Sq vol2

Undergraduate Biological Research Publication Volume 2, Nos. 1, 2, 3 UCSD Division of Biological Sciences http://sq.ucsd.edu

Saltman | Quarterly

Undergraduate Research Publication EDITORS-IN-CHIEF: Cara Cast Caroline Lindsay PRODUCTION EDITOR: Kyle Kuchinsky RESEARCH EDITOR: Ron Alfa FEATURES EDITOR: Eric Chan WEBMASTER: Josh Tan PUBLICITY BOARD CHAIR: Ann Cai POSTER SESSION CO-CHAIRS: Reeti Desai Nicole Gomez REVIEW BOARD: Ron Alfa Brittany Bernik Ann Cai Kristin Camfield Cara Cast Eric Chan Max Chen Reeti Desai Heather Eshleman Daniel Fang Ryan Ferrell Kristine Germar Avanti Ghanekar Nicole Gomez Shruti Jayakumar Peter Kim Stephanie Kinkel Alex Kintzer Kyle Kuchinsky Chi-chung Lee Andrew Lin Nick Lind Caroline Lindsay Laura Lombardi Lauren Ashley Miller Sara Paul Josh Tan Yu-Ting (Alice) Tsai Jennifer Wan Grace Wang STAFF ADVISOR: Patricia Walsh

Copyright@2005. Regents of the University of California. All rights reserved.

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A Message from the Editors In your hands you hold the result of the hard work of many dedicated undergraduate students who, while excelling in their classes, took on the additional responsibility of contributing to scientific research. For many of us, the magic of the lab drew us in—sometimes until late in the evening—to run that extra experiment or discover an amazing result. Science is what motivates us.

When Marika Orlov, Louis Nguyen, and Greg Emmanuel founded Saltman Quarterly last year, we were eager to help with what we knew would become a lasting tradition among biology students. The drive and motivation of those three undergraduates began something totally new at UCSD—an undergraduate-run journal of biology. As the second volume goes to print, we can’t help but reflect on the inspiration of the founders. We knew we had big shoes to fill. But the continuing interest in this journal expressed by our peers reminded us that all the hard work was worth it.

Developing SQ this year was a wonderful experience—one we will never forget. The skills we gained from organizing, editing, managing and delegating are benefits that will serve us well in the future. But in addition to what we have learned, we have had the opportunity to read and review so much incredible research performed by our peers. Often we can’t help but be amazed at the quality of the work done by undergraduates. Our role with SQ has been an honor and an incredible experience.

We would like to thank a few people without whom this journal never would have made it. First, we’d like to acknowledge all the contributing authors. Without their research and excellent communication of their work, we could have never printed Volume 2 of SQ! Second, all the members of the SQ Review Board deserve recognition. They, amidst their own midterms, lab work, and extracurricular activities, were able to give up their time to read and provide feedback for their peers. In addition, we thank those who have held leadership positions with SQ this year. This journal requires delegation by the chief editors to committed fellow undergraduates, many of whom we hope will continue the journal’s legacy in years to come.

We’d also like especially to thank Patricia Walsh, our staff advisor, for her editorial input and advice. And last, but certainly not least, we thank Paul Saltman—an inspiration to all whose lives he touched.

We sincerely hope you enjoy this volume of SQ. ~Cara and Caroline

Cover Image: Confocal microscopy of a section of rat cerebellum that was triple fluorescently labeled. Purkinje neurons are shown in green, the glial cells are in red and cell nuclei are stained blue. Purkinje neurons (branching cells in the cortex discovered by German scientist Jan Evangelista Purkinje in 1837) are some of the largest and most complex cells in the mammalian brain, possessing diameters almost as large as a human hair. Each of the cells exhibits an abundance of very active dendrites and is capable of receiving input from over 200,000 other cells. Yet, Purkinje neurons are the lone source of output from the cerebellum’s cortex. Their primary function is believed to be inhibitory, selectively suppressing and limiting excitatory impulses from other cells and crafting them into a coherent message that the rest of the brain can understand. Image by Thomas Deerinck and Mark Ellisman, The National Center for Microscopy and Imaging Research, UCSD.

Undergraduate Research Publication UCSD Division of Biological Sciences

DEDICATION 5 Dr. Paul Saltman

http://sq.ucsd.edu Volume 2, Nos. 1, 2, 3 23 Analysis of the Manganese Oxidation Capacity of Pseudomonas putida GB-1 and MnB1 Mutant Strains Research Article by Daniel Baron

NEWS 6 Current Events

ORGANISM OF THE QUARTER 7 Xenopus laevis

by Caroline Lindsay

ISSUE 2 - WINTER 31 A Scent-sible Choice of Nobel Laureates Editorial by Cara Cast 32 Toll-like Receptors: An Important Part of Both the Innate and Adaptive Immune Systems Review by Kristin Camfield 33 Nerve Growth Factor Increases Hippocampal Neurogenesis of Adult Rats as seen with Doublecortin Labeling Research Article by Danny Simpson

FEATURED FACULTY

ISSUE 3 - SPRING

9 An Interview with Dr. Nick Spitzer

39 Elevated Intraocular Pressure and Reduced Central Corneal Thickness as Risk Factors for Glaucoma

ISSUE 1 - FALL 15 Francis Crick—A Retrospective Look at a Legacy Editorial by Kyle Kuchinsky 17 Brain-Machine Interfaces: Reinventing Sensory and Motor Functions After Injury or Disease Review by Ronald Alfa 20 Mechanistic Role of Mrf-2 and C/EBPα During Transcription Reseach Article by Nicole Gomez

Review by Pei-Chen (Jennifer) Hsieh 41 Intimate Association of Vibrio splendidus with the Purple Sea Urchin Stronglyocentrotus purpuratus Research Article by Shirin Doroudgar 45 Chemical Influences on Bee Hunger Research Article by Xu (Lloyd) He

SALTMAN QUARTERLY STAFF 48 Biographies and Photographs

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Undergraduate Biological Research Publication UCSD Division of Biological Sciences

http://sq.ucsd.edu

The Saltman Quarterly is UCSD’s undergraduate research publication devoted to providing student researchers here with a place to publish their work.

Become a member of SQ’s peer-review board!!

As a peer-reviewed journal, SQ needs undergraduates willing to participate as members of our review board.

Members of the board will have the opportunity to read and critique research articles submitted for publication by their peers--other undergraduates in Biology at UCSD. Involvement will provide students with valuable experience reading and analyzing scientific literature.

Become a member of our staff !!

Do you have experience with layout, editing, or print production? SQ has limited staff positions open for individuals to help with content and layout decisions and to get the journal into print.

Get published!!

SQ accepts research articles, editorials, feature stories, commentaries, review articles, and “Organism of the Quarter” submissions. If you have an idea for a piece of writing of this type we want to hear from you.

Submissions are accepted on a rolling basis.

If you are interested in becoming involved with this exciting new journal, please visit our website.

http://sq.ucsd.edu 4

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Dedication to Dr. Paul Saltman Positive role models abound in the University of California system. Academic faculty are residents of the contemporary “village” proverbially required to raise a child. Young adults from all walks of life merge briefly during their years of college, choosing to take advantage of an opportunity to have their minds challenged and shaped by university faculty.

This is one benefit given by the university to its undergraduates—trustworthy mentorship. The role is filled by professors who are outstanding teachers.

Some of these people prove to be great instructors not only of science, engineering, humanities and the arts but also of self-awareness, cultural values, and responsibility. Saltman Quarterly is the namesake of one such mentor.

Education of undergraduates was a responsibility Dr. Paul D. Saltman cherished. He believed that once one attains the gift of knowledge, sharing that knowledge becomes a responsibility. Teaching in the classroom, and teaching well, was one of the challenges Professor Saltman met with ardent enthusiasm. One result of his commitment to excellence in education is the Paul D. Saltman Chair in Science Education. The Chair is awarded for a three-year term to a faculty member whose excellence in teaching reflects and embodies the values that Dr. Saltman applied in his own teaching efforts.

During his 32 years at UCSD, Saltman taught many

courses, including introductory biology, metabolic biochemistry, and nutrition. Out of respect for his profession, he always wore a shirt and tie to class, and he took the greatest care to ensure that his students understood the material he presented.

With a lecture style that reflected his charismatic personality, the professor captivated his students.

With extensive knowledge of often abstruse material, Saltman made the details of his course material transparent to undergraduates using some of the best communication skills any classroom has ever seen. His enthusiasm for teaching and learning was contagious. When asked the ingredients of a great teacher, Saltman concluded the keys were knowledge, skill, the ability to comprehend the process of human understanding, and the ability to inspire students and excite them with the notion of learning.

Saltman was responsible for the department opening teaching assistant opportunities to successful undergraduates.

Biology undergraduates who have received credit for BISP 195 have done so because of Dr. Saltman’s initiative and confidence in undergraduate aptitude.

Saltman achieved respect from both the scientific and academic communities through his first-rate research centered on the properties trace metal ions such as zinc, copper, and iron and their utilization

by the body in metabolic processes. An effective researcher and educator, he was also in demand to serve the campus community in administrative roles. He served as Provost of Revelle College and Vice Chancellor of Academic Affairs before finally settling productively into his laboratory and classrooms. Among his lengthy list of honors, he received the first ever Career Teaching Award from the Academic Senate of UCSD. Dr. Saltman died on Aug 27th, 1999 after a tremendous fight with prostate cancer. Sixth College Provost, fellow nutrition professor and dear friend of Dr. Saltman, Dr. Gabielle Wienhausen shared a memory with SQ’s editors about Saltman during the last quarter before he passed away. During his last spring quarter teaching, even with his ailing health, he refused to forego teaching his nutrition course, agreeing with Dr. Wienhausen that he could call her any night, no matter how late, to fill in for him in class the next day. He never called. Another faculty member, Randy Hampton currently holds the Saltman Chair in Science Education and is no stranger to good humor himself. In an interview with SQ, Dr. Hampton quoted Paul Saltman, who in response to an inquiry about his health, stated, “Aaww kid, you know if I die with a piece of chalk in my hand, I’ll be just fine.”

We who did not have the privilege of knowing Dr. Saltman have missed out not only on the mentorship of one of the truly great minds of science and education but also of a truly caring human being.

Yet many aspects of an undergraduate’s positive experience at UCSD are Dr. Saltman’s legacy, which lives on in those who reach for, to quote Paul, “…the limits of their own human potential.”

Photo credits: Top right: © 1994 UCSD Publications Office. All rights reserved.

Photographer Will Guillette Bottom left: © 1987 UCSD Publications Office. All rights reserved.

Photographer Alan Decker Volume 2

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Current Events Saltman Quarterly Faculty Advisory Board Appointed

Division Lecturer Arranges Biotech Education in Baja, California

In the coming year, Saltman Quarterly will have a faculty advisory board to guide students in the peer review process. The committee members, who were recently appointed by the Council of Chairs, consist of a faculty member from each of the four sections of the UCSD Division of Biological Sciences. The faculty members are Laurie Smith from Cellular and Developmental Biology, Lisa Boulanger from Neurobiology, Milton Saier from Molecular Biology, and Chris Wills representing Ecology, Behavior, and Evolution.

Dr. Meredith Gould, a lecturer in the Division of Biological Sciences, organized a week-long workshop on agricultural biotechnology at the Universidad Autonoma de Baja California (UABC) in Ensenada, BC, Mexico, where she has taught courses for many years. This workshop was held June 13-17, 2005 in a teaching lab at UABC, and UCSD professors Maarten Chrispeels and Robert Schmidt, along with volunteer UCSD graduate students, taught twelve UABC graduating seniors and twelve students from elsewhere in Mexico. The students learned about the use of internet resources in nucleotide sequence analysis, genotype analysis using PCR, protein analysis by SDS-PAGE, and detection of transgenes in foods.

Under the Microscope Saltman Quarterly held its second annual undergraduate poster session entitled “Under the Microscope” on June 1st in the Natural Sciences Building. Accompanying the poster session, students were invited to give a short talk about their research. Sophomore Edward Chuong presented his ongoing work with the lab of Dr. Hopi Hoekstra involving evolutionary pressures in deer mice. Neurobiology faculty professor Dr. Nick Spitzer then spoke to the group of aspiring young scientists on making a career in science and related topics. A total of 11 undergraduates discussed their posters with graduate students, postdoctoral fellows, faculty, and, of course, with their peers. The following students presented:

Ron Alfa Linda Boettger Justin Chartron Keri Chen Edward B. Chuong Matt Gielow Peter Kim Ming Na Lee Trina Patel Danny Simpson Man-Un Ung

Kavli Institute for Brain and Mind Inaugurated at UCSD On November 4, 2004, members of UCSD’s Biological Sciences Division joined colleagues from the Cognitive Science Department and other disciplines and organizations in the colorfully lit atrium of the Natural Sciences Building to forge a new relationship among the attending groups.

Thanks to a 7.5 million dollar endowment by Fred Kavli, founder and chairman of the board of The Kavli Foundation, scientists in La Jolla working to bridge the gap in understanding between brain and mind have a new resource for their efforts. Remarks

by Dr. Nick Spitzer opened the evening followed

by Chancellor Marye Anne Fox’s introduction of Fred Kavli. Mr. Kavli’s foundation, which is “dedicated to the advancement of basic science for the benefit of humanity” funds research in cosmology, nanoscience and neuroscience. Dr. Jeff Elman, association dean of Cognitive Sciences and co-director of KIBM with Dr. Spitzer, made closing remarks.

Neurosciences Meeting in San Diego The Society for Neuroscience held its annual meeting at the San Diego Convention Center October 23rd through 27th. Record attendance of over 31,000 scientists and associates filled the venue with poster sessions and numerous lectures and symposia. Many UCSD researchers attended and participated in the event joining neuroscience affiliates from other local research institutes such as The Salk Institute, The Burnham Institute, The Neurosciences Institute, and The Scripps Research Institute as well as universities and science centers around the world. In a featured lecture entitled “Building and Breeding Molecules to Spy on Cells and Networks” Dr. Roger Y. Tsien, UCSD Department of Pharmacology, updated the community on the latest in fluorescent

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protein applications and techniques. Other topics including stem cell research, consciousness, vision, and neurodegenerative diseases such as Lou Gherig’s and Parkinson’s demonstrated the diversity of areas of research presented and discussed at the 34th annual meeting. The Society for Neuroscience is the largest association of neuroscientists in the world.

Donation Program a Worthy Cause for Biology Undergrad UCSD sixth college junior and biology student Alex Quick established “Donor Dudes,” a project to promote awareness of the need for organ, tissue, and blood donors among college students.

The project, which began in 2002, was backed by a $10,000 scholarship from the Donald A. Strauss Public Service Scholarship Foundation.

“The project is an extension of my commitment to life-saving donations,” says Quick, who plans to expand the project to other colleges and make Donor Dudes permanently integrated at UCSD. Quick’s inspiration for the project came from watching Olympic snowboarder and liver transplant recipient Christopher Klug compete in the 2002 Winter Olympics in Salt Lake City.

Hellman Fellows Named for 2005-2006 UCSD assistant professors James Nieh and Pamela Reinagel have received Hellman Fellowships for the 2005-2006 year. The fellowship award was established in 1995 through a generous gift from Chris and Warren Hellman. It is typically given to tenure-track faculty who show potential for great distinction in their work.

Johnson Honored by NSF and Ray Thomas Edwards Foundation UCSD assistant professor Tracy Johnson, one of last year’s Hellman Fellows, received two awards this year. Dr. Johnson received the prestigious National Science Foundation Early Career Development Award, an honor bestowed upon outstanding junior faculty committed to integrating research and education, who are considered most likely to become the academic leaders of the 21st century. Also, the Ray Thomas Edwards Foundation fellowship recognized Dr. Johnson for her commitment to community outreach. Dr. Johnson teaches undergraduate Molecular Biology and is a UCSD Revelle College alumna.

ORGANISM OF THE QUARTER: XENOPUS LAEVIS

Frogs Leap to the Forefront in Biological Research Research in the field of biological sciences could not proceed without the use of many model organisms. These organisms range from tiny bacteria growing on an agar plate to mammals such as mice or rats. In Volume 1, Issue 1 of Saltman Quarterly, the use of the nematode C. elegans as a model organism was discussed.1 C. elegans can be used in many types of experiments, especially neurobiological, since their nervous system has been completely identified and mapped. Because only 302 neurons account for all behaviors that these animals perform, it is much easier to study their brain function than it is to study that of

Figure 1: A female Xenopus laevis in a petri dish full of pond water. Surrounding her are the thousands of eggs she has just laid. (Photo credit: D. Forbes Lab, UCSD)

humans. However, C. elegans is not a sufficient organism on which to perform all neurological, cell biological, or developmental experiments.

In some cases, a vertebrate model organism is needed. For many in vitro analyses of neural and other development in vertebrates, oocytes or embryos from the African clawed frog Xenopus laevis are used.

In the 1940s, Xenopus laevis began being widely used as a useful model organism when it was discovered that Xenopus could be used for human pregnancy tests. The female frog could be injected with a sample of a potentially pregnant woman’s urine. If the sample was positive, it contained the human hormone chorionic gonadotropin (hCG) and this would cause the frog to lay eggs.2,3,4 In the 1970s, it was discovered by John Gurdon et al. that injection of mRNA into Xenopus oocytes causes them to synthesize the foreign proteins7.

His most important discovery, however, was showing that a frog skin cell nucleus, if put in place of the egg nucleus, was able to program full development of the egg into a frog. This was a very novel discovery at the time because it was the first experiment which showed that the genes in all cells were essentially the same. Genes in differentiated cells were basically propagated unchanged through all the cell division and

differentiation required to make an organism.

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Today in many neuroscience labs, the oocyte is effectively used as a living test tube. In the early 1980s, neurobiologists discovered that Xenopus oocytes, if injected with mRNA or genes encoding ion channels from other species, produce these channels and place them in their plasma membrane. The electric current produced by these newly formed ion channels can be studied and quantified. To study the effect of mutations on the channel signaling, mutations can be introduced in the mRNA sequences of channel proteins, and the mutant mRNA can be injected. These mutations will often be a single nucleotide change in a region of interest in the protein structure. When the eggs start producing the channel protein, the effect of the mutation is observed by comparison to the wild type form. In addition, Xenopus has few endogenous ion channels, so the effects of the experimental channels are more easily noticed. At UCSD, Dr. Nicholas Spitzer’s lab uses Xenopus eggs in this way to study calcium channel development and signaling8. Using the oocyte in this way is extremely useful, because the molecular biology can be performed and the physiology observed all in the same system.7 At the same time, scientists realized that they could use Xenopus laevis as a powerful system for easily studying vertebrate development.

Normally, Xenopus lays its eggs in the pond where the frogs live and, if fertilized by a sperm, develop externally into tadpoles. The external development is quite rapid for vertebrates; an egg can develop into a small frog in 6–8 weeks.2 This is a great situation for biologists interested in a frog’s embryonic development because all of the developmental changes can be watched in a petri dish full of pond water. Scientists are able to observe the actual steps in vertebrate development, from embryo to mature tadpole.

This is not very easy to do in mammals since they develop inside the mother. Dr. Christopher Kintner’s lab at The Salk Institute studies the development of the vertebrate nervous system using Xenopus embryos.10 Much of what we know about vertebrate development comes from the use of Xenopus laevis.

Other characteristics of Xenopus eggs make them extremely useful to cell biologists. One Xenopus egg is 1,000 times bigger than a somatic cell and contains huge stockpiles of all the components required to make mature cells. The eggs are extracted, lysed, and their cellular components saved. This is known as a ‘cell-free extract’, and is very useful for analyzing the functions of cellular proteins without having to keep cultured cells.

Caroline Lindsay Since one frog can lay up to 10,000 eggs, there is an abundance of cellular components available for scientists to use. This egg extract can even be frozen on liquid nitrogen, stored at -80°C, and thawed months later for use in experiments.

Surprisingly, these extracts of Xenopus laevis eggs can accomplish in vitro many of the processes studied in cell biology. If genomic DNA is added to the extract, nuclear envelopes will form around the DNA to make a nucleus. The nucleus will import and replicate the DNA just like a normal cell. For this reason, Xenopus egg extract is routinely used for studying cellular processes such as nuclear transport and assembly and DNA replication. Dr. Douglass Forbes’ lab at UCSD uses Xenopus for studies of the assembly of the nucleus and the nuclear pore, as well as to find out which proteins are used to form the nuclear pore.

Xenopus eggs provide the perfect in vitro system for studying these events.

The egg extract can also be used to visualize mitotic events in vitro. The interphase-like extract described above can be converted to a mitotic state by the addition of a mitotic kinase.

In this mitotic extract, when DNA is added, a mitotic spindle will form around the DNA. If a scientist wishes to visualize the spindle, tubulin can be made fluorescent by a technique known as fluorescence tagging. A red fluorescent molecule called rhodamine can be covalently attached to the tubulin protein, allowing easy visualization of the mitotic spindle in vitro. Another fluorescent molecule of a different color can be added to the DNA, allowing visualization of the mitotic chromosomes. Dr. Don Cleveland’s lab at the UCSD Cancer Center uses the Xenopus egg extract in mitotic phase to study the proteins necessary for chromosome alignment. This work has lead to the identification of a protein that, when its function is blocked, causes the cell to arrest in first meiotic metaphase9. If scientists are especially careful when making an egg extract, they can obtain an extract which cycles between interphase and mitotis—up to three times. Researchers find

Figure 2: A closer view of Xenopus laevis eggs. (Photo credit: D. Forbes Lab, UCSD) Volume 2

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it exciting to see the cell cycle occur right before their eyes, even without a cell.

Overall, the discovery that Xenopus can be used as a model organism for neuroscience, developmental biology, and cell biology research has led to many important discoveries in science.

The processes described above are only a few examples of the usefulness of Xenopus laevis oocytes and embryos. Countless labs around the world benefit from their use in many ways. Since it is not always possible or ethical to study the effects of protein mutations on cellular processes in human cells, the eggs from these animals give scientists a great advantage. Because of the use of the Xenopus model system, we now have a much more detailed understanding of vertebrate development, cellular functions and neurological signaling than we otherwise would.

information from a personal interview with the Saltman Quarterly, and especially Dr. Douglass Forbes for her time and editorial advice.

Xenopus Initiative: Advantages.

Accessed October 1, 2004 at http://www.nih.gov/science/ models/xenopus/advantages.html

References

6. Dr. Douglass Forbes; personal interview on October 8, 2004.

1. Orlov, Marika. “Organism of the Quarter: Caenorhabditis elegans.” Saltman Quarterly. 1(1) (2004):6.

2. Garvey, N. (2000). “Xenopus laevis” (online), Animal Diversity Web. Accessed October 1, 2004 at http://animaldiversity.ummz.umich.edu/site/ accounts/information/Xenopus_laevis.html.

3. Kay, Brian K., and H. Benjamin Peng.

(Eds.). (1991). Methods in Cell Biology, Vol. 36. Xenopus laevis: Practical Uses in Cell and Molecular Biology. San Diego: Academic Press.

Acknowledgements

4. “Introduction to Xenopus” (online), Xenbase: A Xenopus Web Resource. Accessed October 1, 2004 at http://www.xenbase.org/intro.html.

I wish to thank Niket Sourabh for his assistance with web research, Dr. Nicholas Spitzer for

5. “Advantages of Xenopus as a Model for Biomedical Research.” (online), Trans-NIH

7. Purves, Dale, et al. 2004. Neuroscience. 3rd ed. Massachusetts: Sinauer Associates, Inc. 773 pp.

8. Dr. Nicholas Spitzer; interview with Cara Cast for Saltman Quarterly on September 1, 2004.

9. “Rebecca and John Moores UCSD Cancer Center: Research / Clinical Summary: Don Cleveland, Ph.D.” Accessed November 7, 2004 at http://cancer.ucsd.edu/Research/summaries/ dcleveland.asp 10. “UCSD Division of Biological Sciences: Faculty Listings: Christopher Kintner, Professor of Biology, The Salk Institute”. Accessed November 7, 2004 at http://www-biology.ucsd. edu/faculty/kintner.html.

Figure 3: The mitotic spindle as visualized using Xenopus laevis cell-free extract. The mitotic chromosomes are stained with a fluorescent blue dye, and the tubulin molecules making up the spindle are labeled with the red dye rhodamine. (Photo credit: D. Forbes Lab, UCSD)

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FEATURED FACULTY

DR. NICHOLAS SPITZER For this fall’s faculty interview, Saltman Quarterly had the pleasure of speaking with Dr. Nick Spitzer, a professor in the Neurobiology Section of the UCSD Division of Biological Sciences. A faculty member since 1972, Dr. Spitzer helped create the reputation UCSD enjoys as a first-rate research center for neuroscience. His lab is working to understand the development of the nervous system using a variety of approaches including imaging and electrophysiological techniques to name a few. “This work is tremendous fun,” he stated. Currently, Dr. Spitzer and Dr. Jeff Elman, associate dean of the Division of Social Sciences, are co-directors of the newly formed Kavli Institute for Brain and Mind at UCSD. KIBM is an exciting new vehicle for bridging the gap between neuroscience and cognitive science. Spitzer discussed his approach to research and KIBM with us at his office in Pacific Hall.

Saltman Quarterly: Can you broadly describe you research for us?

Nick Spitzer: We’re trying to understand how the brain in assembled during the embryonic period of development. This happens for all of us. The magic is that during development when we were all inside our mothers the nervous system was assembling itself. Nerve cells were migrating from one place to another, making connections, matching up with other cells and establishing this fancy computer that we use to think, create, talk, move and enjoy life.

We use a particular model system, the African clawed frog. Several features make it attractive for the work we are pursuing. One is that development is quite rapid. This is a survival issue for the frog. If the eggs are sitting in the pond for too long, somebody’s is going to come along and have lunch. They need to develop rapidly so they can start swimming and get away. That’s useful for me because I’m a little impatient. Because development occurs quickly, we get answers relatively quickly. Experimental manipulability is a second feature that makes them very attractive. It’s possible to take the embryonic nervous system and dissociate the tissue into individual cells that can be placed in a dish and grown in the laboratory. This is cell culture—a very powerful technique. Many neuroscientists exploit this technique to the hilt.

It facilitates experimental perturbation because you can now change the environment around the neurons in a controlled way. We take these nerve cells at very early stages of development and look at the way in which they differentiate.

We try to understand the molecular mechanisms by which that occurs.

SQ: When did your interest in biology arise, particularly your interest in neuroscience?

NS: This is actually an interesting story because my past was quite checkered. I went to Harvard and had had some courses in high school that gave me advanced standing, so I entered as a sophomore. I thought, “This is terrific, I’m going to be a physics major.” I bit off substantially more than I could chew. As a result I turned in a rather shabby performance at the end of my first

year and spent an agonizing time that summer trying to figure out what I was going to do with my life. It was clear that I didn’t want to keep on majoring in physics. Then the pendulum swung all the way in the other direction. The next year I began a major in Slavic languages and linguistics. I took extensive Russian language courses and courses in linguistics. I’ve always been very fond of languages; my parents taught their four children French at a very young age, and I’d had a lot of Latin in high school. I thought, “Well, this is going to be different.” And it WAS!. But by the end of that year, it was clear that I did not want to major in Slavic languages and linguistics.

“I discovered a minus 80 freezer full of bullfrog legs…which of course, cooked and dipped in butter, are quite delicious.”

Fortunately during that year, I took an introductory course in biology. It was the equivalent probably of Bio 1 and 2 here. This was a course taught in its entirety by George Wald.

Wald was a Nobel Laureate for his discovery that the visual pigment in the eye, in the retina, is derived from vitamin A, and he was a wonderfully charismatic figure. A towering scientist and a charming, utterly persuasive lecturer. He must have single-handedly turned on more people to major in biology than any other single person of that period. And he turned me on. So at the end of my second year at Harvard I switched majors from Slavic languages and linguistics to biology. From there it’s been a downhill slide! I approached John Dowling, who at that time was an assistant professor at Harvard, and now a full professor there. I went to him to see if I could do a research project in his lab as an undergraduate.

It was one of the signal events in my early career that John Dowling took me into his lab.

At the time he was working on the problem of dark adaptation: how does dark adaptation occur? We walk out of the bright sunlight into a dark room and we have trouble seeing for a few minutes because we’re adapted to the bright illumination outside. But very quickly, within seconds to minutes, we adjust. We can now see very well, in a dimly lit room or in a movie theatre. How does that occur? Well, it turns out that a lot of dark adaptation has to do with a neurological process that occurs at the level of the retina. John was very interested in this problem and he was working on it with some of his graduate students, studying the rat retina.

