages of using yeast are that they have a fast life
cycle and an easy to manipulate genome. Yeast and
humans have conserved functions in cellular pathways
such as protein folding, protein degradation, and
oxidative stress (Wood et al., 2002). When neurological
diseases strike human beings, one or more of these
pathways malfunctions. Therefore, yeast can serve as
an exquisite model for investigating protein misfolding
neurological disorders.
The process of Lewy body formation from
misfolded α-synuclein was first tested in in vitro
experiments. Studies suggest that α-synuclein
aggregation follows a hypothetical model called
nucleation polymerization (Conway et al., 2000).
Previously, misfolded protein aggregation in many
neurodegenerative diseases, for instance, betaamyloidsin Alzheimer’s Disease, huntingtin in
Huntington’s Disease, and prion proteins in
Transmissible Spongiform Encephalopathies was well
predicted by this model (Caughey and Lansbury, 2003;
Perutz
and
Windle,
2001).
The
nucleation
polymerization
hypothesis
states
that
protein
aggregation begins with the creation of small oligomer
(nucleus) seeds. As time and protein concentration
increases, the oligomers polymerize into polymers
(aggregates). Thus, the formation of protein aggregates
is time and concentration dependent.
Until recently, this hypothesis in PD was
unsupported by in vivo experiments. However, using
live fission yeast, Brandis et al. (2006), showed that αsynuclein aggregates on the basis of the nucleation
polymerization theory in vivo. In the study, there was
evidence of large insoluble clumps of α-synuclein
aggregates which reminds us of the Lewy bodies that
form in the dying neurons of a Parkinson’s disease
patient. At high concentration of α-synuclein in fission
yeast, the aggregates formed in large numbers, but at
low concentration there were no aggregates. In the
presence of multiple α-synuclein aggregates, toxicity in
fission yeast cells was surprisingly absent. Also, the αsynuclein in fission yeast, in contrast to the other family
of yeast called budding yeast, never localized to the
plasma membrane (Brandis et al., 2006).
On the other hand, research with budding
yeast has provided key insight into α-synuclein
misfolding and its ability to disrupt cellular pathways
leading to cytotoxicity (Outeiro and Muchowski, 2004;
Cooper et al., 2006). In studies of budding yeast, when
α-synuclein was expressed at high concentration, it
formed aggregates with toxic effects (Outeiro and
Linquist, 2003). In other studies, α-synuclein
aggregates did not have toxic effects without additional
genetic knockouts causing proteasomal dysfunction
(Sharma et al., 2006).
Lokesh Kukreja*
Lake Forest College
Abstract
Lewy bodies of α-synuclein protein are prominent
characteristics in the Parkinson’s disease (PD)
pathology. The mechanism of Lewy body formation
and consequent cytotoxicity was studied by
Brandis et al. (2006) in a newly developed model
organism of fission yeast. Though, the level of αsynuclein expression studied was either high or
low, the wild-type and A53T familial mutant of αsynuclein followed the nucleation polymerization
theory in the process of misfolding and
aggregating. At high concentration, α-synuclein
formed cytoplasmic aggregates in a concentration
and time-dependent manner. However, these
aggregates appeared to be independent of
cytotoxicity. In this current study, the fission yeast
model is used again but to evaluate α-synuclein
misfolding, aggregation, and non-toxic properties
when expression is moderate. The results indicate
moderate α-synuclein expression to obey the
nucleation polymerization model. In light of this
study, α-synuclein aggregation requires a
necessary threshold concentration. Moderately
expressed α-synuclein forms soluble aggregates,
but at a slightly lower expression. So far, studies in
fission
yeast
cells
show
that
various
concentrations of α-synuclein, neither target the
plasma membrane nor are toxic. Because αsynuclein misfolding and aggregation is linked to
Parkinson’s disease, absence of its toxicity in
fission yeast is paradoxical. We expect that αsynuclein toxicity may require a membrane binding
capacity. In an attempt to induce α-synuclein’s
localization to the plasma membrane, the content of
phospholipids in yeast was increased. Membrane
localization and cytotoxicity were still lacking.
Needless to say, fission yeast shed provocative
insight into α-synuclein’s role in PD pathogenesis.
Introduction
Parkinson’s disease (PD) is the most common
movement disorder in humans characterized by slowed
movement, resting tremors, rigidity and postural
instability. The disabling symptoms of PD result from
dying midbrain striatal dopaminergic neurons. This
neurodegenerative disease affects more than 1 million
Americans over the age of 55 (Collier et al., 2002).
Sporadic occurrences constitute 95% of all PD cases,
while genetic occurrences constitute the other 5%.
There are two well studied familial mutations in the αsynuclein gene in PD: A30P and A53T. In the disease
pathology, the reduction of viable neurons correlates
with the accumulation of misfolded α-synuclein into
cytoplasmic aggregates called Lewy bodies (Giasson,
*This paper was part of an independent study with Dr. Shubhick
DebBurman.
135
Generally, budding yeast and fission yeast
have inequivalent α-synuclein cellular localizations. In
budding yeast models, wild-type and A53T mutant αsynuclein associate with the plasma membrane and
may form aggregates within the cells. A30P mutant αsynuclein, on the other hand, remains cytoplasmically
diffused (Dixon et al., 2005; Outeiro and Lindquist,
2003; Zabrocki et al., 2005; Sharma et al., 2005). In the
fission yeast model, we also observe A30P mutant αsynuclein to be cytoplasmically diffused. However, wildtype and A53T mutant α-synuclein do not associate
with the plasma membrane and form cytoplasmic
aggregates in a time and concentration dependent
manner (Brandis et al., 2006).
So far, the nucleation polymerization
hypothesis can explain why there are more α-synuclein
aggregates when the protein is expressed in high
concentration in contrast to when the protein is
expressed in low concentration (Brandis et al., 2006). In
light of the nucleation polymerization hypothesis, in the
first part of this study, we evaluate the expression of
moderate concentration of α-synuclein in fission yeast.
At moderate levels of α-synuclein, an intermediate
number of α-synuclein aggregate formations are
expected. According to Brandis et al. (2006), these
aggregates should not influence cytotoxicity.
In the second part of this study, we examine
the effect of cellular lipid content on toxicity. Cytotoxicity
has yet to be observed in fission yeast. In budding
yeast,
cytotoxicity
and
-synuclein
membrane
localization coexist (Sharma et al., 2006). The
biochemical property of α-synuclein gives this protein
the specificity to bind to lipid membranes, inviting the
question of why is there no α-synuclein dependent
cytotoxicty in fission yeast (Sharon et al., 2001). It is
possible that toxicity is contingent on α-synuclein
membrane localization. Here, we use dimethyl sulfoxide
(DMSO) to increase the overall lipid content in the
fission yeast. In the Murata et al. (2003) study, upon
exposure to DMSO, there is an induction of membrane
proliferation in budding yeast. The same induction of
phospholipid biosynthesis is expected to occur in fission
yeast. This change in the physiology of fission yeast
may encourage α-synuclein migration towards the cell
membranes. According to Volles et al. (2001),
membranes are the possible locations in the cell where
α-synuclein can form toxic protofibrils (oligomers).
