Sq vol3

Saltman | Quarterly Undergraduate Biological Research Publication UCSD Division of Biological Sciences

Volume 3, Nos. 1, 2, & 3 http://www.sq.ucsd.edu

From the Editor

Saltman Quarterly

Dear Readers, Approximately two years ago, three students, Marika Orlov, Louis Nguyen and Greg Emmanuel, felt the need to create a medium for undergraduates at UCSD to present their research in the biological sciences. Thus, with the enthusiastic support of the Biological Sciences Dean’s Office, Saltman Quarterly was born, named after the late Dr. Paul D. Saltman. This journal was dedicated to him because of his commitment to undergraduates in the classroom and in the laboratory.

His dedication continues to impact students today through the tradition of Saltman Quarterly.

Dr. Paul D. Saltman I say tradition–going into our third volume–as I Scientist & Educator suspect SQ will pass the test of time. The first few years for any student organization, or even a full-fledged science journal, are always the most difficult.

Over these few years we have created dedicated editor positions, established a Faculty Advisory Committee, developed a review workshop for our student reviewers, and changed our layout; these are all steps that I think will help establish SQ as a lasting tradition at UCSD. We’ve even inspired a fellow journal in the Department of Psychology. Through Saltman Quarterly Dr. Saltman, then, is continuing to positively influence students in ways our first three editors probably never imagined.

I hope that along these lines Saltman Quarterly and Dr. Saltman’s educational legacy become a tradition, not only among biology majors and minors but also among all students doing research at UCSD.

Division of Biological Sciences University of California San Diego

.:Staff:.

Editor-in-Chief - Kyle Kuchinsky Managing Editor - Ann Cai Production Editor - Max Chen Features Editor - Eric Chan Research Editor - Nicole Gomez Publicity Chair - Shruti Jayakumar Webmaster - Grace Wang Technical Editor - Reeti Desai Review Board Manager - Nicole Gomez

.:Review Board:.

Katherine Banares Kendra Bettis Linda Boettger Iris Chen Mark Chen Kevin Day

Sincerely, Kyle Kuchinsky Editor-in-Chief Cover Image: The image on the front cover was taken by Kimberly Lo while participating in the EAP Monteverde Tropical Biology and Conservation program in Costa Rica, where she studied the reproductive status and species diversity of bats. For more information, read her article on page 26.

“The publication may have been funded in part or in whole by funds allocated by the ASUCSD. However, the views expressed in this publication are solely those of Saltman Quarterly, its principal members and the authors of the content of this publication. While the publisher of this publication is a registered student organization at UC San Diego, the content, opinions, statements and views expressed in this or any other publication published and/or distributed

by Saltman Quarterly are not endorsed by and do not represent the views, opinions, policies, or positions of the ASUCSD, GSAUCSD, UC San Diego, the University of California and the Regents or their officers, employees, or agents. The publisher of this publication bears and assumes the full responsibility and liability for the content of this publication.”

Copyright © 2005-2006. Regents of the University of California. All rights reserved.

Fernanda Delgado James Duguid Prema Hampapur Cindy Huynh Melanie Kho David Kim Youngjin Kim Eun Kyung Joanne Lee Hyuma Leland Nick Lind Lauren Ashley Miller Sara Paul Ronnie Pezeshk Erica Sanford Ji Woong (John) Suk Koh Tanimoto Kevin Tran

contents

S|Q

Saltman Quarterly - Made for undergrads, by undergrads.

VOLUME 3, Nos. 1, 2, & 3

20 0 5 – 0 6

FEATURES

04

The Challenge of Developing Biological Software

by Noah Ollikainen

18

RESEARCH

Protein-translocating Trimeric Autotransporters of Gram-negative Bacteria

David S. Kim, Yi Chao and Milton H. Saier, Jr.

06 08

26 Mus musculus – Our Genetic Stunt Double / by Reeti Desai Parasitology 101 /

by Grace Wang

10 12

Kimberly L. Lo

32

Wanna Research? / by Ann Cai

16 54

Investigation on the Effect of Age and Group Size on the Simultaneous Production of Bubbles and Calls by Captive Killer Whales (Orcinus orca) at SeaWorld, San Diego Eri Suzukl

Why We Need Medicinal Research / by Shruti Jayakumar

39

Q&A 13

Reproductive Status and Species Diversity of Bats along an Altitudinal Gradient in Monteverde, Costa Rica

Interview: Dr. Milton Saier / by Hyuma Leland

46

Acknowledgements & Saltman Quarterly Staff

2005–06 Volume 3

Mark Chen

Ethidium Bromide Exposure Has Teratogenic Effects on Xenopus Embryos

Jeffrey Cantle

Interview: Dr. Nigel Crawford /

by Max Chen

Review: Olfactory Ensheathing Cells, Bone Marrow Stromal Cells, and the Herpes Simplex Virus for Axonal Regeneration in Spinal Cord Injury Models

50

PIK3CA Mutations Are Rare in Neuroblastoma: Development and Use of a Highly Sensitive dHPLC Assay for the Mutational Analysis of PIK3CA

Youngjin Kim, Alice L. Yu and Mitchell B. Diccianni

UCSD Biological Sciences Saltman | Quarterly

The Challenge by

Noah Ollikainen

W

ith an everincreasing volume of biological data being collected and processed, software developers are continuously presented with the endeavor of figuring out how to best manage and present data.

This task involves developing easyto-use tools such as 3-D protein structure viewers, sequence analysis programs, and web-interfaces that allow biologists to access databases. Unfortunately, this task is tremendously difficult for the following reason: many software developers are not biologists. A developer typically does not have a background in biology, and as a result developers are rarely users of their own software. While the developer understands how to implement a vast array of features, the developer is unaware of significance that these features have towards biological research. Data are just data without any external meaning or value. A fold deviation score might as well be a Chi1 trans dihedral angle. That is not to say the biological data is unimportant

of

Developing

to the developer: sometimes the data is just cryptic. There is an understanding of which features must be provided, but outside of that the developer is oftentimes in the dark.

How then can a developer successfully create software that proves to be useful to a biologist?

If the developer does not become proficient in biology, user feedback will be crucial. The best way to judge the usability of one’s own software is to simply receive comments from the users. Sometimes this feedback consists of trivial issues that the developer can easily fix.

For instance, a user may ask for a color to be changed or a button to be made larger. However, a user may also ask for an option to display all the significant protein-protein interactions in a dimer interface.

This could give the developer a headache, as such a feature may be either difficult to implement or out of synch with the goals of the project. Moreover, adding this new feature may cause a menu to become even more cluttered and confusing, resulting in the displeasure of other users.

In this sense, biologists are demanding customers. They want software to be both simple and robust. A developer must keep this in mind when working on

Saltman | Quarterly UCSD Biological Sciences

a project. Even though a project is simple in the beginning, it may become complicated impressively quickly. It is easy for a developer to lose sight of this and end up with a product that is virtually unusable.

That said, the following is a list of pointers for developers to keep in mind when creating useful and easy to use biological software: 1) Help. There must be clear, wellwritten documentation that is easy to access. What is intuitive to you is not necessarily intuitive to the user. Provide a tutorial to get the user comfortable with using the software.

2) Simple menus. Know which features are most useful to the average user and make these features the easiest to access. Also, understand which features are unnecessary and remove them to avoid cluttering.

3) Customizability. Provide the user with the ability to control how output is displayed.

For instance, the figure on page 5 shows a Color menu of UCSF Chimera, an interactive molecular graphics program.

Users can choose precisely how they want a selected region of a molecule to be colored without having to dig through a maze of menus.

4) Naming.

The names you attribute to features or options are very important. Carefully choose names that accurately reflect the purpose of the feature.

Volume 3 2005–06

Biological Software

5) Error messages. Always provide helpful error messages when a user does something wrong. Clearly explain how the error occurred so that the user understands precisely what happened.

6) Compatibility. Allow users to save files in many different formats. Users will often employ a combination of software in their research and need a way to transfer their results. Make sure to include all the common formats associated with the possible output.

7) Encourage feedback. People often neglect that many of the programs they use are still being developed, so they will be hesitant to report issues that arise.

Allow users to easily report bugs and

2005–06 Volume 3

provide suggestions and assure them that their input is valuable.

Even with these pointers, a developer may still produce confusing software with an enormous learning curve. Without a background in biology, there exists great difficulty for a developer in trying to get inside the mind of a biologist and view software from their perspective.

Fortunately, this difficulty will decrease dramatically in the near future. With the surge of biological data collection and processing comes a new breed of students who are simultaneously being educated in both biology and computer science.

Numerous universities, including UCSD, have initiated Bioinformatics programs in order to provide students with extensive

interdisciplinary training. Within these programs, students learn both how to implement software and how to use that same software for biological research.

These individuals are able to bridge the gap between developers and biologists, as they understand both points of views and can communicate effectively with each one. There is indeed a hopeful future in the development of powerful yet easy-touse biological software.

Noah Ollikainen is a junior majoring in bioinformatics. He is a member of Sixth College.

UCSD Biological Sciences Saltman | Quarterly

Kingdom: Animalia Phylum: Chordata Class: Mammalia Order: Rodentia Family: Muridae Subfamily: Murinae Genus: Mus Species: M. musculus

Mus

musculus

by Reeti Desai

mouse

n. pl. mice (ms) Any of numerous small rodents of the families Muridae and Cricetidae, such as the common house mouse (Mus musculus), characteristically having a pointed snout, small rounded ears, and a long naked or almost hairless tail.

– Our Genetic Stunt Double

Mus musculus, more commonly known as the mouse, is one of the most widely used laboratory animals in biomedical research facilities. This is because as a mammal, mice possess genomes very similar to that of humans, which is of great value in a wide variety of ongoing gene-based studies.

In addition to this genetic homology, their small size, relative inexpensiveness, and short life-spans enable mice1 to become a convenient model organism for experimental manipulation.

Most laboratory mice are hybrids of different subspecies, chiefly Mus musculus domesticus and Mus musculus musculus, which have been inbred in order to make them genetically identical. They are often found to be white in color, with a large occurrence of albinism in the populations. Sequencing of the mouse genome (strain C57BL/ 6J) was completed in 2002; it was found to have

Saltman | Quarterly UCSD Biological Sciences

2.5 billion base pairs that make up approximately 30,000 genes, remarkably similar to the human genome itself.2 This likeness allows experimentation on them that would be unethical if carried out on humans.

Mice were first inducted as research subjects into the world of model organisms by Clarence Cook Little as early as 1909. As an undergraduate at Harvard University, Little studied coat-color inheritance in mice by mating siblings in a group of brown mice to produce a pure strain. In 1929 he went on to establish the Roscoe B. Jackson Memorial Laboratory in Bar Harbor, Maine, where all forms of mouse research began, including the foray into its genetics3.

Many mutant strains of mice are currently available, partly due to the efforts of the now renamed Volume 3 2005–06

Jackson Laboratory4. Ordinary breeding has led to the development of multiple strains, such as mice with an absent immune system (Severe Combined Immunodeficiency or SCID), mice with remarkable regenerative capabilities, and nude mice (hairless mice lacking a thymus and thus unable to create T lymphocytes). “Knockout mice” have also been developed in labs, in which a specific gene has been made inoperable through a gene knockout, allowing the study of a simulated human disease in the mouse2. Finally, transgenic mice with foreign genes added to their genome have been created. Some products of this procedure are large mice (with an inserted rat growth hormone), Oncomice (with an activated oncogene that increases the occurrence of cancer) and Doogie mice (with an enhanced NMDA receptor that results in improved memory and learning).

Research on transgenic mice is generating some of the most groundbreaking discoveries.

Production of the first chimeric mice began in the 1970s, when cells from two different embryos and strains were combined into a single embryo that matured into a chimeric adult displaying characteristics from both strains5. This led to the development of techniques needed to create the first transgenic animals. As a result, DNA microinjection, embryonic stem-cell-mediated gene transfer, and retro-virus mediated transfer5 have become widely used tools. The first transgenic mouse was created in 1981 by J.W. Gordon and F.H. Ruddle6, and since then the number of applications for genetically engineering mice have exponentially increased. The same advantages of small size, short life span, and high degree of homology with the human genome have allowed mice to become the most popular organism to be used in transgenics. UCSD is no exception, where research involving mice is being carried out in on-campus laboratories, the UCSD Cancer Center, and the UCSD School of Medicine.

Ultimately, all of the strains created through these techniques have contributed greatly to our understanding of a variety of human diseases, including diabetes, obesity, sickle cell anemia, cancer, and AIDS. The use of mice as our genetic stunt double has led to revolutionary insights into the human immune system, retroviruses, oncogenes, and the inheritance of complex traits, all of which have been the subjects of substantial studies on mice. Seventeen Nobel prizes have been awarded as a result of studies on mice inspired by Little’s research, as well as two new scientific tools, monoclonal antibodies and gene-targeted strains3. What began as a simple interest in Mendelian inheritance in mice by Little 80 years ago has become the basis for a global standard in biomedical research. The significance of his contribution of engineered inbred strains of mice to our current understanding of the human body is undeniable.

References

1. Mercer, E. Whole Mouse Catalogue. URL: http://www.muridae.com/ wmc. Accessed: 12 February 2006.

2. Animal Diversity Web: Mus musculus: University of Michigan, Museum of Zoology. URL: http://animaldiversity.ummz.umich.edu/site/accouns/ information/Mus_musculus.html. Accessed: 12 February 2006.

3. Festing, M. F. W., & Fisher, E. F. C. Mighty Mice. URL: http://www.bio. unc.edu/courses/2003fall/biol264/Festing1999.pdf. Accessed: 12 February 2006.

4. Jackson Laboratory. URL: http://www.jax.org/index.html. Accessed: 12 February 2006.

5. Buy, M. Transgenic Animals. URL: http://www.acs.ucalgary.ca/~browder/ transgenic.html. Accessed: 12 February 2006.

6. Gordon, J.W., & Ruddle, F.H. Integration and stable germ line transmission of genes injected into mouse pronuclei. URL: http://www.ncbi.nlm.nih. gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_u ids=6272397&adopt=Abstract. Accessed: 12 February 2006.

Reeti Desai is a senior majoring in molecular biology. She is a member of Roger Revelle College.

2005–06 Volume 3

UCSD Biological Sciences Saltman | Quarterly

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4

6 5

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Parasitology 101 by

They are everywhere.

Resistance is futile.

You will be parasitized!

Ignoring the geeky reference, this sweeping generalization is actually true enough to give low-key hypochondriacs perpetual nightmares. Parasites can be found colonizing any organism, anywhere.

In fact, Ralph Waldo Emerson said, “As soon as there is life there is danger.” Granted, he probably wasn’t talking about parasites, much less the bizarre organisms featured here.

This article was originally going to be on the top ten most common human parasites. But instead of potentially resembling a health center’s disease prevention brochure, these next two pages will showcase eight of the creepiest parasites found on Earth.

Grace Wang

#1 Leucochloridium paradoxum Hosts: birds Vectors: amber snails You’ve probably heard the phrase, “The early bird catches the worm!” Well, apparently it’s not always in the bird’s best interest to get that meal. Snails pick up this flatworm by nibbling on infested bird droppings. Then the parasite grows branched brood sacs into the host snail’s eyestalks, causing color changes, swelling, and pulsation. Now, not only do its tentacles look like a pair of tasty caterpillars wriggling about, but the snail also can no longer retract into its shell. In addition, its light sensitivity is impaired, so instead of retreating to the shade, the snail will stay out in the open and scream, “Help me! Heeelp meeeeee!” Okay, maybe it doesn’t talk.

1-> Leucochloridium

#2 Parelaphostrongylus tenuis Hosts: white-tailed deer, cervids Vectors: snails, slugs Location: eastern North America Size: 5-7.5cm Mmmmh, brains! Yes, we have zombie moose. Although this nematode’s natural host is deer, it can

8-> Dracunculus

Saltman | Quarterly UCSD Biological Sciences

paradoxum

2-> Parelaphostrongylus

tenuis

3-> Cymothoa exigua 4-> Toxoplasma gondii 5-> Onchocerca volvulus 6-> Vandellia cirrhosa 7-> Ampulex compressa

medinensis

Grace Wang is a junior majoring in microbiology. She is a member of John Muir College.

Volume 3 2005–06

be transmitted to various other animals including moose, llamas, and alpacas. In deer, the larvae (in the form of infested snail treats) travel up the spinal cord and take up residence in the dura mater, the covering between the brain and the skull. Being in a different host seems to screw up their GPS, though, and they migrate throughout the brain and spinal cord instead. This damage to the central nervous system causes odd behavior and eventually death. If you ever see a llama trying to walk on its hind legs or slapping itself, it just might have meningeal worms. #3 Cymothoa exigua Hosts: red snapper fish Location: Gulf of California Size: 3.5cm You know when the dentist says, “Open wide and say aaaah,” he sure doesn’t expect to find sea critters “hanging” around. Good thing fish don’t have dental plans. This sea dweller climbs in through the gills, attaches itself with its claws to an artery in a snapper fish’s tongue, and feeds on the blood. When the organ eventually atrophies, the little crustacean latches onto the stub and serves as a replacement tongue. Apparently, there is no further damage to the fish. Imagine finding that on your plate. #4 Toxoplasma gondii Hosts: cats Vectors: rats, humans (infects any mammal) Location: worldwide Size: oocyst 10-12µm, tachyozites 3-7µm (pictured) If Tom got his hands on some of these, Jerry would be in big trouble.

This protozoan actually secretes mind-altering substances (not LSD) to get from vector to host. Research showed that while normal rats avoid areas that smell of cats, infected rats lose that fear and explore feline territory. Not only that, their response times are slower and their attention spans shorter. The scary part is that this parasite can also infect just about any mammal, including humans. In fact, a large percentage of relatively symptom-free humans test positive for toxoplasma antibodies. (It does, however, cause problems in people with autoimmune diseases and pregnant women.) Some research suggests there is a link between infection and certain personality traits, and possibly even schizophrenia. #5 Onchocerca volvulus Hosts: humans Vectors: black flies Location: Central and South America, Africa (by water sources where flies can proliferate) Size: 30cm One more reason to dislike blood-sucking insects: you just might go blind! An estimated 18 million people are infected with this nematode, which enters the host as a larva (transmitted through a fly bite) and travels around before congregating and forming nodules just under the skin for maximum itch factor. After maturation and mating, the female worm produces up to a thousand microfilariae a day. These then pack their bags and leave home sweet nodule home to die in rather inconvenient places, the eye in particular, which gives this disease its name “river blindness.”

When the human body mounts an immune response, it causes rashes, skin depigmentation, and blindness. #6 Vandellia cirrhosa Hosts: fish, humans Location: Amazon River Size: 1-5cm

2005–06 Volume 3

Anyone like catfish? Well, this particular one likes you! Especially your urethra. This little critter, commonly known as the candirú, was mentioned for spook factor in the movie Anaconda and an episode of “Law & Order: SVU.” They usually hone in on the nitrogen flowing away from fish gills, where there are thick juicy arteries to feed on, hence the local name “vampire fish.” However, these fish can be attracted by the nitrogen in urine and will swim towards a source (namely, an orifice in your nether regions). Once inside, they latch on with spines and begin feeding on the blood and tissue. The only ways to get them out are extensive surgery and/or inserting a mixture of two poisonous plants that dissolve the fish. Ouch. #7 Ampulex compressa Hosts: cockroaches Size: 2-3cm And you thought Invasion of the Body Snatchers was just some creepy sci-fi flick? This not-so-alien wasp delivers two very precise stings to a roach, one to the thoracic ganglia to temporarily paralyze the front legs, then one to the escape reflex in the head ganglia. After taking away the roach’s free will, the wasp then takes hold of one of its victim’s antennae, using them like reins or a leash, and directs the roach to the wasp’s den. It then covers the entrance with pebbles to prevent unwanted visitors, while the neurotoxins effectively keep the roach in a vegetative state. After the larva hatches, it chews through the roach’s abdomen and feeds off of the internal organs, selecting its meals so that the roach stays alive until the larva is ready to turn into a cocoon. Talk about cruel and unusual punishment. #8 Dracunculus medinensis Hosts: humans Vectors: water fleas Location: sub-Saharan Africa Size: up to 1m length, 1-2mm diameter You’re probably familiar with a symbol used in the medical field—a staff with a snake wrapped around it (the staff of Asclepius, not Hermes’ winged staff with two snakes). One hypothesis suggests that this symbol originated from the “treatment” for the disease caused by this worm.

Contaminated water supplies pass the larvae from water fleas to humans, where the worm matures and travels down towards the legs of its human host. To remove the worm, it is slowly pulled out by wrapping it around a stick as it emerges from the skin. This painfully debilitating process may take weeks, depending on the length of the worm. ***** Long story short, parasites have evolved in many, many weird ways, all with the same goal: to get from host to host, vector intermediate involved or not.

If you’re still interested in learning more, the Internet is a great resource, although one needs to be careful about sources used. The UCSD library also has plenty of information. And, if you’re really stoked about a certain parasite, maybe you can hunt down a faculty member or a postdoc studying that particular organism and ask them about their research (your best bet would be in the ecology, behavior, and evolution or the molecular biology department). The truth is out there! Go forth and parasitize, my pretties! Er, learn. Go forth and learn.

UCSD Biological Sciences Saltman | Quarterly

Wanna Research?

K

by

Ann Cai

nock down doors. Cold call complete strangers. Send out mass emails. Whatever it takes to get into a lab.

For those of us who are so inclined to spend hours upon hours holed up in our laboratories, where results are oh-so-elusive, sometimes the path to get there is even harder than our research. But it really doesn’t have to be. The upside to being a student at UCSD is that there are tons of professors (actually, about 150 of them) with biology labs on campus.

