Saltman | Quarterly
Undergraduate Biological Research Publication UCSD Division of Biological Sciences
Volume 5 Nos. 1, 2, & 3 sq.ucsd.edu
Division of Biological Sciences University of California, San Diego Volume 5 / 2007-2008
Letter From
the
Saltman Quarterly
Editors:
Editors:
Dear Readers, Five years ago, three students, Marika Orlov, Louis Nguyen, and Greg Emmanuel, founded this journal and named it in honor of Dr. Paul D. Saltman. Dr. Saltman was the former Provost of Revelle College and Vice Chancellor of Academic Affairs; but to students, he was more than just an authority figure. Dr. Saltman was an inspirational and dedicated supporter for students. As editors who have been with the Saltman Quarterly for the past two years, we have personally seen the overwhelming amount of support and dedication that our faculty, our deans, and our advisor have shown to us and to this journal. They have not only been there to listen and to guide, but they have also encouraged us to think outside the box, to go for it, and to keep going. Over these past two years, we have also seen your interest in the Saltman Quarterly grow from curiosity into a tangible investment of your participation in the Review Board and/or Staff, your readership, and your efforts in strengthening the breadth and quality of undergraduate research. This year marks the fifth year that the Saltman Quarterly has proudly been able to serve and support undergraduate biological research at UC San Diego. We grew from a simple cause to publish undergraduate research into an organization and journal that wishes to also inspire more undergraduates’ curiosity in the sciences, to inform undergraduates of the different research aspects and opportunities there are available, and to serve as a learning tool on how to conduct research in the sciences. Overall, our goal has been and will always be to support you as a scientist, a researcher, and especially as a fellow student. It is our honor and privilege to carry on this tradition of dedication to students that Dr. Saltman had inspired and still inspires today. And it is our hope that you continue to draw on the journal for inspiration, encouragement, and support for all of your endeavors. Thank you for, what has been for us so far, an amazing journey.
Text:
Technical Editor: Daryush Tabatabai Research Design: Flori Pierri Features Design: Michael Wang
Graphics: Technical Editor: Caitlin Rodriquez
Web: Webmaster: Christine Ha
Publicity: Communications: Yu-li Wang
Staff Writers:
Tyler Green , Emily Hao, Seungleal (Brian) Paek , Marielle Venturino
Review Board Managers:
Christine Cho and Billy Wang
Staff Advisor:
Patricia Walsh
Faculty Advisors:
Best regards,
Editor-in-Chief: Quynh-Anh Mai Research: Brannon Peralta Features: Alex Fortenko Production: Jason Ly
Quynh-Anh Mai
Brannon Peralta
Gabriele Wienhausen, Ph.D. Associate Dean for Education Eric Allen, Ph.D. Molecular Biology
On
the Cover: This photo was taken by Sharon Wong during a trip to the Butterfly Spring in Southern China (Dali) in December 2006. Sharon is a senior majoring in biochemistry and cell biology at Thurgood Marshall College.
Supported by:
Lisa Boulanger, Ph.D. Neurobiology Robert Schmidt, Ph.D. Cell & Developmental Biology Christopher Wills, Ph.D. Ecology, Behavior, & Evolutionary Biology
Feature Articles Discovering Purpose Behind “Junk” a commentary by Tyler Green p.4 New Technology May Lead to Breakthrough in Pancreatic Transplantation by Emily Hao p.6
Minji Cho
HIV Immunity by Seungleal (Brian) Paek p.8 New Treatments May Bring Hope to Millions of Adults and Children Afflicted with Obsessive Compulsive Disorder by Marielle Venturino p.10
Research Articles Effect of Canopy Height on the Microbial Community of Tank Bromeliads Tobin J. Hammer, Winter 2008, p.12
Nicholas Bridalvail
Transient Global Amnesia: An Investigation into Its Pathophysiology Janet Kwok, Spring 2008, p.16 Analysis of Id3 Protein as a Possible Stem Cell Marker in Skin Eun Kyung Joanne Lee, Fall 2007, p.26 The Effects of Cisplatin, a Chemotherapeutic Drug, on the Immune System of Cell Line TRAMPC-1 Halley Park, Spring 2008, p.30
Review Articles Optogenetics: A Novel Approach for Neuroprosthetic Interface Michael Fenlon p.22
Rebecca R. Filbrandt
Neural Prosthetics: Developing a Safe, Rehabilitative Conversation with the Brain Alex Kawas p.24
Senior Honors Thesis Abstracts p.34 Dedication to Dr. Saltman p.37 Staff & Acknowledgments p.38 Division of Biological Sciences, University of California, San Diego 3
Sharon Wong
In This Issue
Discovering Purpose Behind “Junk” A Commentary by: Tyler Green with function in a very purposeful and specific manner. During the past year, headlines were made over the discovered purpose of the human appendix.4 Historically speaking, this organ was once thought to have no significant use or function for humans and to merely exist as a vestigial structure or relic from our evolutionary past. This mode of explanation fits well within an evolutionary framework made popular by the renowned evolutionary biologist Stephen Jay Gould. According to Gould and fellow evolutionary biologist Richard Lewontin, evolution operates not on separate and independent genes or traits, but on the organism as a whole. If, as they say, organisms are “integrated wholes, fundamentally not decomposable into independent and separately optimized parts,” then we should not expect every part of the organism to be useful or optimized.5
E
ver since Aristotle launched what we know today as biology, the belief has been widespread that nature makes nothing superfluous and does nothing in vain.1 So long as this mindset was engrained upon students of nature,2 all parts of organisms were believed to exist for a reason and perform an important and vital function. This mindset affected scientists in that they were unwilling to label any organ, trait, or behavior as lacking a function or as not contributing towards the good of the organism. Ever since the Darwin-
ian revolution in the biological sciences occurred, however, researchers no longer feel compelled to find evidence for design or optimality in the components of life.3 This is because evolution may very well produce and maintain useless artifacts, just as it may likewise produce organisms that exhibit complexity and design under the influence of natural selection. Either view is consistent with evolution. Amidst this disagreement over the process of evolution, recent discoveries seem to indicate that on both large and small scales, from organs such as the human appendix to so-called junk DNA, life is imbued
4 Saltman Quarterly Volume 5 / 2007-2008
Another view within evolutionary research, referred to as the ‘adaptionist program,’ argues that natural selection acts on life in a very robust and specific manner. Essential to this view is the optimizing nature of natural selection and its ability to tailor the individual components of organisms for their special functions. With regards to the human appendix, it seems that believers in widespread functionality and usefulness have finally been vindicated. The appendix’s curious relationship to the immune system has been recognized for a while, but a new study shows it functions as a ‘safe house’ for beneficial bacteria to live and repopulate the bowels in case of an infection.6 Tyler Green is a junior majoring in physiology and neuroscience and in philosophy. He is a student at Thurgood Marshall College.
For example, an extreme case of diarrhea may empty the entire contents of the bowels along with its protective mucus lining. Following this infection, the good bacteria living in the appendix can then take up residence in the bowels before harmful ones have a chance to take hold. The team of researchers from Duke University Medical Center conclude, “The identification of a potential function for the human appendix… lends credence to the idea that the structure is not a vestige, but rather is derived for a specific function.” 7 The discovery of the elusive role of this poorly understood structure only came about after these scientists refused to accept the commonly held view that the appendix has no purpose, and instead insisted that it possesses a function that we had yet to determine. This spirit of discovery extends beyond the level of large-scale organs to what is referred to as ‘junk’ DNA. A team of scientists at the University of California, San Diego (UCSD) medical school have recently demonstrated that a major class of ‘junk’ DNA is not junk, but does in fact have a function.8 It is known that during the course of evolution, once-functional DNA can be rendered non-functional through cer-
tain random biochemical or physical events. Knowing this can happen, researchers have often explained certain portions of the genome as being useless in their own right, but existing and being passed on because of their attachment to a portion of the genome that is functional. However, as UCSD professor William Bechtel points out, ”when dealing with biology, it’s a bad assumption to say something is junk.” 9 In 2007, a team of UCSD researchers headed by Michael G. Rosenfeld studied a highly repetitive sequence of DNA called SINE B2, which is found on the growth hormone gene locus. Rather than being a genetic fossil with no current function, the researchers found that this DNA plays a critical role in organizing gene expression. In order for genes to be expressed, a portion of the genome must contain the appropriate regulatory signals that tell genes when to turn on and off. In their study, Rosenfeld et al. found the SINE B2 sequence functions to bracket or set apart this regulatory region so that when the DNA is being transcribed or read, it is not some long run-on sentence, so to speak. As researcher Victoria Lunyak put it, “Some of the ‘junk’ DNA might be considered ‘punctuation marks’ – com-
mas and periods that help make sense of the coding portion of the genome.” 10 This exciting study contributes to a growing body of literature that identifies functions for most major classes of ‘junk’ DNA. In both of the cases described above, assuming that an unexplained part of an organism is useless impedes scientific advance and brings a halt to scientific inquiry. A review of the history of science demonstrates that scientists should be slow to say a part of an organism has no function, or carries out its function poorly. What researchers need instead is a commitment to the view that life is full of function, whether on the large or small scale. This approach will help researchers to look at things more closely and be hesitant to dismissing an unexplained part as ‘lacking a purpose’, to use the words of Aristotle. All the better, emerging researchers should seek out and study that which is currently deemed useless or badly designed, for great discovery may lie around the bend. References
1. On the Parts of Animals, iii 1. trans. Ogle. This principle is found ubiquitously throughout Aristotle’s writings. Consider, “Nature never makes anything without a purpose and never leaves out what is necessary” On the Soul. iii 9, trans. Smith. 2. The natural scientist (or phusiologos) 3. This period was initiated by the publication of Darwin’s On the Origins of Species in 1859. 4. “Appendix Isn’t Useless at All: It’s a Safe House for Bacteria,” www.dukehealth.org/HealthLibrary/ News/10151. 9 Oct. 2007. 5. Gould, S. J., and Lewontin, R. C. “The Spandrels of San Marco and the Panglossian Paradigm: A Critique of the Adaptionist Programme,” Proceedings of the Royal Society of London, Series B, Vol. 205, no. 1161 (1979), 581-595. 6. Bollinger, R. R., et al. “Biofilms in the large bowel suggest an apparent function of the human vermiform appendix,” Journal of Theoretical Biology 249 (2007) 826-831. 7. ibid. p. 830. 8. Lunyak, V.V., et al. “Developmentally Regulated Activation of a SINE B2 Repeat as a Domain Boundary in Organogenesis,” Science, Vol. 317, no. 5835, pp. 248-251. 9. Bechtel, W. Philosophy of Biology, lecture notes. 4 Dec. 2007. 10. Kain, D. “One Man’s Junk May be a Genomic Treasure,” http://ucsdnews.ucsd.edu/newsrel/health/07-07. GenomicTreasureDK-.asp. 12 July 2007.
Division of Biological Sciences, University of California, San Diego 5
New Technology May Lead to Breakthrough in
Pancreatic Transplantation By: Emily Hao
A
ccording to statistics from the American Diabetic Association, over 20 million people, or about 7% of the population of the United States, are afflicted with diabetes. Though two types exist, recent scientists at the University of San Diego, California have been working on expanding upon a recent European discovery in the hopes that it may replicate pancreatic cells for increasing the pool of patients who would receive life saving cell transplants in the future, thereby eliminating further treatments for Type 1 diabetes. In the laboratory of Dr. Fred Levine at the UCSD Burnham Institute of Medical Research, a team of scientists have been working relentlessly in an attempt to replicate the results documented in the study by German and Belgian scientists published in the premier science and research journal Cell last year.
The pancreas of a mouse during the ligation surgery is dyed with a stain to help locate the pancreatic duct.
In those affected by Type 1 diabetes, the β cells of the pancreas responsible for producing the hormone insulin are suddenly terminated by the body’s own defense mechanisms due to unknown reasons. Though it is unclear as of now why the body’s autoimmunal response targets these cells, we do know that in Type 1 diabetes, the white blood cells and lymphocytes of the blood do not recognize the insulin-producing β cells as part of the necessary components of the body, but instead as a foreign and potentially harmful particle that must be immediately destroyed. The termination of these cells then leaves the individual with an inability to produce insulin, which plays a crucial role in the
regulation of blood glucose levels in the human body. The normal secretion of insulin into the bloodstream stimulates glucose within the blood to be drawn into liver and muscle cells, which then are stored as glycogen or used as energy. When this function does not occur, glucose molecules remain circulating in the bloodstream at abnormally high levels, causing a variety of damaging symptoms that could potentially be fatal to the patient. If left untreated, body tissues begin to die due to lack of energy to carry out their normal functions, leading to cataracts in the eyes, renal function failure, and possible necrosis and amputation among others. In the most serious scenario, Type 1 diabetes
6 Saltman Quarterly Volume 5 / 2007-2008
could be fatal to the patient due to its triggering of kidney failure. The kidneys are the filters of the bloodstream, and without them, ammonia cannot escape but instead circulates to the neurons of the brain, which then cause them to lose their functions due to its toxic properties. Even since the pancreas was first discovered, it has been universally known among the scientific community that once the β cells of the pancreas die, it is not possible for them to regenerate or regain former functions. The currently known treatments for this condition are
Emily Hao is a freshman majoring in biochemistry and cell biology. She is a student at Thurgood Marshall College.
A laboratory mouse recovering from the anesthesia after the completion of surgery.
regular insulin injections or a transplantation of the islet cells of the pancreas, composed primarily of β cells, from organ donors to the patient. The latter is clearly the more permanent and ideal solution, yet a drastically uneven ratio of donors to patients and rising diabetic diagnosis rates have encouraged Dr. Levine’s team to devise alternative ways of generating these cells for transplant. In the study published, scientists had miraculously caused the regeneration of β cells in a mouse by closing off the pancreatic duct with sutures (a form of surgical stitching), causing digestive enzymes which normally flow into the duodenum section of the small intestine to backflow into the pancreas itself. These enzymes contain powerful digestive properties normally used to break down lipids and proteins from our food sources, thus when trapped within the closed duct and flowing back into the pancreas, it causes inflammation known as pancreatitis and the death of nearby β cells. The European scientist who first conducted the study noticed that despite potentially fatal pancreatic damage,
“
the β cells nearest the duct would die but some around it would regenerate and grow more cells to replace them. Such astonishing results inspired a new idea for the Levine team: if they could replicate the results of the mouse model, they would also be able to discover isolate the gene which stimulates the new growth, opening up the possibility of ‘growing’ human β cells outside of the body in a laboratory environment on a larger scale, consequently allowing many more patients to receive life-saving
... over 20 million
people, or about 7% of the population of the United States, are afflicted with diabetes.
“
transplants. Though seemingly simple and brilliantly devised, the experiment is still in its early stages at the Levine lab and scientists within, such Ergeng Hao, whom is directly responsible for performing ligation, the surgery necessary to close the pancreatic duct are optimistic nonetheless about its advancement in the future. “It can most certainly be done, but it will definitely take some time. The surgery is invasive and difficult but we are confident that once we can replicate the results in the mouse, we can then aspire to produce the same results in a human,” says Ergeng when asked of the progress made on the project. With the most advanced equipment and talented personnel available here at the University of California, San Diego, Dr. Levine and his team are confident about the future of their research. Though so far Type 1 diabetes has no known cure, only treatments to alleviate the symptoms of patients, teams such as Dr. Levine’s are the pioneers of diabetic research whom will push the boundaries of medicine in order to discover new and advantageous tactics to ultimately win the battle over the disease that affects so many worldwide. *The author obtained much of her information and insights from interviews with Dr. Fred Levine and Ergeng Hao, from the Levine Laboratory at the Burnham Institute, La Jolla, Calif.
A close up view of the pancreas, the surgery is performed under a high magnification microscope due to its size and difficulty.
Division of Biological Sciences, University of California, San Diego 7
HIV Immunity By: Seungleal (Brian) Paek
I
magine a man, robbed of his health and spirit, desperately searching for a ray of hope he suspects will never come. As the days crawl by, a deadly, unknown force slowly conquers his body. He sees no future, no light, and his dreams are withering away with his health. He soon accepts his somber fate and gloomily awaits his defeat by a veiled foe. This man has lost control of his life to the pervasive and unstoppable Human Immunodeficiency Virus, otherwise known as HIV.
ther partially or completely immune to HIV, that CCR-5 receptor might have a mutation that causes it to seal its openings to the HIV molecule. Consequently, scientists have figured out that the HIV cannot inhibit white blood cells if there are no CCR-5 main “gates” to break in. As a result, scientists have started to decode the DNA sequences for this particular mutation of CCR-5 receptors in an effort to incorporate this discovery into the development of HIV vaccines.
This one man’s devastating ordeal represents that of 36.1 million people worldwide. There have been, however, a few more fortunate people who have managed to survive the disease. As researchers uncover more and more tales of survival, they have begun to look into why some people are resistant, and, in some cases, immune to the virus. Some of their recent discoveries offer promising leads to understanding HIV immunity.
Although this finding has not yet been translated into a new treatment, many pharmaceutical companies are working on drugs that render the CCR-5 receptors inoperative and thus minimize the number of HIV pathogens that can infect cells. Some of these types of drugs are already at the clinical trial stage and may be the most promising HIV treatment to date.