These protofibrils can lead to the formation of
cytoplasmic aggregates (Rochet et al., 2004) or
possibly toxic cytoplasmic aggregates (Lashuel at al.,
2002). Therefore, we inquire if the α-synuclein’s
membrane localization is key to cytotoxicity.
GCCATG-3’. Similarly, PCR was used to amplify GFP
cDNA from GFP-pYES/TOPO S. cerevisiae vectors
constructed by Sharma et al. (2006): forward primer,
5’CCCGGGACCATGGCCAGCAAAGGAGAAG-3’;
reverse primer, 5’-TTTGTAGAGCTCATACATGCCA
TG-3’.
These PCR products were subcloned,
according to the manufacturer’s protocol (Invitrogen),
into each of these three fission yeast pNMT TOPO-TA
expression vectors: pNMT1 (for high expression),
pNMT41 (for intermediate expression), and pNMT81
(for low expression) vectors. Note that these vectors
added a V5-epitope and a 6X histidine epitope at the Cterminal end of the subcloned α-synuclein-GFP
sequence. These vectors were transformed into
Escherichia Coli, again according to the manufacturer’s
protocol (Invitrogen). Positive transformants were
verified for correctly oriented subcloned cDNA by
standard bacteria whole-cell PCR. Plasmid vectors
were purified using a Qiagen miniprep kit, and the
respective subcloned DNA sequences were confirmed
(University of Chicago sequencing facility). The parent
pNMT1, pNMT41, and pNMT81 pREP vectors were
kindly provided by Judy Potashkin (Rosalind Franklin
University of Medicine and Science, North Chicago, IL).
Yeast Strains
The TCP1 strain (h-leu1-32; Invitrogen) of fission yeast
was kindly provided by Judy Potashkin, Rosalind
Franklin University of Medicine and Science.
Yeast Transformation
S. pombe strains were transformed with pNMT vectors
using the lithium-acetate transformation method (Alfa et
al., 1993).
Fluorescence Microscopy
S. pombe cells were first grown overnight at 30oC in
Edinburgh minimal medium (EMM [Invitrogen])
containing thiamine (10µM [to repress α-synuclein
expression]). After 24h, cells were pelleted at 1500g for
5 minutes, washed twice in 10ml dH2O, resuspended in
10 ml EMM without thiamine, of which 125 µL cells
were used to inoculate 25 mL EMM without thiamine (to
express α-synuclein). At desired expression time points
for microscopy, cells were harvested at 1500g (4oC) for
5 min and were washed in 5 mL water. Then cells were
resuspended in 100-1000 uL EMM+T, of which 10 uL
was pipetted onto a slide. Slide of cell culture was
viewed
using
Nikon
TE-2000U
fluorescence
microscope at 1000X magnification. Images were
deconvoluted using MetaMorph software version 4.2. In
order to quantify α-synuclein aggregates, cells were
first viewed under differential interference contrast
(DIC) microscopy, and total cell count in the field was
determined and viewed for GFP fluorescence. The
number of cells in the field containing 1, 2, and 3+
aggregates was determined. The field was then moved
three turns on the field control knob, and the process
was repeated in a new field. At least 750 cells were
evaluated for each treatment. Aggregates were scored
as percent of total cells in the field that expressed 1, 2,
and 3+ aggregates.
S. Pombe Expression Vectors
Human wild-type and A53T mutant α-synuclein cDNAs
were a gift from Christopher Ross (Johns Hopkins
University). A30P and A30P/A53T mutant α-synuclein
were created from wild-type and A53T mutant αsynuclein, respectively, using site-directed mutagenesis
(Invitrogen). Polymerase chain reaction (PCR) was
used to amplify C-terminal green fluorescence protein
(GFP)-tagged α-synuclein (wild-type, A30P, A53T,
A30P/A53T) fusion cDNA from the α-sunclein-GFP
containing pYES2/TOPO S. cerevisiae vectors
constructed by Sharma et al.(2006): forward primer,
5’-GGGGCCAAGCTTGCCATGGATGTATTCATGAAA
GGA-3’; reverse primer, 5’-TTTGTAGAGCTCATACAT
Western Analyses
Yeast cells (2.5x107 cells/ml) were washed in 50 mM
Tris (pH 7.5), 10 mM NaN3 and solubilized in
Electrophoresis Sample Buffer (ESB; Burke, 2000)
containing 2% sodium dodecyl sulfate (SDS), 80 mM
Tris (Ph 6.8), 10% glycerol, 1.5% dithiothreitol, 1 mg/ml
136
bromophenol blue, and a cocktail of protease inhibitors
and solubilizing agents (1% Triton-X 100, 1 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1
mM sodium orthovanadate, 0.7 µg/ml pepstatin A, 0.5
µg/ml leupeptin, 10 µg/ml E64, 2µg/ml aprotinin, and 2
µg/ml chymostatin). Samples were run on pre-cast 1020% Tris-Glycine gels (Invitrogen) using SDS
containing running buffer. SeeBlue (Invitrogen) was
used as the molecular standard. Gels were transferred
to PVDF membranes and Western blot was performed
with anti-V5 AP monoclonal antibody using standard
protocols and detected for alkaline phosphatase
activity.
calculating cell density for inoculation was done
according to the appropriate experiment. The cells had
high expressing α-synuclein pNMT-1 vectors. Before
inoculation, DMSO was added in the range of 0 to 10%
in EMM media. The concentration of DMSO exposure
was increased only up to 10%, technically exceeding
this percent of DMSO would do harm to the cell
(Zabrocki et al., 2005). After the DMSO mixed well in
the media, yeast cells were inoculated, and grown.
Then the cells were observed at the desired time points
for the following examinations: Growth Curves and
Fluorescence Microscopy.
Results
Growth Curve
Cells were grown in 10 ml EMM+T overnight at 30°C in
the incubator which rotates at 200 rpm. Cells were
harvested at 1500 x g for 5 min at 4°C, and were
washed twice in 5 ml H20. Cells were re-suspended in 5
ml H20 and were counted. Flasks with 25 ml EMM were
each inoculated with 2.0x106cells/ml density. At 0, 6,
12, 18, 24, and 36 hours, and in duplicate
measurements, 1 ml of cell culture was removed and
placed in a cuvet to measure absorbance using a
Hitachi
U-2000
Spectrophotometer.
Averaged
absorbance readings were plotted against time points
to produce a growth curve.