The downside is that it might be overwhelming to try to figure out where to start. Who should I work with? How do I contact them? Do I even want to do research?

Start with your own professors. Chances are if you like the professors of your classes, you will continue to like them in labs. These professors are known as the principal investigators (PI) of their labs, since they are in charge of running the lab. Generally, they do not actually run experiments anymore but take care of the administrative end of the research process. Make an effort to visit your professors during their office hours. Ask them about their research. Tell them about your background. If you already have some research experience, talk it up. If their research isn’t interesting to you, ask them if they know anyone else in a more appealing field who might be looking for an undergrad. I casually mentioned research to one of my professors in office hours, and a week later, I was meeting with her colleague about the research project that I have now been working on for two years.

Teaching assistants (TAs) are also great resources. They probably know you as a student better than your professors do and thus might be able to provide a solid reference or recommendation for you. Also, because they may be familiar with other labs on their floor, they might know of a few openings there, too.

More importantly, they can tell you about PIs they know of and whether or not students enjoy working with those PI’s.

Also, you might try asking your friends. I’ve actually been asked by a couple friends if I was interested in joining their labs because their PIs wanted new undergraduates. Your friends may know less than the TAs about what else is happening on their floor and may not have enough clout to recommend you to their PI, but they are nevertheless great resources for information and insights.

If none of these suggestions work, you can try to contact professors other ways.

The Biological Sciences Student Association (BSSA) offers events to bridge the gap between professors and students through faculty/student mixers and lun-

10 Saltman | Quarterly UCSD Biological Sciences

Volume 3 2005–06

cheons. UCSD’s Academic Enrichment Program (AEP) also offers a faculty mentor program for juniors and seniors that links students with professors for research experiences.

Another option is to try http://biology.ucsd.edu/bioresearch/ index.html to get a list of professors and their research interests. While this might be a last resort for some, it might be a starting place for others because it provides the largest single source of information about professors on campus.

Also check out the websites for the Salk Institute for Biological Studies, the Scripps Research Institute, or the Burnham Institute. Many researchers there are affiliated with UCSD and are happy to work with undergraduates. Send them an email and let them know that you are interested in meeting with them regarding the possibility of working/volunteering in their lab. If you decide to send an email, request a meeting regarding any undergraduate opportunities. Let them know why you are interested in the research done in this particular lab so that they don’t think it’s a mass email. Attach a resume if you have one. Talk about your general interests in research. If you don’t get a response, you may need to actually knock down some doors. It’s worked for some students and backfired for others. They might either really like you for taking the initiative, or might think you’re a pest…so it’s a bit of a gamble.

It is also very important to get a sense of what the other people are like in the lab. As an undergraduate researcher, you will need lots of help! If the other people in the lab are very competitive, it may not be the best environment to learn in, and it might end up as a negative experience. Many times, students find themselves fascinated with the research they are doing, but dreading going to lab because of the work environment. While you may not see the PI as regularly as the other people in the lab, he or she is extremely important to your experience. Find a pleasant PI, dedicated to undergraduate education. You really want someone who is interested in helping you learn about research and isn’t just using you for free labor.

The next step after you’ve made the contact is to sit down in a meeting with the PI regarding your potential role in the lab. Make sure that you are actually interested in what that lab is studying. You want to be excited about the research so that you will be motivated to go to lab, instead of dreading your hours there. After talking to the PI, you should be clear as to your level of independence in the lab. Some PIs may initially ask you to spend a quarter washing dishes or the like before they let you actually run an experiment. Others will ask you to volunteer for a quarter without credit or pay before they grant you a BISP 199, which is the independent study course for research at UCSD. You can also get credit for research through BISP 196 (senior honors thesis) and BISP 197 (biotechnology internship), which can also lead to some beneficial research experiences. It all really depends on your past experience, your level of commitment, and the PI. Some students may not feel comfortable working on their own projects with limited help from others and may enjoy taking on some of the smaller tasks such as washing dishes or making solutions to get a taste of what it is like to work in a lab. Others may want to tackle an independent project straight on. Be honest with the PI about your previous lab experience and time commitment to ensure that the lab and your responsibilities will be a good fit for you.

So get out there! Start networking and good luck!

2005–06 Volume 3

As far as answering the question about whether or not you are interested in research, think about whether or not you are curious about how people discovered the things that we study in our textbooks. Do you want to work towards discovering something that no one else has ever known before?

My PI once told me, “Classroom learning is studying the known. Research is exploring the unknown; it gives you the opportunity to be the very first person to ever know something.” If that thought piques your interest, then research may be right for you.

Ann Cai is a senior with a double major in molecular biology and music. She is a member of John Muir College.

UCSD Biological Sciences Saltman | Quarterly 11

Why We Need Medicinal Research

by

(To Highlight Recent Nobel Prize Winners)

A

simple drug, take Advil, for instance, was born out of a mixture of curiosity for a common physical ailment, a need for a straightforward solution, a little bit of capital, and a whole lot of basic research. This kind of research has led us to medicines that have helped prolong our lives and cured us of various maladies.

Many people don’t realize the amount of work, time, and money that goes into the development of a medicinal product.

Oftentimes, researchers base their work on questions and ideas that no one else has asked or even wanted to believe.

It is this kind of perseverance that has allowed us to enjoy the fruits of their labor. The following histories of recent Nobel Prize winners for Physiology or Medicine highlights the importance that research has on the larger community and serves to show how much worse off we would be without them.

Barry J. Marshall and J. Robin Warren: Marshall and Warren1 were awarded the Noble Prize in 2005 for their work in discovering the microbial contribution to peptic ulcer, one of the most common ailments in the stomach. They named this bacterium: Helicobacter Pylori.

Thanks to their findings, antibiotics can now be used to permanently cure the ulcer. Marshall and Warren are good examples of embracing curiosity. After isolating Helicobacter Pylori and realizing that it was probably causing an infection, the two scientists decided to test the bacteria’s effects on an animal. However, an animal could not be found for the purpose of this research, so Marshall drank a bacteria solution himself. As expected, he developed an inflammation in his stomach. With this ultimate proof in hand, Marshall and Warren realized

that the cure was antibiotics and spared discomfort for many today and in the future.

Paul C. Lauterbur and Sir Peter Mansfield: Lauterbur and Mansfield2 won the award in 2003 for their indispensable work in magnetic resonance imaging. They took a more mathematical approach to the imaging systems already in place and improved the resolutions. Lauterbur applied gradients to a magnetic field of two-dimensional images and used the resultant radio waves to allow a more precise image to develop. Mansfield took that idea one step further and applied mathematical equations to the radio waves and found a useful analysis technique for the images, thus making it easier to see irregularities in the body.

He also improved the resonance system so as to increase the speed of image creation. Thanks to their research, magnetic resonance imaging systems have been improved enough to discover diseases well before it is too late.

Leland H. Hartwell, Tim Hunt and Sir Paul Nurse: Hartwell, Hunt, and Nurse3 won the prize in 2001 for their discovery of cyclins that aid in basic cell division.

The scientists discovered the regulators, cyclin dependent kinase (CDK) and cyclin. CDK and cyclin come together to form an enzyme that helps regulate cell division. This research is now present in introductory biology textbooks and has helped to better clarify the different controls of a process that always seemingly knows what to do. Using mutated cells, Hartwell formulated the idea of checkpoints, in which the cell confirms that events are happening when they are supposed to. Using other mutated yeast cells, Nurse discovered

12 Saltman | Quarterly UCSD Biological Sciences

Shruti

Jayakumar

CDK, and realized that it has been evolving for one billion years when he discovered it in other organisms. Hunt discovered cyclin itself, which acts as the switch for CDK, through studying sea urchins. He came to the same evolutionary conclusion, and found that with cyclin, protein degradation occurs to regulate the cell cycle. Their work has been crucial in discovering treatments for cancer which arrest cells during division.

These scientists and researchers have shown what simple basic research can accomplish. While they did not create the resultant drugs and treatments, their work has been vital for their development.

References 1.

2.

3.

Normark, Steffan. “The Nobel Prize in Physiology or Medicine 2005.” Presentation Speech. 2005.

The Nobel Foundation. 14 Feb. 2006. <http:// nobelprize.org/medicine/laureates/2005/ presentation-speech.html> Ringertz, Hans. “The Nobel Prize in Physiology or Medicine 2003.” Presentation Speech. 2003.

The Nobel Foundation. 14 Feb. 2006. <http:// nobelprize.org/medicine/laureates/2003/ presentation-speech.html> Zetterberg, Anders. “The Nobel Prize in Physiology or Medicine 2001.” Presentation Speech. 2001.

The Nobel Foundation. 14 Feb. 2006. <http:// nobelprize.org/medicine/laureates/2001/ presentation-speech.html>

Shruti Jayakumar is a sophomore majoring in human biology. She is a member of Eleanor Roosevelt College.

Volume 3 2005–06

Q&A

Meet Professor Milton Saier, Ph.D. by

Hyuma Leland

ly the same type as the murder weapon. But when he came to trial, the gun couldn’t be found, and he couldn’t be proven guilty.

So he changed his name, went back East and disappeared. That was the end of my botany career.

I then took another advisor, an assistant professor in the department. He was an enzymologist, and under his direction, I came to appreciate the truly remarkable catalytic capabilities of these proteins. But unfortunately, he didn’t get tenure, so that was the end of that project. My last advisor was chairman of the department, a carbohydrate biochemist. Under his direction, I did my Ph.D. thesis on the structure of a bacterial lipopolysaccharide.

Thank you for sitting down with me today, I’d like to start off first with some basic questions. When did you first become interested in biology?

When I was about 4 or 5 years old I started my first garden, and I’ve enjoyed gardening ever since. However, in my sophomore year of high school, I had a biology teacher who was just awful; I couldn’t stand him. He totally turned me off of biology. But I took chemistry the next year from a really cool guy; I thought he was great. Consequently, in college I majored in chemistry. Only years later did I come to realize it wasn’t biology I disliked, it was this one teacher; he’d shaped my direction up to that point. After I graduated from college, I veered towards biology:

first biochemistry (in graduate school), then genetics (as a postdoc) and eventually microbiology.

When I first entered graduate school, I thought I’d become a botanist. There were two botanists in the biochemistry department at UC Berkeley then; one of them was too old to take graduate students, and the other became my advisor. Later that quarter he was murdered…he had liked to pick up hitchhikers since he had been a poor hitchhiker when he was young; apparently it was a hitchhiker who killed him. It was thought by some authorities to have been one of the students in his class who got a poor grade.

This student had a registered gun of exact-

Hyuma Leland is a junior majoring in molecular biology.

He is a member of John Muir College.

2005–06 Volume 3

“

Only years later did I come to realize it wasn’t biology I disliked, it was this one teacher...

”

UCSD Biological Sciences Saltman | Quarterly 13

Only later did I come to appreciate the intricacies and remarkable diversity of microorganisms.

proteins. Its availability has allowed us and other labs to do global analyses that otherwise would have been impossible.

Where did you receive your education? What degrees do you hold?

UC Berkeley, both a B.S. in chemistry and a Ph.D. in biochemistry. Those were exciting times. During the 60’s, Berkeley was a great place to be. We really thought we were going to make a difference. There was this tremendous excitement: we were changing the world!

Idealism was rampant; we really thought we could overcome apathy and involve all of America in progressive movement for world betterment. Then Nixon came along.

What is the larger goal of your research? Who do you foresee benefiting from your progress?

We do basic research…I think of science more as a cultural contribution than as a means to technological advances. It wouldn’t matter to me if I were a composer, writing music, or a writer, entertaining people with stories, or a scientist, contributing to our knowledge of the world. I’m not here primarily to benefit any group of people or to overcome some particular problem. I merely want to contribute to the flow of human culture.

What is the focus of your current research?

What we’ve done over the past has been largely concerned with transcriptional regulation and the phenomenon of catabolite repression in bacteria. Our wet lab has long been involved in studying gene regulation. But about 15 years ago, we started in doing computer work, bioinformatics analyses of protein sequences, and this has become the major focus of the lab. We are interested in transport proteins that bring molecules into and take them out of the cell. We’ve been able to show that they have arisen from small peptides independently of other protein types. Using bioinformatic tools, we can actually go back billions of years and follow the processes by which these proteins evolved. It’s pretty amazing. Now we analyze whole genomes. For this purpose we’ve developed a classification system for transport proteins, a system that two or three years ago was adopted by the International Union of Biochemistry and Molecular Biology. Our Transporter Classification (TC) System is now the universally acclaimed system for classifying transport

“

environmental journals, began writing editorials and essays for them, started a student–faculty group called PREP (Population Reduction and Earth Preservation - www.acs.ucsd.edu/~prep) here on campus, was appointed chair of the San Diego Division of Population Connection, initiated a course entitled “Human Impact on the Environment” and sponsored seminars on various environmental issues, given by experts in their respective fields. Even in this area, my primary purpose is education.

Have you had any memorable successes, highlights, or breakthroughs you’d like to discuss?

In science you never know what’s going to come along. Sometimes you just make an observation that proves to be really important, and then you kind of glide on that discovery for a few years. The first time that I made a truly exciting discovery of that type, I was conducting a very simple experiment. I measured the uptake of a radioactive compound called glycerol by the bacterium E. coli. When I threw in glucose, uptake of glycerol stopped dead within less than a second. So I knew I’d discovered something really exciting and spent the next 10 years figuring out what the mechanism was. I’d started that work as a postdoc and continued it when I came to UCSD as an assistant professor.

Almost every agency was cut, but one went up 30%. Can you guess which one that was? It was administration.

That’s not how to promote discovery and innovation. By cutting research budgets, the Bush administration is sabotaging the American economy.

”

More recently I’ve become convinced that environmental affairs are even more important to mankind than my research or any cultural contribution I might be able to make. This realization hit me one day about 4-5 years ago following a lecture I attended at the UCSD Faculty Club. The speaker was SIO professor Paul Dayton, and he talked about what we humans are doing to the oceans.

What he related was truly shocking, and when I started thinking about what he had said, I realized that my own life experiences confirmed his claims.

At that point I knew I had to devote part of my time to environmental issues. I became associate editor of a couple of

14 Saltman | Quarterly UCSD Biological Sciences

Another time, I was characterizing an enzyme system found in a weird bacterium, Spirochaeta aurantia. Because the activity was very low, I had to change the assay conditions, going down about a thousand-fold in sugar concentration and increasing the specific activity of the radioactivity about a thousand-fold. In Volume 3 2005–06

retrospect, these changes were responsible for the discovery. I was characterizing the phosphoryl donor specificity of an enzyme system called the PEP-dependent phosphotransferase system (PTS).

I tried a bunch of potential phosphate donors. At the time, there was only one known phosphate donor for this system, phosphoenolpyruvate, the end product of glycolysis, and that worked very well as expected. But in this experiment, I tried a sugar phosphate, mannitol-1phosphate. Surprisingly, I found it had tremendous activity. Well I didn’t believe it, but I knew that if it were true it was important. No one had ever reported anything like this before. So I repeated the experiment a second time and got the same result. I still didn’t quite believe it. I did it a third time and again got the same result. By then I was pretty convinced something interesting was going on. This took place on New Year’s Day several years ago, so I was the only one in the lab. Everyone else was on vacation.

I kept on doing experiments, hardly taking time out to eat or sleep, and by the time school started again, I had essentially characterized this novel reaction. After writing it up for publication, I submitted it to a journal, the Journal of Biological Chemistry. When, after about a month, I got the reviews back, I learned that my paper had been rejected for publication; the reviewers simply hadn’t believed it!

The editors indicated a whole series of experiments they wanted me to do to make sure it was real. But by the time I’d gotten the reviews back, I’d already done all the experiments they’d wanted. So I put the new data in, and the paper was finally accepted.

Many of our truly important discoveries are initially rejected by doubting reviewers. Rejection is a true sign that a discovery is novel and unexpected. To be successful, a scientist often has to be stubborn and persistent.

What goals and obstacles do you feel will become predominant in the coming years of biological research?

2005–06 Volume 3

Future goals? I consider microbiology to be a field of the future. There is no area of biology that is less well understood.

Less than 1% of all microbes have been identified and even fewer have been characterized. These tiny organisms have the tremendous potential to cure the ailments that plague our planet due to the excesses of mankind. As we become more aware of how important our environment is, more and more money and effort will be devoted to preserving it. I’m convinced that through bioremediation, toxin metabolism, use of probiotics, and genetic engineering, microbes will prove to be the great recyclers of the future.

Obstacles? The present administration has been consistently cutting the budget of the National Institutes of Health and the National Science Foundation as they did this year. I went through the different agencies of the NIH to see how they had distributed the available funds. Almost every agency was cut, but one went up 30%. Can you guess which one that was? It was administration. That’s not how to promote discovery and innovation. By cutting research budgets, the Bush administration is sabotaging the American economy.

Money is going to be a big problem, particularly for basic science. Unfortunately, you can’t do cutting-edge research without it. Some kinds of research can be done with relatively little money…computational research, for example. You don’t need big bucks once you’ve bought your computer unless you need to hire people to do the work. My students and I can just plink away on our computers and make discoveries. Making novel discoveries is exciting and entertaining. That’s one way to go, to remain productive when funds are scarce. Bioinformatics is really big nowadays, and it’s tremendously fun. There’s so much that needs to be done to reveal the information made available from genome sequencing.

What advice would you give a graduating biologist interested in research?

Do something you’re really interested in.

Choose a field and a subject that you enjoy; you never know the future. I can give you some examples.

When I was in high school, the one secure field you could go into was teaching. But by the time we graduated, the teaching market was flooded. The same thing happened with engineering.

When I started my undergraduate training at Berkeley, most kids were going into engineering because there was such a demand. But by the time we graduated four years later, engineering was flooded; you couldn’t get a job. Then I went into graduate school, and everyone chose what was then called molecular biology, which at that time meant studying transcription, translation and replication.

Once again, by the time we got our degrees, that field was flooded.

I went into carbohydrate chemistry, which was something nobody was doing, and boy, by the time I started looking for a faculty position, there were lots of opportunities. This was because, in the meantime, this slow-moving field had become hot. I just got lucky. But those who followed the fads because they thought it guaranteed their futures were sorely disappointed, particularly if they weren’t doing what they most enjoyed.

And for those who were truly enjoying their work, it didn’t matter so much if they couldn’t get the best jobs. They had not wasted their time doing something they didn’t find satisfying.

Who is he?

Dr. Milton Saier Professor of Biological Sciences, UCSD http://www.biology.ucsd/edu/faculty/saier.html.

What does he teach?

Microbial Genetics (BIMM 122) Human Impact on the Environment BILD 18 Author’s Note. I would like to thank Professor Saier for his enthusiasm and contributions. For more information on Population Reduction and Earth Preservation (PREP) visit: http://acs.ucsd. edu/~prep or email: prep@ucsd.edu. Information on Population Connection can be found at http://www.populationconnection.org.

UCSD Biological Sciences Saltman | Quarterly 15

Q&A

Nigel M. Crawford The path of a biologist is not always one that is lit with the guiding lights of security and predetermination. For many students, the pursuit of biology can be a daunting one. With over 30 years of experience in biology academia, Dr. Nigel Crawford knows this well and shares some words of wisdom with the undergraduates of UCSD.

by

Max Chen

Why biology? Why did you choose to pursue biology?

I always loved biology. I knew when I was in high school that I wanted to go into science; I just didn’t know what field of science. I was initially drawn to biology by the environmental movement.

Was there any particular event that triggered the interest?

It was in the 1970s: there were very intense battles trying to fundamentally change how people viewed the environment and incorporating these ideals into law to protect the environment. It was a very hot topic and drew me into environmental biology.

As advice for the student on campus pursuing biology, if you could make a change in your educational experience, what would it be?

Hum…not sure I would change too much. For me, the critical thing was trying to focus on what I was interested in because often you have so many things that you feel are interesting, fun, and would like to work on. But practically speaking, you need to start focusing on a particular goal. It’s usually a degree for undergraduates and a specific area of biology and projects you’d like to get involved with. So sampling around for what you love and what you’re good at is a very key part in your early stages of education. For some people, their goals are very clear but for most people they are not, and they’ll have to try a couple of things out first. This process also helps clarify your true interests and capabilities. What you think your life will be like in a specific field might be very different from what your experience in the field will actually be.

16 Saltman | Quarterly UCSD Biological Sciences

“

Sampling around for what you love, what you’re good at, is a very key part in your early stages of education.

”

For example?

Well, my wife, for example, thought she might want to go into optometry until she went in to have an eye exam and was reminded of what an optometrist does much of the day.

She said she didn’t like the idea of sitting in a dark room for most of the day.

Haha, yes, I had a very similar experience with my decision to not go into dentistry. What would be the approach to take?

The only way you’re going to know what an experience is like is to go sample it and try it out, be it in an emergency room, in a lab, work as a counselor, or whatever it is you think you might be interested. Go ahead and give it a try to see firsthand how somebody who does that lives everyday.

Was this what you did?

That’s what I did. I worked in a lab, enjoyed it, and began to foVolume 3 2005–06

cus on biochemistry/molecular biology.

And it wasn’t until I was a postdoc that I felt truly committed to my path. When you go from a graduate student to a postdoc you’re making a potentially lifelong career decision because as a postdoc on the academic route, working towards a faculty position, you’re given a job interview based on the science you’ve done as a postdoc. And it is in that field you usually begin your own lab, what you write your grants for, and that’s what you end up doing potentially for the rest of your life. Now people do change. But this is nonetheless a very key juncture in academia, and for me, trying out the different fields in biology really helped to hone in on my interest.

