Sq vol1

COEDITORS-IN-CHIEF Marika Orlov Louis Nguyen Gregory Emmanuel REVIEW BOARD Ann Cai Cara Cast Eric Chan Henry Chen Reeti Desai Gregory Emmanuel Kristine Germar Yee Hung Lorraine Kelley Caroline Lindsay Hao Yi Liu Greg Naughton Louis Nguyen Ian Nicastro Jeff Nichelini Marika Orlov Isaac Pearlman Joshua Tan

In our time as undergraduates at the University of California, San Diego, we came to recognize the extraordinary biological research being conducted at this university by our fellow undergraduates. Though many undergraduates contribute significantly to scientific knowledge before they even receive a degree, we realized that there wasn’t an avenue to broadly present our work in order to share our accomplishments with the community.

To fill this void, we present UCSD’s first undergraduate journal of biological science, the Saltman Quarterly. Our principal goal is to give undergraduates the opportunity to experience writing and submitting articles to a peer-reviewed journal and to create a way to present our work to the UCSD Community.

Dr. Paul D. Saltman, a beloved educator in Nutrition and Biochemistry, was widely praised for excellence in teaching and was widely known in particular for his deep commitment to undergraduates. This journal is dedicated to the memory of Dr. Saltman as it reflects his dedication to the education and the experiences of undergraduates.

With this inaugural issue, we hope to grab the attention of the highly regarded biological community at UCSD and raise the well-deserved awareness of the great accomplishments made by its undergraduates. In the following pages, you will find original work produced exclusively by UCSD undergraduates, including original research articles, review articles, updates, model organism reviews, editorials, and interviews. We hope to make this student-run journal a lasting tradition amongst the biology undergraduates here at UCSD. ~Marika, Louis, Greg

PUBLICITY BOARD Louis Nguyen (Board Supervisor) Jeff Nichelini (Chair) Henry Chen Claudine Heu Lorraine Kelley Kuo Yang SQ POSTER SESSION BOARD Marika Orlov (Board Supervisor) Caroline Lindsay (Chair) Ann Cai Reeti Desai Kristine Germar Ian Nicastro Yu-Ting (Alice) Tsai

WEBMASTERS Gregory Emmanuel Joshua Tan Copyright©2004. All Rights Reserved.

Questions or comments? sq@biomail.ucsd.edu

Many beginnings are humble. Often, an undergraduate’s first important contributions to a laboratory are clean dishes, well-stocked cabinets, and the proper disposal of biohazardous waste. Does it sound simple? Perhaps so, yet executing these responsibilities provides a natural transition during which a student with curiosity and drive becomes accepted and embraced as a member of a laboratory team. This is when the magic can begin.

Moving from knowing the best times to find a free autoclave to debating the best (or missing!) controls in an experiment is but one way in which the transformation from student to scientist begins. Every day in which undergraduates, graduate students, postdoctoral fellows and faculty are mixing things up, technically and intellectually, possibilities for such transformations are ripe.

The Saltman Quarterly provides evidence of transformations complete and mid-metamorphosis. In this forum, UCSD undergraduates document their experiments and experiences in research labs. SQ is a venture initiated and executed by smart, motivated and enterprising undergraduates in the Biological Sciences. Beyond their research, the students are writing, reading and editing critically. SQ will prove to be engaging, informative, and, perhaps occasionally, wickedly amusing. Paul Saltman, in whose memory the journal is named, would have loved all that. The rest of us will read, discuss, and admire the accomplishments of our lab mates and students, and eagerly await more. ~Professor Lorraine Pillus

Undergraduate Biological Research Publication UCSD Division of Biological Sciences

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

NEWS

REVIEWS

4

Undergraduate Research Conference

10

4

VentureForth Biotech Conference

Sir2 Protein: An Important Link in Aging and Metabolism

by Caroline Lindsay

4

SQ Poster Session

11

4

Jane Goodall Receives Nierenberg Prize

Sclerostin: A Novel Bone Morphogenetic Protein Antagonist

by Alex Kintzer

4

Amy Pasquinelli Named Searle Scholar

13

4

Biology Beckman Scholar for 2004-05

Regulated Gene Expression Systems in Gene Therapy

by Ronald Alfa

16

Darwin: On the Origin of Synapses

by Eric Chan

EDITORIALS 5

5

Biological Research - May Cause Some Side Effects

by Louis Nguyen A Potential New Strategy for Preventing Laboratory Animal-Induced Allergic Disease

by Ian Nicastro

RESEARCH ARTICLES 17

Neuroligin-Induced Vesicle Clustering in Rat Hippocampal Neurons

by Greg Naughton

20

Homer: Expression in Chick Ciliary Ganglia

by Arin VanderVorst

23

The Effect of Calcium Intake and Excercise Intensity on Systolic Blood Pressure in Older Men

by Pei-Chen (Jennifer) Hsieh

ORGANISM OF THE QUARTER 6

Caenorhabditis elegans

by Marika Orlov

SALTMAN QUARTERLY STAFF 26

Biographies and Photographs

ACKNOWLEDGMENTS FEATURED FACULTY 7

An Interview with Dr. Randolph Hampton conducted by Cara Cast

27

Couldn’t have done it without you!

News of the Quarter Undergraduate Research Conference The 29th Annual West Coast Biological Sciences Undergraduate Research Conference took place on Saturday, April 24th. UCSD undergraduates Ian Nicastro, Louis Nguyen, Kaori Onozuka and Marika Orlov held seminars on their research projects for student and faculty representatives from 104 colleges and universities. Also, Angela Brooks and Brian Jenkins presented their posters at the conference.

VentureForth and SDBio Connect Entrepreneurship and Biology VentureForth is a UCSD student organization that serves to help students start businesses and forge connections with industry, faculty, and the university. Started by students who were interested in learning applied professional skills, VentureForth has organized many successful events to help students get involved with the challenges of entrepreneurship. Recent events include the UCSD Business Plan Competition, a seminar on the “Importance of Culture and Organizational Development in Building a Successful Company,” and many other businessrelated workshops. VentureForth events aim to help students acquire skills essential to success in business - leadership, initiative, networkingability, professionalism, and entrepreneurship.

SDBio is a biotechnology-focused UCSD student organization that crosses the boundaries of disciplines and brings together students from a wide variety of majors, from molecular biology to mechanical engineering, who are interested in biotechnology. Their main goal is to prepare students for the biotechnology industry by facilitating presentation of topics not focused on in coursework, such as the process of drug discovery and development (how basic science research is translated into a drug), clinical trials and regulatory issues, and manufacturing.

The latest joint endeavor by VentureForth and SDBio is the first Annual Biotechnology Entrepreneurship Conference (ABEC) on Saturday, May 8, 2004 from 10 am to 6 pm.

ABEC will feature many leaders of the San Diego Biotechnology Community speaking about various types of biotechnology companies in a panel discussion format.

A group panel will kick off the conference, discussing the differences between the three major types of biotechnology companies, while the rest of the day will include Biopharmaceutical, Medical Device, Legal Issues and Biotech Tool Products & Services Panels. VentureForth and SDBio anticipate this event will help qualified scientists build successful biotechnology companies based on innovative research from the multitude of biologically-focused labs at UCSD 4

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and in the San Diego area.

For more information about VentureForth, SDBio, and the ABEC Conference, please visit: http: //www.ventureforth.org/biotech.

Under the Microscope The Saltman Quarterly will present the first annual Undergraduate Research Poster Session on May 14th, 2004 from 10 am to 2 pm in the atrium of the Natural Sciences Building. Dr. Randy Hampton will be giving a talk on what drew him to science. Come support your fellow undergraduate researchers: stop by and ask them about their work. If you are conducting research in a lab, this will be an annual event, so plan on presenting a poster next year!

Goodall Receives Nierenberg Prize Dr. Jane Goodall was at the Birch Aquarium on the evening of April 30th, 2004 to receive the distinguished Nierenberg Prize for Science in the Public Interest. The award, honoring the memory of William Nierenberg, was presented to Dr. Goodall by his son, Nico Nierenberg. Dr. Goodall

outstanding individuals in their first appointment to assistant professor. Dr. Pasquinelli’s lab studies the roles of microRNAs in C. elegans development.

Biology Undergraduate Beckman Scholarship

Receives

Laura Lombardi is this year’s recipient of the prestigious Beckman Scholarship, an academic scholarship totaling $17,600 to support research, travel and supplies for one student during the summer of 2004 and 2005, and the 2004-05 academic school year. Laura is one of three Beckman Scholars selected this year at UCSD, and the only recipient in the Division of Biological Sciences. Her work in Professor Brody’s lab will culminate in a presentation at one of the Undergraduate Research Conferences held at UCSD.

Where Else We Have Been Published Khush B. Abid Lahner B, Gong J, Mahmoudian M, Smith EL, Abid KB, Rogers EE, Guerinot ML, Harper JF, Ward JM, McIntyre L, Schroeder JI, Salt DE. Genomic scale profiling of nutrient and trace elements in Arabidopsis thaliana.

Nature Biotechnology 21, 1215-21 (2003).

is world-renowned as a primatologist, ethologist and conservationalist - no small feat, and hardly surprising, considering her revolutionary research on the relationship between humans and animals, and her discoveries that chimpanzees are not so different from humans after all (for example, in their use of tools). It is for all of these accomplishments, and much more, that she is honored with the Nierenberg prize. Her acceptance speech titled “Reasons for Hope” advocated aiding fellow citizens of the world by joining programs such as Roots and Shoots, in addition to general efforts to conserve and better understand the planet on which we live.

Pasquinelli named Searle Scholar Assistant Professor Amy Pasquinelli has recently been selected a Searle Scholar. This annual award was established in 1980 and provides grants to institutions to support independent research of

Jeff Bird Collins JS, Schroer RJ, Bird J, Michaelis RC. The HOXA1 A218G polymorphism and autism: lack of association in white and black patients from the South Carolina Autism Project. Journal of Autism and Developmental Disorders 33, 343-8 (2003).

Rachel E. Bloom Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE, Bodde S, Jones JD, Schroeder JI. NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. European Molecular Biology Organization Journal 22, 2623-33 (2003).

Louis N. Nguyen Murai KK, Nguyen LN, Koolpe M, McLennan R, Krull CE, Pasquale EB. Targeting the EphA4 receptor in the nervous system with biologically active peptides. Molecular and Cellular Neuroscience 24, 1000-11 (2003).

Murai KK, Nguyen LN, Irie F, Yamaguchi Y, Pasquale EB. Control of hippocampal dendritic spine morphology through ephrin-A3/EphA4 signaling. Nature Neuroscience 6, 153-60 (2003).

Biological Research - May Cause Some Side Effects Louis N. Nguyen Every species seeks to survive. What makes humans different is our additional need for happiness. These two goals go hand in hand as we would like to experience happiness for the longest period of time possible. Since our presence on Earth began, we have developed several remarkable innovations to improve our productivity and way of life. These include language, trade, and politics. Innovations in science have created both direct and indirect impacts on our longevity.

Biological scientific research is our solution to the problem of disease. It is what leads us to the development of treatments and pharmaceuticals to save us when we are sick. For example, the current leading treatment for heart disease is the use of statins, which reduce the risk of death by 30%. Dr. Steven Nissen has developed a drug called Apo-A1 Milano that can further reduce the risk of death by cutting the risk in half1. Apo-A1 is an HDL that binds to cholesterol from arterial plaques and carries it away to the liver for disposal. Research sciences don’t necessarily effect a cure, but can change our lifestyles. After the Surgeon General’s Report revealed the side effects of smoking to the public, the percentage of smoking Americans

dropped from 42% to 23% over the course of 40 years2. As of the year 2000, the world population stretched past the 6.1 billion mark3. This steadily increasing number is affected by a rate that is the difference between two other rates: the birth and death rates. Lethal diseases cause 14 million deaths per year4. HIV alone is “set to infect more than 5 million people, and kill at least 3 million” during the year of 20044.

Has mortality been affected by the fruits produced by science? In the developing country of Egypt, the life expectancy has increased by approximately 20 years since 1950. In addition to this, infant and child mortality rates have fallen by 55%. Among the reasons recognized, these trends were also affected by “improvements in immunization.”5 This is not a trend isolated to Egypt alone. According to the Virginia Quality Index, “advances in public health and medicine have increased the life expectancy of countries worldwide.”6 The infant mortality of the world was cut in half, in the past 30 years from 107 per 1,000 live births to 58.7 Trends like this show a decrease in death rates in some form.

Developing countries can be seen as countries that are in a less advanced state of science relative to the developed countries. There is a consistency that science reduces the death rate of countries and the world as a whole, thereby increasing the net global population growth rate. Humanity is

successful at extending our lifespan, but there are some unintended consequences of extending the years we live.

Every person has a cost to the Earth. The average human consumes 45-85 metric tons of natural resources8 per year whether it is due to fertilizer needed to generate brussel sprouts for the person or pollution due to the processes and pollution from the box and address labels used to ship the person’s clothes from a clothing store. Since populations are growing within countries, the volume of natural resources required to support a single country is increasing.

Human load can also be measured in ecological footprints. This footprint is the area of productive land required to support a defined population for an indefinite period of time. In 1900, the ecological footprint of wealthy countries was 1 hectare per capita. In 1994, this expanded to 3-5 hectares per capita. As the ecological footprint grew, the volume of ecoproductive land shrunk. In fact, if every person of the world lived like the average North American, we would need three Earths to support us.9 If medical advancements are decreasing the rate of death, then it is, in turn, increasing the

Continued on page 14

A Potential New Strategy for Preventing Laboratory AnimalInduced Allergic Disease Ian Nicastro The risks of developing allergies and/or allergic asthma to laboratory animals is an often overlooked and yet very real threat to the health of many researchers and laboratory technicians. Can genetic knockout technology provide a novel preventative solution for these allergic diseases?

Researchers and technicians in the fields of biology and medicine are trained to be well aware of the possible health risks associated with their professions, including accidental exposure to carcinogens, corrosives and biohazardous materials. However, the threat of developing allergies and allergic asthma from continual contact with laboratory animals and their byproducts is an often under-acknowledged health risk. A survey of over 5000 researchers and technicians who work with laboratory animals in 137 facilities revealed that 23% had allergic conditions triggered by the animals.1 Of these individuals 82% reported nasal and eye allergy symptoms, 46% reported skin allergy symptoms and 33% were afflicted by allergic asthma.1 In 1998 the National Institute for Occupational Safety and Health (NIOSH) stated

that as many as 56% of individuals who handle laboratory animals may suffer from allergies to them.1 Mammalian laboratory animals are responsible for the vast majority of these reports, with mice, rats and cats being some of the most inducive of allergic reactions.1 Allergic diseases are the result of exaggerated immune responses to typically benign substances, termed allergens, that enter the body from the environment.

Animal allergens are proteins capable of traveling significant distances on airborne particles, making inhalation and dermal contact the most common means of exposure.2 In individuals who suffer from allergies, exposure produces symptoms such as sneezing, a runny nose, watery eyes and skin inflammation.3 Allergic asthma is a more severe allergic disease that affects the airways and is potentially life-threatening.3 The greatest risk factors for developing these diseases are the magnitude, duration and frequency of exposure to allergens.4 These diseases show some familial clustering, and workers with preexisting allergic conditions are more likely to suffer from an adverse reaction to laboratory animals.4,5 Many treatments for these allergic diseases are available; however, the current preventative

Red Fluorescent GloFish available from GloFish.com

measures are limited in there effectiveness. The symptoms of mild to moderate allergies and mild allergic asthma can be alleviated with medication, often allowing afflicted individuals to continue working around laboratory animals.6 Many of these drugs cause unwanted drowsiness, and although the newer classes are relatively free of side effects, multiple medications may be required, which increases expense.6 Continual exposure to laboratory animals, even with medication, can often potentiate the symptoms of afflicted individuals and even provoke new symptoms as well.6 Current preventative measures, such as sophisticated room ventilation systems and various personal protective Continued on page 15

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ORGANISM OF THE QUARTER:

CAENORHABDITIS ELEGANS Humans are very complex creatures who encounter many diseases relating to physiological problems.

While finding cures to these diseases is a necessity, using humans to find these treatments and cures is not always ethical and sometimes is not even possible.

For these reasons, other organisms are used as model systems to study many human diseases. Model organisms need to possess certain characteristics to make them good alternatives to work with. These include a short generation time as well as a sequenced and easily manipulatable genome. Most importantly, a model organism should be able to allow for the study of specific pathologies. Many model organisms are used because different ones are most useful for answering a specific question related to a certain disease. For instance, mice are good model organisms for studying mammalian development, physiology, and the effects of cancers on specific tissues. Singlecelled organisms, such as baker’s yeast, are useful in studying intracellular pathways. To study the signal transduction pathways involved in chemotaxis, social amoeba can be used because they readily respond to chemoattractants.

Sometimes, more complex model organisms can be just as hard to figure out as humans, and when the problem deals with development or the nervous system, single celled organisms simply can’t do the trick. That is why other model organisms, such as the roundworm, are used. Roundworms are eukaryotes with simple structures and can be used in evaluating questions that are too complex to decipher even in flies.

Caenorhabditis elegans are an appealing organism to work with because all 959 cells and 302 neurons have been identified and both a cell lineage map and a complete neuronal wiring diagram exist. In addition, C. elegans are small, rapidly growing multicellular eukaryotes that produce a lot of offspring and are inexpensive to maintain. Additionally, the absence of homologous recombination allows mutations to be tracked easily and there is not a lot of junk DNA, allowing for a higher frequency of mutagenesis as coding regions can be hit more often. These worms are also transparent, so cell divisions can be visualized just by using a high magnification microscope. Cells can be followed throughout development and imaging can be performed on their inner cells.

There are two other characteristics that make these worms unique among other eukaryotes. First, C. elegans can survive starvation by entering a dauer, or larval, stage. Worms can exist in this stage for prolonged periods of time, extending their life span drastically. This is very helpful because when a worm strain is neglected in the laboratory, instead of dying, they simply undergo dauer formation. When re-exposed to food, they will rejuvenate. The other unique characteristic is that these worms are capable of both cross-fertilization and self-fertilization because the two sexes that exist are males and hermaphrodites.

Hermaphrodites can produce both sperm and eggs, and so can self-fertilize. Self-fertilization is very convenient when mutagenizing worms and isolating offspring that are homozygous for a mutated gene.

C. elegans were selected as a model organism in order to understand brain function. Most mammalian behavior is the result of neuronal transmission.

While humans have over one trillion neurons that are responsible for complex behaviors, only 302 known 6

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Marika Orlov

neurons can account for all the behaviors observed in C. elegans hermaphrodites. The protein receptors that are responsible for signal transduction are orthologous to those found in humans, with the exception of sodium voltage gated channels found in many higher eukaryotes, but not in these worms. Due to the notion that the neurons found in worms are a less complex but homologous system to the one found in humans, C. elegans are excellent organisms for studying sensory transduction, especially when lack of a gene product leads to a lack of behavior. One of the behaviors that have been assayed in worms in Dr. William Schafer’s lab at UCSD is the avoidance of touch.

Mutants have been isolated that fail to respond to mechanosensation – an organism’s ability to respond to stimuli such as stretch or pressure – and have been dubbed mec mutants. The neurons responsible for the normal movement caused by touch in mec-2 and mec-4 mutants have been closely examined in order to see what is causing the lack of behavior. It has been shown that in wild type worms the MEC-4 gentle touch mechanoreceptor senses motion rather

calcium influx can be monitored. An advantage to the FRET system is that the imaging is performed on intact organisms, so the connections of the nervous system are kept intact and all the imaging is done in vivo, made possible by the worm’s transparency.

When recordings are done on other eukaryotes, dissections are normally involved to access the neurons of interest, leading to less reliable results.

than pressure because it has a greater response to the computer delivered jab stimulation (harsh touch), than it does to the poke (gentle touch). The MEC-2 and MEC-4 genes are required for gentle touch aversion, but the mutants will still respond normally to harsh touch. Worms are used to decipher the function of these mechanically-gated ion channels and the effects they have on behavior. Because human homologues exist, this can be applied to our species in terms of how we are able to both carry out mechanosensation and how we can discriminate between the different types of stimuli.

the presence of your watch after you have initially placed it on your wrist. This type of habituation allows organisms to pay attention to the stimuli that are continuously changing, and not waste resources to keep track of things that are constant. One of the proteins used in this type of behavior is the dopamine receptor (resembling the mammalian D1-like receptor family); dop-1 is a mutant with a defective dopamine receptor. Analysis of these mutants showed that dopamine is necessary for prolonged habituation. The dop-1 mutant quickly habituates to taps, which are nonlocalized stimuli to the worm culture dish. Although the mutant quickly learns to ignore irrelevant stimuli, it quickly forgets about tap habituation as well. These same phenotypes were observed in cat-2 mutants, which are defective in dopamine synthesis. Aside from its role in non-associative learning, mutants defective in dopamine signaling are important to study because there are many human diseases, such as Parkinson’s, that involve changes in normal dopamine levels in the brain.