Moderately Expressed α-Synuclein Aggregates in Live
Cells
α-synuclein localization in fission yeast is screened as
the expression of the protein, promoted by pNMT-41
vector, is in moderate concentration. Fluorescence
microscopy indicates A30P, A53T, and A30P/A53T
mutant
α-synuclein
localizations.
The
protein
expression level is slightly higher than the expression
by pNMT-81 vector (refer to Figure 5C: Western
Analysis in Brandis et al., 2006). A53T α-synuclein
forms cytoplasmic aggregates. Meanwhile, A30P and
A30P/A53T
α-synuclein
remain
cytoplasmically
diffused. Even in this moderate concentration,
α−synuclein never localizes to the plasma membrane.
These expression characteristics from all isoforms of αsynuclein in live fission yeast match previous findings
(Brandis et. al, 2006) (Figure 1).
DMSO Treatment
Dimethyl sulfoxide (DMSO) was purchased from
Sigma-Aldrich. Cells were grown in 10 ml EMM+T
overnight at 30°C. Steps of cell harvesting and
Figure 1. Fluorescence Microscopy of pNMT-41 Medium Expression: α-Synuclein was expressed in moderate concentration by
pNMT41 promoter vector. These yeast were grown in EMM without thiamine. Images were captured at the indicated times over 36 hours.
th
A53T α-synuclein began to form aggregates at the 18 hour time point. A30P and A30P/A53T exhibited diffuse cytoplasmic fluorescence
throughout the time course.
137
Quantification of Moderately Expressed α-Synuclein
Aggregates
Quantifying the aggregates for A53T mutant αsynuclein is necessary to make qualitative assessment
on how these aggregates form. At moderate
concentrations
of
α-synuclein,
nucleation
polymerization hypothesis predicts a delayed formation
of an intermediate number of aggregates. The data
showed that formation of aggregates was timedependent, as they started appearing at the 18th hour
(Figure 2A). Additionally, when compared to Brandis et
al. (2006), the data supports the predictions made by
Figure 2B. Comparison of moderate to high expression
of
quantified
A53T
α-synuclein
aggregation.
Comparison of quantified data on A53T α-synuclein
aggregates with high expression (red bar) (Quantification
data provided by Brandis et al. 2006) compared to
moderate expression (green bar) over the 36-hr period.
Bars represent percentage of total cells that had 1 or more
aggregates per cell.
Figure 2A. Time course of quantified A53T α-synuclein
aggregation. Cells, cultured in EMM media without
thiamine,
expressed
α-synuclein
in
moderate
concentrations. The number of aggregates formed was
quantized in cells expressing A53T α-synuclein over a 36hr time course. Cells were scored in terms of containing 1
aggregate (blue bar), 2 aggregates (yellow bar), or 3 or
more aggregates (red bar). Bars represent percentage of
total cells counted in each sample that exhibited the
designated number of aggregates per cell.
the nucleation polymerization theory. First, the time of
formation of aggregates at moderate concentration was
six hours later when compared to the time of aggregate
formation at high concentration. Second, throughout the
36 hour time course, the percent of cells forming
aggregates were lower for moderate concentration
compared to high concentration (Figure 2B).
Toxicity Levels of Moderately Expressed α-Synuclein
Aggregates
Brandis et al. (2006) suggest that even at high
concentrations of α-synuclein leading to formation of
many aggregates, the cytotoxicity levels were minimal.
An optical density assay performed in this study
showed no toxicity in the cells expressing α-synuclein
in moderate concentration either (Figure 3). The control
in this growth analysis was a culture of parent vector
(pNMT-41) cells that did not express α-synuclein. None
of the isoforms of α-synuclein was toxic to fission yeast
(Figure 3). Next, we investigated whether membrane
localization was critical for toxicity. The lipid content of
fission yeast cells was induced to promote α-synuclein
to localize to the membrane.
Figure 3. Growth Curve of Cells Moderately Expressing
α-Synuclein. OD600 measurements were obtained over
48 hours for cells containing A30P, A53T, and A30/A53T
forms of α-synuclein that were tagged with GFP in
pNMT41 vector. Cells grown in thiamine (dashed lines)
served as control. Concentration-dependent α-synuclein
toxicity to fission yeast was not observed.
Is α-Synuclein Membrane Localization Gained With
Exposure to DMSO?
Contrary to the prediction, upon DMSO exposure to
fission yeast cells, no α-synuclein lipid binding property
was observed. α-Synuclein aggregates did not
decrease also. The aggregation inducing property of Wt
(Figure 4) and A53T and as well as the cytoplasmically
138
Figure 4. Fluorescence Microscopy of pNMT-1 High expression Cells Exposed to DMSO: Cells were grown in EMM media without
thiamine, expressing α-synuclein in high concentration promoted by pNMT-1 vector. Florescence microscopy cell images were captured at
the 24th hour. Wt α-synuclein did not gain membrane localization and the cytoplasmic aggregates still remain in fission yeast with exposure
to varying concentrations of DMSO.
Figure 5. Growth Curve Analysis on α-Synuclein Expressing cells Exposed to DMSO (1st Trial): OD600 measurements over 48
hours were obtained for cells that express α-synuclein in high concentration. Control cells had simply GFP or parent-plasmid. Cells
were grown in EMM media without thiamine. The growth was measured in exposure to different amounts (0%, 2%, and 10%) of
DMSO. The data showed slight inhibition in growth.
diffused property of A30P and A30P/A53T were
unchanged in exposure to DMSO (data not shown).
These observations were made under fluorescence
microscopy.
findings by Brandis et al. (2006). In turn, we tried to
clarify the inconsistencies by rerunning the DMSO
treatment experiment (Figure 5).
In the second trial, cells were treated with 0 %, and 4.5
% DMSO. This time, the control group with no exposure
to DMSO had normal growth. As the DMSO exposure
climbs to 4.5%, toxicity was not observed. Under toxic
conditions, there would have been a lag in the growth
curves (Figure 6).
Yet, these two trials’ results were
contradictory. In exposure to DMSO, the first trial
Are Toxicity Levels Enhanced in Fission Yeast with
Exposure to DMSO?
All fission yeast in varying concentrations of DMSO
showed a slight inhibition in their growth. We
considered this result anomalous because 0% DMSO
particularly should not be toxic. It contradicts the
139
Figure 6. Growth Curve Analysis on α-Synuclein Expressing cells Exposed to DMSO (2nd Trial): OD600 measurements over 48
hours were obtained for cells that express α-synuclein in high concentration. Control cells had simply GFP or parent-plasmid. Cells
were grown in EMM media without thiamine. The growth was measured in exposure to different amounts (0%, 4.5%) of DMSO. The
control growth curves with no DMSO exposure had normal s-shaped curves. DMSO exposure of 4.5% did not cause toxic effects.
showed slight inhibition of cellular growth, while the
second trial showed normal growth. In the future,
several more trials of DMSO treatment on α-synuclein
expressing fission yeast are necessary.
membrane localization with toxicity (Sharma et al.,
2006). This leads to the hypothesis that membrane
localization might be key to cytotoxicity. We did not
observe α-synuclein dependent toxicity in fission yeast,
but we still suspect that toxicity requires α-synuclein
membrane localization. In a previous study by Rochet
et al. (2004), membrane localization of α-synuclein was
shown to be essential in creating toxic protofibrils.