From your experience as an educator, what would you say is a mistake you commonly see undergrads making?

Well… I wouldn’t call it a mistake, but I see students taking on too much work, too many projects, and, as a result, getting very scattered in their energy, in their focus. It’s a natural thing to do because there’s so much going on around you.

It’s understandable because there are so many fun things to do at the university. There are social activities, extra-curricular activities, clubs, sports, and so forth. But if you get too scattered with your energies, you can become severely drained. Being overwhelmed is a common thing I see in undergrads.

“

tional experience. Whatever it is, adopt it in a healthy way, not in a way that’ll overwhelm you. What it comes down to is focus. The problems arise when students try to take on too much.

Well, now that we’ve talked about the common mistake undergrads make. How about telling our readers the plusses? What characteristics exhibited by a student you’ve came across in the past impressed you the most?

I would say maturity. I see a very wide spectrum when I’m interacting with students. Some are very young and still trying to figure things out, which is fine, while some students already have a sense of where they’re going and what they want. It is impressive for me to see someone who really knows what they want and is going for it. I’d say the maturity to accomplish that in an intelligent, successful manner is the most impressive characteristic a student can exhibit.

I’d say the maturity to accomplish [goals] in an intelligent, successful manner is the most impressive characteristic a student can exhibit.

What’s your advice in helping students avoid making this “mistake”?

They need to pace themselves and ask themselves, “Why am I here? What are my intentions to be here?” It might be focusing on their studies, getting good grades, and going to medical school, or actively incorporating a broader educa2005–06 Volume 3

It is also important for students to accomplish their goals in a kind way. It is not impressive to see students who are forcing their way to the top of the class. It is impressive to see students who are willing to help each other, to volunteer their time to assist other students.

”

UCSD have been experiencing more and more students who take biology as a major but not necessarily to pursue a directly biology-related field. They don’t always want to end up at the lab bench or in a hospital, which the majority of UCSD biology majors still do. What would you say to students who major in biology but who might not want to go into those

two well-established fields?

Every discipline has skills, insights, and history to offer a way to approach life.

Biology offers its own set of tools and understandings. The tools that biology offers allow one to deal with critical issues like medicine and environment, two of the most important issues in biology.

And having a sense of what is going on in those fields and to be able to understand and think critically when people talk about medicine, agriculture, the environment, that is a benefit to everyone.

We have enormous challenges in the future like accessibility and affordability in medicine, the obesity crisis, agriculture, and environmental hazards. Even if one doesn’t directly work in a field like biology, it’s extremely important to be able to think critically and understand the issues at hand when you vote or when you speak to someone else about these issues.

Thank you, Dr. Crawford, for participating in this interview today. Do you have any closing comments for the readers of Saltman Quarterly?

I hope you really enjoy what you’re doing and try to get as much out of it while you can. It’s a great time to be doing biology.

Whatever your interests are, go for it!

Who is he?

Dr. Nigel Crawford Professor of Biological Sciences, UCSD http://www.biology.ucsd.edu/labs/crawford What does he teach?

Fundamentals of Plant Biology (BICD 120) Nutrition (BIBC 120) Max Chen is a junior majoring in molecular biology. He is a member of Thurgood Marshall College.

UCSD Biological Sciences Saltman | Quarterly 17

Protein-translocating Trimeric Autotransporters of Gram-negative Bacteria David S. Kim*, Yi Chao and Milton H. Saier, Jr.

*Roger Revelle College, Senior, Molecular Biology Major (Article Accepted Fall 2005)

The Autotransporter-2 (AT-2; TC #1.B.40) family includes proteins with short (70 residue) C terminal β-sheet domains (AT-2 domains) that form trimeric pores in the outer membranes of Gram-negative bacteria and transport the N-terminal passenger domain from the periplasm to the cell surface. Sixty-nine (69) sequenced homologues that show similarity throughout most of their AT-2 domains have been identified. These proteins derive (almost) exclusively from α, β-, and γ-proteobacteria** and their phage. Analyses reveal an N-terminal amphipathic region, followed by a strongly hydrophobic region. These precede four well-conserved putative amphipathic transmembrane β-strands that are likely to comprise the pore-forming β sheet.

Phylogenetic analyses reveal that the AT-2 domains generally cluster according to organismal type but not according to protein size. Many of these AT-2 domains have a seven residue repeat sequence at their N-termini, and the AT-2 domains may have evolved from multiple such repeated elements. Motif analyses reveal two well-conserved regions that are likely to be of functional significance. This report provides the first detailed bioinformatic analysis of the AT 2 family of autotransporter domains. **The gram-negative Proteobacteria are a large and physiologically diverse group, which are divided into five classes (α, β, γ, δ, and ε), based on 16S rRNA sequences.

Introduction Gram-negative bacteria possess a twomembrane envelope with an outer lipopolysaccharide-containing membrane that provides an effective barrier, protecting these organisms from detergents, organic solvents, drugs and other toxic substances.15 However, the occurrence of an outer membrane poses major problems for the secretion of macromolecules.19 Thus, Gramnegative bacteria have evolved a tremendous diversity of outer membrane systems designed for the export of proteins, complex carbohydrates, nucleic acids and lipids.2,25

Among the well-characterized outer membrane protein secretion systems are the so-called two-partner secretion (TPS; TC #1.B.20) and autotransporter (AT; TC #1.B.12) systems.14,21,35 Following export from the cytoplasm to the periplasm via the general secretory (Sec) system, both TPS and AT translocation domains insert into the outer membrane as β-barrel structures.

They mediate export of virulence proteins or protein domains from the periplasm across the outer membrane to the extracellular medium where the exported protein or domain may either remain attached to the outer membrane or can be released in a free state.35 The exported proteins may serve as adhesins, hemolysins, proteases, cytotoxins or mediators of intracytoplasmic actinpromoted bacterial motility.35

Proteins of the autotransporter family possess C-terminal 250-300 amino acyl residue domains that fold and insert into

the outer membrane to form a β-barrel with 12-14 transmembrane β-strands.12,11,18,20 This structure forms a pore through which the Nterminal virulence factor is presumed to be exported.9,23 There is still some controversy as to the mechanism of protein transport.3,23,34

A second family of autotransporters called “trimeric autotransporters”, “oligomeric coiled-coil adhesins” or “autotransporters2” (AT-2; TC #1.B.40) has recently been discovered.6,12,13,29,36 Among the bestcharacterized members of this family are the multifaceted Yersinia adhesin, YadA6,13,2224, the major adhesin of Haemophilus influenzae that allows colonization of the nasopharynx, Hia16 and the Haemophilus “adhesin and penetration” protein, Hap.7,8,17,33 These proteins define a novel family of autotransporter virulence factors. They are characterized by their ability to cross the outer membrane without the assistance of accessory proteins. A conserved C-terminal domain of about 70 amino acyl residues forms the trimeric β barrel that presumably allows the transport of the N-terminal “passenger” domain to the bacterial cell surface. These proteins form trimeric lollypop-like structures anchored to the outer membrane by their C-terminal autotransporter anchor domain.4,30 The C-terminal 67-76 residue domains are both necessary and sufficient for translocation of the N-terminal adhesin domains.30 They are believed to consist of just four transmembrane antiparallel β-strands.3,4 Deletion of the C terminal domain abolishes outer membrane insertion of YadA31 while

18 Saltman | Quarterly UCSD Biological Sciences

the deletion of the linker region results in degradation of the whole protein.24

The few characterized protein members of the AT-2 family serve as virulence factors in animal pathogens.24 They have been termed invasins, immunoglobulinbinding proteins, serum resistance proteins and hemagglutinins, but all appear to have adhesive properties. The passenger domains sometimes possess multiple (2-8) repeat domains of about 150 residues, and these may consist of smaller repeat units.

For the purposes of this report, however, the characteristic feature that will be used for identification of family members is the presence of the small C terminal domain that is believed to form the outer membrane trimeric β-barrel pore.

The bioinformatic analysis of the AT-2 family presented in this paper entails identifying recognizable sequenced members of the AT-2 family and aligning the sequences of their autotransporter domains. The resultant multiple alignment is used to identify conserved motifs, generate a phylogenetic tree for the family, identify cluster-specific sequence characteristics, and generate average hydropathy, amphipathicity and similarity plots that allow structural predictions. Essentially all of the AT-2 proteins derive from α-, β- and γproteobacteria and their phage. The analyses reveal that phylogeny of the AT-2 domains does not correlate with size or structure of the N-terminal domain. To a considerable degree, however, protein phylogeny correlates

Volume 3 2005–06

with phylogeny of the organisms from which these proteins derive. The results suggest that the genes encoding these proteins have been subject to lateral transfer, but transfer occurred primarily within closely related organisms. This conclusion is substantiated by their occurrence in phage genomes (see below).

Results Established protein members of the AT-2 family Using the PSI-BLAST search tool with YadA of Yersinia enterocolitica as the query sequence and three iterations, about 140 above threshold hits were retrieved from the NCBI database.1 AT-2 family members were identified on the basis of their C-terminal autotransporter-2 domains (hereafter called AT-2 domains). No homologues were identified that appeared to have the AT-2 domain anywhere other than at their extreme C-termini.

Redundancies, very closely related homologues, and hits that showed an insufficient degree of sequence similarity with established members of the family to establish homology (≥9 S.D. using the GAP program)5 were eliminated. This left 69 proteins upon which the analyses reported below were based. These proteins are presented in Table 1 while their aligned AT-2 domain sequences are shown in Figure 1, and the phylogenetic tree, based on this alignment, is presented in Figure 2. The proteins listed in Table 1 are presented according to cluster as shown in the tree presented in Figure 2.

As indicated in Table 1, the homologues exhibit tremendous variation in overall protein size (86 amino acyl residues (aas) to 3674 aas). Even within a single cluster, the size variation is tremendous (see Tables 1 and 2). This degree of size variation was not observed in previous studies of the AT family.35

With the exception of four homologues, all were from proteobacteria.

Two close homologues are from a bacteriophage (p-EibE) and a prophage (pEibA), both of E. coli. These two proteins are annotated as immunoglobulin binding proteins. The two small non-proteobacterial homologues (Dha2, 86 aas and Dha1, 142 aas) are reported to be from Desulfitobacterium hafinense which is a low G+C Gram-positive bacterium with no outer membrane. These proteins could not serve as autotransporters in this organism. Because the genome of D. hafinense has not been completely sequenced and is still being updated, it is possible

2005–06 Volume 3

that these sequences resulted from DNA contamination.

Seven-residue repeat sequences in AT-2 proteins Several of the AT-2 proteins listed in Table 1 exhibit a 7 aa repeat element between the passenger domains and the putative transmembrane regions of the AT-2 domains. For many of these homologues, this repeat element occurs 2 or 3 times at the N terminal end of the AT-2 domain (see Figure 1). However, in other proteins retrieved in BLAST searches, this 7 aa repeat occurred as many as 12 times, creating a domain the length of an AT-2 domain.

An example of this is the Apl protein of Actinobacillus pleuropneumonia with a size of 195 aas. The repeat element, encompassing all but the last 12 residues of this protein, is illustrated in Figure 3 where twelve tandem repeat elements are shown. The consensus for this repeat element is (D E) (Q N) (R K) (F I) (Q D) (Q K) (V L) where the two most prevalent residues at each position are indicated in parentheses. It is possible that the AT-2 domains have evolved from a primordial gene like that encoding the Apl protein, derived from an internally repeated 21 bp genetic element. It is also possible that the repeat element, found in the N-terminal regions of several AT-2 domains (see Figure 1), represents a linker region connecting the passenger domain to the AT-2 domain. Thus, AT-2 domains may have either evolved from a sequence like that shown in Figure 3, or they could have evolved independently of this repeat sequence and become associated with them as a result of gene fusion events.

Phylogenetic clustering according to organismal type All of the proteins in Table 1 exhibit sequence similarity in their AT-2 domains. The phylogenetic tree for these domains, shown in Figure 2, reveals clustering according to organismal type (see also Table 1). Thus, cluster 1a contains only β-proteobacterial proteins, cluster 2a contains only α-proteobacterial proteins, and clusters 2b, 2d and 3a contain only γ-proteobacterial proteins. Moreover, clusters 1b, 2c and 3b contain only β- and γ-proteobacterial proteins with the exception of the two E. coli phage proteins and the two putative Desulfitobacterial proteins, Dha1 and Dha2. Finally, cluster 1c contains only α- and γ-proteobacterial proteins. Thus, to some extent, clustering reflects the organismal type from which these proteins derive. This observation suggests that horizontal transfer

of genetic material encoding AT-2 proteins has been restricted largely to organisms within any one of the proteobacterial subdivisions (see Discussion).

Structural predictions The average hydropathy, amphipathicity and similarity plots, based on the Figure 1 multiple alignment and obtained using the AveHas program37, are shown in Figure 4.

There are five peaks of hydrophobicity (H1H5), and with the angle set at 180º, as is appropriate for a β-strand, there are five peaks of amphipathicity (A1-A5). The average similarity plot (Figure 4, dashed black line) follows the average amphipathicity plot (red line), more closely than it follows the average hydrophobicity plot (blue line).

The first hydrophobic peak (H1) does not show amphipathic character, and the first amphipathic peak (A1) is not appreciably hydrophobic. These regions may not form transmembrane β-strands.

However, A2 overlaps and follows H2; A3 overlaps and follows H3; A4 overlaps and slightly follows H4, and A5 overlaps and precedes H5. Established transmembrane β-strands in outer membrane porins often show overlapping but non-coincident peaks of hydrophobicity and amphipathicity.39 There are therefore four overlapping peaks of amphipathicity and hydrophobicity that serve as excellent candidates for transmembrane, pore-forming β-strands.

Each of these overlapping regions is about 710 aas long as expected for a transmembrane β-strand. Therefore it is predicted that these four strands form a small transmembrane β-sheet. This β-sheet presumably forms the homotrimeric pore through which the passenger domain passes (see Introduction).

Conserved motifs As shown in Figure 4, the most conserved regions of the alignment coincide with hydrophobic peak H1 and amphipathic peak A3. These include the two most conserved motifs among AT 2 domains. These two consensus motifs were: AGIASALALA (motif 1; alignment positions 18-27) and SAVAIGV (motif 2; alignment positions 51-57). Although the majority of the proteins exhibit these conserved residues, no residue position is fully conserved, and the variation at any one position is usually considerable. The best-conserved residue is G56 which is conserved in all but one of the proteins (Hin1) where a V can be found (see Figure 1 and Table 3). Examination of the data in Table 3 reveals that at almost all

UCSD Biological Sciences Saltman | Quarterly 19

Table 1. Recognized proteins of the AT-2 family

Abbreviation1 Cluster 1a Bce1 Bce3 Bce4 Bma1 Rso1 Cluster 1b Dha1 Xca1 Xor1 Cluster 1c Hin1 Rsp1 Cluster 1d Bfu1 Cluster 2a Bhe1 Bhe2 Bme1 Bqu1 Bqu2 Bqu3 Bqu4 Bsu1 Bvi1 Bvi2 Bvi3 Mlo1 Sme1 Cluster 2b Hdu2 Hdu3 Cluster 2c Aac1

Organism

Size2

Database description

Burkholderia cepacia R18194 Burkholderia cepacia R18194 Burkholderia cepacia R18194 Burkholderia mallei ATCC 23344 Ralstonia solanacearum

GI #4

977 1010 276 373 1309

E E E E E

46316503 46315938 46322712 53717377 17549839

Autotransporter adhesin Unknown

142 1328

Clostridia J

23115364 7542317

Outer membrane protein XadA

1265

J

9864182

Haemophilus influenzae R2846 Rhodobacter sphaeroides 2.4.1

Autotransporter adhesin Large exoproteins involved in heme utilization or adhesion

158 411

J D

42630309 46192873

Burkholderia fungorum LB400

Autotransporter adhesin

3068

E

48784624

1747

D

49237768

153 365 1065

D D D

49237769 17988155 49239313

949

D

49239314

950 970 278 1760 3620 1420 1953 1291

D D D D D D D D

51949816 51949818 23502699 52355211 52355212 52355210 13472521 15964211

296 236

J J

33152901 45758814

295

J

19568164

459

J

16923467

257

dsDNA virus

33151932

487

dsDNA virus

7523541

257 686 405

J J E

33151932 18568377 21427129

Desulfitobacterium hafinense Xanthomonas campestris pv. pelargonii Xanthomonas oryzae pv. oryzae

Autotransporter adhesin Autotransporter adhesin Autotransporter adhesin Autotransporter adhesin Putative hemagglutinin-related protein

Bacterial type3

Bartonella henselae str. Houston-1

Surface protein/Bartonella adhesin Bartonella henselae str. Houston-1 Surface protein Brucella melitensis 16M Cell surface protein Bartonella quintana str. Toulouse Surface protein/Bartonella adhesin Bartonella quintana str. Toulouse Surface protein/Bartonella adhesin Bartonella quintana VompA Bartonella quintana VompC Brucella suis 1330 Hypothetical protein BR1846 Bartonella vinsonii subsp. arupensis BrpB Bartonella vinsonii subsp. Arupensis BrpA Bartonella vinsonii subsp. arupensis BrpC Mesorhizobium lot MAFF303099 Hypothetical protein mil2848 Sinorhizobium meliloti 1021 Hypothetical protein SMc01708 Haemophilus ducreyi 35000HP Haemophilus ducreyi

Eco4

Actinobacillus actinomycetemcomitans Escherichia coli

EibA

Prophage P-EibA

EibE

Bacteriophage P-EibE

Hdu1 Mca1 Nme1

Haemophilus ducreyi 35000HP Moraxella catarrhalis Neisseria meningitidis

conserved positions in motif 1, exceptional non-conserved residues can be hydrophilic, hydrophobic or semipolar. Only at alignment position 21 is the residue always semipolar.

This fact suggests that there is not an absolute

Hypothetical protein HD1920 Necessary for collagen adhesion protein Putative adhesin/invasin Immunoglobulin-binding protein EibF Immunoglobulin-binding protein EibA Immunoglobulin-binding protein EibE Serum resistance protein DrsA Ubiquitous surface protein A2 Putative adhesin/invasin

requirement for residue type at most of the positions in putative hydrophobic peak 1 (see Figure 4).

In contrast to conserved motif 1, conserved motif 2 has a characteristic residue

20 Saltman | Quarterly UCSD Biological Sciences

type at each position. Thus at alignment position 51, all residues are semipolar or hydrophilic. At position 52 all residues are semipolar. At position 53, all residues but one are hydrophobic. At position 54, all

Volume 3 2005–06

Abbreviation1 Nme3 Yen1 Yps1 Cluster 2d Ppr1 Cluster 3a Hso1 Cluster 3b Aac2 Apl1 Bce2 Bfu2 Bma2 Dha2 Eco1 Hin2 Hso2 Hso3 Hso4 Hso5 Hso6 Hso7 Nme2 Nme4 Pmu1 Pmu2 Reu1 Sen1 Xfa1 Xfa2 Ype1 Ype2 Cluster 3c Eco3 Eco5 Mca2 Cluster 3d Eam1 Eco2 Ype3 Yps2

Size2 Bacterial type3 GI #4 355 21427156 E 454 1955604 J 434 141104 J

Organism Neisseria meningitidis Yersinia enterocolitica Yersinia pseudotuberculosis

Database description Putative adhesin/invasin Adhesin YadA Adhesin YadA precursor

Photobacterium profundum

Hypothetical protein

288

J

46917051

Haemophilus somnus 129PT

Autotransporter adhesin

452

J

23468079

Actinobacillus actinomycetemcomitans Actinobacillus pleuropneumoniae serovar 1 str. 4074 Burkholderia cepacia R18194 Burkholderia fungorum LB400 Burkholderia mallei ATCC 23344 Desulfitobacterium hafinense DCB-2 Escherichia coli O157:H7 EDL933 Haemophilus influenzae Haemophilus somnus 2236 Haemophilus somnus 2236 Haemophilus somnus 129PT Haemophilus somnus 2236 Haemophilus somnus 2236 Haemophilus somnus 2236 Neisseria meningitidis Neisseria meningitidis Pasteurella multocida subsp.

Multocida str. Pm70 Pasteurella multocida subsp.

Multocida str. Pm70 Ralstonia eutropha JMP134 Salmonella enterica subsp. Enterica serovar Typhi str. CT18 Xylella fastidiosa 9a5c Xylella fastidiosa 9a5c Yersinia pestis CO92 Yersinia pestis KIM

EmaA

1965

J

33578091

Autotransporter adhesin

2600

J

46143665

Autotransporter adhesin Autotransporter adhesin Hemagglutinin family protein Autotransporter adhesin Putative adhesin Adhesin Autotransporter adhesin Autotransporter adhesin Autotransporter adhesin Autotransporter adhesin Autotransporter adhesin Autotransporter adhesin Adhesin NhhA outer membrane protein Hsf

1439 770 831 86 1588 1096 2419 2390 611 3391 1550 3674 591 589 2712

E E E Clostridia J J J J J J J J E E J

46313782 48787852 53717118 536841400 15804146 25359414 46156748 46156040 23467645 32030792 46156755 46156455 15676883 14578023 15602579

Hsf

1299

J

15603435

Autotransporter adhesin Putative autotransporter

465 1107

E J

53761962 16762618

Surface protein Surface protein Putative surface protein (partial) Hypothetical protein y1847

2059 1190 658 144

J J J J

15838130 15838575 16121208 22125740

Escherichia coli Escherichia coli O157:H7 EDL933 Moraxella catarrhalis

IHP1-like Hypothetical protein Z0639 Hemagglutinin

436 338 2314

J J J

29367636 15800223 22000942

Erwinia amylovora Escherichia coli Yersinia pestis biovar Medievalis str.