He suggested that I work on a classic preparation for studies of the visual system—the horseshoe crab, Limulus polyphemus. This is a fabulous, primitive animal that looks sort of like an armored tank. It’s evolutionarily very ancient, found on the east coast of the United States, and easily procured from the Marine Biological Laboratories down at Woods Hole on Cape Cod, close to Boston. As an undergraduate I got my first car, a used Ford Cortina that could barely move. I bumped down the road to the Marine Biological Laboratories, picked up a number of horseshoe crabs, put them in a tank of salt water, and drove them back up to Cambridge. There in the bio labs I did experiments in which I would dissect out the eyes, record from the optic nerve with extracellular electrodes, shine a spot of light on the eye and record the effect of conditioning light stimuli on getting the individual cells to fire. I wrote a senior honors thesis on the work.

It was a fabulous experience! I worked around the clock and on weekends during the summer between my junior and senior years. I applied for a National Science Foundation fellowship and was delighted to receive $600! That’s what you got those days for a summer stipend. I had a room in a boarding house in Cambridge. I lived on whole wheat bread and peanut butter, which, by the way, is almost a complete diet. George Wald’s lab was next to John Dowling’s, where I was working, but Wald was away on sabbatical that summer. He worked on the bullfrog retina, continuing research that he was doing to understand the biology of individual retinal Volume 2

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pigments. He and his colleagues would take out the eyes to study the retina and freeze the rest of the frog. Bullfrogs have great big meaty thighs, and I discovered a minus 80 freezer that was full of these bullfrog legs wrapped neatly in aluminum foil. Wald was away and I thought surely no one would miss a few of them. So I supplemented my diet with some bullfrogs’ legs, which, of course, when cooked and dipped in butter, are really quite delicious.

That was my start doing research as an undergraduate. My father was an astrophysicist,

led me to appreciate that the funding situation for science was way better in the States than in the UK. So I applied for positions in the US and was delighted to get an offer from UCSD. That launched my direction for the ensuing decades.

SQ: Clearly, an optimistic young student interested in research could look at you and say, “That’s a guy that knows this business.”

What kind of characteristics should such a person develop?

NS: One has to really want to do research. I

“The interface between disciplines is tremendously productive.” both theoretical and experimental, so my family background gave me an interest in science, but John was a tremendous inspiration to me. I remember he and his wife had me out for picnics on Cape Ann, which is a nice sandy beach north of Boston. He was very supportive and a wonderful friend. I still see him when I go east, either in Boston or at the MBL at Woods Hole where he is on the Board of Trustees.

So I think it’s fair to say that my path was a little checkered and that I entered college with one expectation and came out with another and a very clear vector for my career. John had done a very interesting thing. He had gone to Harvard Medical School in part to test out whether or not he wanted to be an MD and in part because he felt the breadth of education he would get at a medical school would be very different than the education he would get in a graduate school of arts and sciences. I liked this idea and applied to Harvard Medical School.

I had an interesting time and enjoyed very much my first two years of medical school. However, during the second year one begins to see patients.

You put on a white coat: “This is Dr. Spitzer,” yeah right. Sure, uh-huh. This is Nick Spitzer in a white coat. Going around to see patients confirmed my sense that as a career, being an MD was just not for me. After two years of medical school I did a lateral into the Graduate School of Arts and Sciences at Harvard. A few years later I received my PhD and spent a further year at Harvard as a postdoctoral fellow.

Then I went to England for two years (1970– 1972) as a postdoctoral fellow in the Biophysics Department at University College London, headed by Bernard Katz, and worked with Ricardo Miledi. These gentlemen were giants, and this was a hotbed for ambitious young people wanting to do exciting science. Not only was the science marvelous, but on top of it I was living in London! I mean, goodness does it get any better?

Art galleries, symphonies, theatre, musicals, history. It’s all right there for the taking. Living there was so exciting that I thought, “Gee if I can get a job here, I’ll stay on.” Further reflection 10

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bring this up first just as a cautionary note to make sure that people are aware that it’s not a bed of roses. The hours are long, the progress is slow, always slower than desired, and the pay is much less than you could achieve in many other walks of life. So it’s important for people to get the kind of psychic payoff that makes the work so rewarding. I wouldn’t trade what I do for all the tea in China! This is what I like to do. Left on my own I come in on the weekends to work.

I have to make sure I’m doing the right thing by my family, my wife and my children, to spend time with them because I love them, but I love this, too. There is a tension there.

Given that, some of our mutual friends reading Saltman Quarterly will have research as their passion. Then I think there are some things that one really wants to do to be successful. One of them is to be very persistent. There are going to be failures. It turns out to be a fact that most experiments don’t work. We change the conditions, we try something a little different, change the temperature, the pH, the osmolarity, the age of the animal were using, we switch from optical imaging to electrophysiology. But most experiments don’t work. One has to build up a comfort level with that. It didn’t work.

Why didn’t it work? Jot that down and change parameter X or value Y to get the experiment to work. Persistence is terribly important. That is something that comes out of the strong motivation one has because of discovering something new for the first time. This is about as cool as it gets.

For somebody like me discovering something that nobody knew before is unbeatable.

On top of that there are experimental skills and analytical skills that one has to have. Writing skills are very important. If we can’t communicate what we learn to other people then we’re in trouble. I’ve had a tremendously interesting and pleasurable time over the years refining my writing skills. One of the biggest challenges is writing and publishing one’s work in Nature or Science. These are the premier journals in which to publish because they have the widest reading audience. They are also among the most difficult journals in which to publish. The rejection ratio

for articles submitted to these journals in higher than for almost any other journal. A typical paper is about 8 paragraphs long. Try to tell an entire scientific story in 8 paragraphs. It’s like writing a poem. Every word has to be examined, scrutinized, to make sure it’s in the right place.

Then there’s a word limitation. They’re not going to publish a 20 paragraph article. Sorry.

They’ll either reject it out of hand or force you to reduce it to 8 paragraphs. Learning how to write very concisely, very clearly, to deliver stories in that compact manner, is a fascinating challenge.

It’s helpful to write a lot, to be self-critical, and to give your drafts to your friends and ask them to be merciless with their criticism.

SQ: Thanks for the free, shameless propaganda for involvement with SQ’s peer review process.

NS: Perhaps for long-term success, how is it that one manages to stay in the business and to come out smelling sweet for a sustained period of time?

I think one has to be open-minded in the sense of always looking for the boundaries, looking for the edge, looking for the new, looking for where the questions are. There’s a danger, probably in all walks of life but certainly in science, in getting comfortable with doing something. You just do it again and again. It’s easy to do, I know how to do it, I can do it again. The intellectual excitement of that quickly becomes a little marginal. I think for consistent success one needs always to be figuring out what the new questions are and what the new technologies and techniques are. When I got into neuroscience, the kinds of electrophysiological recording techniques we had were primitive compared to the ones we have now. There was no optical imaging. There was no magnetic resonance imaging (MRI) either. Molecular biology hadn’t been invented. I took a course called Nucleic Acids from Jim Watson when I was a graduate student at Harvard. It was called Nucleic Acids because it was about DNA and RNA, but the term molecular biology had not yet been invented. I’d like to think I’ve got another 20 years in the business if I’m lucky. I’m expecting 10 years from now I’ll be using techniques that I haven’t even thought of, that aren’t available yet.

They’ll be invented in another 2 or 3 years, and in another 3 or 4 years I’ll have some grant money and we’ll get the instruments and my colleagues and I will learn how to use them and we’ll be on our way to making some new discoveries. One needs to be open to and to look forward to that.

SQ: You’ve recently been appointed, along with Jeffrey Elman, the associate dean of the Division of Social Sciences, as co-directors of the new Kavli Institute for Brain and Mind at UCSD. What does this entail for you and for the scientists at UCSD? NS: It’s a fabulous thing to have happen and it came about in an interesting way. Fred Kavli is a Norwegian entrepreneur. He came over from

Norway, started a business making sensors for automobiles, for airplanes, and for rockets, and made his fortune. He sold the company in 2000 and established the Kavli Foundation. Fred’s a wonderful person. Smart, personable, with a great sense of humor and a clear vision of what he would like to leave as his legacy for mankind.

He has established a series of institutes in three different areas of intellectual inquiry: cosmology, nanotechnology, and neuroscience.

He’s launched three institutes for neuroscience.

One of them is at Columbia University, directed

by Eric Kandel. Another one is at Yale where Pasko Rakic is the director. The third is here at UCSD with Jeff Elman in cognitive sciences and myself as co-directors. We are tremendously excited about this because what Fred Kavli has started here with his endowment for the KIBM is an effort to bridge the understanding of the brain, which can be viewed as the hardware, and the mind, which one might say is similar to the software. At the present time there is something of a gap because there are people like myself, neuroscientists, who know a lot about the gray matter, the white matter, circuits and all that jazz. Then there are people like Jeff Elman in cognitive sciences, and colleagues in psychology, anthropology, and linguistics, who treat the brain more like a black box. They are studying fascinating phenomena that are properties of the mind, such as language, consciousness, and memory. Pigeons, mice, rats, monkeys or even volunteers are used to assess some of these interesting properties of mind without getting in there and tinkering around with the brain. But at the end of the day we know that the two have to go together. It’s pretty firmly established that the mind is a product of the activities of the brain.

Understanding just how the one maps onto the other and how they implement each other’s activities is not clear.

La Jolla is the place where this is going to happen.

The reason is that we have a tremendously strong group at the north end of campus in the social sciences who are extremely knowledgeable and working hard and very successfully at understanding the macroscopic properties of the mind. Then we have people on the south end of campus and at the Salk Institute, The Scripps Research Institute, The Neurosciences Institute, and The Burnham Institute who are working on the nuts and bolts of the nervous system. We have these two groups that are poised to bridge the gap of understanding between the two disciplines. We add to this the key factor which I think is the magic of La Jolla. I don’t know if it’s the blue sky or the blue water or the yellow sand or the youth of the institutions, but there is something about La Jolla that makes people not only ambitious but also willing to reach out and try something new. I think this is critical to the success of the KIBM because it means that neuroscientists really want to know how a better understanding of the brain can account for

properties of the mind, and that people studying the mind really want to know how those activities are implemented by the processes that occur in the nervous system. I am publicly skeptical that that kind of reaching out would occur at my alma mater, Harvard. It’s a wonderful place. I love it. I contribute to them every year as an alum and I’m very loyal. But it’s a different intellectual environment, and I don’t think it’s able to promote the kind of interactions that one needs to be successful.

Jeff and I have started a very deliberate program aimed at trying to bridge the gap between brain and mind. The program, in broad brushstrokes, has two parts. The first part is cross-education.

Here what we have to do is to make sure that the neuroscientists who are participating in the KIBM understand what the agenda is, what the language is, what the problems are, what the interests are, of the people who are studying the mind. By the same token, the people who are studying the mind have to be cross-educated to learn about the nervous system, so that they understand the issues, concerns, language and techniques that the neuroscientists are using.

This cross-education process is quite important.

The second part involves using the money from the Kavli endowment as seed money for innovative research. The boundary condition here is that the project for which one applies to the KIBM for seed money must in some way attempt to bridge the gap between brain and mind. We are reasonably confident that if people are sufficiently cross-educated, that if they understand what the problems are and what each other’s agendas are, that they will naturally, because they are hungry and ambitious, come forward with clever and ingenious proposals for experiments. So cross-education and innovative research are the superhighways we have to travel to bridge this gap. Both Jeff and I are stoked about it because it’s new and we are always wanting to redefine the questions and get out on the edge and be doing something interesting and

‘Biophysics’, the discipline of biology and the discipline of physics. They come together, rub and make sparks, and very interesting things happen. In the Physics Department here on campus I have a number of friends who are biophysicists over in Mayer Hall. They were trained as physicists and either did postdoctoral work in biology or undertook collaborations with biologists. Fundamentally they’re bringing these two disciplines together and looking at what happens at the interface. The great thing about knowledge and knowledge acquisition is that the interfaces are always changing. Ten years ago there was no nanotechnology; the word wasn’t even in the dictionaries at the time. Yet now it’s a whole field and there are Kavli institutes that are funding people doing this work. Here in UCSD’s Jacobs School of Engineering there are people doing fabulous nanotechnology.

Working at interfaces is certainly a likely recipe for innovative research.

The KIBM is an explicit effort to bring together these two ends of the intellectual spectrum from brain to mind and mind to brain. Bring them into proximity and rub them together really fast and see if we can’t get a fire going. A hundred years from now the Kavli Institute for Brain and Mind will still be going strong and people will be finding different ways to bridge the gap and getting closer all the time. It will be an interesting process because one of the things that turns out to take place in science is that the deeper one gets into a problem the more one begins to redefine what the problem really is.

For example there was a time when people thought the genetic material was encoded by proteins. Avery, McLeod and did the key transformation experiments that identified nucleic acids as the genetic material. This historical example illustrates how the place to look to solve the problem evolved over a relatively short period of years: James Watson and Francis Crick focused on understanding the structure of DNA, not proteins. Trying to

“There is a wonderful dynamic that comes from the collective mind.” exciting. It’s not something that’s going to play itself out quickly. This is a long-term process and Fred Kavli was very wise to provide an endowment. An endowment gets invested and you spend the income on it every year.

SQ: I’ve heard you state something about how the real innovations happen at the seams between disciplines. Tell us what you mean by that and how it applies to the KIBM.

bridge the gap between brain and mind seems like an enormous and daunting goal. However, science often achieves its successes by taking large problems and breaking them down into smaller, more tractable problems. I’m confident that that’s what will happen with the KIBM; we’ll take some of these big problems and we’ll be able to redefine them as smaller problems. I think that’s a basis for some optimism as we look ahead to this ambitious goal.

NS: I think the interface between disciplines in tremendously productive. When I was a postdoctoral fellow in London in the Biophysics Unit at University College London, the very name tells you what we’re talking about.

SQ: The other research universities involved with the establishment of new neuroscience institutes by the Kavli Foundation are Columbia and Yale. That UCSD stands alongside these institutions with such established academic Volume 2

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reputations speaks volumes about the quality of science happening here.

NS: All of us, from staff to faculty, everyone associated with UCSD, should be very proud of what we have here. It was exciting to come here in the early 70’s at a time when the university wasn’t even a decade old. There was a lot of ambition but there was a lot of uncertainty. How was the university going to unfold? Nothing was guaranteed. There was no 200-year track record of the kind one would find in many of the Ivy League institutions or a place like Stanford or Caltech. Coming back to remarks I made earlier, I don’t know whether it’s the blue sky or the blue water or the yellow sand or something else.

But this place has come together in a way that is phenomenal. One interesting feature of it is that it’s not restricted to UCSD. Look at what’s happened at the Salk Institute. The Salk started about the same time as UCSD. It has had its own uncertainties and ups and downs but boy, what a powerhouse! You can say the same thing for The Scripps Research Institute, The Neurosciences Institute, and The Burnham Institute.

SQ: Some kind of magic here.

NS: Absolutely, there’s some kind of magic here in La Jolla. I see no sign of anything flattening out. The trajectory is still steeply upward. I wouldn’t care to make a prediction about when, if ever, it’s going to get to the top, flatten out or level off in expanding its extraordinary reputation.

SQ: How can undergrads interested in research best take advantage of the opportunities here?

NS: I think that if students are interested in research, testing this as an undergraduate is a great idea. One can do this in a serious way, without committing oneself to going to graduate school, which is a multi-year commitment. One can get a position in a research lab, often starting off with something that isn’t terribly exciting.

But if one is interested and asks questions, and shows up a little early and stays a little late, then your advisor will often say, “Let me show you what you can do.” That way you can find out if research is going to turn into a passion that you can nurture, or if it is interesting but not quite what you want to do.

The other thing to say is that when one comes to college one is looking for a variety of things. One is not always focused immediately on which research lab can I work in as an undergraduate. You come and you figure this out. It took me some time to get that figured out when I was in college. The university is a truly remarkable place. The opportunities for people to do research are extraordinary. I have had and continue to have students work in my lab. Some of them will be authors on papers by the time they leave. That’s a wonderful 12

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opportunity even if they decide later on to go to law school or run a business or something else.

It’s a good experience in using one’s mind and thinking critically. One’s undergraduate years are a wonderful period. I was reluctant to leave college, but in those days it was much more common to go right on from one’s undergraduate career to graduate school. Now, of course, many people knock off for a year or two. But never again will one have the opportunity to do the type of things that one can do in college— work in a lab, take courses in European literature. By my senior year at Harvard I had satisfied all my course requirements and took a lot of my credits from my lab work in John Dowling’s lab. I took a course in European Literature just because I loved reading novels and it was a chance to get rewarded for reading novels and writing papers about them. That is unlikely to happen later on. There are many ways in which college is a unique experience.

“There is something about La Jolla that makes people willing to reach out and try something new.”

I will say one thing here about priorities, though.

In our conversation we’ve talked a lot about my professional interests and just touched a little on my family. Like Paul [Saltman] who was a very active, energetic, athletic individual, rock and ice climbing are what I like to do when I’m not here or at home. My friend Bruce Darling used to be here as Vice Chancellor for University Affairs and is now Senior Vice President of the UC System in Oakland. We enjoy climbing up in the Sierras. He drives over to the east side of the Sierras and I shoot up the 395 and we go into the wilderness for several days at a time. We make a week-long trans-Sierra ski tour each year with other friends that is great fun.

Again, it all comes down to priorities: How do I ration my time? There are times when it helps to take a break. You’re studying and you have a midterm tomorrow. You start at 5 o’clock and you study intensively. It’s 10 o’clock and you think, “Gee, I’m gonna study better for the next two hours if I take half an hour off and talk to friends, watch TV, have a drink, go for a walk.” In the same spirit, getting away and dealing with technical problems, while rock or ice climbing, is a wonderful way for me to clean out the cobwebs and come back fresh and ready to work hard. Last weekend we climbed a fine ice couloir on Thompson, just above South Lake.

We got to the base at noon and topped out at 7 pm in the evening, only to discover that we had a 5 or 6 hour climb down over rotten rock that we didn’t want to do in the dark. So we bivouacked

on the summit. It was a bit chilly. We climbed down the next day and spent the night in Bishop getting caught up on sleep and then drove home.

These are wonderful trips. We often need an outlet, something that takes us away from our professional activities.

SQ: Is there anything else you would like to expand on?

NS: One thing—the value of communicating with one’s peers—I think this is terribly important. There are various ways to achieve this, including lab meetings and another formal way that the Neurobiology Section here has enjoyed for decades, called ‘Neurodinner’.

This is a nice social occasion of a dinner, typically with some ethnic food, followed by an intellectual feast that comes after dining together.

It’s an opportunity to hear about what’s going on in other labs, to learn about new techniques, and be stimulated by the results of our colleagues.

Often there is feedback that affects what we’re doing and thinking. On an informal level, faculty try to create an ambience within their lab whereby interesting discussions take place of which they are unaware. I think I see enough and overhear enough that I know that this is happening in my lab. This is terrific. I try to talk one-on-one with my people, give them my views, energy and motivation – but when they get to the point where they are doing that with each other, this is marvelous! In some ways, it’s a measure of success. It’s not like a paper published in Nature, but it’s what leads to getting papers published in nice places. Stimulating communication among peers is very useful.

To learn more about the research being conducted in the Spitzer lab, visit: http://biology.ucsd.edu/ labs/spitzer/.

Interview conducted by Cara Cast.

Undergraduate Biological Research Publication UCSD Division of Biological Sciences

Francis Crick—A Retrospective Look at a Legacy

by Kyle Kuchinsky / Page 15 Brain-Machine Interfaces: Reinventing Sensory and Motor Functions After Injury and Disease

by Ronald Alfa / Page 17 Mechanistic Role of Mrf-2 and C/EBPa During Transcription

by Nicole Gomez / Page 20 Analysis of the Manganese Oxidation Capacity of Pseudomonas putida GB-1 and MnB1 Mutant Strains

by Daniel Baron / Page 23

Volume 2, No. 1 http://sq.ucsd.edu

Undergraduate Biological Research Publication UCSD Division of Biological Sciences

Volume 2, No. 1 http://sq.ucsd.edu

FALL QUARTER REVIEW BOARD: Ronald Alfa Brittany Bernik Ann Cai Kristin Camfield Cara Cast Eric Chan Max Chen Reeti Desai Heather Eshleman Daniel Fang Ryan Ferrell Nicole Gomez Shruti Jayakumar Stephanie Kinkel Kyle Kuchinsky Chi-chung Lee Andrew Lin Nick Lind Caroline Lindsay Laura Lombardi Lauren Ashley Miller Sara Paul Josh Tan Yu-Ting (Alice) Tsai Jennifer Wan Grace Wang

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Get published!!

In addition to research articles, SQ also accepts editorials, commentaries, review articles, and “Organism of the Quarter” submissions. If you have an idea for a piece of writing of this type we want to hear from you.

If you are interested in becoming involved with the journal, please visit our website: http://sq.ucsd.edu

2004-2005

Francis Crick–A Retrospective Look at a Legacy Kyle Kuchinsky Mention the name Francis Crick and DNA is the first thought that comes to mind. But Crick’s legacy does not end with the discovery of DNA. Rather, this discovery opened the doors for the field of molecular biology, offshoots of which most biology students at UCSD and others around the world are studying today. Crick’s interest in molecular biology diminished during the 1970s. He felt that the most important problems had already been solved and only details remained to be worked out.

Instead, Crick decided to pursue the biological basis for consciousness and, in 1976, moved to the Salk Institute for Biological Sciences.7 While James Watson remained in molecular biology, Crick chose to explore the brain.

Crick’s legacy was already set with the discovery of DNA. But he wanted to look at the big picture. Crick enjoyed thinking about scientific problems more than teaching or working in a lab, which he left to “better experimentalists.” The influence of his position would cause a dramatic shift in the field of neuroscience. “History will judge him certainly as one of the influential biologists of the 20th century, if not the most influential,” said Richard Murphy, president of the Salk Institute. “Because of his stature, he made brain science respectable,” commented Leslie Orgel, also a colleague of Crick’s at Salk. He set a standard for “being a true scientist, … totally dedicated to understanding science and the truth about biology,” added Murphy.8 Crick not only influenced others to explore the field but also indirectly created possibilities for new tools to be used in neuroscience. Prior to the discovery of DNA, neuroscience was limited to what autopsies and psychological deductions could show about human behavior. Now scientists can see inside individual cells and have begun to reveal how our brains process vision, hearing, taste, smell, touch and thought.10 Crick recognized this and stated that neuroscientists should tell molecular biologists what difficulties they have in the hope that new biological tools would be developed.

He suggested the use of recombinant DNA along with rapid DNA sequencing as two of these new tools, both of which are popular techniques today.

Crick hoped that future scientists would work on methods developing specific markers for particular neurons and tools to more precisely measure single pathways.2

Why was Crick so enamored with the neurological processes guiding consciousness? He stated in his book Astonishing Hypothesis1 that “you, your joys

An example of this is how a tennis player swings at a ball before he sees it or how a runner starts running before the sound of the gun.4 Many mammalian brain systems perform complex but routine tasks without conscious input through what are called online systems.5 These systems react quickly to simple input without it ever becoming conscious thought.4 Extreme examples of this type of behavior include sleepwalking, where individuals may avoid obstacles, move furniture, and even drive cars.5 But these behaviors happen throughout the day as well, such as tying the laces on a pair of shoes.9 Crick wondered if it was possible for an animal to run purely on reflex, as a zombie. Before answering his question, it will be necessary to extend our knowledge of neuroanatomy in humans.5

(Photo credit: Marc Lieberman/Salk Institute)

Dr. Francis Crick and sorrows, your memories and ambitions, your sense of personal identity and free will, are, in fact, no more than the behavior of a vast assembly of nerve cells…nothing but a pack of neurons.”

He wanted to know what nerve cells produced consciousness and how they were firing.8 While others thought that consciousness was too tough of a question to tackle, Crick thought it was the greatest challenge in science.7 He began looking for what he called the neuronal correlate of consciousness (NCC), which he believed was the minimal sets of neuronal events—the firing of neurons—that gave rise to conscious perception. He noted that it is even plausible that certain animals have some of the features of consciousness, but it is likely that consciousness itself correlates with the degree of complexity of the nervous system. Crick, along with his colleague Christof Koch of the California Institute of Technology, assumed that different aspects of consciousness (such as pain and visual awareness) used one basic common mechanism or a few such mechanisms. If they could understand one of these mechanisms then it would be possible to understand many of the aspects of consciousness.4 However, consciousness only deals with the slower, broader aspects of sensory input. Many actions in response to sensory input are rapid and stereotyped, what Crick dubbed ‘zombie modes’.3 The information processed in these systems is acted upon but never reaches the level of consciousness.

In one of his later papers, Crick wrote that “neurosurgeons, probing the living human brain on a daily basis, can play a decisive role [in the explanation of consciousness].” This is critical since many questions about consciousness can only be answered in humans. A neurosurgeon is in a rare position to observe clinical states of altered consciousness, the recording of bulk brain activity, and the direct manipulation of brain activity by electrical stimulation. Crick felt that the future of the field would lie in neurosurgery and the use of electrical microstimulation and advocated that neurosurgeons work together with molecular biologists to discover the center of consciousness.6 Until his death, Crick was still working on what he believed in. He had been writing a paper discussing the role a region of the brain called the claustrum plays in consciousness. “He was always willing to revise his own views in light of the actions of a universe that never ceased to astonish him,” said Koch. This may influence the direction of research at the newly established Crick-Jacobs Center for Computational and Theoretical Biology, established in early 2004 at Salk, which will focus on the genes, proteins, and neural networks that make the brain function.7 Work at the center will be driven by computational biology, which will analyze the large amount of genetic information available to researchers. Scientists will then be able to create theoretical models explaining how the brain works, which can then be tested by neuroscientists at Salk.11 Perhaps it will be here, following in the footsteps of Crick, that researchers will decipher some of the Volume 2 Issue 1

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mysteries of consciousness.

References 1. Crick. F. Astonishing Hypothesis: The Scientific Search for the Soul. New York: Charles Scribner’s Sons. 1994.

(317pp.). ISBN: 0684801582.

2. Crick, F. “The impact of molecular biology on neuroscience.” Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 354 (1999): 2021-2025.

3. Crick, F., and C. Koch. “A framework for consciousness.” Nature Neuroscience. 6.2 (2003): 119126.

4. Crick, F., and C. Koch. “Consciousness and neuroscience.” Cerebral Cortex. 8 (1998): 97-107.

5. Crick, F., and C. Koch. “The zombie within.” Nature.

411 (2001): 893.

6. Crick, F., et al. “Consciousness and neurosurgery.”

Neurosurgery. 55.2 (2004): 273-282.

7. Knight, J.. “From DNA to consciousness – Crick’s legacy.” Nature. 430 (2004): 567.

8. LaFee, S. “Francis Crick.” The San Diego UnionTribune. August 2004: B-6.

In 2003 Dr. Francis Crick was awarded the inaugural UCSD / Merck Life Sciences Achievement Award by then UCSD Chancellor Robert Dynes and UCSD Biological Sciences Dean Eduardo Macagno.

9. LaFee, S. “Undead heads.” The San Diego UnionTribune. March 2004: F-1.

10. Lieberman, B. “A brainstorming hub.” The San Diego Union-Tribune. October 2004: B-1.

11. Lieberman, B. “Salk gets $7 million for research on brain.” The San Diego Union-Tribune. December 2003: B-10.

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As a peer-reviewed journal, SQ needs undergraduates willing to participate as members of our review board. Members of the board will have the opportunity to read and critique research articles submitted for publication by their peers—other undergraduates in biological sciences at UCSD. Involvement provides students with valuable experience reading and analyzing scientific literature.

Visit our website: http://sq.ucsd.edu 16

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Brain-Machine Interfaces: Reinventing Sensory and Motor Functions After Injury or Disease Ronald W. Alfa Injury to and disease of the nervous system often result in the loss of sensory or motor skills. While regeneration therapies for repair of damaged neuronal substrates offer promise of reversing sensory-motor deficits, treatments of this type remain far from clinical application. Alternatively, brain-machine interfaces (BMIs) have recently emerged as important and promising technologies to restore hearing, vision, and movement, and to disrupt the pathological brain activity underlying such diseases as Parkinson’s and epilepsy. By circumventing damaged pathways, these systems utilize intact neuronal circuits in conjunction with stimulation or recording devices to establish alternative input and output pathways to the central nervous system (CNS). Although these devices cannot repair damaged systems, their benefit lies in the improvement of associated behavioral deficits.