Under a different study, the destruction of vesicular
membranes by protofibrillar α-synuclein was directly
observed by atomic force microscopy (Volles et al.,
2001). In our lab, the toxicity of budding yeast was
observed when α-synuclein localized at the cell
periphery. In budding yeast, the localization of αsynuclein to the plasma membrane happened prior to
the formation of α-synuclein aggregates. With the
exception of A30P, wild-type and A53T α-synuclein
localized to the plasma membrane before forming
inclusions (Sharma et al., 2006). Conversely, in our
fission yeast model, neither toxicity nor membrane
association was observed. Overall, a connection
between α-synuclein-dependent toxicity and αsynuclein’s association with the plasma membrane is
strongly implicated.
Discussion
From the past study by Brandis et al. (2006) and this
current study, we have successfully developed fission
yeast as a model organism to study the misfolding,
aggregation, and cytotoxic properties of α-synuclein
linked to Parkinson’s disease. Specifically, work with
fission yeast sheds provocative insight into the ideas
that concentration of α-synuclein is important for the
protein’s aggregation and α-synuclein membrane
localization might be critical for the protein dependent
toxicity.
α-Synuclein aggregation: concentration is key
The protein’s polymerization activity is concentration
dependent. At high concentrations, there are many αsynuclein aggregates, while at low concentrations there
are no α-synuclein aggregates (Brandis et al, 2006). In
the current study, at moderate concentration, αsynuclein forms an intermediate number of aggregates.
According to data by Brandis et al. (2006), the protein
expression of moderate concentration was only slightly
higher than the protein expression of the low
concentration. Conversely, the low concentration of αsynuclein was not enough to lead to the formation of
insoluble aggregates. Therefore, the evidence indicates
that slightly higher (moderate) concentration of αsynuclein is required to form aggregates. We consider
the moderate concentration or a concentration near to it
as the threshold point of α-synuclein protein
concentration that must be present to turn the soluble
protein into insoluble aggregates. As protein
concentration increases, the oligomers polymerize to
form greater numbers of aggregates. Moreover, αsynuclein aggregation activity is time-dependent.
Overall, Brandis et al. (2006) and this study display
both concentration and time-dependent α-synuclein
aggregation properties which support the grounds for
the nucleation polymerization model in vivo.
Future Proposal
To more
conclusively
elucidate
α-synuclein’s
membrane association and yeast toxicity, the
approaches of treating fission yeast with DMSO must
be refined and repeated. Further research is crucial
because several yeast models already suggest that
membrane localization of α-synuclein is critical to
pathogenesis (Dixon et al., 2005; Outeiro and Lindquist,
2003; Zabrocki et al., 2005).
Additionally, this future investigation can be
done in budding yeast by reducing the lipid
concentration of the membrane and then measuring
changes in α-synuclein toxicity. Cho1 and Cho2 are
knockouts in budding yeast strain W303 that code for
enzymes critical to making major cell membrane
phospholipids. Cho 1 encodes for phosphatidylserine
synthase enzyme that coverts CDP-DG, a precursor to
two of the major phospholipids in the membranes
namely PI and PS. Cho 2 encodes a PL methyl
transferase to produce PC from PE phospholipids.
Using knockouts of these genes, budding yeast can be
manipulated to give lipid deficient cells (Carman and
Zeimetz, 1996). α-Synuclein must have specificity for
binding to one of the major phospholipids. The
Toxicity: is membrane localization key?
To this date, in a fission yeast model, membrane
localization has not been observed with cytotoxicity. In
the budding yeast model, there is evidence of
140
knockout of one or more of the major membrane
phospholipids may advance the loss of α-synuclein
membrane localization. If this loss occurs, we predict
the reduction in toxicity levels.
We observed the formation of α-synuclein
aggregates in fission yeast that did not acquire the
initial membrane localization. This step may have been
essential to creating toxic protofibrils and/or toxic
cellular aggregates. We predict that the budding and
fission yeast models will facilitate the establishment of
α-synuclein
association
with
the
membrane
phospholipids as a necessary characteristic to increase
cytotoxicity levels. α-Synuclein is an abundant and
broadly expressed protein in the human brain, where it
interacts with membranes and vesicular structures
(Outeiro and Lindquist, 2003). The α-synuclein property
of associating with membranes and its link to
pathogenic consequences makes it critical to future
research.
Cooper A. A., Gitler A. D., Cashikar A., Haynes C. M., Hill K. J.,
Bhullar B., Liu K., et al. (2006) α-Synuclein Blocks ER-Golgi
Traffic and Rab1 Rescues Neuron Loss in Parkinson's Models.
Science. 313: 324 – 328.
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P. T. Jr. (2002) Neurodegenerative disease: amyloid pores from
pathogenic mutations. Nature. 418: 291.
At Lake Forest College, the author likes to thank Dr.
DebBurman for constructive advice on this research
paper and Dr. Karen Kirk for the use of her laboratory
equipments in the experiments. Much thanks to
author’s lab peers for collaborative and technical
assistance. Extra thanks to Lital Silverman for editing
this article. Thanks to Dr. Judy Potashkin (Rosalind
Franklin University of Medicine and Science) for
encouraging us to develop the fission yeast model for
α-synuclein and to Dr. Virginia McDonough (Hope
College, MI) for discussions on lipid physiology in
yeasts. Dr. DebBurman was supported by grants from
NIH, NSF, Campbell Foundation (Michigan), Lake
Forest College, and a MacArthur grant from Kalamazoo
College for fission yeast research.
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into alpha-synuclein biology and pathobiology. Science 302:
1772-1775.
Outeiro T.F., and Muchowski P.J. (2004) Molecular genetics
approaches in yeast to study amyloid diseases. J. Mol.
Neurosci. 23: 49-60.
Perutz M. F. and Windle A. H. (2001) Cause of neural death in
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diseases attributable to expansion of glutamine repeats. Nature
412: 143-144.
the author.
Rochet J. C., Outeiro T. F., Conway K. A., Ding T. T., Volles M.
J., Lashuel H. A., Bieganski R. M., Lindquist S. L., and
Lansbury P. T. (2004) Interactions among alpha-synuclein,
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neurodegeneration in Parkinson's disease. J Mol Neurosci. 23:
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J. Mol. Neurosci. 28(2): 161-178.