91001 Yersinia pseudotuberculosis IP 32953

Autoagglutinating adhesin STEC autoagglutinating adhesin Hypothetical protein HP1206

494 516 364

J J J

38638179 16565696 45441033

Hypothetical protein pYptb0018

416

J

51593960

The cluster refers to the clustering pattern in the phylogenetic tree shown in Figure 1.

Size of the protein in amino acyl residues.

3 Greek letters refer to the subcategory of the proteobacteria.

4 GI number: Genbank Index number.

1 2

residues are semipolar, and at positions 5557 no residue is strongly hydrophilic. Thus, motif 2 has the highest degree of conservation in terms of the variable residue types found at the various aligned positions. This suggests

2005–06 Volume 3

that motif 1 in hydrophobic region H1 may have evolved to serve dissimilar functions within the differing AT-2 domains, while motif 2, in putative transmembrane β-strand 2, serves a single function, which is common

to all family members.

Discussion This paper experimental

summarizes the available evidence and reports

UCSD Biological Sciences Saltman | Quarterly 21

Motif1 Nme1 Nme3 Aac1 Eco4 EibA EibE Mca1 Yps1 Yen1 Hdu1 Hdu2 Hdu3 Bhe1 Bvi3 Bqu1 Bqu2 Bqu4 Bqu3 Bhe2 Bvi2 Bvi1 Sme1 Mlo1 Bme1 Bsu1 Bce2 Dha2 Bma2 Bfu2 Reu1 Xfa1 Xfa2 Sen1 Eco1 Ype1 Ype2 Mca2 Nme2 Nme4 Hin2 Apl1 Aac2 Hso2 Hso5 Hso6 Hso7 Hso4 Hso3 Pmu2 Pmu1 Hso1 Eco3 Eco5 Eco2 Eam1 Yps2 Ype3 Xca1 Xor1 Dha1 Bce3 Bma1 Rso1 Bce1 Bce4 Bfu1 Rsp1 Ppr1 Hin1

Motif

____ ______ ______ RLNGLDKTVSDLRKETRQGLAEQAALSGLFQPYNVGRFNVTAAVGGYKSESAVAIGTGFR-FTENFAAKAGVAVGTSSGSSAAYHVGVNYEW-RIDSLDKNVANLRKETRQGLAEQAALSGLFQPYNVGRFNVTAAVGGYKSESAVAIGTGFR-FTENFAAKAGVAVGTSSGSSAAYHVGVNYEW-RIDRLDSRVNELDKEVKNGLASQAALSGLFQPYNVGSLNLSAAVGGYKSKTALAVGSGYR-FNQNVAAKAGVAVSTNGGS-ATYNVGLNFEW-RLDSQQRQINENHKEMKRAAAQSAALTGLFQPYSVGKFNATAAVGGYSDQQALAVGVGYR-FNEQTAAKAGVAFSDG---DASWNVGVNFEF-RLDSQQRQINENHKEMKRAAAQSAALTGLFQPYSVGKFNASAAVGGYSDEQALAVGVGYR-FNEQTAAKAGVAFSDG---DASWNVGVNFEF-RLNSQQRQIRENHEEMKRAAAQSAALAGLFQPYSVGKFNATAALGGYSDKQAVAVGVGYR-FNEQTAAKAGIAASDG---DVSYNMGVNFEF-KVNAFDGRITALDSKVENGMAAQAALSGLFQPYSVGKFNATAALGGYGSKSAVAIGAGYR-VNPNLAFKAGAAINTSGNKKGSYNIGVNYEF-KFSQLDNRLDKLDKRVDKGLASSAALNSLFQPYGVGKVNFTAGVGGYRSSQALAIGSGYR-VNESVALKAGVAYAGS--SNVMYNASFNIEW-KFRQLDNRLDKLDTRVDKGLASSAALNSLFQPYGVGKVNFTAGVGGYRSSQALAIGSGYR-VNENVALKP-VWLIGS--SDVMYNASFNIEW-MMEQNTHNINKLSKELQTGLANQSALSMLVQPNGVGKTSVSAAVGGYRDKTALAIGVGSR-ITDRFTAKAGVAFNTYN-GGMSYGASVGYEF-IQQIDQRILHQFRKEMHMNTANTAAMSSLNFGNGYG-VSVGAAIGGHKGQYSLALGTAYTDYQTQVNVKIALPVKQPKPSNITYGVGFVYNFQILKQVNQKVHELRKETYMNTANTAAMSSLNFGNSQG-ISFGAAIGGHKGQHSLALGTAYTDYQTQVNVKIALPVRQPKPSNITYGIGFVYNFQKFEALNYSIENVRKEARQAAAIGLAVSNLRYNDTPGKLSVGFGSGLWRSQSAFAFGAGYTSESGSIRSNLSITTSGG---HWGIGAGFNMTLNKFEALNYNIENVRKEARRAAAIGLAVSNLRYNDTPGKLSVAFGSGLWRSQSAFAFGAGYTSEKGNIRSNLSVTSSGG---HWGIGAGLNMTLNKFEALNYGIEGARKEARQAAAIGLAVSNLRYNDTPGKLSIAFGSGLWRSQGAFAFGAGYTSESGAIRSNLSVTSSGG---HWGIGAGLGLTLNKFEALNYGIEGARKEARQAAAIGLAVSNLRYHDTPGALSVAFGSGLWRSQGAFAFGAGYASEDGKILSNGSITTSSG---HWGIGSGLGLTLNKFEALNYGIEGARKEARQAAAIGLAVSNLRYNDTPGKLSIAFGGGLWRSQGAFAFGAGYASEDGKILSNGSITTSSG---HWGIGAGLSLKLKS KFEALNYGIEGARKEARQAAAIGLAVSNLRYHDTPGALSVAFGSGLWRSQGAFAFGAGYASEDGKTLSSVSITTSGG---IWNISAGLSLKLKS KFETLSYVVEDVRKAARQAVAMGLAVSNLRYYDIPGSLSLSFGTGIWRNQSAFAIGVGYTSEDGNIRSNLSITSADS---HWDIGAGLRIKLNKFETLSYVVEDVRKEARQAAAIGLAVSSLRYYDIPGSLSVSFGTGTWRSQSAIAFGAGYTSEDGNIRSNISVTSAGG---HWGVGAGVTLRLRKFNILSYDIKSVRKEARQAAAVGLAVSNLRYFDDPGSLSVSFGSGAWRGQSAFALGAGYTSENGKIRSNISATSAGG---HWGVGGAITLKIKRFAQLSGEIGQVRSEARQAAAIGLAAASLRFDNEPGKLSVALGGGFWRSEGALAFGAGYTSEDGRVRANLTGAAAGG---NVGVGAGLSITLNKLSQLNSDLGGIRDEARQAAAIGLAAASLRYDDRPGKLSVAAGGGFWRDSSALAFGAGYTSEDGRIRGNVSGTAAGG---HVGVGAGISFTLNKFGKLNEDIVATRIEARQAAAIGLAAASLRYDDRPGKISAAIGGGFWRGEGAVALGLGHTSEDQRMRSNLSAATSGG---NWSMGAGFSYTFNRVDGLQGQINSARKEARAGAANAAALSGLRYDNRPGKVSIATGVGGFKGSTALAAGIGYTSKNENARYNVSVAYNEA---GTSWNAGASFTLNRIGQVYNSFNDLKKDMYGGVASAMAVAGLPQPTGAGRSMVSAATSNYHGQQGFAAGYSYVTESNRWVVKASVTGNTRSDFGAVVGAGYQF---GNQLMRNEIGRLDDKASAGVASAMAVAGLPQSYMPGKSMAAIAASSFRGESGFAIGISTITEDGRYVYKISGNSNSKGDVGVTVGAGIVW---ANQYTDQKVDHLRREMNGGVAAAMAVAGLPQPTAPGKSMVAIAGSTWQGQQGFALGVSTISENGKWLYKGSLTTSTRGGTGAVLGAGYQW---ANSYTDDQIRSARRDSYGGTASAMAMAGLPQAVLPGHGMVAMAGGTYAGQSAFAIGVSQLSETGKWVYKLQGTTDSRGQFGASIGAGMHW---AISNLSNRIDGAQRDANAGTASAMALAGLPQSVLPGKGMVALAGSTYSGQSALALGVSKLSDSGRWVFKGGVTSNTRRNVGATVGAGFHW---AKQYTDGMVGNLRRETSGGVAAAIATANLPQAYVQGRGMTSVGVSSYQGQSAIAVGVSAVSESGHWVFKFSGSANTRSHVGVGAGVGYQW---AKQYTDGVVGSLRRDTDGGVAAAIATANLPQAYIPGRGMTSVGVSSYRGQSAIAVGVSSVSESGRWVFKFSGSANTRSQVGIGAGVGYQW---KMGEMNSKIKGIENKMSGGIASAMAMAGLPQAYAPGANMTSIAGGTFNGESAVAIGVSMVSESGGWVYKLQGTSNSQGDYSAAIGAGFQW---RMVEMDNKLSKTESKLSGGIASAMAMTGLPQAYTPGASMASIGGGTYNGESAVALGVSMVSANGRWVYKLQGSTNSQGEYSAALGAGIQW---RYSELKQDLRKQNSVLSAGIASAMSMASLTQPYTSGSSMTTIGAASYRGQSALSLGVSSISDSGRWVSKLQASSNTQGDFGIGVGVGYQW---RYSALKEDLKKQDSTLSAGIAGAMAMASLTQPYTPGASMATIGAASYRGQSALSVGVSSISDSGRWVSKLQASSNTQGDMGVGVGVGYQW---ATNELDHRIHQNENKANAGISSAMAMASMPQAYIPGRSMVTGGIATHNGQGAVAVGLSKLSDNGQWVFKINGSADTQGHVGAAVGAGFHF---VAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW---VAQNLNNRIDNVNGNARAGIAQAIATAGLAQAYLPGKSMMAIGGGTYLGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGTSASVGYQW---QVNNLEGKVNKVGKRADAGTASALAASQLPQASMSGKSMVSIAGSSYQGQSGLAIGVSRISDNGKVIIRLSGTTNSQGKTGVAAGVGYQW---NVANIDNRVSKLDKRVRGIGANAAAASSLPQVYIPGKSMVALAGGAYSGASAVAVGYSRASDNGKVILKVNGTANSAGHYSGGVGVGYQW---RIDNIDKRVKKMDKRRKAGTASALATAGLMQPHRDGQSALVAAVGQYQSETAVAVGYSRISDNGKYGVKVSFSTNSQGEVGGTAGAGYFW---KFNQLENRFDAFSKESRAGIAGSNAAAALPTISIPGKSVLSVSAGTYKGQSAVALGYSRVSDNGKVLLKLHGNSNSVGDFGGGVGIGWAW---AVNRLDNVISTNNRTLQAGIAGANAAAALPTVTMPGKSTIALSAGTYKGRNAVAIGYSRLSDNGKITLKLQGNSNSAGDFGGGVGVGWTW---RNNELRTQLNNTDRNLRAGIAGANAAAGLTSVSMPGKSMLAISAAGYGGENAMAIGYSRMSDNGKIMLKFQGNRNSQGKMAGSVSIGYQW---QNNALRTQIHHADRRLRAGIAGANAAAALASVSMPGKSMVAIAAAGHDGESALAIGYSRISDNGKVMLKLQGNSNSQGKVSGAVSVGYQW---KLNNLEHKFDMSNKNLRAGIAGANAAAGLASVSMPGKSMLAISAAGYDGENAVAVGYSRMSDNGKVMLKLQGNSNSRGKVGGSVSVGYQW---KLSNLNNKLDMSNKELRAGIAGALATSGLPMSSVPGKSMFAASAGSYKGQSAVALGYSRVSDNGKITLRLQGTRSSTGDVGGSVGVGYQW---NYNILNNRINKVDKDLRAGIAGANAAAGLPQAYIPGKSMVAVAAGTYKGQNAIALGMSRISDNGKVIIKLTGNTNSRGDFGASIGAGYQW---AINKLGDHINKVDKDLRAGIAGATAVAFLQRPNEAGKSIVSLGVGSYRSESAIAVGYARNSDNNKISIKLGGGMNSRGDVNFGGSIGYQW---GLVNVNKRVDTLDKNTKAGIASAVALGMLPQSTAPGKSLVSLGVGHHRGQSATAIGVSSMSSNGKWVVKGGMSYDTQRHATFGGSVGFFFN--NFSSLKHEVEDNRKEANAGIASAVAIASQPQVKTGDFMMVSAGAGTFNNESAVSVGAS-FNAGIHTVIKAGVSADTQSDFGAGVGVGYSF---HFSSLKNEVDDNRKEANAGTASAIAIASQPQVKTGDVMMVSAGAGTFNGESAVSVGTS-FNAGTHTVLKAGISADTQSDFGAGVGVGYSF---QFRQLRDQINKNRKRSDAGIAGAMAMTAIPMID-GKQYSFGMAASNYRDEQAIAAGIIFRTSEN-TVVRLNTSWDTQHGTGVATGMSIGW---KFSELNDRVNRNESRANAGIAGAMAMSAIPYLNNYVDNSFGMATSTFRGETAIASGYQRQINPY-VNVRLSSSWDTSNGVGVAAGVALGW---RVNDLSNKVDRNYKRANAGIAGAMAQAAIPQQFGYKYN-FGMALGNYRDGTAIAAGGSFQVKKN-VVSKTAVSWDAEGGVGVSAGVSVGW---KYNQLSDKVNKNFNKTNAGISGAMAMSGIPQKFGYEKS-FGMAIGAYRGQSALAVGGDWNINHK-TITRVNVSADTEGGVGVAAGFAFGIN--DIEDRLRRQNRRLDRQGAMSSAMLNMSASVAGIAS-QNRVGAGVGFQNGESALSVGYQRAISPRATVTVGGALSGDDSSIGVGAGFGW-----DIEDRLRRQNRRLDRQGAMSSAMLNMSASVAGIAS-PNRIGAGVGFQNGESALSVGYQRAISPRATVTVGGALSSGDSSIGVGAGFGW-----EVNDRFEDLDRRIRRNGAMSAAMSQMSANSAYAKPGRGRLAVGAGFQDGESGLAIGYGRRINENVSVSIGAAFSGSESSGGVGFGVDL-----TAGQLQQGINDTARKAYSGVAAATALTMIPDVDKDKVLSVGVGVGSYQGYSAVALGATAR-ITENIKMRAGASLGGSG-TAIGMGASMQW---RIGDLQQSITDTARDAYSGVAAATALTMIPDVDRDKRVSIGVGGAVYKGHRAVALGGTAR-INENLKVRAGVAMSAGG-NAVGIGMSWQW---QIGMVRQGISQVARGAYSGIAAATALTMIPDVDQGKSIAIGIGSATYKGYQAVALGASAR-ISHNLKAKMGVGYSSEG-TTVGMGASYQW---RVGAIQQGVNDLARNAYSGIAIAGALAGMPQVDPGKVISVGAGFGNYGGYTAIAVGGSAR-IAQNTVIKLGVGTVNGSRMMVNGGIGHSW---AHADAAADPADRFDGAR-GIAATAGMASIPHMDRDSSFAMGGGTATFQGRKAMAVGVQAR-ITENLKATVNVGFAGSQ-RVVGAGMLYQWK--AMGNMSNSINNVDRNAAKGIASASALN-IVTPYLPGRTTLNAGVANYRGYQSVGLGVSRWNEKGTINYNLGVSTSGGNSTIVRAGIGIVLGN-NDAVNVGQLNDGLREVSAGVAMSMAMAQLPAPLDGSNHSFGVAVGGFDGQEALALGGTAIVNNNVTLRGALSHAGGKTGAGVGVGWSF-----RQDNFEKRLDKMDKKMDGVMAGTHAVTNARPFAGNGQTAMGVGTGFAGSAQAVAIGVSHNFQDSAWSMSATTNVSTGSGVKTDVSGGVGAHYVF LSLVGSYKNAQAMAMGAVFKPAENVLLNVAGSFSGSEKIVGAGVSWKFGSKSKPAVSTQSAVNSAEVLQLRQEISAMQKELAELKKALRK---1........10........20........30........40........50........60........70........80........90...

Figure 1. Multiple alignment of the sequences of 69 putative AT-2 domains. The alignment was generated using the CLUSTAL X program32. The positions of conserved motifs 1 and 2 are indicated above the alignment. The horizontal lines at the top left-hand side of the alignment indicate the position of the 7 residue repeat sequences, present in several homologues as illustrated in Figure 3. The positions of motifs 1 and 2 are indicated above the alignment. Residue alignment position for the AT-2 domains is indicated below the alignment.

bioinformatic analyses of the newly discovered autotransporter 2 domains, thought to form trimeric structures in the outer membranes of Gram-negative bacteria. These trimers are thought to form 12-β-strand transmembrane pores that allow export of the N terminal passenger domain from the periplasm to the external milieu (see Introduction).

The analyses have led to several important evolutionary conclusions or suggestions: (1) AT-2 domains are found (almost) exclusively in proteobacteria of the α-, β- and γ-

subdivisions and their phage. (2) Only two homologues were found outside of these bacterial subkingdoms, and they were from a low G+C Gram-positive bacterium with an incompletely sequenced genome. These two sequences may be erroneous, resulting from DNA contamination. (3) Several paralogues can be present in a single organism; for example, Haemophilus somnus 2336 has 5 paralogues of similar AT-2 domain sequence, while Burkholderia cepacia R18194 has 4 AT-2 domain paralogues, three of which

22 Saltman | Quarterly UCSD Biological Sciences

are similar in sequence. (4) AT-2 sequence similarity does not imply similarly sized passenger domains, as phylogeny of the AT-2 domains does not correlate well with protein size. (5) Although there is a poor correlation between AT-2 domain and protein size, there is a reasonably good correlation between AT-2 protein domain phylogeny and the source organismal type (with a few potential exceptions).

Points 3, 4 and 5 imply that the shuffling of AT-2 domains relative to their

Volume 3 2005–06

Table 2. Organismal types and average sizes of the twelve phylogenetic clusters of the AT-2 family

Cluster 1a 1b 1c 1d 2a 2b 2c 2d 3a 3b 3c 3d

Organisms represented E J (Clostridia) D, J E D J E, J (E. coli phage) J J E, J (Clostridia) J J

Average size ± S.D.1 789 ± 445 911 ± 667 285 ± 179 3068 1271 ± 907 266 ± 42 422 ± 118 288 452 1468 ± 992 1029 ± 1113 448 ± 70

Average size of the proteins in a cluster in terms of numbers of amino acyl residues ± standard deviation (S.D.).

1

transmembrane β-strand 2 (peak A3 in Figure 4). The former proved to be more hydrophobic than the latter. Most interestingly, motif 1 exhibited AT 2 domain-specific residuetype differences that were lacking in motif 2. Motif 2 exhibited conservation in the different clusters typically characteristic of the entire AT-2 family. Since only in motif 1 was there a suggestion of residue (and hence functional) specialization, and since full residue conservation was not observed at any one position, these results suggest that the pores formed from AT-2 domains are fairly flexible and non-specific, accommodating a range of passenger proteins. It is possible, however, that substrate protein selectivity is a function performed by motif 1.

The analyses reported in this communication make several predictions concerning the structures, functions and evolutionary origins of a novel family of autotransporter domains. A four transmembrane strand β-sheet is likely to serve as the pore-forming element, and oligomerization is likely to be required for function as is the case for all well-characterized channel-forming peptides.26,27,28 The functional significance of conserved motifs 1 and 2 has not been investigated. Further studies will be required to understand the structure/function relationships of these interesting virulence-related transport proteins.

passenger domains and/or that passenger provided the basis for formation of the ATdomain size modification during recent 2 domain, extensive sequence divergence evolution has occurred repeatedly, even though had to have occurred in order to form the horizontal transfer of these proteins across more hydrophobic, strongly amphipathic, β bacterial phylogenetic groupings has been structured AT-2 domains that mediate pore relatively rare. It also appears that recent AT- formation.

2 domain-encoding gene duplication events Two particularly well-conserved have given rise to most of the paralogues in sequence motifs in the AT-2 domain organisms such as H. somnus and B. cepacia, that must be of structural and functional even though duplication of the entire protein significance have been identified. One proved has not occurred. Detailed analyses of the to be in the N-terminal region of the AT-2 passenger domains with respect to AT-2 domain in a strongly hydrophobic region domains are likely to be revealing. (peak H1 in Figure 4), while the other was

Sequence analyses led to a very in a strongly amphipathic region in putative tentative but plausible suggestion that AT-2 domains may have evolved Table 3. Residue composition of the two most conserved motifs in proteins of the AT-2 family from domains that arose by repeated duplication of a genetic element Residue Position Frequency of amino acid residues of 21 nucleotides, encoding a 7 18 A29 Q13 G9 R4 S4 K3 N2 M2 T1 V1 amino acyl residue peptide. This 19 G46 A15 M3 N2 I1 V1 F1 peptide had the probable sequence 20 I25 A15 V9 T7 L6 S3 M2 G1 K1 of: (DE) (QN) (RK) (FI) (QD) 21* A64 S4 P1 (QK) (VL). This is a strongly 22 S17 G14 A12 I11 N5 Q5 E2 M2 V1 Motif 1 hydrophilic heptapeptide with only 23 A37 G12 S7 Q5 T4 M3 E1 two hydrophobic residue positions.