In the classical experiments on nerve activation using the squid giant axon, electrodes were placed directly into the neurons to measure currents and voltages in order to assess the activity in the cells. Since worms have an inner hydrostatic pressure keeping them inflated, electrodes cannot be placed into the neurons to measure activation voltages, as this would rupture the cuticle and pop the worm. But because worms are transparent, levels of calcium can be monitored visually in neurons by using a strain of worm carrying the cameleon construct, which is a genetically encoded fluorescent calcium sensor that can be expressed cell or tissue specific. Cameleon is a fusion protein consisting of a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) connected by calmodulin, which is a calcium binding protein, and M13, a substrate for calmodulin. When neurons are stimulated, calcium quickly accumulates inside the neuron causing the calmodulin to bind M13 and bring CFP and YFP into close enough proximity for fluorescence resonance energy transfer (FRET) to occur and emit yellow light. Without calcium, CFP and YFP are too far apart for the CFP to transfer the energy to YFP so cyan light is emitted by CFP. By monitoring the changes in the yellow:cyan ratio,

Upon stimulation of the mec mutants with gentle touch (a poke or a buzz), FRET does not occur showing that their neurons do not become activated, which explains why no behavioral response is observed. Even though the neurons are fully functional, as established with cell culture experiments, they do not depolarize, subsequently causing no behavior in response to gentle touch.

Another behavior that can be easily studied in worms is non-associative learning, or habituation. This mode of learning helps humans do things such as ignore irrelevant stimuli, for example, not noticing

C. elegans are a very powerful tool in understanding how behavior is modulated by signaling molecules.

They are also widely used in many other fields of biology including development and aging. Overall these little roundworms have allowed us to study events in eukaryotes that had never before been possible. So the next time someone tells you that they work with worms, don’t cringe and picture an earthworm!

References

1. Suzuki H, Kerr R, Bianchi L, Frokjaer-Jensen C, Slone D, Xue J, Gerstbrein B, Driscoll M, Schafer WR. “In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation.” Neuron. 39(6) (2003): 1005-17 2. Sanyal S, Wintle RF, Kindt KS, Nuttley WM, Arvan R, Fitzmaurice P, Bigras E, Merz DC, Hebert TE, Van Der Kooy D, Schafer WR, Culotti JG, Van Tol HH. “Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans.” Embo Journal. 23(2) (2004):473-82

FEATURED FACULTY

DR. RANDOLPH HAMPTON The Saltman Quarterly will include in each issue an interview with a UCSD faculty member who plays a role in the shaping of undergraduates into scientists and researchers. For our pilot interview, we sought a faculty member who could both advise and relate to undergraduates and lend some inspiration and guidance to our readers and contributors. We had the privilege of speaking with Dr. Randy Hampton, an associate professor in the Division’s Section of Cell & Developmental Biology. Dr. Hampton’s lab in Pacific Hall is working on understanding aspects of how the cell chooses proteins for degradation and destroys them for purposes of regulation and quality control using, as Dr. Hampton describes it, “a gamisch of genetics, cell biology, molecular biology, biochemistry, and lot’s of rain dancing and prayer.” A favorite lecturer among students, Dr. Hampton is well known for his dynamic lecture style, sense of humor, and commitment to teaching. He was recently selected for the Paul D. Saltman Chair in Science Education and was kind enough to spend an hour with a member of our staff.

Saltman Quarterly: At what age did you know you were interested in science?

Randy Hampton: I was interested in science my whole life, but I think for the wrong reasons. A child who goes, “I wanna be a fireman!” has never crawled through a burning house or jumped on a truck going 93 miles per hour in a terrifying firestorm. The reasons young people want to do things are often not that well-informed. My theory is that my interest in science came from when I watched TV in the late 50’s or early 60’s. Whenever you watched a horror movie, whether the monster was a radioactive spider or some kind of blob, or creatures, the terrified people would go to the police, and the police would call the scientists. The thing I noticed about the scientists was that everyone listened to them, they never had to sit down, they were always running around with clipboards figuring out what to do with the radioactive spider or the behemoth that came up from the lagoon.

SQ: What musical instrument do you play?

RH: I play the five-string banjo and a little bit of guitar.

SQ: Many undergraduates who come to UCSD work in a lab. Can you tell us about what your first lab experience was like?

RH: I was actually incredibly lucky in this, and didn’t realize it at the time. I was an undergraduate at a little liberal arts college called Ohio Wesleyan, not a research place. But, there were chemistry professors there and I learned that you could do research over the summer. I got a little research project with a guy named Larry Wilson who was a chemist and just a great, great teacher, that whole department was full of great teachers. Wilson had this very simple problem where they discovered you could make this caged compound that had sulfur molecules--it’s called an adamantane skeleton. It’s easier to draw than to say.

“I play the five string banjo and a little bit of guitar.”

So I said, “That is definitely the job for me!” I think that was my initial inclination and then it refined. I remember in junior high and elementary school I was very interested in science. I think the thing that drew me to it was that it was very physical. In science it seemed like you actually did stuff. Except now that I’m a faculty member all I do is read and write things and sit on my butt and think, so it’s all gone to hell.

And I haven’t seen a radioactive spider for years and years.

SQ: Hmmm...Lot’s of reading and writing, so perhaps being on a editorial and writing staff of an undergraduate publication such as the SQ might be of benefit to a person who’s going into science?

RH: Absolutely!! (Shameless propaganda to inspire reader to get involved with the SQ) SQ: Did you have any distinctly different goals at any point?

RH: Well I’ve done a bunch of things. For a while I played music pretty seriously, and did other sorts of performing arts. I was very interested in that.

Also I ran an office at a very active restaurant/food emporium for a while. But the science was always sort of percolating in the back.

It’s this pretty, box shaped nested set of six membered rings. You can make it out of two dithioacetone structures that are condensed. You just throw it in a flask and it forms crystals of this stuff. The idea was to try to make derivatives of that to use as a carrier molecule to drag metal ions around--basically a pure organic chemistry problem. This was a great research project for a new young person, because the way you do organic is you try reactions out. If they work, you get a product; if they don’t, you try something else.

Organic chemists who do it for a living work at a very high level can really see through a molecule and think about how to manipulate it. But you could be a total ignoramous like I was and try stuff. I wasn’t that successful because I didn’t know what I was doing but what was cool was that it was an authentic research experience. I would go to the library and discover how to do certain reactions; add a bromine to this carbon or put a metal ion here or there. I would set up the apparatus and follow the instructions and just try it.

If I didn’t get it, I’d just ponder it, be confused. What I realized was that Dr. Wilson was just as baffled as I was. It wasn’t like I was doing an assignment for him.

We were working on this problem. Then I would go back to the library. It was great because within a few weeks I knew as much as anyone in the world about

this molecule. That’s a totally authentic research experience. I quickly established the standards of what I was doing. It was totally internal, totally selfmotivated. I was there 10, 16, 18 hours a day and I would work Saturdays/Sundays--I just loved it. I now realize that by some lucky fluke I ended up having an extremely authentic research experience at a place you wouldn’t necessarily expect one to fall in your lap. Years later when I was working in labs cloning genes, it felt the same way.

There is this important transition that students have to go through to become scientists. The basic job of the student is to redeliver answers in the correct form for the people who ask them and to learn to think about how those answers were obtained. Take the Millikan oil drop experiment, I can now describe that many years later from that class because it was taught to me. I’ve been redelivering the answer to, “How do we know what the charge of an electron is?”

“Well the first experiment was…” That’s incredibly important. We need that base of information. What answers do we know?

But a scientist has to divine questions that could be answered. They deliver questions to nature as opposed to delivering answers to professors. So students are doing assignments and scientists are making discoveries and they are different but one could become the other. I love to see an undergraduate come into the lab. At first they’re all about doing the assignment. “Do this and this and this,” and they do it. But very quickly they start saying, “Well I did this, but that happened, and you didn’t expect that.” And I’ll say, “No, I didn’t.” And suddenly they’re not delivering the answer to me that I knew about. They’re delivering a question. And that’s what research is: formulating questions and asking how to find out the answers.

SQ: Do you remember the first time the experiment worked or that you got the result that you had hypothesized? What that felt like?

RH: It was during this organic chemistry thing. I decided I was going to brominate this compound, and my expectation was to get mostly monobromnated tetrathiotetramethyl adamantane or whatever the hell it was. I set up the reaction and got this sludge at the bottom, recrystalized it a few times and came up with these beautiful needle shaped crystals that had different properties from the starting material so I was pretty sure I got the brominated compound.

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And I was thrilled!! I went and read about how you brominate these things and went and DID it! It was like, “all right!”

Then I remember I was very excited about a little beaker of crystals and for God knows what reason I dropped them on the floor! So that was the first lesson that things can go awry. I tried to pick the crystals up and re-dissolve them, but there was all this floor wax on them. I just let them go. See the thing about science is, I then knew how to do it. It took me another day to make more. Science has a certain permanence. You learn something that’s transmissible and permanent.

When you look around at what we have and what we know it’s just amazing how much there is.

Humanity is basically this 6 billion and growing beehive of incredible curiosity and activity; that’s what the human experience is. And science is one of the activities. There are other things too; culture, philosophy, poetry, music, and we’re all so productive! Walk into Tower Records, walk through there and look at all those CD’s!! There are thousands of them! Those are all the intense effort of 10’s to 100’s of people who wanted to make a piece of music!

And you take Lehninger, the metabolic textbook, and open it. There’s probably over a million person hours of experimental science. And that’s excluding all the early stuff like Copernicus and Newton. There’s billion’s of person hours to get up to that solid information but now it’s there!

We’ve all moved irrevocably forward every time somebody drops those crystals on the floor and can figure out how to make them again. It’s a change in the culture. Science to me is not about individuals getting the Nobel prizes. It’s about many people being a part of something bigger than themselves. I love things like the Nobel awards, and I think they’re important because they make people aware of the high points in science. Those are the arches on the building, but every brick is critical to the edifice.

And I think the spiritual part of science, which I know is sort of a weird thing to hear, in the same sentence, but the spiritual part is being part of something much bigger than yourself. That’s a very deep and powerful thing that draws people in. I think there really is this sort of solidarity people have by being a part of the scientific community. That’s what to me is powerful about it.

teach really well.

They give you this title, which is an honor, and some money. You can use it any way you want. I try to devote it to making my teaching better--things I need to teach a class better, things that I might read, experiences I might have, I might travel somewhere to meet someone to talk to them about what research they’re doing. It’s a sum that makes things a little easier. If for instance I want to buy a device, some kind of microphone or a laser, some teaching aid…

SQ: Like a big bright green laser?

RH: Like a big-ass bright-ass green laser! I bought that (used for Metabolic Biochemistry last fall) with the Saltman Chair funds.

Also being designated has made me think more seriously about teaching. I want to try to start thinking about ways to communicate biology to people who are not biologists. Most of the time when you communicate biology it is to would-be biologists or students in a class. One of the nicest things that ever happened to me teaching was getting a long email from a student who was a literature major who took my Bio 1 class because it was a distribution requirement. She wrote this really touching, long email. I had the privilege of being the one to tell her about DNA and proteins and the textual basis of life that is highly digital and extremely precise. I’m sure it would have happened in any Bio 1 class because it’s such interesting material. It’s remarkable in its density and its richness and it is completely unintuitive. You wouldn’t look at an amoeba swimming and think it’s full of many gigabases of textual information that would allow it to make an exact copy of another amoeba that works perfectly. When Leeuwenhoek was looking at amoebas he was amazed that they would produce other things that looked the same way.

He was one of the first to realize that there’s a fidelity at the cellular level, that you could take a drop full of paramecia and they all look like each other and are distinct from the other microorganisms. He realized there was an informational fidelity to life but didn’t phrase it that way. He was able to describe and draw these beautiful animals--animalcules, he called them because they were little. But that has gone to realizing the textual basis, a digitally formatted textual basis to life that then folds into a sort of an analog basis, that’s how proteins work. It’s a very deep thing.

I had the privilege of teaching her that. She so enjoyed this that she went and signed up to be what

“We can’t know what questions to ask unless we know what answers have already been obtained.”

SQ: You were recently selected the Paul D. Saltman Chair in Science Education. Can you tell us what that appointment entails, and if you have any goals during that tenure?

RH: Well, first, I think it’s a little arbitrary because every teacher I know is a great teacher. I take what I get, and I’m happy that they honored me that way, but every professor I know is a great teacher. They’re also all great researchers. They blow my mind because they do so much science and then they also 8

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she called a lab monkey, in the lab of Susan Taylor, a famous researcher in the Department of Chemistry and Biochemistry. She worked in Susan’s lab and got a research project and did an honors thesis and went off to graduate school and became a scientist!

Another aspect of teaching that is so precious to me is working with the grad students because science is like a guild. The way we learn science is by being around people who do it day to day. They teach us

how to think by watching them think. It’s just like all the great painters; painters always taught people by working in a guild. They would start by imitating other painters and then work by themselves. Most of the painters we love and study worked with people that you’ve never heard of. You might think science is much more mechanical and technical but there’s a lot of it that’s more like feeling about, evaluating options with very little information. There’s a lot of feel in it. Like if you’re presented with one of those little eureka moments, then have to decide from 50 things that you might then ask. Which one do you do?

That’s an intuitive thing, that’s a feeling thing. You learn how to make those priorities by watching other people who are more experienced. Also watching people work at the lab bench is definitely something you gain by watching, by example. People who have good technique, it infuses itself when you watch it.

It’s a guild. So, another huge part of what I think is important in education is educating other scientists, the grad students I work with. I’m lucky. They’re all incredibly talented people. They’re all super high performance people who are driven. They’re there right now while I’m sitting here getting interviewed (it’s nighttime). It’s a real gift to me to be allowed to work with that, that they trust me to teach them something, that they are so energetic. Earlier I made it sound like research and classroom teaching are different, but the way we do research is completely an educational phenomenon. Bringing people up through it by working with them, teaching them to think for themselves. That’s part of education, too.

One of my goals is to just think about things with that modulus--that research is instructive.

Another is to just try to be as good a teacher as I can be. Paul Saltman was totally into teaching. I remember when I knew he was ill I made a point of going to visit him to ask him about teaching this metabolic biochemistry course because he’d taught it for years. I knew that our styles of teaching would be very different but I knew I could learn something from him. Also I wanted to see him because I knew he was ill and I wanted to make sure and connect with him a few more times. I went to his office and we had this great chat, and he gave me some notes. I finally said, “Well, so, how are you doing?” And he goes, “Aww kid, you know if I die with a piece of chalk in my hand, I’ll be just fine.” And I said, “Well you’d get really good CAPE scores.” He laughed, “That’s great!” He was an amazing guy. So part of getting that chair is that it reminds me of his value system, which I want to integrate into my value system.

SQ: To what characteristics do you attribute that success, and by success I mean the ability to keep having a job like this, to continue to work in this field. What are the characteristics that a young person in science needs to develop?

RH: I think there’s a lot more in persistence than anything else. Persistence and hard work. Science is a lot of work. It’s very pleasant work; you’re running around with the clipboard dealing with the radioactive spiders. It’s very dynamic work, but it’s work. I think one of the things that’s really neat for an undergraduate to learn while they’re in a lab is how much goes into doing just one experiment, which makes you all the more amazed when you look at a book like Lehninger.

SQ: Two sentences in one paragraph might summarize something that took weeks and weeks.

RH: Or even one word in a sentence, like “folate”,

could have been someone’s PhD thesis--the proof that that was folate that did this. There are eureka moments in discovery, but it’s important to remember there’s a lot of hard work to get up to them and a lot of hard work to reveal them and it’s a matter of persistence.

I think the key is you have to decide whether or not you like the process. If you only like the discoveries, stick to reading textbooks. Discoveries are great, and they’re important but you have to love the process by which you lead up to discovery and deal with it once you have it. Science is not 9am-5pm. Science is more 5am-5am. It’s very demanding. The classic cliché scientist will say to someone, “I’ll be an hour, it’ll take me about an hour in the lab,” and four hours later they’ll come out, still not done. I used to live with someone and when I would do my science, we would joke about the vortex of the lab. If you go near the lab and say, “Oh, I’ll just do this,” and it turns into, “Well, now I need to do this, and I’ll put a blah blah in this and a blah blah in this…” and four or five hours later it’s like, “Hellooooo, are you in there?” “Oh, I fell into the vortex.” The point is if you find yourself experiencing it as a vortex that sucks you in, you’re in the right place. If you find that you don’t like that it takes two or three hours or two or three daaaaaays to set up an experiment that you might need to take a single measurement then you’re maybe not in the right place. What’s cool is you’re job as an undergraduate is to experience a smorgasbord of opportunities to see how you want to fill your plate. Experience enough of the things you think you’re interested in to decide what you’re really interested in. That’s one thing.

Another thing is, it’s incredibly important to understand the difference between product and process. All you ever see when you’re an undergraduate is product. You open a textbook, you see these amazing things, the structure of DNA, how a transcription factor works, plant vascular systems, the immune system, the brain, the way a neuron grows, all this incredible stuff. That’s all product--the product of many people working very hard. And it doesn’t just extend into science. You go to a music class and hear symphonies, you hear arias, you hear harpsichord pieces, you hear the entire body of Handel’s work, you listen to operas, and it’s all product. It’s all finished. It’s all beautiful. It’s all incredible. But you don’t learn process. In science, too, it’s process. It’s doing this, trying that. (Imagine) someone sitting at a desk with hundreds of balls of paper that have missed the wastebasket, and it’s their fiftieth attempt at the chapter of a book. That’s process. That’s the way things grow, an iterated reevaluation of what is going on, adjustments, moving forward, backwards, that’s what process is. Anything anyone does is like that. It permeates all the things we do. This beehive we live in of incredible productivity, of music and literature and art and science and thinking and philosophy and construction and mechanisms and technology--it’s all iterative readjustment, two steps forward and one and a half steps back. Students have to learn what process is about. Going to a lab is a great place to experience the process that’s behind product.

There was a new building built outside my lab, my office. It was an incredible thing because I’ve only looked at buildings. But to watch a building get constructed, it’s pure process. And you can’t even imagine the product. It’s like, “How is that big hole going to be turned into a building full of pristine labs?” Then three months later, “How are these

girders going to turn into a pristine set of 50 new laboratories?” The way you get there is very different than what you end up with. This is allegorical. To be an undergraduate in a laboratory is to understand that process is something you have to experience and see if you like it. We all like the product. Who doesn’t want to make an earth-shattering discovery? Who doesn’t want to figure out something about a disease that’s scourging mankind, or a biological phenomenon

The more you know about something you’re doing the better you are. I love it when an undergrad will be in the lab and they’ll go to the scientific literature and learn something that pertains to the work they’re doing. Everyone eventually learns to do that, but that’s an incredible thing to see. They’re basically trying to find out where that interface is between question and answer. They’re climbing all the way out to the end of Answer Street and that’s where

“...it’s important to understand the difference between product and process.” that’s interesting? But what working in a lab will do is teach you that it’s about the process. If you like the process, if you like the day to day, doing something, evaluating, regenerating hope, trying something new, doing it again, and occasionally a product comes out, then you’ll be fine as a researcher.

So my advice to undergrads is if the product turns you on, if you like reading these textbooks and going to class and hearing about these cool things, that’s a step in the right direction. The next thing to do is to decide if you like the process. I’ve seen OK students who love the process who become complete lab rats who do great. I see people who are fantastic students, like A+ students, who don’t like the process. And that’s not a value judgment! People that smart, there’s a place for them. Science, because it’s a physical art in a way of asking questions of nature, has its own special feel. One has to decide whether they like that, and if they do they have to decide whether they want to really work hard at it. It’s a privilege to do what I do in science. The only reason that people don’t line up around the block to do science is because it is hard work.

SQ: Any skills outside science that one needs to develop?

RH: There are a couple of things I would say are worth starting to think about. One is, learn how to communicate verbally and in writing. A good 50% of science is the communicating of science, both casually and formally. Very casually, like in interpersonal discussions, like at a poster session or a meeting, on the phone, or more formally like giving a seminar, or extremely formally like at a large meeting or in writing. I think communication skills, and that includes writing, are critical. A lot of scientists are not very good writers. I had a sojourn away from science and I ran an office in a multi-million dollar a year business. I had to write tons of business letters.

I had no idea it was training for when I eventually got back into science. It was so useful because I had to learn to concisely, clearly state what I wanted to say.

It really helped a lot.

Another one is, learn a lot about what you are doing.

Become an expert. Develop an appreciation that you can have expertise by gradual acquisition of knowledge. I said that students deliver answers, and I don’t think that’s bad, that’s critical, but every time we enter a new field we become students again until we know most of the answers that have already been delivered. We can’t figure out what questions to ask unless we know what answers have already been obtained. We must know where those boundaries are.

the territory of questions begins. So my advice is, become expert at the things you’re working on, and learn to communicate.

There is something else I’d like to touch on, too, and that is the communicating of your work with your non-peers. Communicating your work with your peers has obvious value because they usually have input, and criticism is useful. What’s less understood is how unbelievably important it is to communicate your work to your non-peers--the people who aren’t in the lecture, aren’t in the hallway, aren’t in your division, who aren’t in your department, people who aren’t in your university.

Who pays for science in America? Every single taxpayer. A big, BIG chunk of science is paid for by the public coffers, the tax burden. Every time we do an experiment, we are doing something because the taxpayers agreed to let us do it. To me, what that translates to is that anytime I’m out in the world and someone says to me, “What are you doing (in the lab)?” it is my absolute responsibility to tell them what I’m doing in a way that will make them happy that they’re paying for my research. Every single person who’s in the lab from undergraduate dish monkey all the way up to the PI has an almost sacred responsibility to make the science they’re doing as exciting and important and upfront and understandable as they can to anybody who asks them, because those people are paying for it! They are our employers and we have to answer to them. They are the people who make it possible.