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141
Primary Article
α-Synuclein Causes Non-Specific Toxicity in vps34 Yeast
and remodeling (Kahle et al., 2000). Recently, αsynuclein was shown to protect nerve terminals against
injury in conjunction with other synaptic proteins
(Chandra et al., 2005). Over the past 10 years, three
missense point mutations in α-synuclein have been
discovered in families with PD: A30P, A53T, and E46K
(Polymeropoulos et al., 1997; Kruger et al., 1998;
Zarranz et al., 2004). These familial mutations on
chromosome 4 are associated with early-onset PD and
may cause the misfolding and subsequent aggregation
of α-synuclein in Lewy bodies (Zabrocki et al., 2005).
The duplication or triplication of the α-synuclein gene is
also known to cause PD (Ibanez et al., 2004). In 2001,
McNaught et al., found that protein degradation is
impaired by 33-42% in PD patients, providing evidence
that PD is caused by impaired degradation of αsynuclein leading to the formation of protein-rich Lewy
bodies containing misfolded α-synuclein.
A possible way of treating PD is by degrading
misfolded and aggregated α-synuclein. There is genetic
and chemical evidence to show that α-synuclein is
degraded by the ubiquitin-proteasome system (UPS;
Thrower et al., 2000; Holtz and O’Malley, 2003;
McNaught 2002; Webb et al., 2003). Familial mutations
in parkin, an E3 ubiquitin ligase, and mutations in
ubiquitin carboxyL-terminal hydrolase L1 (UCH-L1)
inhibit the UPS in PD patients (Kitada et al., 1998;
Leroy et al., 1998). Furthermore, McNaught et al.
(2002), provided strong evidence that defects in the
UPS underlie PD pathology and toxicity.
Studies have shown that the UPS is not the
only organelle involved in α-synuclein degradation
(Webb et al., 2003). In fact, monomeric and aggregated
α-synuclein has been shown to inhibit the UPS, rather
than defects in the UPS causing α-synuclein
aggregation (Snyder et al., 2003). Further,
pharmacological inhibition of the UPS did lead to an
increase in cellular levels of α-synuclein (Rideout and
Stefanis, 2002; Biasini et al., 2004). This suggests that
another
pathway
of
protein
degradation—the
endosome/lysosome pathway—may be involved.
The yeast vacuole acts as the lysosome,
which degrades extracellular molecules, membrane and
endo-membrane proteins, and organelles. It also
degrades nuclear and cytoplasmic proteins, making it a
possible target for α-synuclein degradation. Webb et
al., provided evidence that in addition to the
proteasome, the lysosome also degrades α-synuclein.
Moreover, inhibition of the lysosome leads to an
increase in the intracellular levels of α-synuclein (Webb
et al., 2003; Lee et al., 2004; Cuervo et al., 2004).
Proteins are sent to the lysosome via
endocytic pathways. The multivesicular body (MVB)
sorting
pathway
to
degradation
by
the
lysosome/vacuole sorts proteins that are to be
degraded by the lysosome by targeting them into the
lumen of endosomes (Katzmann et al., 2001). Proteins
that are to be recycled or sent elsewhere are kept at the
limiting membrane of the endosome (Katzmann et al.,
2002). The fusion of the endosome with the lysosome
delivers the contents of the MVBs to the lysosome for
hydrolysis, while proteins on the limiting membrane of
the MVBs remain on the limiting membrane of the
lysosome (Katzmann et al., 2001).
The MVB sorting pathway is composed of
more than 15 vacuolar protein sorting (vps) proteins
which work alone or in complex. Monoubiquitination
Mithaq Vahedi*
Lake Forest College
Summary
α-Synuclein is implicated in Parkinson’s Disease, a
neurodegenerative disease that destroys midbrain
neurons.
The
misfolding
and
subsequent
aggregation of this protein is the likely cause of cell
death. A major hypothesis in the field is that
increasing α-synuclein’s rate of degradation may
prevent its aggregation and toxicity. The prevalent
model for α-synuclein degradation is via the
proteasome, and malfunctions in this pathway have
been shown to increase α-synuclein accumulation
and toxicity. However, increasing evidence
suggests that the Multivesicular Body (MVB)
sorting pathway is involved in protein degradation
via the lysosome. To test the role of the MVB
sorting pathway for the degradation of wild-type
and mutant α-synucleins, we asked if α-synuclein
would accumulate and increase toxicity in yeast
that lacked one of the MVB proteins. Previously,
Price and Shrestha showed that the absence of
vps28, an MVB protein caused toxicity in yeast
expressing α-synuclein (Eukaryon). We tested
another protein, vps34, a PI 3-kinase acting
upstream in the proteins involved in the MVB
pathway. The absence of vps34 was toxic to yeast
and this toxicity was severely exacerbated in the
presence of any foreign protein, including αsynuclein. Future research will examine several
other essential lysosomal pathway factors in
mediating α-synuclein toxicity.
Introduction
Parkinson’s disease (PD) is the second most common
neurodegenerative disease affecting about 1 million
people in North America (Greenamyre and Hastings,
2004). In PD, dopaminergic neurons in the substantia
nigra pars compacta (SNpc), in the mid brain die.
These basal ganglia neurons secrete dopamine which
is necessary for smooth and coordinated muscle
movement (Giasson and Lee, 2003). Loss of dopamine
results in most of the clinical symptoms of PD which
include resting tremors, slowness of movement, rigidity,
postural instability, and depression (Wolters and Braak,
2006). Round eosinophilic inclusions comprised of a
halo of radiating fibrils, known as Lewy bodies, found
within SNpc neurons and dystrophic neurites (Lewy
neurites) are the pathological hallmarks of PD (Giasson
and Lee, 2003; Dawson and Dawson, 2003). Misfolded
and aggregated α-synuclein is the primary filamentous
component of Lewy bodies (Spillantini, 1997; Spillantini
et al., 1998).
α-Synuclein is a 140 amino acid protein
found in pre-synaptic nerve terminals in neurons
(Spillantini, 1998; Mclean et al., 2000; Choi et al.,
2004). Although the precise function of α-synuclein is
unclear, it is known to play a role in synaptic plasticity
*This article was written as part of an independent study with Dr. Shubhik
DebBurman.
143
serves as a recognition signal for proteins to be
degraded through the MVB sorting pathway (Katzmann
et al., 2001). The ESCRT-I complex, a 350kDa protein
complex composed of vps23, vps28, and vps37
functions downstream of vps27, while vps15 and vps34
which form a complex are upstream of vps27
(Katzmann et al., 2003). Vps34 is a PI 3-kinase. Vps27
is then recruited to the enriched phospholipid layer of
the endosome membrane, where it can bind
ubiquitinated MVB cargo and recruit and activate
ESCRT-I (Katzmann et al., 2003). ESCRT-II and
ESCRT-III are composed of other vps proteins and act
downstream of ESCRT-I. Since α-synuclein is known to
bind membranes (Choi et al., 2004), it may be
degraded through the MVB sorting pathway to the
lysosome/vacuole.