One such discovery lies in a genetic mutation found in some people’s hereditary ethnic backgrounds as well as the illnesses experienced by their farflung ancestors. The HIV infects and overtakes white blood cells through the CCR-5 receptors on the surface of human cells. Without these receptors, the HIV cannot gain access into the cells. In the cases of a few people who are ei-
Another mutation that could ensure natural immunity against HIV was found in a recent study of Gambian prostitutes. Researchers from England, Gambia, and Japan worked with 20 female prostitutes in the western African nation. These women had been constantly exposed to HIV, yet have not developed AIDS. Interestingly, their immunity seems to stem not from blockage of CCR-5 receptors, but from constant exposure to the virus. Due to the frequen-
8 Saltman Quarterly Volume 5 / 2007-2008
cy of exposure, these women’s genes had mutated to contain highly specific cytotoxic T-lymphocytes (CTLs) that counterattack HIV infected cells in the body. Scientists now believe that CTLs work as protective agents and resisters against the spread of the HIV. A combination of these counterattacks along with the immunity genes of the CCR-5 mutation could eventually lead to an effective cure or treatment for the HIV. In addition, scientists took interest in infected Gambian women whose children were born free of the HIV. As David Ho, a researcher at the Aaron Diamond AIDS Research Center in New York explains, “these rare patients provide a ray of hope to researchers and infected patients that it is possible to co-exist with HIV.” Studies are underway to determine if there is a possibility of inducing these CTLs without the exposure to HIV, in an effort to build immunity. Another treatment may lie in what are called the “jumping genes.” Long-term research has shown that the HIV developed from a chimpanzee retrovirus named SIV, or Simian Immunodeficiency Virus. For a virus to travel ef-
Seungleal (Brian) Paek is a sophomore majoring in biochemistry and cell biology. He is a student at John Muir College.
fectively from one species to another, it has to overcome the new host’s innate immune system, also known as TRIM5. This system doesn’t allow retroviruses to enter from other species. Unfortunately the human TRIM5 fails miserably at its task and allows the HIV infection to enter. However, a research team led by Professor Greg Towers and the UCL Infection and Immunity Group found that a species of Rhesus Macaque, a type of Asian monkey, has a TRIM5 that successfully protects it from retroviruses. By studying these systems closely, the scientists discovered core genes responsible for the immunity, which are collectively referred to as ‘jumping genes’. These jumping genes are capable of creating new DNA codes that are not descended from one’s ancestral background, but are made by new genes being placed in the body and then merge with existing genes. One specific case has been closely examined in Rhesus monkey species. Rhesus monkeys have exhibited a tremendously remarkable ability for merging existing genes with jumping genes to form genes that defend against the HIV. These new mutat-
ed cells, called TRIMPCyp cells, have the combined distinctive characteristics of the genes to detect viruses such as HIV and destroy them the moment they break into a species’ immune system. Scientists are now occupied with applying the discovery of this unique fusion of jumping genes and normal genes to possible synthesis of TRIMPCyp cells and human stem cells. A treatment involving these altered stem cells might have the potential to ensure immunity to HIV for humans. While most HIV immunity researchers believe the genetic mutations have high potential for providing a cure or effective treatment for HIV, there are also a those who oppose this line of research. Dr. Donald Mosier,a professor of immunology at The Scripps Research Institute in La Jolla, California, published a report in the journal Nature which disproved the plague theory. The plague theory states that those who survive a deadly disease create their own way of combating the plague; therefore, in the future, it is theorized that they will not be affected by a similar epidemic. During Mosier’s experiment,
mice injected with the AIDS-protective gene mutation ended up contracting the plague. Such findings lead researchers to believe that the plague theory could be false. With such conflicting positions, professionals claim that even though numerous researchers are working on the subject, a cure or effective treatment for HIV is far off. With greater attention and support for HIV research it is hoped that it is just a matter of time until scientists develop a vaccine for HIV. References: 1. Dotinga, R. “Genetic HIV Resistance Deciphered.” Wired 7 January. 2005. <http://www.wired.com/medtech/health/ news/2005/01/66198> 2. Jones, R. “Natural Immunity To HIV.” Arctica January. 1995. <http://www.accessexcellence.org/WN/SUA04/natural_immunity_HIV.php> 3. Oxford University. “Prostitutes Lose HIV Immunity.” BBC News 26 Jan. 2000. http:// news.bbc.co.uk/1/hi/health/619316.stm 4. University College London. “Imitating Monkey’s ‘Jumping Genes’ Could Lead To New Treatments For HIV.” ScienceDaily 19 February. 2008. <http://www.sciencedaily. com/releases/2008/02/080218172305.htm>
3-D rendered HIV virus.
Division of Biological Sciences, University of California, San Diego 9
New Treatments May Bring Hope to Millions of Adults and Children Afflicted with Obsessive Compulsive Disorder By: Marielle Venturino
I
magine being afflicted with a disorder that hindered the very fundamental functions that are performed on a routine daily basis. What if leaving your house does not simply mean dimming the lights and locking the door? What if instead, you are coerced by an invisible force to flip the light switch15 times, and check the lock 22 times, before finally being able to leave? Sadly this is the reality for one in 40 adults and one in 200 children in the United States who are plagued with obsessive compulsive disorder (OCD).1 OCD is characterized by obsessions (involuntary repeated thought impulses and undesired im-ages that cause anxiety) and compulsions (voluntary behaviors performed in response to obsessions as to reduce anxiety or neutralize unwanted thoughts). Compulsions may include hand washing, checking all the locks in the house, counting steps or turning lights on and off. 2 In addition to clinical OCD, there are
multiple obsessive compulsive related disorders that often overlap with OCD. These may include major depressive disorder, anxiety and anorexia nervosa. Others include body dysmorphic disorder where a person imagines that there is a substantial defect with their body other than weight; depersonalization disorder where one feels detached from oneâ&#x20AC;&#x2122;s own body or mind; and Touretteâ&#x20AC;&#x2122;s syndrome which is characterized by a combination of motor and vocal tics, sexual compulsions, impulsive personality disorder, and pathological gambling.3
10â&#x20AC;&#x192; Saltman Quarterly Volume 5 / 2007-2008
OCD ranks fourth among neuropsychiatric disorders in the United States and thus is a major topic of recent research. In the January 2008 issue of Molecular
Mari Venturino is a sophomore majoring in physiology and neuroscience. She is a student at Eleanor Roosevelt College.
weeks prior to, as well as throughout, the study.4
“
Obsessive compulsive disorder ranks fourth among neuropsychiatric disorders in the United States.
indicate that as few as four weeks of intensive daily cognitive and behavioral therapy (CBT) produces signifi-cant changes in activity in certain regions of the brain. Saxena’s study examined 10 adult patients with OCD (mean age 40.6 years), and 12 normal con-trols (mean age 46.4 years). Diagnostic testing was performed on the OCD patients prior to treatment to ensure a clinical diagnosis of OCD.4 Medications were kept constant in the OCD patients for the 12
A 3D magnetic resonance imaging (MRI) scan was also completed to compare changes in glu-cose metabolism in different regions of the brain. These findings were based on previous studies on OCD.4 The treatment component for the OCD patients consisted of daily sessions with a cognitive be-havior therapist, five days per week for 90 minutes. The same therapist was used for all patients. In addition to this therapy, four hours of homework was assigned daily as a supplement to the treatment. This homework was a way for the patients to monitor their progress outside of the daily sessions. There was no treatment administered to the normal controls.4
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Psychiatry, an article was published that described a significant treatment breakthrough for OCD., The study’s principal inves-tigator was Sanjaya Saxena, M.D., director of the Obsessive-Compulsive Disorders Program, of the University of California, San Diego School of Medicine. Saxena’s findings
Positron emission tomography (PET) scans were performed prior to treatment, as well as after four weeks, for the OCD patients, and after 10-12 weeks for normal controls in order to mini-mize habituation to the scanning procedures. PET scans take 3D image maps of functional sys-tems of the body after an injection of a radioactive substance is administered, typically through the bloodstream. These scans measured the cerebral glucose metabolism. The results prove sig-nificant in expressing the positive effects of the intensive therapy.4
The results of the study indicate that the OCD patients’ response to the intensive cognitive be-havior therapy (CBT) was very high. Nine of the 10 subjects were classified as responders, or showed significant improvement in symptoms as seen in a second administration of the diagnos-tic tests performed at the beginning of the study and an increase in brain activity.4 Normal con-trols did not exhibit any significant changes.4 CBT has been shown to have some effects similar to those of medications used by OCD patients, but has also resulted in some different effects on the brain. Current treatments for OCD include varying combinations of weekly CBT and selec-tive serotonin inhibitor medications,4 a common class of antidepressant medications often used to treat depression and anxiety disorders.3 A high percentage of subjects in the student responded well to treatment, as seen in other inten-sive CBT studies. Thus, a new approach to treating Obsessive Compulsive Disorder and Obses-sive Compulsive related disorders makes for a brighter outlook for treatment of patients with OCD.4. The small sample size of this study was a limiting factor. Future studies with larger sample sizes could measure the long-term effects of intensive CBT, as well as compare this method to longterm weekly CBT sessions. References: 1. Questions and Answers about Obsessive Compulsive Disorders. 2008. Obsessive Compulsive Foundation. 17 March. <http://www. ocfoundation.org/UserFiles/File/Questions-Answers-About-OCD.pdf>. 2. Frank Tallis. 1995. Obsessive Compulsive Disorder, (John Wiley & Sons, New York). 3. Eric Hollander et al. 1994. Current Insights in Obsessive Compulsive Disorder, (John Wiley & Sons, New York. 4. S. Saxena et al. 2008. Molecular Psychiatry.
Division of Biological Sciences, University of California, San Diego 11
Effect of Canopy Height on the Microbial Community of Tank Bromeliads Tobin J. Hammer
Junior, General Biology major, Eleanor Roosevelt College Education Abroad Tropical Biology and Conservation Program Abstract Epiphytic bromeliads trap water and organic matter, forming miniature ecosystems suspended in the canopy of forests. While these microcosms are rich in animal life, the most abundant and diverse organisms—the unicellular microbes—have long been overlooked in scientific research. Little is known about what biotic and abiotic factors affect the microbial communities in bromeliad tanks. This study tested the effect of above ground tank height on bacterial, fungal, and protozoan abundance in 24 bromeliads. Only fungi differed in abundance between low and high bromeliads. In addition, bromeliad size and canopy height affected fungal growth and protozoan abundance, respectively. Future research on the roles of microbes in bromeliad tank water is necessary for understanding basic ecological dynamics in this unique environment.
Introduction
affect bacterial community composition.8
Epiphytic bromeliads often function as phytotelmata, holding bodies of water that can support a diversity of organisms. These “tanks” support miniature ecosystems that can include amphibians, macro-invertebrates, microscopic metazoans, fungi, protists, and bacteria.12 Bromeliads serve as easily studied and experimentally tractable model systems for investigating larger-scale tropical habitats.1 Most studies of bromeliad tanks, however, have focused on aquatic insects and have ignored less conspicuous components of tank microcosms.4 Unicellular microbes such as bacteria and protozoans are key components of aquatic food webs, providing most of their biomass, respiration, nutrient cycling, and productivity.6 To properly examine the ecological community of tank bromeliads, these microbes should be taken into account.
One factor that could affect nutrient input is the altitude of an epiphytic bromeliad in a tree; plants lower to the ground should be subject to more falling detritus and nutrients compared to those high in the canopy. Higher plants are also exposed to heavier rainfall and higher winds, which can wash out tanks more frequently, suppressing long-term growth. Lastly, bromeliads high in the canopy experience strong temperature fluctuations, which can disrupt microbial communities. Supporting this idea, species richness of macrofauna decreased with increasing height of sampled bromeliads in a previous study.9 In addition, colonization experiments with artificial containers demonstrated that those placed closer to ground level received more protist species.12
Studies of organisms in Costa Rican bromeliads have found some environmental factors that affect variation in abundance and community composition. For example, a positive relationship between turbidity of tank water and species richness of protists has been demonstrated.2 High turbidity can be a correlate for a high amount of detritus, the most important resource for insect and microbial food webs in bromeliads.15 In another study, detritus was shown to increase the alpha diversity of invertebrates.5 It is reasonable to assume that conditions affecting the amount of organic matter input will also affect the composition of microorganisms in phytotelmata. In fact, previous studies have reported that these environmental factors, especially the availability of organic matter and nutrients,
To date, the effect of natural bromeliad height on contained microbes has not been considered. Bacteria, though integral in tank ecosystems as the basal trophic level, have only been studied as ecological components of bromeliad tanks twice, and in both cases only coliform bacteria were analyzed.9,14 Bacterial content can be used to infer information about the total biomass and productivity of the tank. Microfungi are another important link in phytotelm food webs and are present in bromeliads with a high diversity, but have also been understudied.11 To determine how canopy height affects microbial tank communities, epiphytic bromeliads sampled at two different heights on trees were tested for differences in protozoan, fungal, and bacterial abundance. It was hypothesized that bromeliad tanks at lower heights would con-
12 Saltman Quarterly Volume 5 / 2007-2008
tain a higher abundance of microbes, owing to increased detrital input and sheltering from extreme conditions. In addition, other physical factors that influence nutrient input to the ecosystem were analyzed for their effect on tank biota. Canopy cover, wind speed, tank water turbidity, and bromeliad diameter were also measured. Bromeliad size is positively related to total organic matter, faunal abundance, and tank volume, all of which may have effects on microorganism communities.13
Materials and Methods Twenty-four (24) bromeliads were sampled from three trees near the University of Georgia Station in San Luis, Puntarenas, Costa Rica. The trees were located in disturbed secondary forest or pasture, at an elevation of 1200 m. Sampling occurred on three separate days between November 15th and November 21st, 2007, in the morning (10:30-11:30am). Using ascension devices including rope, harness, and jumars, water samples were collected from four bromeliads at both 5 m and 10 m. At each bromeliad, canopy cover was measured with a canopy densiometer, height and plant diameter measured with measuring tape, and wind measured with a wind speed meter. Using a sterile syringe, 10 ml of water contained in multiple axils of each bromeliad tank were extracted. After withdrawal the water was deposited into a sterile urine sample container and placed in a container of ice. Within 24 hours the samples were returned to the laboratory and centrifuged for five minutes to separate particulate matter. 1 ml of the supernatant of each sample was extracted with a sterile syringe onto two petri dishes. One plate contained Mueller-Hinton agar medium, which supports the growth of a wide range of bacteria, and the other plate contained Potato Dextrose agar, designed to grow fungi (Alfonso Calvo, personal communication). Using a sterilized Grijalsky spreader, even coverage of each plate with sample was ensured. The petri dishes were incubated at 37°C for 24 hours for growth of bacteria and fungi.
Figure 1: Average abundance of fungi and bacteria, measured in percent growth, at each height on a tree (n=24). Error bars show standard deviations.
1200
% Growth
1000 800 Fungi Bacteria
600 400 200 0 5m
10m
Height on Tree
Since large amounts of carpet growth made it impossible to count colonies, a method was developed to measure the percent growth of bacteria or fungi on a plate. First, three 1/8th sections of a standard petri dish were covered in equal sized dots from the tip of a permanent marker, and the average was taken to get a value for the total area of a 1/8th section in dots. After plates were removed to room temperature, the surface of each was divided into eight sections. For each section analyzed, the area covered by growth was counted in dots and divided by the total average number of dots for a section. This percent growth was measured for each 1/8th section, and then averaged for all eight sections to get a value for percent growth on the whole plate.
ment and then shaken to homogenize the debris and organic matter. A drop of water was placed on a hemacytometer, an instrument with laser engravings that allows measurable and repeatable counting of cells. Protozoans were then analyzed under a compound microscope at 400X. Total abundance was estimated by counting protozoans in two 1 mm2 squares in the chamber, averaging them, and extrapolating the value to a per ml volume. Protozoan abundance was only measured from the last 16 bromeliads sampled, as water samples from the first eight were retained for too long before analysis of live organisms was possible. These water samples were then used to test turbidity with an instrument in the LaMotte Water Monitoring Kit.
The amount of bacterial and fungal growth was used as a correlate for the original abundance of organisms in the natural bromeliad tank. Controls were done for each media type using sterile water and an identical methodology, but no growth was observed.
The statistical software package JMP IN v4.04 was used to analyze data. T tests were conducted to determine the significance of height on bacterial and fungal growth and protozoan abundance, both overall and for individual trees. In addition, a t test was used to compare the four factors measured (canopy cover, plant diameter, turbidity, and wind speed) between heights. Regression analyses were run to test the effect of
Number of Protozoans (millions) per ml
The remaining amounts of water samples were frozen for 2-3 hours to slow protozoan move-
5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
5m
Height on Tree
10m
Figure 2: Average abundance of protozoans, extrapolated to a per ml level, at each height on a tree (n=16). Error bars show standard deviations.
these factors on microbial abundance.
Results Bromeliad tanks at lower tree heights displayed higher amounts of fungal growth (T=1.74, df=22, p=0.09; Fig. 1). There was no difference in bacterial growth (T=-0.669, df=22, p=0.51; Fig. 1) nor protozoan abundance (T=1.079, df=14, p=0.29; Fig. 2) between tanks sampled at the two heights. When each tree was analyzed separately, no height-related differences existed for any of the microorganism measurements (Table 1). This result allowed each tree to be treated as a replicate. From the four tree factors measured, there was a positive correlation between diameter and fungal growth (Table 2, Fig. 3). There was also a positive trend between canopy cover and protozoan abundance (Table 2, Fig. 4). However, there were no other effects of any factor on any of the microbial measurements (Table 2). None of the factors measured was different between tanks sampled at 5m and 10m (Table 3).