I think it’s important to do this for other reasons.

There’s massive understandable ignorance about science because scientists are not very good at communicating what they do to non-science people.

It’s not something we’re taught to do. There’s an aesthetic separate from who’s paying for it: it’s worth learning about the world. Knowledge, every time we tell someone else, becomes a bigger part of the culture. The effectiveness is measured by how many people know what you’re talking about. Knowledge becomes part of the culture when the culture knows it. If it’s a secret, it’s not knowledge. For all these reasons, I think that communicating to your non-peers is critical.

To learn more about the research being conducted in the Hampton lab, visit http://hamptonlab.ucsd.edu.

Interview conducted by SQ staff writer Cara Cast.

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Sir2 Protein: An Important Link in Aging and Metabolism Caroline Lindsay Introduction The “Silent Information Regulator” (SIR) proteins are a class of proteins with many diverse functions.

The Sir2 protein (Sir2p) is of particular interest, as it has been recently linked with coordination of aging mechanisms in yeast (Saccharomyces cerevisiae) and the nematode (Caenorhabditis elegans). Sir2p was first discovered in regulation of transcription at the silent mating-type loci. The repression at these loci is important for formation of wild-type haploid yeast that can mate normally. Sir2p (along with other proteins including Sir1, Sir3, Sir4, and Rap1) also binds yeast telomeres and helps to repress transcription by deacetylating the N-terminal tails of histones H3 and H4, which help package DNA in the cell. The removal of acetyl groups from the histone tails causes them to become positively charged, causing a stronger interaction with negatively charged DNA molecules. Tightly packed DNA/histone complexes contribute to tighter packing of chromatin in this region, which causes transcriptional repression. In addition, Sir2p functions in double-stranded DNA break repair and suppression of mitotic recombination in ribosomal DNA (rDNA). This review will summarize some of the research that has been done on the SIR2 (“SIR2” denotes the wild-type allele of the gene in yeast, and “sir2” indicates the mutant allele) gene in S. cerevisiae, and its homolog, sir-2.1 (sir-2.1 indicates the gene in nematodes) in C. elegans, especially the recent discovery of Sir2p as a protein involved in aging and metabolism.

Analysis Recently, a link between Sir2p and yeast aging has been discovered. Kaeberlein et al. (1999) showed that a sir2 mutation has been shown to have a severe effect on life span, shortening it by ~50%.

This effect is due to the inability of the mutants to suppress generation of small circles of rDNA in the cell, called extrachromosomal rDNA circles (ERCs). Sinclair and Guarente (1997) have shown that ERC accumulation in yeast is a cause of aging.

Sir2p was shown to localize to the rDNA and repress homologous recombination. Homologous recombination at the rDNA is essential for the formation of ERCs. Therefore, the sir2 mutants are unable to suppress ERC generation, causing them to age faster and have a shorter life span. Sir2p effectively inhibits formation of the ERCs that normally leads to aging in yeast (Fig.1).

In diploid yeast, homozygous SIR2 mutants (sir2/sir2) were found to have a significantly shorter life span as compared to wild type. Heterozygous mutants (SIR2/sir2) also had a shortened life span, but not to the same extreme as the double mutant.

When an additional copy of SIR2 was integrated into the wild type strain, a ~30% longer life span was detected. This evidence showed that low levels of Sir2p limited yeast life span. The more Sir2p available, the longer the yeast were able to survive 10

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Figure 1. Sir2p linked to yeast aging. Active Sir2p represses homologous recombination. Normally, homologous recombination is required for ERC formation and aging. However, this function is knocked out in yeast that express Sir2p. Therefore, ERC formation does not occur and cannot stimulate this particular aging pathway.

(Kaeberlein et al., 1999).

Lin et al. (2000) have shown that Sir2 protein is activated in yeast that are raised on a restricted-calorie “diet”. The yeast were either grown on media containing a lower concentration of glucose, or were subjected to lower protein kinase A (PKA), which normally helps increase glucose levels in the cell. It was proposed that Sir2p was activated either by increasing the NAD+/NADH ratio, or by regulating the levels of the Sir2p inhibitor nicotinamide. Glucose begins to break down to produce energy during the glycolysis phase of cellular respiration, and the electron carrier molecule NAD+ is reduced to NADH. These NADH molecules are then fed through the electron transport chain to ultimately produce ATP. NADH levels accumulate when the cell is under anaerobic conditions and is not able to utilize the electron transport chain to re-oxidize NADH to NAD+. When calories are restricted, fewer molecules enter the electron transport chain, which limits oxidation of NADH to NAD+. Since Sir2p is NAD+-dependent (Landry et al., 2000), Guarente (2000) proposed that the increase in concentration of NAD+ molecules activates Sir2p.

Lin et al. (2004) recently showed that it was indeed the NAD+/NADH ratio that contributed to the activation of Sir2p, but it was actually a decrease in NADH, not an increase in NAD+, that caused the activation. Caloric restriction was successful in extending yeast life span when Sir2p was active, which occurred only when NAD+ levels were high and NADH levels low. This discovery showed an important link between aging and metabolism.

When the possibility of Sir2p involvement in aging in higher eukaryotes arose, Tissenbaum and Guarente (2001) analyzed the homolog of SIR2 in C. elegans: sir-2.1. sir-2.1 is the gene that is most homologous to the yeast SIR2 of the four homologies identified. The duplication of a large region of the C. elegans chromosome IV containing the sir-2.1 gene caused a significant increase in life span. When the sir-2.1 gene was mutated in this duplicated region, the life span increase was not detected, demonstrating that sir-2.1 functions in longevity of C. elegans.

It was also shown by the same group of researchers that increasing the dosage of the sir2.1 gene extended the C. elegans life span, as overexpression of SIR2 extended the life span in yeast. In C. elegans, longevity is partially controlled by the insulin-like signaling pathway (Dorman et al., 1995, Larsen et al., 1995). The hormone insulin signals the cell to produce more glucose,

which stimulates more cellular respiration and therefore more NADH is produced. Since insulin is important in regulation of the NAD+/NADH ratio needed for Sir2p activity, it was hypothesized that the sir-2.1 protein might be acting in the insulin-like signaling pathway. Through study of double mutants of sir-2.1 and various genes in the signaling pathway, it was determined that sir-2.1dependent life span extension is also dependent on daf-16, one of the downstream genes in the insulinlike pathway. Without daf-16, the effect of sir-2.1 on life span was not observed. From this and other experiments, it was determined that sir-2.1 plays a role in the insulin-like signaling pathway (Tissenbaum and Guarente, 2001).

Tissenbaum and Guarente (2001) have also suggested that sir-2.1 may play a role in determining development and survival in response to environmental conditions in C. elegans as well. Further research may discover a more specific role for sir-2.1 in development.

As research on the SIR2 gene continues, it will be interesting to discover if there exists a Sir2p homolog that functions in humans in the same way as in S. cerevisiae and C. elegans. If so, this could help us better understand the process of aging in humans. Perhaps in the distant future, a human form of Sir2p could be the target of medication that will slow the aging process and extend our life span.

References

1. Guarente, L. “Sir2 links chromatin silencing, metabolism, and aging.”

Genes & Development. 14 (2000)1021–1026.

2. Kaeberlein, M., McVey, M., and Guarente, L. “The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms.” Genes and Development. 13.19 (1999):2570-2580.

3. Landry, J, Sutton, A, Tafrov, ST, Heller, RC, Stebbins, J, Pillus, L, and Sternglanz, R. “The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases.” Proc. Natl. Acad. Sci. 97 (2000): 5807–5811.

4. Lin, SJ, Defossez, PA, and Guarente, L. “Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae.”

Science. 289 (2000):2126–2128.

5. Lin SJ, Ford E, Haigis M, Liszt G, Guarente L. “Calorie restriction extends yeast life span by lowering the level of NADH.” Genes Dev. 18.1 (2004):12-6.

6. Sinclair, DA and Guarente, L. “Extrachromosomal rDNA circles –a cause of aging in yeast.” Cell. 91 (1997):1033-1042.

7. Tissenbaum, HA and Guarente, L. “Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans.” Nature. 410.6825 (2001): 227-230.

Caroline Lindsay is an SQ staff member.

Her bio can be found in the staff bio section on page 26.

Sclerostin: A Novel Bone Morphogenetic Protein Antagonist Alex Kintzer Sclerosteosis is a progressive bone dysplasia that is characterized by hyperostosis and sclerosis, leading to thickening and deformation of the skull, mandible, ribs, clavicles, and all other long bones. Uncontrolled ossification often causes narrowing of the foramina of the cranial nerves, causing facial nerve palsy, deafness, and impaired vision. Two mutations in the sclerostin gene (SOST) that result in distinct, malformed translation products have been observed in the Afrikaner population of South Africa. These mutations encode truncated and incorrectly spliced sclerostin transcripts, respectively, which may be responsible for the improperly regulated bone growth exhibited by Sclerosteosis victims. Sclerostin has been shown to bind bone morphogenetic proteins (BMPs) with relatively high affinity. BMPs regulate osteocyte proliferation, differentiation, and coordinated destruction via TGF-β receptors. Sclerostin was shown to competitively bind BMPs with the Dan family of BMP antagonists, inhibiting the downstream phosphorylation of Smads, which regulate the transcription of other regulatory proteins involved in bone growth. Sclerostin shares a common cystine-knot motif with Dan antagonists, noggin, and other proteins. The structural similarity with other characterized BMP antagonists makes sclerostin a potentially valuable target in the development of therapeutic treatments for many bone disorders, including Van Buchem’s disease, Sclerosteosis, and diseases involving bone loss.

Introduction Sclerosteosis is an autosomal-recessive disease that is characterized by hyperostosis, which is characterized by increasing bone formation that usually surrounds the cranial nerves, leading to pain and other associated disorders [1-4]. Sclerosteosis has been studied extensively over a 38-year period in 63 affected members of the Afrikaner population in South Africa. However, cases in Brazil, USA, Switzerland, Japan, Senegal, and Spain have also been observed [5-12]. Hyperostosis commonly causes facial deformities, syndactyly, gigantism, and unusual dental strength. Crowding of the cranial nerves, which commonly shows symptoms by 4 years of age, causes facial palsy (paralysis), hearing loss, speech impediment, and chronic headaches [13-20]. Surgical procedures have been fairly successful in relieving these symptoms [21, 22].

However, death is a common result due to the early onset of these symptoms.

Sclerosteosis was mapped to the 17q12-21 chromosomal region and two mutations in the sclerostin gene (SOST) have been identified [23, 24]. The most common mutation in SOST is a cytosine to thymine substitution that produces a premature termination codon in the second exon of the coding region. This results in a completely novel transcript that lacks proper functionality. An adenine to thymine substitution was found in a cterminal intron that encodes a non-spliced transcript, which is presumably not translated. These mutations are sufficient to produce identical symptoms in unrelated subjects from South Africa and Senegal, respectively. Similar intronic mutations occur in Brazilian, and American patients that encode incorrectly spliced sclerostin transcripts [25].

Sclerostin belongs to a superfamily of secreted, cystine-knot proteins. Cystine-knot proteins incorporate conserved CxC and CxGxC motifs, which were first discovered to form the cystine ring motif in mammalian endothelin and insect neurotoxins [26-28]. An additional cysteine pair forms a disulfide bond that threads through the cystine ring [29]. This forms the exceptionally stable cystine knot. Cystine knot proteins are usually secreted, heterodimeric or homodimeric signaling proteins. Sclerostin belongs

to the family of cystine-knot-containing, secreted ligands such as bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), TGF-βs, gonadotropins, platlet-derived growth factors, and BMP antagonists [30], some of which have been show to be involved in tumorigenesis [31-33]. Sclerostin belongs more specifically to the family of cystine knot containing BMP antagonists, which includes noggin, Dan, cerebrus, chordin, follistatin, and others, most of which are expressed in osteocytes.

Sclerostin as a Negative Regulator of Bone Growth Sclerostin was believed to be involved in the regulation of bone proliferation from the symptoms of its dysfunction, namely sclerosteosis [1, 11, 14, 18-20, 22, 23, 25]. Recently, Sclerostin was shown to be expressed in cartilage and bone, though the determined expression levels in various tissues differ significantly [23, 25]. These observations

confirm the longstanding idea that sclerostin may be directly involved in bone formation. It has been also demonstrated that sclerostin and SOST are also expressed in osteoblasts and that sclerostin is localized in differentiating bone cells [34]. However, the specific mechanism of sclerostin action was not known.

Symptomatic observations indicated that sclerostin could be a novel BMP antagonist. It was recently confirmed that sclerostin competes for BMP-2, 4, 5, 6, and 7 binding to BMP type I and II receptors with nanomolar affinity [35]. Winkler et al. also showed competitive inhibition of BMPs by Dan, a related cystine-knot containing antagonist.

The competitive interplay of Dan with sclerostin has not been investigated. However, sclerostin was able to compete with noggin for binding to BMPs, for which the mechanism of BMP antagonism has been well studied (Figure 1). The molecular mechanism of BMP antagonists has been elucidated for noggin [36, 37] and human chorionic gonadotropin (HCG) [38] by structural analysis.

Until recently, sclerostin was assumed to act as a conventional BMP antagonist, effectively sequestering BMP dimers through binding, thereby inhibiting BMP receptor signaling. Winkler et al. confirmed that sclerostin expression in osteoblasts inhibits Smad phosphorylation by sequestering BMP dimers. However, Kusu et al. previously observed that the simple binding of sclerostin to BMPs was not sufficient to inhibit Smad phosphorylation completely. Recently, the presence of a BMPinduced cofactor was proposed to mediate binding of sclerostin to BMPs [39]. This is based on the observation that inhibition of Smad phosphorylation by sclerostin requires a minimum BMP induction time. Winkler et al. also pointed out the difference in affinity between noggin and sclerostin for BMPs. The presence of a cofactor may raise sclerostin’s affinity for BMPs near to the picomolar affinity observed for noggin. Thus sclerostin is a novel BMP

Figure 1: Accepted mechanism of BMP signaling antagonism Uninhibited BMP signaling proceeds by binding of a BMP to BMP Type-I receptor.

A Type-II receptor is then recruited, which constitutively activates the Type-I receptor through phosphorylation. The Type-I receptor then typically phosphorylates downstream targets such as Smads, for which a co-Smad is recruited.

The active Smad complex then enters the nucleus and induces transcription of addition differentiation and growth factors. A BMP antagonist such as sclerostin may bind to BMPs, thereby inhibiting the propagation of a growth signaling through BMP receptors.

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probably facilitates covalent dimerization. Dan has evolved a tenth cysteine that is predicted to form a second intra-monomer disulfide linkage, thereby inhibiting its ability to covalently dimerize.

However, the ninth cysteine that appears to link dimeric subunits is simply not present in sclerostin and USAG-1, thus setting these two proteins apart as monomeric, eight-membered cystine knotcontaining antagonists. Thus the predicted structure is a monomeric, cystine-knot protein antagonist with an additional intra-loop disulfide bridge (Figure 2).

Implications and Future Directions

Figure 2: Predicted CAN Family Cystine – Knot Structure The six-membered cystine ring is represented here by cysteines C1-C6. Intra-loop cysteines are denoted by C’s. This structure was predicted by [43] based on genomic analysis and comparison with cystine-knot proteins whose structures are known. The importance of highly conserved cystine knot motifs CxGxC and CxC is illustrated by their participation in the four-membered cystine ring.

antagonist that may be operating with an inducible affinity cofactor, extending the regulation of bone growth into a Tsg-chordin/Sog paradigm. In this system, twisted gastrulation (Tsg) binds a complex of chordin, Sog, and BMP-4, forming a tertiary inhibitory complex [39-42].

Experimental observations implicate sclerostin as a regulatory inhibitor of BMP signaling.

Researchers have wondered whether sclerostin was expressed in osteocytes undergoing degradation.

Recent expression studies indicate that sclerostin is expressed in osteoblast and osteocytes, but not in osteoclasts [39]. This does not come as a surprise, given that sclerostin inhibits bone growth and differentiation in vivo.

Structural Insights and Predictions Sclerostin displays the common features of secreted, dimeric, cystine knot proteins [25]. The presence of a secretion peptide and a highly conserved cystine knot, which has been thought to facilitate dimerization, leads to the supposition of sclerostin as a dimeric, secreted antagonist. Sclerostin also displays the hydrophobic characteristic of cystine knot proteins, two N-glycosylation sites, and a heparin-binding domain [34, 39] following the last cysteine. However, sclerostin was observed as a monomer on gel filtration chromatography and a non-reduced acrylamide gel [35]. Kusu et al. also observed two variable Mr forms of sclerostin, which are thought to be variable glycosylation forms. These distinct forms have not been characterized thus far.

The presence of a heparin-binding domain was confirmed recently by the purification of sclerostin through heparin affinity chromatography [34, 39].

However, an explanation for the monomeric form of sclerostin remained elusive until recently.

Genomic analysis reveals that sclerostin belongs to the CAN family of eight-membered cystine knot-containing proteins, which includes Dan, gremlin, coco, PRDC, cerebrus, and USAG-1 [43]. All cystine knot proteins utilize six conserved cysteines to form the cystine knot. Two additional cysteines are believed to form an intra-subunit disulfide linkage in Dan, gremlin, sclerostin, USAG1, coco, PRDC, and cerebrus. A ninth cysteine is found in gremlin, coco, cerebrus, and Dan that 12

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Despite the convincing evidence from genomic analysis and gel filtration chromatography, the true nature of BMP antagonism by sclerostin must be characterized by x-ray diffraction crystallography or by analytical ultracentrifugation. Analytical ultracentrifugation will give information about the nature of sclerostin monomers in native setting, indicating whether polymeric states exist. Additional centrifugal analysis of the sclerostin-BMP complex would elucidate the stochiometry of the sclerostinBMP interaction.

The mechanism of BMP antagonism is of interest to researchers who seek therapeutic remedies for the common bone diseases osteoporosis, dysplasia, osteopetrosis, osteomalacia, and sclerosteosis. Sclerostin has been shown to act as a primary antagonist for bone differentiation and growth in osteoblasts and osteocytes, respectively.

In diseases that involve bone loss, small molecules may be designed as sclerostin inhibitors to improve bone growth. Obtaining structures of sclerostin in complex with BMPs would assist rational drug design. Additionally, the interplay of co-expressed BMP antagonists should be investigated to reveal other sources of osteogenic regulation. Dan’s competition with sclerostin for BMPs is a notable example, whose interactions may uncover additional causative mechanisms for common bone diseases.

Acknowledgments I thank Dr. Senyon Choe for support in my efforts to characterize Sclerostin.

Inquiries may be sent to: Senyon Choe, PhD, Structural Biology Laboratory, The Salk Institute for Biological Studies, Post Office Box 85800, San Diego, California 92186-5800.

References

1. Barnard, A.H., Sclerosteosis. S Afr Med J, 1979. 55(25): p. 1015.

2. Beighton, P., Genetic disorders in Southern Africa. S Afr Med J, 1976. 50(29): p. 1125-8.

3. Beighton, P., et al., Sclerosteosis - an autosomal recessive disorder.

Clin Genet, 1977. 11(1): p. 1-7.

4. Truswell, A.S., Osteopetrosis with syndactyly; a morphological variant of Albers-Schonberg’s disease. J Bone Joint Surg Br, 1958.

40-B(2): p. 209-18.

5. Sugiura, Y. and T. Yasuhara, Sclerosteosis. A case report. J Bone Joint Surg Am, 1975. 57(2): p. 273-7.

6. Stein, S.A., et al., Sclerosteosis: neurogenetic and pathophysiologic analysis of an American kinship. Neurology, 1983. 33(3): p. 267-77.

7. Tacconi, P., et al., Sclerosteosis: report of a case in a black African man. Clin Genet, 1998. 53(6): p. 497-501.

8. Freire Paes-Alves, A., et al., Sclerosteosis: a marker of Dutch ancestry? Rev Brasil Genet, 1982: p. 825-834.

9. CH, K. and L. JW, Albers-Schonberg disease: a family survey.

Radiology, 1946. 47: p. 507-513.

10. Bueno, M., et al., Sclerosteosis in a Spanish male: first report in a person of Mediterranean origin. J Med Genet, 1994. 31(12): p. 976-7.

11. NL, H. and A. SF, Osteopetrosis: four cases in one family. Am J Surg, 1941. 53: p. 444-454.

12. Pietruscka, G., Weitere Mitteilungen uber die Marmorknochenkrankheit (Albers-Schonbergsche Krankheit) nebst Bemerkungen zur Differentialdiagnose. Klin Monatsbl Augenheilkd, 1958. 132: p. 509-525.

13. Hamersma, H., J. Gardner, and P. Beighton, The natural history of

sclerosteosis. Clin Genet, 2003. 63(3): p. 192-7.

14. Beighton, P., L. Durr, and H. Hamersma, The clinical features of sclerosteosis. A review of the manifestations in twenty-five affected individuals. Ann Intern Med, 1976. 84(4): p. 393-7.

15. Barnard, A.H., et al., Sclerosteosis in old age. S Afr Med J, 1980.

58(10): p. 401-3.