Previously, our lab showed that knocking out
vps28, a component of ESCRT-I lead to modest toxicity
(Price and Shrestha, 2005). We hypothesize that
knocking out vps34 and expressing wildtype and
mutant forms of α-synuclein, will cause toxicity, since
vps34 is required for the proper functioning of the MVB
sorting pathway to protein degradation by the
lysosome.
An S. cerevisiae model developed in our lab
(Sharma et al., 2006) was used to assess the lack of
vps34 with expression of α-synuclein, as yeast have
already been shown to be useful model systems for the
study of study neurodegenerative diseases, including
Huntington’s disease and PD (Outeiro et al., 2003). The
toxicity of wildtype and mutant (A30P, E46K and A53T)
α-synucleins was assessed in vps34 mutants using
growth curve analysis through optical density data and
dilution serial spotting. Western blotting was done to
examine the levels of wildtype and mutant α-synucleins
in vps34 strains. These data shed light on the role of
vps34 in the degradation of wildtype and mutant αsynucleins.
Figure 1. Optical density analysis of α-synuclein in vps34
yeast. The top graph serves as a control, as cells were grown in
non inducing media (top). In the bottom graph, cells were grown
in galactose (inducing media). Yeast lacking vps34 hardly grew
at the 24 hour time point, compared to the wild type control.
Results
Wildtype α-synuclein is toxic to vps34 yeast
To assess the effects of knocking out vps34 and
expressing α-synuclein in the cells, an optical density
analysis at 600 nm was done. Untagged α-synuclein
was expressed in vps34 yeast by growing cells in
galactose media. As seen in figure 1, cells lacking
vps34 showed moderate toxicity. At 24 hours post
induction, yeast lacking vps34 had barely grown, while
those with vps34 had an absorbance value of 1.5.
Strains not expressing any α-synuclein are shown at
the top graph and serve as controls. These strains were
grown in SC-Ura glucose (Refer to methods).
Wildtype α-synuclein tagged to GFP is also toxic to
vps34 yeast
In order to assess the localization of α-synuclein in
vps34 yeast, a GFP tag was ligated to the C- terminal
of the α-synuclein gene (Refer to methods). As seen in
figure 2, α-synuclein-GFP is highly toxic to vps34 yeast.
This strain did not grow at all, until about 48 hours,
however, the parent strain, expressing vps34 and αsynuclein-GFP had already reached saturation point by
24 hours. A sharp increase in growth was observed
after 48 hours in vps34 yeast expressing α-synucleinGFP.
Figure 2. Growth curve showing growth of wildtype αsynuclein-GFP in vps34 yeast. When expressing α-synucleinGFP, cells lacking vps34 do not grow until about 48 hours, while
cells with vps34 reach saturation point by 24 hours.
GFP alone is extremely toxic to vps34 yeast
Since the toxicity associated with α-synuclein-GFP was
greater than that associated with α-synuclein, an optical
144
density analysis was done in a vps34 strain expressing
GFP alone. Surprisingly, GFP was extremely toxic to
vps34 yeast. There was virtually no growth even after
72 hours. However, the parent strain expressing vps34
reached saturation density before 24 hours (figure 3).
Another Foreign Protein, LacZ, is also toxic to vps34
yeast
Green fluorescent protein (GFP) is a reporter gene
which is used extensively by scientists to study protein
localization in model systems. The toxicity associated
with GFP in vps34 yeast led to the analysis of another
reporter gene; LacZ. As seen in figure 4, the vps34
strain expressing LacZ, is highly toxic. There is barely
any growth in this strain up to 36 hours post induction;
however, the parent strain reaches saturation in only 36
hours. In vps34 yeast, toxicity due to expression of
LacZ is less than the toxicity associated with GFP.
Spotting analysis of vps34 yeast shows high toxicity
To further analyze the toxicity associated with the
vps34 strain, a dilution series spotting assay was
performed. The three familial α-synuclein mutants,
A30P- α-synuclein, E46K- α-synuclein and A53T- αsynuclein were transformed in wild type parent strain
and vps34 yeast. The empty pYES2 plasmid and GFP
were used as controls. Cells were plated on inducing
and non-inducing media (figure 5). Vps34 transformants
can be seen to have grown less than parent strain
transformants on non-inducing media. In inducing
media, parent strain transformants grow well. However,
vps34 transformants do not grow at all, with the
exception of some growth in the empty pYES2
transformant. Growth curve data showing toxicity in any
vps34 transformant expressing foreign proteins
supported the dilution series spotting data.
Figure 3. Vps34 yeast expressing GFP are extremely
toxic. No growth is observed even after 72 hours. Yeast
expressing vps34 reach saturation by 24 hours post induction.
Vps34 affects α-synuclein expression
Western analysis was done on parent strain and the
vps34 transformants to confirm α-synuclein expression.
Cell lysates were prepared after 24 hours of protein
induction. Lysates were run on two gels, one for the
blotting membrane (figure 6A) and one for the
Coomassie control (figure 6B). As expected, no protein
expression was seen in cells transformed with the
pYES2 vector (lane 1). Lane 2 showed the expected
size of the green fluorescent protein at about 36 kDa.
For wildtype and mutant forms of α-synuclein (lanes 36), bands were seen at 60 kDa, which is about 8 kDa
greater than the standard size of monomeric αsynuclein. In our S. cerevisiae model, α-synuclein was
previously shown to consistently migrate 6-8 kDa higher
than expected (Sharma et al., 2006). Band intensities of
wildtype and mutant α-synucleins in the parent strain
(4741) are comparable. In 4741 transformants, GFP,
wildtype and mutant α-synucleins showed multiple
bands, as seen in lanes 2 through 6. This indicates that
proteins are degraded either during cell lysis or in vivo
(Sharma et al., 2006).
In the empty pYES2 vps34 transformant, no
protein was seen, as expected (lane 7). Importantly, the
lack of vps34 completely suppressed α-synuclein
expression (lanes 8-12).
Figure 4. Growth curve analysis of LacZ in vps34 yeast. In
inducing media (bottom graph), vps34 yeast expressing LacZ
do not grow till about 36 hours post induction. At 36 hours,
cells expressing vps34 and lacZ reach saturation.
Galactose media is toxic to vps34 yeast
A growth curve analysis was done with the parent strain
and vps34 strain grown in SC-Ura glucose and SC-Ura
galactose (figure 7). Both strains reach saturation
Density by the 24 hour time point when grown in non-
145
Figure 5. Dilution series spotting.