Discussion Microorganism abundance was expected to be higher in low height tanks, as they should receive more nutrient input and have a more stagnant aquatic environment, conditions that would favor the growth of a richer microbial community. However, only fungal growth exhibited a trend supporting this hypothesis. It is possible that fungi are more sensitive to changes in nutrient availability or fluctuations in the local environment compared to bacterial and protozoan populations. There are both natural and methodological explanations for the lack of a major difference between the microbial communities of tanks at low and high heights. The environment of epiphytes is heterogeneous, with varying branch or trunk cover and proximity to other epiphytes even among a similar height. This heterogeneity lends itself to varying microclimates and nutrient input available to individual bromeliad tanks. It is possible that this local heterogeneity was more important than canopy height in its effect on the biotic community. Another possibility is that the location of sampled trees minimized the variation in resource availability between high and low bromeliad tanks. The trees were located in disturbed forest or pasture and thus were isolated from surrounding vegetation and its nutrient contribution. In the absence of surrounding forest the whole
â&#x20AC;&#x192;â&#x20AC;&#x192; Division of Biological Sciences, University of California, San Diegoâ&#x20AC;&#x192; 13
Table 1: T test by tree of the difference in abundance from bromeliads tanks sampled at 5m and at 10m, for each group of microorganism analyzed.
Tree # 1 2 3 1 2 3 2 3
Microorganism Fungi Bacterial
df 6 6 6 6 6 6 6 6
T 0.83 1.40 1.00 -1.20 -0.06 1.60 0.63 1.50
P 0.44 0.21 0.35 0.28 0.95 0.17 0.55 0.18
transient sources of food, and is not necessarily conducive to the growth of organisms common in the environment.7 Studies using cultivationindependent methods, such as epifluorescence microscopy or DNA sequencing (for taxa identification), are recommended.
Physical factors also influenced microbial abundance in certain cases. For example, bromeliad diameter was positively associated with fungal Protozoans growth. Large bromeliad diameter allows for larger tanks that would catch and hold more water and detritus, contributing to a more active ecosystem. Again, it is possible that only fungi Table 2: Regression analyses of the effect of the four measured factors on the abundances of bacte- were sensitive enough to changes in nutrient levels to display a correlation with plant diamria, fungi, and protozoans. eter. Canopy cover also 2 had an effect on the Factors Equation R F N Microorganism P abundance of protozo0.21 24 0.07 1.63 y = 0.70x + 0.002 Canopy Cover Bacteria ans in the bromeliad 24 0.22 0.07 1.56 y = -0.15x + 0.009 Fungi tanks sampled. More 0.07 16 0.22 3.85 y = 784908x + 37330 Protozoans canopy cover relates to 0.20 24 0.07 1.71 y = 0.81x + 0.17 Diameter Bacteria more leaves, branches, 24 0.09 0.13 3.19 y = 0.11x + 0.78 Fungi or other nutrient sourc16 0.17 0.13 3.85 y = 2598338x + 1548007 Protozoans 24 0.19 0.08 1.87 y = 0.84x + 0.007 Turbidity Bacteria es over the bromeliad, 0.78 24 0.004 0.08 y = 0.48x + 0.0005 Fungi increasingly the likeli0.69 16 0.01 0.16 y = 3979551x - 5826 Protozoans hood of detritus falling 24 0.62 0.01 0.26 y = 0.89x + 0.003 Wind Bacteria into the tank. In addi0.98 24 0.00002 0.0005 y = 0.52x + 0.0004 Fungi tion, protozoans may 0.73 16 0.009 0.123 y = 3473887x - 23167 Protozoans be more susceptible to sunlight or temperature changes than bacteria or fungi. Further studies tree is exposed, which, in accordance with the microorganism abundance between heights lies should investigate in more detail the effect of results, could have effected a lack of difference in the sensitivity of the methods used to detect bromeliad size, canopy cover, and other biotic in wind strength and canopy cover between the it. The culture method used to analyze bacteria and abiotic factors on microbial communities. canopy and lower heights. Therefore, testing the and fungi may not accurately represent their aceffect of canopy height on bromeliad tanks in tual composition in bromeliad tanks. Plating on An especially interesting result from this study primary, undisturbed forest would be of value. a nutrient-rich medium selects for the organisms was the sheer number of protozoans extracted Another explanation for the overall similarity of that are adapted to take advantage of new and from bromeliad tanks. One tank contained more than 4.5 million protozoan organisms per ml of water, an incredibly high number. If one extrapolates this value to a gallon, a volume common in the tanks of large bromeliads, there can be over 17 billion individuals in one tank. This abundance illustrates the importance of protozoans as members of bromeliad tank ecosystems, and should emphasize the need for their inclusion in any further study of bromeliad ecology. Future research on the role of microorganisms in bromeliad tank ecosystems will shed light on basic ecological processes, such as energy flow and the role of microbes in aquatic food webs. These investigations also have implications for human health, as exemplified by
1.200
% Fungal Growth
1.00 0.800 2
R = 0.1265
0.600 0.400 0.200 0.000 0.3
0.4
0.5
0.6
0.7
0.8
Bromeliad Diameter (m) 14â&#x20AC;&#x192; Saltman Quarterly Volume 5 / 2007-2008
0.9
1
Figure 3: Regression analysis of the effect of plant diameter on fungal growth (n=24).
Figure 4: Regression analysis of the effect of canopy cover on protozoan abundance (n=16).
100
% Fungal Growth
80 R2 = 0.1265 60 40 20 0 0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Bromeliad Diameter (m)
Table 3: T test of the difference in factors between bromeliads at low and high heights. Measurement of each factor is given as an average, with standard deviation in parentheses.
Factor
5m
10m
T
df
P
Canopy cover (%)
68.97 (9.15)
72.10 (8.52)
-0.87
22
0.40
Diameter (m)
0.55 (0.20
0.49 (0.04)
1.09
22
0.29
Turbidity (JTU)
66.67 (43.76)
86.67 (29.95)
-1.31
22
0.20
Wind (mph)
2.08 (3.42)
1.92 (3.00)
0.13
22
0.90
a recent study that identified a novel antibiotic from the DNA of Costa Rican bromeliad tank microbes.3
trescine, an antibiotic isolated from the heterologous expression of DNA extracted from bromeliad tank water. Journal of Natural Products 67:1283-1286
Acknowledgments
4. Carrias, J., Cussac, M., and Corbara, B. 2001. A preliminary study of freshwater protozoa in tank bromeliads. Journal of Tropical Ecology 17:611-617.
Thanks to Ramsa Chaves for help with the project and data analysis, and to Carlos Alfonso Calvo for advice on microbiology and the use of his lab. I would also like to acknowledge Ruth Salas, Brian Lonquich, Federico Chinchilla, and Erick McAdam for assistance with various aspects of the project.
5. Castillo, J. 2006. Invertebrate diversity in epiphytic bromeliads: a comparison between San Luis and Monteverde. University of California Education Abroad Program Spring Course Book, Monteverde, Costa Rica.
References
6. Cochran-Stafira, D. L., and von Ende, C.N. 1998. Integrating bacteria into food webs: studies with Sarracenia purpurea inquilines. Ecology 79:880-898.
1. Armbruster, P., Hutchinson, R.A., and Cotgreave, P. 2002. Factors influencing community structure in a South American tank bromeliad fauna. Oikos 96:225234.
7. Dyer, B.D. 2003. A Field Guide to Bacteria. Cornell University Press.
2. Bernal, S. 2002. The effects of volume and turbidity on protist communities in tank bromeliads (Vriesea spp.). Council on International Educational Exchange Fall Course Book, Monteverde, Costa Rica. 3. Brady, S.F., and Clardy, J. 2004. Palmitoylpu-
sand dune ecosystems of southeast Brazil. Canadian Journal of Microbiology 39:973-7. 10. Henry, D.M. 1999. Effects of canopy height on macrofaunal diversity in bromeliad communities of Guzmania nicaraguensis. University of California Education Abroad Program Spring Course Book, Monteverde, Costa Rica. 11. Landell, M.F., Mautone, J.N., and Valente, P. 2006. Biodiversity of yeasts associated to bromeliads in Itapuã Park, Viamão/RS. Biociência. 14:144-149. 12. Maguire, B. 1971. Phytotelmata: biota and community structure determination in plant-held waters. Annual Review of Ecological Systematics 2:439-464. 13. Richardson, B.A. 1999. The bromeliad microcosm and the assessment of faunal diversity in a neotropical forest. Biotropica 31:321-336. 14. Rivera, S.C., Hazen, T.C., and Toranzos, G.A. 1988. Isolation of fecal coliforms from pristine sites in a tropical rainforest. Applied and Environmental Microbiology 54:513-517. 15. Srivastava, D.S. 2006. Habitat structure, trophic structure and ecosystem function: interactive effects in a bromeliad–insect community. Oecologia 149:493–504.
8. Gao, X., Olapade, O.A., and Leff, J.G. 2005. Comparison of benthic bacterial community composition in nine streams. Aquatic Microbial Ecology 40:5160. 9. Hagler, et al. 1993. Yeasts and coliform bacteria of water accumulated in bromeliads of mangrove and
Division of Biological Sciences, University of California, San Diego 15
Transient Global Amnesia: An Investigation into Its Pathophysiology Janet Kwok Junior, Physiology and Neuroscience major, Cognitive Science minor, Revelle College Abstract Many studies have attempted to determine the pathophysiology behind Transient Global Amnesia (TGA). While many different precursors of TGA have been hypothesized, the precise pathophysiological mechanism is still unknown. This may be due in part to the fact that its transient nature makes it difficult for experimental work to be performed during the episode. In this review paper, TGA and the medial temporal lobe memory system will be described, and four of the most popular proposed etiologies will be discussed: transient ischemic attack (TIA), migraine, epilepsy, and emotional stress. Two case studies will be presented, for which the likely pathophysiology appears to be emotional stress and TIA.
Introduction Transient global amnesia, or TGA, has existed as a recognized neurological disorder since 1964. TGA is a type of amnesiac memory loss characterized by its short-lived nature; the attack always spontaneously resolves itself within 24 hours of onset. TGA is considered by researchers in the field as clinically dramatic but essentially benign in nature.6,12 The syndrome involves a sudden onset of anterograde amnesia and is most often accompanied by repetitive questioning, disorientation to time and place, and some impaired cognitive functioning. Those suffering from TGA are, however, still conscious and alert. The prognosis is such that there is a complete recovery of memory and cognitive abilities within 24 hours, thus characterizing the disorder as â&#x20AC;&#x153;benignâ&#x20AC;?. Because the attack is so short-lived, there is not a wealth of scientific literature (as compared to other amnesia types) regarding the pathophysiology of the disorder. There are many different hypotheses, and these will be discussed in this article. Several studies have been conducted, with various neuropsychological measures used to assess cognitive dysfunction. TGA is not a widely known disorder, and it should be noted that when TGA does occur, it can be an extremely frightening experience to the loved ones of the patient, as shown by a personal account that will be included below.
Diagnosis The symptoms of TGA are: repetitive questioning, disorientation to time and place, but no loss of consciousness or personal identity.5 Patients remain alert, attentive, and cognition is hardly impaired.11 TGA primarily affects the middle-aged and elderly population. One study
surveyed 246 TGA patients, and the mean age was 60.322; all other studies demonstrate a similar age group. The recurrence rate is very low; TGA episodes typically only occur once in a lifetime.22 In terms of the amnesia types, TGA is characterized by severe anterograde amnesia for verbal and nonverbal material and a temporally graded retrograde amnesia for personal and public events dating back 30 years.3 TGA always resolves within 24 hours, with no persistent damage, as shown by a follow-up study 4-6 months after onset,6 and treatment has thus not been indicated.11
The Role of the Medial Temporal Lobe/Hippocampus in Memory Memory is considered to be directly associated with the medial temporal lobe system. Some forms of memory are specifically and exclusively linked to the activity of the hippocampus. Drs. Larry Squire and Stuart Zola (1996), prominent neuroscientists and researchers in the field of memory, put forward a model explaining the brain structures involved in the formation and retrieval of long-term declarative memories (see Figure 1.) As shown in this model, declarative memory is a subtype of long-term memory, characterized by the ability to recall specific facts and events that have occurred in the past. Other types of memory include non-declarative memory, such as remembering how to carry out certain tasks, using certain learned skills. The medial temporal lobe system consists of the hippocampal formation and the adjacent perirhinal (PR) and parahippocampal (PH) cortices. The hippocampal formation is composed of the hippocampus proper (CA1, CA3), the dentate gyrus (DG), the subiculum (S), and the entorhinal cortex. The entorhinal cortex directly projects to the hippocampal region, and also
16â&#x20AC;&#x192; Saltman Quarterly Volume 5 / 2007-2008
projects to the surrounding PR and PH cortices. These cortices are also the major source of cortical projections to the entorhinal cortex. As such, these structures are all strongly inter-linked collectively, accounting for a major contribution by the system to memory formation and retrieval. The projections within the system allow the transmission of information that will constitute the memories to be formed. Selective damage to these brain structures has been shown to produce deficits in memory, demonstrating that the medial temporal lobe system is crucial to memory.1 Of these hippocampal structures, the CA1 region has been the most implicated in memory loss.
Identifying the Pathophysiology of TGA Amnesia, which is the general umbrella term for the disturbance of memory, is classified into two forms: anterograde and retrograde. Both types of amnesia are believed to arise from selective damage to brain structures in which declarative memory is impaired.1 Global amnesia is the total loss of memory; there is an inability to recall events from before the precipitating event, and the inability to form new memories. Transient global amnesia features both anterograde amnesia and temporally-graded retrograde amnesia.2 This refers to the combined inability to form new memories and to recall recent memories immediately preceding the attack. This memory loss is similar to that of HM and EP,4 in which memory loss is progressive, with childhood memories being the last to go. In their case studies, Stefanacci et al discovered that both HM and EP suffered extensive damage to their medial temporal lobe. Thus one possibility is that TGA may arise from selective damage to the medial temporal lobe also, specifically targeting the hippocampus. Indeed, recent studies have confirmed that during the occurrence of TGA, the CA1 section of the hippocampus is selectively affected.5,6 These studies utilized the method of MR-signal diffusion-weighted imaging (DWI), carried out within either two or four days after onset, which detected a high frequency of small high-signal abnormalities in the lateral portion of the hippocampus corresponding to the CA1 region.5 Bartsch et al6 found the same results, with 94% of the DWI damage studied to be found selectively in the CA1 region.
There are many different ways the hippocampus and its pyramidal neurons could be damaged, including the release of the neurotransmitter glutamate, and these proposed pathophysiological mechanisms will be discussed below. The mechanisms of the precursors are all wellestablished; their link to TGA onset, however, is open to discussion. The pathophysiology will be discussed under the assumption that the cell body of CA1 neurons themselves or their synapses may be selectively damaged, which would modify glutamergic transmission.
TIA as a Precursor to TGA--the Most Supported Theory Transient global amnesia (TGA) has frequently been associated with transient ischemic attacks (TIA). Ischemia is a restriction in blood supply and is often caused by arterial blockage. A TIA is a mini-ischemic stroke, caused by an embolus (calcification or fatty plaque) that becomes lodged in an artery to the brain. Although it dissolves and breaks up, arterial thromboembolic ischemia temporarily blocks blood supply and thus causes temporary oxygen deprivation to the brain.11 TIA provides a likely pathophysiological mechanism for TGA; both TIA and TGA are
transient (lasting about a day on average), and TIA immediately precedes TGA. Also, vasculature and neural function are highly linked; neurons, like all cells, require an oxygenated blood supply for functioning. Of important note is the fact that the CA1 region of the hippocampus is unusually susceptible to ischemia, suggesting that disturbances could selectively affect CA1 neurons and thereby disrupt memory.5 The posterior cerebral artery supplies blood to the occipital and inferior temporal lobes, which includes the hippocampus. A TIA in the posterior cerebral artery could thus result in TGA15; blocking the blood supply to the hippocampus would temporarily disrupt hippocampal function. Specifically, the lenticulostriate branch of this artery has been implicated in the etiology of TGA.14 Also, aside from hippocampal dysfunction, the caudate nucleus (in the basal ganglia) may play a role in TGA; the lenticulostriate branch of the posterior cerebral artery ends in the caudate nucleus, which has been implicated in memory functioning. It is thus possible that disruption of the blood flow through the lenticulostriate branch of the posterior cerebral artery is a cause of transient global amnesia, via a TIA mechanism targeting the caudate nucleus.
Other potential vascular precursors of TGA are hypertension and hyperlipidemia (Chen et al, 1999).9 These are not directly linked with TIA, but produce the same results as TIA, in that they share the same symptoms that have been implicated in TGA pathophysiology. Arterial hypertension, or high blood pressure, was found to be a common risk factor for TGA by several different groups; Chen et al9 found that 39% of the TGA patients also had hypertension, and Santos et al10 similarly found 58.62% of their TGA patients with high blood pressure. However, Pinol-Ripoll et al8 found that TGA was not a symptom of arteriosclerosis, so this theory of TGA’s pathophysiology is not as concrete. Hyperlipidemia was reported in 25% of patients,9 and this relates to TIA and hypertension in that the excess of lipids in the blood may accumulate and cause a blockage (TIA), or may accumulate on the walls to increase the blood pressure. These other vascular precursors, however, are not as supported as the TIA etiology, as illustrated by Tuduri et al.13 They compared clinical data between two groups of patients with and without vascular risk factors, all who had TGA. There was a lack of significant differences between the two, which rules out a vascular etiology and it may only be a trigger. Instead, they hypothesized that Leao’s propagated depression was more likely in the pathophysiology.