This repeat unit could be identified 24 L17 M15 A14 N7 I5 T4 S3 V2 G1 H1 in the N-terminal “linker” regions 25* A63 N2 S1 Q1 G1 V1 of several AT-2 proteins. It is 26 L19 M16 V14 A11 T6 I2 Q1 possible that this hydrophilic 27 A31 S25 T8 N3 G1 L1 “linker” connects this domain with 51 S32 Q11 T7 G7 N4 A2 K2 Y1 H1 R1 E1 the passenger domain in some of the 52 A58 G7 S4 homologues. Surprisingly, it could 53 V21 L21 F12 I9 Y2 M2 T1 K1 be found throughout most of the C 54* A61 S6 G1 P1 Motif 2 terminal regions of other proteins 55 I19 V19 L16 F9 A5 S1 that exhibit certain characteristics 56* G68 V1 of AT-2 proteins and were retrieved 57 V19 Y16 A14 T5 G5 S4 I3 L1 M1 with PSI-BLAST iterations (see, for example, Figure 3). It is clear *The most highly conserved residues are indicated by asterisks. The positions of these two motifs in the that if this repeated heptapeptide multiple alignment can be found in Figure 1.

2005–06 Volume 3

UCSD Biological Sciences Saltman | Quarterly 23

Figure 2. Phylogenetic tree of the AT-2 C-terminal autotransporter domains. The clusters (1a-d, 2a-d and 3a-d), analyzed for sequence conservation (see text), are indicated in the figure. The tree is based on the CLUSTAL X-derived multiple alignment shown in Figure 1. It was drawn with the TreeView program.39

Acknowledgements This work was supported by NIH grant GM64368 from the National Institute of General MEdical Sciences. I would like to thank Professor Milton Saier for his guidance throughout the project and Mary Beth Hiller for her assistance in the preparation of this manuscript.

References 1.

2.

3.

4.

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402 (1997).

Busch, W., and Saier, M.H., Jr. The Transporter Classification (TC) System. CRC Crit. Rev. Biochem. Mol. Biol. 37: 287-337 (2002).

Cotter, S.E., Surana, N.K., and St. Geme, J.W., 3rd.

Trimeric autotransporters: a distinct subfamily of autotransporter proteins. Trends Microbiol. 13: 199205 (2005).

Cotter, S.E., Yeo, H.J., Juehne, T., and St. Geme, J.W., 3rd.

Architecture and adhesive activity of the Haemophilus influenzae Hsf adhesin. J. Bacteriol. 187: 4656-64

5.

6.

7.

8.

9.

10.

11.

(2005).

Devereux, J., Haeberli, P., and Smithies, O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387–395 (1984).

El Tahir, Y., and Skurnik, M. YadA, the multifaceted Yersinia adhesin. Int. J. Med. Microbiol. 291: 209-218 (2001).

Fink, D.L., Buscher, A.Z., Green, B., Fernsten, P., St. Geme, J.W., 3rd. The Haemophilus influenzae Hap autotransporter mediates microcolony formation and adherence to epithelial cells and extracellular matrix via binding regions in the C-terminal end of the passenger domain. Cell. Microbiol. 5: 175-186 (2003).

Fink, D.L., and St. Geme, J.W., 3rd. Chromosomal expression of the Haemophilus influenzae Hap autotransporter allows fine-tuned regulation of adhesive potential via inhibition of intermolecular autoproteolysis.

J. Bacteriol. 185: 1608-1615 (2003).

Guyer, D.M., Henderson, I.R., Nataro, J.P., and Mobley, H.L.T. Identification of Sat, an autotransporter toxin produced by uropathogenic Escherichia coli. Mol. Microbiol. 38: 53–66 (2000).

Henderson, I.R., Cappello, R., and Nataro, J.P. Autotransporter proteins, evolution and redefining protein secretion.

Trends Microbiol. 8: 529–532 (2000).

Henderson, I.R., and Nataro, J.P. Virulence functions of autotransporter proteins. Infect. Immun. 69: 1231– 1243 (2001).

24 Saltman | Quarterly UCSD Biological Sciences

12.

Henderson, I.R., Navarro-Garcia, F., Desvaux, M., Fernandez, R.C., and Ala’Aldeen, D. Type V protein secretion pathway: the autotransporter story. Microbiol.

Mol. Biol. Rev. 68: 692-744 (2004).

13. Hoiczyk, E., Roggenkamp, A., Reichenbecher, M., Lupas, A., and Heesemann, J. Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins. EMBO J. 19: 5989-5999 (2000).

14. Jacob-Dubuisson, F., Fernandez, R., and Coutte, L. Protein secretion through autotransporter and twopartner pathways. Biochim. Biophys. Acta 1694: 235257 (2004).

15. Kumar, A., and Schweizer, H.P. Bacterial resistance to antibiotics: active efflux and reduced uptake. Adv. Drug Deliv. Rev. 57: 1486-1513 (2005).

16. Laarmann, S., Cutter, D., Juehne, T., Barenkamp, S.J., and St. Geme, J.W, 3rd. The Haemophilus influenzae Hia autotransporter harbours two adhesive pockets that reside in the passenger domain and recognize the same host cell receptor. Mol. Microbiol. 46: 731-743 (2002).

17. Liu, D.F., Mason, K.W., Mastri, M., Pazirandeh, M., Cutter, D., Fink, D.L., St. Geme, J.W., 3rd, Zhu, D., and Green, B.A. The C-terminal fragment of the internal 110-kilodalton passenger domain of the Hap protein of nontypeable Haemophilus influenzae is a potential vaccine candidate. Infect. Immun. 72: 6961-6968 (2004).

18. Loveless, B.J., and Saier, M.H., Jr. A novel family of autotransporting, channel-forming, bacterial virulence

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Repeat Number 1 2 3 4 5 6 7 8 9 10 11 12

Residue position 100 107 114 121 128 135 142 149 156 163 170 177

Consensus residues:

Sequence E D D D D D E D E E N D

G K L Q Q Q D I S E N N

K K R R R R K R R K K K * D Q R E N K

F F F F F F I I L I F F * F I

F E Q Q Q Q H G D D D D

19.

N Q Q Q Q Q K K V V K K

I V I V V V L V V L L M

Q Q V D K L

Figure 3. The 7-residue repeat element comprising the C-terminal region of the Apl protein from Actinobacillus pleuropneumoniae (GI #32035081). This region, exhibiting 12 repeat units in Apl, is about the same length as a typical AT-2 domain. Corresponding repeat units can be detected in the N-terminal region of many AT-2 domains as revealed by overlined positions in Figure 1. The C-terminal twelve (1-12) 7-residue repeats in Apl are presented in column 1 while the residue position at the beginning of each repeat is indicated vertically in column 2. The residues present in each of the 7 positions of the 12 repeat elements are shown to the right. The two dominant residues are presented in the consensus sequence at the bottom.

20.

21.

22.

23.

24.

25.

proteins. Mol. Membr. Biol. 14: 113–123 (1997).

Ma, Q., Zhai, Y., Schneider, C.J., Ramseier, T.M., and Saier, M.H., Jr. Protein secretion systems of Pseudomonas aeruginosa and P. fluorescens. Biochim. Biophys. Acta 1611: 223-233 (2003).

Maurer, J., Jose, J., and Meyer, T.F. Characterization of the essential transport function of the AIDAI autotransporter and evidence supporting structural predictions. J. Bacteriol. 181: 7014–7020 (1999).

Newman, C.L., and Stathopoulos, C. Autotransporter and two-partner secretion: delivery of large-size virulence factors by gram-negative bacterial pathogens. Crit. Rev. Microbiol. 30: 275-286 (2004).

Nummelin, H., Merckel, M.C., Leo, J.C., Lankinen, H., Skurnik, M., and Goldman, A. The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel beta-roll. EMBO J. 23: 701711 (2004).

Oomen, C.J., van Ulsen, P., van Gelder, P., Feijen, M., Tommassen, J., and Gros, P. Structure of the translocator domain of a bacterial autotransporter. EMBO J. 23: 12571266 (2004).

Roggenkamp, A., Ackermann, N., Jacobi, C.A., Truelzsch, K., Hoffmann, H., and Heesemann, J. Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA. J. Bacteriol. 185: 3735-3744 (2003).

Saier, M.H., Jr. A functionalphylogenetic classification system for transmembrane solute transporters. Microbiol. Mol. Biol.

Rev. 64: 354-411 (2000).

26.

27.

28.

29.

30.

31.

32.

33.

Saier, M.H., Jr. Families of proteins forming transmembrane channels.

J. Membr. Biol. 175: 165-180 (2000).

Saier, M.H., Jr. Answering fundamental questions in biology with bioinformatics. ASM News 69: 175-181 (2003).

Saier, M.H., Jr. Tracing pathways of transport protein evolution. Mol. Microbiol. 48: 1145-1156 (2003).

St. Geme, J.W., III, and Cutter, D. The Haemophilus influenzae Hia adhesin is an autotransporter protein that remains uncleaved at the C terminus and fully cell associated. J. Bacteriol. 182: 60056013 (2000).

Surana, N.K., Cutter, D., Barenkamp, S.J., and St. Geme, J.W., 3rd.

The Haemophilus influenzae Hia autotransporter contains an unusually short trimeric translocator domain. J. Biol. Chem. 279: 1467914685 (2004).

Tamm, A., Tarkkanen, A.M., Korhonen, T.K., Kuusela, P., Toivanen, P., and Skurnik, M. Hydrophobic domains affect the collagen-binding specificity and surface polymerization as well as the virulence potential of the YadA protein of Yersinia enterocolitica.

Mol. Microbiol. 10: 995-1011 (1993).

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G. The CLUSTAL X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876–4882 (1997). van Ulsen, P., van Alphen, L., Hopman, C.T., van der Ende, A.,

Figure 4. Average hydropathy, amphipathicity and similarity plots for the AT-2 domains of the 69 autotransporter-2 proteins included in this study. The plots were generated with the AveHas program.37 H1-5, five peaks of hydrophobicity; A1-5, five peaks of amphipathicity when the angle is set at 180º as is appropriate for a β-strand. Hydropathy, heavy solid blue line; amphipathicity, red line; similarity, dashed black line.

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UCSD Biological Sciences Saltman | Quarterly 25

34.

35.

and Tommassen, J. In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters.

FEMS Immunol. Med. Microbiol. 32: 53-64 (2001).

Voulhoux, R., Bos, M.P., Geurtsen, J., Mols, M., and Tommassen, J. Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299: 262-265 (2003).

Yen, M.R., Peabody, C.R., Partovi, S.M., Zhai, Y., Tseng, Y.H., and Saier, M.H. Protein-translocating outer

36.

37.

membrane porins of Gram-negative bacteria. Biochim.

Biophys. Acta 1562: 6-31 (2002).

Yeo, H.J., Cotter, S.E., Laarmann, S., Juehne, T., St. Geme, J.W., and Waksman, G. Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter. EMBO J. 23: 1245-1256 (2004).

Zhai, Y., and Saier, M.H., Jr. A web-based program for the prediction of average hydropathy, average amphipathicity and average similarity of multiply aligned homologous proteins. J. Mol. Microbiol. Biotechnol. 3:

38.

39.

285–286 (2001).

Zhai, Y., and Saier, M.H., Jr. The β-barrel finder (BBF) program, allowing identification of outer membrane β barrel proteins encoded within prokaryotic genomes.

Prot. Sci. 11: 2196-2207 (2002).

Zhai, Y., Tchieu, J., and Saier, M.H., Jr. A web-based Tree View (TV) program for the visualization of phylogenetic trees. J. Mol. Microbiol. Biotechnol. 4: 69–70 (2002).

Reproductive Status and Species Diversity of Bats along an Altitudinal Gradient in Monteverde, Costa Rica

Kimberly L. Lo

Earl Warren College, Senior, General Biology Major Education Abroad Program (EAP) Monteverde Tropical Biology and Conservation (Article Accepted Fall 2005)

Bat diversity can be an indicator of environment in terms of food supply, available roost sites, and overall quality. I investigated the patterns of reproductive activity and diversity of bats along an altitudinal gradient in Monteverde, Costa Rica. I evaluated and compared diversity by looking at abundance, species richness, and composition. I also compared distributions and abundance to previous studies done at different times of the year. Between species and families, I searched for trends in reproductive activity and for female bats, a relationship between elevation and reproductive status. Mist-netting occurred for 13 nights over a one-month period in four different elevational ranges. The study sites ranged from 750m to 1,850m in elevation, and I used three nets each night to capture the bats. In all, I captured 68 individuals, representing five feeding guilds. I found evidence that species abundance is highest at the lowest elevation, especially of small frugivores. Sturnira and Carollia were the two most abundant genera captured. The greatest difference in composition was between highland species and lowland or intermediate-lowland species. I found 10 of the 19 species sampled, as well as more females (70%) than males (22%), to be reproductively active. Overall, females tended to be more advanced in their reproductive cycles at higher elevations; however, the trend within specific genera was different. As diversity and climate shift over time, species may migrate as dictated by their food supply, which could also have effects on their reproductive status.

Introduction The tropics are home to myriad bat species.

Costa Rica alone is inhabited by at least 108, which makes up 11% of the bat species in the world.10 Species richness and abundance of bats are commonly affected by elevation and latitude. Previous studies have indicated that species richness shows a classic latitudinal gradient in bats worldwide.7 The diversity of many species declines as you reach higher elevations.

Yet, scientists worldwide are beginning to notice dramatic shifts in the distribution and abundance of many taxa.17 One reason is temperature, which limits the altitudinal ranges of many animals.4 Birds, for example, have been noted to move to higher elevations, eventually establishing breeding populations where the birds were not previously present.16 Other factors involved that could affect bat diversity include climate changes and habitat loss due to deforestation. When large hollow trees are cut, species that are dependent on tree hollows may find the lack of roost space limiting on their populations, even if the

forest is allowed to regenerate.10

The reproductive cycles of many neotropical Chiroptera (the order of bats) could also be affected by altering food availability5 by changing environmental conditions along latitudinal gradients. In tropical regions, births in both Megachiroptera and Microchiroptera tend to coincide with times of greatest food availability.12 Female bats need ample food supply for gestation and lactation; therefore, reproductively active females could be important indicators of environment, in terms of insect and fruit abundance. A greater understanding of their geographic distribution is valuable for monitoring the conditions of both bat populations and their surrounding habitats.

In the Monteverde area of Costa Rica, studies on species abundance and distribution were conducted in 2000.11 This study will be useful to compare the change in diversity over time. A study was also done at the end of the rainy season1, which could be helpful in searching for evidence of species migration throughout the year.

I will examine the diversity of bats

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in different areas of Monteverde in order to address two questions: (1) how species abundance, richness, and composition vary at different elevations during this period of the year (the beginning of the rainy season) (2) how reproductive activity and stages vary between species along an altitudinal gradient.

Bat reproductive cycles are precisely timed, and pregnancy can be delayed after copulation in many species to synchronize birth with the abundant food available during the wet seasons.2 I would expect many bats to be reproductively active at this time of year due to the fruiting peaks associated with the rain. Bats in lower elevations should be more advanced in their reproductive cycles since primary biological productivity increases with temperature2, and higher temperatures are generally characteristic of lower elevations.

In terms of diversity at different elevations, I would expect to see greater species abundance and different species composition at lower elevations as in Adams’s study1 at the end of the rainy season. The environmental conditions found in lower areas are more ideal for many floral and fauna

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Figure 1. Species richness of bats at different elevations in Monteverde, Costa Rica.

9 8 7 Number of species captured

6 5 4 3 2 1 0

Low (750-850m)

Intermediate-Low (1,150-1,250m)

Intermediate-High (1,350-1,450m)

High (1,750-1,850m)

Altitudinal Range species to thrive. Species richness should similarily increase at lower altitudes.3

each altitudinal range for three nights with three 3 x 12 m mist-nets, with the exception of the Intermediate-High elevation, which Materials and Methods I sampled for four nights. Each night, I I conducted this study in Monteverde, checked nets at 20 or 30-minute intervals Costa Rica in four different altitudinal between 18:00 and 22:15, depending on ranges: Low (750-850m), Intermediate-Low weather.

(1,150-1,250m), Intermediate-High (1,350 I removed bats from mist-nets with 1,450m), and High (1,750-1,850m). I used gloves and placed them in cloth bags to be five sites, one at each of these elevations: the weighed. For each captured bat, I noted Premontane Moist Forest in the Peñas Blancas the location, time, gender, weight, forearm Valley at 820m, the Premontane Moist Forest length, reproductive status, and species. I in San Luis at 1,200m, the Lower Montane identified individuals to species level using Moist Forest in Bajo del Tigre at 1,300m and a hand lens and ruler according to Timm near the Monteverde Institute at 1,350m, and LaVal’s key.14 Forearm length, dental and the Lower Montane Rain Forest in the arrangement, and the presence of stripes or Elfin Forest above the Estación Biológica at tails were important in this identification 1,800m.11 I mist-netted for thirteen nights process. between 1 May and 27 May 2005. I sampled For females, I diagnosed reproductive status by looking at nipple morphology, the presence or absence of hair around the nipples, and belly size. I classified females as either not reproductively active (no signs), pregnant-earlier stage (I) (large belly and hairy nipples), pregnant-later stage (II) (large belly and hairless nipples), or lactating (normal belly, nipples hairless and elongated). I considered males Figure 2. Percentages of male bats in different reproductive sta- to be reproductively active if tus at the beginning of the wet season in Monteverde, Costa Rica either one or both of their testes (n = 35).

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were at the scrotum, since testes are scrotal only during the reproductive season.10 I classified males who had both testes hidden in their abdomen as inactive.

After all data was taken, I trimmed off a small patch of back hair from each bat with scissors to avoid double-counting recaptures. Data analysis was performed in two parts to address my questions.

Diversity I evaluated species diversity according to abundance, richness, and composition. I used the Shannon-Weiner diversity index (log base 10) to compare relative abundances in the four altitudinal ranges. I calculated relative abundance both overall and for each Holdridge Life Zone8 visited to compare distribution and abundance with previous studies. I defined relative abundance according to the frequency that the species were caught over the thirteen nights (Table 1). When evaluating distribution, I used Zone 6 to represent Low elevations, Zone 1 for Intermediate-Low elevations, Zone 2 for Intermediate-High elevations, and Zone 4 for High elevations. I also calculated Sorensen’s similiarity coefficient to compare species composition between the four altitudinal ranges.

Guilds included large frugivores (those species whose weights averaged 30g or more), small frugivores (less than 30g), nectarivores, gleaning insectivores, and

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Table 1. Definitions of relative abundance used for Table 2.

Category

Within Same Elevational Range

Overall

Abundant (A)

Recorded all three nights

Recorded at all four elevational ranges

Common (C )

Recorded on two of the nights

Recorded at three of the elevational ranges

Uncommon (U)

Recorded on one of the nights

Recorded at two of the elevational ranges

Rare (R )

Never recorded during three nights

Recorded at only one elevational range

Table 2. The relative abundances and distributions in zones of each species. Abundance and Distribution 1 are from this current study. Abundance and Distribution 2 are from the Mammals of Monteverde species list,15 while Abundance and Distribution 3 are from a study done at the end of the wet season.1

Species Anoura geoffroyi Artibeus intermedius Artibeus lituratus Artibeus phaeotis Artibeus toltecus Carollia brevicauda Carollia castanea Carollia perspicillata Glossophaga commissarisi Glossophaga soricina Lonchophylla robusta Lonchorina aurita Myotis keaysi Myotis oxyotus Phyllostomus discolor Platyrrhinus helleri Platyrrhinus vittatus Sturnira ludovici Sturnira mordax

Overall Abundance Abundance 1 Distribution 1 Abundance 2 Distribution 2 Abundance 3 Distribution 3 Rare Common 4 Common 1,2,3,4,5 Common 2 Rare Uncommon 1 Uncommon 2 n/a n/a Uncommon Uncommon 2,4 Uncommon 1,2,3,4 Common 2 6 Uncommon 6 n/a n/a Rare Common 2 Abundant 1,2,3,4,5,6 4,6 Rare Common A, U Common C, C, A 1,2,6 Common 1,2,3,4,5,6 6 Uncommon Rare Common 6 Uncommon 1,2,6 C, R, A 2,4,6 1 2,6 Rare Uncommon Rare 1,2,6 C, R Rare Common Rare Rare Uncommon Rare Rare Rare Common Common Rare

Common A, C, C Uncommon Common U, C Uncommon Uncommon Uncommon C, U, U A, C, U Abundant

1 1,2,6 6 6 2,4 2 2 1 1,2,6 2,4,6 4

Common Common Rare Uncommon Abundant Rare Common Uncommon Common Abundant Common

1,2,3,4 1,2,3,6 2,3,6 5,6 2,3,4,5 1,2 1,2 6 1,2,3,4,5 1,2,3,4,5,6 2,3,4,5,6

Common n/a n/a n/a Common n/a n/a Common U, C U, C, A n/a

2,4 n/a n/a n/a 2 n/a n/a 6 4,6 2,4,6 n/a

Table 3. X2 Contingency table for abundance of female bats, organized by reproductive condition, at different elevations in Monteverde, Costa Rica.

Elevation Low Intermediate-Low Intermediate-High High Total

Inactive 6 2 1 1 10

omnivores. I distinguished between large and small frugivores using my own definition. In order to look at the diversity of bats in terms of their feeding guilds, I ran an X2 Contingency test to see if the abundance of species varied between elevations. I calculated the composition of bats by feeding guild as well to give an overall picture of their diversity in Monteverde. In order to see the presence of feeding guilds at different elevations, I also calculated their relative abundances.