This review will present a concise summary of BMIs currently under development and their clinical progress.

Brain-machine interfaces (BMIs) have recently emerged as important systems to circumvent neuronal damage and return behavioral function. These systems may be classified into three general categories: input, inhibitory, and output BMIs. The primary function of input BMIs is the delivery of sensory information to the CNS using prosthetic stimulus detection devices such as a camera.

As such, these systems return sensory function to impaired individuals, e.g., vision in blind patients. Inhibitory BMIs are important in diseases involving aberrant neural activity, such as Parkinson’s or epilepsy. These BMIs deliver intense electrical stimulation to underlying neurons, altering their pathological activity patterns.

Introduction Until recently, the concept of bionic humans, a synergy of biological systems with computerized robotic components, was an idea reserved for science fiction and pop culture. However, scientists are now beginning to realize both the value and practicality of such systems in treating diseases of the central nervous system (CNS).

Moreover, groups combining expertise from engineering to molecular and systems neuroscience are demonstrating that the technology to rebuild sensory and motor systems should not be considered science fiction. Some classes of this technology, such as auditory and visual prosthesis, have already achieved widespread success in the clinic.13 Before introducing these systems, some preliminary remarks on diseases of the CNS should be made.

Figure 1: Heirarchical organization in a typical sensory system allows BMIs to bypass damaged neuronal substrates and restore sensation in impaired individuals.

In a normal system, (a) stimuli are detected by specialized cells (photoreceptors shown) and transduced into electrical activity patterns. These activity patterns are then relayed via afferent nerves (retinal ganglion cell shown) and higher order cells to the appropriate cortical region for initial procession (V1 for vision). If sensory receptor cells are damaged, (b) stimuli can be detected using a prosthetic device (camera) and transduced into electrical activity patterns which are delivered to intact cells along the pathway or directly to cortical centers (dotted lines).

CNS diseases are immense in number and present a diverse array of symptoms. Nevertheless, most involve the degeneration or death of specific populations of neurons.

Though repair of affected systems is an important goal for regeneration-based research, clinical therapies of this type remain in their early stages. With millions of individuals coping with CNS disease-related physical debilitation, many researchers have shifted focus from regeneration to therapies designed to return behavioral function without neuronal repair. Such a paradigm is conceivable because neuronal input (sensory) and output (motor) pathways involve hierarchically organized networks of neurons that communicate through electrical activity. In a network of this type, information is transmitted in a step-wise manner through the communication of populations of neurons along a pathway. For example, consider the production of a simple movement of some type. Cells responsible for the planning of the movement activate the appropriate populations of cells for the initiation of the movement. These cells then signal motor neurons (that control muscles) to produce muscle contractions, with the end result being a movement. In the case of damage or disease, affected neurons generally impair transmission of information along some step(s) in such a pathway.

Conversely, transmission along unaffected elements remains functional.

Therefore, by circumventing damaged cells and utilizing intact pathways, treatments may be developed to improve behavioral deficits.

Lastly, output BMIs include a variety of systems that utilize normal neural activity to control computer cursors (for communication) or devices such as a robot arm (for movement) in paralyzed individuals.

cells along the auditory pathway) and are finally received by specialized auditory processing centers in the cortex.

Many cases of hearing impairment involve the loss of auditory hair cells; meanwhile, spiral ganglion cells that constitute the auditory nerve remain intact. Therefore, an important feature of auditory prosthetic devices is their ability to utilize functional nerve fibers to deliver sound information to auditory cortex. These devices involve three basic components: an external microphone, a speech processor, and a stimulation device implanted within the cochlea.5 Sound is captured by the microphone and converted to digital output signals for the implanted stimulator. The speech processor is used to further decompose features of speech into signals for optimal comprehension. Lastly, the stimulator–a multi-channel electrode array–electrically stimulates spiral ganglion cells, which subsequently relay activity patterns to the auditory cortex for processing.

Cochlear implants are currently approved for use in both hearing impaired children and adults and have shown immense success in restoring functional hearing. Though the range of performance is dependent on many features (e.g., age, duration of hearing loss), applications using bilateral implants report significant speech perception and sound localization.6 Though a major challenge of these systems was speech perception over telephone conversations, Qian et al. recently reported a telephone adaptor that, through Bluetooth wireless technology, routes audio signals directly to the implant, thus, significantly increasing efficacy of cochlear implants for telephone conversation.7 Although a major challenge of these systems remains their high cost ($20,000–$25,000), inexpensive systems are under development.8 In addition, development of background noise reduction algorithms, increasingly advanced electrodes, and better speech processors remain important goals.2, 3, 9 Visual Prostheses

Cochlear Implants

The immediate goal of visual prosthetic devices is to restore functional, but limited, visual perception in blind patients through electrical stimulation at different locations along the visual pathway. In the visual system, light is detected and transduced by photoreceptor cells along the outer retina. The subsequent patterns of electrical activity are then sent to the visual cortex via retinal ganglion cells, whose processes comprise the optic nerve (signals are also relayed by neurons of the lateral geniculate nucleus). Since many cases of blindness involve loss of photoreceptors, an effective visual prosthesis should exhibit the ability to capture and transform photons of light into electrical signals which, when presented to functional neuronal substrates, lead to perception. Current approaches utilize a camera to convert light into electrical signals and various types of CNS interfaces to stimulate neuronal targets.10 These interfaces involve the placement of stimulating electrodes at precise locales along the visual pathway, such as behind the retina (subretinal), on the inner retina (epiretinal), on the optic nerve, or directly in the visual cortex.11-15 The basic premise is to bypass damaged neural systems and deliver visual information directly to functional units for processing.

In the auditory system, hair cells of the inner ear transduce sound information into electrical activity patterns. These signals traverse the auditory nerve (and subsequent relay

Promising preliminary results suggest retinal implant stimulatory devices may be close to clinical applications.

Chow et al. recently demonstrated improvement in

Input BMIs Input BMIs use electrical stimulation of the CNS to restore sensation in patients who suffer from loss of sensory function.4 In a normal sensory system, stimuli are detected by specialized cells (e.g., retinal photoreceptors) and converted into electrical activity patterns, a process called transduction. These activity patterns are then delivered via afferent nerves to a region of the brain specialized for the processing of a particular type of sensory information.

Although the subsequent computational processes leading to perception are vastly complex, BMI systems are primarily designed to facilitate the input of sensory information to the CNS. Moreover, the activity patterns used by sensory receptor cells to encode stimuli are well characterized, thus allowing researchers the ability to design BMI devices that can encode sensory information for appropriate CNS processing (Figure 1).

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frontal cortex. In brief, degeneration of SNpc neurons results in decreased activity of VA/VL thalamus circuits and, subsequently, diminished excitability of upper motor neurons. As a result, patients suffer from severe movement disorders with symptoms including slowness of movement, tremors at rest, and rigidity.26

Figure 2: The basal ganglia encompass a set of diverse nuclei lying deep within the cerebral hemispheres. These nuclei play an essential role in the modulation of movement.

In Parkinson’s disease, neurons of the substantia nigra pars compacta (SNPC) undergo degeneration. (a) Motor movements initiated in the cortex are subject to modulation by projections from the ventral anterior and ventral lateral thalamus. The VA/VL thalamus is modulated by tonic inhibitory signals from the globus pallidus internus (GPi).

During movements, inhibition of the thalamus from the GPi is blocked by inhibitory activity of the putamen (P) on the GPi; this activity is subsequently regulated by activity from the SNPC. Moreover, the SNPC further modulates GPi activity indirectly through the caudate nucleus, globus pallidus externus (GPe), and subthalamic nucleus (STN). In this indirect route, activity of STN positively regulates inhibitory projections of the GPi. Degeneration of substantia nigral neurons in Parkinson’s disease results in diminished ability to disinhibit the VA/VL thalamus by decreasing GPi activity. (b) Inhibitory BMIs for deep brain stimulation involve the placement of a stimulating electrode into the STN, or, more recently, the GPi. High-frequency stimulation of these areas disrupts their activity. In the former, GPi activity is reduced indirectly by disrupting STN activity. In the latter, GPi activity is directly inhibited by stimulation. Reduced activity of GPi, decreases tonic inhibition of the VA/VL thalamus, permitting thalamocortical activity in the generation of movements.

functional vision and positive long-term biocompatibility (6–12 months) in six patients implanted with subretinal prosthetic devices.1 In addition, recent studies in rabbits using the epiretinal implant have reported stable longterm implantation and the ability to effectively monitor the electrical properties of the implant.16, 17 Despite these positive results, obstacles such as implant spatial resolution and surgical methods will require further research before such devices reach widespread use.18 Inhibitory BMIs Inhibitory BMIs, including deep brain stimulation therapies, are currently being utilized in many clinical applications for the treatment of symptoms associated with Parkinson’s disease, chronic pain, migraines, and epilepsy.19-23 In these applications, chronic stimulating electrodes are implanted at various locations along neural pathways or within CNS nuclei. The delivery of continuous electrical stimulation to neurons at distinct centers blocks aberrant electrical activity patterns and subsequently leads to the therapeutic effects of the treatments. Though the diverse applications will not be discussed here, deep brain stimulation for Parkinson’s disease is currently an important clinical therapy and serves as an excellent example of these treatments.

Parkinson’s disease is a progressive neurodegenerative disorder characterized by degeneration of neurons in the substantia nigra pars compacta (SNpc).24,25 Neurons of the SNpc, together with those of nuclei in the basal ganglia, serve essential roles in modulating the activity of upper motor neurons responsible for the initiation of movements (Figure 2). The SNpc exerts control over regions of the basal ganglia that regulate activity of the ventral anterior / ventral lateral (VA/VL) thalamus. The VA/VL thalamus, in turn, is a positive regulator of motor neurons in the 18 Volume 2 Issue 1 2004-2005

Deep brain stimulation therapy stems from fundamental studies demonstrating that animal models of Parkinson’s disease exhibit increased neural activity in brain areas responsible for imparting tonic inhibition (subthalamic nucleus, globus pallidus internal segment) on the VA/VL thalamus.27 Moreover, studies have shown that lesions to these regions can improve motor function by promoting disinhibition of thalamic centers.27,28 However, surgical brain lesions often result in undesirable and irreversible effects. High frequency deep brain stimulation can selectively decrease activity of neural targets and, therefore, simulate the effects of a lesion without irreversibly damaging the brain 29 In human trials, bilateral implantation of stimulating electrodes into the subthalamic nucleus or internal segment of the globus pallidus substantially improves motor function in patients with Parkinson’s disease.19 To reiterate the mechanisms of such improvements, these regions indirectly inhibit motor control centers and, in Parkinson’s disease, cells responsible for modulating these inhibitory areas degenerate. Therefore, implanted electrodes partially fulfill the role of lost cells by deactivating inhibitory areas or permitting disinhibition of motor areas. Though this procedure is widely used, neuroprotection of degenerating neuronal populations has yet to be demonstrated. Moreover, additional challenges of this treatment are reports of psychiatric complications, including depression and aggression, in some patients with implants into the subthalamic nucleus.30,31 Despite these caveats, deep brain stimulation can significantly improve the quality of life for patients with advanced Parkinson’s disease and is therefore an important therapy where few are available.30 Output BMIs Injury or degeneration of the CNS often results in impairment of motor functions and in severe cases, full body paralysis. In the United States alone, over 200,000 patients live with permanent paralysis due to spinal cord injuries.32 Although regeneration researchers have demonstrated success in inducing growth of neurons across an injury site in spinal cord injury models, such methods remain far from delivering a clinical therapy for useful restoration of motor function in paralyzed patients.33 In recent years, neuroscientists have conceived approaches utilizing output brain-machine interfaces to restore communication and motor functions in paralyzed patients. These systems record neural signals from intact populations of neurons and, subsequently, use these extracted signals to drive prosthetic actuator devices (e.g., robot arms, gripping tools), a patient’s own musculature, or on-screen communication programs.33,34 Electroencephalography (EEG) and microelectrode implantation are two widely used techniques for sampling and monitoring neural activity.4,34-36 EEG records the summed activity of large populations of neurons using electrodes placed on the scalp. Though this technique allows for visualization of large activity fluctuations in response to cognitive processes, or event related potentials (ERPs), EEG cannot be used to follow the activity of individual cells. For precise neuronal recording, microelectrodes are surgically implanted beneath the skull in brain regions of interest. Though the latter technique offers greater spatial resolution, microelectrode implantation is highly invasive. Nonetheless, in BMI applications

the advantages of each of these systems are exploited to different ends: motor control and communication.

In addition, functional electrical stimulation (FES), a technique used to move paralyzed muscles, will be discussed in brief. FES uses skin surface electrodes or implanted electrodes to electrically stimulate muscles and thereby induce contractions.37 EEG-Based Brain-Computer Interfaces (BCIs) Output systems utilizing an EEG based interface, or BCIs, have been the subject of much research because they offer the potential of a relatively simple, inexpensive and non-invasive communication device for individuals with complete loss of motor control.35 These systems convert the user’s scalp-recorded EEG activity into a computer output, creating a direct feedback loop through which a user can observe and correct neural activity to achieve the desired result. EEG-based systems therefore require the user to develop and maintain the skill of controlling neural activity.35 Several classes of BCIs have been developed; each is centered on a distinct type EEG activity.35 To sample current progress in this field, slow cortical potential and P300 based systems will be discussed.

BCI-based communication systems are important tools for patients suffering from amyotrophic lateral sclerosis (ALS). ALS, or Lou Gehrig’s disease, is a severely debilitating neurodegenerative disease characterized by slow and irreversible degeneration of motor neurons in the spinal cord, brainstem, and later, motor cortex. With impairment limited to motor systems, ALS patients retain cognitive function throughout the progression of the disease.26 The gravity of this element must be realized as, at later stages of ALS, patients become ‘locked-in’ , i.e., cognitively proficient with no means of communication or movement. Nonetheless, it is precisely these patients’ intact cognition that forms the foundation of BCI systems for communication.

Two types of EEG-recorded activity patterns have shown much success in BCI systems thus far: slow cortical potentials (SCP) and P300 event-related potentials (ERP).

Slow cortical potentials (SCPs) are among the lowest frequency signals of scalp-recorded EEG.35 Recently, Birbaumer et al. reported the development of a spelling device based on the user’s ability to modulate SCPs when provided appropriate feedback.38 Two patients with advanced ALS were trained to produce voluntary changes in SCPs. Following initial training sessions, subjects were able to accurately select desired letters and construct sentences with a spelling device by making two choice selections via SCP modulation.38,39 The P300 is a wellcharacterized EEG peak recorded over parietal cortex when a subject encounters an infrequent or significant stimulus interspersed within routine stimuli.40 In a P300-based BCI described by Donchin et al., the user is presented a grid of letters in which columns and rows

Figure 3: (a) A 10x10 microelectrode array (Bionic Technologies, Inc., Salt Lake City, UT) for sampling individual activity of cortical neurons (length = 1.5 mm). (b) In microelectrode-recording BMIs, microarrays are implanted in the motor cortex, cortical activity is extracted, then decomposed into desired movement information to control prosthetic actuators such as a robot arm.

undergo a series of flashes.41 As the user attends to a desired selection, P300 event-related potentials are recorded when the selection is among the flashing characters, and through a series of such rounds, an algorithm identifies the user’s selection. A significant difference between this system and the former lies in that the P300 BCI requires little training; the subject simply attends to the selection.

In contrast, the SCP system requires the subject to first learn to modulate neural activity independent of the spelling device.

Though these systems have shown progress in providing a communication pathway for individuals suffering from complete paralysis, further development is needed to allow for increased efficiency and speed. Clinical tests using SCP and P300 BCIs have reported speeds of up to 3.0 and 7.8 characters per minute, respectively.38 Nonetheless, with the use of a lexicon encompassing a user’s vocabulary and algorithms to select words based on relatively few letter choices, quicker communication may be achieved with EEG-based BCIs. Despite the speed limitation of these systems, BCI systems present an immense communication breakthrough for ‘locked-in’ patients without other means of communication.

Motor Cortex Microelectrode-Recording BMIs Direct recording of the activity of individual and populations of neurons via implanted microelectrode arrays is a procedure extensively used in neuroscience research (Figure 3). The introduction of this technique in developing motor BMIs stems from the premise that desired movements can be extracted in real time from the electrical activity of populations of cortical neurons and thereby, used to drive artificial actuators (e.g., robot arms, gripping tools) or a patient’s musculature 34 In contrast to systems in which subjects must learn to manipulate neural activity patterns for control of on-screen cursors, the goal of these systems is to effectively decode desired movement information and thereby allow users nearly effortless control over the actuator device.

Information regarding desired movements such as direction, distance, speed and force are encoded by the simultaneous activity of populations of cortical neurons.4250 Therefore, the necessary substrate for a neuroprosthetic movement device, motor information, lies just below the skull, accessible to surgically implanted microelectrode arrays. These neuronal activity patterns can be decoded, and useful movement information extracted to control computer-modulated actuator. To this end, a group led by Miguel Nicolelis previously reported real-time movement of a robotic arm using cortex-extracted movement information in rats and primates.51,52 Moreover, with the aid of visual feedback, subjects were able to effectively monitor and improve movement accuracy.53,54 In addition, similar technology has been applied to movement of a computer cursor in both monkey and humans.55,56 Though this technology is promising, much remains to be explored before such systems deliver widespread clinical treatments. First, the invasive nature of the required implants demands that issues of long-term biocompatibility and performance be reasonably assessed.

For this purpose, Koeneman et al. recently demonstrated a system using rat brain slice cultures that could effectively be used to assay the biocompatibility of potential electrodes.57 In addition, implanted systems will require further research into components such as microelectrode arrays and telemetry devices (to transmit information between implant and processor). Finally, the development of increasingly advanced algorithms for extracting and processing neural activity will further improve control of robotic actuators.

NeuroControl Freehand System The NeuroControl Freehand System was developed by Hunter Peckman as a means for patients with C5 and C6 spinal cord injuries to regain hand function.42,44 Injury at cervical levels 5 and 6 are the most common forms of spinal cord trauma and result in tertraplegia, or the loss of muscle strength in all four extremities. However, the success of this system relies on the ability of these individuals to retain control of shoulder muscles. Using functional electrical stimulation (FES) of hand muscles, the freehand system permits individuals to initiate various types of grasps. To achieve such movements, FES electrodes are placed under the control of a shouldermounted position detector; patients are then trained to use shoulder movements to initiate FES-induced grasps.

Furthermore, the system allows users to control tightness and duration of grasps as a function of different features of the shoulder movement (e.g., trajectory, speed). This system is one of the few BMIs that is currently FDA approved and has been shown to improve quality of life in tetraplegic individuals in many clinical studies.37,43-45 Conclusion Despite much progress in medical technologies, overcoming damage to the CNS due to injury or degenerative disease remains a major clinical obstacle.

Considering the incredible diversity (and complexity) of neuronal networks, regeneration may require populationspecific fine tuning far beyond the scope of current research. Therefore, the incredible progress of brainmachine interfaces, with devices such as the cochlear implants and the neurocontrol freehand system currently being widely used in the clinic, yields exciting news for patients worldwide.

Though BMIs do not offer solutions to repair diseased neuronal substrates, their potential to return function to debilitated behavioral conditions may be more immediately important to a patient. In addition to the immediate physical benefits, the psychological benefits of increased independence and quality of life make BMIs highly desirable treatments. Moreover, the nature of this technology allows BMIs to be constantly improved and upgraded through the replacement of processing components. As research continues, the future may bring neuroprosthetic actuators that retain the full range of motion as their evolutionary counterparts. In conclusion, until damaged pathways can be regenerated to the level of functional recovery, BMIs have the potential to bring patients closer to overcoming the physical debilitation caused by neurological diseases.

Acknowledgements My very special thanks to UCSD biologist Dr. Dan Feldman for inspiration and guidance throughout the writing of this manuscript.

References 1. Chow, A.Y., et al. The artificial silicon retina microchip for the treatment of vision loss from retinitis pigmentosa. Arch Ophthalmol, 2004. 122(4): p. 460-9.

2. Chatelin, V., et al. Cochlear implant outcomes in the elderly.

Otol Neurotol, 2004. 25(3): p. 298-301.

3. Cullen, R.D., et al. Cochlear implantation for children with GJB2-related deafness. Laryngoscope, 2004. 114(8): p. 1415-9.

4. Donoghue, J.P. Connecting cortex to machines: recent advances in brain interfaces. Nat Neurosci, 2002. 5 Suppl: p.

1085-8.

5. Linstrom, C.J. Cochlear implantation. Practical information for the generalist. Prim Care, 1998. 25(3): p. 583-617.

6. Tyler, R.S., et al. Three-month results with bilateral cochlear implants. Ear Hear, 2002. 23(1 Suppl): p. 80S-89S. 7. Qian, H., P.C. Loizou, and M.F. Dorman. A phone-assistive device based on Bluetooth technology for cochlear implant

users. IEEE Trans Neural Syst Rehabil Eng, 2003. 11(3): p.

282-7.

8. Wilson, B.S., et al. Design for an inexpensive but effective cochlear implant. Otolaryngol Head Neck Surg, 1998. 118(2): p. 235-41.

9. NIH consensus conference. Cochlear implants in adults and children. Jama, 1995. 274(24): p. 1955-61.

10. Maynard, E.M., Visual prostheses. Annu Rev Biomed Eng, 2001. 3: p. 145-68.

11. Peachey, N.S. and A.Y. Chow. Subretinal implantation of semiconductor-based photodiodes: progress and challenges. J Rehabil Res Dev, 1999. 36(4): p. 371-6.

12. Eckmiller, R. Learning retina implants with epiretinal contacts. Ophthalmic Res, 1997. 29(5): p. 281-9.

13. Veraart, C., et al. Visual sensations produced by optic nerve stimulation using an implanted self-sizing spiral cuff electrode.

Brain Res, 1998. 813(1): p. 181-6.

14. Normann, R.A., et al. A neural interface for a cortical vision prosthesis. Vision Res, 1999. 39(15): p. 2577-87.

15. Chowdhury, V., J.W. Morley, and M.T. Coroneo. Surface stimulation of the brain with a prototype array for a visual cortex prosthesis. J Clin Neurosci, 2004. 11(7): p. 750-5.

16. Walter, P., et al. Successful long-term implantation of electrically inactive epiretinal microelectrode arrays in rabbits.

Retina, 1999. 19(6): p. 546-52.

17. Nadig, M.N. Development of a silicon retinal implant: cortical evoked potentials following focal stimulation of the rabbit retina with light and electricity. Clin Neurophysiol, 1999.

110(9): p. 1545-53.

18. Zrenner, E. Will retinal implants restore vision? Science, 2002. 295(5557): p. 1022-5.

19. Deep-brain stimulation of the subthalamic nucleus or the pars interna of the globus pallidus in Parkinson’s disease. N Engl J Med, 2001. 345(13): p. 956-63.

20. Matharu, M.S., et al. Central neuromodulation in chronic migraine patients with suboccipital stimulators: a PET study.

Brain, 2004. 127(Pt 1): p. 220-30.

21. Ness, T.J., et al. Low intensity vagal nerve stimulation lowers human thermal pain thresholds. Pain, 2000. 86(1-2): p. 81-5.

22. Novak, C.B. and S.E. Mackinnon. Outcome following implantation of a peripheral nerve stimulator in patients with chronic nerve pain. Plast Reconstr Surg, 2000. 105(6): p. 196772.

23. Forrest, D.M. Spinal cord stimulator therapy. J Perianesth Nurs, 1996. 11(5): p. 349-52.

24. Forno, L.S. and R.L. Norville. Ultrastructure of Lewy bodies in the stellate ganglion. Acta Neuropathol (Berl), 1976. 34(3): p. 183-97.

25. Klockgether, T. Parkinson’s disease: clinical aspects. Cell Tissue Res, 2004.

26. Purves, D., et al. Neuroscience. Second ed. 2001: Sinauer Associates, Inc. 27. Wichmann, T., H. Bergman, and M.R. DeLong. The primate subthalamic nucleus. III. Changes in motor behavior and neuronal activity in the internal pallidum induced by subthalamic inactivation in the MPTP model of parkinsonism.

J Neurophysiol, 1994. 72(2): p. 521-30.

28. Bergman, H., T. Wichmann, and M.R. DeLong. Reversal of experimental parkinsonism by lesions of the subthalamic nucleus. Science, 1990. 249(4975): p. 1436-8.

29. Benabid, A.L., et al. Combined (thalamotomy and stimulation) stereotactic surgery of the VIM thalamic nucleus for bilateral Parkinson disease. Appl Neurophysiol, 1987. 50(16): p. 344-6.

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Mechanistic Role of Mrf-2 and C/EBPα During Transcription Nicole Gomez and Robert Whitson, Ph.D.* *Division of Molecular Biology, Beckman Research Institute of the City of Hope Modular Recognition Factor-2 (MRF-2) is a transcription factor belonging to the AT-Rich Interaction Domain (ARID) protein family. Mice that lack Mrf-2 have a lean phenotype as well as craniofacial abnormalities. For the past several years, we have been attempting to determine how the absence of Mrf-2 leads to this lean phenotype by identifying the genes that it regulates. Previous work has shown that embryo fibroblasts derived from Mrf2-/- mouse embryos are deficient in adipogenesis. Using that model system, we identified several genes whose expression was significantly reduced when comparing Mrf-2-/- and wild-type cultures. One of these genes, C/ EBPα, is of particular interest because it is known to be essential for normal fat development. Examination of the C/EBPα gene sequence reveals the presence of a cluster of putative Mrf-2 binding sites about 1.5-1.9 kb upstream of the transcriptional start site. In the work described here, we test whether C/EBPα is regulated by Mrf-2.

To do this, we made two luciferase reporter constructs; pCAPE-Luc contained the previously characterized C/EBPα promoter (1.2 kb upstream of the transcriptional start site, plus an additional 700 bases containing the cluster of Mrf-2 binding sites). A second construct, pCAE-Luc, had only the region containing the Mrf-2 binding sites, and this was placed upstream of the heterologous SV40 promoter. These constructs were transfected into a rat hepatoma cell line (McA RH7777) either in the presence or absence of expression plasmids for two splice variants of Mrf-2 (Mrf-2A and Mrf-2B) or the closely related ARID protein Mrf-1. We found that the pCAPE construct was transcriptionally silent, either in the absence or presence of any expression plasmids. In addition, the presence of the Mrf-2 binding sites gave a strong repression of the SV40 promoter in the pCAE-Luc construct.

These results suggest that the Mrf-2 recognition element in the C/EBPα gene is a transcriptional repressor.

Introduction In recent years, patients and doctors have been battling a rising medical epidemic: obesity and obesity-related diseases such as type II diabetes.

Due to the devastating nature of these diseases, scientists began investigating the genetic and environmental causes of this phenomenon. Most of this research has focused on factors that affect growth and development of adipose tissue. Once thought to be rather passive cells engaged only in the storage and release of lipids, adipocytes are now known to release a variety of hormones that regulate food intake and energy consumption and modulate the response to insulin in liver, muscle, and other tissues. Adipocyte differentiation has been studied intensively using in vitro model systems, including 3T3 pre-adipocytes and primary cultures of mouse embryo fibroblasts. These studies have revealed that adipocyte differentiation is a regulated process that entails sequential expression of multiple

transcription factors, including members of the C/ EBPα family. This, in turn, leads to the expression of adipose-specific enzymes, including fatty acid synthase, which facilitate the synthesis and storage of lipids. C/EBPα refers to CCAAT/Enhancer Binding Protein alpha. It belongs to a family of transcription factors that facilitates the interaction between transcription factors and promoters or enhancers of target genes. This particular family of proteins regulates cell proliferation and differentiation, primarily in adipocytes and liver cells.5

Whitson et al. identified Modular Recognition Factor-2 (Mrf-2), a transcription factor belonging to the AT-Rich Interaction Domain (ARID) protein family.3,6,7 ARID transcription factors have a unique DNA binding domain and are responsible for the regulation and differentiation of gene expression in various living systems. Although Mrf-2 is expressed in nearly all tissues, its normal cell targets are unknown.

Mrf-2 is expressed in two splice variants, Mrf-2A and Mrf-2B. Both variants contain the ARID DNA binding domain.

However, the ratios of these variants differ depending on the tissue that is it being expressed in.