S
Migraine/Cortical Depression as a Precursor to TGA
CA1 CA3
HIPPOCAMPAL FORMATION
DG
ENTORHINAL CORTEX
PERIRHINAL CORTEX
OTHER DIRECT PROJECTIONS
PARAHIPPOCAMPAL CORTEX
UNIMODAL AND POLYMODAL ASSOCIATION AREAS (Frontal, temporal, and parietal lobes)
Migraine is a neurological disease characterized by the symptom of severe, unilateral headache. Its prevalence has been observed in TGA patients, and it is thus a possible precursor. Following a migraine, a patient is more susceptible to a TIAcaused TGA, which suggests two things: that migraine and TIA are inextricably linked, and that TIA is the most common theory.10 Indeed, migraine is not as likely a mecha-
Figure 1: Schematic view of the medial temporal lobe memory system; Squire, Larry R. and Zola, Stuart M. (1996) Proc. Natl. Acad. Sci. USA 93, 13515-13522 The diagram illustrates the different brain structures involved in the formation and retrieval of declarative memories, and the projections amongst these brain regions. The hippocampal formation is the primary area associated with memory, with adjacent cortices that have been shown to impact the hippocampal activity.
Division of Biological Sciences, University of California, San Diego 17
Figure 2: Doctor’s report: MRA Brain & Neck examination of my mother’s TGA attack. All arteries were patent (unobstructed), and no stenosis (narrowing) was observed. This demonstrates the transient nature of the TGA episode, such that if TIA were the precursor, the abnormality resolved within 24 hours since the MR angiogram scans were taken the day after the episode. Another possible explanation is that TIA was not a precursor at all, thus there were no disturbances to the blood flow. This ambiguity demonstrates the difficulty in pinpointing the precise pathophysiology of TGA.
to the previously mentioned etiologies. Unlike with TIA, which may immediately trigger TGA, epilepsy often occurs several years before the onset of TGA.
nism as TIA, due to the contradictory evidence put forward by different groups. Schmidtke and Ehmsen18 showed that the expression of key TGA features was not affected by comorbidity with migraine, and vice versa, arguing against the interaction between migraine and TGA. On the other hand, other groups have suggested that migraines are indeed triggers for TGA, as shown by analyzing the pathophysiology of migraine as the pathophysiology of TGA. Migraine has been characterized by cortical spreading depression, also known as the spreading depression of Leao,16 which is potentially relevant to TGA. This spreading depression in the hippocampus can cause transient amnesia with full recovery, due to the binding of glutamate to NMDA receptors.16 Spreading depression has also been implicated in decreased blood flow through the bilateral medial temporal lobe.17 This again suggests that migraine and vasculature cannot be separated, and that TGA may arise from vascular mechanisms activated during migraine. Furthermore, the depression that characterizes migraines may also precipitate strong emotional events that possibly liberate glutamate and bring about TGA.
Epilepsy as a Precursor to TGA Epilepsy, the neurological condition characterized by the recurrence of seizures, has been hypothesized to be a precursor to TGA, but there is not as much literature examining this, compared
Epileptic seizures can often occur selectively in the CA1 region of the hippocampus. Wittner et al (2002) offered a detailed mechanism of epilepsy in this area, focusing on the distribution, morphology, and postsynaptic targets of inhibitory calbindin-containing interneurons. He found epileptic CA1 neurons to have spiny dendrites, and that curvy, distorted dendrites replace the radially-oriented dendrites in samples from patients with epilepsy. In non-sclerotic epileptic samples, with intact pyramidal cells, the interneurons received more inhibitory synaptic input, and the calbindin-positive excitatory afferents from the CA1, CA2 and the dentate gyrus were sprouted. The increase in inhibition by inhibitory interneurons resulted in abnormal synchronization in the output region of the hippocampus. In sclerotic tissue, with apoptosis of pyramidal cells, the interneurons and other excitatory afferents changed their synapses. The synaptic reorganization and morphological alterations to the neurons within the CA1 region could thus impair memory function in the temporary manner of TGA. Furthermore, TGA was indeed found in one recent study to occur after a surgery consisting of anterior temporal lobectomy for refractory medial temporal lobe epilepsy associated with hippocampal sclerosis.19
Stress has been shown to enhance long-term depression (LTD) selectively in the CA1 region of the hippocampus,21 indicating that the synapses formed by the CA1 neurons can be weakened by stress. Since damage to CA1 has already been shown to produce TGA, emotional stress may cause TGA through depression in CA1. It should be noted that “long-term” depression refers to a length of time from minutes to hours, which is in agreement with the duration of TGA. The pathophysiological mechanism of LTD in the hippocampus has been outlined by Yang et al.21 The study involved the extraction of tissue slices containing glutamergic NMDA receptors from stressed animals. It was observed that behavioral stress induced a decrease in the amount of glutamate detected by the NMDA receptors of the CA1 pyramidal neurons, and this decreased glutamergic transmission is what normally characterizes LTD. This weakening of the synapses within the CA1 could lead to temporary memory dysfunction. Therefore, emotional stress (as with migraine), a precursor of TGA, may cause selective LTD in the hippocampus, which may ultimately lead to a transient global amnesia attack.
Case Studies Case Study of Patient YH Patient YH experienced a transient global amnesiac attack on Friday, September 24, 2004. It lasted for approximately six hours. She is Chinese and was 54 at the time.
Emotional Stress as a Precursor to TGA
That day, YH was at church leading the worship service. At that time in her life, she was experiencing much stress. Yet while she was singing, she reported that she was feeling very happy and spiritually high. Immediately afterwards, her friend asked her for a copy of the song. YH responded with confusion and told her friend that she didn’t know where it was, thus marking the onset of the TGA episode. Thereafter, YH showed memory decline. Initially, she was aware that something was wrong, and asked to be sent to the hospital. To this day, she remembers making this request. The last thing YH remembers was telling her friend to notify her husband; she could recite both his office and cellular phone numbers.
There are examples in the scientific literature of other case studies featuring emotional stress as the precursor for the TGA attack (Chen et al, 1999). Out of the 28 patients in that study, 32% of the cases were associated with a potential precipitating factor such as emotional stress.
Her friend reported that during the taxi ride to the hospital, YH was very confused, and kept repeating, “What am I doing in this taxi?” Her friend would answer, only to have YH repeat the same question seconds later. YH appeared to recognize and trust her friend, since she was
18 Saltman Quarterly Volume 5 / 2007-2008
able to ask that question. Also, while she was in the hospital room, she repeatedly asked, “Why am I in this bed?” and while the doctors were running the CT scans, she similarly kept asking, “What am I doing in here?” YH’s daughter went to visit YH just before her TGA resolved. According to her daughter, YH was very unlike her normal self, and was completely oblivious to the seriousness of the situation. She continued to repeat questions, such as “You are my daughter, right?” The entire situation was extremely frightening, because none of YH’s family knew what was happening, and neither did the specialists; TGA had not yet been diagnosed. During the attack, her daughter reported that she felt like she had lost her mother forever. This sheds light on the nature of TGA: it cannot be predicted. Medical history At the time of the attack, YH had a slight history of migraines. She had no history of hypertension, diabetes, heart or lung disease, seizures, stroke, or traumatic loss of consciousness. At the time of TGA attack, she was on no medication. She does not consume alcohol, and exercises almost daily. Neurological examination YH’s neurological status has been stable ever since the TGA attack. Neuropsychological tests were administered both during and after the attack. During the TGA episode, they tested her
memory, by presenting her with lists of simple words to recall, of which she could recall only some. When the lists were lengthened by adding more words to recall, she could not recall any. All of this information has been provided by YH’s friend who was present with her at the time. YH has no recollection of any of this. (figure 2) Computed tomography CT scans were taken during the TGA attack. They were normal. Magnetic resonance imaging MRI scans of both the brain and the cerebral arteries were taken after the TGA had resolved. YH had fully recovered and was able to go to the MRI center herself to get the scans done. They were also normal (see figure 3).
Case Study of WS (YT’s mother) WS had a TGA attack in the year 1991, at the age of 64. At the time of attack, she was taking care of her three year old granddaughter. The granddaughter had done something very bratty and refused to listen to WS, and this greatly upset WS. WS was very angry, and immediately after this, she experienced a sudden total memory loss. Her husband was with her and she did not recognize him. He recalls her having a completely blank look on her face and she was disoriented to her surroundings. However, after around 10 minutes, she recovered totally, and
Figure 3: Coronal Non-Contrast and Saggital Non-Contrast MRI images of patient’s brain after the TGA episode. No abnormalities were observed.
resumed her normal activities. She did not see a doctor or go to the hospital. Medical history WS now has Alzheimer’s disease. She was diagnosed in 2003. She also has colon cancer and a history of high blood pressure. At the time of TGA attack, she was on medication for high blood pressure. She does not consume alcohol, and maintains a healthy lifestyle, through diet and exercise. In both cases of patients YH and WS, TGA appeared to be triggered by emotional stress, but resolved within 24 hours. Both patients exhibited the characteristic disorientation to time and place, and in addition, patient YH demonstrated repetitive questioning on multiple occasions. With YH, she lost her more recent memories (i.e. where the location of the sheet music was), but appeared to maintain an unconscious recognition of familiar faces, as with her friend, her husband, and her children. The repetitive questioning demonstrates the sudden onset of anterograde amnesia and the consequent inability to retain new information—to remember that she had already asked the question. In YH’s case, migraine may have played a role due to her medical history, but at the time, she was not experiencing a migraine. The TGA episode appeared to be associated with intense emotional fluctuations; she see-sawed between anxiety and euphoria. Although the MR images of her arteries were normal after the attack, she may have experienced stresstriggered or LTD-triggered TIA during the TGA. In WS’s case, hypertension may have been implicated, and this may have been linked with TIA. Both examples of TGA thus seem to result from emotional stress, perhaps selectively targeting the CA1 region of the hippocampus, and both cases implicate vascular risk factors. Vascular mechanisms are even more likely than the other suggested etiologies in that they are more likely to lead to global dysfunction, by means of global oxygen deprivation, targeting not one specific cell but a group of cells (the CA1 of the hippocampus). Although, for YH, all arteries were patent (unobstructed), and no stenosis (narrowing) was observed, this may be due to the transient nature of the TGA episode, such that if TIA were the precursor, the abnormality may
Division of Biological Sciences, University of California, San Diego 19
have resolved within 24 hours since the MR angiogram scans were taken the day after the episode (see figure 2). It is also possible that TIA was not a precursor at all, thus there were no disturbances to the blood flow. This ambiguity demonstrates the difficulty in pinpointing the precise pathophysiology of TGA. It is interesting to note that both YH and her mother WS experienced the same TGA attack, both apparently stimulated by intense emotional stress. From this, it would be natural to assume that TGA could be genetic, which is an aspect of the syndrome that has never been researched. This isolated account of both a daughter and her mother experiencing the same TGA is not sufficient to generalize the results to the general public, but it provides an alternative perspective. A likely explanation is that TGA itself is not heritable, especially because the recurrence rate is extremely low, and only occurs once per lifetime.22 Instead, the precursors of the disorder may be genetically determined. Hypertension has genetic roots, as demonstrated by findings from the Family Blood Pressure Program in the United States 23. Also, migraines are a hereditary condition affecting 12% of the US population, and UCLA researchers have discovered that the q23 region of chromosome 4 has been consistently linked to migraines 24. Also, epilepsy is a genetic trait, and inheritance may increase the likelihood of TGA onset 25. Finally, emotional stress may not be genetically implicated per se, but personality certainly is, and one’s tendency towards behavioral stress may be inherited from the parent 26. On a side note: WS’s case may cause others to assume that transient global amnesia and Alzheimer’s disease may be linked somehow, but this is definitely not the case.
Discussion Transient global amnesia (TGA) is a spontaneous and sudden attack of anterograde amnesia that resolves within 24 hours. It has existed as a clinical disorder for over 40 years, but due to its short nature and rare occurrence of 3-10/100,000 cases per year, insufficient research has been carried out to identify the exact pathophysiology. Many potential etiological and pathophysiological mechanisms have been put forward by research groups from all over the world. Specifically, transient ischemic attacks, cortical depression, migraines, epilepsy, and emotional stress have all been implicated in the unknown etiology of the syndrome. All of these mecha-
nisms were discussed from the perspective of affecting the CA1 region of the hippocampus, a brain structure that is critical in the formation and recall of declarative memory. Damage to this region has been shown to result in both anterograde and retrograde amnesia, and similar to TGA. In light of patient YH and her mother’s experiences of TGA, The author believes that TGA is most likely caused not by epilepsy or migraine, but instead by emotional stress.
References
TIA has been at the forefront of hypothesized pathophysiological mechanisms, and TIA may be linked to emotional stress. What this means is that emotional stress may trigger the ischemic blockage of blood supply in the posterior cerebral artery, which may then set off TGA. Emotional stress thus may result in TGA via an indirect neurological route. Although TGA is considered “benign” by the scientific field, it is one of the most frightening things anyone could ever experience—the prospect of losing a loved one, since one cannot know right away that it is only a transient amnesia. In no way should TGA be undermined as a lesser disorder.
3. Kritchevsky, M., Squire, L.R., and Zouzounis, J.A. 1988. Transient global amnesia: Characterization of anterograde and retrograde amnesia. Neurology Feb. (3):213-219.
Further work should be carried out to confirm the pathophysiology of transient global amnesia, by focusing on specific known mechanisms of potential precursors and their relation to TGA. This would allow for quicker diagnosis and perhaps even provide information on susceptibility, despite its seemingly random nature. Other research groups would also benefit from studying an animal model based on the four different hypotheses, by teaching the animals a task and then inducing forgetting, via the framework set forth by this review. Animal models would be ideal as they would eliminate the time-limiting factor of TGA in humans, as its short duration prevents studying of the disorder. TGA is a random and quick phenomenon. It is not a wellknown syndrome to the public, and so people need to be well-informed.
Postscript After these case studies were carried out, we incidentally know of one other case where two sisters in the 70s age range both experienced TGA. Both sisters are Mexican. This provides very interesting insight into the pathophysiology of TGA. Future studies would benefit from looking at the potential genetic aspect of TGA, and also whether culture and ethnicity factor into TGA vulnerability. Such investigation would shed light on the genetic and environmental disposition for this disorder, and would thus help in predicting whether TGA would occur in certain individuals.
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1. Squire, L.R., and Zola, S.M. 1996. Structure and function of declarative and nondeclarative memory systems. Proc. Natl. Acad. Sci. USA. Nov. 93:131513522. 2. Kritchevsky, M., and Squire, L.R. 1989. Transient Global Amnesia: Evidence for extensive, temporally graded retrograde amnesia. Neurology. Feb. 30(2):213-218.
4. Stefanacci, L., Buffalo, E.A., Schmolck, H., and Squire, L.R. 2000. Profound amnesia after damage to the medial temporal lobe: A neuroanatomical and neuropsychological profile of patient E. P. J Neurosci. Sep 15. 20(18):7024-36. 5. Lee, H.Y., Kim, J.H., Weon, Y.C., Lee, J.S., Kim, S.Y., Youn, S.W., and Kim, S.H. 2007. Diffusionweighted imaging in transient global amnesia exposes the CA1 region of the hippocampus. Neuroradiology. Jun. 49(6):481-7. Epub 2007 Feb 14. 6. Bartsch, T., Alfke, K., Stingele, R., Rohr, A., Freitag-Wolf, S., Jansen, O., and Deusch, G. 2006. Selective affection of hippocampal CA-1 neurons in patients with transient global amnesia without longterm sequelae. Brain. Sep. 129(11):2874-2884. 7. Stroke. 2007. COGS 172 Course Readings, Univ. of California San Diego, Dr. Andrea Chiba, Fall. 8. Pinol-Ripoll, G., de la Puerta Gonzalez-Miro, I., Martinez, L., Alberti-Gonzalez, O., Santos, S., Pascual-Millan, L.F., Mauri-Llerda, J.A., Mostacero, E. 2005. A study of the risk factors in transient global amnesia and its differentiation from a transient ischemic attack. Rev Neurol. Nov 1-15. 41(9):513-6. Spanish. 9. Chen, S.T., Tang, L.M., Hsu, W.C., Lee, T.H., Ro, L.S., and Wu, Y.R. 1999. Clinical features, vascular risk factors, and prognosis for transient global amnesia in Chinese patients. J Stroke Cerebrovasc Dis. Sep-Oct. 8(5):295-9. 10. Santos, S., López del Val, J., Tejero, C., Iñiguez, C, Lalana, J.M., and Morales, F. 2000. Transient global amnesia: a review of 58 cases. Rev Neurol. Jun 16. 30(12):1113-7. 11. Owen, D., Paranandi, B., Sivakumar, R., and Seevaratnam, M. 2007. Classical diseases revisited: transient global amnesia. Postgrad Med J. Apr. 83(978):236-9. Review. 12. Uttner, I., Weber, S., Freund, W., Schmitz, B., Ramspott, M., and Huber, R. 2007. Transient global amnesia--full recovery without persistent cognitive
impairment. Eur Neurol. 58(3):146-51. Epub 2007 Jun 29. 13. Tudurí, I., Carneado, J., Fragoso, M., Ortiz, P., and Jiménez-Ortiz, C. 2000. Transient global amnesia and vascular risk factors. Rev Neurol. Mar. 30(5):41821. 14. Ravindran, V., Jain, S., Ming, A., and Bartlett, R.J.V. 2004. Transient global amnesia in a patient with acute unilateral caudate nucleus ischemia. J Clin Neuro. Aug. 11(6):669-672. Case report. 15. Imamura, K., Kowa, H., Maeda, M., Nakano, T., and Nakashima, K. 2002. A case of bilateral posterior cerebral artery embolism with transient global amnesia. Neurol Med. 57(3):281-283. Japanese.
spreading depression in the hippocampus explains transient global amnesia. A hypothesis. Acta Neurol Scand. Feb. 73(2):219-20. 17. Jovin, T.G., Vitti, R.A., and McCluskey, L.F. 2000. Evolution of temporal lobe hypoperfusion in transient global amnesia: a serial single photon emission computed tomography study. J Neuroimaging. Oct. 10(4):238-41. 18. Schmidtke, K., and Ehmsen, L. 1998. Transient global amnesia and migraine: A case control study. European Neurology. 40:9-14. 19. Dupont, S., Samson, S., and Baulac, M. 2007. Is anterior temporal lobectomy a precipitating factor for transient global amnesia? J Neurol Neurosurg Psychiatry. Nov 21. [Epub ahead of print]
20. Wittner, L.E., Eross, L., Szabo, Z., Toth, S., Czirjak, S., Halasz, P., Freund, T.F., and Maglozky. Z. 2002. Synaptic reorganization of calbindin-positive neurons in the human hippocampal CA1 region in temporal lobe epilepsy. Neuroscience. Dec. 115(3):961-978. 21. Yang, C.H., Huang, C.C., and Hsu, K.S. 2005. Behavioral stress enhances hippocampal CA1 long-term depression through the blockade of the glutamate uptake. J Neurosci. Apr. 25(17):4288-4293. 22. Quinette, P., Guillery-Girard, B., Dayan, J., de la Sayette, V., Marquis, S., Viader, F., Desgranges, B, and Eustache, F. 2006. What does transient global amnesia really mean? Review of the literature and thorough study of 142 cases. Brain. Jul. 129(Pt 7):164058. Epub 2006 May 2. Review.