Pregnant-I 3 0 0 0 3

Pregnant-II 2 0 3 2 7

Reproductive status I compared the abundance of female bats altitudinally with a X2 Contingency table. To evaluate the proportions of reproductive activity, I looked at females and males separately and noted their specific reproductive conditions. I also compared reproductive activity between subfamilies.

I looked at the most abundant genera in closer detail to test for elevational trends in reproductive status. I used Fisher exact probability tests to see if reproductive status

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Lactating 0 4 6 3 13

Total 11 6 10 6 33

(pregnant or lactating) is affected by elevation in the genera Sturnira and Carollia. I used the statistical program JMP IN 4 to analyze all data.

Results I captured a total of 68 bats, representing 19 species, ten genera, and two families (Table 2). I found the number of species at all altitudinal levels to be fairly consistent except for the amount found at higher elevations, which was noticeably less with only five

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Relative Abundance

Figure 3. Presence and relative abundance of bats grouped by guilds at different elevations in Monteverde, Costa Rica.

(X2 = 22.18, df = 12, p = 0.036)

Feeding Guild species (Figure 1). Small fruit-eaters were the majority (50%), followed by large frugivores (19%), nectarivores (19%), gleaning insectivores (10%), and one omnivore (0%).

Of the 33 females and 35 males caught, 45% were reproductively active (70% of the females and 22% of the males) (Tables 3 and 4, Figure 2).

Diversity and distribution No one species was considered abundant overall. The most abundant species I captured were Carollia brevicauda, Glossophaga soricina, Platyrrhinus vittatus, and Sturnira ludovici, and I considered them common species. I found 13 species to be rare in general since they only occurred in one of the altitudinal ranges, but all 13 were more common when looking at their relative abundances in their specific life zones (Table 2).

The most extreme altitudinal difference in diversity according to their Shannon-Wiener diversity index scores was between bats in high elevations (0.66) and intermediate-high elevations (0.88). Bat abundances at low and intermediate-low elevations were almost exactly the same.

Overall, there was no altitudinal trend in abundance, but low elevations had the greatest number of individuals (Figure 3).

In terms of diet and the abundance of bats in specific feeding guilds at different elevations, every guild was present in all elevational ranges with the exception of gleaning insectivores, which were not found

2005–06 Volume 3

in Intermediate-Low and omnivores, which were only found in Intermediate-High (Figure 3). Elevation significantly affected the abundance of bats belonging to certain guilds, especially due to the high number of small frugivores present in Low and Intermediate-High elevations (Figure 3). I captured ten C. brevicauda in Peñas Blancas out of the 25 individuals sampled there.

The Sorensen coefficient showed greatest similarity in species composition between low and intermediate-high elevations (0.47). Comparisons in composition between other altitudinal ranges were similar, except for species in high compared with low and intermediate-low elevation species (0.15 and 0, respectively).

later stages of pregnancy or lactating (Table 3).

In terms of reproductive activity, the subfamily Stenodermatinae had the highest ratio of active to inactive individuals (Figure 4). On the other hand, Phyllostominae had no reproductively active members caught.

A closer look at the more abundant genera Sturnira and Carollia reveals that elevation has no significant effect on female reproductive condition (Fisher p = 0.36, Fisher p = 0.14, respectively). Most female Sturnira were reproductively active and more were captured in higher elevations. The majority of female Sturnira at higher elevations were pregnant and the majority at lower elevations were lactating (Table 4).

Reproductive status Ten of the 19 species I caught had at least one reproductively active individual. The most reproductively active species during this time of year, when looking at its ratio of active to inactive individuals, was S. ludovici.

For female bats, 30% were in some stage of pregnancy and 40% were lactating (Table 3). The proportion of reproductively active male individuals was much lower (Figure 2), since most of the males were reproductively inactive. All genera of females tended to be more advanced in their reproductive cycles at higher elevations (X2 = 18.70, df = 9, p = 0.028). Most female bats in lower elevations were either reproductively inactive or in the first stages of pregnancy. Females in the highest elevation sampled were mostly in the

Discussion The four most abundant species in this study, Carollia brevicauda, Glossophaga soricina, Platyrrhinus vittatus, and Sturnira ludovici, were also abundant or common in previous studies.15 However, there was a greater difference in relative abundances with that study than when compared to Adams’s study1 at the end of the wet season. In comparison to Timm and LaVal’s study15, nine species had the same relative abundance, seven species increased in abundance, and three species decreased. In comparison to Adams’s study1 at the end of the wet season, I found eight species of bats during this time of year that were not captured then. Species found in both studies remained the same in relative abundance (Table 2), with the exception of

UCSD Biological Sciences Saltman | Quarterly 29

C. brevicauda. This suggests that species abundance in some Holdridge Life Zones is changing over time, but not seasonally.

These results could be due to ongoing changes in climate each year, but the reasons remain unclear. Monteverde has experienced an increasing number of dry days during the dry season from January through May.16 Changes in rainfall and temperature, which affect food supply, could account for changes in distribution and migration patterns.

Future studies should follow shifts in diversity in response to changing climate. I noticed that nights with colder temperatures and increased rainfall reduced the relative abundance caught. For this reason, I combined data from two of the nights spent mist-netting in intermediate-high elevations to count as one night’s worth of data.

Changes in species abundance and distribution could also be a result of deforestation. Habitat loss can be extremely limiting on bat populations, and much has already occurred. Only 2% of the original dry forest that covered western Central America remains, and most of the Pacific premontane and montane habitats are now gone.10 The loss of available roosting sites would contribute to both relocation and the possibility of extinction.

The high abundance of C. brevicauda in the Peñas Blancas valley, which Adams1 found to be uncommon, could be due to the arbitrary placement of mist-nets.

One C. brevicauda was captured carrying a fruit in the Actinidiaceae family, which was identified as Saurauia. If, by chance, I had placed mist-nets near Saurauia plants and C. brevicauda feed on them, their relative abundance in this particular spot would be explained. This could also happen if I unknowingly placed the nets near other fruiting plants. Members of the genus

Elevation High High High High Intermediate-High Intermediate-High Intermediate-High Intermediate-High Intermediate-High Low Low

Carollia are specialists on plants of the genus Piper and also eat species like Cecropia, which are both present in the Peñas Blancas valley.10 I could have estimated certain species to be more common than they actually are in the area because of their increased concentration at the plants. C. brevicauda abundance greatly contributed to the abundance of bats in low elevations (25). It was interesting that the only notable difference in abundance at different elevations was the high amount of individuals in low elevations.

The C. brevicauda could also be greatly contributing to the high abundance of small frugivores captured at lower elevations.

The 17 small frugivores accounted for the significant effect of elevation on feeding guild abundance (Figure 3). Frugivores in general could have also composed 80% of the individuals caught because mist-nets were placed at ground-level and failed to capture higher-flying species of some insectivores.

Frugivores could also be abundant at this time of year since increasing rainfall generally triggers peaks in fruit abundance.6

In terms of species richness, the only altitudinal difference was a decreased number of species captured at high elevations. The similarity in richness among low and intermediate elevations, which contradicts previous research showing that species diversity gradually declines in Costa Rica at increasingly higher elevations10, could be due to environmental disturbances.

Differences in species richness as compared to Adams’s study1 also suggest the possibility of bat migration. Many bats undergo lengthy seasonal migrations to different locations 801600 km away.2

Species composition was most variable between high elevations and the two lowest elevational ranges, which makes sense because those were the most extreme altitudes

Species Sturnira ludovici Sturnira ludovici Sturnira mordax Sturnira mordax Sturnira ludovici Sturnira ludovici Sturnira ludovici Sturnira ludovici Sturnira ludovici Sturnira ludovici Sturnira ludovici

in the study. Some species were restricted to only one or two elevational ranges. For example, Sturnira mordax, is not found below 1400m on the Pacific slope of Costa Rica. The species Artibeus phaeotis, which I only captured in my lowest altitudinal range, is similarily restricted to elevations mostly below 1000m throughout the country.10

Reproductive activity overall occurred in only ten of the 19 species captured, so about half of the species caught synchronized their reproductive activity with each other. Yet, 70% of females were reproductively active compared to only 22% of males. This could be due to the fact that the testes of some species of males are withdrawn from the scrotum into the wide inguinal canal when the bats are disturbed, and they descend in response to elevated environmental temperatures.13 Male reproductive activity and bat reproductive activity in general during this time of year could therefore be higher than calculated.

Future studies could more accurately monitor the reproductive status of females by running immunological tests to measure progesterone levels.13 This would allow the detection of pregnancies in the earliest stages and perhaps also indicate higher levels of detectable reproductive activity. Patterns of reproductive status also varied with elevation (Table 3). Females were more advanced in their reproductive cycles at higher elevations, which was opposite of my expected results.

One explanation for this could be the smaller amount of females caught in higher elevations as opposed to lower elevations (Table 3).

When looking at the specific frugivorous genera Sturnira and Carollia, no significant relationship was found between elevation and female reproductive status.

This contradicts the finding that female bats are more advanced in their reproductive

Reproductive status Inactive Lactating Pregnant-II Pregnant-II Lactating Lactating Lactating Lactating Pregnant-II Inactive Pregnant-II

Table 4. Reproductive status of Sturnira at different elevations in Monteverde, Costa Rica.

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Volume 3 2005–06

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Relative Abundance

Figure 4. Number of reproductively active and inactive bats, organized by subfamily in Monteverde, Costa Rica (Umax= 17, Ucrit= 23, p > 0.05)

Family or Subfamily cycles at higher elevations (Table 3). In fact, the Sturnira showed an opposite trend (Table 4). Perhaps reproductive status is more influenced by other factors like fruiting patterns. Dinerstein6 found that lactation schedules for A. toltecus overlapped neatly with peak fruit abundance and in S. ludovici, lactation periods overlapped completely with the 1981 wet season and the 1982 dry season peaks in fruit abundance. In turn, synchronizations of flowering, fruiting, and other phenological events change with Costa Rica’s seasonal variations in rainfall.5 From this, one could hypothesize that frugivores share similar reproductive cycles.

Acknowledgements My thanks to Federico Chinchilla for teaching me all I know about mist-netting and identifying bats. He made long nights bearable with his humor, stories, and smile.

I am also forever grateful for the company of Pamela Valle, with whom I had many sabor extremo picnics in the dark and adventures

2005–06 Volume 3

excelentes. Thanks to Arturo Cruz for his help in Peñas Blancas, to Andres Chinchilla for never being tired, and to Aníbal Torres for the rides, laughs, and ability to count the students. Thanks also to Ruth Salas for her input and amazing personality. For use of their land, thank you to the Refugio Eladio, San Luis EcoLodge, Frank Joyce, Monteverde Institute, and the Estación Biológica. Lastly, thank you to everyone who gave me support or put up with my nocturnal lifestyle.

References 1.

2.

3.

4.

5.

6.

Adams, A. Species composition and diversity of bats at different elevations in the Monteverde region of Costa Rica. Unpublished. UCEAP, 2003.

Altringham, J.D. Bats: Biology and Behaviour. Oxford, New York, 1996.

Begon, M., Harper, J.L., and Townsend, C.R. Ecology: Individuals, Populations, Communities. Blackwell Scientific Publications, 1990.

Bogan, M.A. 1997. Potential Effects of Global Change on Bats.

URL: http://geochange.er.usgs.gov/sw/ impacts/biology/bats. Accessed: 8 June 2005.

Coen, E. Climate. In: Janzen, D.H. (Ed.) Costa Rican Natural History, pp.43-45. The University of Chicago Press, Chicago and London, 1983.

Dinerstein, E. Reproductive ecology of fruit bats and the seasonality of fruit production in a Costa Rican cloud

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

forest. Biotropica 18: 307-317 (1986).

Findley, J.S. Bats: A Community Perspective. Great Britain: Cambridge University Press, 1993.

Fogden, M. An Annotated Checklist of the Birds of Monteverde and Peñas Blancas. San José, Costa Rica, 1993.

Hayes, M., and LaVal, R.K.. The Mammals of Monteverde.

San José, Costa Rica: Tropical Science Center, 1989.

LaVal, R.K., and Rodríguez-H, B. Murciélagos de Costa Rica: Bates, Editorial INBio, San José, Costa Rica, 2002.

Nadkarni, N.M., and Wheelwright, N.T. (Eds.).

Monteverde Ecology and Conservation of a Tropical Cloud Forest. New York: Oxford University Press, 2000.

Nowak, R.M. Walker’s Bats of the World. Baltimore and London: The Johns Hopkins University Press, 1994.

Racey, P.A. Reproductive Assessment in Bats. In: Kunz, T.H. (Ed.). Ecological and Behavioural Methods for the Study of Bats, pp. 31-42. Washington, D.C. and London: Smithsonian Institute Press, 1988.

Timm, R. M. and LaVal, R.K. A field key to bats of Costa Rica. Kansas: Center of Latin American Studies, Univ. of Kansas, 1998.

Timm, R.M., and LaVal, R.K. Mammals of Monteverde.

In: Nadkarni, N.M., and Wheelwright, N.T. (Eds.).

Monteverde: Ecology and Conservation of a Tropical Cloud Forest, pp. 553-557. New York: Oxford University Press, 2000.

Pounds, A. J., Fogden, MP., and Campbell, J.H. Biological response to climate change on a tropical mountain.

199. Nature 398: 611-614 (1999).

Walther, G.R., Post, E., Convey, P., Menzel, A., Parmesan, C., Beebee, T.J.C., Fromentin, J.M., Hoegh-Guldberg, O., and Bairlein, F. Ecological Responses to Recent Climate Change. Nature 416: 389-395 (2002).

UCSD Biological Sciences Saltman | Quarterly 31

Investigation on the Effect of Age and Group Size on the Simultaneous Production of Bubbles and Calls by Captive Killer Whales (Orcinus orca) at SeaWorld, San Diego

Eri Suzuki John Muir College, Senior, General Biology Major (Article Accepted Winter 2006)

Emission of bubbles has been widely used as a visual cue for the detection of calling dolphins. Fripp found that a whistle simultaneously produced with bubble streams is influenced by age and social context in bottlenose dolphins (Tursiops truncatus).4 We studied bubble emissions and call productions among killer whales (Orcinus orca). We expected that bubble emissions in killer whales are not random but affected by age and social context similar to what was found in bottlenose dolphins since they both belong to the same family, Delphinidae. Data was collected from previously recorded videos of captive killer whales at SeaWorld, in San Diego, California. The subjects produced three types of bubbles: streams, clouds, and rings.

Out of a sample of 429 attributed bubble emissions, 77% of bubbling corresponded with calls. The percentage of simultaneous bubble emissions with calls varied with age, types of bubbles produced, social state, and call types. The rate of bubbling with calls changed with age. A month old calf produced 30% of the bubbling without calls. At 7, 13, and 43 months old, the same calf reduced the amount of bubble emissions without calls from 46% to 10%, and eventually to 7%. Of all types of bubbles observed, bubble streams were most likely to be produced with calling (88%).

Only 3% of bubble production by a single adult male occurred without calls. However, a mother and calf pair produced 9% of bubble emissions without calls, while a trio of socializing whales produced 27% without calls. Pulsed calls (62%) were often associated with bubbling but rarely with clicks (1%) and whistles (0%). Overall, it is shown that the simultaneous production of bubbles and calls was dependent on the age, bubble structure, group size and call types.

Introduction Marine mammals, especially whales and dolphins, use sound as their primary sense. Their highly evolved acoustic senses, an adaptation to their aquatic environment, allows them to better understand their surroundings. This is due to the fact that sound is transmitted more efficiently and effectively underwater than on land; thus, the use of sound increases their capacity to “see” better in their environment where vision can be inhibited.

Cetaceans (whales and dolphins) depend on sounds to see their world as well as communicate with each other. Hence, in order to fully appreciate the behavior and communication between these animals, it is essential to understand their acoustic signals. Many extensive studies on the acoustics of dolphins and killer whales have been conducted (see references). Researchers studying acoustic behaviors of dolphins and killer whales have been searching for the optimal technique to identify the calling individual within a study group. Investigations on the acoustic behavior of these animals requires knowledge on when and which animal is calling in order to determine context and functions of sound being used. Visual identification of the caller, however, is very challenging since dolphins and killer whales do not necessarily produce

a visual sign when calling.4 Therefore, researchers have resorted to several methods to attribute a call to a specific animal. One of the commonly used techniques is a hydrophone array that localizes the origin of the sound within an open area.10,11,12 In this method, sounds are recorded by a linear hydrophone array towed in the direction of whale movement and are analyzed along with visual observation of the behavior to identify calling individuals. Dudzinski also introduced a “mobile video/acoustic system” which simultaneously records vocalizations and behaviors of dolphins.3 This system facilitates the identification of a calling animal by providing a general direction of the sound origin. In addition, some researchers isolated an animal to record vocalization individually in the air or through a suction cup.13 Of all the methods, bubble emission is most commonly used as a visual cue of vocalization.1,4,5,6,7,8 Dolphins and killer whales are known to produce bubbles when calling; however, not all calls are associated with bubbling. Previous data on our subjects have shown that the calls produced with bubbles made up only 4% of all calls produced (unpublished data from Hubbs-SeaWorld Research Institute). In terms of types of calls concurrently produced with bubbling, Mc-

32 Saltman | Quarterly UCSD Biological Sciences

Cowan reported that there is no significant difference between the whistles accompanied with bubble streams and whistles without bubble streams.6 The conclusion was made form the comparison of 20 whistles with bubble streams and 57 whistles without bubble streams. Fripp, on the other hand, described that only 1 % of the whistles were associated with bubble streams while bubble streams were produced with the whistles 79% of the time, and bubble streams were context dependent.4 For killer whales, no study has been done on the context of bubble emissions and calls associated with bubbles; therefore, we attempted to establish the correlation between production of bubbles and calls.

The exact function and mechanism of bubble emission is unknown, yet bubble emissions have often been observed among very active and excited whales as well as among infants.1,5 From observations on bubbling behaviors of dolphins, scientists have suggested that bubbles are used as a means of visual communication.4,5 Herzing suggested that the use of bubbles was a sign of aggression while Fripp described them as a visual signal between a mother and a calf as well as an indication of “distress, location, or excitement.”4,5 We examined the bubbles produced

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Figure 1. Killer whale calf producing bubble stream.

by captive killer whales to investigate factors affecting bubble emissions and functions of these bubbles. It is believed that bubble emission is not random but affected by the age of the animal and the social state in which bubbles are being produced. If bubble emission is random and is simply a by-product of call production, we would not see any significant factors affecting bubble emission or variations among calls corresponded with bubbles. The result of this study will allow us to better understand the acoustics of killer whale as well as provide insight into their ontogeny, communication, behaviors and their view of the world.

nia (Figure 1 shows a bubbling killer whale calf ). Total of eight whales were observed and six of them provided relevant data. Each video contained the recordings of whale calls collected through an array of hydreophones embedded in the bottom of the observation pool. The video recorded whales’ activities and their positions were determined using an array of four cameras. The cameras provided a view of the observation pool from the left and right corners, a view of a channel between pools, and an overhead view of the observation pool. Figure 2 provides a view of the observation pool and positions of hydrophones and cameras. Nine videotapes were used for data collection. The video was systematically named according to the date of the recording. Detailed information on each video is shown in Table 1.

the former researchers and three additional videos were scored in this study. The scoring of vocalizing animals and their activities were conducted through video monitoring while the data logging was facilitated by The Observer software. The software recorded time of data input in seconds from the start of the logging session. Data consisted of a sequence of events, which were recorded using previously designed keys, with information on the presence of bubbling, name of bubbling whales (focal whale), and their positions.

Data was exported from The Observer to Microsoft Excel and time recorded in The Observer was converted into the time recorded in the video. Killer Whale Video Observation Files provided times and focal whales of each calling event, which facilitated locating the data of interest within the videos.

Video Scoring In order to locate bubbling events within a video, each video was scored using The Observer Basic version 3.0 to create Killer Whale Video Observation Files, which are systematic video observation logs. Six videos used for data collection were previously scored by

Video observation and data sampling Using Killer Whale Video Observation Files as a reference, bubbling events were extracted.

A bubble emission was assigned to a call only when the bubble was produced simultaneously with a call without any delay. For some videos, real-time observers recorded the ver-

Methods Data collection Bubbling events were collected from previously recorded videotapes of captive killer whales at Sea World in San Diego, CaliforFigure 2. View of the observation pool. Positions of cameras are indicated with circles and positions of hydrophones are marked with numbered squares.

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Table 1. Video Information.

bal narration of the activities of whales at the moment of videotaping. They provided the information on bubble emissions from the whales, which were out of the visual range of cameras. In these occasions, simultaneity of bubbling and calling was determined only from the sound of a bubble and a call production. For each event, we recorded the identity of the focal whale, position of focal whale, types of bubble produced, presence of simultaneous calling, types of calls, and the

position of other whales relative to the calling whale in the observation pool. The observation pool was divided into six sections, and the location of each whale was assigned to one of the six sections. The image of the observation pool provided by an array of four cameras included blind spots at the entrance of the channel and the area between two observation windows. Figure 3 shows the view provided by the cameras. Bubbles were categorized into three types: bubble streams,

bubble clouds, and bubble rings. Bubble streams are characterized with a continuous trail of bubbles from the whale’s blowhole while bubble clouds are one massive formation of bubbles produced with a single exhalation. Bubble rings are the formation of a single disc produced from whale’s blowhole.