Nevertheless, the consequences of the splice variation between Mrf-2A and Mrf-2B remain Figure 1: The first vector contains the C/EBPα promoter and enhancer- The triangular region unknown. A second illustrates an amplified region of the plasmid. Within this region we see various restriction ARID transcript, sites in addition to the cluster of Mrf-2 binding sites, indicated within the box. To the right Mrf-1, was also of the amplified region is the multi-cloning site, followed by the luciferase gene, SV40 poly cloned in the Itakura A signal, and an ampicillin gene. The second vector contains the C/EBPα enhancer- Here, lab.

The DNAthe amplified region with the Mrf-2 binding sites resides within the multi-cloning site. In binding domain addition to the luciferase gene, SV40 poly A signal, and amplicillin gene, we have added the of Mrf-1 is nearly SV40 promoter. identical to that of 20

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Nicole Gomez is an SQ staff member. Her bio can be found in the staff bio section on page 48.

Mrf-2 and recognizes the same DNA sequence, but there is little homology elsewhere in these proteins.

This suggests that Mrf-1 and Mrf-2 may control the same gene in response to different stimuli, but as of yet there is no evidence of this.

The Itakura lab produced transgenic mice propagated with homozygous deletions in the Mrf-2 gene. The knockout mice have a high neonatal mortality rate, and surviving adults exhibited a lean phenotype and also suffered from craniofacial abnormalities.8 Leanness in these transgenic mouse strains may result from a number of causes, including reduced food intake, mal-absorption of lipids, or accelerated metabolic rate. Many of these potential causes have been eliminated (8, and unpublished data). A further finding that is highly relevant to the current research is that fibroblasts derived from embryos with other transcription factor knockouts had significant deficits in adipogenesis assays (8, and unpublished data). Since adult mice with these same knockouts were also lean, these experiments provided a satisfying mechanistic link between transcription factor expression, adipocytes differentiation, and the development of a lean phenotype.

We have investigated whether a similar link exists between Mrf-2 expression and adipogenesis. We established primary fibroblast lines from Mrf-2-/, Mrf-2+/- and Mrf-2+/+ embryos, and showed that Mrf-2-/- embryo fibroblast had a significant deficit in adipogenesis. These results strongly suggest that Mrf-2-/- mice are lean due to a defect in in vivo adipogenesis (8, and unpublished data).

In subsequent work, we exploited this mouse embryo fibroblast system as a means of identifying target genes for Mrf-2. Fibroblast cultures were treated with a cocktail of adipogenic hormones, and RNA was isolated from the cells at 0, 2, 4, 6, 8, 10 and 12 days. We examined the expression of several key enzymes and transcription factors using northern blots. This analysis provided an important breakthrough in identifying a potential mechanism of the lean phenotype. Although the levels of several early response genes (most notably PPAR-γ) were normal, several late response genes, including

The first vector, pCAPE-Luc (i.e., C/EBPα-promoter-enhancer luciferase vector), contains a fulllength C/EBPα promoter which is 1912bp upstream and 41 bp downstream from the start site.

This fragment, which contains multiple Mrf-2 binding sites, was ligated into the pGL3-basic luciferase reporter vector. This reporter vector also contains an ampicillin resistance gene, SV40 poly A signal, and the luciferase gene. The second vector contains a Hind III/Stu I fragment of the C/EBPα gene. This fragment contains the cluster of Mrf-2 binding sites as well as other transcription factor binding sites Figure 2: that indicate this fragment has *The pCAPE-Luc plasmid is transcriptionally silent. an enhancer. The fragment was *The 5 left-most bars indicate co-transfection with the pGL3-Control-Luc ligated into the pGL3-promoter plasmid and the indicated expression vectors. *The 5 right-most bars indicate co-transfection with the pCAPE-Luc plasmid vector which has an ampicillin and the indicated expression vectors. resistance gene, SV40 poly A signal, the luciferase gene, and the SV40 promoter. This PEPCK, FAS and C/EBPα, were significantly reduced in Mrf-2-/- fibroblast cultures [unpublished reporter vector is our pCAE-SV-Luc vector (C/ data from our lab]. The fact that PPAR-γ, whose EBPα-enhancer-SV40-luciferase). (Constructs are expression is essential for adipogenesis, expresses shown in Figure 1.) at normal levels shows that the phenotype of the Mrf-2 knockouts is not due to a defect in PPAR- Tissue Culture and Transfections γ expression. Surprisingly, the levels of Mrf-2 did not change appreciably in Mrf-2+/+ cultures McA RH7777 cells were cultured and used for during adipogenesis. These results suggest that transfection analysis. The McARH7777 cells are Mrf-2 is essential for the later stages of adipocyte known to have difficulty adhering to the surface development, and that its role is “permissive”, of culture flasks and plates. For this reason they meaning that it is not induced by adipogenesis, were grown on gelatin-coated plates. These cells but is required. The northern blot analysis also are unusual in that they prefer to grow in acidic suggested that C/EBPα may be a direct target of conditions, maintaining a low pH. Also, they require modified DMEM which is high in glucose.

Mrf-2, which is the primary focus of this work.

When the cell cultures reached approximately 90% Previous work on the C/EBPα promoter revealed confluence they were passed into 6 well plates and the presence of multiple activating and suppressing prepared for transfection. elements in the region 0-1.2 kb upstream of transcriptional start site (unpublished data from our Prior to adding the co-transfection mixes, the lab). We examined the sequence further upstream cells were incubated for an hour in antibiotic free and identified a cluster of canonical Mrf-2 binding medium, which was a mixture of modified DMEM sites in the region from ~1.5 to 1.9 kb upstream. medium, fetal bovine serum, and donor horse serum.

These preliminary results led us to formulate Cells were co-transfected with a reporter vector, the hypothesis that C/EBPα is a direct target for which was either pCAPE-Luc, pCAE-SV-Luc, activation by Mrf-2. To test this hypothesis, we pGL3-control, or the pGL3-promoter. The second cloned a luciferase reporter that contained the entire reporter was always pCMV-β-galactosidase. The sequence for 0-1.9 kb upstream from the start site. In order to determine that the Mrf-2 binding sites had an inhibitory effect, we prepared a second reporter that contained the Mrf-2 binding sites upstream from the SV40 promoter.

We then transfected these reporter plasmids into rat hepatoma cells in the presence or absence of expression plasmids for Mrf-2A, Mrf-2B and Mrf-1. Our results suggest that the Mrf-2 recognition element in the C/EBPα gene is a strong transcriptional repressor.

substrate in these transfections was lipofectamine (2mg/ml). The transfections were carried out with a 5:1 lipofectamine to DNA ratio. In addition to the reporter vectors, cells contained one of 5 effector plasmids which included opti-MEM (i.e., no effector), pCMV-0, pCMV-Mrf 2A, pCMV-Mrf 2B, and pCMV-Mrf-2B. Cells were harvested 48 hours after transfection and prepared for luciferase and β-gal assays.

Transfection with T3 We carried out a series of transfections as outlined above; however, half of the transfected cells received a dosage of T3. T3 (thyriod hormone) is a regulatory hormone which interacts with specific receptors to inhibit or enhance transcription rates of certain genes. Menendez-Hurtado et al. have reported that T3 is involved in the regulation of C/EBPα mRNA.5 To fully understand how C/ EBPα expression is regulated, T3 was transiently transfected into our cell cultures. Twenty-four hours after the transfection mixes were added, the medium was changed to serum free medium and 100 nM T3 was added to the appropriate well.

Twenty-four hours after the addition of T3, the cells were harvested for luciferase and β-gal assays.

Luciferase Assay In this assay, light is produced by converting chemical energy of luciferin oxidation through an electron transition, which forms oxyluciferin.

Firefly luciferase catalyzes luciferin oxidation

by ATP Mg2+ as a co-substrate. The luciferase assay was chosen mainly for two reasons. First, reporter activity can be recorded directly following translation. Second, the assay itself is extremely sensitive because the light production has high quantum efficiency.

After preparing cell lysates, opaque vials were labeled and used in a scintillation counter. 20 μl aliquots of each cell suspension were added to a tube for the assay. In 30-second intervals, 100 μl of luciferase reagent was added to each sample.

The sample was counted for 30 seconds. After the sample had undergone the reaction, an average CPM value (counts/minute) was recorded by the scintillation counter. The first sample to be read was always a blank, which contained 20 μl of lysis buffer and 100 μl of luciferase reagent. Each cell

Methods Cloning Luciferase Reporter Vectors The generation of two luciferase reporter vectors was the aim of the cloning studies. In order to do this, the C/EBPα promoter was amplified by PCR.

Figure 3A Figure 3B *T3 does not stimulate luciferase transcription in the pCAPE-Luc vector. *McA RH7777 cells were transfected with pCAPE-Luc, plus the indicated expression vectors, then treated with T3 (red bars) or with nothing (blue bars) *A and B represent duplicate experiments.

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degree (Figure 2).

Figure 4: The Mrf-2B Recognition Element represses transcription. The pCAE-Luc vector contains the cluster of Mrf-2 binding sites, as well as the SV40 promoter.

This is significant because, in the absence of the Mrf-2 binding sites, expression is substantially higher. *McA RH7777 cells were transfected with either pSV-Luc (red bar) or pCAE-Luc (blue bar). Note that luciferase activity was decreased by 70% in pCAE-Luc compared to pSV-Luc. *Figure 1 shows pCAE-Luc and its contents. Keep in mind that this vector contains the Mrf-2 binding sites, whereas the pSV-Luc does not.

extract was measured for luciferase activity in duplicates. After the data was obtained, the raw values were normalized by subtracting the average of the blank values. These values were then used to calculate a specific luciferase activity. β-gal Assay Using the same cell lysates as the luciferase assay, a β-galactosidase assay was conducted as a control. The assay was intended to measure the efficiency of transfection. A ratio of luciferase to β-galactosidase was calculated to account for transfection efficiency.

Stocks were made of 100x magnesium buffer (100mM MgCl2, 5 M 2-mercaptoethanol), 0.1 M sodium phosphate buffer, and ONPG substrate solution [ONPG (o-nitrophenyl-β-Dgalactopyranoside) in 4mg/ml in sodium phosphate buffer]. Reagents were combined in a 96-well plate: 10-150 μl of cell extract, 3 μl of 100X magnesium buffer, 66 μl of ONPG solution, and sodium phosphate buffer for a final volume of 300 μl. This reaction mix was incubated at 37˚ C for approximately 2 hours. Once a yellow color appeared in the samples, 0.5 ml of 1M Na2CO3 was added, and the color was measured in an ELISA reader at 410 nm.

Results The 1.9 kb C/EBPα promoter is inactive in McA RH7777 cells.

When the pCAPE-Luc vector was transfected in the absence of effector plasmids, (i.e., opti-MEM), we detected little or no luciferase activity in McA RH7777 cell extracts. In fact, the luciferase activity ranged from 0.1–0.5% of the activity obtained with the luciferase control vector, in which the luciferase is controlled by the moderately-strong SV40 promoter/enhancer (Figure 4). We also found that co-transfection with Mrf-2A, Mrf-2B and Mrf-1 did not activate this construct to any significant 22

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Since the 1.2 kb C/EBPα promoter construct is not fully active in the absence of hormones, we then tested to see whether the addition of T3 would also activate the 1.9 kb promoter construct, either in the presence or absence of the effector plasmids.

Figure 3 shows that there was no consistent increase in luciferase activity after T3 treatment. Initially, Mrf-2B appeared to increase the luciferase activity in a non-hormone dependent fashion (Figure 3A).

However, subsequent transfections were highly variable, and the results were inconclusive (Figure 3B).

The Mrf-2 recognition element in the far upstream sequence of the C/EBPα gene may contain a transcriptional repressor.

The 1.2 kb C/EBPα promoter fragment is quite active in the presence of T3; it appears that the addition of the upstream sequence that contains the Mrf-2 sites acts as a suppressor. This is supported by our experiments with the pCAE-Luc construct.

This construct contains the 0.4 kb fragment upstream of the heterologous SV40 promoter. The SV40 promoter shows relatively weak activity on its own, but the activity is reduced by more than 70% by the presence of the putative Mrf2B recognition element, (Figure 4). The addition of effector plasmids did not affect the activity of either the pSV promoter plasmid or the pCAE-Luc plasmid (not shown).

Discussion Our hypothesis is that C/EBPα is a target for activation by Mrf-2. Therefore, we expected that co-transfection of the expression plasmids for Mrf-2A or Mrf-2B would stimulate transcription from the pCAPE-Luc plasmid, which contains 1.9 kb of upstream sequence from the C/EBPα promoter. Instead, we found that this construct is almost completely inactive, either in the presence or absence of Mrf-2-expressing plasmids or T3.

There are several possible explanations for this inactivity, the first being that our hypothesis is incorrect. The most probable explanation is that the McA RH7777 is not a suitable test system. There are several possible explanations for the failure of Mrf-2A and Mrf-2B to stimulate pCAPE-Luc in these cells. One possibility is that these cells lack the appropriate steroid hormone receptors to allow the appropriate response from T3. McA RH7777 cells probably do not express the thyroid receptors at high levels, or even at all. Thus far, the cell line that we tried does not contain the necessary factors to allow Mrf-2 to stimulate C/EBPα. It is also possible that the effects of Mrf-2 on C/EBPα cannot be demonstrated with a reporter assay, because Mrf2 must act in the context of chromatin. Even though the transfected plasmids may be packaged into nucleosomes, this does not begin to resemble the chromatin structure that surrounds normal cellular DNA (personal communication, Robert Whitson).

Another possible explanation is that the expression of the effector plasmids was not sufficient to see an effect on this pCAPE plasmid, and that the assay itself may be flawed. This is supported by our failure to detect β-galactosidase in the cell extracts,

despite the fact that it was co-transfected with the luciferase reporter in every experiment. Since βgal expression was driven by the very strong CMV promoter/enhancer, this was somewhat surprising.

To test this further, we transfected the cells with both pCMV-CAT and pSV-CAT vectors and measured CAT (chloramphenicol acetyl CoA transferase) activity in the cell extracts. CAT activity was barely detectable in extract from cells transfected with 1 ug of pCMV-CAT and not detectable in cells transfected with 1 ug of pSV-CAT, despite the fact that both these vectors give easily detectable CAT activity when transfected into other cells. Finally, we found that β-gal was readily detectable in cell extracts when the cells were transfected with 3 ug of either pSV-β-gal or pCMV-β-gal, and a more efficient transfection agent was used. These results suggest that McA RH7777 cells are unusually resistant to transfection.

Our results also suggest that the cluster of Mrf-2 binding sites upstream of the start site (which we refer to collectively as an Mrf-2 recognition element or Mrf-2 RE) constitutes a strong transcriptional repressor. The evidence for this is that the addition of this sequence appears to completely inactivate the C/EBPα promoter construct. Additionally, we showed that this element acts as a strong repressor when placed upstream of the heterologous SV40 promoter. Although these preliminary results are intriguing, we need to confirm them, first, by showing that the deletion of this sequence restores the activity of the reporter construct and, second, by showing that specific mutations in the Mrf-2 binding sites eliminate the suppressing activity.

References 1. Bioalternatives.

4.asp.

http://www.bioalternatives.com/pages5_5_

2. Darlington G.J., S.E. Ross, and O. MacDougald. The role of C/EBPα genes in adipocyte differentiation. J Biol Chem.

273,46:30057-30060; 1998.

3. Gregory, S., R.D. Kortschak, B. Kalionis, & R. Saint.

Characterization of the dead ringer gene identifies a well, highly conserved family of sequence-specific DNA binding proteins.

Mol Cell Biol. 16:792-799; 1996.

4. MacDougald, O.A., & M.D. Lane. Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem. 64; 345-373; 1995.

5. Menendez-Hurtado, A., A. Santos, & A. Perez-Castillo.

Characterization of the promoter region of the rat CCAAT/ Enhancer Binding Protein α gene and regulation by thyroid hormone in rat immortalized brown adipocytes. Endo. 141, 11:4164-4170; 2000.

6. Shandala, T., R.D. Kortschak, S. Gregory, & R. Saint. The drosophila dead ringer gene is required for early embryonic pattering through regulation of argos and buttonhead expression.

Development. 126: 4341-4349; 1999.

7. Whitson, R.H., T. Huang, & K. Itakura. The novel Mrf-2 DNA binding domain recognizes a five-base core sequence through major and minor groove contacts. Biochem Biophys Commun.

258: 326-331; 1999.

8. Whitson, R.H., W. Tsark, T.H. Huang, & K. Itakura. Neonatal mortality and leanness in mice lacking the ARID transcription factor Mrf-2. Biochem Biophys Res Commun. 26; 312(4):9971004; 2003 9. Yuan, Y.C., R.H. Whitson, Q. Liu, K. Itakura, & Y. Chen. A novel DNA binding motif shares structural homology to DNA replication and repair nucleases and polymerase. Nat Struct Biol.

5: 959-964; 1998 .

Analysis of the Manganese Oxidation Capacity of Pseudomonas putida GB-1 and MnB1 Mutant Strains D.B. Baron Manganese (Mn) oxides of biological origin have been shown to possess the ability to reduce the toxicity of contaminated sites, including those containing heavy metal pollutants. However, relatively little is known about the actual process by which biological manganese oxidation occurs. This study focused on examining the phenotypic effects of knockout mutations on 11 genes of interest, especially ones involved in loss of soluble oxidized Mn production in Pseudomonas putida strains already deficient in the production of insoluble Mn(IV).

The purpose was to gain clues about the roles these genes play in the enzymatic pathway where Mn(II) is oxidized to Mn(IV). The data gathered in this study resulted in the discovery that three proteins coded for by a cytochrome maturation complex F homolog, an undefined gene, and sdhc might be responsible for the production of detectable soluble or precipitated oxidized Mn, even Mn(III). Knockout mutants for 7 other genes, including one coding for a multicopper oxidase, showed some production of oxidized Mn that was judged to be Mn(III) because visible dark brown or black Mn(IV) oxide precipitates did not appear in the liquid cultures. This observation gives credence to the prospect that the products of these 7 genes play a role in the oxidation of Mn(III) to Mn(IV). In addition, evidence indicating that ccm mutant cells lack an exopolymer matrix suggests that Mn oxidizing enzymes embedded in a mucous layer on the cell surface are involved in the oxidation of Mn(III) to Mn(IV). The data from this study have also revealed a number of additional distinguishing characteristics of each mutant strain that can be used in further pursuits to advance the understanding of proteins involved in P. putida manganese oxidation.

Introduction Manganese Oxide Background Manganese is the fifth most abundant metal in the Earth’s crust and the second most common trace metal after iron.

An important characteristic of manganese is its ability to exist in several different oxidation states ranging from 0 to +7, although it is almost always found in nature in its +2 state as Mn(II), +3 state as Mn(III), or +4 state as Mn(IV).

Of these, Mn(II) is readily soluble in water while Mn(III) is more unstable and has a tendency to precipitate or dissociate to Mn(II) or Mn(IV) unless chelated to another molecule. Mn(IV) is insoluble and can be detected by the presence of a visible brown or black precipitate in neutral solutions. Mn(IV) is often found as part of the compound MnO2.

The gram-negative bacterium Pseudomonas putida (P.putida) has been observed to precipitate Mn(IV) oxides

cycling of several other elements such as C, Fe, N, S, etc., usually by acting as an electron acceptor at the end of the electron transport chain during the production of ATP. Evidence has shown that Mn(III) and Mn(IV) compounds function in the geochemistry of water bodies by acting as electron acceptors in anoxic environments.24 This same electron accepting capability has led to the introduction of oxidized manganese precipitates in bioremediation projects at sites contaminated with heavy metal pollutants.

The high reactivity of manganese oxides due to their high sorptive capacities for heavy metals combined with their strong oxidizing nature means that they have the capability to help in the degradation of various toxic molecules.

Research on the immobilization of oxidized heavy metals by binding with manganese oxides of different origins suggests that cellular produced oxidized manganese performs better in this capacity than manganese oxides of abiotic nature.40 Past uses of biological manganese oxides include their application in the remediation of water polluted with heavy metals. One project involved filtering contaminated water through a layer of sand surrounded by an insoluble adsorbent Mn oxide coat of biological origin which absorbed hazardous heavy metals out of the water.17 Mn oxides have also been shown to be capable in controlling arsenic levels in a water body using their absorptive properties to reduce the element’s toxic effect. Improving techniques for the treatment of tainted sites and water stocks is a major goal in studying enzyme catalyzed manganese oxide formation.

Daniel Baron is a graduating senior in Environmental Systems: Ecology, Behavior, Evolution in Thurgood Marshall College. He has interned both at the Tebo lab at Scripps Institution of Oceanography and at the biotechnology company Amgen and plans to apply to graduate school in molecular biology for fall 2005 with a specific interest in studying biopharming and genetic engineering. and how they function during its course. The main focus of this project was to compare oxidized manganese levels, the presence of an exopolymer matrix, and other characteristics of the liquid cultures of these 11 mutants with wild type liquid cultures to clarify the function of each gene in biological manganese oxidation.

Of the 11 mutant strains studied during the course of this research, 4 were MnB1 mutants created by Ron Caspi: MnB1-UT 303, MnB1-UT 402, MnB1-UT 403, and MnB1-UT 3501, and contained Tn5 insertions in the cytochrome maturation (ccm) complexes F (UT 303), A (UT 402), E (UT 403), and succinate dehydrogenase subunit c (UT 3501). 9 The remaining 7 strains were GB1 mutants created by Johannes PM de Vrind: GB-1-003, GB-1-004, GB-1-005, GB-1-006, GB-1-007, GB-1-008, and GB-1-009.15 The main GB-1 mutant strains of interest were GB-1-003 and 004, which are mutants for ccm F homologs, and GB-1-007 which is a mutant for CumA,

Manganese Oxidation in Pseudomonas putida Wild Type and Mutant Strains Figure 1A: MnB1 wild type surrounded by layer of Mn(IV) oxide fibers.

on its cell surface (Figure 1A) by a presumed cascade of action by enzymes either acting with haem porphyrins or enzymes embedded in either the plasma membrane or a exopolymer matrix surrounding those theorized to contain polysaccharides.44 The mechanisms involved in catalyzing such a reaction, as well as the purpose of the oxides, are not well known and are under investigation.

Environmental Uses of Mn Oxides Oxidized manganese is a strong redox reagent in anoxic and suboxic environments, where it influences the redox

Many mutant Pseudomonas putida of the strains GB-1 and MnB1 6,11 have been made deficient in the production of Mn(IV) by performing transposon (Tn5) mutagenesis on wild type cells.34 The mutant strains in this study all possess a gene involved in manganese oxidation that has been knocked out via the insertion of the Tn5 transposon into their genome. As a result, these mutants are negative for the Mn(IV) oxide film surrounding the cell surface (Figure 1B).

Making distinctions between the phenotypes of the different mutant strains is a good method for determining what molecules and proteins are involved in the cascade of protein action during manganese oxidation in P. putida

Figure 1B: MnB1 mutant lacking surface Mn(IV) oxides.

a multicopper oxidase proven to function in manganese oxidation in 6. GB-1-007, a cumA mutant, has also been of interest in past studies.

As well as being a manganese oxidizer, P. putida is well documented as being a siderophore producer under iron limiting conditions.12 Stress induced by low Fe(III) concentration around the cell results in the secretion of the metal complexing siderophore pyoverdine, a fluorescent ligand detectable by the presence of a neon green color.

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Turbidity vs. Time Patterns of Mutant Cultures

in the bacterium. Research demonstrating the ability of Mn(III) to compete robustly with Fe(III) for chelation with pyoverdines produced by P. putida indicates that pyoverdines can also function to stabilize Mn(III) produced as an intermediate during the oxidation of Mn(II) to Mn(IV) 41,44 thus preventing its dissociation and prolonging its existence.32 The implications of a longer lifespan of strongly oxidizing species like biologically produced Mn(III) in the natural environment would require reconsidering some currently held views on many types of elemental cycling in nature such as iron and chromium.41,19 The production of pyoverdines in P. putida has been thought to be related to the capability of the cell to synthesize Mn oxides. This relationship involves a link between pyoverdine production and the formation of an extracellular-polysaccharide matrix surrounding the cell.43 The gene prfA, coding for a single polypeptide, is a positive regulator involved in transcriptionally regulating alginate polysaccharide biosynthesis in Pseudomonas aeruginosa and is believed to carry over into P. putida.

This can affect the oxidation of manganese because the extracellular mucous matrix in P. putida, thought to consist of polysaccharides, has also been believed to form an extracellular matrix in which one or more manganese oxidizing enzymes are embedded.37 If the formation of this exopolymer matrix was prevented, it could therefore be assumed that manganese oxidation would be likewise impeded. The presence of this matrix or capsule is also believed to allow cells to attach to one another, causing a precipitation of both viable and dead cells in a liquid culture shortly after inoculation. This theory has been given some credence by evidence that the cells of some GB1 mutants deficient in Mn(IV) production intracellularly contain oxidized Mn, thought to be Mn(III).14 The failure to convert this intracellular Mn(III) into extracellular Mn(IV) could be due to the lack of the necessary catalytic enzymes on the cell surface as a result of the absence of the aforementioned extracellular matrix.

Methods Preparation of Cells and Liquid Cultures Eleven mutant strains were grown on Lept + Kanamycin plates to select for mutant colonies. Lept media was prepared using the recipe 0.5g Yeast Extract, 0.5g Casamino Acids, 1L MQ H20, 0.5ml 1M CaCl2, 0.83 ml 1M MgSO4, 5ml 1M Glucose, 10 ml 1M Hepes pH 7.5, and 1ml 1M Lept trace elements. Colonies taken from these plates were used to inoculate Lept + Kan liquid medium 24

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to establish a base set of cultures from each mutant strain that were of about equal density. 20 µL of cells from these stock liquid cultures were used to inoculate duplicate sets of either Lept or Minimal Salt + Glucose (MSG) medium in 20 ml trace metal acid washed tubes to initiate various experiments such as LBB, spectrophotometer, and exopolymer assays.23 Assays of Oxidized Mn by the LBB Method Mn oxides in liquid cultures can be detected by reactions with the colorimetric dye leucoberbelin blue (LBB). LBB is a redox dye that is oxidized by a two electron reaction with Mn(IV) or a single electron transfer reaction with Mn(III).

Mn+3 + LBB(reduced) > Mn+2 + LBB(oxidized) Oxidized LBB is dark blue in color while non-oxidized LBB is lighter blue. The degree of coloration is a function of the number of electrons transferred in the reaction to the LBB and therefore can be used to quantify the amount of Mn oxides being reduced. The strong redox potential of LBB means only very strong oxidizing agents like Mn oxides can react with it.13 Therefore, a positive LBB reaction in a sterile medium can be inferred as the result of the presence of Mn(III).

Testing for oxidized manganese in these cultures was done using several methods. Daily observations of the mutant and wild type liquid cultures were performed to check for the visible appearance of a brown or black Mn(IV) precipitate. Leucoberbelin blue colorimetric dye (LBB) assays20 were also utilized to test for the production of Mn(III) over time. Cells centrifuged for 3 minutes at 14,000 rpm and non-centrifuged mutant and wild type liquid culture sample, respectively, were reacted with 0.4% LBB test solution in a 1:3 ratio. The purpose of centrifugation was to observe if solubalized or colloidal Mn(IV) was present in the supernatants of the centrifuged cultures by comparing LBB results between centrifuged and non centrifuged samples. The LBB + liquid culture solution was then aliquoted in 96 well plates, incubated in the dark for 15 minutes, then recorded for absorption at 620 nm on a Perkin Elmer HTS 700 Bio Assay reader.

A permanganate standard curve was created by measuring the absorbances at 620 nm of 0, 5, 10, 15, and 20 µM permanganate stocks in order to quantify the levels of oxidized Mn present in the liquid cultures. It was assumed that centrifugation removed any LBB-reacting Mn(IV) oxide precipitates or any cell-bound Mn(III,IV); colloidal Mn(IV) does occur under some circumstances.24

Turbidity of the mutant liquid cultures was measured by recording the absorbances of liquid culture samples at 620 nm using a Milton Roy Spectronic 20. The data were used with the LBB data to create a LBB reaction vs.

Growth plot that might offer insight into the differences in the Mn(III)-producing capabilities of the mutant strains.