16. Cremin, B.J., Sclerosteosis in children. Pediatr Radiol, 1979. 8(3): p. 173-7.

17. Epstein, S., H. Hamersma, and P. Beighton, Endocrine function in sclerosteosis. S Afr Med J, 1979. 55(27): p. 1105-10.

18. Itin, P.H., B. Keseru, and V. Hauser, Syndactyly/brachyphalangy and nail dysplasias as marker lesions for sclerosteosis. Dermatology, 2001. 202(3): p. 259-60.

19. Wood, R.E., et al., Jaw involvement in sclerosteosis: a case report.

Dentomaxillofac Radiol, 1988. 17(2): p. 145-8.

20. Stephen, L.X., et al., Dental and oral manifestations of sclerosteosis. Int Dent J, 2001. 51(4): p. 287-90.

21. Beighton, P., B.J. Cremin, and H. Hamersma, The radiology of sclerosteosis. Br J Radiol, 1976. 49(587): p. 934-9.

22. Beighton, P., Sclerosteosis. J Med Genet, 1988. 25(3): p. 200-3.

23. Brunkow, M.E., et al., Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet, 2001. 68(3): p. 577-89.

24. Balemans, W., et al., Localization of the gene for sclerosteosis to the van Buchem disease-gene region on chromosome 17q12-q21. Am J Hum Genet, 1999. 64(6): p. 1661-9.

25. Balemans, W., et al., Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet, 2001. 10(5): p. 537-43.

26. Tamaoki, H., et al., Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations. Protein Eng, 1991. 4(5): p. 509-18.

27. Kobayashi, Y., et al., The cystine-stabilized alpha-helix: a common structural motif of ion-channel blocking neurotoxic peptides.

Biopolymers, 1991. 31(10): p. 1213-20.

28. Tamaoki, H., et al., Folding motifs induced and stabilized by distinct cystine frameworks. Protein Eng, 1998. 11(8): p. 649-59.

29. Hearn, M.T. and P.T. Gomme, Molecular architecture and biorecognition processes of the cystine knot protein superfamily: part I. The glycoprotein hormones. J Mol Recognit, 2000. 13(5): p. 223-78.

30. Vitt, U.A., S.Y. Hsu, and A.J. Hsueh, Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules. Mol Endocrinol, 2001. 15(5): p. 681-94.

31. Risma, K.A., et al., Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc Natl Acad Sci U S A, 1995. 92(5): p. 1322-6.

32. Matzuk, M.M., et al., Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice.

Biol Reprod, 2003. 69(1): p. 338-46.

33. Chang, H., C.W. Brown, and M.M. Matzuk, Genetic analysis of the mammalian transforming growth factor-beta superfamily. Endocr Rev, 2002. 23(6): p. 787-823.

34. Winkler, D.G., et al., Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. Embo J, 2003. 22(23): p. 6267-76.

35. Kusu, N., et al., Sclerostin is a novel secreted osteoclast-derived bone morphogenetic protein antagonist with unique ligand specificity.

J Biol Chem, 2003. 278(26): p. 24113-7.

36. Groppe, J., et al., Structural basis of BMP signaling inhibition by Noggin, a novel twelve-membered cystine knot protein. J Bone Joint Surg Am, 2003. 85-A Suppl 3: p. 52-8.

37. Groppe, J., et al., Structural basis of BMP signalling inhibition by the cystine knot protein Noggin. Nature, 2002. 420(6916): p. 636-42.

38. Lapthorn, A.J., et al., Crystal structure of human chorionic gonadotropin. Nature, 1994. 369(6480): p. 455-61.

39. Van Bezooijen, R.L., et al., Sclerostin Is an Osteocyte-expressed Negative Regulator of Bone Formation, But Not a Classical BMP Antagonist. J Exp Med, 2004. 199(6): p. 805-14.

40. Canalis, E., A.N. Economides, and E. Gazzerro, Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev, 2003. 24(2): p. 218-35.

41. Larrain, J., et al., Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling.

Development, 2001. 128(22): p. 4439-47.

42. Larrain, J., et al., BMP-binding modules in chordin: a model for signalling regulation in the extracellular space. Development, 2000.

127(4): p. 821-30.

43. Avsian-Kretchmer, O. and A.J. Hsueh, Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists. Mol Endocrinol, 2004. 18(1): p.

1-12.

Alexander Kintzer is currently a senior at UC San Diego who is pursuing degrees in chemistry and biology. He works for Senyon Choe in the Structural Biology laboratory at The Salk Institute. After obtaining degrees from UC San Diego he intends to pursue a doctorate in chemistry.

Regulated Gene Expression Systems in Gene Therapy Ronald Alfa Genetic-based therapies hold the potential to treat a milieu of diseases. Inasmuch, central to such applications are regulated gene expression systems. These systems allow one to precisely control gene expression in a spatiotemporal manner. Here, I will review current progress in chimeric, regulatable systems and application of site-specific recombination methods to the regulation of gene expression.

Introduction

Tetracycline Regulated Systems

Gene therapy is best generalized as the introduction of a gene into target cells to replace a disease-causing gene or modify production of specific cellular factors. Genes are introduced into the host genome via a carrier molecule, or vector. Due to their inherent ability to deliver genetic information into cells, viruses are the most commonly used vectors.

Such viral vectors are, however, modified and deficient in their replication machinery – allowing for efficient gene delivery without pathogenesis.

As genetic therapy research continuously progresses, the need for regulatable gene expression systems becomes evermore apparent. In such applications, it is essential that vectors have the ability to control expression of transgenes both reversibly and dose-dependently in response to exogenously administrable pharmacological agents.1 Thus far, multiple regulatable systems have been developed. Through introduction of such systems into viral vectors, scientists may exert spatiotemporal regulation of transgene products.

Regulatable systems may be classified into the following categories: naturally occurring physical stimuli responsive promoters; chimeric, ligandinducible regulated systems (reviewed in [3]); and ligand-activated site-specific recombination2 systems.

Each group exhibits exclusive characteristics and thus is more or less suitable for future genetic therapy based applications. Here we will look at current progress in such regulatory systems.

The tetracycline system is based on the Tn10 tetracycline resistance operon of Escherichia coli.

The presence of tetracycline blocks the binding of the tet repressor (tetR) to the tet operator (tetO) sequence and thereby allows transcription of resistance mediating genes. The chimeric “Tetrepressed,” tetracycline transactivator (tTA) was created through fusion of the tetR with the C-terminal domain of herpes simplex virus (HSV) viral protein 16 (VP16). In absence of tetracycline, the tTA binds with high affinity to a promoter region consisting of a minimal promoter downstream from a repetition of tetO sequences. The binding of tTA thereby permits transcription of the target gene through formation of a transcriptional initiation complex.

In addition, a “tet-on” system was reported shortly after the tTA, “tet-off” system. Utilizing a mutant tetracycline repressor protein, rtTA, Gossen et al. reported a system in which doxycycline (tet analog) positively induces transcription from tetO containing promoters.9 This system presented the benefit of rapid induction and as such, one could quickly stimulate gene expression whereas, with the tTA system, transcription is delayed by the rate of tetracycline clearance from the organism.10 The tetracycline system has shown great efficacy in the control of multiple genes. Baron et al. previously reported the regulation of two genes could be achieved through simple placement of two minimal promoters, in opposite orientation, flanking the tetO sequences.11 Multiple gene regulation was also achieved through use of both tTA and rtTA sequences on a single plasmid. Utilizing both systems, a novel system was introduced whereby regulation of two genes could be achieved, individually, by simple variation of doxycycline concentration.12

Early Systems Early systems were based on naturally occurring inducible promoters. Such promoters were found to be responsive to heat shock3 or heavy metal4 ions.

However, these systems suffered major drawbacks in that inducers were either toxic to mammalian cells or interfered with endogenous gene expression.5 Nevertheless, these systems have not dissipated as of yet. Progress has been made in naturally occurring, physically-inductive promoters yielding systems responsive to electric pulse6 and light.7 However, these systems still appear to be quite far from playing a role in mammalian genetic therapies and as such, they will not be examined here.

Chimeric, Ligand-Inducible Systems1, 8 The next generation of regulatable systems utilized a combination of prokaryotic and eukaryotic elements in their construction in order to overcome much of the difficulties encountered by the early systems.

These systems all exhibit a transactivator consisting of ligand binding, DNA binding, and transcriptional activation domains. As such, these systems have been designed to function specifically in response to an effector ligand.

Figure 1: Tetracycline Regulated System

sequences) and subsequent transcription from a downstream minimal promoter corresponding gene.

This system has also undergone some degree of modification from its original form. Wang et al. reported an additional mutation to the PR-LBD resulting in a novel regulator, which exhibits greater sensitivity to its ligand. Additionally, exchanging the VP16 activation with a KRAB transcriptional repressor, a negatively regulated mifepristone system was created.5 Although these improvements increase its versatility for gene therapy applications, the inherent drawback of the mifepristone system lies in its slow de-induction rate (as a function of the long half-life of RU-486).14

Figure 2: Mifepristone Regulated System

Ecdysone Regulated System15 The ecdysone regulated system (ERS) utilizes the insect hormone ecdysone and its related receptors.

Unlike the previous systems, the ERS requires expression of two components; the modified ecdysone receptor (VpECR) and retinoid X receptor (RXR). The modified ecdysone receptor consists of a fusion between HSV VP16 and a mutant ecdysone receptor with an altered DNA binding specificity.

RXR is the mammalian homologue of the product of the ultraspiracle gene (USP). As with the other systems, a gene of interest is controlled by a minimal promoter, here downstream from 4 repetitions of a unique response element (ECRE). In the presence of ecdysone (or muristerone) a heterodimeric DNA binding complex (consisting of 2 elements and ligand) is activated, initiating transcription.

The ecdysone system offers significant benefit in gene therapeutic application over other systems. First, the steroids such as ecdysone are quickly distributed and cleared from biological systems, resulting in lower basal activity and higher inducibility than tetracycline systems. In addition, No et al. report that the system’s inducing agent, muristerone, exhibits very little toxicity in mice. However, this system requires two separate components, making it less attractive in application where one must condense various genes into a single vector.

Mifepristone (RU486) Regulated Systems13 The mifepristone system begins with a mutant human progesterone receptor (hPRB891) ligand binding domain exhibiting a 42-amino acid deletion at its C-terminal domain (PR-LBD). This mutant receptor can no longer bind progesterone, though it retains the ability to bind progesterone antagonist, mifepristone. The system’s transactivator (GLVP) was constructed through fusion of the mutant PRLBD with yeast GAL4 DNA-binding domain and HSV viral protein 16 (VP16). Herein, mifepristone induces binding of GLVP to a GAL4 binding domain (consisting of a repitition of 4 GAL4 binding

Figure 3: Ecdysone Regulated System

Rapamycin Regulated System16 The rapamycin system is one of many dimerizerregulated gene expression system.

Since the rapamycin system is most commonly used, and the analogous systems follow the same basic functional composition, we will look at it in detail. This system utilizes a chimeric DNA binding domain consisting Volume 1 Issue 1

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of the composite ZFHD1 (2 DNA binding modules – a zinc finger pair & a homeodomain) fused to three copies of human FK-506 binding protein (FKBP). The activation domain consists of a portion of the p65 unit of human NF-kB protein fused to a truncated copy of FKBP rapamycin-associated protein (FRAP). As rapamycin is introduced into the system, it stimulates dimerization of the ZFHD1FKBP/p65-NfkB proteins, which subsequently bind to a 12 repetition ZFHD1 sequence and initiate transcription of a minimal promoter.

Once again, the rapamycin system exists as one of many dimerizer-regulated systems (for a complete listing see Pollock et al.16) These systems exhibit high transgene expression, although rapamycin is immunosuppressive, warranting investigation into alternative dimerizers for genetic therapy applications. Similar to the ecdysone system, these systems require expression of multiple components – lending difficulty in integrating all components of a proposed therapy into a single vector.

Figure 4: Rapamycin Regulated System

Ligand-Activated Site-Specific Recombination Systems Cre-ERT – Lox Mediated Recombination This system is based upon the capacity of Cre (cyclization recombination) recombinase to catalyze recombination between two loxP (locus of X-over of P1) recognition sites. Metzger et al. previously reviewed the ability of Cre to invert sequences flanked by inverted loxP sites, and additionally, excise sequences flanked by repeated loxP sequences, or integrate/translocate sequences downstream from single loxP sites (on separate DNA molecules).17 Though these findings work well for permanent genetic manipulation, the system required modification if it could be used for the spatiotemporal genetic manipulation sought by genetic therapies.

To achieve appropriate spatiotemporal regulation using the Cre / lox system, Cre was fused to a mutant ligand binding domain of human estrogen receptor.18 This chimeric Cre-ER could be activated by the synthetic ligands tamoxifen and 4-hydroxytamoxifen, however not by endogenous estrodiol. Thereby, an effector ligand may be used to control Cre mediated loxP recombinations.

Thus far the Cre/lox system has found the most utility in the study of gene functions as a tool for creating permanent gene manipulations. In regards to gene therapy, the system suffered from a major drawback in that it requires the use of separate vectors. However, Kaczmarczyk recently reported a modified version of Cre, CREM, which is not expressed in prokaryotic cells but is expressed in eukaryotic cells.19 Hence, this system allows transformation of bacterial cells without excision and therefore one may successfully construct a single vector with both Cre and lox sequences.

Alternatively, it has been shown that when working with lentiviral vectors, one can simply introduce the loxP sequence into the U3 region (which is 14

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replicated upon integration) and thereby achieve complete excision of the vector.20 Conclusion This paper reviewed the mode of action of the most frequently used regulated gene expression systems of late, though many variants do exist. As genetic therapy research progresses, researchers approach a goal of exhibiting the ability to safely and effectively introduce genes in vivo and meticulously regulate their expression. The importance of the latter lies in that the therapeutic effects of introduced factors may largely be affected by prolonged expression. Thus, the ability to induce transient and regulated gene expression is a valuable asset in the development of genetic based therapies. As systems are improved, and the regulatory tool-belt expanded, progress will continue towards gene based cures for hundreds of diseases.

References

1. Agha-Mohammadi, S. and M.T. Lotze, Regulatable systems: applications in gene therapy and replicating viruses. J Clin Invest, 2000. 105(9): p.

1177-83.

2. Feil, R., et al., Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci U S A, 1996. 93(20): p. 10887-90.

3. Bienz, M. and H.R. Pelham, Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter. Cell, 1986. 45(5): p. 753-60.

4. Mayo, K.E., R. Warren, and R.D. Palmiter, The mouse metallothionein-I gene is transcriptionally regulated by cadmium following transfection into human or mouse cells. Cell, 1982. 29(1): p. 99-108.

5. Wang, Y., et al., Positive and negative regulation of gene expression in eukaryotic cells with an inducible transcriptional regulator. Gene Ther, 1997. 4(5): p. 432-41.

6. Rubenstrunk, A., et al., Transcriptional activation of the metallothionein I gene by electric pulses in vivo: basis for the development of a new gene switch system. J Gene Med, 2003. 5(9): p. 773-83.

7. Shimizu-Sato, S., et al., A light-switchable gene promoter system. Nat

Biotechnol, 2002. 20(10): p. 1041-4.

8. Gossen, M. and H. Bujard, Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A, 1992. 89(12): p. 5547-51.

9. Gossen, M., et al., Transcriptional activation by tetracyclines in mammalian cells. Science, 1995. 268(5218): p. 1766-9.

10. Miller, N. and J. Whelan, Progress in transcriptionally targeted and regulatable vectors for genetic therapy. Hum Gene Ther, 1997. 8(7): p.

803-15.

11. Baron, U., et al., Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res, 1995. 23(17): p. 3605-6.

12. Baron, U., et al., Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes. Proc Natl Acad Sci U S A, 1999. 96(3): p. 1013-8.

13. Wang, Y., et al., A regulatory system for use in gene transfer. Proc Natl Acad Sci U S A, 1994. 91(17): p. 8180-4.

14. Serguera, C., et al., Control of erythropoietin secretion by doxycycline or mifepristone in mice bearing polymer-encapsulated engineered cells.

Hum Gene Ther, 1999. 10(3): p. 375-83.

15. No, D., T.P. Yao, and R.M. Evans, Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A, 1996.

93(8): p. 3346-51.

16. Pollock, R. and T. Clackson, Dimerizer-regulated gene expression. Curr Opin Biotechnol, 2002. 13(5): p. 459-67.

17. Metzger, D. and R. Feil, Engineering the mouse genome by site-specific recombination. Curr Opin Biotechnol, 1999. 10(5): p. 470-6.

18. Metzger, D. and P. Chambon, Site- and time-specific gene targeting in the mouse. Methods, 2001. 24(1): p. 71-80.

19. Kaczmarczyk, S.J. and J.E. Green, A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissuespecific amplification of gene expression. Nucleic Acids Res, 2001. 29(12): p. E56-6.

20. Berghella, L., et al., Reversible immortalization of human myogenic cells by site-specific excision of a retrovirally transferred oncogene. Hum Gene Ther, 1999. 10(10): p. 1607-17.

Ronald Alfa is a first-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 Neurosciences department. His interests include neural regeneration, plasticity and neuroimmunology.

Side Effects

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human load on the world and accelerating us to the total consumption of natural resources. This is a problem that is at our doorstep. An article in Science Daily comments that our population growth rate is slowing, which is evidence that we are nearing the total consumption of the planet’s ecosystems.10 The author also commented on the chain reaction of how when populations grew, “the rise of medicine… allowed birthrates to rise faster as populations got more crowded.” We may already be crossing humanity’s limit. Human demand has exceeded nature’s supply since the 1980s and was at 120% of the capacity of the Earth in 1999.11 The convergence of medical advancement, death rate decreases, and expanding ecological footprints points a finger at science. Medicine has contributed to the excessive growth and introduces a rate change that works against sustainable development. But this is a drawback we cannot negotiate with since the pursuit of new treatments eases pain, increases lifespan, and leads back to the extension of happiness. What can be done to counter the growing global ecological footprint, impacted by science, to ensure the future of the human race on earth?

Not all aspects of science add to unsustainable development. Biological science has other applications that are positive for the goal of sustainable development. Ecology is the use of biologically generated tools to study and remedy the effects of civilization on the environment.

It is the portion of biological science that uses knowledge from the medical science and applies it towards the goal of reducing the ecological footprint of humans. This discipline is still underdeveloped mostly because federal funds don’t support ecological biological science as much as the medical sciences are supported. Nevertheless, ecological biological science is in sustainable development’s best interest.

The application of ecological biological science can, in itself, help reduce our ecological footprint.

One objective of the ecological sciences is to study the contaminants that end up in the natural environment. Currently, the EPA studies river bed, forest, and ocean samples using chemistry methods, which use expensive and non-reusable solutions and chemistry devices. Biological methods of detecting contaminants reduce cost and eliminate the use of environmentally unfriendly chemicals and devices from the chemistry analysis.12 Analysis of contaminants in the air and drinking water will make the findings more physiologically relevant to humans. Policies resulting from the findings of contaminant studies will suit a more biologically relevant ecosystem, as it should. Ironically, the mode of contaminant detection described above originated from medical biological science research. Use of biotechnology is not limited to studying contamination, but can also be reducing and preventative of chemicals in manufacturing.

DuPont and Cargill-Dow are currently producing durable fibers by using genetically engineered

bacteria to carry out the job instead of “traditional chemistry.”13 Other companies are working on similar biological generation of fabrics and fibers.

Another plus is that since these products are being synthesized using bacterial enzymes, they are also biodegradable.

Ecological biotechnology can also serve to reverse the damage already done to the Earth. In the case of the Exxon Valdez oil spill, the bacteria Pseudomonas, a microbe that breaks down oil, was used to aid in the clean up. This is a bacteria originally identified by medical science as an infective bacterium. Currently, detoxifying plants are being engineered to pull contaminants from areas destroyed by the chemicals. There are lots of other organisms out there that can be used for our benefit. Methanotrophs are a class of bacteria that can convert the methane from manure into protein, therefore providing a source of food supply. These all lead to remedies for cleanup and regeneration of nature where we humans have left our poison.

The biodetection and bioremediation technology described here is not far from home. The UCSD Superfund Basic Research Program (SBRP) is a model research program that brings nine basic and biomedical science research laboratories together to develop such tools for ecological applications.

Dr. Robert Tukey, program director of UCSD’s SBRP, is working on generating detection methods of arsenic and polycyclic hydrocarbons (PAH), toxicants known to cause cancer. The Tukey lab has studied the human mechanism of exposure of PAHs and has used this knowledge to generate the P450 Reporter Gene System, which is a HeLa cell line that links luciferase to this mechanism14.

Dr. Palmer Taylor heads another research project focused on Acetylcholinesterase inhibitors15.

Organophosphates are such inhibitors that are a component of pesticides. Exposure to this class of toxins causes paralysis and brain damage. Taylor is using the knowledge generated from his research to generate a user-friendly organophosphate detection system that can be placed in the field. Dr. Julian Schroeder has been working with Phytochelatin Synthase, a protein that is responsible for worm, fungus, and plant resistance to harmful heavy metals. The Schroeder lab found that phytochelatins chelate these metals and sequester them from their mechanism of exposure. He is now generating Arabidopsis plants that transgenically express phytochelatins that can pull out these toxins from soil16. This is an excellent model institution that incorporates the fruits of basic research into assays that can be used to improve environmental health with relevance to mankind.