16. Olesen, J., and Jorgensen, M.B. 1986. Leao’s
Division of Biological Sciences, University of California, San Diego 21
Research Review
Optogenetics: A Novel Approach for Neuroprosthetic Interface Michael Fenlon BISP 194, Spring Quarter 2008 / Professor: Dr. Gert Cauwenberghs
Neuroprostheses: The Promise Like a real life 6 Million Dollar Man or Bionic Woman, the field of neural prosthesis aims to meld man and machine. Their aim is to integrate electrical machines into human bodies to replace diseased or missing parts. To someone unfamiliar with the field, the idea of combining a human body with mechanical parts sounds like something out of Star Wars rather than real science. But this field has already gone from concept to clinic, with the most famous and successful example of this is the cochlear implant, which is approved for use in certain deaf patients. But restoring hearing is not the only possible use of such technology, for there is no end to potential applications for these devices. Neural prosthetics have promising potential in fields such as limb replacement, sight restoration, repairing spinal cord damage, Parkinson’s disease and depression. And there is no shortage of researchers working on these projects. The science underlying these advances rests in tapping into the way our nervous system works: electricity. Scientists have known for a very long time that the functional units of the nervous system, neurons, transmit information via electrical propagation. And this knowledge has been used to tap into the workings of electrical physiological systems, such as the heart and the brain with electrocardiograms and electroencephalograms respectively. But scientists only recently began controlling these systems rather than listening to them, using electrical stimulation to incite muscle and nervous system actions. Neuroprostheses: The Reality For the entire span of its very short existence, neuroprosthetic design has been reliant on direct electrical stimulation of nerves. This method of exciting nerves has been successful both in vitro and in vivo for many applications, including neuroprosthetics. But there are problems associated with this classical approach. For one, targeting the correct and specific nerves in a system is a herculean task. For, as great as a neurosurgeon may be, localizing a microwire or single electrode onto a specific neuron is impossible. Even if this were possible, electrical stimulation propagates nonspecifically in all directions, which is the second major problem. In the ideal implantation, where the stimulating electrode is as close to the neuron as possible, activation would still travel to neighboring neurons, leading to unintentional excitation. The final and most serious problem is biocompatibility of electrodes in clinical applications. In sites such as the brain, which is bathed in highly corrosive cerebral spinal fluid, electrode longevity is an issue. Every stimulation sent through the electrode causes an oxidation-reduction reaction on the metal tip that creates toxic breakdown product, which inherently leads to chronic corrosion. At a certain point, the corrosion will render the electrode inoperative, which is a serious complication for a device that is implanted for chronic use (several decades). In addition to device degradation, there are also problems with the biocompatibility of implantation, namely with the scar tissue formed around the device. This gliosis further
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exacerbates the problem of nonspecific excitation, for this insulating tissue forces the device to increase the voltage because of an increase in what is necessary to elicit a response. With all of the problems of electrode-based neural prostheses in mind, it is amazing that a successful electrode device exists. But this is not the only interface possible for neural prosthetic applications. To circumvent the problems inherent in electrode-based neuroprosthetics, scientists have developed optical-based interfaces to serve as alternatives. This incipient branch of biology is known as optogenetics. Optogenetics: Optogenetics is an emerging field that uses methods from genetics and optics to visualize and control neurons. With the use of lentiviral vectors, scientists are able to specifically target neurons and transfer certain genes into them. To date, many different types of genes have been transferred or transfected into neurons, both in vitro and in vivo. For visualization, many types of fluorescent proteins have been inserted, with amazing results. Using these optically active proteins is important for neuronal visualization. But the most interesting and consequential for the future of neural prosthesis are optically active channel proteins, which insert into the membrane. In Palo Alto California, the laboratory of Karl Deisseroth M.D., Ph.D. is pioneering the field of neuronal channel protein transfection. With lentiviral transfection, they are able to simultaneously insert two types of membrane channel proteins: channelrhodopsin-2 (ChR2) and N.pharaonis halorhodopsin (NpHR). Both ChR2 and NpHR are optically activated membrane proteins originally found in unicellular organisms, ChR2 from algae and NpHR from an archaea. But Deisseroth is not merely transfecting neurons with random membrane proteins. Both ChR2 and NpHR have great consequence on theactivity of neurons. ChR2 and NpHR: Temporally Precise Neuron Control As we all remember from introductory physiology, the basis of signaling in the nervous system is rooted in neuron membrane potentials. The electric gradient across the membrane is depolarized, then repolarized in response the opening of specific ion channels. Through this mechanism, the signal (action potential) is regeneratively propagated down the length of the neuron. This occurs until it reaches the end (presynaptic terminal), where the signal is transduced into neurotransmitters and carried across the synapse to the next neuron. Optogenetic methods allow scientists to tap into this process and control it. Specifically, the implementation of ChR2 and NpHR in neurons comprises the “Yin and Yang” of control. This antagonistic combination is able to activate or inhibit neuron signaling by either depolarizing or hyperpolarizing the neuron membrane. ChR2 is a cation channel that depolarizes the membrane, leading to action potential firing. Conversely, NpHR is an
anion channel (Cl-), which causes the membrane to hyperpolarize, resulting in the suppression of action potential firing. Overall, the combination of these channels allows for the precise control of neuron firing. Multimodal Optical Interrogation The first order of business in the investigation of ChR2 and NpHR control was to confirm that these channels were compatible with mammalian cells and did not interfere with neuron health or survival. Using lentiviral infected neurons, the group was able to show that gene transfection did not interfere with neuronal stability. This was shown by using the DNA-binding dye propidium iodide which labels cells with broken plasma membrane. Also, the membrane stability was shown by recording its resistance two weeks after transfection. Next , the Deisseroth lab determined that the action spectra of the NpHR channel was sufficiently distinct from that of the ChR2 channel. To be able to selectively control which ion channel was being opened, it was necessary for the action spectra to be far away from each other. This was confirmed by recording the NpHR activation versus wavelength. The final result was that the NpHR peak (593 nm, yellow) was compatible with the ChR2 peak (460 nm, blue). The most important part of their experimentation was to determine if the ChR2 and NpHR combination could effectively work antagonistically. Using dual-transfected cultured hippocampal cells, the group effective showed that this was possible. Trains of blue light (activating) were shined on the cells, faithfully reproducing action potentials. At intermittent times, concomitant yellow light (deactivating) was applied. The result was that yellow light effectively inhibited the generation of action potentials, validating the antagonistic function of the ChR2/NpHR combination.
Considerations and Future Direction Although this powerful technology demonstrates the potential of optogenetics, its application in neuroprosthetics is decades away. For an optogenetic interface to be effective, it must be selective in its control of neurons. Only certain tracts must be activated or inhibited, so gene regulators for specific cell types must be elucidated. In addition to this, the gene transfection delivery method is problematic in humans. This type of gene therapy has a poor history in clinical applications, so further development of this is necessary before any type of human studies are to be done. Testing must be done to ensure the specificity, efficiency, and safety of such technology in clinical applications. In conclusion, optogenetics is changing the interface of machines and neural tissue at the basic science level, and it is reasonable to imagine that this could translate to clinical applications as well. If safe and effective methods are developed to introduce this technology into humans, optogenetics could revolutionize neuroprosthesis development. Bibliography 1. Feng, Z. et al. 2007. Multimodal fast optical interrogation of neural circuitry. Nature. 446, 633-639. 2. Boyden, E. et al. 2005. Millisecond timescale, genetically targeted optical control of neural activity. Nature Neuroscience. 8, 1263-1268. 3. Evanko, D. 2006. Optical excitation yin and yang. Nature Methods. 4, 384. 4. Miller, G. Shining new light on neural circuits. 2006. Science. 314, 1674-1676.
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Research Review
Neural Prosthetics: Developing a Safe, Rehabilitative Conversation with a Brain Alex Kawas BISP 194, Spring Quarter 2008 / Professor: Dr. Gert Cauwenberghs Abstract Starting a conversation with a stranger can be a difficult process. Neuroscience has spent over a century in conversation with the brain, learning how it works. Neural prosthetics, picking up the conversation where neuroscience leaves off, is learning how to work with the brain in a rehabilitative manner. Current prosthetics techniques are faced with a common set of challenges to engaging in the conversation. For example, electrodes are assaulted by glial cells which diminish the electrode’s longevity, implanted power supplies require replacement, and invasive implant procedures risk infection. Prosthetics in development may not share in these challenges, but will likely face their own unique set. This review identifies the common challenges faced by current neural interface designs, discusses how these challenges are being faced by current prosthetics and how they can be addressed by future prosthetics, and explores the unique challenges that may arise for the next generation of devices. Introduction As with any conversation, the participants must agree on a language. Neuroscience has taken on the mission of understanding the natural, biological language of the brain. The goal of neural prosthetics research is to create machinery that can speak that language in order to spark that deep conversation that will repair and augment long-term neural function in a safe and effective manner. In information theory, the simplest conversation model involves a transmitter, a receiver, and a channel over which information travels between them. To fit the needs of a device conversing with a brain, this simple conversation model need only be modified slightly to include a transmitter and a receiver at both ends for feedback control purposes. This modified model approximates the majority of the information architecture of current prosthetics. That addressed, the biggest remaining challenges are in designing the channel and determining how the engineered transmitters and receivers push and pull information. Current neural interfaces, while enticing in their capabilities, have one or more of the following costly design repercussions: destructive, shortlived, unidirectional, recurrently costly, noisy, and life-threatening. Any of these can be challenging when you’re trying to have a conversation with a patient’s brain. There are several potential interfaces in the early stages of development which may not be challenged in these ways. Electrodes Electrode implantation has proven to be quite useful in repairing impaired function in several clinical populations. Cochlear implants for patients with hearing impairments once suffered from low-resolution but have since advanced greatly (Wilson et al, 1991). Retinal implants for patients with retinal damage are in nascent stages (Weiland et al., 2005). Deep brain stimulation, involving electrode implantation in the deep recesses
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of the brain to modulate regional neural activity, has been explored for a variety of clinical applications, including Parkinson’s disease (Kumar et al, 1998) and treatment-resistant depression (Mayberg et al, 2005). The electrodes and electrode arrays used in these prosthetics vary in their mass, shape, metallic composition, electrical properties, and cellular adhesive properties. The diversity of construction leaves room for site-specific specialization and modifications to address challenges as they arise. While electrode-based techniques have already demonstrated their value and still hold further promise in treating clinical populations, there are non-negligible costs, both potential and definite, to the patient in invasive electrode and electrode array implantation procedures. Often, the batteries which drive the implanted device need to be replaced at regular intervals. Electrode corrosion and insulation by neuro-protective glial cells eventually interferes with electrical signal conductance, requiring the replacement of electrodes. Re-implantation reintroduces the already significant risk of infection, due to the removal of the physiological barrier provided by epithelial tissue, and the potential for severe complications that go along with any invasive neurosurgery. Implantable wireless telemetry addresses some of the aforementioned costs by separating the power source from the sensing or actuating electrode (Sauer et al., 2005). As many of the risks are probabilistically proportional to the physical size of the implanted device, this approach allows for a reduction in risk by enabling the replacement of just the electrode compartment without the additional risks involved in replacing a larger assemblage. Also, by removing the need to tether wires between the power source, processors, and electrodes, the total area of modification can be minimized to a degree not possible otherwise. Additionally, wireless telemetry can be advantageous in cases where space is at a premium and impinging upon nearby neuro-anatomy would create additional risks. Carbon nanofiber-reinforced polycarbonate urethane has been presented as an improvement upon traditional electrodes (Webster et al., 2004). This material addresses the issue of glial scarring by diminishing astrocyte adhesion, which prolongs the function of the nano-electrode. Also demonstrated is an increase in nerve cell adhesion. While this approach prolongs the life of the electrode, it is still challenged by the need for invasive implantation and for eventual replacement of power source and electrode. Optogenetics Light-gated channel proteins such as Channelrhodopsin-2 (ChR2) and a halorhodopsin from Natronomas pharaonis (NpHR) can be packaged inside viruses for delivery into neurons. Boyden et al. (2005) demonstrated ChR2, delivered this way, could reliably drive the spiking of neurons on a scale of milliseconds. Later, NpHR, with its Cl- channel conductance, was used to inhibit neuron spiking. NpHR and ChR2 have different absorption spectra, that is, they are sensitive to different ranges of light, allowing for the differential up—and down—regulation of the electrical activity of single neurons by transfecting both channels (Han et al. 2007). Viral-delivery approaches have unique challenges and the current pro-
posed optogenetic devices share challenges with electrical implants. The prosthetic involves implantation of an RF-receiver coupled to light-emitting diodes specific to the spectra of the photoreceptors. While risking infection through the implant procedure, the inevitable glial scarring is unlikely to induce recurrent risks through a need for replacement because the glial tissue, which is electrically insulating, is thin enough to be lightpermeable. Virus elimination needs to be explored as well, as the presence of a mutable virus in the brain is potentially life-threatening should it conjugate with a more harmful virus. It’s currently unclear how the virus will specify its target cells, so as not to transfect the proteins throughout the nervous system. Nano-scaffolds In acutely damaged neural tissue, cellular regeneration processes are challenged in guiding the tissues to re-associate in such a way which re-institutes the pre-existing topography. Recently, Ellis-Behnke et al. (2006) demonstrated a non-invasive, self-assembling peptide nanofiber scaffold (SAPNS) could act as a prosthetic scaffold to support the existing regenerative process of a severed hamster optic nerve. In cases like this, where the prosthetic is only needed transiently, the persistence problems are less of an issue. While exciting, there are numerous potential challenges with nano-scaffolds. Immune response to the SAPNS peptides could prevent the support process before it begins. Transient use of immunosuppressants could be used in conjunction with the SAPNS injection. Unexpected binding properties of the peptides could lead to disturbances in function elsewhere. Because the SAPNS is only necessary as a transient support, questions naturally arise about the mechanism to remove or eliminate the SAPNS when regeneration is complete. Animal model tests will be necessary to sort these latter two challenges out. Future Directions The future of neural prosthetics research is promising. Recent advances in molecular engineering, such as the duroquinone-based logic device, hint at a virtually unexplored path for the further development of neural prosthetics. Bandyopadhyay and Acharya’s nano-scale logic devices are able to perform 16-bit parallel processing using only 17 molecules. The scale of these logic devices confers great advantages over current approaches. The assembly could evade complications of glial scarring, similar to the nano-electrodes previously mentioned, and avoid the risks of infection from transcutaneous implantation procedures as it could conceivably be injected or transdermally diffused into the bloodstream. The largest practical hurdle in implementing a logic-device-based prosthetic is the current requirement for a scanning tunneling microscope (STM) to act as an external controller. In order for an STM to interact with the logic device, the single-atom-tipped tunneling needle must come within a few atomic diameters of the logic device. Further investigation is necessary to determine whether the STM can be replaced by a more appropriately sized device that could change the logic device’s state like a phosphorylating kinase changes the conformation of proteins. At a larger scale, findings in viral delivery of light-gated ion channels for light-activated neuronal regulation hold great potential and want for
deeper exploration (Bandyopadhyay and Acharya, 2008). Should the ChR2 or NpHR proteins, for example, be modified to be sensitive to other frequency bands in the electromagnetic spectrum (eg, by lowering the energy of the receivable photons by several orders of magnitude), the need for a radio-mediated, light-activator implant could be removed entirely, nullifying the associated risks of neurosurgery. ChR2, if so designed, could still be delivered virally, but activated directly by an RF transmitter external to the patient’s body. These aforementioned early technologies would benefit from the further development of cell-type-specific targeting. A shared catalogue could be created of cell types and their associated membrane-bound molecules to be used as viral targeting markers. Such a catalogue could serve to assist in the development of molecular targeting devices, be they manufactured biologically-derived proteins or nano-engineered molecules, with refined distinguishing capabilities designed around detecting unique patterns in specific membrane-bound molecules. As other techniques, beyond membrane-bound molecule pattern detection, are developed, the catalogue could be expanded accordingly. With the variety of potential routes open to communicating with the brain, neural prosthetics share with us an encouraging vision of the future of our conversation with the brain and ourselves. Bibliography 1. Bandyopadhyay, A., and Acharya, S. “A 16-bit parallel processing in a molecular assembly.” 2008. Proceedings of the National Academy of Sciences. 105:10, 3668-3672, Mar. 2. Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., Fawcett, J.W., and McMahon, S.B. 2002 “Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature. 416(6881):636-40. 3. Ellis-Behnke, R., Liang, Y.X., You, S.W., Tay, D., Zhang, S., So, K.F., and G. Schneider. 2006. Nano neuro knitting: Peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision. Proc. Natl. Acad. Sci. U. S. A. 103:5054–5059. 4. Evanko, D. 2007. Optical excitation yin and yang. Nature Methods, 4:384. 5. Han, X., and Boyden, E.S. 2007. Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution. PLoS. ONE Vol. 2, e299. 6. Sauer, C., Stanacevic, M., Cauwenbergh, G., and Thakor, N. 2005. Power harvesting and telemetry in CMOS for implanted devices. IEEE Transactions on Circuits and Systems. 52:12, 2605-2613, Dec. 7. Webster, T., Waid, M., McKenzie, Price, R., and Ejiofor, 2004. J. Nano-biotechnology: carbon nanofibres as improved neural and orthopaedic implants. Nanotechnology. 15:48-54. 8. Weiland, J.D., Liu, W., and Humayun, M.S. 2005. Retinal prosthesis. Annu. Rev. Biomed. Eng. 7:361-401, Aug. 9. Wilson, B.S., Finley, C.C., Lawson, D.T., Wolford, R.D., Eddington, D.K., and Rabinowitz, W.M. 1991. Better speech recognition with cochlear implants. Nature. 352:236-238, Jul.