Before absorbance was recorded the tubes were tapped lightly to suspend any clumps of cell material that had accumulated at the bottom of the tube. Results of LBB and turbidity assays were presented as positive LBB reaction vs. time, turbidity change vs. time, as well as positive LBB reaction vs. turbidity in order to establish the relationship between the growth stage of the cells and their ability to produce Mn(III). These turbidity vs. time plots were also used to record the presence of extracellular polymers that form a mucous layer, thought to be polysaccharides surrounding P. putida cells.43,11 Such cell surface polymers/polysaccharides are thought to be involved in both the attachment of cells to one another during stationary phase to produce precipitated clumps of cell material, and have also been hypothesized to form a cell capsule containing proteins involved in the oxidation of Mn(II) or Mn(III).

The positive LBB reaction datum gathered from each mutant was divided by the recorded turbidity datum for each time point and plotted against days of growth for the purpose of comparing mutant strain trends of cell material accumulation [a measure of the production of Mn(III)]. It is hypothesized that the absence of a cell surface polymer matrix comprised of extracellular polysaccharides hinders the clumping of dead and stationary phase cells together, which in turn increases the turbidity of a liquid culture because cell material can no longer precipitate at the bottom of the growth tube.

Testing for Exopolymers in Mutant Strain Liquid Cultures The presence or absence of extracellular polysaccharides was further tested by negative and positive staining of liquid culture samples. Samples taken from strains of interest were mixed with India black ink and observed under a light microscope at 10x magnification. The black ink provided at negative background to highlight the presence of polymer structures and precipitated Mn(IV) oxides.

In a separate assay, 200 µL crystal violet dye was added to liquid culture from each strain after three days of growth in Lept medium without shaking conditions.2 This was done to positively stain any pellicle that had formed on the surface of the liquid culture which would provide additional evidence for the presence of an exopolymer matrix that possibly contains Mn oxidizing enzymes.

This procedure was based on the hypothesis that the same structures that function to cause an agglomeration of cells during stationary phase also results in pellicle formation.

Mn(III) Chelation with Pyoverdines in Mutant Strains The effect that each of the 11 point mutations had on the production of pyoverdine and Mn(III) was tested by recording absorption spectrums between 300 nm and 500 nm on a Perkin Elmer Lamda Bio 20 from liquid culture samples of each mutant strain. Samples were centrifuged at 14,000 rpm for 30 seconds and added in

a 10:1 ratio with EDTA pH 8.0 prior to recording the absorption spectrum to remove potential pyoverdine binding competitors with Mn(III). Absorption values were measured at three time points: after the inoculation of the medium with cells during growth phase, another during the onset of stationary phase early on day three, and a final one on day 5 well after the cells had entered stationary phase. The presence of Fe(III) bound pyoverdine, Mn(III) bound pyoverdine, and unbound pyoverdine in a mutant liquid culture was identified by examining the liquid culture sample for absorption spectrum for peaks which will occur at about 400 nm (with a large shoulder at 460 nm) for Fe(III)-pyoverdine and at 410 nm when Mn(III)pyoverdine is present.32 Examination of Mutant Strains for Complementation by Cross Streaking To test the hypothesis that one of the proteins involved in the redox cascade of Mn oxidation might be membrane diffusible, each mutant strain was cross streaked with each of the other 10 mutant strains as well as with both wild types on Lept + Kan plates. The intersections of the two streaks were examined for the production of visible brown Mn(IV) oxides in order to test for complementation.9 Several comparisons of single colonies from mutant strains GB-1-007 (CumA mutant), MnX-G, and wild type strains GB-1 and MnB1 spaced 1 cm apart were also conducted to test for diffusible proteins complementary in the overall oxidation of Mn(II) to Mn(IV).

Results Assays of Oxidized Mn by the LBB Method Results of this assay revealed varying levels of positive LBB reaction between the mutant strain cultures in comparison to the wild type cultures. Examination of LBB vs. time data from mutant and wild type cultures allowed the strains to be separated into 3 categories, denoted a, b, and c, based on their LBB curve characteristics. Category (a) strain liquid cultures show both positive LBB reaction visible and visible Mn oxide precipitates within 2.5 days of growth. This category includes only the wild type strains GB-1 and MnB1. The two wild type LBB vs. time plots are shown in Figure 2A. Category (b) encompasses strains with liquid cultures showing significant positive LBB reaction but no visible Mn precipitates. This category includes the mutant strains GB-1-006, GB-1-007, GB1-008, MnB1-UT 303, MnB1-UT 402, and MnB1-UT 403. All category (b) strains possess LBB vs. time plots are similar in structure to the plot shown in Figure 2B. Mutant strains in this category also have at least 70% the LBB reacting material as the wild types after several days of growth. Category (c) consisted of strains with liquid cultures showing lower amounts of positive LBB reaction and includes of mutants GB-1-003, GB-1-004, MnB1 UT 3501, and GB-1-005. Of the strains in category (c), the liquid culture samples from GB-1-004 and MnB1 UT 3501 showed severely reduced levels of positive LBB reaction, with positive LBB reaction measurements close to 25 % that of the wild types after two days of growth. An example of a LBB vs. time plot characteristic of a category (c) strain is listed as Figure 2C. Samples from GB-1-003 and GB-1-005 cultures also exhibited liquid cultures with reduced levels of positive LBB reaction, although they possessed somewhat more LBB reacting material than GB-1-004 and MnB13501. The positive LBB reaction levels of GB-1-003 and GB-1-005 ranged from 33% to

50% of the wild types after two days of growth.

Centrifuging the culture samples prior to assaying noticeably influenced the amount of positive LBB reaction recorded in each strain. The recorded LBB reaction value of a liquid culture sample was consistently lower following centrifugation than the absorbance recorded from a duplicate uncentrifuged sample. This result was observed in all liquid cultures.

Turbidity vs. Time Patterns of Mutant Cultures Absorbance data recorded during the turbidity assays were analyzed by organizing the mutants and wild type strains into three additional categories based on the growth patterns of the liquid cultures: normal growth, delayed growth, and abnormal growth. Strains in the normal growth category had liquid cultures with turbidity vs. time plots in which absorbance at 620 nm peaked between 48 and 72 hours before decreasing by about 25% and then remained somewhat constant over the remaining time. The strains included in this group are: GB-1-003, GB-1-004, GB-1-005, GB-1-006, GB-1-007, GB-1-008, MnB1 UT 3501 and both wild types. A turbidity vs. time plot characteristic for strains of this category is shown in Figure 3A. (Ed. Note: For Figures 3A - 5C refer to http:// sq.ucsd.edu/volume2_issue1.html.)

Strains in the delayed growth category had liquid cultures showing a much higher absorbance 620 at 24–36 hours and a turbidity peak at a much later time point than strains with normal growth. The only strain that exhibited these characteristics was GB-1-009 (Figure 3B), which was the only strain that required re-inoculation of both liquid culture tubes due to a lack of visible growth after about 42 hours. This failure to cultivate GB-1-009 mutants in Lept medium was not observed in other assays requiring GB1-009 grown in Lept.

The strains belonging to the abnormal growth category showed irregular turbidity curves that reached an absorbance plateau rather than a peak a little after 48 hours and also exhibited a turbidity plot with no large drop in absorbance after about 72 hours. A typical turbidity vs. time plot for this category of strains is shown in Figure 3C. Furthermore, the liquid cultures of strains from this group were observed to be much cloudier than the cultures of normal growth strains and also lacked dead cell deposits that usually accumulated at the bottom of the tubes in wild type cultures following the stationary phase.

Plots of LBB/Turbidity vs. Time The liquid cultures of several wild type and mutant strains produced a bimodal curve when their positive LBB values for each time point was divided by turbidity and then plotted against days of growth (Figure 4C-4E). The plots made from data gathered on both duplicate liquid cultures for each mutant strain were then combined to produce an average LBB/turbidity vs. time curve (Figure 4A-4B).

The LBB/turbidity vs. time plot of wild type GB-1 culture shows a large bimodal curve with peaks at both 24 and 84 hours (Figure 4A). The LBB/turbidity vs. time plots of most GB-1 mutant strains show a similar bimodal shape but with the second LBB/turbidity peak appearing closer to 72 hours than 84 and also severely reduced in magnitude compared to the wild type. The exception to this is strain GB-1-009, which possesses a LBB/turbidity vs. time plot

with a peak at 24 hours that matches that of wild type GB-1 (Figure 4B). Also, GB-1-003 liquid culture does not show a bimodal curve structure, but instead a gentle rise in LBB/turbidity over the five day growth period (Figure 4A). Furthermore, strains GB-1-004 and to a lesser extent GB-1-005 exhibit LBB/turbidity vs. time plots with the bimodal curve pattern but of smaller size than mutant strains GB-1-006 and GB-1-007 (Figure 4A).

Wild type MnB1 did not show a bimodal LBB/turbidity vs. time plot, and like wild type GB-1, the LBB/turbidity values of its plot were much larger than those of the four MnB1 mutants (Figure 4B). Interestingly, mutant strains MnB1 UT 303 and MnB1 UT 402 both possess LBB/ turbidity vs. time curves showing a faint bimodal pattern even though wild type MnB1 does not. Strain MnB1 UT 3501 exhibits one LBB/turbidity peak like wild type MnB1 and MnB1 UT 403, but the peak is much lower in magnitude than the other MnB1 mutants (Figure 4B).

Testing for Exopolymers in GB-1 and MnB1 Mutants Crystal violet staining revealed the production of a large pellicle by cells from all mutant and wild types with the exceptions of strains GB-1-004, MnB1-UT 303, MnB1UT 402, and MnB1-UT 403. It should be noted when viewing this image that GB-1-009 and wild type GB-1 did produce pellicles, but unfortunately both were lost while washing the liquid culture from the tube with ddH20.

Using a light microscope to view cultures of strains GB1-009, UT 303, and UT 402 under 10x magnification resulted in the detection of whitish exopolymer clumps in strain GB-1-009 and wild types GB-1 and MnB1 that are likely to be alginate extracellular polysaccharides.

Each of these three mutant and wild type strains found to possess the white exopolymers was also observed to be pellicle producers. No evidence of these white clumps was found in samples from strains UT 402 and UT 403.

The liquid cultures of strains UT 402 and UT 403 did not contain deposits of cell material usually found at the bottom of the liquid culture tube following the onset of stationary phase.

Mn(III) Chelation with Pyoverdines in Mutant Strains A total of three sets of each mutant and wild type P. putida strain in Lept medium were grown during the course of this study. During this time, it became visually apparent from the fluorescent color of pyoverdine that cells from 4 strains — GB-1-004, GB-1-009, MnB1 UT 402 and MnB1 UT 403 — consistently produced very few or no pyoverdines compared to wild type cells. However, the pyoverdine production of cells from a fifth strain, MnB1 UT 303, ranged from low to comparable to the levels produced by wild type cells.

Absorption spectrum results revealed very similar absorbance peaks at 410 nm in mutant strains GB-1005, 006, 007, 008, 009, MnB1-UT 3501 and wild type strain MnB1, all of which had absorbance readings of approximately 0.32 (Figure 5A-C). Unexpectedly, the samples from the duplicate GB-1-003 liquid cultures showed peaks at about 403 nm instead of 410 nm with higher absorbances of magnitudes 0.4174 and 0.5144 compared to the average GB-1 mutant strain absorbance peak value of 0.2896 (Figure 5A-C). Strains GB-1-004 (Figure 5A), MnB1 UT 403 and MnB1-UT 402 (Figure 5C) showed no visible absorbance peaks, although the Volume 2 Issue 1

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sample from one of the duplicate MnB1 UT 402 cultures had a much higher range of absorption values over its entire absorption spectrum than its duplicate (Figure 5C).

The absorption spectrum of wild type control strain GB-1 exhibited no peaks, but did show a low shoulder at 350 nm (not pictured in Figure 5). The absorbance spectrum data from one of the duplicates from both strains UT 303 and UT 403 was lost.

Examination of Mutant Strains for Complementation by Cross Streaking While none of the 62 cross streaks of mutant strains resulted in the appearance of a brown or black precipitate at the intersection of the two strains, it was noted that colonies and the plate around the colonies from strains GB-1-004 and UT 3501 consistently turned bright yellow in color, an indication of an overproduction of pyoverdines when grown on Lept + Kan plates. This observation was replicated in a liquid culture experiment with these same strains in which Lept + Kan medium was mistakenly used.

None of the plates containing adjacent single GB-1-007 and MnX G mutant colonies showed complementation nor a renewed ability to produce Mn(IV) oxides.

Discussion Assays of Oxidized Mn by the LBB Method Any manganese product with an oxidation state higher than +2 will result in a color changing reaction when combined with Leucoberbelin blue. Since no brown precipitate was visible in any mutant strain liquid culture during the course of several experiments, it is probable that the positive LBB reaction in the mutant cultures resulted from the presence of Mn(III). However, there is also a chance that a small amount of Mn(IV) is present that is cell associated or in the form of tiny precipitates that are not visible to the human eye. Therefore, the exact nature of the oxidized manganese detected by the LBB assay cannot be fully confirmed.

One explanation of the uniform decrease in positive LBB reaction levels following centrifugation is that much of the oxidized manganese produced by the cell is associated with the cell’s surface or exopolymer matrix and is removed from the solution by centrifugation. The effect of centrifugation on the levels of positive LBB reaction in culture samples suggests a possible technique for separating background LBB reacting material out of a solution prior to recording absorption. This technique would require one to pellet the cells in a liquid culture to remove the cell surface bound Mn(III) intermediates from the solution and dispose the supernatant, then resuspending the cells and recording absorption to obtain a true measure of cell-bound oxidized manganese in the culture.

By analyzing the results of the LBB vs. time plots created for each mutant strain it can be proposed that strains GB1-004, MnB1-UT 3501 are deficient in the production of oxidized manganese (most likely Mn(III)), while the capacity of strain GB-1-005 is only fairly retarded for its production comparatively (See Results: Assays of Oxidized Mn by the LBB Method). Every other mutant strain had positive LBB reactions similar to those of the wild types and appear to produce levels of Mn(III) similar to that of their respective wild types.

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These experiments also support the theory that three genes: the ccm homolog mutated in GB-1-004, the unclassified gene mutated in GB-1-005, and the sdhc gene mutated in MnB1 UT 3501 are responsible for the oxidation of Mn(III) to Mn(IV). Substantiating this theory is the observation that the three mutant strains that lack functioning copies of these genes possess a diminished or near absent capability for the production of both Mn(III) and Mn(IV) (See Results: Assays of Oxidized Mn by the LBB Method). It can also be postulated that the product of the unclassified disrupted gene in strain GB-1-005 is involved in, but is not essential to, the catalyzed process by which Mn(II) is oxidized to Mn(III). This idea is supported by the LBB assays which document only moderately reduced oxidized manganese levels in the GB-1-005 liquid culture compared to the wild type (see Results: Assays of Oxidized Mn by the LBB Method).

Interpretation of Results from Turbidity, Pellicle, and Exopolymer Assays The irregular turbidity vs. time curves exhibited by mutant strains GB-1-009, MnB1-UT 303, and MnB1UT 402 do not show the expected drop in turbidity after 48 hours corresponding to the onset of stationary phase (Figures 3A, 3B). The abnormal turbidity vs. time data recorded from the GB-1-009 culture in this assay can be explained by a failure of both GB-1-009 cultures to show significant growth after 48 hours and their subsequent reinoculation. An unknown degree of error is also thought to be inherent in the collection of turbidity data due to the process of flicking each tube prior to reading absorption to re-suspend cells that had settled at the bottom of the tube.

The examinations under 10x magnification of these three mutants in comparison with other mutants shows similarities in the characteristics of the mutant strains that concur with those seen in the turbidity assay. The white clumps visible under the light microscope at 10x magnification in most tubes are not found in tubes with strains MnB1-UT 303, 402, and 403. These white exopolymer clumps, most likely extracellular aggregates on the surface of the cell, probably aid in the amalgamation of individual cells to one another to form large clumps.

The abnormal turbidity vs. time curves and visual microscopic observations of the two mutant strains UT 303 and UT 402, together with an observed lack of cell material deposits at the bottom of the tubes of these same mutant strains liquid cultures has led to speculation that an inability to produce extracellular polymers (See Results: Testing for Exopolymers in MnB1 and GB-1 Mutants) is the cause for the abnormal growth curves. This research has provided strong evidence for the multiple roles of cell surface exopolymer matrix in the formation of a surface pellicle and the agglomeration and precipitation of cells during stationary phase. Another mutant strain similar to UT 303 and 402 was MnB1 UT 403, despite lacking evidence of an abnormal turbidity vs. time curve or white exopolymers under 10x magnification, mirrored many of the characteristics of strains MnB1 UT 303 and MnB1 UT 402. The consistent visible cloudiness of the UT 403 liquid cultures to the eye following the onset of stationary phase and consequential lack of cell material deposits suggest a further similarity between UT 403, UT 303, and UT 402.

In addition UT 403 was also comparable to strains UT 303 and UT 402 in the respect that none of the liquid cultures of these three strains formed a pellicle. Thus, it is reasonable to theorize that the function of the knocked out gene ccmE

is likely to be similar to that of ccmF and ccmA. It should be noted that there is a small possibility that the turbidity vs. time plot for UT 403 did not correlate to those of UT 303 and UT 402 because of potential inaccuracy of the turbidity assay due to the imprecise nature of the data collection as previously stated.

The absence of a pellicle and unusually high turbidity due to the lack of cell material deposits in strains UT 303, UT 402, and UT 403 (See Results: Turbidity vs. Time Patterns of Mutant Cultures and Testing for Exopolymers in GB-1 and MnB1 Mutants), which contain mutations for ccm F, A, and E, respectively, provides evidence for the view that these ccm gene products are responsible for the secretion of porphyrin groups that form an exopolymer matrix containing enzymes involved in the oxidation of Mn(II). These three ccm mutants are also deficient in pyoverdine production, which suggests that the cytochrome maturation proteins have a dual function that could include the transportation of a pre-pyoverdine complex during their maturation.

Analysis of Wild Type and LBB/Turbidity vs. Time Plots

The presence of bimodal LBB/Turbidity vs. time curves constructed from data collected from wild type GB-1 and many GB-1 and MnB1 mutant strain liquid cultures (See Results: Plots of LBB/Turbidity vs. Time) suggests two separate spurts of Mn oxidation, perhaps the oxidation of Mn(II) to Mn(III) early, or the oxidation of Mn(III) to Mn(IV) later. The recording of a spike in Mn(III) levels by day one is fairly surprising considering that it is well known that Mn(IV) oxides only begin precipitating on the cell surface following the onset of stationary phase.9 The lower magnitude of almost all mutant strain curves compared to the wild type reflects the greater amounts of positive LBB reaction observed in wild type liquid culture samples due to a degradation of the Mn(II) oxidizing capacity of GB-1 and MnB1 mutants. The lack of any LBB/Turbidity peaks in the plots for both the duplicate cultures of GB-1-003 (Figure 4C) and its absorbance spectrum peak at 403 nm characteristic of Fe(III)-PVD (See Results: Mn(III) Chelation with Pyoverdines in Mutant Strains) highlight a major difference from other mutant strains in the timing and/or the mechanism of Mn oxidation.

The presence of a bimodal pattern in the LBB/Turbidity vs. time curves constructed from GB-1-004, GB-1-005, and MnB1 UT 3501 liquid culture data suggests that disrupting the function of the mutated gene of these three strains does not result in a complete inability of the cell to oxidize Mn(II) into Mn(III). The mutated genes in these three strains are: a ccmF homolog, an undefined gene and sdhc. Combined with the observation that all mutants were observed to produce less Mn(III) than the wild types, this finding would lend itself to the theory that the products of these three genes produce components of a system that is directly involved in catalyzing a Mn(II) to Mn(III) oxidation reaction in GB-1 and MnB1. However, this system may still function at a degraded capacity following the disruption of these genes.

Pyoverdine Production and Binding of Mn (III) to Pyoverdines The slightly altered absorption peak centered at about 400 nm instead of 410 nm exhibited by mutant strain GB-1-

003, despite showing strong positive LBB reaction on the plate reader scan, indicates an altered fate of Mn(III) produced by cells. A peak at 400 nm has been demonstrated to be characteristic of Fe(III) bound to pyoverdine, implying that the Mn(III) was no longer present in the sample. The pyoverdine absorbance spectrum only shifts from 400 nm to 410 nm when Mn(III) is added, and this phenomenon has been attributed to the chelation of Mn(III) by pyoverdine.32 This is interesting because Mn(III) is normally competitive with iron in binding pyoverdines in wild type cells and thus the mutated ccmF homolog GB-1-003 must be responsible for the failure to detect Mn(III) bound pyoverdine, perhaps by somehow causing an increase in the competitiveness of Fe(III) against Mn(III). An absorbance spectrum of liquid culture samples of GB-1-003 grown in iron deficient media such as MSG to check for a Mn(III)-pyoverdine peak would be a useful next step in resolving this issue.

Interestingly, the absence of pyoverdines in several cultures strongly correlates to a complete lack of exopolymers. Strains MnB1-UT 303, UT402, and UT403 all lack pyoverdine production and test negative for the accumulation of solid cell material. This is likely an effect of the disrupted production of extracellular polymers that act to aggregate cells together. This correlation would be explained if the chaperone action of the ccmF, A, and E proteins was also involved in the export of pyoverdines outside of the cell. Another explanation for the relationship between lack of pyoverdines, absence of an extracellular polysaccharide matrix, knockout mutations in ccm genes, and an inability to oxidize Mn(III) to Mn(IV) is that the expression of the gene responsible for positively regulating both alginate polysaccharides and pyoverdines synthesis, prf A43, is dependent on a fully functioning cytochrome c complex. Furthermore, it has been suggested that the presence of incompletely matured cytochrome c complexes in mutant strains containing degraded ccm genes could be the cause of the lack of pyoverdine production because of the iron required for the matured cytochromes haem group.36 With intracellular Fe(III) no longer being depleted by cytochrome c maturation, the regulatory mechanisms that respond to iron starvation would not be activated and pyoverdines would not be synthesized.

The cause of the pyoverdine overproduction in mutant GB-1-004 and MnB1-UT 303 grown in the presence of Kanamycin is completely unknown but is quite possibly linked to the similar nature of the mutated gene in each strain: ccmF in MnB1 UT 303 and a ccmF homolog in GB-1-004. Subsequent studies on this matter should include tests to further clarify the results gathered in this assay would be to compare the absorbance spectrums of liquid cultures of cells from GB-1-004 and MnB1-UT 303 grown in Lept + Kan medium with cells grown in Lept medium in order to better quantify the level of pyoverdine overproduction.

Analysis of the Rresults of Cross Streaking Mutant Strains Failure to induce the production of Mn(IV) by cross streaking the mutant strains or by growing single colonies of mutant cells side by side shows an inability of the mutant strains to complement the degraded gene functions in other strains (See Results: Examination of Mutant Strains for Complementation by Cross Streaking). An inability by the proteins responsible for catalyzing the electron

transfer performed during Mn oxidation to diffuse through the plasma membrane of the cell is a possible cause of this observed failure to complement gene function. Another possibility could be that the Mn(III) intermediate itself is unable to pass through the plasma membrane and interact with cells from other mutant strains that have retained the ability to oxidize Mn(III). A situation in which the Mn(III) intermediate is embedded in the cell’s exopolymer matrix could account for such an event. This explanation would only be relevant in cross streaks involving a mutant strain deficient in Mn(III) production such as GB-1-004 and MnB1 UT 3501.

Acknowledgements I offer my deepest thanks and appreciation to my internship mentor Dorothy Parker for supplying invaluable technical aid of many forms and for providing information sources on additional procedures and techniques, to my internship supervisor and lab director Dr. Brad Tebo for his direction in the planning of my research strategy and aid in analyzing my results, as well as to Flip McCarthy, Rachael Howard, Mylene Jacobson and the members of the Haygood-Tebo lab for numerous fruitful discussions and helpful insights on my work. In addition, I thank Rebecca Verity for her early help in establishing correct laboratory procedure and providing a wealth of background information on this interesting and distinguished field of research. Finally, the members of the Saltman Quarterly Review Board deserve a great deal of thanks for their hard work and many constructive recommendations regarding the composition and format of this paper.

8. Brouwers, G-.J, de Vrind-de Jong, E.W., Corstjens, P.L.A.M., Jong, E.W.V. Involvement of genes of the two-step protein secretion pathway in the transport of the manganese-oxidizing factor across the outer membrane of Pseudomonas putida strain GB-I. Am. Mineral. 83 157382 (1998) 9. Caspi, R. Molecular Biological Studies of Manganese Oxidizing Bacteria. Published M.S. thesis, University of California San Diego (1996) 10. Caspi R. Haygood M.G., Tebo B.M., Unusual ribulose1,5-bisphosphate carboxylase/oxygenase genes from a marine manganese-oxidizing bacterium. Microbiology 142 2549-59 (1996) 11. Caspi, R., Tebo, B. M., and Haygood, M.G. c-Type cytochromes and manganese oxidation in Pseudomonas putida MnB1. Appl. Environ. Microbiol. 64 3549-3555.

(1998) 12. Cornelis P., and Matthijs, S. Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines. Environ Microbiol.

12 787-98. (2002) 13. DePalma, S. R. Manganese Oxidation by Pseudomonas putida. PhD theses, Harvard University (1993) 14. de Vrind, J., de Groot, A., Brouwers, G.J., Tommassen, J., de Vrind-de Jong, E. Identification of a novel Gsprelated pathway required for secretion of the manganeseoxidizing factor of Pseudomonas putida strain GB-1. Mol. Microbiol. 47 993-1006. (2003)

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43. Taylor, P., J. Esnouf, and J. Hobby. The functional impact of the Freehand System on tetraplegic hand function. Clinical Results. Spinal Cord, 2002. 40(11): p. 560-6.

44. Taylor, P., J. Esnouf, and J. Hobby. Pattern of use and user satisfaction of Neuro Control Freehand system. Spinal Cord, 2001. 39(3): p. 156-60.

45. Rupp, R. and H.J. Gerner. Neuroprosthetics of the upper extremity--clinical application in spinal cord injury and future perspectives. Biomed Tech (Berl), 2004. 49(4): p. 93-8.

46. Georgopoulos, A.P., A.B. Schwartz, and R.E. Kettner.

Neuronal population coding of movement direction. Science, 1986. 233(4771): p. 1416-9.

47. Hatsopoulos, N.G., et al. Information about movement direction obtained from synchronous activity of motor cortical neurons. Proc Natl Acad Sci U S A, 1998. 95(26): p. 15706-11.

48. Fu, Q.G., J.I. Suarez, and T.J. Ebner. Neuronal specification of direction and distance during reaching movements in the superior precentral premotor area and primary motor cortex of monkeys. J Neurophysiol, 1993. 70(5): p. 2097-116.

49. Thach, W.T. Correlation of neural discharge with pattern and force of muscular activity, joint position, and direction of intended next movement in motor cortex and cerebellum. J Neurophysiol, 1978. 41(3): p. 654-76.

50. Moran, D.W. and A.B. Schwartz. Motor cortical representation of speed and direction during reaching. J Neurophysiol, 1999. 82(5): p. 2676-92.

51. Chapin, J.K., et al. Real-time control of a robot arm using simultaneously recorded neurons in the motor cortex. Nat Neurosci, 1999. 2(7): p. 664-70.

52. Wessberg, J., et al. Real-time prediction of hand trajectory by ensembles of cortical neurons in primates. Nature, 2000.

408(6810): p. 361-5.

53. Taylor, D.M., S.I. Tillery, and A.B. Schwartz. Direct cortical control of 3D neuroprosthetic devices. Science, 2002. 296(5574): p. 1829-32.

54. Carmena, J.M., et al. Learning to control a brain-machine interface for reaching and grasping by primates. PLoS Biol, 2003. 1(2): p. E42.

55. Serruya, M.D., et al. Instant neural control of a movement signal. Nature, 2002. 416(6877): p. 141-2.

56. Kennedy, P.R., et al. Direct control of a computer from the human central nervous system. IEEE Trans Rehabil Eng, 2000.

8(2): p. 198-202.

57. Koeneman, B.A., et al. An ex vivo method for evaluating the biocompatibility of neural electrodes in rat brain slice cultures.

J Neurosci Methods, 2004. 137(2): p. 257-63.