In some cases, bubbling which was outside of the camera view was recorded by a realtime observer talking on the tape. In these cases, the bubble type was not noted and

Figure 3. Video monitor showing a view provided by four cameras.

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Whale ID number

Individual variations in a proportion of bubbling with or without calls.

With a call Without a call

Total

5

0127 (calf)

3

9801

0

9426

0

9126

0

8826

0

7804

2

Unknown

0%

100%

50%

Proportion of bubbling with or without calls.

Figure 4. Proportion of bubbling without call differs significantly among different individuals (Chi-square test: χ2 = 11.6287, p = 0.0402).

these occurrences were scored as “unknown bubble types.” Calls associated with bubble emissions were either “stereotyped” pulsed calls or clicks for the adult whales and stereotyped calls, whistles or scream-like calls, termed “variables,” for the calf. For each focal whale, the relative location of the calling whale with other whales in the same pool was evaluated using three scales: X, Y, and Z. “X” is defined as two whales in the same section, “Y” as two whales in the adjacent sections, and “Z” as two whales more than one section apart from each other.

Statistical analysis Microsoft Access was used to process data and make counts of various situations with specific interests. Relevant percentages were obtained and chi-square tests were applied to determine the significant difference among variables. Chi-square tests provide whether the results are different from the expected values. The expected values are calculated in the assumption that each variable being examined (age, bubble type, social state and call type) has no effect on the proportion of bubble produced without calls. The p-value of less than 0.05 indicates that the results are significantly different from the expected; hence, the difference observed in each situation did not occur by chance and the variable

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had a significant effect on the results.

Results Individual variation A total of 429 attributed bubble emissions were observed. Out of the total bubbling, 330 (77%) samples corresponded with a call and 94 (22%) did not. Of all, 1% of the calls were considered unknown since the presence of a call could not be confirmed because of overlapping calls from other whales. The results show that a proportion of bubble emissions without a call differs significantly among each individual (chi-square test: χ² = 11.6287, df = 5, p = 0.0402) (Figure 4).

Age effect A relationship between proportion of bubbling without calls and the age of a calf was examined. The chi-square test showed that the proportion of bubbles without a call varied significantly at different ages (chi-square test: χ�² =���������������������� 22.5571, �������������������� df = 3, p< ������������� .0001). When the calf was a month old, 30% of his bubbling was made without calls. At 7 months, 46% of the bubbling did not correspond with calls while at 13 months, only 10% of the bubbling occurred without vocalization.

After 13 months, the ratio of bubbling without calls was consistent through 43 months (7%) (Figure 5). The linear regression line in-

dicated that with each month of age, the calf reduced its proportion of bubbling without calls by 0.7%, and age explained 47% of the variance in percent bubbling without calls (Figure 6). However, the linear regression was not significant (p>0.05) and more data for the calf at different ages is needed.

Effect of type of bubble There was a significant difference in the proportion of bubble emissions with or without calls among the types of bubbles associated with calling (chi-square test: χ²= ������������� 129.8090, df = 2, p <.0001). Out of three types of bubbles, bubble streams were the most commonly cooccurring with calls (87%). Bubble clouds, on the other hand, never occurred with calling. Bubble ring was observed only once.

Bubble types for 11% of total samples were unknown because the focal animal was out of the camera view and data was provided by .he real-time observers as audio information; thus, the types of bubble could not be confirmed during video observation (Figure 7).

Effect of social states The proportion of bubble emission without calls differed significantly when different numbers of whales were present in the same pool (chi-square test: χ² =������������������� 50.1209���������� , df = 2, p<.0001). For a solo adult whale, only 3% of

UCSD Biological Sciences Saltman | Quarterly 35

Proportion of bubbling with or without calls by 0127 (calf) at different age

the bubbles occurred without calls. A mother and a calf pair produced bubbles without calls 9% of the time, while trio of socializing whales produced bubbles without calls 25% of the time (Figure 8).

Effect of position of other whales Relative locations between the focal whale and surrounding whales were analyzed for each bubbling event. Overall, the location of the other whales did not affect the bubbling of the mother-calf pair (chi-square test: χ² ��= 0.0073������������������������������������ , df = 2, p = 0.9964���������������� ���������������������� )��������������� . When the spatial relationship was X (two whales were in the same section) or Y (two whales were in the adjacent section), 86% of bubble emissions co-occurred with calls. When the spatial relationship was Z (whales were two or more sections apart from each other), 83% of bubbling occurred with calls. When the focal whale was surrounded by two whales, there was no significant difference in the co-occurrence of bubbling with calls among different

0127 (calf) age in months

Types of calls associated with bubbling Types of calls that corresponded with bubble emissions were categorized into stereotyped calls, clicks, calf stereotyped calls, calf whistles, and calf scream-like calls. Of all bubble emissions, stereotyped calls were most often associated with bubbling (99% for adults and 76% for a calf ) while clicks (1% only by an adult) and whistle (0% by adult and 18% by a calf ) were rarely associated with bubbling. The calf was also observed to produce bubbles during characteristic variable calls (Figures 9, 10).

0 2 1 0

With a call Without a call Unknown

Proportion of bubble with or without calls (actual counts written in the column)

Figure 5. Proportion of bubbling without call differs significantly at different ages for a calf (Chi- square test: χ2 = 22.5571, p< 0.0001).

location relationships (chi-square test: χ² = 0.4032, df =5, P = 0.5255).

Discussion General discussion In this study, 77% of the bubble emissions were found to co-occur with calls. These results correspond with research conducted on bottlenose dolphins.4 Fripp reported that 79% of the bubble streams were associated with call productions.4

% bubbling without a call

Effect of age upon 0127 (calf) bubbling without a call

Age (in months) Figure 6. Linear fit on the relationship between proportion of bubbling without call and calf age. Equation shows that with each month of age, the calf reduced its percentage of bubbling without a call by 0.7% and age explained 47% of the variance in % bubbling without a call.

36 Saltman | Quarterly UCSD Biological Sciences

Results from this study showed several factors which influence the association of bubbling with calls: age, type of bubbles, group size and type of calls. If bubble emission is simply a random by-product of call production, bubbling should be independent of these variables. A significant difference was observed in the proportion of bubble emissions without calls among subjects. One whale produced bubbles without calls only 3% of the time while the other whales produced bubbles without calls up to 33% of the time.

A calf showed a significant difference in bubble emissions without calls at different developmental stages. The result indicates that the bubble emission is not consistent over a period of time but rather fluctuates as a calf develops. Though age is a variable affecting bubble emission, due to a lack of data from a calf at different ages, regression analysis failed to demonstrate that the age of a calf was a significant factor for the variation observed (p>0.05). Additional data on calves at different stages of development is required to confirm the importance of age on the proportion of bubble emission without calls.

A significant difference in the relationship between types of bubbles and proportion of bubbles without calls was found.

Most of bubble streams were produced with calls while no bubble clouds were produced

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with calls. The results indicate that bubble stream most effectively serves as a visual cue for the calling. “Surprised, curious, or excited” bottlenose dolphins have been observed to produce a rapid exhalation termed “bubble bursts”, which correspond to the behavior defined as “bubble clouds” in this study.9 The fact that there were no particular calls associated with bubble clouds may suggest that the bubble clouds are not by-products of call productions and that they may serve as a form of visual communication. Detailed study on the context of the use of bubble clouds would aid in better understanding of the function of this type of bubble. Regarding bubble rings, no conclusion can be drawn at this point since only one bubble ring was observed during the entire study. However, bubble ring production has been suggested as a play activity among dolphins.2,9 Thus, it is expected that bubble rings are intentionally produced.

Variability in the proportion of bubble emissions without calls was observed among different social states, yet the data may have been biased by the small sample

size. Data collected characterizing the solo whale was obtained from a single male; thus, it is hard to determine whether the result regarding simultaneous production of bubbles and callings was driven by the fact that the animal was alone or that it was merely a reflection of individual variation. Additional data from different solo adult individuals will be needed to clarify this matter. In addition, the higher proportion of bubbling without calls observed in the mother-calf pair might have been derived from skewed representation since 90% of the data was obtained from a single calf. The results, however, showed that the production of bubbles without a call increased from when a calf was only with a mother (10%) to when he was with the mother and an additional whale (26%). It may suggest the importance of group size and social activity regarding the bubble emission which is independent of a call production.

In order to demonstrate a stronger correlation between bubble emissions without calls and social states, acoustic behaviors of whales in different trios should be investigated. In addition, the whale examined should include

more adults and more calves to minimize bias by the calf.

A majority of calls associated with bubble emissions were stereotyped calls for both adult whales and the calf. It is noteworthy that the bubbles were hardly accompanied with clicks. If bubble emission is a random by-product of a call production then we should have seen it correspond with any type of call. No detailed information on the loudness of each call type is available; therefore, we cannot exclude the possibility that the loudness of the call is a determinant of bubble production. However, the strong bias toward stereotyped calls suggests that a mechanism of a specific call production rather than simply the loudness of a call is more likely to be a determinant. Even though the mechanism of bubble production is still unknown, the fact that the bubble emission is most likely to be accompanied by stereotyped calls suggests that the samples obtained relying primary on bubble emissions as a visual cue for callings may not represent the whole acoustic repertoire of killer whales. In this study, however, the categorization of calls was done based

Proportion of bubbling with or without calls

Difference in proportion of bubbling with or without calls between bubble streams and bubble clouds Bubble streams = a continuous trail of bubbles from the whale’s blowhole Bubble clouds = one large formation of bubbles produced with a single exhalation

Unknown Without a call With a call

100% 80% 60% 40% 20% 0%

Stream

Cloud

Total

Types of bubbles (actual counts are written in the column) Figure 7. Proportion of bubbling without call differs significantly among different bubble types (Chi-square test: χ2 = 129.8090, p <0.0001).

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Social states

Proportion of bubbling with or without calls in different social contexts

Proportion of bubbling with or without calls (actual counts are written in the column)

With a call Without a call Unknown

on the individual decision of the researcher.

Though the calls are fairly distinct to each other, more systematic, detailed categorization is required to identify specific calls associated with bubble emission.

Future research The results indicated the dependency of simultaneous bubble and call production on the age, bubble structure, group size and call types, yet the study was inconclusive due to the limited availability of samples. Further investigation on the relationship between calf ages and bubbling occurrences may provide insight regarding the ontogeny of calf calls. Furthermore, additional data on the social states including samples of bubble emissions from different solo whales as well as reactions following bubble emissions may illustrate their functions as a form of visual communication among killer whales. Since bubble clouds never occurred concurrently with calls, further investigation on the contexts of bubble clouds may provide valuable information on the function of these visual signals. Most of the calls associated with bubbling were stereotyped; thus, calls attributed only by bubble emissions should not be used as a representative of the killer whale vocal repertoire. Analysis on the specific feature of calls which frequently occurs with bubbles among stereotyped calls will aid in providing crucial information on vocal behavior when

Figure 8. Bubbling and number of whales in a tank *1: Samples are only from one adule male *2: 90% of the sample comes from 0127 (calf) and 10% from the mother (7804) *3: 54% of the sample comes from 0127 (calf) and the rests are from four indivudual whales (Chi-square test: χ2 = 50.1209, p<0.0001).

using bubble emission as a visual cue. It will also improve our knowledge on the mechanism of bubble production. If bubbles are only associated with a certain type of call, the determinant on the bubble production may be a physical characteristic of the way the call is produced. Though calling does not often occur concurrently with bubble emission, acquiring knowledge on specific types

of calls that are associated with bubbles will allow researchers to use bubbling as a cue for particular purposes. For example, the study of the development or the learning mechanism of a specific call type can be facilitated by bubble emissions. Further understanding of the bubble use by killer whales in relation to their vocal behaviors will help us appreciate the acoustics of killer whales. Insights into the acoustics will be vital for further exploration of their development, learning, behaviors, and cultures among killer whales.

Acknowledgements I would like to thank Dr. Ann Bowles and Jennifer Keating for all their support in every aspect of this research. This work could not have been done without their advice and guidance. I would also like to thank Dr. James Nieh for his assistance as a faculty advisor, and Hubbs-SeaWorld Research Institute for providing the materials and facilities for this project.

References 1.

2.

3.

4.

5.

6.

Bowles, A.E., Young, W.G., and Asper, E.D. Ontogeny of stereotyped calling of a killer whale calf, Orcinus orca, during her first year. Rit Fiskideildar 11: 251-275 (1988).

Fabienne, D., and Aulagnier, S. Bubble blow in Beluga Whales (Delphinapterus lleucas) a play activity? Behavior Processes 40: 183-186 (1997).

Dudzinski, K.M., Clard, C.W., and Wursig, B. A mobile video/ acoustic system for simultaneous underwater recording of dolphin interactions. Aquatic Mammals 21(3): 187-193 (1995).

Fripp, D. Bubblstream whistles are not representative of a bottlenose dolphin’s vocal repertoire. Marine Mammal Science 21(1): 29-44 (2005).

Herzing, D.L. Vocalizations and associated underwater behavior of free-ranging Atlantic spotted dolphins, Stenella frontails and bottlenose dolphins, Tirsiops truncates. Aquatic Mammals 22(2):61-79 (1996).

McCowan, B. A new quantitative technique for categorizing

Proportion of types of call associated with bubbling produced by adult whales.

Stereotyped call Click Other

Figure 9. Bubble is produced with stereotyped calls but rarely with clicks.

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Proportion of types of calls associated with bubbling produced by a calf

7.

8.

9.

10.

11.

Calf stereotyped call

12.

13.

Calf whistle

whistles using simulated signals and whistles from captive bottlenose dolphins, Tursiops trucatus. Ethology 100: 177-193 (1995).

McCowan, B., and Reiss, D. Quantitative comparison of whistle repertoires from captive adult bottlenose dolphins, Tursiops truncatus: a re-evaluation of the signature whistle hypothesis. Ethology 100: 194-209 (1995).

McCowan, B., and Reiss, D. Whistle contour development in captive-born infant bottlenose dolphins (Tursiops truncatus): role of learning. Journal of Comparative Psychology 109(3): 242-260 (1995).

McCowan, B., et al. Bubble ���������������������������������������� Ring Play of Bottlenose Dolphins (Tursiops truncatus): Implications for Cognition. Journal of Comparative Psychology 114(1): 98-106 (2000).

Miller, P.J.O. Mixed-directionality of killer whale stereotyped calls: a direction of movement cue? Behavioral Ecology and Sociobiology 52: 262-270 (2002).

Miller, P.J.O., Shapiro, A.D., Tyack, P.L., and Solow, A.R. Call-type matching in vocal exchange of free-ranging resident killer whales, Orcinus orca. Animal Behavior 67: 1099-1107 (2004).

Miller, P.J.O., and Tyack, P.L.. A small towed beamforming array to identify vocalizing resident killer whales (Orcinus orca) concurrent with focal behavioral observations. Deep-Sea Research 45(2): 1389-1405 (1998).

Sayigh, L.S., Tyack P.L., Wells, R.S. and Scott, M.D. Signature whistles of free-ranging bottlenose dolphins Tursiops truncatus: stability and mother-offspring comparison. Behavioral Ecology and Sociobiology 26:247-260 (1990).

Calf variable

Figure 10. Bubble is produced with stereotyped calls and with whistles some of the time.

Review: Olfactory Ensheathing Cells, Bone Marrow Stromal Cells, and the Herpes Simplex Virus for Axonal Regeneration in Spinal Cord Injury Models Mark Chen

Earl Warren College, Senior, Animal Physiology & Neuroscience Major (Article accepted Spring 2006)

The inhibitory growth environment of the spinal cord and a lack of growth in adult neurons are two obstacles in the treatment of spinal cord injury. Transplantation of ex vivo genetically modified cells has shown some success in dealing with these two issues. This review examines the current research on olfactory ensheathing cells and marrow stromal cells with respect to axonal repair, and assesses the potential for using the herpes simplex virus to produce transient transgene expression and enhance the reparative effects of these cells.

Introduction The growth-inhibiting environment of the adult central nervous system is a major obstacle in the treatment of neural damage.

Several cell types have been used as grafts in spinal cord injury (SCI) models to provide a more permissive substrate for axonal regeneration. Another important barrier in spinal repair is the failure of neurons in the adult central nervous system (CNS) to grow sufficiently after injury. Neurotrophic factors can often enhance the growth and survival of adult neurons when transplanted into the site of injury (reviewed in 23). Cells that appear to enhance axonal regeneration in their native state have also been modified ex vivo to secrete neurotrophic factors. Such modified cells provide a growth-permitting environment in addition to eliciting regeneration

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and survival of damaged neurons (Figure 1). Ideal cellular candidates for ex vivo genetic modification should be easily obtainable from donors, capable of surviving in vitro, and multiply in vitro such that there are enough cells for transplantation and sufficient viral transfection can occur. Additionally, target cells should be able to survive after transplantation for extended periods of time and not form tumors60. Primary fibroblasts61, Schwann cells (SCs),63 and stem cells have been discussed and examined as targets for ex vivo gene therapy.23

Two additional cell types have been the topic of numerous reports in the past decade regarding the treatment of spinal cord injury and general neural repair. Olfactory ensheathing cells (OECs) and bone marrow stromal cells (MSCs) have various properties

that make then distinctive candidates for ex vivo gene therapy and the treatment of CNS damage.

Olfactory Ensheathing Cells Olfactory ensheathing cells are believed to play a role in directing axons of newly formed olfactory receptor neurons in the periphery to their targets in the olfactory bulb. Researchers have examined the ability of OECs to stimulate axonal regeneration in SCI models and also in clinical trials.19,38 Isolation and purification of olfactory ensheathing cells Two major sources of OECs are the olfactory bulb (OB)30,49 and the olfactory mucosa.20,33,34 Cells from the two sources have been shown to exhibit slightly different properties in

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Figure 1. Ex vivo genetic modification. Primary cells are isolated from donors. Cells are then expanded in cell culture and genetically modified. Genetically modified cells overexpressing neurotrophic factors can then be transplanted back into the donor, or into another host.

vivo51. The olfactory mucosa, which consists of the olfactory epithelium (OE) and the olfactory lamina propria (LP), is the more clinically relevant source of OECs, since the removal of cells from the olfactory bulb requires extensive surgery, while the cells from the olfactory epithelium can be removed relatively easily20. However, much of the current research continues to examine the utility of OECs from the olfactory bulb.

Purified olfactory ensheathing cells have been used to promote axonal regeneration. Several methods have been used to purify OECs from the olfactory mucosa or the olfactory bulb including immunopanning49, fluorescence activated cell sorting (FACS)3, magnetic bead purification4, cytotoxic elimination12, and cell separation through differential adhesion rates40. Immunopanning, FACS and magnetic bead sorting typically use antibodies against the low affinity nervegrowth factor receptor (L-NGFR), also known as p75, or the O4 surface protein3.

In cytotoxic elimination, anti-Thy 1.1 is used to remove several contaminating cell types.

Purification of OECs from the human nose with Neurotrophin 3 (NT-3) protein has also been described5. After purification, the purity of the mixture is commonly determined by positive staining for p75, glial fibrillary acidic protein (GFAP), and S10021. There is significant variation between reports regarding the growth-promoting effects of OECs in SCI models, but since the isolation and purification methods vary, it is difficult to make a firm conclusion regarding the true potential of OECs to produce neuronal regeneration.

It is therefore important to be cognizant of the different purification and/or isolation techniques used in obtaining OECs for ex-

perimentation.

Olfactory ensheathing cells and neural repair in the spinal cord In initial reports, it appeared that OECs had tremendous potential for regenerating damaged spinal axons. Early reports suggested that OECs could regenerate axons into and beyond the lesion, produce functional recovery, and remyelinate axons. Based on these observations, OECs might be considered superior to Schwann cells (SC), a well-studied alternative cell type (reviewed in Oudega et al., 2005), due the ability of OECs to permit long-distance axonal growth beyond the region of the cell graft69.

Adult rats with unpurified OBOECs, transplanted immediately into a focal electrolyte spinal cord lesion between the C1 and C2 level, showed axonal elongation that extended through the OEC graft and into caudal host-tissue 6 days after transplantation30. Functional recovery, determined with the forepaw reach task65 ipsilateral to the lesion, accompanied the apparent axonal regeneration. Under similar experimental conditions31, OECs reduced the formation of the astrocytic scar32 and increased vascularization of the injury site compared to controls. Long-distance axonal regeneration was also observed, with some axons found almost 10 mm from the lesion site. It was noted in Li et al.31 that these axons were most likely cut axons regenerating, as opposed to spared axons, because none of the lesion-only control animals exhibited axonal elongation or sprouting and because axons appeared to extend further into the caudal host-tissue with increasing post-lesion survival times.

In another example of long-dis-

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tance regeneration, highly purified adult OB-OEC suspension injected at the transition between host tissue and a SC guidance cable69 appeared to assist axonal regeneration into and through the SC cable into caudal host-tissue50. This was surprising because the SC cable alone was only shown to enhance axonal extension into the SC channel, but not back into the host tissue69. Since the host tissue was completely removed from the cable area, axon extension was believed to be authentic.

Lamina propria derived OECs have also been reported to elicit extensive neural repair and functional recovery. After complete transection of the spinal cord, either cultured lamina propria cells or 1mm2 chunks of lamina propria cells were injected into the lesion site33. Animals with both forms of lamina propria cells recovered movement of their hind limbs and joints 10 weeks after transection. Control animals, injected with culture medium, collagen, or cells from the respiratory lamina propria, did not recover hind limb movement. Additionally, the depression of the H-reflex57 was recovered in experimental animals, suggesting that modulatory serotonergic axons had reinnervated the H-reflex spinal circuit; this hypothesis was supported by the observation of serotoninpositive axons distal to the transection site.