Division of Biological Sciences, University of California, San Diego 25
Analysis of Id3 Protein as a Possible Stem Cell Marker in Skin Eun Kyung Joanne Lee
Senior, Biochemistry and Cell Biology, Earl Warren College Abstract Id proteins inhibit differentiation through their capacity to inhibit DNA binding. As a member of the basic Helix-loop-helix (bHLH) family, Id proteins inhibit activity of transcription factor proteins such as E-protein and tissue-specific bHLH protein by forming heterodimers with their HLH domain. It has been suggested, in Dr. Cornelius Murre’s experiments with Id3-GFP knock-in mice on hematopoietic stem cell (HSC) differentiation and development, that Id3 may be another stem cell marker. Finding a new stem cell marker would be useful for future experiments of various fields, as it will provide another means to purify stem cell population. In this experiment, level of expression and location of various Id proteins in skin were investigated using techniques such as RT-PCR, Western Blotting, immunofluorescence, and in situ hybridization. The results indicated that Id3 is present primarily in the hair matrix region, which is essentially not a stem cell region. Therefore we have concluded that the Id3 protein cannot be used as a stem cell marker in skin. However, it is shown in the adult mouse brain that the localization of different Id proteins varies. 11 Therefore the localization of the other isoforms of the Id proteins cannot be predicted according to the results for the Id3 protein. In the future, the study of Id proteins will continue using in situ hybridization to locate Id1, Id2, and Id4 in skin.
Introduction Id protein is an inhibitor of differentiation and DNA binding protein. Currently, four isoforms have been identified and are denoted Id1-4. In the mouse (Mus Musculus family), Id3 is also referred to as HLH462 (Helix-Loop-Helix 462) and Idb3 (Inhibitor of DNA binding 3).12 The expression of Id3 protein in skin was the focus of our study for six months. Id proteins belong to a basic Helix Loop Helix (bHLH) family of transcription factors along with E-proteins and tissue-specific bHLH proteins.13 E-proteins and tissue-specific bHLH proteins contain a basic region that is necessary for DNA binding and a HLH domain that
is necessary for facilitating protein-protein interaction via dimerization. 13 E-proteins, such as E2A gene products (E47/E12) and tissuespecific bHLH proteins form dimers that bind to a promoter site of a target gene through its DNA-binding domain to induce transcription. Id protein inhibits bHLH protein-dependent cellular activity primarily by interfering with DNA binding. This occurs because they lack DNA-binding domains but retain the ability to interact with HLH domains of related proteins (see figure 1).1 The primary reason for studying the Id3 protein is due to unpublished results obtained in the laboratory with Id3-GFP knock-in mice on hematopoietic stem cell (HSC) differentiation and
Figure 1: The function of E- and ID proteins in lymphocyte development. (Engle, I., and Murre, C. 2001. Nature Reviews Immunology. 1:193-199.)
26 Saltman Quarterly Volume 5 / 2007-2008
Figure 2: Human skin layers.
development. The data suggest that Id3 may be used as a marker for HSCs. Finding a new stem cell marker would be useful for future experiments in various fields, as it will provide another means to purify the stem cell population. However, it is unclear whether Id3 protein expression can be utilized as a stem cell marker only in blood or if it can be utilized in other types of cells as well. In this set of experiments, mouse epidermis and dermis were used as the model system to test the universality of Id3 protein as the stem cell marker in different cell types. Skin cells are easy to access and skin sections of the Id3-GFP knock-in mice from the lab could be easily visualized under the microscope to pinpoint Id3’s exact cellular localization. Overall, the project consisted of three main goals: (1) Test whether Id3 protein is present in skin cells. (2) Localize Id3 expression to specific cells of the skin. And if it is localized in the stem cell region, (3) study how Id3 expression is regulated in skin. As we would be performing all of our experiments using mouse skin cells, it is important to be able to recognize different parts of the skin. Skin has three main layers: epidermis, dermis, and subcutaneous tissue. Within these three layers, we are focusing mostly on epidermis and dermis.
The epidermis is a stratified tissue comprised of biochemically distinct layers: starting from the layer closest to the dermis is the basal cell layer, which is a thin layer of epidermal stem cells that perform rapid proliferation and thus push the cells above them further upwards.8 Cells that leave the basal cell layer enter a genetic program called the terminal differentiation, i.e., cells that are destined to die.8 The second deepest layer in the epidermis is the spinous cell layer, which is named for its spiny, irregular appearance due to its keratin penetrating through the plasma membrane and making connections (via desmosomes) with the cells in its vicinity.8 Pushed further away from the basal layer by the cells in spinous cell layer are the ones in granular cell layer.8 Lastly, there is stratum corneum, which is the outermost layer of our skin that we normally see and consists of dead skin cells packed very tightly on top of each other with high amounts of keratin accumulated in them. In about two week’s time, stratum corneum sloughs off and results in what is called the epidermis turnover (see figure 2).9 Stem cells need to keep a good balance between number of cells that perform self-renewal and terminal differentiation for turnover to take place at a normal pace. Dermis contains blood vessels that supply nutrients to the basal cell layer, and fat, collagen, and elastic fibers to give strength and flexibility to the skin.8 Also, the hair follicle penetrates deep into the dermis and its own stem cells localize at its bulge. Stem cells in the bulge of the hair follicle move into the matrix of the hair follicle, where it begins rapid proliferation. In the experiments, we used Id3-Green Fluorescent Protein (GFP) Knock-in mice. Knock-in mice are “an experimental line of mice, produced from embryonic stem cells in which a reporter gene has been inserted into the genome in such a position that the expression, or activity, of a gene under investigation can be monitored.”2 In our case, GFP is the reporter gene that we used to monitor Id3 expression in skin cells.
Methodology Mouse strains Id3-GFP knock-in heterozygote (+/-) mouse strain and Id3-GFP/Noggin knock-out heterozygote mouse strain is used to determine the expression pattern of Id3 with and without its regulator, respectively. Reverse transcription - PCR (RT-PCR) mRNA is purified from the whole mouse, the whole skin, the epidermis and the dermis to
test whether Id proteins are beTable 1: OligoDT Primers Used for RT-PCR Experiment ing transcribed from genomic DNA and where it is generally Id1 sense, 5′-TCAGGATCATGAAGGTCGCCAGTG-3′; localized. Whole mouse puriId1 antisense, 5′-TGAAGGGCTGGAGTCCATCTGGT-3′; fied RNA is used as the posiId2 sense, 5′-TCTGAGCTTATGTCGAATGATAGC-3′; tive control and GAPDH as our Id2 antisense, 5′-CACAGCATTCAGTAGGCTCGTGTC-3′; loading control. Also, samples Id3 sense, 5′-CCTCTCTATCTCTACTCTCCAACA-3′; Id3 antisense, 5′-TGACCAGCGTGTGCTAGCTCTTCA-3′; that are processed without the Id4 sense, 5′-GCGATATGAACGACTGCTACAGTC-3′; reverse transcriptase in making Id4 antisense, 5′-ACTTAGCAGTCTGGTCGACAACAC-3′ (6) the cDNA are used as our negative control. The isolated mRNA is used as templates to make cDNA by reverse transcriptase and oligoDT further homogenized using sonication. primers. PCR is performed to amplify cDNA using several primer sets (see Table 1). The sample is then boiled for five minutes to denature proteins in solution, and then is elecThe total reaction volume is 25 μl, containing 1 trophoresed on stacking and running gel. Proμl of cDNA sample, 2.5 μl of 10× PCR buffer, teins are then moved electrophoretically from 0.50 μl of 10mM dNTPs, 0.25 μl of Taq poly- the gels onto a nitrocellulose membrane. Memmerase, and 0.50 μl of each 10mM primer, and branes are then treated (blocked) using non-fat diluted with 18.5 μl of dH2O. dry milk powder dissolved in PBS-tween 20 to prevent non-specific binding of antibody to the PCR was programmed as follows: membrane. Then primary antibody is used to 1. incubate for 1 min. at 94.0°C identify the proteins of interest and is visualized 2. denature for 30 sec. at 94°C by using secondary antibody with horseradish 3. anneal for 30 sec. at 65°C peroxidase (HRP) that produces fluorescence 4. elongate for 40 sec. at 72°C when reacted with chemiluminescent working 5. repeat amplification steps 2-4 35 times solution and hydrogen peroxide. 6. incubate for 5 min. at 72°C The fluorescence produced by HRP attached to 10 μl of the PCR product is gel electrophoresed the secondary antibody binding to the primary through 1.0% or 1.5% agarose gel with ethidi- antibody varies in its intensity depending on the um bromide. amount of protein (or antibody) available. The reaction is exposed to a light-sensitive sheet to In situ hybridization develop the image of the blot. PCR product for the RT-PCR is cloned into a PCR II vector containing SP6 and T7 primary Immunofluorescence/epifluorescence microssites. The sites are used to generate cRNA in an copy, cryosections in vitro transcription reaction. RNA is labeled An immunoflourescence technique is used to with digoxigenin-UTP in DIG RNA labeling kit pinpoint the localization of the protein and to (Roche). compare with the in situ result. Back skin and tail samples are frozen down in OCT embedding A DIG RNA labeling kit is used to create RNA media. The frozen samples are then sectioned labeled with digoxigenin-UTP by in vitro tran- into 8 µm sections on a cryostat and mounted on scription with either SP6 and T7 RNA poly- microscope slides. Samples on slides are fixed merase (Roche). DIG labeled RNA probes are using 4% formaldehyde for 10 minutes, washed detected with an anti-digoxigenin antibody, with 1% PBS twice, 5 minutes each, then incuwhich can be visualized with highly sensitive bated in blocking solution for 10 minutes. The chemiluminescent substrates such as NBT/ samples are then stained with primary and secBCIP (Roche). ondary (in case of immunofluorescence) antibodies. Keratin 5 (a marker of the basal layer) is co-stained with most sections to visualize the Western blot Tail samples from Id3-GFP knock-in heterozygote mice are frozen rapidly using liquid nitrogen and are pounded with a metal hammer. Lane: 1 2 3 4 5 6 7 8 Broken tissue samples are then put in Laemmli Figure 3: RT-PCR results. In lanes 1, 3, 5, 7: without sample buffer solution containing 4% SDS, 20% RT. In lanes 2, 4, 6, 8: with RT. Lanes 1, 2: whole glycerol, 10% 2-mercaptoethanol, 0.004% bro- mouse; lanes 3, 4: whole skin; lanes: 5, 6: epidermis; mophenol blue and 0.125M Tris HCl. It is then lanes 7, 8: dermis. RT: Reverse-Transcriptase.
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Division of Biological Sciences, University of California, San Diego 27
Figure 4a
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Figure 4: Western blot to detect Id3-GFP in knock-in heterozygote mice.
boundary between the epidermal and dermal layers. DAPI dye is used for nucleus staining; GFP antibody is used along with checking for knocked-in GFP expression.
Results / Discussion Since Id3-GFP knock-in heterozygote mice are Lane 1checking 2 3 4 5 6 of Id37 available, for epi-fluorescence GFP in skin cryo sections seemed to be the ideal first experiment for the study. Co-staining with K5 antibody to make a boundary between the epidermis and the dermis, localization of Id3GFP in wild type mouse skin sections (negative control) and Id3-GFP heterozygote mouse skin sections were tested. In either skin section, there seemed to be no epi-fluorescence localization anywhere. A conceivable explanation could be that the amount of Id3 expressed in any specific types of cells in skin may be too small to be detected solely based on epi-fluorescence of GFP. Thus, amplification of GFP expression via various GFP antibodies was utilized. Wild type skin sections were used as negative controls once again. On each slide, two sections of the skin samples were arranged, and while one sample was stained using immunofluorescence (ex-
posed to the primary antibody that targets the GFP and then to the secondary antibody that targets the first antibody), the other was kept as epi-fluorescence staining. In every case, however, all the sets of slides showed too much background and the negative control slides and the test slides looked the same. After experimenting with different dilution factors of the antibody and with varying antibody-washing protocols, we concluded that the GFP antibodies available today are not specific enough for our purposes. Since the immunofluorescence failed to give us a specific localization of Id3 protein, we retreat4b very basics of the experiment. We first edFigure to the decided to test whether the mRNA for the Id3 protein was being produced in skin via visualization using Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR). Whole mouse cDNA templates were used as positive control, since it includes bone marrow, where the presence of Id3 protein was confirmed. cDNA “made” without the reverse transcriptase were used as negative controls, since mRNA without the enzyme cannot produce cDNA. Also, whole skin and separated dermis and epidermis cDNA were tested using the Id3 primer sets. The result (figure 3) confirmed that only the template cDNA made with reverse transcriptase was able to function properly, showing a faint band for whole mouse (lane 2) and whole skin (lane 4) PCR and a strong band for epidermis (lane 6) and an even stronger band for dermis (lane 8). Only one microliter of cDNA template was used and the reason that there was a faint band for whole mouse and whole skin verses the dermis and epidermis was that the relative proportion of Id3 cDNA to the whole amount of different cDNA present in solution may be much lower for whole mouse than for epidermis or dermis alone. There was, however, contamination between the dermis and the epidermis during the separation step, but the data was sufficient enough to confirm the presence of Id3 mRNA in skin.
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Figure 5A: Id3-SP6 Sense Control (Pedro Lee). 5B: Id3-T7 antisense (Pedro Lee).
28 Saltman Quarterly Volume 5 / 2007-2008
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Then, as the presence of mRNA was confirmed, presence of the actual protein must also be confirmed. If the protein were to be regulated in the translation process and not in the transcription process, there may be abundance of mRNA present in solution without making any active protein. To test whether there are any proteins made, a western blot was performed on genotype-confirmed Id3-GFP knock-in mice. Tail samples were obtained from each of the mice to be homogenized and be used in western blotting. A known amount of GFP was also used in the experiment as the positive control. After the proteins were transferred to the nitrocellulose membrane and blocked with the blocking solution (non-fat dry milk+ PBS tween-20), GFP antibody was used to detect the level of GFP present. Though the GFP antibody was not specific enough for immunofluorescence, western blotting does not require the same degree of antibody specificity. Figure 4a shows the photograph taken for the western blot with the GFP antibody. All the tail samples seem to have a distinct band for GFP except for the sample 5206, loaded on lane 4. Samples 5201 and 5202 in lane 1 and 2 respectively, appear to give a much stronger signal, while samples 5203, 5207, and 5208 in lane 3, 5, and 6 respectively, produced signals of similar intensity. For comparison, GFP alone was also loaded onto lane 7. To determine whether lane 1 and 2 produced a stronger signal due to an unequal loading of the samples into the gel or because a higher concentration of Id3 protein was present in the sample, the nitrocellulose membrane was washed with the stripping solution for ten minutes before it was treated with E7 tubulin antibody (figure 4b). Tubulin is a globular protein that assembles into microtubules, a necessary component of cell cytoskeleton, therefore is found in every mammalian cell. The amount of tubulin indicates the
amount of sample loaded into each lane and thus serves as a loading control: meaning the band strength for the target protein can be adjusted relative to the band strength for tubulin. Since band strength for tubulin in lanes 1 and 2 are also much stronger than the others, it is clear that the differences in signal strength for GFP in lanes 1 and 2 are due to the differences in loading volume. Also, this experiment ensured that there was a sample loaded onto lane 4 where no visible band for GFP was found (figure 4a). Overall, the general presence of GFP was confirmed and therefore, we believe that in sample 5206, the transgene was present but was not expressing the actual protein. Overall, expression of GFP in samples other than 5206 was confirmed by this experiment. Lastly, in situ hybridization was used to determine more precisely in which regions of the skin cell layers the Id3 gene is being expressed. Id3-SP6 sense control (Figure 5a) showed no reaction, while the Id3-T7 anti-sense showed clean localization of Id3 mRNA in the matrix region of hair follicle (figure 5b). A clean control slide confirms the specificity of the RNA probes for Id3 mRNA. There are two stem cell regions in the skin, one is the basal layer of the epidermis, and the other is the bulge of the hair follicle. The area in which the Id3 gene showed localized expression was not the stem cell region, but rather the region of
the hair follicle where the stem cells from the bulge migrate to and begin rapid proliferation, called the matrix of the hair follicle. Therefore, we have concluded that the Id3 protein cannot be used as a stem cell marker in skin. However, it was shown in the adult mouse brain that the localization of different Id proteins varies.11 Therefore the localization of the other isoforms of the Id proteins cannot be predicted according to the results for the Id3 protein. In the future, the study of Id proteins will continue using in situ hybridization to locate Id1, Id2, and Id4 in skin.