The innovation of biological science is a double bladed sword for the goal of sustainable development. On one hand, the part devoted to medicine increases the load on the ecosystem and increases the already too large human ecological footprint. On the other, ecological science generates more efficient use of natural resources and remedies the destruction we have implemented on the environment. The current policy favors the medical sciences. This is fine, since it fulfills our species’ goals. However, if we wish to continue developing the way we are, we will need to apply

more effort towards the ecological approaches of improving our lives.

References

1. Lemonick, Michael D. “Drano for the Heart.” Time 17 November 2003: 60.

2. Gorman, Christine. “Stub Out That Butt!” Time 19 January 2004: 147.

3. Urbanization Facts and Figures. United Nations Human Settlements Program. 20 January 2002 <http://www.unchs.org/mediacentre/documents/ backgrounder5.doc>.

4. El Feki, Shereen. “Getting a grippe.” The Economist: The World in 2004 December 2003: 126.

5. Egypt: Recent Changes in Population Growth, Their Causes and Consequences.

Electronic Newsletter on Population, Health and Nutrition Issues. The World Bank. 19 April 1995. <http://www.worldbank.org/html/extdr/hnp/hddflash/issues/ 00083.html>.

6. Halfon, Leah. “Population Growth.” Virginia Environmental Quality Index: VCU Center for Environmental Studies. Virginia Environmental Quality Index. 20 January 2004 http://www.veqi.vcu.edu/pop_indic.htm 7. World Development Report 2003. Washington, DC: The International Bank for Reconstruction and Development / The World Bank, 2003.

8. World Resources 1998-99. Oxford University Press, 1998. <http://www.wri.org/ wri/trends/wasting.html>.

9. Rees, William, et al. “Urban ecological footprints: Why cities cannot be sustainable and why they are a key to sustainability.” Environmental Impact Assess Review. 16.4-6 (1996): 223-248.

10. “Human Population Growth Already Slowing.” Science Daily. 7 August 2001. <http://www.sciencedaily.com/releases/2001/08/010807080055.htm>.

11. Wackernagel, Mathis, et al. “Tracking the ecological overshoot of the human economy.” Proceedings of the National Academy of Sciences. 99.14 (2002): 9266-71.

12. Inouye et al. “Biomarker-based analysis for contaminants in sediments/soil: Review of cell-based assays and cDNA arrays.” DOER Technical Notes Collection (ERDC TN-DOER-C19), U.S. Army Engineer Research and Development Center, 2000.

13. Anderson, Alun. “Meet industrial biotech.” The Economist: The World in 2004.

December 2003: 122-123.

14. Jones, J., et al. “Using the metabolism of PAHs in a human cell line to characterize environmental samples.” Environmental Toxicology Pharmacology.

8.2 (2000):119-126.

15. Jennings, L.L., et al. “Direct analysis of the kinetic profiles of OrganophosphateAcetylcholinesterase adducts by MALDI-TOF mass spectrometry. Biochemsitry.

40.37 (2003): 11083-11091.

16. Gong, J-M., et al. “Long-distance root-to-shoot transport of Phytochelatins and cadmium in Arabidopsis.” Proceedings of the National Academy of Sciences. 100 (2003): 10118-10123

For more information on SBRP, please see superfund.ucsd.edu.

Animal Allergies Continued from page 5

devices, remain relatively unhelpful since the minute amounts of allergens able to bypass these barriers are still capable of inducing disease.2,7,8 Due to the failings of these preventative measures, new steps need to be employed to prevent allergic diseases in researchers and laboratory technicians.

A novel strategy to severely reduce the risk of allergic diseases to mammalian laboratory animals would be to develop strains of animals in which the allergen encoding genes have been genetically knocked out. A knockout animal is one in which the genomic copies of a specific gene have been replaced with desired mutant copies by taking advantage of the rare but natural event of homologous recombination.9 Since all laboratory animal allergens are proteins, substituting mutant genes that code for proteins incapable of eliciting allergic reactions in humans could lead to allergen free animals. Besides their utility in the lab, allergen knockout animals would also be a boon to those in the general public who desire a furry pet but suffer from allergic reactions to those animals.

A commercial effort was underway a few years ago to develop allergen knockout cats for the general public. The company, Transgenic Pets LLC, was started by a New York couple that had always wanted a cat, but both suffered from allergies.10 The couple recruited Dr. Xiangzhong “Jerry” Yang, who is head of the University of Connecticut’s Transgenic Animal Facility and successfully lead the first effort in the U.S. to make a clone from an adult farm animal.10 The aim of the cat project was to knockout a single protein present in the skin and saliva of felines, Fel d1, which is responsible for the majority of allergic responses

to the animals.2 Although not vital to the health of the animal, the normal biological function of Fel d1 still remains elusive.2 It is important to note that some individuals afflicted with cat allergies are responsive to at least one of the 12 minor allergenic proteins produced by felines and therefore would garner no benefit from these Fel d1 free animals.2 The cats were originally slated to be available in 2003; however, Dr. Xiangzhong Yang has stated that the project was halted due to a loss of funding (author’s personal correspondence). Although the cat project collapsed, the first genetically engineered pet in history, named GloFish,™ was recently introduced to the North American markets.

These are zebra fish, a popular aquarium pet and valuable research model organism, engineered to express red fluorescent protein (RFP) throughout their bodies; RFP is normally found in sea anemones and coral.11 Despite numerous efforts by public interest groups to outlaw GloFish™ they remain legal in the United States, excluding California.11 The legal success of GloFish™ offers great hope for those who desire other genetically engineered pets to become available to the public, such as allergen knockout animals.

The mouse would be the ideal first target for an allergen knockout animal since the technology and knowledge required for the complicated process are already widely available. Mice are the most widely utilized mammalian model organisms in biological and medical research and therefore are responsible for most reported allergic reactions.1 Two proteins found in the urine, blood serum and epithelium (skin and hair) are responsible for the majority of allergic responses to mice.12 Research has also shown that there could be as many as six other minor allergen proteins that affect smaller subsets of allergic individuals.12 One of the major mice allergen proteins is transthyretin, a prealbumin responsible for carrying thyroid hormones and retinol in the blood plasma as well as cerebrospinal fluid.12,13 Transthyretin null knockout mice (JAX® GEMM® strain B6;129S6-Ttrtm1Wsb/J) have already been developed for neuroscience and nutrition research and are viable and fertile.14 These mice do not show observable abnormalities in thyroid signaling; however, they do exhibit some increased locomotor activity and slightly elevated levels of norepinephrine in their limbic forebrain.13,15 The mice have never been tested to see if they have a reduced allergenic potential. To successfully knockout a single mouse gene it takes around two to three years, therefore the existence of a null mutant for one of the two major murine allergens is an extremely useful starting point for anyone embarking on the mouse allergen knockout project.

The task of creating allergen free knockout animals would be a very difficult, time consuming and expensive undertaking, due to the sheer amount of genes that would need to be rendered nonfunctional. Research on mammalian laboratory animals, other than mice and cats, has shown that multiple major and minor allergenic proteins are present in these species as well.2 A potential setback to this monumental project is the fact that knockout techniques in mammals other than mice are not widely used, and are completely Volume 1 Issue 1

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nonexistent for most species. Also, researchers who study behavior, morphology or function would be unable to perform experiments on the knockout animals if any of these traits were altered. Another important consideration is the fact that some of these allergen proteins might play a vital role in the health or well being of the animal and therefore could not be inactivated. However, even knocking out just the major and non-essential minor allergen proteins in laboratory animals would still be helpful in preventing allergic diseases in many researchers and technicians. These reduced allergenic potential animals could also be made available to members of the public afflicted with allergic diseases so that they could enjoy animal companionship without the need for medication or drastically risk making their conditions worse.

Acknowledgments I would like to thank Dr. Xiangzhong “Jerry” Yang

for his correspondence and Lorraine Kelley for her editorial insight.

References

1. “Preventing Asthma in Animal Handlers.” NIOSH Alerts. No. 97-116 (1998).

2. Wood, Robert MD. “Laboratory Animal Allergens.” Institute for Laboratory Animal Research Journal. 42.1 (2001).

3. Campbell, Neil, Jane Reece, Lawrence Mitchell. Biology: 5th Edition.

Menlo Park: Benjamin Cummings, 1999.

4. Bush, Robert MD. “Mechanism and Epidemiology of Laboratory Animal Allergy.” Institute for Laboratory Animal Research Journal. 42.1 (2001).

5. Laitinen, Tarja MD PhD. “The value of isolated populations in genetic studies of allergic diseases.” Allergy and Clinical Immunology. 2.5 (2002): 375-8.

6. Bush, Robert MD. “Assessment and Treatment of Laboratory Animal Allergy.” Institute for Laboratory Animal Research Journal. 42.1 (2001).

7. Harrison, DJ. “Controlling Exposure to Laboratory Animal Allergens.”

Institute for Laboratory Animal Research Journal. 42.1 (2001).

8. Wood RA, Laheri AN, Eggleston PA. “The aerodynamic characteristics of cat allergen.” Clinical & Experimental Allergy. 23.9 (1993):733-9.

9. Alberts et al. Molecular Biology of the Cell: 4th Edition. New York: Garland Science, 2002.

10. Transgenic Pets, LLC. Transgenic Pets, LLC. April 19 2004 <http: //www.transgenicpets.com/default.htm>.

11. Choi, Charles, Steve Nash. The Scientist: GloFish™ Draw Suit. The

Scientist. April 19 2004. <http://www.biomedcentral.com/news/20040107/ 01/>.

12. Price JA, Longbottom JL. “Allergy to mice. Identification of two major mouse allergens (Ag 1 and Ag 3) and investigation of their possible origin.”

Clinical Allergy. 17.1 (1987): 43-53.

13. Sousa JC, Grandela C, Fernandez-Ruiz J, de Miguel R, de Sousa L, Magalhaes AI, Saraiva MJ, Sousa N, Palha JA. “Transthyretin is involved in depression-like behaviour and exploratory activity.” Journal of Neurochemistry. 88.5 (2004): 1052-8.

14. JAX® Mice Database - B6;129S6-Ttr<tm1Wsb>J. The Jackson Laboratory. April 19 2004 <http://jaxmice.jax.org/jaxmice-cgi/jaxmicedb.c gi?objtype=pricedetail&stock=002382>.

15. Palha, Joana PhD. We have been studying the role of Transthyretin…

University of Minho, Health Sciences School. April 19 2004 <http://ecs20 02.ecsaude.uminho.pt/postgrad/2004/gn/Joana%20Palha-description.htm>.

Ian Nicastro is an SQ staff member. His bio can be found in the staff bio section on page 26.

Darwin: On the Origin of Synapses Eric Chan In this age of biotechnology and synthesis of various areas of scientific study, one dynamic field stands out as a driving force behind a wave of exciting new discoveries. This field is neurobiology, the study of the nervous system. With the deciphering of the human genetic code, one of the last remaining great challenges in human biology is that of the mind, which is by far the most complex system in the body. However, through the application of more and more sophisticated techniques, technology, and ideas, even the extraordinary challenge posed by an organ with over one hundred billion neurons (which by themselves form more connections than there are stars in the universe), can be deciphered. At UCSD, cutting edge research into this intricate science is carried out by the world-class Neurobiology and Computational Neurobiology Research Section.

From left to right: Darwin Berg, Greg Naughton, Arin VanderVorst, and Bill Conroy

One of the leading researchers in this section is Professor Darwin Berg. Under his expert guidance, the Berg lab carries out experiments in the field of synaptic components on neutrons. More specifically, his lab investigates the regulation of these synapses, the tiny gaps between the ends of nerve fibers that are responsible for the transmittance of information from one neuron to the next. Greg Naughton and Arin VanderVorst, two undergraduate researchers working at the Berg Lab under the guidance of senior investigator Bill Conroy, have instrumental roles in Berg’s research in this field. Naughton’s Neuroligin Induced Vesicle Clustering in Rat Hippocampal Neurons and VanderVorst’s Homer: Expression in Chick Ciliary Ganglia not only detail important discoveries on these two young researchers’ parts, but also pave the way for more research, 16

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undergraduate or otherwise. The importance of undergraduate research can be seen by taking a look at both these articles and understanding how they connect with Berg’s main goals.

Naughton’s article is an investigation into the role of a protein, neuroligin. This protein is a postsynaptic cell adhesion molecule of excitatory synapses, and is often found in the brain and spinal cord. In his examination of the role that neuroligin plays, Naughton grew rat hippocampal neurons with HEK293 (human embryonic kidney) cells that expressed neuroligin and compared them to a control group of rat hippocampal neurons that were grown with GFP linked to actin/ EphB2, which did not express neuroligin. The results were dramatic and profound: the rat neurons that were exposed to neuroligin had more numerous and larger vesicle clusters and axon connections than the rat neurons that were not exposed to neuroligin. In one particular assay, there were six times as many vesicles in the neuroligin group as compared to the EphB2 control group. Thus, Naughton demonstrated that neuroligin has an important, if not central, role in getting neurons to develop vesicles and axon connections, as well as synapse formation.

VanderVorst’s research had a decidedly different tack.

VanderVorst examined a different group of proteins, the Homer family of proteins. Unlike neuroligin, the specific role of Homer proteins is better known; it acts as a link between glutamate receptors and neural activity by modulating intracellular calcium stores. However, VanderVorst examined whether or not Homer protein 1a could have a role involving a different type of receptor: nicotinic receptors, which are important in the study of various phenomena such as Alzheimer’s and nicotine addiction. This examination was done by seeing if the formation of Homer 1a protein could be induced in a system with nicotinic synapses- namely, chick ciliary ganglion neurons. Thus, these chick ciliary ganglion neurons were subjected to varying treatments, including addition of nicotine, membrane depolarization via addition of extracellular potassium, and addition of forskolin to increase cyclic AMP levels, all of

which are believed to have an influence on Homer 1a protein levels. Then, VanderVorst examined the effect of Homer 1a expression by subjecting the ciliary ganglion neurons to three different DNA constructs: myc-tagged-Homer1a, with the two control groups being myc-tagged-Homer1c and EGFP-C1. In the end, Homer 1a was not detected despite the use of the treatments. However, the subjection of neuron cells to Homer 1a resulted in Homer 1a being spread across the entire neuron, which only served to underline the puzzling question of exactly what role Homer 1a might have on nicotinic receptors.

Both the research of Naughton and VanderVorst, already interesting in their own right, complement Professor Berg’s overall research objective of trying to understand synaptic regulation and neuronal signaling. Both lines of research delve into the question of synaptogenesis, as both Neuroligin and Homer proteins are instrumental in the creation of synapses. Past that, VanderVorst’s research into nicotinic receptors and Naughton’s research into the postsynaptic aspects of neuroligin are important to the Berg lab because the synthesis of these two (up until now) separate lines of research can lead directly to an answer to the question, “What role do these proteins play in nicotinic signaling?” Even further down the line these two lines of research started

by Naughton and VanderVorst might ultimately lead to an answer to the question, “What role do nicotine receptors have in the hippocampus?” As the hippocampus is believed to be instrumental in the formation of memories, the answer to such a question could very well lead to a more complete understanding of memory and human consciousness itself.

Eric Chan is an SQ staff member. His bio can be found in the staff bio section on page 26.

Neuroligin-Induced Vesicle Clustering in Rat Hippocampal Neurons Greg Naughton Neurons within the central nervous system communicate with one another through connections known as synapses. Little is currently understood about the molecular mechanisms mediating synapse formation within the central nervous system. Our research primarily involves the investigation of postsynaptic cell surface proteins that can interact with presynaptic partners to induce synaptogenesis. Specifically, we are interested in a protein, neuroligin, expressed in a variety of systems. Neuroligin is believed to interact with both the presynaptic and postsynaptic sides of a synapse, mediating presynaptic differentiation, as well as controlling intracellular signaling on the postsynaptic cell. To study the effects of neuroligin, we transfect various DNA constructs into HEK(human embryonic kidney)293 cells. Among these constructs are full length neuroligin and a variety of neuroligin isoforms, as well as pEGFP-actin and other simple cell adhesion molecules to provide a negative control. The transfected HEK cells are cocultured with rat embryo hippocampal neurons. The resulting cell cultures are imaged via a fluorescence imaging microscope. Large bright clustering, above background level illumination, of presynaptic vesicles is indicative of a newly formed active synapse. Neuroligin transfected cells clearly show large vesicle clustering when compared to the levels of clustering in control cells. Generally, neurons within these cultures would simply extend neurites over the HEK cells with no mechanism for communication between the two. Neuroligin, however, seems to induce an “urge” in these neurons to contact and communicate with the transfected HEK cells.

Introduction The external behaviors and internal homeostasis of an organism are coordinately regulated and controlled by the central nervous system (CNS).

This complex “spider web” of millions of cells known as neurons uses electrical conduction and chemical signaling to control simple actions such as breathing, all the way to complex integration of emotions. These neurons connect to one another through connections known as synapses. Chemical signals, known as neurotransmitters, pass from presynaptic neurons to postsynaptic neurons, and in doing so communicate a signal from cell to cell.

It’s the integration of millions of these signals in complex circuits that generate what we have come to understand as the human mind.

Little is currently understood about the molecular mechanisms that underlie synapse formation in the central nervous system. When neurons in the CNS are damaged, they are unable to regenerate new connections due to a variety of factors, including production of chemicals which inhibit cell growth. A solution to this problem in the CNS could mean much help for paralyzed individuals and those with extensive brain or spinal cord damage. The regrowth of CNS neurons is a hot topic in research across the world and involves the study of these cells from the molecular level up to larger cellular interactions.

Our research is concerned with a cell adhesion molecule, neuroligin, concentrated in neurons.

Neuroligins 1-4 constitute a family of cell adhesion membrane bound proteins found primarily in the brain and spinal cord (Ichtchenko et al. 1990, 1996). Expression is low in embryonic brains, but increases rapidly and plateaus when most synapses of the CNS are formed. These molecules have been localized at the postsynaptic side of an excitatory synapse and have been proven to interact with the β-neurexins on the presynaptic side resulting in synapse formation (Dean et al. 2003). Often

colocalizing with PSD-95 and NMDA receptors, neuroligin may play a role in the formation of synapses and the maintenance of synaptic terminals. The protein is enriched in postsynaptic plasma membranes, clustering within synaptic clefts and postsynaptic densities (Song et al. 1999).

Neuroligins consist of an extracellular component, a short transmembrane segment, and a cytoplasmic tail which sits within the postsynaptic membrane.

The extracellular domain is largely homologous to acetylcholinesterases, though neuroligin lacks the necessary amino acids for esterase activity (Ichtchenko et al. 1995). The intracellular domain contains a PDZ binding motif, and is believed to be involved in the scaffolding of cellular signaling proteins. The interactions between neuroligin and β-neurexin, which mediate synapse formation, are not yet fully understood.

Neuroligin is only one of many proteins expressed in developing neurons and mature synapses. By culturing transfected non-neuronal HEK293 cells with neurons, we hope to show the involvement of neuroligin in synapse formation and maintainence. Through a variety of mutants, critical areas of the neuroligin sequence are beginning to be elucidated. Amino acids within the acetycholinesterase-homologous domain are likely involved with β-neurexin binding. Between this domain and the transmembrane segment, the amino acids may be involved in forming multimers with other neuroligins.

Formation of these multimers is likely a necessary step in synapse formation and transduction of a cellular signal to the presynaptic cell. By altering these areas of interest in the protein, the specific actions of the neuroligin extracellular domain may be revealed.

We are a long way from understanding the specifics of neuroligin interactions, but we are beginning to prove that neuroligin is a vital player in synapse formation in a variety of circuits within the central nervous system.

Greg Naughton is an SQ staff member. His bio can be found in the staff bio section an page 26.

Methods and Materials Plasmids: The cDNA encoding rat Neuroligin-1 was subcloned into a FLAG-tagged vector for detection and purification, resulting in NL-fullFLAG DNA. The FLAG peptide was localized to the amino terminus of the protein. A neuroligin isoform was generated in which the intracellular portion of the protein was replaced with a GPI moiety. The cells transfected with this neuroliginGPI linked protein contain an intact extracellular segment anchored to the plasma membrane through the GPI linkage. This protein should form multimers with native neuroligin, effectively tying those proteins up and limiting the signal sent to the presyanptic cell in neurons. Thus, the GPI-linked isoform of neuroligin may be a good candidate for future dominant-negative assays. In HEK cells, this isoform can isolate the effects mediated solely by the extracellular portion of neuroligin. A variety of other control DNAs were used throughout the experiment, ranging from pEGFP-tagged actin to FLAG-tagged EphB2, a tyrosine kinase receptor.

EphB2 is an effective negative control since it is expressed and embedded in the plasma membrane, but is thought to serve different functions.

Cell Culture and Transfection: HEK293 cells were maintained at 37˚ C under 10% CO2 in Dulbecco’s modified Eagle’s medium. The HEK cells were cultured in 10 cm plates until they reached approximately 30% confluency. At this time, the HEK cells were transfected with our various constructs. Cells were transfected via a calcium phosphate precipitation with 16 µg of DNA per plate. The transfected cells were placed in a 3% CO2 incubator for a period of 24 hours.