4741 and vps34 transformants were grown on non-inducing (glucose) and inducing (galactose) media. Growth of vps34 yeast was much
less than parent strain on non-inducing media. When expressing GFP and α-synuclein, vps34 yeast did not grow at all. Slight growth was
seen in the pYES2 control.
Figure 6. α-Synuclein expression in vps34
(A) Western Blotting. α-synuclein was probed with a Anti–V5 AP 1° antibody. As expected, the empty pYES2 transformant showed no
protein. A band was seen at 36 kDa corresponding to expected size of GFP. Bands were seen for wildtype and mutant α-synucleins at about
60 kDa. Multiple bands are seen in lanes 2-6. No protein was seen for any of the vps34 transformants.
(B) Coomassie Staining. For parent strain 4741, band intensities are comparable, corresponding to equal amounts of protein being loaded.
However, for vps34 transformants, bands 7, 8 and 10 are darker, indicating that more protein was loaded in these lanes.
146
when GFP was tagged to the C- terminal of the αsynuclein gene. This increase in toxicity was shown to
be caused by the addition of GFP, as α-synuclein alone
was less toxic to vps34 yeast. This may have occured
due to some adverse effect of GFP on metabolism in
vps34 yeast. It can also be possible that α-synuclein
has a protective function, as α-synuclein-GFP was less
toxic than GFP alone. Research will have to be
conducted to study the interactions of GFP in vps34
yeast.
Studies using vps34 yeast have previously
been done (Katzmann et al., 2003); however, parent
strain 4741 was not used. The extreme toxicity
observed when expressing wildtype or mutant αsynuclein, GFP or LacZ is most likely a strain specific
sensitivity of the 4741 parent strain. Different strains of
yeast are known to be sensitive to different proteins.
Unpublished results from our lab show that the E46Kα-synuclein mutant is moderately toxic in the 4741
parent strain, but not in other isogenic strains like 5-1.
α-Synuclein expression in vps34 yeast
The absence of α-synuclein in the western blot
analysis, suggests that the cells decreased α-synuclein
expression in order to survive. Unpublished data from
our lab, show that in yeast knocked out for αketoglutarate dehydrogenase, a mitochondrial enzyme,
α-synuclein expression is also greatly reduced. Thus,
reduction in α-synuclein expression may be used by the
cell to survive.
Figure 7. Growth curve analysis of vps34 grown in glucose
and galactose. When grown in glucose, both strains reach
saturation density by 24 hours. In galactose, vps34 do not grow
until 24 hours post induction, however, the parent strain attains a
density of about 1.5 at the same time point.
Toxicity associated with galactose media
SC-Ura galactose caused toxicity in untransformed
vps34 yeast. Yeast prefer glucose, and are known to
grow slower in galactose media. However, SC-Ura
galactose caused more than usual toxicity in the
untransformed vps34 strain. It is possible that vps34
plays a role in galactose metabolism. This toxicity may
also be strain specific. The effect of galactose could be
studied in vps34 knockouts in other yeast strains to
determine if galactose toxicity is a general
phenomenon.
The MVB sorting pathway to the lysosome
has been implicated in α-synuclein degradation
(Willingham et al., 2003). Knocking out vps28, a
component of the ESCRT-I complex of the MVB
pathway was shown to cause toxicity in cells
expressing wildtype and mutant forms of α-synuclein
(Price and Shrestha, 2005). In this study, we have
shown that α-synuclein causes non-specific toxicity in
vps34 yeast. Future research will examine several other
vps proteins, like vps22 and vps27 in mediating αsynuclein toxicity.
inducing media. However, the vps34 strain grown in
inducing media hardly grew by the 24 hour point, while
the parent strain reached a density of 1.5. This shows
that galactose media causes toxicity in vps34 yeast.
Discussion
There is evidence to suggest that impaired degradation
of α-synuclein leads to the formation of α-synuclein rich
Lewy bodies (McNaught et al., 2002). Therefore,
increasing the rate of protein degradation could serve
as a potential therapy. Genetic and chemical evidence
points to the role of the ubiquitin proteasome system in
degrading α-synuclein. However, studies have shown
that the lysosome also degrades α-synuclein (Webb et
al., 2003; Lee et al., 2004; Cuervo et al., 2004).
Absence of vps28, a component of the MVB sorting
pathway to degradation via the lysosome has been
shown to increase α-synuclein toxicity (Willingham et
al., 2003; Price and Shrestha, 2005). In this paper, we
examined the role of vps34 a PI 3-kinase, in the MVB
pathway, in mediating α-synuclein toxicity.
Acknowledgements
I would like to thank my advisor, Dr. Shubhik K.
DebBurman, who guided and supported me throughout
this project. I would also like to thank Dr. Karen Kirk for
allowing the use of her laboratory and equipment. This
work was supported by a grant from the National
Institutes of Health.
Foreign proteins including α-synuclein are toxic to
vps34 yeast
Vps34 is a PI 3-kinase which phosphorylates PI on
endosomes to PI(3)P. Endosomal membranes
containing PI(3)P, then target vps27 which binds to
ubiquitinated cargo on endosomes and recruits and
activates the ESCRT-I complex (Katzmann et al.,
2003). The kinase activity of vps34 is probably not
restricted to endosomal membranes and is essential for
survival when foreign proteins, like α-synuclein, GFP
and LacZ are expressed.
The toxicity associated with untagged
wildtype α-synuclein in vps34 yeast was exacerbated
Methods
Strains and Transformation:
A30P and E46K mutant α-synuclein were created using
site directed mutagenesis from human wild type αsynuclein (Invitrogen). A53T mutant alpha-synuclein
147
and human wild type α-synuclein cDNAs were gifts
from Christopher Ross (John Hopkins University). In
order to tag the synuclein cDNAs with GFP, the
synuclein cDNAs were subcloned into the mammalian
pcDNA3.1/C-terminal
GFP
expression
vector
(Invitrogen). After amplification, the synucleins were
subcloned
into
pYES2.1/V5-His-TOPO
yeast
expression vector (Invitrogen). α-Synuclein expression
plasmids were transformed into competent E. coli
grown on LB ampicillin media for selection. Plasmids
were then isolated and transformed as described
(Burke, 2000) into URA-3 deficient S. cerevisiae 4741,
and vps34 strains. Yeast cells were grown on syntheticcomplete media lacking uracil (SC-Ura) for selection.
PCR was used to confirm the presence of α-synuclein
in the pYES2.1 expression plasmids. α-Synuclein
expression was controlled with the galactose inducible
promoter(GAL1) in the pYES2.1 vector. The parent
pYES2.1expression plasmid (Invitrogen) and GFP in
pYES2.1 vector were used as controls.
proteins of interest. Membranes were placed in 10mL of
blocking solution (Western Breeze) and incubated for
30 minutes on a rotary shaker set at 1 revolution/sec.