Acknowledgments I am grateful for the opportunity, support and encouragement provided by Dr. Kees Murre and Dr. Colin Jamora. I thank Pedro Lee and Luke Denly for their work with the in situ hybridization and for their mentorship. This project was supported by the Faculty Mentor Program at UCSD.
References 1. Engle, I., and Murre, C. 2001. The function of Eand ID proteins in lymphocyte development. Nature Reviews Immunology. 1:193-199. 2. QIMR, glossary of terms. http://www.qimr.edu.au/ qimr_glossary.html. 3. Ying, Q- L., et al. 2003. BMP induction of id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell. 115:281–292.
4. Groppe, J., et al. 2003. Structural basis of BMP signaling inhibition by Noggin, a novel twelve-membered cystine knot protein. J Bone Joint Surg Am. 85:A suppl 3:52-8. 5. Kawamoto, H., Ikawa, T., Ohmura, K., Fujimoto, S., and Katsura, Y. 2000. Immunity. 12:441-450. 6. Ikawa, T., Fujimoto, S., Kawamoto, H., Katsura, Y., and Yokota, Y. 2001. Commitment to natural killer cells requires the helix-loop-helix inhibitor Id2. PNAS. 98:5164-5169 7. Figure 2: MyDr, 2002, skin biology and structure. http://www.mydr.com.au/default.asp?Article=3718. 8. Fuchs, E., and Raghavan, S. 2002. Getting under the skin of epidermal morphogenesis. Nature Genetics Review. Mar; 3(3): 199-209. 9. Taylor, G., et al. 2000. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell. 102(Aug. 18): 451-461. 10. Fuchs, E., and Segre, J.A. 2000. Stem cells: A new lease on life. Cell. 100(Jan. 7):143-155. 11. Kitajima, K., et al. 2006. Localization of Id2 mRNA in the adult mouse brain. Brain Res. Feb; 1073-1074. 12. NCBI. <http://www.ncbi.nlm.nih.gov/entrez/ viewer.fcgi?db=protein&val=6680341> Inhibitor of DNA binding 3 [Mus Musculus] 13. Massari, M.E., and Murre, C. 2000. Helix-loophelix proteins: regulators of transcription in eucaryotic organisms. Mol. Cell. Biol. 20, 429-440.
Division of Biological Sciences, University of California, San Diego 29
The Effects of Cisplatin, a Chemotherapeutic Drug, on the Immune System of Cell Line TRAMPC-1 Halley Park
Junior, Biochemistry and Cell Biology, Revelle College Abstract One of the roles the immune system plays in the development of cancer is not actively participating in eliminating the cancer cells. It is hypothesized that immunoparticles produced by the tumors prevent the immune system from recognizing the tumor as harmful. Vaccine approaches that are actively being implemented in clinical trials focuses on reactivating the immune system, a potent tool that is innate in organisms which will prove to be useful in eliminating the cancerous growth. In this experiment, the chemotherapeutic drug cisplatin was used in order to induce stress in the TRAMPC-1, murine-based, prostrate cancer cell line. IC50 of cisplatin was determined to be in the range of 0.66 µM and 14.19 µM with the average being 4.50 µM. In order to induce an immunological response, three different concentrations of cisplatin were selected: 0.13, 0.41, and 3.7 µM. These concentrations were administered on 10,000 TRAMPC-1 cells at 24, 48, and 72 hour increments and the effects were analyzed. At both 24 hour and 48-hour treatments, the number of viable cells increased. However, the number of viable cells decreased dramatically 72 hour posttreatment. These results indicate the possibility of the immune system playing a more active role in the elimination of cancerous cells, which suggests subsequent experiments should be performed.
Introduction The immune system within living organisms is a necessary and a potent solution to protecting the organism against prominent biological dangers in the organism’s environment. Exploiting this system to treat cancer is therefore a viable and effective approach amongst many other treatment options to curing cancer. The exact mechanisms of how the immune system functions in the protection against, or the development and persistence of, cancer are unknown and controversial. One can easily appreciate the confusion that is present when reviewing the literature. For example, Sheffler et al.1 and Melcher et al.2 propose two opposing results and conclusions in explaining the role of the immune system in combating tumor growth. While Sheffler argues that apoptotic cell death created a more potent immune response in comparison to necrotic cell killing, the results of Melcher suggests the exact opposite conclusion showing that non-apoptotic cell death creates a more potent immune response. Examples such as this highlight the ambiguity surrounding the role of immunological response and cancer persistence. Hence, additional studies are required to clarify our understanding. Vaccine approach is the clinical end of our understanding in the role the immune system plays on cancer development. Vaccines contain al-
tered cancer cells undergoing either necrosis or apoptosis that are injected into the area of cancer cell growth in hopes of increasing the patient’s immune response, which is suspected to be tolerant or suppressive of the ongoing tumor growth. The relative efficacy of these vaccines is often minimal having shown to be effective only 2.6% of the time in clinical trials.3 This poor rate is an indication of the need for further studies. One common approach to design anti-cancer vaccines is the use of chemotherapy agents. Low-dose chemotherapy or sub-lethal dose chemotherapy is geared to stimulate one’s innate immune system via production of endogenous “danger signals” such as cytokines. Other modes of stress-causing agents include temperature shock and radiation, both of which are currently being employed as cancer treatments. The focus of this experiment was to induce an adequate amount of stress in cells to produce endogenous “danger signals” to affect the immune reactivity of the cancer cell line TRAMPC-1. TRAMPC-1 cell line is derived from the primary tumor in the prostate of a transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse and is a good model. To elicit endogenous danger signals from TRAMPC-1, the chemotherapeutic drug cisplatin was utilized. Cisplatin is a commonly prescribed chemotherapeutic drug that is often used to treat metastatic testicular
30 Saltman Quarterly Volume 5 / 2007-2008
or ovarian tumors. Its IUPAC name is cis-diamminedichloridoplatinum (II) and is written as H6Cl2N2Pt with molecular mass of 300.05 g/ mol. Cisplatin causes cells to undergo apoptosis by interfering with mitotic cell division. More specifically, cisplatin causes irreparable DNA damage through the displacement of chloride ligands by cross-linking two bases in the DNA, which in turn causes the cell to become apoptotic. This particular drug was chosen because TRAMPC-1 would react sensitively being that it is a prostrate cancer cell line of mouse. Furthermore, because cancer cells usually replicate massively in numbers via mitosis, they are likely to be affected the most in comparison to noncancerous cells. Cisplatin is the most effective when the cells are in the growth phase. In this experiment, it was hypothesized that sublethal doses of cisplatin will induce endogenous danger signals that will enhance immune reactivity. To investigate this hypothesis, the IC50 value of cisplatin was first established for the TRAMPC-1 cell line; IC 50 refers to the dosage of chemotherapeutic drug that kills 50% of the cells. This information was necessary in order to determine the amount of cisplatin to administer to achieve the low-dose chemotherapy effects. Secondly, varying sublethal dosages of cisplatin were used in order to determine the growth effects of cells under low-dose conditions. The duration of the chemotherapy treatments were also varied. Although not completed, the immune reactivity of TRAMPC-1 after cisplatin is to be checked using bone marrow-derived dendritic cells (BMDC) from mice and running an immunological assay or ELISA.
Methods and Materials The primary cell line that was utilized for this experiment, was TRAMPC-1, murine transgenic prostrate cancer cells. The cells were cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose concentration, glutamine, penicillin, and fetal bovine serum. These cells were cultured in a sterile environment following all standard protocols. To count the cells as well as to check for their viability, a
GUAVA cytometer was utilized. The standard procedures for a GUAVA cell viacount were followed. To determine the IC50 value of cisplatin on TRAMPC-1, different quantities of the cells (5000, 2500, and 500 cells/plate) were first plated onto a 96-flat bottom well plate. A 24-hour period was allowed for the cells to adhere and double in quantity. Varying concentrations of cisplatin were made starting at 300μM initially with1:3 serial dilutions onward. Two controls were made, one with just the cell medium and the other with the organic solvent DMSO, which was used to dissolve the cisplatin. Aqueous solvents were utilized initially; however, because of the solubility issues, the organic solvent DMSO was used instead. Furthermore, a fresh stock of cisplatin in DMSO was prepared every time the experiment was performed due to its instability in a liquid form when left at room temperature. The effect of the drugs was observed at 24, 48, and 72-hour intervals.
An MTT cell proliferation assay using yellow tetrasolium MTT reagent was performed to quantify cell death. The standard procedures as outlined by ATCC were followed. In this assay, cells pick up MTT through the plasma membrane potential and then reduced to formazan or the purple crystals by intracellular NAD(P) H-oxidoreductases. Therefore, only viable cells will be able to pick up MTT and form the purple crystals. Briefly, the epithelial cells were allowed an hour period to settle on the flat-bottom wells. Different quantities of cisplatin were added to each well. The effect of the drugs was observed at 24, 48, and 72-hour intervals. MTT reagent was added to each well in a 1:20 ratio of MTT reagent to the volume of each well. One day was allowed for crystal formation and lysis buffer was added to dissolve all the crystals that were formed. The absorbency of each well was read at 560nm using the GENios program and the machinery.
Results (1) IC50 Determination and Cell Death Curve Three-day administration of the drug cisplatin decreased the number of viable cells in each well. This can be visibly inferred from Figure 1 and Figure 2 below. These figures of the same well before and after the administration reveal fewer round-shaped cells, indicative of cell death, before treatment (Figure 1) compared to experimental treatments (Figure 2). Out of nine total trials for total nine trials for IC50 value determination, only seven trials produced reliable results due to the problems with cisplatin solubility and stability. As shown in Table 1, IC50 values were out to be 2.039, 14.19, 0.66, 1.168, .973, 5.859, and 3.650 µM which all fall in the range of 0.66 and 14.19 with an average IC50 of 4.50 µM. Figure 3 shows the cell death curve of trial 5 and 6, which produced the closest IC50 values to the average value. The same procedures were followed using a different drug called pacletaxol instead of cisplatin. However, IC50 values could not be estimated due to technical problems. Based on the values shown in Figure 3, three varying concentrations of cisplatin were chosen as being sublethal to TRAMPC-1. These values are 0.13, 0.41, and 3.7 µM, respectively. These chosen values are indicated by the arrows displayed in Figure 3. OD values at these concentrations indicate that the cell viability should not be affected due to cisplatin being present.
Figure 1: TRAMPC-1 in DMEM media at 37oC prior to cisplatin treatment (10,000 cells/plate in 96well flat-bottom plate)
(2) The Effect of Sub-lethal concentrations of Cisplatin on TRAMPC-1 Figure 4 indicates the result of the sublethal experiment. At the 24-hour period of cisplatin administration, cell viability was unaffected. At the 48-hour period, cell proliferation was observed indicating active cell replication, despite the presence of cisplatin. However, the effect of the cisplatin became more visible for all three concentrations at the 72-hour period post-administration. The rapid decreases in OD values indicate presence of cell death.
Discussion Figure 2: TRAMPC-1 in DMEM media at 37oC three days after cisplatin treatment (10,000 cells/ plate in 96-well flatbottom plate)
Nine trials were performed to determine the average IC50 value of 4.50 µM for cisplatin against TRAMPC-1. The range of IC50 values, 0.66-14.19 µM, varied due to several reasons. Foremost, due to the chemical nature of cisplatin, its solubility in DMSO was not optimal. Furthermore, the solution had to be made fresh
Division of Biological Sciences, University of California, San Diego 31
Table 1: IC50 values of cisplatin in µM on TRAMPC-1 determined from 7 different trials.
Trial # 1 2 3 4 5 6 7
µM 2.039 14.19 0.66 1.168 3.973 5.859 5.859
OD
every time the experiment was performed due to instability upon prolonged storage. Despite measures to minimize these technical effects, it is possible that heterogeneity in the solubility and/or stability of cisplatin occurred during the experiments. In addition, problems with differential evaporation within wells on the 96-well plates may have occurred. This may have added to the discrepancy when the absorbency of the MTT crystals was being read. Nevertheless, the cell death curve and IC50 value helped to select appropriate concentrations of the drug to be used in the subsequent experiment of sublethal dosage effect on TRAMPC-1. IC50 of a different chemotherapeutic drug pacletaxel was attempted to be determined. However, pacletaxel has similar solubility and instability issues as cisplatin. Not enough trials were performed to be able to conclude its IC50 value on TRAMPC-1.
2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.001
Theoretically, the cells duplicate themselves every 24 hours. However, the cell growth curve should have been made to confirm this assumption since all the cells have slightly different rates of growth and duplication. In this experiment, it was just assumed that TRAMPC-1 duplicates every 24 hours, and this assumption was taken into account when plating the cells initially and waiting 24 hrs before the drug administration.
of OD values, the number of viable cells at all three concentrations of cisplatin decreased. Because sublethal amounts of cisplatin were purposely selected, the cell numbers should have kept increasing and remained viable. However, the results indicate otherwise. Perhaps this is due to sub-lethal doses of cisplatin inducing endogenous danger signals that will enhance the immune reactivity. This enhancement would result in a lower number of viable cancerous cells, TRAMPC-1, as shown in the lower value of OD. To further prove this statement, the solution will have to be extracted and be checked for any en-
During the sublethal experiment, the number of viable cells went up during the second day of adminSublethal killing curve 1.2 istration despite 1.0 the ad0.8 dition of cisplatin. 0.6 0.13 µM This re0.41 µM 3.7 µM 0.4 sult was to be ex0.2 pected 0 since low 24 hr 48 hr 72 hr concentrations Duration of drug administration of cisplaFigure 4: Effects of varying concentrations and time of administration of cisplatin on TRAMPC-1. tin based 0.13, 0.41, and 3.7 µM of cisplatin were selected from the previous experiment as indicated by on the Figure 3. 10,000 cells were plated per well. cell death c u r v e earlier were selected in order to not completely dogenous danger signals such as cytokines and halt the cell growth but to rather solely elicit a other immunoreactivities. Mouse bone-marrow stress response. However, on the third day of derived macrophages or dendritic cells both in the administration, as indicated by the rapid fall vitro and in vivo can be utilized in checking the immunoreactivities of the cells and their media. If successful, in vivo experiments can be done with mice and vaccine injections at the site of the cancer. In theory, sublethal amount of drugs in vaccines should induce endogenous danger signals, enhance the immune reactivity, and help the mice to survive. 5th Trial
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1000
In conclusion, the results of the experiments
Figure 3. Cell Death Curve of TRAMPC-1 with cisplatin from the 5th and the 6th trial. IC50 vales were 3.973µM and 5.869µM, respectively. OD represents optical density. Three day administration of cisplatin starting with 300 µM and 1:3 serial dilution onward was performed with 10,000 cells per plate. The graph was curve-fit to cell-death sigmoidal curve using logarithmic values of the drug concentration. The three arrows indicate three different concentrations of cisplatin 0.13, 0.41, and 3.7 µM. As shown by the OD value for each concentration, the effect of drugs on TRAMPC-1 at these values is insignificant.
with sublethal amounts of cisplatin used on TRAMPC-1 indicate that although sublethal to TRAMPC-1, the drugs elicits dramatically different effects on the 2nd versus the 3rd day of the administration. The results suggest further experiments need to be done to prove increased immunoreactivities being present upon the addition of sublethal amounts of cisplatin including in vivo experiments.
Acknowledgments I would like to give special thanks to my faculty mentor Gregory A. Daniels at Moores Cancer Center for his intellectual guidance, his vast
source of knowledge, and his patience throughout the experiment. Without his assistance, this project could not have been done. Thanks to him, I have developed much appreciation for research, and I am truly thankful for that. Also, I would like to thank Rommel Tawatao for helping me tremendously with hands-on knowledge regarding the experiment as well as getting acquainted with different resources available within the lab.
References
T. 2003. Apoptotic, but not necrotic, tumor cell vaccines induce a potent immune response in vivo. Int. J. Cancer. 103:205-211. 2. Melcher, A., Todryk, S., Hardwick, N., Ford, M., Jacobson, M., and Vile, R. 1998. Tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Nature Medicine. Vol 4:5;May. 3. Rosenberg, S., Yang, J., and Restifo, N. 2004. Cancer immunotherapy: moving beyond current vaccines. Nature Medicine. 10:909-915.