The cells were then washed, provided with fresh media, and given a few hours to recover before re-plating. Some HEK cells were transfected using a Superfect Reagent (QIAGEN) according to the manufacturer’s protocol. Upon transfection, Volume 1 Issue 1

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Figure 1: Rat Neuroligin-1 Schematic The signal sequence is marked; acetylcholinesterase homologous domain is shown in grey; the transmembrane region is shown in black; and the cytoplasmic region is marked. Splice variants result from alternative splicing in 2 sites within the acetylcholinesterase homologous domain.

the cells were treated in the same fashion as the calcium phosphate protocol.

Neuronal Cocultures and Immunocytochemistry: Cocultures with the transfected HEK293 cells were performed with E18 (embryonic day 18) rat hippocampal neurons that had been in culture for 1-2 weeks. Hippocampal neurons were cultured as described by Kawai et al. (J. Neuroscience 2002). A drop of transfected HEK cells was added to each well of neurons and then given 48 hours of incubation. After 48 hours of incubation, the cells were fixed with 2% formaldehyde and primary antibodies were then added to the cocultures.

After another 24 hours of 4Ë&#x161; C incubation, secondary antibodies were then added to image the cell stainings. Both primary and secondary antibody incubations were carried out in PBS containing 0.05% Triton X100 and 5% donkey serum. The following antibodies were used for the immunocytochemistry of the cocultures: polyclonal anti-goat-DDDK (Novus) and monoclonal antimouse-Synaptophysin (Sigma).

Image Acquisition and Quantification: Images were acquired via a deconvolution microscope using a 63x objective. Three-dimensional Zsectioned images were collected using a CCD digital camera, and clustering of fluorescence in both the green (FITC) channel and red (CY3) channel were analyzed. For quantification, all images were taken at the same exposure settings.

The images were deconvolved, and 2-D projections through the Z-series were made. Clusters only above a specific threshold value were quantified.

This threshold was well above background levels and was the same value for all images. Areas, pixel intensities, and number of total clusters were all measured. HEK cells were selected at random, but cells of poor health were excluded.

Figure 2: Imaging of HEK and Neuronal Cocultures HEK293 cells were cultured with rat hippocampal neurons and stained for FITC (right) and CY3 (middle). The two images were superimposed (left). Transfections done were neuroliginfull (top), neuroligin-gpi (middle), and GFP-actin (bottom).

Neuroligin-full and gpi-linked neuroligin transfected cells induced vesicle clustering surrounding the plasma membrane, while no such bordering is seen in the GFP-actin transfection used as a negative control.

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Results Neuroligin Induces Presynaptic Vesicle Clustering in Hippocampal Neurons To determine the effects of neuroligin (Figure 1) on presynaptic differentiation, we cocultured rat hippocampal neurons with transfected HEK293 cells expressing neuroligin. The cultures were stained for synaptophysin, a component of synaptic vesicles in presynaptic terminals and imaged using a fluorescence microscope. Images were taken of neuroligin-transfected cells, as well as a neuroligin construct with the extracellular domain linked to a sequence that directs the attachment of a GPI moiety. Control cells were transfected with GFP linked to actin. Neuronal cells and axons were identified through their basic morphology. The hippocampal neurons proliferated well in culture, spreading out abundant processes and growth cones throughout. Healthy HEK cells not fully enveloped in axonal processes were selected for imaging. The control cells showed little clustering of synaptic

vesicles (Figure 2). Axons often contacted these HEK cells, but the contacts were limited and the axons seemed to be simply crossing over them to other destinations within the culture. Occasionally, bright clusters were found on control cells, but this clustering was limited and appeared to occur at random. Neuroligin-transfected cells showed vesicle clustering in large numbers and increased size. Axons formed extensive contacts with these cells, often wrapping around the border of an entire HEK cell. The synaptophysin label was much more prevalent on the neuroligin-transfected cells than on the controls. The neurons, rather than simply passing over the cells, formed an array of connections across the entire portion of exposed plasma membrane. In many cases, these clusters were much larger than anything imaged in the control cell cultures. The GPI-linked neuroligintransfected cells showed levels of vesicle clustering above those of the control. The protein was able to get out into the membrane and induce presynaptic differentiation, despite a complete lack of the native intracellular sequence. Neuronal axons formed extensive connections and often bordered the entire plasma membrane of these cells. These effects are similar to that seen in the full-length neuroligin transfections.

Figure 3 shows the development of hippocampal presynaptic terminals on neuroligintransfected cells and control EphB2-transfected cells. Upon imaging, the effects of neuroligin versus the control were very clear. The clusters on transfected HEK cells were much more plentiful than on the control. The hippocampal neurons formed an extensive border of synaptic connections around the entire HEK cell as seen in Figure 3.

This trend of neuroligin induced vesicle clustering on presynaptic terminals was visualized in 8 of 10 assays performed. The 2 assays in which this trend was not seen, likely had a defect in staining, rather than a lack of neuroligin activity. A distinctive trend

Figure 3. Cocultures of HEK and Hippocampal Neurons The column designations are: left is both FITC and CY3, middle is CY3, and right is FITC. The top row is neuroligin-transfected cells, while the bottom row is the control, EphB-2. An intense bordering of synaptic vesicles can be visualized wrapping around the neuroligin cell.

of neuroligin’s actions in synaptic formation is observed here. In the control, EphB2 transfections, little to no vesicle clustering occurs. Neurites that contact these cells extend to other areas of the culture, unlike the extensive connections found in the neuroligin-transfected cells.

In a pilot study, we attempted to quantify data for one assay in which two transfections were performed, neuroligin-full and EphB2. A six fold increase in the number of vesicles was observed between the control, 3.8 +/- 1.5 (mean +/- S.E.M.), and the neuroligin transfections, 24.8 +/- 10.4 (mean +/- S.E.M.). Less significant differences were observed for both area of clusters and fluoresence intensity. The data here does not appear to be statistically significant (p value of 0.08, students t test), but a trend of neuroligin induced vesicle clustering definately exists. The sample size for quantification of this assay was 5 cells per transfection, and an increase in sample size would likely prove this data to be significant.

Discussion We have shown in our assays that neuroligin is capable of inducing presynaptic differentiation in CNS axons. Specifically, non-neuronal cells transfected with neuroligin are capable of clustering synaptic vesicles in hippocampal neurons. Vesicle clustering is an indication of a forming synapse.

The acetylcholinesterase-homologous domain appears to be the primary sequence of the peptide involved in interactions with the presynaptic cell. This domain on the amino terminus of the protein is believed to be functionally important in synaptogenesis. This region most likely is involved with binding to a presynaptic receptor. Upon binding, a signal can be sent within the presynaptic terminal to trigger vesicle clustering and other cellular responses to synaptogenesis.

In hippocampal neurons, neuroligin induced vesicle clustering above control levels. The ability of neuroligin to induce presynaptic differentiation depends solely on the extracellular sequence.

When the intracellular portion of the protein is removed, as in the GPI-linked transfections, large bright clustering of synaptophysin is still seen in culture. The acetycholinesterase-homologous domain is interacting with the presynaptic cell in a manner that attracts the formation and clustering of vesicles of neurotransmitters, effectively forming a functional presynaptic terminal.

Addition of soluble β-neurexin inhibits the activity of neuroligin (Scheiffele et al. 2000). Thus, a reasonable model for neuroligin action is the binding of the acetylcholinesterase-homologous domain to presynaptic bound β-neurexin. This binding may act to line up pre- and postsynaptic terminals in preparation for synapse formation.

Through a series of protein -protein interactions, when activated by neuroligin, β-neurexin may recruit neurotransmitter vesicles to the newly forming synapse. The cytoplasmic tail of βneurexin can interact with the synaptic vesicle protein synaptotagmin, as well as other intracellular signaling molecules such as CASK/Lin-2 (Hata et al. 1996). The molecular mechanisms of this, however, have yet to be fully explained.

The addition of neuroligin to non-neuronal cells clearly induces vesicle clustering in neurons of the CNS. The process by which this occurs, both presynaptically and postsynaptically is not fully understood. At times, control HEK cells seem to induce levels of clustering associated with neuroligin-transfected HEK cells.

This sometimes results when the cultures are overgrown with numerous neuronal processes. The increased numbers of axons produce the effect of what appears to be vesicle clustering on non-neuronal control cells. Another consideration, however, is

that neuroligin may not be the only player in CNS synapse formation. While HEK cells transfected with neuroligin appear to induce vesicle clustering above control levels, other cellular molecules may be at work here. The increased expression of neuroligin may increase the likelihood of synapse formation, but it may not be the determining factor for whether or not a synapse will form at that specific area on a cell. Neuroligin itself did not appear to cluster dramatically in transfected HEK cells directly opposing presynaptic vesicle clustering. Rather, the protein appeared to spread evenly across the membrane. Neuroligin may be “sensed” by the growing axon via interactions with β-neurexin and induce synaptogenesis. It may not be required in large concentrations at very specific sites in the postsynaptic cell. These are mechanisms which need to be revealed and are actively being researched.

Discovery of the mechanism by which neuroligin operates and CNS synapse formation occurs will be a giant step in molecular biology research, as well as clinical studies. While perhaps a long way off, elucidation of the molecular mechanisms of synapse formation could lead to clinical applications. A severely paralyzed or brain-damaged individual could have gene expression altered to produce the necessary players to help regenerate CNS synapses within the human brain and spinal cord. The possibilities of this are exciting to many scientists, and perhaps life altering to many others.

Acknowledgments I thank everyone in Dr. D.K. Berg’s lab for assistance with my assays and the writing of this paper. I also thank B. Conroy for development of the DNA constructs, assistance throughout the development of the neuronal coculture assay, and for much help in generation of this text.

References

1. Dean, C., Scholl, F.G., Choih, J., DeMaria, S., Berger, J., Isacoff, E., and Scheiffele P. (2003). Neurexin mediates the assembly of presynaptic terminals. Nat. Neurosci. 6, 708-716.

2. Gilbert, M., Smith, J., Roskams, A.J. and Auld, V.J. (2001). Neuroligin 3 is a vertebrate gliotactin expressed in the olfactory ensheathing glia, a growth-promoting class of macroglia. Glia 34, 151-164.

3. Hata, Y., Butz, S., and Sudhof, T.C. (1996). CASK:a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependant protein kinase domain identified by interaction with neurexins. J. Neurosci. 16, 24882494.

4. Hata, Y., Davletov, B., Petrenko, A.G., Jahn, R., and Sudhof, T.C. (1993). Interaction of synaptotagmin with the cytoplasmic domains of neurexins. Neuron 10, 307-315.

5. Hirao, K., Hata, Y., Ide, N., Takeuchi, M., Irie, M., Yao, I., Deguchi, M., Toyoda, A., Sudhof, T.C. and Takai, Y. (1998). A novel multiple PDZ domain-containing molecule interacting with N-methyl-D-aspartate receptors and neuronal cell adhesion proteins. J. Biol. Chem. 273, 2110521110.

6. Ichtchenko, K., Hata, Y., Nguyen, T., Ullrich, B., Missler, M., Moomaw, C., and Sudhof, T.C. (1995). Neuroligin 1: a splice site-specific ligand for beta-neurexins. Cell 81, 435-443.

7. Ichtchenko, K., Nguyen, T., and Sudhof, T.C. (1996). Structures, alternative splicing, and neurexin binding of multiple neuroligins. J. Biolo. Chem. 271, 2676-2682.

8. Kawai, H., Zago, W., Berg, D.K. (2002). Nicotinic alpha 7 receptor clusters on hippocampal GABAergic neurons: regulation by synaptic activity and neurotrophins. J Neurosci. 22(18), 7903-7912.

9. Scheiffele, P., Fan, J., Choih,J., Fetter, R., and Serafini, T. (2000).

Neuroligin expressed in non-neuronal cells triggers presynaptic development in contacting axons. Cell 101, 657-669.

10. Song, J.Y., Ichtchenko, K., Sudhof, T.C. and Brose, N. (1999).

Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses. Proc. Natl. Acad. Sci. U.S.A. 96, 1100-1105.

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Homer: Expression in Chick Ciliary Ganglia Arin VanderVorst The Homer family of proteins are scaffold proteins that have been demonstrated to affect the localization of metabotropic glutamate receptors and interact to modulate the intracellular signaling cascades that involve calcium stores. There are three forms of Homer proteins that are named: Homer 1a, Homer 1b, and Homer 1c. Homer 1b and 1c protein sequences are substantially longer than Homer 1a and contain two leucine zipper motifs that have a coiledcoil structure, which is thought to induce multimerization. Homer 1a was the protein of interest in this experimentation. It lacks the coiled-coil structure and exhibits dominant negative activity. It is also classified as an immediate early gene, which means it can be rapidly induced by outside stimulation. The experimental goal was to see if formation of the Homer 1a protein could be induced in chick ciliary ganglion neurons, a model system for nicotinic synapses.

Ciliary ganglion neurons were cultured and used to test for the presence of this protein with different treatments. Treatments included nicotine to activate nicotinic receptors, membrane depolarization by increasing extracellular potassium, and forskolin to increase cyclic AMP levels. Induction of Homer protein after each treatment was determined by polyacrylamide gel electrophoresis and western blotting. No Homer 1a band was detected. Problems arose in visualization of Homer protein bands because of interference from serum protein bands that were the same size. The effect of Homer 1a expression in these neurons was also tested by over- expressing Homer 1a by transfection of Homer DNA into the neuron. A myc-tag on the expressed protein allowed for visualization by fluorescence microscopy. Homer 1a was highly expressed and diffusely distributed throughout the neuron and all its processes.

Introduction The thickening of the postsynaptic membrane of excitatory synapses in the CNS is termed the postsynaptic density (PSD). The PSD at glutamatergic synapses contains glutamate receptors and other proteins that are anchored in a matrix of signaling molecules and cytoskeleton (Sala et al., 2001). Scaffold proteins make up part of this dense material. Scaffold proteins allow assembly of polypeptides with different catalytic activities as one unit. The arrangement of polypeptides in this way allows the products of one reaction to be channeled straight to the next enzyme in a pathway (Lodish et al., 2004). Protein scaffolds are necessary for facilitating downstream intracellular signaling at synapses and organizing postsynaptic structures. Protein scaffolds increase the rate and spatially restrict signaling cascades by organizing components into closely located groups that can be easily activated. Many neurotransmitter and ion channels are assembled onto protein scaffolds at the synaptic membrane (Fagni, Worley, and Ango, 2002).

The Homer protein family are scaffold proteins that assist in the assembly of receptors and calcium channels at the plasma membrane. It has been shown that they bind clusters of proteins and glutamate receptors at these postsynaptic locations. Homer proteins prevent or promote spontaneous activation of glutamate type 5 or 1 receptors (Fagni, Worley, and Ango, 2002).

There are three possible hypotheses to explain the function of Homer proteins. The signaling hypothesis states that Homer regulates the coupling of membrane receptors by using intracellular stores of calcium. The Homer proteins could play a role in synaptogenesis and spatial placement of metabotropic glutamate receptors. Receptor trafficking could also involve Homer proteins (Xiao, Cheng Tu, and Worley, 2000).

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Homer 1a is 186 amino acids long. It is encoded by a 6.5 kb mRNA (Fagni, Worley, and Ango, 2002). It has been shown that Homer 1a is an immediate early gene expressed rapidly after electrical stimulation or seizure in rat brain.

Its production leads to long term potentiation of hippocampal glutamatergic synapses. Expression of Homer 1a can be induced by various reagents: such as potassium channel blockers and high concentrations of KCL in rat brain. Neuronal activity only induces the expression of Homer 1a, not Homer 1c or Homer 1b. Induction of Homer 1a expression increases the number of mGlu5 receptors in the dendrites and axon of rat neurons.

In cerebellar cells the expression of Homer 1a increases latency and duration of receptor mediated calcium responses. This Homer protein may be involved in coupling of intracellular IP3 or ryanodine sensitive calcium stores (Fagni, Worley, and Ango, 2002).

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed throughout the nervous system and help to influence many events in the cell, but few scaffold proteins have yet been identified for this type of receptor in neurons.

Recently, the PSD-95 family of scaffold proteins has been found to be associated with certain nicotinic receptors. The PSD-95 proteins were first identified at glutamatergic synapses and may play a role in maturation at these sites. The postsynaptic PSD-95 complex may also be essential for nicotinic synapses; disruption of PDZ interactions reduces synaptic activity and nicotinic signaling (Conroy et al., 2003). Homer may be a direct player in the scaffolding organization involved with nAChRs. nAChRs are excitatory, ligand-gated, cation selective ion channels. They can permit significant amounts of calcium entry and are involved in different types of calcium signaling events as a consequence. The α7-containing nicotinic receptors participate in transmission as well as

Arin Marie VanderVorst is a 4th year General Biology Major in Muir College. Arin has been assisting in Dr. Darwin K. Berg’s neurobiology laboratory for two and a half years. She is pursuing a career in medicine and wishes to specialize in obstetrics.

regulatory signaling in chick ciliary ganglion (Berg and Conroy, 2002). The PDZ proteins at nicotinic synapses also function to organize cellular components that are required for calcium signaling.

Disrupting PDZ interactions decreases the ability for receptors to generate long-term pCREB (Chang and Berg, 2001). Calcium release from intracellular stores is also necessary for induction of long-term pCREB so, PDZ scaffold could also be involved in organization of calcium reservoirs (Chang and Berg, 2001). The PSD-95 family could be only the beginning of a vast array of scaffold proteins that are involved in intracellular signaling. The ability of Homer proteins to bind glutamate receptors and effect intracellular calcium suggests that it may also be involved in intracellular signaling.

An appropriate system for analyzing nAChRassociated signaling is the chick ciliary ganglion (CG). These neurons express high numbers of receptors and organize them into easily identifiable and distinctive structures. Culturing of these neurons is understood so they can be easily kept alive in vitro. Heteromeric receptors containing α3, α5, β4, or β2 subunits have been found to concentrate in the postsynaptic density and on somatic spines. α7-nAChRs are more abundant, but are not located on postsynaptic densities.

Each receptor has been found to contribute to fast synaptic transmission and drive downstream signaling (Conroy et al., 2003). The function of

Figure 1: Blot: Pan Homer Ab Western blot showing presence and size of Homer proteins in CG. Homer 1c is present in E14 CG sample, but Homer 1a is not. Homer 1c and Homer 1a protein samples came from transfected HEK293 cells. CG sample came from a dissociated E14 culture. Arrows indicate position of Homer 1c and Homer 1a proteins. Molecular weight markers are shown to the left.

Homer as a scaffold protein is explored here.

Experimentation strove to characterize the role of Homer and it‘s effects on downstream intracellular signaling involved with nicotinic receptors.

Materials and Methods CG cultures: Ciliary ganglion cultures were made from dissociated E8 and E14 (embryonic day 8 and 14) ciliary ganglia. Each E8 culture was maintained for 5-7 days and plated on glass coverslips with poly-D-lysine, fibronectin, and lysed fibroblasts.

E14 cultures were maintained freshly dissociated for 1-24 hours on coverslips with no fibroblasts.

Transfections: CG neurons were allowed to attach to the coverslip for one hour then DNA constructs were transfected in using Effectene according to the manufacturers protocol (Qiagen) and allowed to incubate for 24 hours. Culture media was then replaced and cells were analyzed 5-7 days later. DNA constructs: myc-tagged-Homer1a (mycH1Awt), myc-tagged-Homer1c (mycH1Cwt), EGFP-C1. The myc-tag is an epitope tag that codes for a gene sequence that is specific for the 9E10 antibody used and allows for visualization with fluorescence microscopy.

Polyacrylamide gel electrophoresis (PAGE) and Western Blotting: 10% PAGE gels were run, blotted to nitrocellulose, and probed with a rat Homer primary antibody (1:1000) and an antirat secondary (1:1000). Blots were treated with ECL substrate applied to membrane and film was exposed and developed.

Fluorescence Microscopy: Long and short term treatments were washed in a Ringer’s solution and fixed with 4% paraformaldehyde (PFA) for 20 minutes. To label cells for the presence of DNA transfected constructs, E8 and E14 cultures were first treated with a 9E10 primary antibody (1:1000 concentration) which was incubated over night in PBS with Triton and 5% donkey serum. 9E10 is a monoclonal antibody that is specific for the myctag. After washing with PBS, cells were incubated with donkey Cy3 secondary antibody. After washing with PBS, each coverslip was mounted with DAPI (to stain the nucleus) and visualized using a 63X, 1.4NA objective on a Zeiss Axiovert equipped with CCD camera and digital imaging with Slidebook deconvolution software.

Figure 2: Blot: Pan-Homer Ab Western blot to detect Homer 1a in treated E8 CG cultures. Arrows indicate the approximate size of Homer 1a and 1c proteins. The presence of Homer 1a can not be determined because a protein band in the culture media is present at approximately the same size. A protein band that is approximately the same size as Homer 1c can be seen. None or slight inhibition (1a form) and no increased stimulation of either form is detected because all bands are at relatively the same intensity. This blot is representative of blots from 3 experiments.

Eggs: All embryos used were White leghorn chick embryos. They were maintained in a humidified incubator until use.