Undergraduate Biological Research Publication UCSD Division of Biological Sciences

A Scent-sible Choice of Nobel Laureates

by Cara Cast / Page 31 Toll-like Receptors: An Important Part of Both the Innate and Adaptive Immune System

by Kristin CamďŹ eld / Page 32 Nerve Growth Factor Increases Hippocampal Neurogenesis of Adult Rats as seen with Doublecortin Labeling

by Danny Simpson / Page 33

Volume 2, No. 2 http://sq.ucsd.edu

Undergraduate Biological Research Publication UCSD Division of Biological Sciences

Volume 2, No. 2 http://sq.ucsd.edu

WINTER QUARTER REVIEW BOARD: Ronald Alfa Ann Cai Cara Cast Eric Chan Daniel Fang Kristine Germar Shruti Jayakumar Alex Kintzer Kyle Kuchinsky Caroline Lindsay Grace Wang

Become a member of the SQ staff !!

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A Scent-sible Choice of Nobel Laureates Cara Cast Nobel Laureate selection is a respected tradition with a long history. The Nobel Foundation, an organization founded by a directive in the last will and testament of Scandinavian Alfred Nobel began awarding prizes from its base in Stockholm, Sweden in 1901.2 To many the prizes have come to exemplify valuable work in various science and non-science fields. The 2004 award in physiology or medicine, given to Linda Buck and Richard Axel for their study of olfaction, adhered to established standards of excellence of the Nobel Foundation.

Alfred Nobel was a man of great vision. Born in Stockholm in 1833, his parents saw to his firstrate education, broad in its inclusion of the natural sciences, languages and literature. He found great successes in business, invention and science. His perhaps best-known achievement brought a patent for dynamite. By his death in 1896 Nobel had amassed great wealth. In his will he designated a large portion of his assets to forming the fund managed by a Nobel Foundation, whose mission would be to award yearly prizes to individuals who “have conferred the greatest benefit to mankind.”

Since the Foundation’s inception in 1901, Nobel Prizes have been bestowed in several categories.

Those set forth by Alfred Nobel include chemistry, physics, literature, peace and the aforementioned award for physiology or medicine. The Bank of Sweden prize in economics was created in memory of Alfred Nobel later, in 1968.

While the Nobel Foundation manages the funds, the Nobel Assembly at the Karolinska Institute in Stockholm oversees selection of the awardees for physiology or medicine. Confidential nominations are taken from a defined group of individuals including members of the Nobel Assembly, various medical faculty, and previous Nobel Laureates.

The pool of nominees, sometimes numbering in the hundreds, is reduced by careful consideration and recommendations of the 16-member Nobel Committee, the assembly’s working body whose membership changes each year. This adjudication process is kept secret and is usually completed by mid-October of each year. The committee then makes its formal recommendation to the assembly, which makes final approval of the recommendations.

Winners are notified and announced immediately.

A grand ceremony follows in early December. The 2004 prize winners split a sum of ten million in Swedish Crowns, an amount equal to nearly 1.4 million U.S. dollars.

In recent years, distinguished research has garnered

the award for physiology or medicine for a spectrum of diverse discoveries. Recent discoveries that have proven worthy of Nobel Prizes include elucidation of the details of signal transduction in the nervous system, so-called “signal sequences” contained in proteins which act as cellular zip codes, and prions, the proteins proven to elicit a previously unknown route of infection. One UCSD faculty member whose work has been recognized is Dr. Sydney Brenner.* He pioneered the use of Caenorhabditis elegans as a model organism. He and colleagues H. R. Horovitz and J. E. Sulston took the 2002 award for work identifying genes that program cell death and organ development in C. elegans.

Brenner’s contribution, according to the Nobel Committee, “laid the foundation” for the further accomplishments of Horovitz and Sulston.2 These examples illustrate that the diversity of scientific breakthroughs found worthy of this award is vast, but a unifying characteristic is the widespread implication in solving problems in basic sciences that relate to biomedical discovery.** Richard Axel and Linda Buck received the most recent Nobel Prize in physiology or medicine “for their discovery of odorant receptors and the organization of the olfactory system”.2 A keystone paper published in Cell in 1991 2detailed their discovery of the gene family containing the proteins responsible for odorant detection in the specialized olfactory cells of nasal epithelium. A mechanistic understanding of the process by which scents are perceived eluded scientists until Axel and Buck discovered a set of more than 1,000 genes encoding the specialized odorant receptor proteins and further found that these proteins operate as G-protein coupled receptors.****3 Both researchers have contributed numerous publications to the scientific literature detailing related experiments.*** The work that warranted their recognition by the Nobel Committee examined and defined the set of genes that code for the receptor proteins and worked toward understanding the signaling pathway from initial binding of an inhaled scent particle through the cellular circuitry stimulated by this binding.1,2 In an era in which neuroscience is a dynamic field, ripe for groundbreaking discovery, the elucidation of details of the function of this important sensory pathway is an obvious choice for recognition by award of the Nobel Prize. Study of what allows the elaborate nervous system of humans able to exhibit such higher order functions as the sense of smell, memory and cognition are topics on which great scientific effort is currently focused. From molecular events taking place on the nanometer scale, through intracellular signaling and transport, to interconnectedness of cellular circuitry, on up

to physiological function of tissues, organs and organ systems, the study of the physical brain and its operation takes the best efforts of some of the world’s brightest investigators. While the potential for positive impact on humanity is difficult to quantify, it is easy to see how a better understanding of one of our major sensory systems stands to lay a foundation for application to many human health interests. Furthermore, any person who values the pursuit of understanding would agree that the ability to coax nature into revealing the mechanisms of one of its most complex systems is meritorious enough for recognition in and of itself.

Notes * A complete list of UCSD faculty awarded Nobel Prizes can be found at http://academicaffairs.ucsd.edu/faculty/ awards/nobel.htm. ** To learn more about past Nobel laureates in physiology or medicine or other categories, visit http://www.

Nobelprize.org . *** A sampling of articles and reviews detailing the work of Axel and Buck’s work related to olfaction as well as other researchers follows. Both researchers have had long, productive and successful careers in the field. A comprehensive description of their work would be quite lengthy. **** G-protein coupled receptors are a large class of membrane-bound receptors that, when activated, engage a cascade of intracellular events which ultimately lead to transduction of an extracellular signal. For a succinct description, see Purves, D., Neuroscience. p. 346 and figure 12-6.

References 1. Buck, L., and Axel, R. “A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.” Cell. 65:1 (Apr 5, 1991): 175-87.

2. Nobelprize.org. The Nobel Foundation. 18 Oct 2004. http://nobelprize.org/index.html 3. “Sensing Smell.” Howard Hughes Medical Institute 1999 Annual Report. Howard Hughes Medical Institute, 2000.

Further Reading Buck, L. “The molecular architecture of odor and pheromone signaling in mammals.” Cell. 2000:100: 611618.

Mombaerts, P., Wang, F., Dulac, C., Vassar, R., Chao, S.K., Nemes, A., Mendelsohn, M., Edmondson, J., and Axel, R. “The molecular biology of olfactory perception.” Cold Spring Harb Symp Quant Biol. 1996;61:135-45.

Sullivan, S.L., Ressler, K.J., and Buck, L.B. “Spatial patterning and information coding in the olfactory system.” Curr Opin Genet Dev. 1995; Aug;5(4):516-23.

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Toll-like Receptors: An Important Part of Both the Innate and Adaptive Immune Systems Kristin Camfield Toll-like receptors play an important role in our innate immune system. They are able to bind and recognize many different bacterial and viral components. This binding leads to the initiation of signal transduction pathways that lead to the production of inflammatory cytokines and interferons. Toll-like receptors have effects on the adaptive immune system as well as the innate immune system, which can be seen when the signaling pathway for the Toll-like receptor is inactivated. Mutations in the Toll-like receptors have been linked with a wide variety of conditions ranging from prostate cancer to periodontitis.

Although often overlooked, the innate immune system plays a key role in the body’s defense against pathogens. This system focuses on a few key elements of microorganisms that are highly conserved and therefore found in most pathogens. Because the ligands that are recognized do not change, the elements of the innate immune system that identify them are encoded by the DNA. In contrast, the adaptive immune system uses DNA rearrangements and a selection process to find the best receptor to create flexibility and diversity for the immune system in order to match that of microorganisms. The Toll-like receptor is an important part of both of these systems. Homologs of this receptor have been found in many different types of organisms including plants, invertebrates, and mammals.

Its conservation over such a wide range of species shows the evolutionary advantage it imparts in protecting a host from invading pathogens.

The original Toll receptor was discovered in the well-studied fruit fly, Drosophila melanogaster. Its initial function appeared to involve the regulation of differentiation of the Drosophila embryo. The first hint of its role in the innate immune system came in 1996, when Lemaitre et al. showed that when the gene for Toll was knocked out in Drosophila, the fly was unable to effectively combat fungal infection.1 Soon, homologs of these receptors were found in many species, including humans. They were named Toll-like receptors because they have a similar structure to their complements in

Drosophila, and because they also have the function of combating infections.2 There are nine different variations of Toll-like receptors found in humans, TLR1 through TLR9, and they all bind to different components of microbes (Figure 1).

For example, both TLR2 and TLR4 bind to cell wall components of bacteria and fungi. However, while TLR2 binds to the peptidoglycan and lipoteichoic acid found in Gram positive bacteria, as well as to zymosan found in fungi, TLR4 binds to the lipopolysaccharide from Gram negative bacteria. TLR5 binds to flagellin, which is the subunit for flagella used by both Gram positive and Gram negative bacteria for movement. Some Toll-like receptors also bind to viral components. For example, TLR7 binds to single-stranded RNA, which is the means by which many viruses transfer their genetic material. The combination of all nine receptors leaves very few microbes that can remain undetected.3 Once a receptor binds its corresponding ligand, it initiates a signal cascade that causes the production of inflammatory cytokines. There are two major pathways that can be initiated by the binding of a protein to a Toll-like receptor and each of them leads to the production of different transcription factors. If a bacterial or fungal component is present, it will bind to TLR2, TLR4, and/or TLR5. This will start a signal cascade that ends in the production of the transcription factor NF-кB. This transcription factor regulates the translation of many different genes which function in antimicrobial and inflammatory responses.

However, if a virus is present, its components will bind to TLR3, TLR7, and/or TLR8. This will start a second signal cascade that leads to the production of Type I Interferons (IFN α / β), as well as other cytokines. The interferons halt viral replication4 and signal the presence of the virus to neighboring cells so they can start producing their own Toll-like receptors and interferons.5 Both of these pathways help to initiate the start of the innate immune response and allow the body to begin defending itself.

The adaptive immune system has also been shown to benefit from Toll-like receptors. Schnare et al. showed that mice which had part of their TLR signaling cascade knocked out were unable to generate a CD4 TH1 response. CD4 TH1 cells are needed to help activate CD8 effector T cells to kill microbial invaders and to help activate macrophages and other components of the innate immune system so they can begin clearing the infection. It has also been shown that a lack of the signaling cofactor MyD88 prevents dendritic cells, which are involved in activating T cells, from maturing.6 The binding of MyD88 activates the signaling pathways needed to turn on important transcription factors such as NFκB. Without the signaling cascade turned on by the Toll-like receptors, the adaptive Figure 1: Some of the well-known Toll-like receptors. They bind a immune response is severely weakened. variety of highly conserved microbial components, which leaves very few microbes that go undetected.

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Although much is already known about the functions of the Toll-like receptor, there is still much to be resolved.

Mutations in Toll-like receptors have been shown to have a role in various conditions, ranging from the potentially deadly prostate cancer7 to the relatively mild periodontitis9, a gum disease in which teeth are lost. They have even been shown to play a role in rheumatoid arthritis8, a painful condition suffered by many in the world today. Further studies of the effects of mutations in Toll-like receptors could lead to better treatments and possibly prevention of these conditions. In addition, there may be more genes controlled by or associated with these receptors that have yet to be discovered. Their discovery would be of interest to both clinicians and academics.

References 1. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.M., and Hoffmann, J.A. “The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults.” Cell. 86(6) (1996): 973-983.

2. Medzhitov, R., Preston-Hurlburt, P. and Janeway, C.A., Jr. “A human homologue of the Drosophila Toll protein signals activation of adaptive immunity”. Nature. 388 (1997): 394-397.

3. O’Neill, L.. “TLRs: Professor Mechnikov, sit on your hat”.

TRENDS in Immunology. 25.12 (2004): 687-693.

4. Alexopoulou, L., Holt, A.C., Medzhitov, R. and Flavell, R. A. “Recognition of double-stranded RNA and activation of NF-κB by Toll-like Receptor 3”. Nature. 413(2001): 732-738.

5. Meittinen, M., Sareneva, T.,Julkunen, I., and Matikainen, S. “IFNs activate toll-like receptor gene expression in viral infections”. Genes and Immunity. 2 (2001): 349-355.

6. Schnare, M., et al. “Toll-like Receptors control activation of Adaptive Immune Responses” Nature. Published online 4 September 2001. DOI: 10.1038/ni712.

7. Sun, J., Wiklund, F., Zheng, S.L., Chang, B., Balter, K., Li, L., Johansson, J.E., Li, G., Adami, H.O., Liu, W., Tolin, A., Turne,r A.R., Meyers, D.A., Isaacs, W.B., Xu, J., and Gronberg, H. “Sequence variants in Toll-like receptor gene cluster (TRL6TLR1-TLR10) and prostate cancer risk.” J Natl. Cancer Institute.

97(7) (2005): 525-532.

8. Radstake, T.T., Lieshout, T.V., Riel, P.V., van den Berg, W., and Adema, G. “Dendritic cells, Fc gamma receptors and Tolllike receptors; potential allies in the battle against rheumatoid arthritis.”Ann. Rheum. Dis. Published online 5 May 2005; doi:10.1136/ard.2004.033779.

9. Schroder, N.W., Meister, D., Wolff, V., Christan, C., Kaner, D., Haban, V., Purucker, P., Hermann, C., Moter, A., Gobel, U.B., and Schumann, R.R. “Chronic periodontal disease is associated with single-nucleotide polymorphisms of the human TLR-4 gene.”

Genes Immun. Published online 2005 May 5. doi: 10.1038/ sj.gene.6364221.

Kristin Camfield is an SQ staff member. Her bio can be found in the staff bio section on page 48.

Nerve Growth Factor Increases Hippocampal Neurogenesis of Adult Rats as seen with Doublecortin Labeling Danny Simpson, Helena Frielingsdorf, Donald P. Pizzo, and Leon J. Thal* *Department of Neurosciences, University of California, San Diego and Neurology Service, Veterans Affairs Medical Center San Diego Neurogenesis occurs in the dentate gyrus of the adult hippocampus, where neuronal precursors divide and mature into neurons in the granule cell layer (GCL). Nerve growth factor (NGF) is required for survival and maintenance of neurons in the cholinergic basal forebrain (CBF), which innervates the hippocampus via projections originating in the medial septum and diagonal band. NGF delivery has been proposed as a therapeutic strategy for treating Alzheimer’s disease because NGF protects the CBF neurons. We hypothesize that NGF may enhance survival of newly born neurons in the dentate gyrus, possibly through increased cholinergic activity in the hippocampus. Neurogenesis is thought to play a role in cognitive functions such as learning and memory, with new neurons playing a role in the consolidation of new memories. Thus a method to augment neurogenesis may enhance cognitive function. Doublecortin, a microtubule-associated protein expressed in immature neurons, was used to analyze neurogenesis in the dentate gyrus. Animals treated with NGF showed increased neurogenesis in the GCL measured two weeks after the last injection of BrdU, an indicator of adult neurogenesis. This suggests that NGF increases neurogenesis by increasing the survival of newly formed neurons in the GCL. Introduction The adult dentate gyrus of the hippocampus is a site of active neurogenesis. Precursor cells in the granular cell layer give rise to both glial cells and neurons with approximately 60-70% of these precursor cells becoming neurons. However the percentage of precursors that undergo neuronal differentiation can vary with age, species, and strain.6,7 Adult neurogenesis is different from embryonic neurogenesis. Neuronal development in an adult requires inhibition of anti-neurogenic factors and new neurons develop independently, unlike the unified fashion found in the embryo.5 Neuronal precursors are formed in the subgranular zone (SGZ) of the hippocampus, and they differentiate into neurons in the GCL. Then they migrate outward toward the molecular layer, where they extend axons into the CA3 region of the hippocampus.9

treatment with growth factors also enhances adult neurogenesis in the hippocampus.3 Nerve growth factor (NGF) is a neurotrophic factor responsible for maintenance and survival of the adult cholinergic basal forebrain (CBF) neurons.16 It is well established that NGF increases levels of acetyl choline in CBF projection areas.2 CBF neurons have a high density of high affinity NGF receptors on their surface, making them very responsive to NGF. CBF neurons residing in the medial septum and diagonal band of Broca project to the hippocampus. It is our hypothesis that NGF enhances survival of newly born neurons in the adult hippocampus, either by increasing levels of acetylcholine, or through a direct effect on the precursor cells in the dentate gyrus.

Danny Simpson is a fourth-year Animal Physiology and Neuroscience major in Revelle College. He is currently involved in studying the effects of growth factors on neurogenesis in the adult mammalian brain with Dr. Helena Frielingsdorf of Dr. Leon Thal’s lab.

Danny will attend medical school at UCSD and is looking towards a career in clinical neuroscience research as an MD/PhD.

There are several methods by which newly born neurons in the dentate gyrus can be labeled for detection. Bromo-deoxyuridine (BrdU), a thymidine analog which after systemic or intracerebroventricular (ICV) administration is incorporated into cellular DNA during S-phase of the cell cycle, is the most common marker used to assess adult neurogenesis. To determine the phenotype of a BrdU-labeled cell, co-labeling with specific cell type markers such as NeuN, a neuronal marker, is necessary. However, BrdU has some limitations. One concern regarding BrdU labeling as a marker for proliferating cells is that some labeled cells might have incorporated the BrdU during DNA repair rather than during division.

However, the amount of BrdU incorporated during DNA repair is likely to be very small compared to the amount incorporated in cell division, and would not be detected.8 Doublecortin (DCX), a

Adult neurogenesis has been implicated as an important part of hippocampal-mediated learning and memory. It is speculated that the formation of new neurons in the hippocampus plays an important role in the consolidation of new memories.15. Many of the newly formed neuronal precursors in the GCL die within the first two weeks after they are born. Net neurogenesis can be increased by either increased proliferation or increased survival of newly born neurons, or a combination of both. In aging, there is a decline in neurogenesis due to decreased proliferation in the granular cell layer7, which may in part explain reduced learning and memory capacity in aging. Alterations in neurogenesis have also been implicated in neurodegenerative disorders such as Alzheimer’s disease.4 There are several behavioral and pharmacological factors that Figure 1: DCX-immunoreactive cells in the dentate gyrus of NGF treated rat. Panel can increase neurogenesis. A shows the dentate gyrus in a section in which no primary antibody was used. There Voluntary exercise in adult was no specific staining in the absence of primary antibody. Panel B shows specific rodents increases survival staining of neurons in the granule cell layer of the dentate gyrus at low magnification of newly born neurons in (inset magnified in Panel B). Panel C shows the same cells at higher magnification in the mouse dentate gyrus.12 which the neuronal morphology can be distinguished. Individual cell bodies can be clearly seen with apical dendrites projecting outward (arrows).

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Figure 2: Effects of NGF on the number of DCX positive cells per bilateral GCL in the rat dentate gyrus. The average number of cells per GCL for the NGF and vehicle treated groups were 21027 ± 4085 and 14986 ± 4227, respectively. Thus, there was a 28.7% increase in the average number of DCX positive cells per GCL in the NGF treated group compared to the vehicle treated group. A t-test was done, and the p-value was determined to be 0.0115.

microtubule-associated phosphoprotein involved in neuronal migration and differentiation, has been used as an alternative to detect newborn neurons. In a study by Brown et al. ,1 it was shown that, in the hippocampus, DCX is expressed only in neurogenic regions, such as the GCL. Furthermore, DCX is expressed in newborn neurons from two hours to 30 days post-mitosis. Previous studies have shown that DCX labeling alone is an effective way of quantifying all newborn neurons in the dentate gyrus.1,13 Thus, quantification of DCX positive cells can be used as an alternative or complement to BrdU labeling to analyze neurogenesis in the dentate gyrus.

In a companion study, the effects of NGF on cell proliferation were examined. Rats were implanted with osmotic mini pumps infusing NGF intracerebr oventricularly (ICV) and then given BrdU injections on days two to six after pump implantation. The animals were euthanized two hours after the last BrdU injection. Cell proliferation was analyzed by quantifying the number of BrdU-positive cells.

There was no difference in BrdU-labeled cells between the groups, indicating that the NGF does not alter proliferation.

In this study, rats were implanted with osmotic mini pumps infusing NGF ICV, and then given BrdU injections on days two to six after pump implantation. The animals were then euthanized 21 days after pump implantation. This allows sufficient time for BrdU-labeled, newly born cells to differentiate into neurons.5 Thus, the goal of this study was to determine whether NGF might alter neuronal survival rather than proliferation.

Materials and Methods Animals, NGF Infusion, and BrdU Injections Fourteen adult male Fisher 344 rats were implanted with Alzet 2004 osmotic mini-pumps delivering 0.25 µl/h of NGF (5 µg/day) or vehicle, which consisted of the same solution in which NGF was dissolved. Animals received intraperitoneal 34

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injections of BrdU daily (100mg/ kg) for the first five days after pump implantation.

(Figure 1A) of primary antibody. The neuronal morphology was apparent at high magnification (Figure 1C).

Histology

Stereology

On day 14 after the last BrdU injection, the animals were lethally anesthetized and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde in 0.05 M phosphate buffer pH 7.4. The brains were dissected out and post-fixed in 4% paraformaldehyde overnight. Then the brains were transferred to 20% sucrose in phosphate buffer until equilibration. The brains were sectioned at 40 µm using a sliding microtome. The sections were kept in cryoprotectant (30% ethylene glycol: 30% glycerol: 40% H2O 0.05 M phosphate buffer pH 7.4) at -20°C until they were stained.

For each animal, DCX positive cells in every 12th section (out of 72) from the dorsal hippocampus ranging from –2.50 to –5.30 (bregma) according to the atlas of Paxinos and Watson10 were counted using the Bioquant Nova Prime system (Bioquant Image Analysis Corporation, Tennessee). Slides were blinded prior to counting. This software program controlled the movement of the motorized stage (ASI-XYZ, ASI Inc. Eugene, Oregon) attached to an Olympus (Melville, New York) BH2 microscope. The images were captured using a Sony DCX-390 video camera (Tokyo, Japan). First, the SGZ was delineated as a 2 cell body thickness on the border of the granular cell layer. Over this region a grid (60x60µm) was randomly overlaid by the software program. For each section, all DCX positive cells were counted within the dissector box (40x40µm), which was moved to every intersection within the delineated area. The program gave an estimated count of the total number of neurons in the entire SGZ. Once all the animals were counted using the stereological method described above, the slides were deblinded so that the animals could be identified as NGF or vehicle. Since there were two groups, the numbers were analyzed using a T-test. P values <0.05 were considered significant.

Every twelfth section was collected for DCX immunohistochemistry. Sections were washed three times in TBS solution and incubated in 0.6% H2O2 for 30 minutes to quench endogenous peroxidase.

The sections were then rinsed three more times in TBS for 15 minutes each. Sections were then incubated in TBS/5% normal donkey serum (NDS) (Sigma-Aldrich, Saint Louis, MO, USA)/0.2% Triton X-100 for one hour. After that, the sections were incubated in goat anti-DCX primary antibody (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in TBS/NDS/TX overnight at 4°C. The sections were then rinsed three times in TBS/2% NDS/0.2% Triton X-100 and incubated in donkey anti-goat (biotinylated) secondary antibody (1:250, Jackson Immunoresearch, West Grove, PA, USA) in TBS/DS/TX for one hour at room temperature. After secondary incubation, the sections were rinsed three times in TBS and incubated in ABC-Elite Standard Solution (Vector Elite; Vector Labs, Burlingame, CA, USA) for one hour. The sections were then rinsed three times in TBS and incubated in DAB solution (TBS/0.025% diaminobennzidine/0.01% H2O2/0.6% NiCl2) for five minutes.

Next, the sections were rinsed four times in TBS and once in 0.05 M phosphate buffer solution.

Finally the sections were mounted on gelatin-coated glass slides, dehydrated through increasing concentrations of ethanol, and cover-slipped using DPX. As part of our immunohistochemical analysis, we first confirmed antibody specificity. Specific neuronal staining was only observed in sections incubated with primary antibody (Figure 1B).

No specific staining was seen in sections incubated in the absence

Results In the hippocampus, DCX-immunoreactive cells were found only at the inner border of the GCL, which is consistent with known expression of DCX.1 As shown in Figure 2, the average number of DCX positive cells per bilateral GCL for the NGF and vehicle treated groups was 21027 ± 1444 and 14986 ± 1494 (mean ± SEM), respectively.

Thus, there was a 28.7 % increase in the number of DCX-immunoreactive cells in the bilateral GCL in the NGF group, p = 0.0115 compared with the

Figure 3: The effects of NGF on rat bodyweight. Both vehicle-treated and NGF-treated rats lost approximately 10% bodyweight within the first 5 days after pump implantation. After day 5 the bodyweight of the animals in the vehicle-treated group increased steadily until perfusion. The bodyweights of the NGF-treated animals showed little change after day 5. Ten days after pump implantation, the percentage of original bodyweight for the vehicle group was 7.5% higher than the NGF group (p <0.05). On day 11, the vehicle-treated group was 8.9% higher (p <0.005). By day 17, the vehicle-treated group was 12.4% higher (p <0.0005).

vehicle treated group.

The percentage of original body weights were also analyzed for each group. As shown in Figure 3, the bodyweights of the animals in both groups dropped by approximately 10% in the first 5 days.

After that the vehicle-treated animals showed a steady increase in bodyweight up until the time of perfusion. The NGF-treated animals showed little change in bodyweight after the first six days.

Ten days after pump implantation, the percentage of original bodyweight for the vehicle group was 7.5% higher than the NGF group (p <0.05). On day 11, the vehicle group was 8.9% higher (p <0.005).

By day 17, the vehicle group was 12.4% higher (p <0.0005).

Discussion The animals were euthanized 14 days after the last BrdU injection, allowing sufficient time for BrdUlabeled cells to mature into neurons. Since most newly born cells in the dentate gyrus die between one and two weeks after they are born3, the primary effects of NGF in this case were on cell survival. As seen in the companion study analyzing the effects of NGF on proliferation, there was no increase in BrdU-labeled cells in the NGF-treated animals when animals were euthanized immediately after the last BrdU injection. Thus, it is likely that NGF does not change the basal proliferation rate of neural precursors. Therefore, the increase in neurogenesis seen with the NGF-treated animals is most likely due to NGF acting to increase the survival of the newly formed neurons.

Since the percentage of BrdU cells co-expressing NeuN was approximately equal in the vehicle and NGF rats, NGF does not increase the ratio of new neurons, but by increasing survival of all newly born cells in the GCL, it leads to an overall increase in net neurogenesis. The increase in neurogenesis measured as DCX-immunoreactive cells is consistent with the BrdU/NeuN co-localization as a marker for neurogenesis. When the number of BrdU positive cells was analyzed there was a 27% increase in neurogenesis in the bilateral GCL of the NGF treated animals. The increase in net neurogenesis was 1.7% higher with DCX-positive

cells compared to the number of BrdU positive cells. Since, DCX is expressed in new neurons for the first four weeks post-mitosis1, any neurons that formed one week before pump implantation and up until the day of euthanization would be positive for DCX. Furthermore, the decrease in weight with NGF is also consistent with the decrease in weight seen in previous studies11 and confirms that the pumps were delivering the NGF into the lateral ventricle.

Our data indicate that NGF acting as a survival factor can cause a significant increase in the number of newly formed neurons in the bilateral GCL of the rat dentate gyrus when it is infused for three weeks. Thus, NGF may play an important role in cognitive functions such as learning and memory by increasing hippocampal neurogenesis. This NGF-dependent increase in neurogenesis may be a mechanism by which NGF improves cognitive function, and it could also serve as an attractive treatment for other neurodegenerative diseases or mood disorders associated with a decrease in neurogenesis.

References 1. Brown, J.P., Couillard-Despres, S., Cooper-Kuhn, C.M., Winkler, J., Aigner, L., and Kuhn, H.G. 2003. Transient expression of doublecortin during adult neurogenesis. J Comp Neurol. 467(1), 1-10.

2. Gnahn et al. Unpublished communication.

3. Gould, E., Beylin, A., Tanapat, P., Reeves, A., and Shors, T.J. 1999. Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci 2, 260–265.