1. Scheffler, S., Nave, H., Korangy, F., Schilote, K., Pabst, R., Jaffee, E., Manns, M., and Greten,
Division of Biological Sciences, University of California, San Diego 33
Senior Honors Thesis Research (BISP 196)
T
UC San Diego Division of Biological Sciences
he Division of Biological Sciences Senior Honors Thesis Program (BISP 196) is open to undergraduate biology majors who have an overall, and major, GPA of 3.7 or higher, have senior standing, and commit to three consecutive quarters of research during their senior year.
In spring quarter each year, students in the honors thesis program participate in a poster session which showcases their research and gives them the opportunity to discuss their research with faculty and their fellow students. The Biological Sciences Student Services Office oversees the program.
The goals of the program are to increase one-to-one interactions between students and faculty and to encourage more biology majors to pursue independent research. Each student in the program has a faculty mentor who provides guidance to the student during the year.
Below are abstracts of some of the outstanding research projects conducted by undergraduates in the program during the 2007-2008 academic year.
Estrogen Dependence of Mammary Tumors in PyMT Mice Rachel Poythress
Earl Warren College, General Biology Major Principal Investigator: Dr. Carole Banka Polyoma middle T antigen (PyMT) will cause spontaneous mammary tumors to form in virgin female PyMT mice. These tumors parallel early human mammary tumors in their initial development (1). It has been shown (2) that estrogen promotes mammary tumor explant growth in culture and in vivo. At this point, no one has looked at estrogen requirements for spontaneous mammary tumor development in PyMT mice. We hypothesized that PyMT mice with estrogen will produce mammary tumors quicker and more fully than PyMT mice without estrogen. PyMT mice were bred in house. Two weeks after their birth, females were genetically tested for the presence of the tumor virus long terminal repeat that induces the spontaneous mammary tumors. At 4 weeks, the mice with a positive genotype for the virus underwent surgery to remove their ovaries, the main source of estrogen in mice. Half of the mice were then given estrogen pellets. After 8 weeks, there was no sign of tumor growth in any of the mice. After 13 weeks tumors were developed, but no difference was observed between the mice with and without estrogen. The mice were sacrificed after 15 weeks with an average of 3 mammary tumors per mouse. After our study, research on mammary tumors in rats was published suggesting a link between mammary tumor development and the combination of estrogen with progesterone. Further research could explore the relationship of estrogen and progesterone in PyMT mice. With our virally driven model, we can conclude that the presence or absence of estrogen may or may not have an effect on mammary tumors. To look at this further we would need to explore the relationship of estrogen receptors on these tumor cells.
The Contribution of microRNA to Interferon downregulation of CXCR4 Peggy Leung
Earl Warren College, Biochemistry and Cell Biology Major Principal Investigator: Dr. Michael David, UCSD Division of Biological Sciences In 1998, a process of post-transcriptional silencing of gene expression (RNA interference or RNAi) was demonstrated and has now become an important tool in understanding eukaryotic biology. RNAi through microRNAs (miRNA), ~22 nucleotides long single stranded non-coding RNA molecules, has been recognized to play a vital role in innate antiviral immune response in plants, invertebrates and recently even mammalian organisms. It is also well accepted that interferons play a crucial role in antiviral immunity. Here we aim to describe the contributions of miRNA in the process of interferon downregulation of CXCR4, a well documented receptor used by T-tropic Human Immunodeficiency Virus type 1 (HIV-1) to gain entry in the cell. We demonstrate that interferon &#946; (IFN&#946;) induces the expression of miRNAs that have sequence-predicted targets within the gene CXCR4, specifically mir-519e and mir-224. We have also shown that the introduction of synthetic miRNA-mimics corresponding to these IFN&#946;-induced miRNA reproduces the effects of IFN&#946; on CXCR4. Thus, our data shows the likely contributory effects of miRNA in the interferon downregulation of CXCR4.
34â&#x20AC;&#x192; Saltman Quarterly Volume 5 / 2007-2008
Interactions between RasGEF proteins in Dictyostelium Shifeng Xue
Revelle College, Biochemistry and Cell Biology Major Principal Investigator: Dr. Richard A. Firtel, UCSD Division of Biological Sciences Ras proteins are small GTPases involved in many signaling pathways. In Dictyostelium discoideum, Ras proteins are involved in the control of cell movement, chemotaxis and multicellular development. The activation of Ras requires guanine-nucleotide exchange factors (GEF). Here we examined the interactions between 3 RasGEF proteins with LisH domains, GefF, GefI and GefJ. Through co-immunoprecipitation experiements, we showed that these proteins were able to pull down one another. We purified and identified the complex in which these proteins are found and find no other GEF proteins in the complex, indicating that the LisH domains are important in the interactions between these 3 proteins. To understand the functions of these proteins, we attempted to make knockout cell lines of each of the genes. gefF could not be disrupted even after multiple attempts, suggesting that it is essential to cell growth. A gefI-null cell line was made successfully but there was no phenotypic difference between wild-type and gefI-null cells. Because GefI and GefJ are very similar in structure, we propose that GefI and GefJ are used interchangeably in the cell and one can rescue the loss of the other.
Evidence That the Lipid Fraction of Serum Contains an Activity That Forms Apatite Crystals Valerie Gerriets
Earl Warren College, Biochemistry and Cell Biology Major Principal Investigator: Dr. Paul Price, UCSD Division of Biological Sciences The goal of the present study is to understand the process by which a matrix gets mineralized so that we can better understand the mechanism of bone mineralization and artery calcification. Previous studies have shown that collagen, demineralized bone and the elastic lamellae of arteries all calcify when incubated in serum. A recently proposed mechanism of mineralization involves the generation of small calcium-phosphate crystals in solution by an as yet unidentified serum nucleation factor when in the presence of alkaline phosphatase. Most of the crystals are immediately bound by fetuin, a known inhibitor of mineral growth, so only the crystals that diffuse into a matrix, such as collagen, are able to grow. The presence of fetuin therefore allows the preferential growth of mineral nuclei within a matrix while inhibiting formation in the surrounding solution. Density gradient ultracentrifugation was used to separate calf and rat serum into fractions based on density and these fractions were tested for the ability to nucleate calcium-phosphate crystals in a pH 7.4 Tris buffer containing calcium and phosphate at physiological concentrations as well as alkaline phosphatase. The lipid fraction of serum of both species were found to have this ability; mineral precipitate that formed in vitro was confirmed to contain both calcium and phosphate. In the same physiologically relevant buffer, presence of 2% of the lipid fraction of serum by volume was shown to preferentially mineralize a matrix when fetuin was present, allowing for growth of the mineral crystals within the matrix while excluding growth outside. This suggests that the serum nucleation factor is present within the upper density fraction of serum. Identification of the specific lipid or protein involved in the process would greatly add to the understanding of the mechanism of mineralization, whether as a normal process in bone or in human vascular disease.
Comparisons of Type IV and Type VI Protein Secretion Systems Michael Stockin
Revelle College, Biochemistry and Cell Biology Major Type vi bacterial secretion (T6SS) is a recently discovered method of protein translocation through the membrane. Several kinds of multimeric complexes have been discovered that utilize type vi secretion to release toxins into eukaryotic host cells. T6SS is associated with virulence in several pathogenic species including Edwardsiella tarda and Vibrio cholerae. An operon coding for several proteins called the VAS (virulence associated secretion) genes has been sequenced, but its structure remains largely unknown. In order to better understand the structural biochemistry of its subunits, we test the VAS genes for homology to components of various type iv secretion systems (T4SS) which are much better characterized than T6SS. We find that 5 of the 12 VAS genes show regions that are homologous to proteins of known T4SS. We create hydropathy plots to look for transmembrane segments (TMS) in the Vas genes and their type iv homologues. There appears to be poor correlation between number and placement of TMS in subunits showing homologous regions of the two types of systems. Our results suggest that T6SS and T4SS derived from proteins sharing domains but they have diverged in sequence and possibly function. Further bioinformatic research may be able to find homology between two other VAS genes and T4SS—as well as uncover other structural motifs in common between two types of systems.
Division of Biological Sciences, University of California, San Diego 35
Correlation of the Expression Levels of Key Components, Targets and Associated Proteins of the wnt Signaling Pathway in the Functional Outcome of Arthritis in Mutant Mice Nicole Flores
Revelle College, Animal Physiology & Neuroscience Major Principal Investigator: Maripat Corr, M.D. Professor, UCSD School of Medicine Rheumatoid arthritis is a destructive autoimmune disease that is characterized by synovitis and proliferation of synovial tissue cells. The rapidly dividing cells that contribute to this swelling make destructive cytokines and proteases that invade and erode the adjacent bone. We hypothesized that the joint-lining cells might express embryonic proteins in an attempt to recapitulate embryonic programming to repair the damage. Arthritis was induced in cyclooxygenase1 (COX1), COX2, TRIFLPS, and interleukin6 (IL6) mutant mice. Using quantitative PCR, the expression levels of key receptors, ligands, downstream targets and associated proteins of the wnt signaling pathway was measured from RNA extracted from the joints of mice sacrificed on specific days following arthritis induction, then correlated with the presence and/or severity of joint swelling in these mice. The wnt signaling pathway is involved in the abnormal growth of tumors, joint formation, and important developmental events in the growing embryo. Levels of wnt3a in COX2 mice were highly upregulated on day 1 and day 4 in the COX2 mutant mice. It was also observed that levels of the wnt pathway components were consistently downregulated in the TRIFLPS mutant mice. The lower levels of the wnt pathway components correlated with increased levels of the pro-inflammatory cytokines interleukin-1β, interleukin-6, and TNF. The expression levels of the pro-inflammatory cytokines IL1β and IL6 directly correlated with the severity of arthritis and paw swelling observed in the mutant mice which increase from COX1 mutant mice, to COX2, IL-6, then LPS mice, which had the most severe arthritis. This suggests that the molecules these mice are deficient in play increasingly important roles in controlling the expression of pro-inflammatory cytokines which contribute to the severity of murine arthritis. Understanding how gene expression and protein levels contribute to a possible repair mechanism for rheumatoid arthritis could aid in the development of effective treatment for this destructive disease.
Augmenting the CD8 T Cell Response Using IL-15 Cytokine Complexes Pauline Filippou
Eleanor Roosevelt College, Human Biology Major Principal Investigators: Dr. Ananda Goldrath and Dr. Mark Rubenstein, UCSD Division of Biological Sciences The role of interleukin 15 (IL-15) is pivotal for natural killer and CD8 memory T cell survival. This cytokine, released by mononuclear phagocytes in response to infection, is known to regulate T and natural killer cell activation and proliferation. When administered in vivo as soluble IL-15, poor half-life and limited biological activity hinder the therapeutic effect of this molecule. Recent studies have shown that the biological activity of IL-15 can be greatly enhanced through association with its receptor alpha subunit, in the form of IL-15/IL-15sRα complexes. We have shown that these IL15 complexes selectively and strongly increase proliferation of antigen-specific CD8+ T cells in response to bacterial and viral infection. A significant augmentation in the response is seen when examining the endogenous CD8 T-cell response, as well donor CD8 T cell populations. Interestingly, IL-15 complex administration selectively increases the survival of a subset of the CD8+ memory population, marked by increased expression of the KLRG1 cell surface marker, despite these cells being typically described as short-lived. These findings demonstrate the applicability of this reagent to augment T cell-directed vaccination strategies.
Purification of a Novel GFP-fused E. coli Membrane Protein Containing Four Transmembrane Segments Martin Krupa
Revelle College, Bioinformatics Major Dr. Senyon Choe, Structural Biology Laboratory, The Salk Institute for Biological Studies About 40 percent of therapeutic drugs in use today target integral membrane proteins (IMPs), yet, compared to the 40,000 soluble proteins, only about 150 IMP structures have been deposited into the Protein Data Bank. The main reason for this discrepancy is that IMPs are difficult to express, solubilize, and crystallize for X-Ray diffraction. In 2005, the von Heijne group experimentally determined the topologies for 601 E. coli IMPs using a GFP fusion construct, and as a byproduct, they gauged the expression levels of these proteins using whole cell fluorescence. Among the best expressing targets was the four-transmembrane E. coli IMP YcjF, with an unknown function. We have found that the YcjF-GFP fusion does indeed express well and accumulates in the membrane fraction. Here we report that the fusion can be efficiently solubilized by most non-denaturing detergents, but that its stability after GFP cleavage is highly dependent on detergent type and that it is most stable in DM, which has been previously used to crystallize other IMPs. We have also found that the protein can be extracted by the novel polysaccharide-based polymer NVOY, which, as we have determined, increases protein stability by decreasing degradation products. Additionally, using NMR we have found that NVOY can exchange many highly hydrophobic detergents. Our results lay a strong foundation for future structural analysis of YcjF and we are currently setting up crystallization trials with different detergents and NVOY.
36 Saltman Quarterly Volume 5 / 2007-2008
Dedicated to the memory of
Paul D. Saltman, Ph.D.
W
hen speaking to anyone who has had the pleasure of working with him, interacting with him, or seeing him interact with other students, one can feel the enthusiasm that the memory of Dr. Paul D. Saltman still brings to those who knew him. As Dr. Gabriele Wienhausen, Associate Dean for Education, says, “He was committed to students, he never compromised
truth for silence, and he always had an open door. Students really felt the respect he had for them and he changed many lives.” Judging by the support that we have received from the faculty members who take their time to personally review each research article with the author, who are always thinking and suggesting a plethora of new features to include in the journal to spark student interest, who submit their
students’ works for publication, and who always have an open mind and open door for the staff here at Saltman Quarterly, it is clear that Dr. Saltman’s exemplary enthusiasm and dedication has not been lost, but continues to show through today in the people he has touched. As a family of faculty, students, and staff, we hope that we may carry on his remarkable example.
Division of Biological Sciences, University of California, San Diego 37
Behind the scenes
Saltman | Quarterly 2007-2008 Staff Advisor Patricia Walsh│ We are so grateful for your infinite patience and trust in us. You have been incredible with your encouragement in all that we aspired to do. Your ability to laugh with us, think with us, and explore with us has made you an unparalleled guide for us these past few years. You’ve been here since the beginning, and we really couldn’t have continued to grow so strong and vibrant without you. Thank you!
Staff Members
Not Pictured: Daryush Tabatabai, Technical Editor for Content
Quynh-Anh Mai
Brannon Peralta
Alex Fortenko
Senior, Molecular Biology major, Eleanor Roosevelt College
Senior, Human Biology major, Earl Warren College
Senior, Microbiology major, Eleanor Roosevelt College
Editor-in-Chief
Flori Pierri
Research Design Editor
Junior, Molecular Biology major, John Muir College
Research Editor
Michael Wang
Features Design Editor
Sophomore, Human Biology major, Revelle College
Features Editor
Caitlin Rodriguez
Technical Graphics Editor Freshman, Physiology & Neuroscience major, Thurgood Marshall College
Jason Ly
Production Editor
Senior, Human Biology major, Sixth College
Christine Ha Webmaster
Christine Cho
Review Board Manager
Sophomore, Human Biology major, John Muir College
Billy Wang
Review Board Manager
Sophomore, General Biology major, Sophomore, Human Biology major, Revelle College Earl Warren College
Staff Writers
Yuli Wang Publicity
Senior, Animal Physiology & Neuroscience major, Eleanor Roosevelt College
Tyler Green
Marielle Venturino
Seungleal (Brian) Paek
Emily Hao
Acknowledgments
Review Board Members Allison Ashley Aka
Junior, Biochemistry & Cell Biology major
We would like to acknowledge the guidance and support of the members of the Saltman Quarterly Faculty Advisory Committee and of guest faculty reviewer David Holway.
Alan Naim
Senior, Human Biology major
Hossein Ayazi
Junior, General Biology major
Sarah Cooper
Junior, Human Biology major
Matt Croskey
Freshman, General Biology major
Fernanda Delgado
Senior, Molecular Biology major
Emily Grauel
Senior, Human Biology major
Kyla Holmes
Junior, Animal Physiology & Neuroscience major
Christina Homer
Gabriele Wienhausen Ph.D. Associate Dean for Education
Eric Allen, Ph.D.
Section of Molecular Biology
Junior, Biochemistry & Cell Biology major
Connie Hong
Junior, General Biology major
Lauren Ashley Miller
Junior, General Biology major
Nicole Mehaffie
Junior, Ecology, Behavior, and Evolutionary Biology major
Mimi Nguyen
Sophomore, Biochemistry & Cell Biology major
Tuongman (Tiffani) Phan
Junior, Human Biology major
Patrick Shih
Senior, Microbiology major
Bao Truong
Junior, Microbiology major
Lisa Boulanger, Ph.D. Section of Neurobiology
Robert Schmidt, Ph.D.
Section of Cell & Developmental Biology
Ryan Vander Werff
Junior, Molecular Biology major
Jennifer Vo
Freshman, General Biology major
Robert Vo
Sophomore, Biochemistry & Cell Biology major
Victor Wang
Freshman, Biochemistry & Cell Biology major
Qingmeng Zhang
Sophomore, Human Biology major
David Holway, Ph.D.
Section of Ecology, Behavior, and Evolutionary Biology
Christopher Wills, Ph.D.
Section of Ecology, Behavior, and Evolutionary Biology
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