Membranes were rinsed twice with 20 mL of H2O for 5
minutes each time. Primary antibody solution was
prepared by diluting the Anti–V5 AP 1° in 7mL of H2O,
2mL Blocker/Diluent (Part A) and 1mL Blocker/Diluent
(Part B). The dilution was 1:2000 (5 µL in 10mL of
Primary Antibody Diluent). Membranes were incubated
with 10mL of Primary antibody diluent for 1 hour.
Antibody was removed and saved, and then
membranes were washed four times with 20 mL of
Antibody Wash for 5 minutes each time. For PGK
controls, the membranes were incubated in 10mL of
Secondary Antibody Solution for 30 minutes, and
washed four times with 20 mL of Antibody Wash for 5
minutes each time. Membranes were then rinsed with
20mL of water for 2 minutes, thrice. 5mL of
Chromogenic Substrate was used to incubate the
membranes until bands of desired intensity could be
seen. Membranes were washed again with 20 ml of
water three times after which they were dried on a
clean piece of filter paper under infrared light.
Cell Lysates:
BY 4741, and vps34 knockout strains with wild-type αsynuclein-GFP, A30P-GFP, E46K-GFP, A53T-GFP,
GFP and parent plasmid were grown overnight at 30˚C
in a shaking incubator at 200rpm in 10mL SC-URA
glucose. Cells were washed three times with 10mL H2O
and resuspended in 2ml H2O. To induce protein
expression, 1mL of cell suspension was used to
inoculate 25ml of SC-URA galactose. Cultures were
then incubated for 24 hours at 30˚C, in a shaking
incubator at 200rpm. Cells in each culture were counted
to determine the cell density. 2.5 x 107 cells were taken
from each culture and washed with 1ml 50mM Tris (pH
7.5) and 10mM NaN3 [ 100ml: 95mL of H2O; 5mL of
Tris 1M pH 7.5; 0.06501g NaN3]. Cells were
resuspended in 30µL Electrophoresis Sample Buffer
[(ESB) 2% SDS, 80mM Tris (pH6.8), 10% glycerol,
1.5% DTT, 1 mg/mL bromophenol blue], and various
protease inhibitors and solubilizing agents [1% Triton-X
100, ImM phenylmethylsulfonyl fluoride (PMSF), 1mM
benzamide, 1mM sodium orthovanadate, 0.7µg/mL
pepstaton A, 0.5µg/mL leupeptin, 10µg/mL E64,
2µg/mL aprotinin and 2µg/ml chymostatin]. The cell
mixtures were then vortexed and heated at 100˚C for 3
min. 0.3 grams of 0.5mm glass beads were added to
the cell mixtures and vortexed for 2 minutes. 70 µL ESB
was added to each tube and samples were heated
again at 100˚C for 1 minute.
After protein samples were run on the 10-20% TrisGlysine SDS gel, the gel was stained with Coomassie
Blue [Coomassie Staining Solution:50%
(v/v)
methanol,0.05% (v/v) Coomassie brilliant blue R-250
(Bio-Rad or Pierce), 10% (v/v) acetic acid, 40% H2O].
The solution was prepared in deionised water, by
dissolving the Coomassie brilliant blue R-250 in
methanol before adding acetic acid and water. Staining
was carried on for two hours. The gel was then
destained with Destaining Solution [7% (v/v) acetic
acid, 5% (v/v) methanol, 88% H2O], overnight on a
rotary shaker. The gel was then washed three times, for
two minutes each time in deionised water (50mL).
35mL of Gel-Dry Drying Solution was added to the geltray and shaken for 5minutes in the StainEase gel
Staining Tray. A sheet of cellophane was immersed in
the Gel-Dry Drying Solution for 20 seconds, after which
it was placed on one side of the DryEase Gel Drying
Frame. Another wetted cellophane was put on top of
the gel and air bubbles and wrinkles were removed.
The frame was aligned and the plastic clamps were
fastened onto the four edges of the frames. The gel
dryer assembly was allowed to sit upright on a bench
top for 48 hours.
Western Analysis:
Toxicity Analysis:
20µL cell lysates were loaded into 10-20% Tris-Glysine
SDS gels (Invitrogen) and electrophoresed in 1x TrisGlycine SDS running buffer [(diluted to 1x from 10x):
29.0g Tris Base, 144.0g Glycine, 10.0g SDS, 1.0L Di
H2O, pH 8.3], at 130 volts.10µL of SEEBLUE protein
ladder was used. Gels were then transferred onto
polyvinylidene difluoride (PVDF) membranes, in 1x
transfer buffer [(diluted to 1x from 25x): 18.2g Tris base,
90.0g Glycine, to 500mL DiH2O, pH 8.3]. PVDF
membranes were presoaked in methanol, H2O and 1x
transfer buffer. The PVDF membrane was placed on a
foam pad immersed in 1x transfer buffer. The gel was
placed on the PVDF membrane and another foam pad
soaked in transfer buffer was placed onto the gel. The
protein was transferred for 1.5 hours at 30 volts.
Western
Breeze®
Chromogenic
Immunodetection protocol was used to probe for the
Growth Curve: For the OD600 analysis, transformed
knockouts and the 4741 parent strain were grown
overnight in 10ml of SC-URA glucose at 30°C in a
shaking incubator at 200 rpm. Cells were harvested at
1500 x g for 5 min at 4°C, and were washed twice with
5 mL H2O. Cells were resuspended in 10 mL H2O and
were counted. Flasks with 35mL SC-URA galactose
and 35ml SC-URA glucose (for controls) were
inoculated to a 2.0 x 106 cells/mL density. Absorbance
readings were taken at 0,3,6,12,18,24,36 and 48 hours
at 600nm using a Hitachi-U-2000 Spectrophotometer.
Absorbance readings were plotted against time points
to produce a growth curve.
Coomasie Blue Staining:
Spotting:
Transformed knockouts and 4741 parent strain were
148
grown in 10mL SC-URA glucose overnight at 30°C in a
shaking incubator at 200rpm. Cells were harvested at
1500 x g for 5 min at 4°C, and were washed twice with
5 mL H2O. Cells were resuspended in 10 mL DI H2O
and counted. 2.0 x 107 cells/mL were removed from the
cultures and resuspended in 1mL H2O. 100µl of this
culture was added to the first lane of a microtiter plate.
The next 5 lanes contained 80µl H20. 20µL of the
100µL of culture was removed from the first lane and
added to the second. After mixing, 20 µL from the
second lane was pipetted and added to the third lane
and so on, until there were 5 lanes with five-fold serial
dilutions for each cell culture. These cells were plated
by inserting a frogger into the microtiter plate and
plating cells onto SC-URA glucose and SC-URA
galactose media plates. Plates were grown at 30°C for
3 days and pictures were taken.
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Alpha-Synuclein Aggregation and Membrane

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