Homer detection in E8 cultures: Dissociated E8 cultures were transfected and allowed to incubate for 5-7 days. Upon which time the culture was treated with a number of different stimulations to detect an increase or a decrease in Homer 1a expression including: TTX (tetrodotoxin) which is meant to stop all cell receptor membrane activity, 10μM nicotine to activate nicotinic receptors, 25 mM potassium to depolarize the membrane, 100μM d-tubocurarine (dTC) which blocks all nAchRs, and 10μM forskolin to increase cyclic AMP levels. Short term stimulations were washed with a ringers solution (containing 150mM NaCl, 2.5mM KCL, 2mM CalCl2, 1mM MgCl2, 10mM glucose and 10mM HEPES). Stimulations were added for 5 minutes, washed in ringers solution, incubated with dTC for 20 minutes-4 hours, and detection of Homer was done by polyacrylamide gel electrophoresis and western blotting or the coverslips were fixed and stained to visualize with fluorescence microscopy.

Homer detection in E14 cultures: Freshly dissociated E14 cultures were prepared on the same day as treatment. Stimulations were added immediately after culturing and removed at 1, 2, and 4 hour timed intervals. In this method, each sample was tested for Homer 1a by polyacrylamide gel electrophoresis and western blotting. Testing with fluorescence microscopy required each sample to be washed with ringers solution and cadmium, stimulated for 5 minutes, and washed and fixed for short term or long term stimulation. Treatments included: 5μg/mL cyclohexamide to inhibit protein synthesis, 10μM U0126 to inhibit MEK, 1μM BAPTA-AM to chelate intracelluler free calcium, 10μM nicotine to activate nicotinic receptors, 25μM potassium to depolarize the membrane, and 10μM forskolin to increase cyclic AMP levels.

Results Detection of Homer with western blotting: Dissociated E14 CG neurons were collected from culture and processed with SDS-PAGE and western blotting to test for the presence of Homer proteins.

Figure 1 illustrates the size of Homer 1a (30kD) and Homer 1c (50kD). This sample of Homer protein was taken from HEK293 cells (human embryonic kidney) transfected with rat Homer 1a and 1c cDNA constructs, as a positive control. Figure 1 verifies that the rat Homer antibody used to probe each blot recognized both the Homer 1a and 1c proteins in the HEK293 sample. The antibody used was not specific for one type of Homer. It is able to recognize all forms because it binds to the aminoterminus of each Homer protein, which is relatively homologous in all sizes (Fagni, Worley, and Ango, 2002). In the CG sample no bands the size of Homer 1a could be detected, but a band the size of the larger Homer isoform can be seen.

Homer 1a is an immediate early gene that can have expression induced by various stimulation.

Figure 2 shows the results from a western blot examining the expression of Homer proteins of an

Figure 3: Blot: Serum Band Western blot demonstrating the presence of immunoreactive serum protein bands that are similar in size to Homer 1a. The rat antibody used to probe for the Homer constructs also recognized a band in the serum of the medium used to culture the CGs. The antibody did not recognize protein in the eye extract or pure medium. Testing for the presence of Homer 1a was hindered by this interfering band. Arrows indicate serum proteins in pure serum sample and in culturing media.

E8 CG culture that was stimulated with tetrodotoxin (TTX) to block action potentials, d-tubocurarine (dTC) to block synaptic responses, and nicotine (10μM) to stimulate nicotinic receptors. The cultures were treated overnight, washed and collected the next day. The western blots of these cultures showed intense bands at the approximate size expected for Homer 1a. The intensity of the Homer 1a sized band seems to be equal or slightly inhibited throughout the gel. The culture medium, with no CG present, also had an immunoreactive band at the same size. The presence of Homer 1a can not be ruled out. The horse serum in the media (Fig.2 lane 5) and Homer 1a production could be equally contributing to the band seen in figure 2. Further testing is required to conclude on this possibility. Time periods of treatment were extended from 4 hours to overnight to possibly give Homer protein more time to be translated, still no Homer 1a expression was detected. Other treatments to stimulate Homer 1a expression were tried with similar results, these included incubation in high extracellular potassium (25 mM) to depolarize the membrane cells and stimulation of cyclic AMP by incubation with forskolin. Larger Homer forms were also present in the E8 treated culture. Double bands at the 45kD marker were repeatedly found in trials. The intensity of each band did not seem to change, so any stimulation or inhibition was inconclusive.

The components of the complete medium were tested to see where the immunoreactive band originated and whether the culture conditions could be changed to prevent further disruption.

The rat antibody that was used to probe the gels also recognized a protein in the medium (Cellgro Minimum Essential Medium). This protein was found to be in the horse serum that was added to the media (Fig.3). Pure eye extract (a growth supplement that is also added to medium) and medium alone did not produce a reactive band at the 30kD size for Homer 1a. Pure horse serum and horse serum added to growth medium did produce a band of relatively the same size as Homer 1a. The neurons were tested to see if they could grow in the absence of serum, but they could not. Chick and rat serums supported the growth of neurons, but the rat serum also produced an interfering band. Future experiments can now be done by substituting the Volume 1 Issue 1

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chick serum for the horse serum. A mouse antibody was purchased and tested to see if it also reacted with the medium being used. This antibody did not react with other medium bands, but it could not be used because it was not specific for Homer 1a. Interfering media bands that were the same size as larger Homer forms were not recognized by the rat antibody used. Figure 3 shows that no bands are present in any sample lane at the 50kD size of Homer 1c.

Figure 4: CG neurons transfected with rat Homer constructs Both Homer isoforms are distributed throughout the neuron and it’s processes. Clusters of Homer 1c are more concentrated in spines and filopodia. Homer 1a is more clustered intracellularly. Yellow arrows indicate processes from the transfected neuron. White arrows indicate processes from other transfected neurons.

Over-expression of Homer in CG Neurons: Over expression of Homer 1a in CG neurons is another method that was used to examine the functions of these proteins in nicotinic synapses and nicotinic receptor signaling. The first step in the experiment was to see if the neurons could be transfected with DNA that would express the protein, whether the protein could be detected, the exact distribution of the over-expressed protein, and whether the over-expression had any effect on the health of the neuron. Over-expression of rat Homer 1a and 1c in CG neurons was done by transfection of E8 ciliary ganglion cultures using Effectene (Qiagen). Cultures were allowed to incubate for 5-7 days after transfection to allow the protein to be expressed. The Homer cDNA constructs allowed for visualization by tagging the expressed protein with a myc epitope that can be detected with a specific monoclonal antibody (mAb 9E10). Homer 1a was highly expressed. It was distributed throughout the neuron and all of its processes. Figure 4 demonstrates the distribution of Homer 1a throughout the neuron. The intracellular concentration of Homer 1a is more abundant than the distribution of Homer 1a in the processes of the neuron. Small intracellular clusters were found in the soma (cell body). Figure 4 also verifies that transfected neurons were still healthy after transfection and still had their processes intact.

Over expression of larger forms of Homer (Homer 1c) was also done by transfecting E8 CG cultures with Homer 1c DNA. Homer 1c was also widely expressed, but distributed slightly different than Homer 1a (Fig. 4). Homer 1c had similar sized clusters to Homer 1a but it was distributed more toward the outer plasma membrane of the neuron and into it’s processes. Homer 1c can be seen in the spines and filopodia (a thin, foot-like process protruding from a cell) of the neuronal processes.

Processes of neighboring transfected neurons can also be seen which might be synapsing with the transfected neuron in figure 4.

Discussion The initial characterization and participation of Homer proteins as scaffold proteins in nicotinic receptor signaling and nicotinic synapses was explored here. The expression of a long form of Homer was detected by western blotting, but the expression of the short form, Homer 1a was inconclusive. The over-expression of Homer isoforms in CG neurons, tried here for the first time, will allow future experiments to investigate the roles of Homer proteins in nicotinic signaling and synapses.

The large form of Homer (Homer 1c) was expressed in the CG sample of the E14 culture 22

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and in treated E8 cultures. The rat antibody used was not specific for certain forms of Homer so the correct form can not be detected here. The antibody could detect Homer 1, Homer 2, and Homer 3. The next step for the detection of larger Homer forms could be to obtain a specific antibody and test for the presence of each long form.

Homer 1a was not present in the E14 CG sample. This could be due to the dominant negative activity that it expresses. It has been found to be expressed in “stressful” situations, like electroconvulsive seizure (Fagni, Worley, and Ango, 2002). It was hypothesized that the stress the CG experiences when being cultured may be enough to induce the formation of Homer 1a. This hypothesis proved to be false. More interaction must be needed to produce the protein.

Homer 1a might have been expressed in the treated E8 culture, but the interfering protein originating from the horse serum used in culturing did not allow a conclusive test. Steps to remove the interfering serum band must be taken before further analysis. In the future, chick serum can be used as an alternative to the horse serum because it supports the growth of the neurons and does not have interfering bands. A new, more specific antibody could be obtained and tested to see if it interacts in the same way as the one used. A new technique for testing the presence of Homer 1a could also be used that doesn’t require probing with antibodies.

The function of Homer proteins in CG neurons was tested by over-expressing the rat Homer proteins. Neurons transfected with Homer cDNA constructs and cultured for 5-7 days showed high expression of Homer proteins. Fluorescence microscopy was used to test Homer 1a and Homer 1c distribution. Interestingly, the two isoforms had different distributions within the neuron. The difference in distribution observed for the two forms could have been due to their differences in structure. The coiled-coil domain of the Homer 1c form may have had some interaction with the cytoskeleton of the neuron that caused it to cluster towards the plasma membrane. The spines and filopodia of the neuronal processes might be stabilized by the presence of the Homer 1c sequence, which caused it to cluster there. The Homer 1a protein, lacking this coiled-coil domain, showed clustering that was highly concentrated in the interior of the cell body. Homer 1a is not thought to normally be expressed continuously so it may just appear where it is produced and when it is needed, a different situation from the overexpression over days seen here.

Future experiments will be to test the effects

Homer 1a has on nicotinic receptor distributions and intracellular signaling cascades. Future experiments should try to correlate nAChR distributions with Homer protein expression. This could be done by using antibodies to stain for endogenous Homer and nAChRs or transfecting neurons to overexpress Homer proteins. Does over-expression of Homer proteins alter the distribution of nAChRs? Do Homer 1a and Homer 1c have different effects?

The type of nAChR distribution could also be examined with a pCREB assay in order to correlate the effects that intracellular calcium has on Homer expression and function. Experiments dealing with the over-expression of Homer on nAChR function (electrophysiology) and downstream signaling (activation of transcription/pCREB activation) will provide interesting new information about the roles of these proteins.

Acknowledgments Special thanks to Bill Conroy for all the guidance and supervision with my research project and article editing; without his help none of this would be possible. Thank you to Dr. Darwin K. Berg for opening up his lab to undergraduates and allowing them to pursue their interests.

Thanks to Lynn Ogden for all of her assistance with techniques and protocols, Xaio-Yun Wang and Heather Eshleman for help with blots and gels, Cara Cast for encouraging me to publish my project, Nolan Campbell for help with fluorescence imaging, David Ko and Wagner Zago for help with formatting, and everyone in the lab for making it an inviting environment for learning.

References

1. Berg, Darwin K., and Conroy, William G. “Nicotinic 7 Receptors: Synaptic Options and Downstream Signaling in Neurons.” Journal of Neurobiology. 53 (2002): 512-23.

2. Chang, Karen T., and Berg, Darwin K. “Voltage-Gated Channels Block Nicotinic Regulation of CREB Phosphorylation and Gene Expression in Neurons.” Neuron. 32 (2001): 855-65.

3. Conroy, William G., Liu, Zhaoping., Nai, Qiang., Coggan, Jay S., and Berg, Darwin K. “PDZ-Containing Proteins Provide a Functional Postsynaptic Scaffold for Nicotinic Receptors in Neurons.” Neuron. 38 (2003): 759-71.

4. Fagni, Laurent., Worley, Paul F., and Ango, Fabrice. “Homer as Both a Scaffold and Transduction Molecule.” Science’s STKE (2002), http://www.stke.org/cgi/content/full/sigtrans;2002/137/re8 5. Lodish, Harvey, Arnold Berk, Paul Matsudaira, Chris A. Kaiser, Monty Krieger, Matthew P. Scott, S. Lawrence Zipursky, and James Darnell. ed. Molecular Cell Biology. New York: W.H. Freeman and Company, 2004.

6. Sala, Carlos, Piech, Valentin, Wilson, Nathan R., Passafaro, Maria, Guosong, Liu, and Sheng, Morgan. “Regulation of Dendritic Spine Morphology and Synaptic Function by Shank and Homer.”

Neuron. 31 (2001): 115-30.

7. Xiao, Bo., and Cheng Tu, Jian., and Worley, Paul F. “Homer: a link between neural activity and glutamate receptor function.”

Neurobiology. 10 (2000): 370-74.

The Effect of Calcium Intake and Exercise Intensity on Systolic Blood Pressure in Older Men Pei-Chen (Jennifer) Hsieh To determine whether a higher intake of calcium and overall exercise intensity are associated with lower systolic blood pressure in older men, a cross-sectional study was executed on a total of 301 healthy men aged 54-89 years living in San Diego County, California, who volunteered for a prostate cancer primary prevention trial. Dietary and supplementary intake (by the Selenium and Vitamin E Cancer Prevention Trial food frequency questionnaire), medical history, body mass index (BMI), total energy expenditure (met/week) and blood pressure were examined. We found that a higher intake of calcium from diet and supplements was not associated with a lower systolic blood pressure. The calcium effect was not modified by exercise intensity. While vitamin A, vitamin C, vitamin E and beta-carotene were not associated with systolic blood pressure, BMI and nicotine have a direct relation to systolic blood pressure.

These results suggest that, regardless of total calcium intake and physical activity, systolic blood pressure increases with BMI and nicotine consumption.

Introduction Hypertension is a major cardiovascular risk factor affecting nearly 50 million adults in the United States [1,2]. People with high blood pressure are prone to have cardiac disorders such as angina pectoris, myocardial infarction and heart failure [3]. The deficiencies of principal nutrients such as calcium, potassium and protein, are associated with the prevalence of high blood pressure in the United States based on the analysis of the first National Health and Nutrition Examination Survey (NHANES I) reported in 1984 [4]. It is also known that obesity, sodium intake, alcohol intake, caffeine intake, diabetes, smoking, lack of physical activity and African American descent influence blood pressure [3,5,6].

Systolic blood pressure increases with age, particularly after age of 50 [1]. Many elderly need more than 1 drug to lower systolic blood pressure to below 140 mm Hg [1]. Therefore, it is difficult to achieve the optimal blood pressure level in accordance to any guidelines. This poses a great infliction for “the health and well-being of our society,” and a costly burden for the healthcare payers [11].

Hypertension has been associated with a increased risk of cardiovascular disease in many epidemiologic studies [12]. We know that diet and physical activity are two important determinants of blood pressure [13, 14]. However, while exercise and physical activity are known to reduce the incidence of coronary heart disease, calcium’s blood pressure lowering effect is still uncertain.

However, it is not known whether physical activity is an important adjunct to calcium intake for achieving and maintaining the optimal blood pressure level. The goal of our study was to determine whether non-pharmacologic approaches of a higher intake of calcium and elevated overall exercise intensity can lower blood pressure in older men, and thereby reduce the level of a primary cardiovascular risk factor with possible implications for reducing cardiovascular morbidity

and mortality [15].

A cut-off of 140/90 mm Hg is more widely accepted as a threshold level for hypertension [7,8]. Nonetheless, the Joint National Committee 7 (JNC 7) recently published a new guideline for hypertension prevention and management on May 21, 2003. JNC 7 suggested that the risk of cardiovascular disease (CVD) begins at 115/75 mm Hg and individuals with a blood pressure ³120/ 80 should be considered as prehypertensive and required “health-promoting lifestyle modifications to prevent CVD [9].” Unlike the blood pressure level—140/90 mm Hg— established by the Sixth Joint National Committee (JNC 6) in 1999 [10], the new blood pressure level established

by JNC 7 significantly increased the fraction of people needed to be treated and controlled for hypertension. Therefore, the relative effectiveness of calcium and exercise in many blood pressure reports may be subjected to dispute since the optimal blood pressure level has always been a controversial moving target.

Pei-Chen (Jennifer) Hsieh is a Biology major and Psychology minor at UCSD. She has studied glaucoma with Dr. Pamela A. Sample and was selected by the American Heart Association to work on her own research project. She is the Vice-President of Insight. Her career goal is to become an optometrist.

Coumadin or aspirin over 175 mg were excluded.

The inclusion criteria included participants who were cancer free for 5 years and in good physical and mental health.

This multi-center national clinical research trial is coordinated by the Southwest Oncology group (SWOG). Informed consent was obtained from all participants. This study was approved by the Human Subjects Committee of the University of California, San Diego and its protocol adhered to the Declaration of Helsinki.

Dietary and Supplementary Intake: Usual dietary intake over the past year was assessed by using a self-administered food and frequency questionnaire (FFQ). The questionnaire assessed the average frequency of intake over the previous year. The questionnaire listed 125 individual food items classified into 9 food categories: cereals, breads and snacks, meat, fish and eggs, spaghetti, mixed dishes and soups, vegetables and grains,

Subjects and Methods Subjects: Between August 2001 and June 2003, the Selenium and Vitamin E Cancer Prevention Trial (SELECT) enrolled 865 male participants aged 54-89 years from San Diego County, California.

For this study, we extracted the data from 301 randomly selected subjects. Details of the subjects are given in Table 1.

The purpose of the SELECT study is to determine whether taking supplementary Selenium and Vitamin E can prevent prostate cancer and other cancers. For our study, we extracted the data from the SELECT Dietary Supplement and Food Questionnaire, Health Related Habits Form and Medical History Form.

Exclusion criteria for this study included systolic blood pressure ³160, diastolic blood pressure³90, prostate-specific antigen³4.0, prior prostate cancer, abnormal Digital Rectal Exam (DRE) or stroke. Participants who used Warfarin,

Table 1

Subject Characteristics

n Age (yr) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) Nicotine (pack yrs) Reported physical activity (Met/week) Calcium intake (mg/day) Dietary Calcium intake (mg/day) Supplementary Calcium intake (mg/day) Vitamin A (IU/day) Beta-carotene not from multivitamins (IU/day) Vitamin C (mg/day) Vitamin E (IU/day) a

301 64.26 ± 6.82 28.51 ± 4.80 126.36 ± 13.17 78.07 ± 7.37 10.862 ± 19.502 8.06 ±9.81 602.270 ± 537.68 404.695 ± 440.60 197.575 ± 294.69 3000.88 ± 3811.46 1000.866 ± 2739.051 234.28 ± 361.13 132.97 ± 204.18

Mean ± standard deviation

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Table 2 Pearson Bivariate Correlation Coefficientsa Table 3 Variables SBP and calcium SBP and dietary calcium SBP and supplementary calcium SBP and BMI SBP and weight SBP and exercise SBP and Vitamin A SBP and Vitamin C SBP and Vitamin E SBP and beta-carotene SBP and nicotine a

Coefficient -.018 .0049 -0.0418 .2600 .1880 .0013 -.0425 -.0564 -.0646 .0316 .1444

SPSS Windows Version 6.

dairy products, sauces and condiments, sweets, and beverages and alcohol. Subjects were required to recall the frequency of consumption of each food items (number of times per month, per week or per day), and the serving size (small, medium or large) determined by a standardized proportion size for each food category.

For each participant, we calculated the total dietary calcium intake by multiplying the reported frequency that each of the calcium-rich food was consumed by the calcium content for the specified portion size. The calcium-rich food sources included milk, yogurt, low-fat cheese, other cheese and cooked greens such as spinach and collards.

The conversion from individual food source to its calcium content in mg was based on the Calcium Information Center handout [16] from the certified dieticians in the clinic.

We then added the calcium intake from multivitamins and supplements. The supplement section of the questionnaire estimated the calcium intake in the multivitamin (brands, number of times in the last 10 years, in the last 3 months, days per week, and amount per day if the brand of the multivitamin taken by the participant is not given in the questionnaire) and in the individual calcium supplement (number of times in the past 10 years, days per week, and amount per day). Total calcium intake in mg per day was calculated as dietary and all supplementary intakes.

Antioxidants such as vitamin A, vitamin C, vitamin E, beta-carotene from the FFQ were also assessed as covariates. These variables were calculated from multivitamin and supplement intakes only in a manner similar to that for calcium. Vitamin A, vitamin E and beta-carotene were estimated in IU, and vitamin C was estimated in mg.

Estimation of Physical Activity: We collected the data on physical activity from the Personal Habits Form, which asked the participants to estimate the types of exercise (strenuous, moderate or mild), number of times per week and the time duration of the exercise in minutes. For each participant, we calculated the total energy expenditure by multiplying the reported frequency they exercise per week by the duration. The conversion from the exercise time for each type of exercises to metabolic equivalent was based on the © 2000 Family Practice Notebook, LLC [17].

For the exercise intensity calculation, we used a standardized classification of energy expenditure 24

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p value .744 .933 .470 .000 .001 .983 .462 .329 .264 .585 .012

Partial Correlation Coefficientsa

Variables SBP and calcium controlling for BMI controlling for weight controlling for exercise controlling for nicotine a

Coefficient -.0304 -.0388 -.0189 -.0260

p value .607 .504 .744 .654

SPSS Windows Version 6.1

associated with physical activities and calculated a weekly energy-expenditure score in metabolic equivalents (MET score) for total physical activity [18].