4. Jin, K., Peel, A.L., Mao, X.O., Xie, L., Cottrell, B.A., Henshall, D.C., and Greenberg, D.A. 2003. Increased hippocampal neurogenesis in Alzheimer’s disease. PNAS. 101(1), 343-7.

5. Kempermann, G., Jessberger, S., Steiner, B., and Kronenberg, G. 2004. Milestones of neuronal development in the adult hippocampus. Trends in Neurosciences. 27(8), 447-52.

6. Kempermann, G., Kuhn, H.G., and Gage, F. 1997.

Genetic influence on neurogenesis in the dentate gyrus of

adult mice. Proc Natl Acad Sci U S A. 94(19),10409-14.

7. Kuhn, H.G., Dickinson-Anson, H., and Gage, F. 1996.

Neurogenesis in the dentate gyrus of the adult rat: agerelated decrease of the neuronal progenitor proliferation.

J Neuroscience. 16(6), 2027-2033.

8. Kuhn, C.M., and Kuhn, H.G. 2002. Is it all DNA repair?

Methodological considerations for detecting neurogenesis in the adult brain. Brain Res Dev Brain Res. 134(1-2),1321.

9. Markakis, E.A., and Gage, F.H. 1999. Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol 406, 449–460.

10. Paxinos and Watson, 1998.

11.Pizzo, D.P., and Thal, L.J. 2004. Intraparenchymal nerve growth factor improves behavioral deficits while minimizing the adverse effects of intracerebroventricular delivery. Neuroscience. 124(4), 743-55.

12.Praag, H.V., Kempermann, G., and Gage, F.H. 1999.

Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci. 2(3), 26670.

13. Rao, M.S., and Shetty, A.K. 2004. Efficacy of doublecortin as a marker to analyze the absolute number and dendritic growth of newly generated neurons in the adult dentate gyrus. Eur J Neurosci. 19(2), 234-46.

14. Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee, J., Duman, R., Arancio, O., Belzung, C., and Hen, R. 2003. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science. 301(5634), 805-9.

15. Shors, T.J., Miesegaes, G., Beylin, A., Zhao, M., Rydel, T., and Gould, E. 2001. Neurogenesis is involved in the formation of trace memories. Nature. 410, 372-376.

16. Winkler, J., Fisher, L.J., Thal, L.J., and Gage, F.H. 1998. Cholinergic strategies for Alzheimer’s disease. J Mol Med. 76, 555-567.

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Want to do some research of your own?

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BISP 196 or BISP 199 Benefits Gain hands-on lab experience Study with outstanding faculty Meet new people who share your interests Explore a new field of study or enhance your current one Get a head start in your career Earn elective credit towards your major (may be used one time only, as an upper division elective) Requirements BISP 196 You must have an overall and major GPA of 3.7 or better, and must have senior standing (final three quarters at UCSD).

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Undergraduate Biological Research Publication UCSD Division of Biological Sciences

Elevated Intraocular Pressure and Reduced Central Corneal Thickness as Risk Factors for Glaucoma

by Pei-Chen (Jennifer) Hsieh / Page 39 Intimate Association of Vibrio splendidus with the Purple Sea Urchin Stronglyocentrotus purpuratus

by Shirin Doroudgar / Page 41 Chemical InďŹ&#x201A;uences on Bee Hunger

by Xu (Lloyd) He / Page 45

Volume 2, No. 3 http://sq.ucsd.edu

Undergraduate Biological Research Publication UCSD Division of Biological Sciences

Volume 2, No. 3 http://sq.ucsd.edu

SPRING QUARTER REVIEW BOARD: Ronald Alfa

Become a member of SQ’s peerreview board!!

Ann Cai Kristin Camfield Cara Cast Eric Chan Reeti Desai Heather Eshleman Daniel Fang Avanti Ghanekar Nicole Gomez Shruti Jayakumar Peter Kim Kyle Kuchinsky Caroline Lindsay Lauren Ashley Miller Sara Paul Josh Tan Grace Wang

As a peer-reviewed journal, SQ needs undergraduates willing to participate as members of our review board.

Members of the board will have the opportunity to read and critique research articles submitted for publication by their peers--other undergraduates in biology at UCSD. Reviewers will be invited to participate in a learning workshop with our faculty advisory board early in fall quarter. Involvement will provide students with valuable experience reading and analyzing scientific literature.

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Elevated Intraocular Pressure and Reduced Central Corneal Thickness as Risk Factors for Glaucoma Pei-Chen Hsieh Introduction Glaucoma is an eye disease associated with gradual vision loss. An increase of pressure inside the eye is a major risk factor for developing the disease.

This eye pressure is known as intraocular pressure (IOP). When IOP is too high, the pressure on the optic nerve may damage the nerve and slowly cause vision loss and a reduction in the visual field. Many recent studies have used central corneal thickness (CCT) measurement as an important variable in detecting glaucoma. Patients who have undergone refractive or corneal surgeries have a reduced corneal thickness and intraocular pressure. The underestimated IOP can cause significant difficulty for the ophthalmologist to accurately assess any signs of glaucoma. Therefore, the incorporation of CCT-adjusted IOP is a useful measurement to diagnose the onset of glaucoma.3,6,9 Determining Glaucomatous Progression I. Intraocular Pressure Glaucoma is a common disease of the aging eye and a leading cause of blindness in the world.

This eye condition affects approximately 3 million Americans, of whom 120,000 have become blind.13 The cause of glaucoma is not well understood at the sub-cellular level. However, many studies have confirmed the significant effect of intraocular pressure on glaucoma. In general, the pressure within the eye becomes elevated because the anterior chamber of the eye cannot provide sufficient drainage of fluid into the retina as shown in Figure 1.1 The pressure within the vitreous chamber rises and damages the blood vessels of the optic nerve.

As a result, the nerve cell axons cannot carry visual information from the retina to the brain.4 In open-angle glaucoma, vision loss usually starts in the periphery and moves toward the central vision as shown in Figure 2.8 A person’s visual field will get smaller and smaller if the condition is not

Figure 1: Retinal photography of a glaucoma patient taken by the Ophthalmoscope (Wooldridge, 2004).

treated early.5,6 Because most people with glaucoma do not experience noticeable symptoms, it is a great challenge for ophthalmologists to fight this dangerous disease, which is sometimes called the “sneak thief of sight.”7 Steadily, ophthalmologists are developing new methods for early detection of glaucoma and of glaucoma progression.

In the Ocular Hypertension Treatment Study,5,6 an elevated intraocular pressure (IOP) was determined to be a significant risk factor for progression of visual field loss and nerve damage caused by glaucoma. For every 1 mm Hg increase in IOP, the risk of glaucomatous progression increased by approximately 13%. In addition, every 1 mm Hg reduction in IOP would reduce the patient’s risk of developing glaucoma by approximately 10%.12 More recently, other studies have confirmed that elevated IOP as a fundamental risk factor in glaucoma, even for patients with normal intraocular pressure.12 II. Central Corneal Thickness Central corneal thickness (CCT) provides additional information about the likelihood of developing glaucoma. From previous studies, it was determined that corneal thickness served as a predictor for people who would develop visual field loss.9 Patients with thicker than average corneas were prone to have higher IOP. This resulted in an overestimation of the true IOP for these patients.

Similarly, patients with thinner than average corneas were prone to have lower IOP, which resulted in an underestimation of the true IOP. The thickness of the cornea can be used as a predictor variable for the development of visual field loss among patients.

The CCT measurement is crucial in clinical evaluation of patients with high IOP, known as ocular hypertension. For patients who have already lost some visual function, corneal thickness serves as an independent risk factor for further glaucomatous progression.8 Unlike applanation tonometry, which is an instrument used to measure the IOP, CCT examines the potential risk factors for patients with thicker and thinner corneas.12 It has been shown from previous studies that, when the applanation tonometry was used to measure IOP, progression of the patients with thicker corneas may be overestimated and patients with thinner corneas may be underestimated. Thus, some patients classified as having ocular hypertension may be at a lower risk for glaucomatous development than originally predicted.10 Therefore, patients with thick corneas may be at a lower risk for the IOPrelated glaucomatous defects while patients with thinner corneas may be at considerably greater risk for development of IOP-related damages than would be predicted by this measurement.

Elevated intraocular pressure and reduced central corneal thickness are important risk factors for

glaucomatous onset and progression. In Table 1 the risk level of developing glaucoma caused by the different values of IOP and CCT was examined. Patients with IOP >25.75 mm Hg and CCT <555µm are in the high risk group. Patients with IOP >23.75 to ≤ 25.75 mmHg and CCT >555 to <558 µm are in the moderate risk group. Lastly, patients with IOP ≤ 23.75 and CCT >588µm are in the low-risk group. Based on the values given in the table, the ophthalmologist can easily evaluate his or her patient’s level of risk to develop glaucoma as a function of IOP and CCT.5,6 5,6

Refractive Surgery and Its Effect on Glaucoma Evaluation Today, many people consider surgery to correct their vision. Refractory surgery is composed of a variety of different surgical procedures, such as using knives, lasers, or other techniques to alter the curvature or the thickness of the cornea.11 These procedures restore an individual’s perceptual ability and reduce dependency on eyeglasses and contact lenses. These surgical procedures are, however, irreversible and alter cornea shape and thickness permanently. Although refractive surgeries such as LASIK may restore 20/20 vision, some vision and ocular complications might result from the damaged cornea. For example, the thinning of the cornea caused by laser-based refractive surgery would lower IOP measurement.2 After surgery the cornea loses its rigidity, and thus, an artificially lowered IOP is commonly observed in patients who have undergone refractive surgery.

The primary eye care providers need to be careful when they are examining patients who have had refractory surgery done on their eyes. The applanation tonometry readings are typically normal for these patients, but their actual IOP readings could be in the mid 20 mm Hg. If the practitioner fails to notice the flap resulting from the surgery in these patients during the eye exam, over time, these patients will have a higher chance of getting aggravated visual field defects and develop severe glaucoma.11 As a result, patients with histories of refractory surgery need more intensive examination and treatment to reduce IOP and prevent glaucoma development.

When establishing the standard IOP for glaucoma treatment for patients who have developed glaucoma already, the clinician may often overlook the error related to corneal thickness by calculating the desired reduction level of IOP based on the rate at which glaucomatous damage had occurred.11 Therefore, LASIK patients with thinner than normal corneas may be provided a false sense of assurance and the clinician may stop further treatment since the IOP reading in these patients has become relatively low. Although it is crucial for primary eye care practitioners to keep in mind corneal thickness when evaluating glaucoma patients, it is difficult to determine the amount of adjustment needed to Volume 2 Issue 3

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risk factors associated with this condition.

References 1. Cole, R.M. Glaucoma. Review of Optometry, 2004; 142: 5.

2. Fanelli, L.J. (2001, June 15). IOP Reads Lower After Lasik. Review of Optometry. Retrieved on May 1, 2005, from http://www.revoptom. c o m / b o d y / a r t i c l e s / 6 _ 2 0 0 1 / r o 0 6 0 1 g g r. h t m .

3. Gaasterland, D.E., et al. Advanced Glaucoma Intervention Study. Ophthalmology, 2004; 111: 651-64.

4. Goldstein, E. B. Sensation & Perception. Belmont: Brooks/Cole Publishing Company, 1999.

5. Gordon, M.O., et al. The Ocular Hypertension Treatment Study. American Medical Association, 2002; 122: 813-820.

6. Gordon, M.O., et al. The Ocular Hypertension Treatment Study: Baseline factors that predict the onset of primary openangle glaucoma. Arch Ophthalmol, 2002; 120: 714-720.

7. Heijl, A., et al. Essential Perimetry. Dublin: Carl Zeiss Meditec Inc., 2002.

Figure 2: The progression of visual field loss in a glaucomatous patient.4

compensate for corneal thickness when measuring IOP. Alternative Diagnostic Tools In addition to using adjusted IOP based on CCT measurement, some ophthalmologists endorse using visual field tests, nerve fiber analysis, and optic nerve tomography as diagnostic tools for glaucoma.11 The pressure inside the eye changes throughout the day so it might not be reliable to evaluate the development of glaucoma based on a fickle IOP value. As the population of postrefractive surgery patients increases, it is pertinent to place less emphasis on the IOP reading as the primary diagnostic indicator of glaucoma. It has not been proven whether or not it is safe for glaucomatous patients to undergo LASIK surgery. However, the practitioner must be aware of the distorted diagnostic test results caused by the surgery.

Other Risk Factors Several independent variables were analyzed as potential risk factors for development of glaucomatous damage. As one ages, the chance of developing glaucoma increases. People with different racial backgrounds have disproportionate likelihood for glaucoma development. Many population-based studies have shown that people of African descent have a higher risk of developing glaucoma than Caucasians.13 People of African descent often have thinner corneas than Caucasians; they also have an increased risk for glaucoma. Family history is also a common risk factor for glaucoma.

However, in the Ocular Hypertension Treatment

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Study (OHTS), about 42% of the participants reported having a relative with glaucoma, but only 8.5% of these participants developed glaucoma.

The percentage of participants who did not report any family history of glaucoma but developed glaucoma was very similar: 7.3 %. Based on the OHTS, these two values were statistically the same. The Early Manifest Glaucoma Trial (EMGT) study also confirmed the insignificant effect of family history on the development of glaucoma.

13 Nonetheless, if the patient feels that his or her family history puts him at higher risk of developing glaucoma, it is critical for the clinician to include family history in the patient’s management as the possible risk factor.13

8. Medeiros, F.A., et al. Corneal Thickness as a Risk Factor for Visual Field Loss in Patients with Preperimetric Glaucomatous Optic Neuropathy.

Am J Ophthalmol, 2003; 136: 805-813.

9. Medeiros, F.A., Sample, P.A., & Weinreb, R.N. Corneal Thickness Measurements and Visual Function Abnormalities in Ocular Hypertensive Patients. Am J Ophthalmol, 2003; 135: 131-7.

10. Sample, P.A., et al. Using Machines Learning Classifiers to Identify Glaucomatous Change Earlier in Standard Visual Fields. Invest Ophthalmol Vis Sci, 2002; 43: 2660-2665.

11. Samuelson, W. T. Refractive Surgery in Glaucoma.

Current Opinion in Ophthalmology, 2004; 15: 112-118.

12. Shih, C.Y., et al. Clinical Significance of Central Corneal Thickness in the Management of Glaucoma.

Archives of Ophthalmology, 2004; 122: 1270-1275.

13. Wooldridge, P. R. Risk Factors for Glaucoma: The Usual and Unusual Suspects. Review of Optometry, 2004; 141: 9.

Conclusion Glaucoma is an eye disease characterized by abnormally high IOP, damaged optic disk, and partial to total vision loss. However, the causes of glaucoma at the sub-cellular level are not well understood. Many studies have confirmed that IOP and CCT measurements can be elusive factors for correctly identifying true risk of glaucoma progression. Patients with thinner corneas as a result of refractive surgery are more likely to develop early glaucomatous functional damage and that adjusted CCT measurements should be taken into account when assessing risk for the development of glaucoma. In addition to IOP and CCT, age and race can also serve as risk factors for the progression of glaucomatous damage. Glaucoma ranks as one of the leading causes of blindness in the world. In order to prevent and reduce the damage caused by glaucoma, it is important to accurately evaluate the

Pei-chen (Jennifer) Hsieh is a graduating senior from Revelle College.

She is a Biology major and Psychology minor.

She is the vice-president of InSight, pre-optometry club. Jennifer currently works at LensCrafters as a Frame Stylist and Patient Pre-tester. She has won the American Heart Association Undergraduate Summer Fellowship to perform research with Dr. Robert D. Langer from the UCSD School of Medicine.

Jennifer’s research article on the Effect of Calcium Intake and Exercise on Systolic Blood Pressure was published in Volume 1, Issue 1 of Saltman Quarterly. Jennifer’s most rewarding experience during her college career was working on glaucoma with Dr. Pamela A. Sample from UCSD Shiley Eye Center. After graduating from UCSD, Jennifer will attend University of California Berkeley’s School of Optometry.

Intimate Association of Vibrio splendidus with the Purple Sea Urchin Stronglyocentrotus purpuratus Shirin Doroudgar Isolates from the purple sea urchin Stronglyocentrotus purpuratus were identified through 16S rRNA sequencing. Seven of the 8 isolates identified were strains of Vibrio splendidus; one was a strain of Pseudoalteromonas haloplanktis. Because fertilization in sea urchins occurs outside the body, the effects of the bacterial species associated with sea urchins on the gametes were investigated. Isolated strains were used to inoculate sea urchin eggs and sperm in an infectivity assay performed using gametes. It was observed that presence of the isolates facilitates decay of gametes, but differences between interactions of gametes with each strain were not assayed. Because strains of V. splendidus were isolated from eggs, intestines, and cadavers of sea urchins, an intimate association of these Vibrios with S. purpuratus was established.

Introduction Belonging to the phylum Echinodermata, class Echinoidea, sea urchins (genus Stronglyocentrotus) are only found in marine environments. More than 6,000 species exist today. Sea urchins are used in saltwater aquariums to indicate water quality because when water quality is poor, urchins are the first organisms to show signs. In poor-quality water, their movement stops and their spines lay down.

Bacteria are among the predators of sea urchins.

Populations of sea urchins are most susceptible in the years of bad weather and poor food supply. This project is focused on Stronglyocentrotus purpuratus, the purple sea urchin. Stronglyocentrotus species experience attacks from bacteria of the genus Rhodospirillum. These bacteria are purple and cause necrotic spots to appear on the outside of the urchin. At the spots, the spines and tube feet fall off and the infection eats its way inside eventually killing the sea urchin. However, the infected urchin can live for months.

As stated in a study done at Hiroshima University, “infectious diseases have occurred in sea urchin hatcheries, and a filamentous bacterium resembling Flexibacter maritimus was isolated from juveniles of Red sea urchin (Pseudocentrotus depressus) and Ezo sea urchin (Strongylocentrotus intermedius).

As investigated by Hamaguchi et al.,2 Flexibacter sp., causing spotting disease in sea urchins, and fluctuates seasonally, only being detected when sea water temperature reaches 20oC or higher, as the bacteria enters into a viable but not culturable (VBNC) state during low-temperature seasons. At low water temperatures, (11-13oC), Vibrio sp. was isolated as a pathogen in Ezo sea urchin.8 Furthermore, Stronglyocentrotus species experience attacks from bacteria from the genus Rhodospirillum.4 Rhodospirillum are phototrophic bacteria that belong to the α-1 proteobacteria. These spiral-shaped, purple non-sulfur bacteria have great phenotypic diversity.

This study sought to discover microbial associations specifically with the purple sea urchin Stronglyocentrotus purpuratus. It was believed that healthy sea urchins would have different microbes associated with them than sick sea urchins. Since

fertilization happens outside of the body in sea urchins, it was believed that the bacteria associated with the sea urchins would affect the gametes.

Furthermore, isolated strains were believed to have different effects on eggs than on sperm.

Materials and Methods Isolation The sea urchins used in this experiment were obtained from the aquarium at Scripps Institution of Oceanography (SIO) from mid-May to the beginning of June. All were kept in the same tank at SIO. Two aquariums were set up at UCSD: one for the apparently healthy urchins (those that were intact and maintained their spines) and the other for those urchins that either had lost spines or had spots on the surfaces and were thus labeled ‘sick’. Three urchins were placed in the ‘healthy’ aquarium and two were placed in the ‘sick’ aquarium. An apparently healthy urchin was dissected and swabbed with sterile cotton swabs. The samples on the swabs were then plated on TCBS plates, which select for Vibrios.

The two sick urchins died after two days and were swabbed and plated on TCBS and marine (2216) plates. Before its death, one of the sick urchins spawned naturally. Its sperm were collected in sea water and saved at 4˚C. Attempts were made to spawn the healthy sea urchins. Only one was successfully spawned. Its eggs were collected in sea water and also saved at 4˚C. All three healthy urchins died within hours of being induced to spawn.

The first set of spawning attempts was not as sterile and developed as what was later used to obtain gametes to inoculate at the end of the experiment.

The gametes obtained from the first set of urchins were eventually fouled by bacterial growth. The perfected method of spawning was as follows: A 0.5M potassium chloride solution was prepared (about 4 ml per urchin). The urchin to be spawned was first washed with sterile sea water. It was then placed on a paper towel with the mouth side up.

With a syringe with a fine needle, about 1.5 ml KCl was injected into the soft tissue around the mouth.

Shirin Doroudgar is a graduating senior, majoring in Molecular Biology, at Muir College. Previously, she has worked on purification and crystalization of Thermus thermophilus proteins at TSRI. At SIO, she has worked on constructing a cDNA library of the barophilic Photobacterium profundum. Following graduation, she will work with Dr. Jean Y. J. Wang at UCSD’s Cancer Center, investigating c-Abl tyrosine kinase, its adaptors, and the inhibitors of its downstream effector pathways. She plans to attend graduate school in Fall 2006.

The needle was inserted almost horizontally in the part where the soft tissue joins the spines. Another similar injection was made across from the first injection site. The urchin was then placed with its mouth on the bottom and watched. If the urchin is a male, the sperm come out of the openings on the top. The sperm were collected ‘dry’ with a Pasteur pipet and placed in a micro-centrifuge tube at 4˚C. If the urchin is female, orange-colored eggs come out of the openings at the top. The urchin must then be inverted into a beaker of sterile sea water and where the eggs are dropped. The eggs are kept at 4˚C. The first set of gametes obtained as well as the urchins placed in the two aquaria were swabbed and the samples were plated on TCBS and Marine (2216) plates. These isolates grew at room temperature for two days. Twenty-three colonies were then selected from the eggs, sperm, intestines, cadaver, and aquaria waters. The selected colonies were streaked on Marine plates and left to grow for two days at room temperature. A colony from each of the 23 plates was then transferred to 5 ml of Marine broth. This was set to grow for two days at room temperature.

Infectivity At this point, the gametes collected from the first set of urchins brought to UCSD had degraded and could not be used for infectivity tests. Urchins were then spawned at SIO and another set of gametes was obtained. The ratio of males to females was very high (10 males: 1 female), and many spawned before eggs were obtained.

After obtaining fresh eggs and sperm, gametes were inoculated with 8 selected isolates out of the 23 growing in Marine broth. 200 μl of ‘dry’ sperm were mixed with 50 ml of sterile sea water. The eggs were in a sterile sea water solution. As much sea water as possible was decanted and about 200 μl of eggs were re-suspended in 50 ml of sterile sea water.

One ml of each gamete type was inoculated with 10 μl of isolate. There were 8 inoculated samples and one un-inoculated control for each gamete.

The samples were kept at room temperature for 15 hours, at which point observations were made and Volume 2 Issue 3

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Figure 1: 16S rRNA sequence identification of unknown isolates including color of each unknown colony, source of isolate, medium used to enrich for each isolate, and identity of the isolate.

microscopic images recorded.

16S rRNA Sequencing The 8 isolates were also prepared for 16S rRNA sequencing. Universal bacterial primers were used for gene amplification through a two-hour PCR reaction. PCR products were run on 0.08% agarose gel at 50V for one hour. All isolates were successfully amplified. The PCR products were then purified using aPCR clean-up kit. The purified PCR products were then submitted for 16S rRNA sequencing. Obtained sequences were searched for matches on BLAST and the Ribosomal Database Project.

Results Sequencing Seven of the eight isolates of S. purpuratus from the eggs, intestines, and cadavers were identified as strains of Vibrio splendidus, while one strain from the eggs was identified as Pseudoalteromonas haloplanktis subsp. Tetraodonis. Three of the four unknowns obtained from dead sea urchins were V. splendidus (str. 2-100 C9).

Strains 1, 2, and 3 (Figure 1) were isolated from S. purpuratus eggs. With 0.98 accuracy on the RDP sequence match search, strain 1 was identified as Vibrio splendidus (str. 1-140 C33). Several strains of V. splendidus came up for this strain’s search on BLAST with E values of 0 with scores between 1770 and 1673. However, none of these was str. 1140 C33. The BLAST result with the highest score (1871) for this search and an E value equal to zero was Vibrio sp. Sk1. Strain 2 was identified as Vibrio splendidus (str. 2-100 C9) with 0.95 accuracy. The V. splendidus strains on the BLAST search appear between 1939 and 1828 bits with E equal to 0. The top result for strain 2 on the BLAST search, with E value equal to 0 and a score of 2062, is Vibrio sp.

ED4. Strain 3 was identified as Pseudoalteromonas 42

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haloplanktis subsp. Tetraodonis (str. MED8) with 0.98 accuracy on the RDP search and at a score of 1826 with an E value of 0 on the BLAST search.

The top result on the BLAST for strain 3 was Pseudoalteromonas atlantica at 1897.

Strains 4 and 5 were isolated from dead urchins.

Strain 4 was identified as V. splendidus (str. 2-100 C9) with 0.95 on RDP. On the BLAST search for this strain, the top result was Vibrio sp. QY101 with a score of 1953. The strains of V. splendidus showed up between 1840 and 1744 with E values of 0 on the BLAST search. Strain 5 was identified as V. splendidus (str. 1-140 C33) on the RDP search with 0.96 accuracy. The highest match for strain 5 on the BLAST was Vibrio sp. Sk1 at 1867 bits.

Strains of V. splendidus showed up between 1752 and 1673 bits with E values of 0 on the BLAST search.

Strains 6 and 7 were isolated from cadavers of sea urchins that were believed to be sick because of spots on their surfaces. Strain 6 was identified as V. splendidus (str. 2100 C9) with a 0.96 accuracy on the RDP search. The top match for this strain on BLAST was Vibrio sp. ED4 at 1972, and strains of V. splendidus appeared between 1850 and 1786 with E values of 0. Strain 7 was identified as V. splendidus (str. 2100 C9) with 0.96 on the RDP search. As with strain 6, the top BLAST match was Vibrio sp. ED4. Strains of V. splendidus appeared with E values equal to 0 between 1798 and 1748 with E values equal to 0.

Infectivity All 9 culture tubes containing eggs inoculated with the isolates were compared to a control with no inoculate added. The same was done for the sperm. Fifteen hours after inoculation, the uninoculated eggs in the contro, were observed to be in the process of lysing. The control eggs were in different stages of lysing, with almost perfectly round eggs to eggs with very deformed shapes observed. The 8 other infection experiments on eggs were compared with this control. In all the cases, lysing eggs were observed in all inoculated cultures (Figures 2 and 4). However, the numbers of near perfect (round) and intact eggs were very small. Most eggs observed were very advanced in the process of lysing.

The deformations observed in the eggs of the control were the results of osmosis and looked like the break down of the cell membrane, leading to the eggs losing their shape and becoming deformed (Figure 3). In the inoculated samples, microbes were observed crowding around the eggs (Figure

Strain 8 was obtained from the intestine of S. purpuratus. It was identified as V. splendidus Vibrio ANG.218 str. ANG. strain 218) on the RDP search (0.85).

This was also the top match on BLAST Figure 2: Egg lysis facilitated by V. splendidus (str. 1-140 C33) in with a score of 1945 bits and an E value sample 5 at T=15 hours at room temperature, 1000x magnification. of 0.

Shown is part of the jagged outline of the lysing egg. Bacteria are clustered around the egg and are breaking off pieces of the egg.

P. haloplanktis with the gametes compared with that of V. splendidus with the gametes.

Some V. splendidus are luminous bacteria. V. splendidus are globally distributed in marine environments and can colonize marine animals as symbionts and parasites. The numbers of these Vibrios are low in water; only in association with animals can the numbers be very high.

almost all cases observed, the theory of tails simply falling off as sperm begin to die may not hold. It is not clear whether in inoculated sperm, the tail seemed to have broken off with a piece of the head, or whether the head had broken into pieces after losing the tail. Nevertheless, many sperm observed were damaged and destroyed.

Strains of Flexibacter and Rhodospirillum, known pathogens of sea urchins, were not obtained as any of the isolates in this study.

V. splendidus was found associated with eggs, isolated from cadavers, as well as found in the intestines of S. purpuratus. V. splendidus strains do not appear especially pathogenic to S. purpuratus. However, the infectivity assay done in this study was on gametes and cannot be used to make inference about infectivities of isolates, since it is known that colonization by microbes in general facilitates gamete fouling.3 Future studies testing Koch’s postulates must be done in order to determine pathogenic capabilities of these isolates.