Statistics:The sample population of 301 participants was determined by the power calculation, which was based on an effect size of 5 mm Hg reduction in systolic blood pressure (SBP), a standard deviation of 400 mg in daily calcium intake and two sided alpha value of 0.05 to achieve a power of 0.80.

Statistical analyses were performed by using SPSS Windows version 6.1. For correlations, Pearson’s Bivariate Correlation coefficients and Partial Correlation coefficients were used.

Linear trend was analyzed with simple scatterplot using SPSS. A regression line was drawn to show the association between the predictor variable and outcome variable. R2 more than 0.10 was considered statistically significant.

All tests were two-tailed, and p values less than 0.05 were considered statistically significant.

Results The study population consisted of White, African American, Hispanic, Asian, and others with a mean age of 64 years. The mean BMI and blood pressure of the participants in our study was 28.51 kg/m2 and 126/78 mm Hg respectively. Estimated dietary and supplementary intakes from the food frequency questionnaire are shown in Table 2. For all calcium-rich foods and supplements, intakes varied widely among individuals. The ranges for total calcium intake, dietary calcium intake and supplementary calcium intake are 25.6 - 3992.2 mg/day, 16.5 - 3992.2 mg/day and 0.0 - 1600.0 mg/day accordingly.

Figure 1 presents the best fitting regression models for the association of total calcium intake and systolic blood pressure. The slope of the regression line was very flat, which means that there was no significant association between total calcium intake and systolic blood pressure.

Table 2 shows that there was an inverse relation between total calcium intake and systolic blood pressure. As calcium intake increases, systolic blood pressure decreases. However, this result was statistically insignificant. Therefore, our primary test of whether calcium influences blood pressure was negative.

There were also no significant associations between dietary calcium intake and systolic

blood pressure, supplementary calcium intake and systolic blood pressure, and exercise and systolic blood pressure (see Table 2). Our secondary test of whether any calcium effect was modified by exercise was negative.

Although calcium intake and exercise were not associated with systolic blood pressure, the covariates BMI, nicotine and weight has a direct relation to systolic blood pressure. Conversely for covariates such as vitamin A, vitamin C, vitamin E and beta-carotene, there were no associations found between these variables and systolic blood pressure (see Table 2).

Table 3 illustrates the association between total calcium intake and systolic blood pressure after adjusting for BMI, weight, nicotine and exercise. Although the partial correlation coefficients for three of the four relations were more negative than the coefficient obtained for our original hypothesis on the calcium effect on systolic blood pressure, the inverse association between total calcium intake and systolic blood pressure was not enhanced by body mass index, weight or nicotine because these results were statistically insignificant.

Discussion Our results suggest that there is no statistically significant association between calcium intake and systolic blood pressure or exercise and systolic blood pressure. The effect of calcium on systolic blood pressure was also not modified by exercise.

However, we still need to acknowledge the discrepancies caused by the difficulty to recall and answer the questionnaire. For further investigation, we can enlarge the sample size to increase power.

Many epidemiological studies suggested that diet and physical activity play two important roles in determining the risk of cardiovascular diseases associated with blood pressure [19,20].

Nonetheless, while adequate physical activity is evidently critical to optimal blood pressure regulation, the results on calcium’s blood pressure lowering effect are inconsistent [12,21]. Some evidences suggested that increasing calcium intake protects against hypertension and other diseases, but little is known about how other factors can modify calcium’s effect [22].

It is recommended that exercise 3 times per week for 30 minutes [23, 24] and taking >800 mg of calcium from diet and supplements daily can substantially lower blood pressure [25].

However, for feeble seniors, while moderately intense exercise can reduce the risks of getting

nutrients in food.

6. People who are taking antihypertensive medication have lower blood pressure in comparison to those who are not taking medication, but with similar lifestyle.

7. Blood pressure is determined by one’s overall dietary and physical pattern over an extended period of time and not by the intake of a single nutrient.

Figure 1: Simple scatterplot for Systolic Blood Pressure vs. Total Calcium Intake

a coronary heart disease [17], strenuous exercise acutely increases the risk of sudden cardiac death and myocardial infarction [26].

“Historically more emphasis has been placed on systolic than diastolic blood pressure as a predictor of cerebrovascular and coronary heart disease [27].” Systolic blood pressure (SBP) elevates throughout the adulthood, where diastolic blood pressure (DBP) plateaus at around age 60 years in men and 70 in women, and falls gradually after that [27]. The recent JNC 7 report also suggested that SBP more than 140 mm Hg is a stronger indicator than DBP on cardiovascular disease for people older than 50 years of age [9,28]. Since we know that there is a significant association between systolic blood pressure and cardiovascular disease [3,12], we chose to use systolic blood pressure in our study to determine calcium and exercise’s effect on blood pressure, and their additional benefit for reducing cardiovascular disease.

Many studies have found that dietary and physical patterns can treat and prevent hypertension, but additional study is needed to investigate the potential joint effect between calcium and exercise. Although calcium’s hypotensive action is not fully understood, several possibilities have been considered. These include “a calcium-induced natriuretic (urinary sodium excretion) effect, a decrease in calcium regulating hormones and a reduction in intracellular free calcium [4].” Other studies also postulate that due to calcium’s role in “vascular contraction in relation to the metabolic changes influencing vascular smooth muscle contraction,” this mechanism dilates arteries and decreases blood pressure [4].

Other possible explanations for the inconsistent results on calcium’s blood pressure lowering effect observed in our study and other similar studies are: 1. Blood pressure lowering effects of a single nutrient such as calcium, may be too small to detect in small-scale observational study [11].

2. When several nutrients such as certain minerals and fiber, are consumed together, their additive effect may interfere with the primary predictor variable, calcium intake [11].

3. Interactions among nutrients could amplify or compress the effect of calcium [11].

4. Untested or unknown nutrients in the diet may elevate or lower blood pressure [11].

5. Nutrients in supplements may not affect blood pressure to the same extent as do the same

We were also aware of factors such as age, high blood calcium, osteoporosis, which are critical confounders for hypertension [22]. However, when we ran the bivariate statistic test, the association between systolic blood pressure and these individual factors were not statistically significant. Therefore, we did not include them as covariates when we ran the Partial Correlation coefficient test due to their lack of effect on systolic blood pressure. As a result, although calcium may be associated with a small reduction of blood pressure [22], we do not have adequate evidence to advise the public to increase calcium intake in substitution for antihypertensive medication [13] or as means to reduce cardiovascular morbidity and mortality.

Acknowledgments This study was supported by the American Heart Association Undergraduate Summer Research Program. I also want to thank Dr. Langer and Dr. Allison for their guidance and support.

References

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2. Svetkey LP, et al. Effects of dietary patterns on blood pressure: Subgroup analysis of the dietary approaches to stop hypertension (DASH) randomized clinical trial. Arch Intern Med. 1999; 159: 285-293.

3. Slama M, Susic D, Frohlich ED. Prevention of hypertension.

Lippincott Williams & Wilkins Inc. 2002; 17: 531-536.

4. McCarron DA, Reusser ME. Finding consensus in the dietary calcium-blood pressure debate. Journal of American College of Nutrition 1999; 18: 398S-405S. 5. Krauss RM, et al. AHA Dietary guidelines: A statement for healthcare professionals from the nutrition committee of the American Heart Association. Stroke 2000; 31: 2751-2766.

6. Appel LJ, et al. A clinical trial of the effects of dietary patterns on blood pressure. The New England Journal of Medicine 1997; 336: 1117-1124.

7. Sacks FM, et al. Rationale and design of the dietary approaches to stop hypertension trial (DASH): A multicenter controlledfeeding study of dietary patterns to lower blood pressure. Elsevier Science Inc. 1995; 5: 108-118.

8. Brown MJ, Haydock S. Pathoaetiology, epidemiology and diagnosis of hypertension. Drugs 2000; 59: S1-S12.

9. Chobanian AV, et al. The seventh report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure. JAMA 2003; 289: 2060-2572.

10. Moser M. World Health Organization-International Society of Hypertension Guidelines for the Management of HypertensionDo These Differ From the U.S. Recommendations? Which Guidelines Should the Practicing Physician Follow? Journal of Clinical Hypertension 1999; 1: 48-54.

11. Izzo JL, et al. Importance of systolic blood pressure in older Americans. Hypertension 2000; 35: 1021-1024.

12. Cappuccio FP, et al. Epidemiologic association between dietary calcium intake and blood pressure: A meta-analysis of published data. American Journal of Epidemiology 1995; 142: 935-944.

13. Yao M, et al. Relative influence of diet and physical activity on cardiovascular risk factors in urban Chinese adults. International Journal of Obesity 2003; 27: 920-932.

14. Hamet P, et al. Interactions among calcium, sodium, and alcohol intake as determinants of blood pressure. Hypertension

1991; 17: S150-S154.

15. Glynn RJ, et al. Development of predictive models for longterm cardiovascular risk associated with systolic and diastolic blood pressure. Hypertension 2001; 106: 105-110.

16. Calcium Information Center (2003). The best sources of calcium in the four food groups. Retrieved July 16, 2003, from http://www.cereusmed.com/MyBones/best_source_of_calcium_ from_foods.html 17. Moses S. (2000). Family Practice notebook.com. Retrieved July 29, 2003, from http://www.aroundcharlotte.com/SPO34.htm 18. Manson JE, et al. Walking compared with vigorous exercise for the prevention of cardiovascular events in women. The New England Journal of Medicine 2002; 347: 716-725.

19. Kottke TE, Stroebel RJ, Hoffman RS. JNC7—It’s more than high blood pressure. JAMA 2003; 289: 2573-2574.

20. Criqui MH, Langer RD, Reed DM. Dietary alcohol, calcium, and potassium. Independent and combined effects on blood pressure. Circulation 1989; 80: 609-614.

21. Krauss RM, et al. AHA Dietary guidelines: A statement for healthcare professionals from the nutrition committee of the American Heart Association. Stroke 2000; 31: 2751-2766.

22. Weaver CM. Calcium requirements of physically active people. American Journal Nutrition 2000; 72: S579-S584.

23. Langer RD. Exercise and survival in the very old. American Journal of Geriatric Cardiology 1994; 2: 23-34.

24. Thompson PD, Lim V. Physical Activity in the Prevention of Atherosclerotic Coronary Heart Disease. Curr Treat Options Cardiovasc Med 2003; 4: 279-285.

25. Zozaya JL. Nutritional factors in high blood pressure. Journal of Human Hypertension 2000; 14: S100-S104.

26. Thompson PD, et al. Exercise and physical activity in the prevention and treatment of Atherosclerotic cardiovascular disease. Circulation 2003;107: 3109-3116.

27. Guidelines Committee. 2003 European society of hypertension guidelines for management of arterial hypertension. Journal of Hypertension 2003; 21: 1011-1053.

28. Lenfant C, et al. Seventh report of Joint National Committee on the prevention, detection, evaluation, and treatment of high blood pressure (JNC7): Resetting the hypertension sails.

Hypertension 2003; 41: 1178-1179.

29. Al-Delaimy WK, et al. A prospective study of calcium intake from diet and supplements and risk of ischemic heart disease among men. American Journal Clinical Nutrition 2003; 77: 814-818.

30. Hermansen K. Diet, blood pressure and hypertension. British Journal of Nutrition 2000; 83: S113-S119.

31. Beta Carotene Cancer Prevention Study Group The AlphaTocopherol. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The New England Journal of Medicine 1994; 330: 1029-1035.

32. Clark LC, et al. Decreased incidence of prostate cancer with selenium supplementation: Results of a double-blind cancer prevention trial. British Journal of Urology 1998; 81: 730-734.

33. McCarron DA, Reusser ME. Finding consensus in the dietary calcium-blood pressure debate. Journal of American College of Nutrition 1999; 18: 398-406.

34. Jorde R, et al. Serum calcium and cardiovascular risk factors and diseases: The Tromsø Study. Hypertension 1999; 34: 484490.

35. Ascherio AA, et al. Intake of potassium, magnesium, calcium, and fiber and risk of stoke among US men. Circulation 1998; 98: 1198-1204.

36. Jorde R, et al. Relation between low calcium intake, parathyroid hormone, and blood pressure. Hypertension 1999; 35: 1154-1159.

37. Wright JD, Wang CY, Kennedy-Stephenson J, Ervin RB. Dietary intake of ten key nutrients for public health, United States: 1999-2000. Advance data from vital and health statistics; no. 334. Hyattsville, Maryland: National Center for Health Statistics. 2003.

38. Neaton JD, et al. Treatment of mild hypertension study. Final results. Treatment of mild hypertension study research group.

JAMA 1993; 270: 713-724.

39. Vanhees L, et al. Effects of antihypertensive medication on endurance exercise capacity in hypertensive sportsmen.

Hypertension 1991; 9: 1063-1068.

40. Langer RD. The epidemiology of hypertension control in populations. Clin Exp Hypertens 1995; 17: 1127-1144.

41. Lindblad U, et al. Metabolic Syndrome and Ischemic Heart Disease in Elderly Men and Women. American Journal of Epidemiology 2001; 153: 481-489.

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The staff of the Saltman Quarterly Ann Cai is a second year student studying Molecular Biology and Music. She is currently researching in Partho Ghosh’s lab investigating the reverse transcription of Bordetella pertussis by looking at the functions of parts of the genome associated with reverse transcriptase.

She has also worked in Robert Page’s lab at UC Davis researching population genetics of termites to determine kinship. Outside of biology, her interests include ballroom dance, piano, and violin.

Cara Cast is a transfer student in her fourth year of the General Biology major. She is assisting with research in Darwin Berg’s laboratory where she helps explore roles of various synaptic components in synapse formation using molecular and cell biological techniques.

Cara’s interest in neurobiology stemmed from previous military service in the electronics field, which sparked an interest in understanding the importance of proper connectivity in biological circuits. She hopes to attend graduate school in neuroscience.

Eric Chan is a second year General Biology and History major with a Music minor in Marshall College. He is interested in neurobiology, immunology, oncology, and aging research, especially the effects of various antioxidants on DNA oxidation and human health as a whole. He also has a side interest in the fusion of Chinese and Western medical techniques and practices, as he has done work in traditional Chinese medical centers in Taiwan and the U.S. Henry J. Chen is a fourth year student in Warren College majoring in Biology – Animal Physiology & Neuroscience with Psychology and Chinese Studies minors. He currently works in Dr. Elena Pasquale’s lab at The Burnham Institute. Henry is also an active member of the Alpha Kappa Psi Professional Business Fraternity. He enjoys many sports including basketball and swimming. Henry plans to be involved with neuroscience research in the near future and a career in medicine.

Reeti Desai is a second year Molecular Biology

major in Revelle College. Currently, she is not doing any research, but hopes to begin doing so in the 2004-05 academic year. Reeti plans to apply to the Biological Sciences MS/BS program at the end of her junior year and is going to study abroad, in England, fall semester of her senior year.

Gregory Emmanuel is a senior majoring in Animal Physiology & Neuroscience and Psychology. He has contributed to projects in the V.S. Ramachandran and Schmidt labs. He currently performs microsurgery and organ transplantation research at the San Diego Microsurgical Institute and is conducting an epidemiological study on vascular disease with Dr. Matt Allison in the Department of Preventive Medicine at UCSD’s School of Medicine. Greg is searching for an opportunity in a lab on campus for the MS program in Biology!

Kristine Germar is a third year Molecular Biology major in Earl Warren College. She has previously worked in Dr. Yang Xu’s laboratory, which is investigating the function of tumor suppressor p53. She is currently working at Gen-Probe, where she is helping to improve their current technology in HIV diagnostics.

Kristine hopes to continue her education at UCSD next year through the BS/MS program.

Claudine Heu is a third year student in the Biochemistry & Cell Biology major in Warren College.

Yee Hung is a second year Biochemistry & Cell Biology major in Sixth College. She is currently conducting immunological research in Dr. Nicholas Gascoigne’s laboratory at The Scripps Research Institute. She is studying two proteins in T-cells, called CD8 and TCR, by using a technique called fluorescence resonance energy transfer. Yee has applied to The City of Hope Summer Student Research Program for the summer of 2004. There, she hopes to continue her research in immunology.

Caroline Lindsay is a third year Molecular Biology major in Revelle College. She is

Top row left to right: Gregory Emmanuel, Ian Nicastro, and Marika Orlov.

Bottom row left to right: Reeti Desai, Jeff Nichelini, Lorraine Kelley, and Kristine Germar 26

Volume 1 Issue 1

currently participating in the Biotechnology Internship Opportunities Program, working at Cibus Genetics. Her project involves screening plant genome mutations for increased resistance to herbicides. Next year she will be working in Amy Pasquinelli’s lab, researching the function of specific microRNAs in C. elegans. She hopes to attend graduate school in molecular biology and eventually would like to work in the biotechnology industry.

Henry Chen and Caroline Lindsay

Hao Yi Liu is a second year Microbiology major in Revelle College. Her interest is growing in the research field after taking a quarter of Biochemical Techniques lab at UCSD. She is interested in gene research and DNA extraction.

Taking classes relating to biology lab research is her first step in the research field, and being an SQ staff member is her second step. During the summer, she plans to work part time in a research institute as a lab technician.

Lorraine Kelley is a senior majoring in Biochemistry & Cell Biology in Revelle College. She is currently working in Tama Hasson’s laboratory studying the regulation of Myosin VI in cellular trafficking for both the 2003-04 Beckman Scholars Program and her senior honors thesis. Lorraine will be attending UCLA medical school in the fall of 2004 and is looking towards a career in orthopedic surgery.

Greg Naughton is a fourth year Animal Physiology & Neuroscience major and has conducted research on developmental Drosophila genetics and neuronal cell surface proteins. He is currently working on his Master’s research through the BS/MS program in Dr. Berg’s lab, investigating synapse formation and cell signaling in chick embryo ciliary ganglia.

Upon completion of his master’s work, Greg plans on attending medical school and specializing in neurology.

Ian Nicastro is a fourth year Neuroscience & Animal Physiology major, minoring in Psychology. He is interested in behavioral neuroscience and is currently performing research on dopamine and cannabinoid receptors in nematodes in the Schafer Lab. Ian is also currently assisting the Laboratory of

morphology in the mature nervous system.

From left to right: Eric Chan, Alice Tsai, and Ann Cai

Neurophenomics in a study of the murine DBP clock gene. Next year he will be heading to the NIH for a year-long fellowship before applying to graduate school.

Jeff Nichelini is a fourth year Revelle College Molecular Biology major with three years experience in the Mayfield lab at The Scripps Research Institute. Scientific words of wisdom: always have a fall out guy...that way, it’s never your fault.

Louis Nguyen is a fourth year Biochemistry & Cell Biology major who is currently conducting neurobiological research in Elena Pasquale’s laboratory at The Burnham Institute. He has done work under the 2002 Howard Hughes Summer Research Program, and has been working under the Chancellor’s Research Scholarship since Fall 2002. These projects are concerned with Eph receptors, receptor tyrosine kinases responsible for axon guidance in the developing nervous system and dendritic spine

Marika Orlov is a fourth year Molecular Biology major in Marshall College. She is conducting research in William Schafer’s laboratory on the characterization of a gene for both the 2003 Howard Hughes Summer Research Program and as part of her senior honors thesis. Marika will be starting the Postbaccalaureate program at the NIH this July.

She hopes to enter a Medical Scientist Training Program in the fall of 2005.

Joshua Tan is a third year Marshall College student with a major in Biochemistry & Cell Biology and a minor in Economics. He is currently working as a lab assistant in the Hasson Lab. He hopes to begin getting involved in a research project this summer and plans on pursuing a degree in the biomedical sciences.

Joshua is also a member of the Biological Sciences Student Association.

Alice (Yu-Ting) Tsai is a second year Biochemistry & Cell Biology major in Warren College. She has participated in the 2003 Howard Hughes Summer Research Program and is continuing her project in Dr. Schmidt’s plant biology lab here at UCSD with the Ronald E. McNair Postbaccalaureate Achievement Program. Her project involves the isolation and characterization of similar maize and sorghum genes that will contribute to the understanding of evolution in grass family.

Kuo Yang is a fourth year Animal Physiology & Neuroscience major at UCSD with a minor

in music. He is working on his master’s degree under Dr. Ryan’s auditory research lab. His current research involves investigating various molecules/methods that guide rat spiral ganglia nerve growth. He plans to attend medical school after completion of his Master’s degree.

Top row: Claudine Heu, Josh Tan, and Louis Nguyen.

Bottom Row: Cara Cast and Greg Naughton

The Saltman Quarterly is accepting manuscript proposals on a rolling basis. Please write to sq@biomail.ucsd.edu

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

Darwin Berg for his time and patience with the feature review.

Randolph Hampton for the faculty feature interview.

Katie Kindt for the worm picture in the table of contents.

Elena Pasquale for her advice in reviewing and editing.

Lorraine Pillus for advice, support, and a photo shoot.

Zachary Sellers for his help in holding the review workshop.

Linda Strause for holding the review workshop and the pictures of Dr. Saltman.

Kevin Ford for being our wonderful photographer.

And most importantly Mrs. Barbara Saltman for permission to dedicate this journal to further Dr. Saltman’s commitment to the undergraduate experience here at UCSD.

Volume 1 Issue 1

27

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