Members of the marine bacterial genus Pseudoalteromonas have been found in association with living surfaces and are thought Figure 3: Two stages of the continuum of the breakdown of eggs observed to produce bioactive compounds in sample 7, inoculated with V. splendidus (str. 2-100 C9). 400x magnificathat work against the settlement of tion. spores of algae, invertebrate larvae, fungi, and bacteria. The growth 2). The microbes were very motile, competing of fungi and bacteria are the initial fouling agents with each other to get closer to the eggs. The on marine organisms.3 Because eggs contain microbes closest to the eggs were the most active many nutrients essential for living things, they are Because isolates obtained from dead urchins were and energetic. The closest ones to the eggs would susceptible to many infections. It is very important all strains of V. splendidus, it was thought that make contact and “feed” on the eggs. Furthermore, for reproduction that eggs remain intact and perhaps these strains of Vibrios are opportunistic it was observed that some microbes were wedging healthy. Hence, the symbiotic relationship between pathogens of S. purpuratus. As was observed, their way into the eggs and slowly, but persistently, P. haloplanktis and eggs is very critical. However, microbial infections facilitate the lysis and breakbreaking off pieces of the eggs. This led to the eggs because in the infection assay done on the eggs in down of gametes. Hence, microbial infections being broken into pieces of different size. Some this study no major difference was observed between are limiting factors in sea urchin reproduction. V. microbes were observed attaching to the broken-off the interactions of P. haloplanktis and the eggs and splendidus, which is very closely associated with pieces of the eggs and, as the pieces floated away, V. splendidus and the eggs (both bacteria facilitated S. purpuratus, may play an especially important seemingly using the pieces as mobile sources of the lysis of eggs), it would be very interesting to see role as a limiting factor in gamete survival and food (Figure 2). if there indeed is a difference in the interactions. If sea urchin reproduction. This may become a P. haloplanktis facilitates egg lysis just as much as problematic issue in the case of sea urchin nurseries The control sperm observed at 15 hours at room V. splendidus does, it would be very interesting to where the urchins are kept confined and bred for temperature were still alive and moving (Figure see why this happens because P. haloplanktis should 5). The sperm were intact; tails and heads had commercial purposes. be protecting the eggs through its own production not separated. Furthermore, the number of sperm of bioactive compounds. Certain environmental One possible control for this experiment would observed under 1000X magnification did not seem conditions, such as changes in temperature, could have been to sequence isolates obtained from the to have decreased from what was observed at T=0 be causes. waters of the aquariums in which the sea urchins (15 hours before, when the inoculations were made).

Almost no live sperm were observed in any of the The effects of the isolates on eggs were different than lived both at SIO and at UCSD. It would be inoculated samples. Most of the observed sperm the effects on sperm. The eggs, being larger, and interesting to see if the very close association of the in inoculated samples were not intact; the tails had containing more nutrients, were directly “attacked” Vibrios with V. splendidus is because these strains separated from the heads (Figure 6). The heads by the microbes, and damage could be observed are so prevalent and maybe even selected for in the were no longer motile. The intact sperm were not as microbes attached and wedged themselves into confined tanks and aquariums where urchins were very motile. Only very rarely would a live sperm the eggs. Although be observed in any of the inoculated samples. The the sperm count had numbers of sperm had greatly decreased in these dropped 15 hours samples; whereas instead of hundreds of sperm after inoculation, observed in a viewing field in the control at T=15 no direct interaction hours, there were only a few (5-10) sperm observed could be observed at T=15 hours in inoculated samples. between sperm and microbes.

It was Discussion not clear exactly what had caused Seven of the eight unknowns identified by 16S the detachment of rRNA sequencing were strains of Vibrio splendidus. tails in these sperm.

From this, the observation was made that V. It might be that as splendidus can be very closely associated with S. sperm began to die, purpuratus. Three of the four isolates obtained from the tails dropped off sea urchin cadavers were identified as V. splendidus as the normal cellular (str. 2-100 C9). However, further studies must be functions of the cells completed in order to provide additional evidence stopped. However, for this association. All of the isolates facilitated because the breakthe break down of the gametes. The observations off of the tails was Figure 4: Egg sample 3 inoculated with P. haloplanktis subsp. Tetraodonis (str. MED8) at made were not specific enough for a conclusion to not a ‘clean break’ in 1000x magnification. Egg contents are in lower half of picture. Observed part of egg does not have its normal smooth outline. be made about the difference in the association of Volume 2 Issue 3

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This is an important factor that could have striking effects on the activity and infectivity of microbes as well as on susceptibility of sea urchins to infection.

The combination of the sea urchins being environmentally stressed at temperatures outside of their biological range and most microbes growing faster at temperatures close to room temperature may have had an effect on the intimate association of the Vibrio strains observed with S. purpuratus.

Labs’ faculty, Lorlina Almazan’s wonderful lab management, and SIO for supplying the sea urchins.

If in the future it can be demonstrated that strains of V. splendidus are indeed not pathogens of healthy sea urchins, further studies could be done to examine the capabilities of these strains as opportunistic pathogens that infect the urchins during periods of stress such as during changes in temperature or poor water quality. It would be intriguing to examine at what point in the development of the purple sea urchin association with strains of V. splendidus begins. If the relationship begins very early on, the microbes could be living in symbiosis with the urchins. The urchins’ use for these bacteria could then be studied. If the Vibrios and urchins live symbiotically, could changes in the environment, such as fluctuations in temperature, have an effect on the capabilities of the Vibrios as pathogens?

Also, it would be interesting to examine whether the same results can be obtained if urchins are ‘fresh’ from the ocean.

4. Muroga, K. 2001. Viral and bacterial diseases of marine fish and shellfish in Japanese hatcheris. Aquaculture 202: 23-44.

Figure 5: Control sperm sample after 15 hours at room temperature, 1000x magnification. Sperm are alive and motile.

being kept. Furthermore, the temperature of the aquariums at UCSD, to which the sea urchins were brought from SIO, was warmer than the temperature in which they lived at SIO. For future studies, the temperatures should be kept the same throughout the experiment, and preferably kept close to the actual temperature in urchin’s natural habitat.

Acknowledgements Figure 6: Sperm sample 2 inoculated with V. Splendidus (str. 2-100 C9) at 1000x magnification. Very few sperm are intact.

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The author would like to thank Dr. Doug Bartlett for his guidance and advice, UCSD’s Division of Biological Sciences Undergraduate Teaching

References 1. Bartlett, D. BIMM 127 Course Reader, Spring 2004, 141-143.

2. Hamaguchi, M., Kawahara, I., and Usuki, H. 1993.

Mass mortality of Pseudocentrotus depressus caused by a bacterial infection in summer. Suisan Zoshoku 41, 189193.

3. Holmstrom, C., Egan, S., Franks, A., McCloy, S., and Kjelleberg, S. 2002. Antifouling activities expressed by marine surface associated pseudoalteromonas species.

FEMS Microbiology Ecology. 41: 47-58.

5. Nippon Suisan Gakkaishi-Bulletin of the Japanese Society of Scientific Fisheries. 68: 46-51.

6. The Prokaryotes. http://141.150.157.117.8080/ prokPUB/index.htm. Release 3.16 (5/3/2004).

7. Sogabe, Y., Tajima, K., Tanaka, R., Sawabe, T., and Ezura, K. 2002. Development of 16S rRNA targeted PCR for the identification of Vibrio spp., the causative bacteria of the diseae in cultured sea urchin Stronglyocentotus intermidius occurring at lo water temperatures. Nippon Suisan Gakkaishi-Bulletin of the Japanese Society of Scientific Fisheries. 68: 210-206.

8. Tajima, K., Takeuchi, K., Iqbal, M.M., Nakano, K., Shimizu, M., and Ezura, Y. 1998. Studies on a bacterial disease of sea urchin Stronglyocentrotus intermidius occurring at low water temperatures. Fisheries Science 64: 918-920.

9. Taniuchi, Y., Tajima, K., Shimono, I., and Ezura, Y. 2002. Survival of Flexibacter sp strain F-2, the causative bacterium of spotting disease of sea urchin Stronglyocentrotus intermidius at low temperatures.

Chemical Influences on Bee Hunger Xu (Lloyd) He In honey bees, the method of measuring hunger levels is determined by the bees’ sucrose intake level. The common method to measure the sucrose intake through proboscis extension response (PER) is to apply sucrose solutions of various concentrations to the antenna of a bee. Through research, the biogenic amines octopamine, tyramine and dopamine, are found to be able to modulate sucrose responsiveness. The experimental method was carried out through injection and feeding of octopamine. The result led to a significant increased sucrose responsiveness, leading to frequent and immediate proboscis extension response. However, dopamine decreased sucrose responsiveness when injected into the thorax, but the feeding of dopamine had no effect. Thus, all of these chemical factors indicate their importance for honey bee metabolism and growth. One of the most important chemical factors, the Juvenile Hormone analog, has been hypothesized to have some effect on hunger level thresholds, in determining honey bee labor division, and in promoting development in forager growth. Methoprene is an analog to the Juvenile Hormone that is often recognized as an insect growth regulation hormone. Its primary function is often seen as to stunt the insect’s development, preventing larvae from reaching adulthood.14 Introduction In the honey bee, Apis mellifera L., researchers have determined its feeding process is influenced by either odor or sucrose.7 Further research points out that the structures involved in the detection and integration of these two factors are bilaterally symmetrical in the bee brain.10 Thus, in addition to stimulating proboscis extension through the octopamine induced sucrose intake, the anatomic structure associated with proboscis extension also helps to improve the bee’s brain’s signal by forming a bilateral signal input differentiating from the unilateral signaling of each antenna.5 This leads to an assumption that the hunger threshold could be viewed as a result of collective organization of bee structures and chemical signals. Thus, the modulation of bee hunger through measuring proboscis extension also plays an important role when analyzing bee motivation threshold that influences foraging and divisions of labor among bees.1 In addition to the octopamine, another type of chemical factor, the Juvenile Hormone (JH), has

been hypothesized to have a certain influence on the hunger response thresholds. Through extensive research conducted by Robinson, Schultz and Sullivan, bees treated with the JH and octopamine indicated precocious forager development in worker bees. “JHA treatment is active even as a single dose administered when bees are not even capable of flight, let alone foraging”.13 Through a comparison trial between octopamine and Juvenile Hormone, the result indicated that the JH affects the initiation of foraging at least in part by increasing brain levels of octopamine, but octopamine can act independently of JH.13 They also used methoprene, a Juvenile Hormone analog, to test its effects and found out that it actually caused slower foraging development compared to the Juvenile Hormone.13 Based on other contemporary researches, Juvenile Hormones seem to maintain the modulation of larval growth and development, regulation of reproductive physiology and caste determination.9 In his research, Prestwich also pointed out that chemical analogs are among the tools used to modify catalytic sites by allowing manipulation of physiologically important interactions.9 His suggestion hinted that there could be a relationship between the Juvenile Hormone analog, methoprene, and insect hunger regulation, as feeding contributes to an insect’s catabolic metabolism. In our prediction, the introduction of methoprene might be a hunger level depressant. This leads to the primary goal on this experiment: to find out the role methoprene plays in modulating the honey bee’s hunger threshold.

Materials and Methods

Figure 1: Measurement of sucrose intake in control honey bees fed with only sucrose. The series 1 represents the pattern of sucrose intake at different time periods of a day.

Experiment 1: Repeat the Effect of Handling Methods on Proboscis Extension Response to Sucrose

Xu (Lloyd) He is a graduating senior in Revelle College majoring in Animal Physiology and Neuroscience. He will be pursuing a master’s degree beginning Fall 2005 in UCSD’s integrated BS/MS program. His current research involves the study of hunger levels in the honey bee species Apis mellifera with Professor James Nieh.

He plans on completing his Master’s thesis in this field in Spring 2006.

In Pankiw and Page’s research,7,8 they have confirmed the procedure of testing proboscis response using sucrose as seen in this following experiment: “To determine the effect of handling methods on proboscis extension response (PER) to sucrose, we prepared 30% sucrose solution and water ad libitum and maintained at 33C degree, 55% RH and 24-h light cycle.3 Next, they chilled and anesthetized groups of 5 worker bees for 4 minutes at 5C degrees. After waiting for 30-60 minutes recovery time for the bees and placing the bees in holders,6 they established a control group of bees with 30% sucrose solution control, with 3 levels of dosage given at 0.2 microgram, 2.0 microgram and 20 microgram, all dissolved in 30% sucrose in at least 50 bees.7 Later, they tested the bees under PER assay in 15-, 30-, and 60-minute ranges after feeding for both the OA and the control group.7 Experiment 2: Effect of Methoprene on Hunger Threshold In the common cutworm species, Yoshiga and Tojo14 have discovered that methoprene is capable of blocking larval-pupal transformation by blocking the BP proteins, a key molecule in larva transformation. Although non-lethal to adult and pupal insects, methoprene is considered a larvicide because it inhibits the maturation of insects from larvae to adult. It is primarily used to control mosquitos, beetles, moths, ants and a variety of aquatic animals such as frogs and some fishes.2 Proposed Method In this experiment, honey bees are used as the species to be tested with the Juvenile Hormone analog methoprene. To determine the effect of methoprene on honey bee hunger threshold, we must first sedate the bees for better results under a chilled ice box with temperatures varying between 15 to 20 degrees Celsius for approximately 3 minutes.

We then establish three groups with 4 bees in each group labeled numerically. The control groups will Volume 2 Issue 3

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specifically assigned 1M sucrose solution and record the amount of sucrose each bee consumed between the groups. For a better result, only hungry bees will be used as subjects. On a daily basis, each bee will be fed twice within a 6-hour interval with the introduction of methoprene and acetone in the 1st feeding only. After obtaining these results, graphs will be designed with group name, bee number, amount of sucrose intake, and how frequently each bee consumed in its lifetime.

Results and Discussion

Figure 2: Measurement of sucrose intake in honey bees fed with methoprene in acetone before sucrose. The series 1 represents the pattern of sucrose intake after methoprene was applied at different time periods of the day.

be a sucrose-only and sucrose with acetone. The experimental group will be an acetone, methoprene and sucrose group. The experimental group will be treated with approximately 200 µg methoprene with density of 0.9261 (g/ml) dissolved in 5 µl acetone.

As for the control group, half of it will be treated with just 5 µl acetone while the other half is left untreated. We apply the methoprene and acetone mixture on the abdomen from top to bottom. After allowing 15 minutes for the methoprene to begin its influence, we will begin to treat the bees with

The result of this experiment was as expected.

The acetone treatment in the honey bees did not have significant effects on honey bee hunger levels, which is seen through their constant daily 1M sucrose consumption indicated by the Figures 1-3. In Figure 1, the sucrose-only control group showed a consistent level of sucrose intake while the induction of methoprene caused sucrose intake levels to drop (Figure 2). The acetone treatment, shown in Figure 3, was used as a secondary control group. Although most of the bees could recover within a five to six hour period, some still were influenced by methoprene and did not consume enough 1M sucrose to survive. The small number of bees that survived was able to gradually resist a small portion of methoprene and retained increased sucrose consumption level. Overall, the results were satisfactory as they clearly indicated that the Juvenile Hormone analog methoprene does indeed affect the hunger level threshold of honey bees.

Acknowledgements I wish to thank UCSD biology professor James Nieh and all the members of his wonderful lab team.

References 1. Barron, and Schulz, D.J., and Robinson, G.E. (2002) Octopamine modulates responsiveness to foraging-related stimuli in honey bees (Apis mellifera). J Comp Physiol A 188:603-610.

2. Bennet, S.M., 2003.

3. Bitterman, M.E., Menzel, R., Feitz, A., and Schafer, S. (1983) Classical conditioning of proboscis extension in honeybees (Apis mellifera). J Comp Psychol 97:107-119.

4. Bloch, G., Sullivan, J.P., and Robinson, G.E. (2002) Journal of Insect Physiology volume 48, issue 12,Pages 1123-1131.

5. Galizia, C.G., and Menzel, R. (2001) The role of glomeruli in the neural representation of odours: results from optical recording studies. J Insect Physiol 47:115130.

6. Menzel, R., and Muller, U. (1996) Learning and memory in honeybees: from behavior to neural substrates.

Annu Rev Neurosci 19:379-404.

7. Pankiw, T., and Page, R.E. (2002) Effect of pheromones, hormones, and handling on sucrose response thresholds of honey bees(Apis mellifera. L) Behav Ecol Sociobiol 189:675-84.

8. Pankiw, T., and Page, R.E. (2001a) Brood pheromone modul,ates sucrose respo,nse thresholds in honey bees(Apis mellifera L.). Behav Ecol Sociobiol 49:206213.

9. Prestwich, G.D. (1987) Chemistry of Pheromone and Hormone Metabolism in Insects. Science 237: 999-1006.

10. Robinson, G.E., et al. 1989.

11. Schulz, D.J., and Ronson, G.E. (1999) Biogenic amines and division of labor in honey bee colonies: behaviorally related changes in the antennal lobes and age related changes in the mushroom bodies. J Comp Physiol A 184:481-488.

12. Schulz, D.J., and Ronson, G.E. (2001) Octopamine influences division of labor in honey bee colonies. J Comp Physiol A 187:53-61.

13. Schulz, D.J., Sullivan, J.P., and Robinson, G.E. (2002) Juvenile Hormone and Octopamine in the Regulation of Division of Labor in Honey Bee Colonies. Hormones and Behavior 42, 222–231.

14. Yoshiga, T. and Togo. (2001) Effects of a juvenile hormone analog, Methoprene, on the hemolymph titers biliverdin-binding proteins in the common cutworm, spodoptera litura (Lepitoptera: Noctudae).

Figure 3: Measurement of sucrose intake in honey bees fed with acetone before sucrose. The series 1 represent the pattern of sucrose intake after acetone was applied at different time periods of the day.

Saltman | Quarterly Acknowledgements

In addition to the SQ contributors and review board, the editors-in-chief would like to thank:

>>Jennifer McEntee and the San Diego Daily Transcript for the interview and publicity in their paper.

>>Patricia Walsh, Barbra Blake, Dean Eduardo Macagno and the Division of Biological Sciences for their support.

>>Jonathan Lazarus for artistic input on the layout.

>>Nick Spitzer for the feature faculty interview and for speaking at Under the Microscope. >>Gabriele Wienhausen for sharing her memories of Paul Saltman with the editors-in-chief. >>Lorraine Pillus for advice and encouragement in the early stages of planning this year.

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>>Marika Orlov, Louis Nguyen, and Greg Emmanuel for founding Saltman Quarterly, and for encouragement and advice this year. >>And Mrs. Barbara Saltman for allowing us the opportunity to continue her husband’s legacy of commitment to undergraduate education.

Notes:

Staff of Saltman Quarterly Ronald Alfa is a second-year Revelle College transfer student in the Animal Physiology and Neuroscience major. Ron is currently involved in gene therapy projects (for treatment of Alzheimer’s Disease) under Dr. Armin Blesch of Dr. Mark Tuszynski’s lab in the Department of Neurosciences. His interests include neural regeneration, plasticity, and neuroendocrinology.

Ann Cai is a third-year Molecular Biology and

was working on. Currently, she is working in Dr. Ananda Goldrath’s lab studying transcription factors involved in T Cell regulation. Kristin is planning to apply to the BS/MS program and would like to continue working in immunology.

Cara Cast is a transfer student and graduating senior majoring in General Biology in Revelle College. She is currently researching genetic interactions affecting chromatin remodeling in S. cerevesiae in the Pillus lab. Cara plans to attend graduate school beginning in fall 2006.

Eric Chan is a third-year student in Marshall College with majors in History and Political Science, as well as a minor in Biology.

He has previously Left to right: Alexander Kintzer, Ryan Ferrell, Kyle Kuchinsky worked in the Schafer Lab as a lab assistant Music major from Muir College. She is currently and was also a TA for BILD 2. Outside of researching in the Partho Ghosh lab, investigating academia, he enjoys tennis, fencing, piano, and the role of Bordetella reverse transcriptase in clarinet.

Bordetella bacteriophage variability generation through the Chancellor’s Research Scholar Reeti Desai is a third-year Molecular Biology Program. This summer, she will be researching major in Revelle College.

Currently, she myocardial infarction in the Mary Gray lab at works as an intern at the pharmaceutical UCSF School of Medicine. Following graduation, company Neurocrine Biosciences, as part of the Endocrinology department. Next fall, as a she plans to go to medical school. senior, Reeti plans to study abroad in England.

Kristin Camfield is a senior majoring in After graduating, she hopes to pursue a Master’s Molecular Biology with a minor in Psychology. degree in Molecular Biology.

Previously, she worked at Cibus Genetics through the Biotechnology Internship Opportunities Heather Eshleman is a fourth-year Molecular program. Her project involved sequencing the Biology major in Revelle College. She is control regions of Canola genes that the company currently working on her senior honors thesis in Matthew Weitzman’s lab at the Salk Institute studying viral interactions with cellular DNA damage response machinery. She will be traveling to the NIH after graduation for a postbaccalaureate research fellowship and plans to attend graduate school.

Kristine Germar is a senior Molecular Biology major in Earl Warren College. Last year she worked at Gen-Probe through the Biology Internship Oppotunities program, and has previously worked in Dr. Yang Xu’s laboratory investigating the function of the tumor suppressor p53.

Shruti Jayakumar and Reeti Desai 48

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2004-2005

Avanti Ghanekar is a third-year Biochemistry and Cell Biology major with a minor in Psychology (and possibly dance). She currently works at Pfizer Pharmaceuticals in the Department of Research

Pharmacology.

Avanti is considering ma pursuing either optometry or opthamology after finishing her u n d e rg r a d u a t e education at Clockwise from top left: UCSD. Outside of Stephanie Kinkel, Daniel school, she enjoys Fang, Josh Tan, Brittany Berrunning, playing nik, Eric Chan raquetball, and cooking gourment/ethnic food.

Nicole Gomez is a Molecular Biology major and Psychology minor. She currently works in Robert Dutnall’s lab in the Division of Biological Sciences as an EXPORT Scholar. She has interned with the City of Hope research program for the past two summers, and presented her research twice at the Southern California Conference of Undergraduate Research (SCCUR). Nicole plans to go to Spain this summer.

Shruti Jayakumar is a freshman in Eleanor Roosevelt College. She is majoring in Human Biology because the human body has always been a deep interest of hers. She plans on graduating in four years and then going to medical school.

Shruti is also Student Life Editor of the first-ever Eleanor Roosevelt College yearbook.

Peter Kim is a third-year student studying Biochemistry and Neuroscience. He is working with professor Sung Ho Jin at UCSD to locally induce cell physiology in a controlled manner using magnetic nanoparticles. In his spare time, he enjoys listening to music and drinking a good cup of coffee.

Stephanie Kinkel is a fourth-year transfer student studying Molecular Biology. A recipient of the Merck undergraduate fellowship, she is currently conducting research in Dr. Yitzhak Tor’s lab.

Her research focuses on the biological application of modified aminoglycosides.

These molecules have relevance both as antibiotics and HIV therapeutics.

She hopes to pursue her PhD in biochemistry.

Peter Kim

by Johnson & Johnson.

She is currently working on gene expression profiling in many types of cancer.

Grace Wang, Laura Lombardi and Ronald Alfa

Alexander Kintzer is a graduating senior at UC San Diego. He works for Senyon Choe in the Structural Biology laboratory at The Salk Institute. After obtaining his degree from UC San Diego, he will pursue a doctorate in chemistry at Cornell University.

Kyle Kuchinsky is a second-year Biochemistry and Cell Biology major in Sixth College. He has previously worked as an intern in the Williamson Lab at The Scripps Research Institute studying the synergistic effects of antibiotics, and has also worked as a lab assistant for the Hunter Lab at The Salk Institute. He is interested in studying the biochemical basis for human disease, the mechanisms for mental disorders, and pharmacogenomics.

Laura Lombardi is a transfer student who is graduating this year with a degree in Biochemistry and Cell Biology from Thurgood Marshall College.

She works in the Brody lab researching the multi-oscillator system responsible for the circadian rhythm of Neurospora crassa, a filamentous fungus. She has applied to PhD programs in Cellular and Molecular Biology.

Association.

Alice Tsai is a Warren third-year double majoring in Biochemistry & Cell Biology and Music. She currently researches in the Glass Lab, UCSD School of Medicine, working on a project to identify possible ion channels that expressed through the interaction of peroxisome proliferator-activated receptor gamma and rosiglitazone. She is also the current Treasurer of the Biological Sciences Student Association and will be the Senior Events Chair next year. This summer, she will be interning at the National Cancer Institute.

Jennifer Wan is a first-year studying Biochemistry and Cell Biology in Muir

Lauren Ashley Miller is a first year General Biology major in Revelle College.

She is interested in both molecular biology and marine biology. She has not done any research yet, but hopes to have a research position next year.

She’s also a member of the Biological Sciences Student Association, and enjoys volunteering at the Convalescent Children’s Hospital. Her future plans include doing research and attending grad school.

Nick Lind is a fourth-year student at UCSD pursuing a double major in Biochemistry and Political Science. He currently works in the immunology lab run by Dr. Ananda Goldrath, which studies regulation of naïve and memory T-cell populations. After graduation, Nick plans to go to law school and pursue a career in environmental law.

Sara Paul is a third-year transfer student working on a General Bio major/Economics minor at Warren College. She is interested in pursuing a career in marine biology and plans on studying abroad in Australia next year at James Cook University. She also plans on continuing her education at Scripps Institution of Oceanography.

She is currently not involved in any research, however upon return from Australia she plans on getting involved with lab/research work.

Caroline Lindsay received her B.S. in Molecular Biology from UCSD in March 2005, but has since continued her duties as SQ co-editor-inchief. During the fall and winter quarters, she worked in Dr. Amy Pasquinelli’s lab researching the regulation of the miRNA let-7 in C. elegans.

Following graduation, she took on the position of Scientist I at Veridex, a biotech company owned

Joshua Tan is a fourth-year Marshall College student with a major in Biochemistry & Cell Biology. He is currently working on a research project in Tama Hasson’s lab involving studies of mutations in Myosin 6. He hopes to continue his research in the M.S./B.S. Program. Joshua is also a member of the Biological Sciences Student

Lauren Ashley Miller, Nick Lind, Kristin Camfield and Sara Paul

Alice Tsai and Ann Cai

College. She is interested in neurobiology and immunology. She has worked previously at UCI, studying the inhibition of Herpes Simplex Virus Type-1 through RNA interference methods. She loves playing the violin, working out, dancing, and painting ceramics. Jennifer is currently studying French and hopes to go to France in two years.

Grace Wang is a second-year Microbiology major in Muir College. She previously assisted with research in Dr. Ethan Bier’s lab to identify the L5 enhancer in Drosophila wing vein development. She plans to minor in Japanese Studies and enjoys watching basketball and hockey.

Additional review board members: Brittany Bernik Max Chen Daniel Fang Ryan Ferrell Chi-Chung Lee Andrew Lin Volume 2

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ACTS AS THE POST-SYNAPTIC RECEPTOR REGION BINDS TO MEMBRANE RECEPTOR OR ION CHANNEL CROSSING OF OPTICAL NERVES NEUROTRANSMITTER RELEASED AT AUTONOMIC SYNAPSES AND NEUROMUSCULAR JUNCTIONS MEASUREMENT OF ELECTRICAL ACTIVITY IN THE BRAIN (ACRONYM) NEUROTRANSMITTER RELEASED BY MOST SYMPATHETIC NERVE TERMINALS NEUROTRANSMITTER OFTEN RELEASED AT EXCITATORY SYNAPSES CELLS RESPONSIBLE FOR PROVIDING MYELIN SHEATH

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HINDBRAIN INVOLVED WITH BALANCE, HEARING, SLEEP, ETC. MASTER GLAND CELL BODY OF AXON MATTER THAT CONTAINS NEURONAL CELL BODIES PLAYS PART IN EMOTIONS, PART OF LIMBIC SYSTEM FOUND IN THE POSTERIOR FOSSA BEHIND THE BRAINSTEM, IMPORTANT FOR LEARNING GROUP OF FUNCTIONALLY RELATED CELL BODIES POPULAR BRAIN IMAGING TECHNIQUE (ACRONYM) MATTER THAT CONTAINS NERVE FIBERS AND GLIAL CELLS RELIES INFORMATION TO CEREBRAL CORTEX AREA IN THE POSTERIOR PART OF DOMINANT PREFRONTAL CORTEX, IMPORTANT FOR SPEECH WHERE NEURONS COMMUNICATE

http://sq.ucsd.edu Saltman Quarterly

University of California, San Diego 9500 Gilman Drive La Jolla, CA 92093-0376 sq@biomail.ucsd.edu