In a chronic SCI model, the rat spinal cord was transected at the T10 level34. Four weeks later, the scar tissue was removed, leaving a 3-4 mm gap, and pieces of lamina propriaderived cells were inserted. Experimental animals given the LP cells showed significantly greater motor recovery, as determined by the Basso-Beattie-Bresnahan (BBB) locomotor scale. Serotonergic axons were also observed

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an average of 3.2 mm from the caudal interface of the graft and host-tissue. OB-OECs have also been reported to generate significant functional recovery after complete transection of the spinal cord48.

Immunohistochemistry, electron microscopy, and electrophysiological evidence suggest that OECs can form myelin around damaged spinal cord axons. OECs from E18 embryonic rats selectively formed myelin around large dorsal root ganglion axons16. Electrophysiological analysis showed that OECs could recover rapid conduction after spinal cord transection, suggesting that cells were remyelinated24. Electron microscopy of the tissue from the same experiment showed axons that were wrapped in myelin, in a peripheral, Schwann cell-like pattern, as opposed to an oligodendrocyte-like fashion normally observed in the CNS. In another study of the myelinating potential of OECs, purified OB-OECs from GFP-transgenic mice were transplanted into lesioned dorsal funiculus, a component of the spinal cord (Sasaki et al., 2004). GFP-positive cells appeared to wrap around axons longitudinally.

These presumptive OECs also expressed P0, a peripheral myelin protein. Furthermore, GFP-OECs were recently shown to modify the sodium and potassium channel distribution along demyelinated axons, form nodes of Ranvier, and increase axonal conduction55.

Evidence against the ability of OECs to mitigate extensive neural repair As Boyd et al.8 note, there are problems with identifying OECs in culture with p75, GFAP and S100; one issue is that both Schwann cells and OECs express all three markers, and another problem is that Schwann cells have been reported to enter the CNS after injury59. Boyd et al.8 compared the insoluble protein expression of OECs isolated from embryonic rats (E18), which are not contaminated with other cell types16, with purified Schwann cells. They found that antibodies against calponin, a smooth muscle marker, could uniquely identify OECs8. Using this marker, they showed that calponin-positive OECs did not directly associate with unmyelinated axons, but that OECs formed tunnels around phagocytic cells and around axons that were already myelinated. Examining OEC cultures purified with the p75 antibody, the same lab showed that these OEC cultures contained GFAP+/S100+/LNGFR+/calponin- cells, which they concluded were Schwann cells, and GFAP+/S100+/ L-NGFR+/calponin+ cells, which they concluded were OECs52. Rizek et al.52 showed

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that only about 30% of the p75 FACS-immunopurified cells expressed calponin, even though 100% expressed p75 after one week of culture after purification. As noted in Rizek et al.52, it is often assumed that there is little SC contamination in the areas from which OECs are isolated. However, this recent evidence suggests that Schwann cells contaminate OEC cultures before and after purification.

The possibility that SC contamination is present in purified OEC cultures affects the interpretation of previous research.

For example, in the Plant et al.45 paper described above, the authors note that they could not differentiate between SCs and OECs in vivo, and therefore, their observations might be explained by SC contamination or invasion. Although others have used GFP-labeled OECs from transgenic mice55,56, OEC purity was only determined with p75 and S100 reactivity. Therefore, the GFP-labeled cells seen myelinating axons may actually be Schwann cells.

Other results support the possibility of SC contamination in immunopurified OECs. One report showed that SCs enhanced myelin formation around cocultured dorsal root ganglion neurons, whereas adult OECs purified through immunopanning formed myelin at the same level as DRG neurons cultured alone46. This suggests that previously observed presumptive OEC-induced myelination was in fact the result of cocultured SCs isolated with the DRG and/ or the previously unidentified contaminating SCs. In another study comparing SC and OECs, animals were injected with SCs, SCs + OECs, OECs alone, or culture medium one week after a contusion injury at T959.

OECs were isolated from adult rats and purified through p75 immunopanning. Relative to culture medium alone, all cells grafts reduced the loss of tissue (cavity formation) typically observed after spinal cord injury.

However, the number of brainstem axons beyond the graft was elevated only in SC and SC + OEC grafts. Furthermore, animals with SC grafts were the only ones to show significantly functional recovery of hindlimb locomotor performance 8-11 weeks after injury. It is therefore possible that the OECs themselves have very little effect on neural repair. These results, taken with the evidence that purified OEC cultures are commonly contaminated with SCs, and that SCs may invade the injured spinal cord even when pure OEC cultures are applied, suggest that the various forms of axonal regeneration, remyelination and functional recovery may be

due to contaminating Schwann cells.

The ability of OECs to elicit axonal growth into and well beyond the lesion site has also been contested. One article in press58 discusses an attempt to replicate the findings reported in Lu et al.34 In both studies, lamina propria derived OECs were transplanted 1 week after a complete transection of the spinal cord at the T10 level. Yet, in the second study, the authors found no regeneration of Fluorogold labeled axons through the graft site, and BBB analysis showed no statistically significant recovery of locomotor function with OEC transplantation. There was, however, a slight modification to the procedure; because of a high incidence of incomplete lesions with the original lesion method in the second study, the spinal cord was lifted 1-2 mm with a curved needle to produce a more consistent and complete lesion. The authors argue that this manipulation should have little effect since the distance was quite small and there was no evidence of contusion injury. Therefore, it is possible that the long-distance regeneration described in Lu et al.34 might be explained by incomplete transection of the spinal cord.

Overall, the beneficial effects of OECs have all come into question. Continued investigation of the reparative effects of OECs is required to determine if OECs are truly capable of inducing long-distance axonal regeneration, remyelination and functional recovery. Future research should employ the recently discovered OEC marker, calponin, to differentiate the effects of OECs and SCs. In all SCI experiments, the lesioning procedure should also be as complete and consistent as possible. The dorsal column lesion with the Kopf wire-knife device (David Kopf Instruments, Tujunga, CA) is one method for producing consistent lesions.

Bone Marrow Stromal Cells Bone marrow contains non-adherent hematopoietic stem cells, which produce blood, immune effector cells, and adherent stromal cells62. The adherent bone marrow cell population contains a subset of cells that can selfrenew and also give rise to osteoblasts, chondroblasts, and adipocytes under appropriate conditions44. These adherent cells are often referred to as marrow stromal cells (MSCs) or mesenchymal stem cells, due to their ability to differentiate into cells of mesenchymal origin. Within MSC cultures, it is believed that there is a subset of pluripotent stem calls termed multipotent adult progenitor cells, or MAPCs. These cells have been reported to differentiate into cells of all three germ layers.

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Perhaps the most attractive aspects of MSCs and MAPCs is the possibility that these cells can differentiate into neurons in vitro and in vivo.

Isolation and purification of marrow stromal cells Bone marrow is easily removed from the iliac crest of living human donors17 in a common medical procedure. Lu et al.36 noted that MSCs rapidly expand in culture, and can be passaged more than 25 times (more than 50 population doublings)6 without visible signs of developing tumors.

To isolate MSCs from rats, the tibia and femur are removed, and the marrow extruded with DMEM. After culturing the cell mixture, the non-adherent hematopoietic cells are washed away by changing the cell culture medium2. Undesired cells can also be removed with magnetic beads conjugated to antibodies against CD45, specific to nucleated hematopoietic cells, and CD11b, which is specific to myelomonocytic cells27. Purified cells can also be tested for adipogenesis and chondrogenesis, to ensure that pluripotency is intact27.

In vitro neuronal transdifferentiation of MSCs MSCs were shown to transdifferentiate (to give rise to cells outside of their established differentiation program) in vitro into cells with a neuronal morphology that express various neural markers. Unmodified MSCs express endodermal, mesodermal and ectodermal genes66. Exposing rat MSCs to β-mercaptoethanol (BME) or DMEM/2% dimethylsulfoxide (DMSO)/200 μM butylated hydroxyanisole (BHA) produced cells with neuronal morphology that showed significantly greater immunoreactivity for neuron-specific enolase (NSE), neuron-specific nuclear protein (NeuN), neurofilament-M, and tau (a neuron-specific protein associated with microtubules), all within 5 hours of exposure66. DMSO/BHA treated MSCs showed a decrease in nestin reactivity and an increase in nerve growth factor receptor TrkA expression over a six-day period. Nestin is expressed in neuronal precursor stem cells, and its expression decreases as neurons mature29.

Woodbury et al.66,67 also showed that human marrow stromal cells react similarly to the induction protocol.

Other induction protocols have been used for neural induction of MSCs. Treatment of human MSCs with isobutylmethylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) increased the number cells

labeled with microtubule-associated protein 1B (MAP1b), neuron-specific tubulin (TuJ1), NSE, and vimentin (an early marker for glia)15. These observations were interpreted to show that an increase in cAMP plays a role in MSC differentiation into neurons and glia, since IBMX is a phosphodiesterase blocker and dbcAMP is a cAMP analogue.

Notably, IBMX/dbcAMP neural induction protocol acts more slowly than the Wood et al.66 protocol, since the earliest signs of neuronal morphology were first identified 2 days after IBMX/dbcAMP treatment.

In vitro transdifferentiation of MAPCs MAPCs have also been reported to differentiate into cells of endodermal, mesodermal and ectodermal tissue in rats, mice and humans26. MAPC differentiation into neural tissue occurs over a period of days, rather than hours. This is again in contrast to the rapid differentiation observed by Woodbury et al.66,67. For example, for MAPCs grown on fibronectin and in medium lacking platelet derived growth factor (PDGF) and epidermal growth factor (EGF) and supplemented with 100 ng/ml of basic fibroblast growth factor (bFGF), a larger portion of the cells became neurofilament-200 positive after a period of 14 days, indicating neuronal phenotype. MAPCs also exhibit an enormous reproductive potential: after 100 population doublings, there was no sign of telomerase shortening, differentiation, or morphological changes.

MAPCs developed a “more mature” neuronal phenotype when the original treatment was followed by a combination of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH) for 7 days, followed by brain-derived neurotrophic factor (BDNF) for another 7 days25. RT-PCR analysis showed that mRNA for GABA, dopamine, and specific neurotransmitter biosynthetic enzymes (tryptophan hydroxylase and tyrosine hydroxylase) all increased by 1.7- to 120fold by days 10 and 14 after the additional treatment. These cultured neuron-like cells also exhibited a decrease in resting membrane potential from days 5 to 9, as well as a rapidly inactivating sodium current in voltage-clamp analysis, potentially the result of functional voltage-gated sodium channels.

In vivo properties of MSCs and MAPCs Marrow stromal cells have been reported to show migration and differentiation into neuronal phenotypes in vivo. 5-bromo-2deoxyuridine (BrdU) labeled MSCs were injected into the lateral ventricles of neonatal

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mice. These cells appeared to participate in the normal development of the central nervous system, including migrating along established pathways, proliferating, integrating into various parts of the brain, and possibly differentiating into astrocytes and neurons, as indicated by GFAP and neurofilament reactivity27.

In a similar study of MSC differentiation in neonatal rats, adult MSCs from a male rat were injected into the lateral ventricle of female PU.1 mutants 24 hours after birth. PU.1 mutants lack a transcription factor found only in cells of hematopoietic lineage. The injected MSCs derived from adult rats, which provided the necessary cells for the animals to survive after birth, were detected through fluorescence in situ hybridization to the Y chromosome. The Y chromosome-positive cells accounted for 2.3 to 4.6% of all identifiable nuclei throughout the body. Furthermore, the marrow-derived Y chromosome-positive cells also frequently expressed NeuN39.

Similarly, Brazelton et al.9 reported that injection of GFP-expressing adult rat MSCs into the tail vein of lethally irradiated adult rats eventually yielded labeled cells in the brain expressing neuronal markers. Specifically, the GFP-positive cells that were found throughout the brain frequently expressed the neuron specific markers NeuN and TuJ1. In addition, phosphorylation of the transcription factor cAMP response element-binding protein (CREB) was found in GFP positive cells at a frequency expected of endogenous tissue. This can be interpreted as evidence that injected MAPCs can integrate into the nervous system and participate in neural functioning. Furthermore, the original donor cells highly expressed CD45 and CD11b; flow cytometry analysis of the GFP-positive cells in the recipient showed that these cells lacked CD11b and CD45, which is more evidence of differentiation after injection into an irradiated host9.

Phenotypically neural MSCs have also been found to integrate into the spinal cord and dorsal root ganglion, and these cells contain single nuclei with standard ploidy, which makes cell fusion an unlikely explanation for these observation13,14. In another in vivo experiment, ROSA26-derived β-gal expressing MAPCs were injected into an early blastocyst. Labeled cells were found in the brain, retina, lung, myocardium, skeletal muscle, liver, intestine, kidney, spleen, bone marrow, blood, and skin. This shows the wide range of cell types that MAPCs appear to differentiate into within a realistic devel-

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opmental environment26. Overall, several lines of evidence suggests that marrow stromal cells can migrate, integrate, and develop a neural phenotype in the central nervous system.

Evidence against Transdifferentiation in vivo and in vitro A reexamination of the potential for transdifferentiation for rapid neural induction of MSCs66 showed that exposure of primary rat fibroblasts, HEK293 cells, human keratinocytes and rat PC-12 cells to BME or BHA and DMSO produced neuron-like cells with pyramidal morphology, the same appearance as BME or DMSO/BHA treated MSCs35.

Furthermore, exposure to various unrelated stressors, including pH extremes, high concentrations of sodium chloride, or detergents (e.g., Triton-X-100), was sufficient to induce a neuronal phenotype in the MSCs. Timelapse photography showed that the presumptive neurite extension might actually be due to cell body shrinkage. Since RT-PCR anlaysis showed that NSE mRNA levels were similar before and after the neural induction protocol, Lu et al. 35 hypothesized that the observed increase in NSE staining66 may be due to the increase in concentration of surface markers as cells shrank, rather than upregulation of these proteins. Finally, blocking protein synthesis with cycloheximide did not block the rapid transition to a neuronal morphology, suggesting that changes in protein synthesis were not required for the observed cellular changes.

A major source of confusion regarding the ability of MSCs to migrate, integrate and proliferate is probably due to cell fusion. One study specifically examining MSC differentiation into Purkinje-like neurons reported unnatural ploidy64. As noted

by Takami et al.59, identifying MSC-derived cells solely with the Y chromosome or GFP expression will not reveal whether or not cell fusion occurred. To illustrate this point, they cocultured MSCs from transgenic mice expressing GFP and puromycin resistance with male mouse embryonic stem (ES) cells from a different strain. GFP positive and puromycin resistant cells that that had the physical appearance of embryonic stem cells (bone marrow-derived, embryonic stem-like or BMESL) were isolated. BMESL cells divided and reacted to differentiation protocols like regular ES cells. However, genetic analysis showed that these cells were tetraploid and exhibited genetic sequences from both animal strains, demonstrating that they were the result of fusion between host and donor cells.

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These results were confirmed in a second independent study70.

Marrow stromal cells and spinal cord injury Functional recovery in SCI models has been reported with MSC injections. 1 week after contusion injury, injection of MSCs into the injury site yielded higher scores on the BBB scale compared to controls11. The authors hypothesize that this effect is due to sparing of injured neurons through the release of factors such as cytokines or trophic factors.

Similarly, injection of MAPCs yielded higher scores on the BBB scale 5 weeks after contusion injury71. Surprisingly, immediate MAPC implantation after injury was less effective at supporting cell sparing compared to delayed implantation. Injection of MAPCs one week after injury appeared to form bridges that crossed the center of the injury site, forming a matrix permissive to cellular regeneration, and also increased the total number of surviving cells.

The role of marrow stromal cells in functional recovery from spinal cord injury is not clearly defined. However, marrow stromal cells appear to integrate well into the host environment and produce a permissive substrate for axonal regeneration. Also, considering their putative ability to form neurons in vivo, MSCs and MAPCs are unique cells worthy of further evaluation.

OEC, MSCs, and Neurotrophin Delivery for Spinal Cord Injury Repair OECs and MSCs have been reported to elicit some levels of neural repair in the adult

CNS. Neurotrophic factors can be added to augment the inherent beneficial properties of these cells. Direct injection of protein into the cell graft, or viral insertion of the neurotrophin genes into the cells or tissue of interest are two methods for combining the benefits of neurotrophin delivery and growth-permissive cell grafts. One of the major benefits of genetically modified cells is that they can provide long-term, localized delivery of trophic factors in the CNS environment60. Numerous cell types have been modified ex vivo to express neurotrophic factors before transplantation into the injured spinal cord10,36,53,54,61,63.

Genetic modification of OECs Ruitenberg et al.54 isolated OECs from adult female rats and purified these cells through anti-p75 immunopanning. These OECs were modified to express LacZ, BDNF, NT3, or BDNF and NT-3 with the adenoviral vector. In a rubrospinal tract lesion, injection of BDNF-expressing OECs increased axonal regeneration compared to LacZ expressing OECs. NT-3 plus BDNF expression did not have any additional benefit over BDNF alone. A subsequent study by Ruitenberg et al.53, using the same transfection, isolation, and purification methods, showed that OECs modified to express NT-3 promoted additional regeneration of corticospinal tract (CST) axons compared to OEC controls.

Cao et al.10 used OECs from the olfactory bulbs of adult rats as well. Unlike the Ruitenberg paper, Cao used a leukemia virus-based retroviral vector pN2A. They re-

Figure 2. Attracting regenerating axons towards a new source of neurotrophic factors. 1) A cell graft expresses neurotrophic factors transiently. 2) After the concentration of neurotrophic factors within the cell graft decreases, a new source of neurotrophic factors attracts the axons that have passed through the cell graft.

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searched the effects of glial cell line-derived neurotrophic factor (GDNF) expression in unilateral cervical (C4) CST transections.

Like OECs modified to overexpress BDNF and NT-3, GDNF expressing OECs promoted axonal regeneration to a greater extent than than control OECs10.

Combinatorial therapies and MSCs Genetically modified MSCs have benefits over native MSCs36. Injection of native MSCs into a cervical level lesion site produced sensory and motor growth into the cell graft. However, MSC grafts that were genetically modified to express brain derived neurotrophic factor (BDNF) allowed more axons, with greater molecular diversity, to enter the lesion site. Specifically, the number of serotonergic, coerulospinal, and dorsal column sensory axons was significantly greater in the BDNF-MSC group than in the MSC group. Interestingly, Enzyme-Linked Immunosorbent Assay (ELISA) analysis was unable to detect BDNF, NT-3, GDNF or NGF expression from MSCs in vitro. However, in vivo grafts of native MSCs showed substantial quantities of BDNF, NT-3 and NGF. BDNF-MSCs not only expressed significantly greater amounts of BDNF, but also NT-3 and NGF. The authors note that there are important differences between in vivo and in vitro expression characteristics of these cells, and the invasion of Schwann cells into the graft may partially explain these observations.

MSCs were also utilized in a combinatorial therapy approach to SCI repair37.

5 days before a C4 dorsal column wire knife lesion, cAMP was injected bilaterally into the L4 dorsal root ganglia. Pretreatment with cAMP had been shown to counter the inhibition of neurite outgrowth in the CNS47.

NT-3 was injected into the cell graft at the lesion site and also 1.5 mm rostral to the lesion site. With the full combination of treatments, multiple axons were observed 2 mm rostral to the lesion site. This is in contrast to NT-3 injections or cAMP treatment alone, where axons did not reenter the host tissue.

A potential drawback of long-term transgene expression The high concentrations of neurotrophic factors expressed at the graft site may actually reduce the ability of axons to exit the graft and grow towards another target. The option of dynamically regulating transgene expression would be especially useful after regenerating axons have entered the cell graft, at which time the downregulation of neurotrophic

transgene expression in the graft zone could, hypothetically, promote axon extension up a concentration gradient towards new source (Figure 2).

One method of downregulating transgene expression is to use a regulatable promoter. A Lentiviral-transduced “tet-off” tetracycline-regulated promoter was recently described as a method for reducing transgenic nerve growth-factor expression in vivo7. Survival of NGF-sensitive cholinergic neurons overexpressing NGF under the control of a tet-off promoter after fimbria-fornix lesions was reduced to control levels with the addition of doxycycline (a tetracycline analogue).

However, as the authors note, an immune response to the tetracycline transcription factor has been observed18,28, which can reduce the ability to control expression and may introduce unpredictable immune effects.

Another method for dynamically regulating transgene expression is through the use of different combinations of vectors, promoters, and cellular targets. For example, adenoviral vector-mediated gene transfer yielded OECs that expressed high levels of the transgene at 7 days post-transfection, but expression gradually decreased from day 7 to day 30, whereas lentiviral transfected OECs continued to express the transgene for at least 4 months54.

Herpes simplex virus for temporally and spatially limited transgene expression The herpes simplex virus is another vector that can give a certain degree of temporal control. HSV is a double stranded DNA virus surrounded by an icosadeltahedral capsid and a lipid envelope with several glycoproteins (gB, gC, gD, gH and gL) that mediate cell entry (reviewed in Glorioso and Fink22).

The deletion of several immediate early genes renders the virus functionally incapable of replication or entering the lytic cycle, the phase when the host cells are destroyed 43.

There are several benefits to using HSV (reviewed in Glorioso and Fink22; Hendriks et al.23). Firstly, unlike the lentivirus, the HSV genome does not integrate into the genome of the host but rather forms an intranuclear episomal element. This reduces the risk of undesired mutagenesis such as insertion into a tumor-suppressor gene. Secondly, HSV can be propagated to a high titer, such that vector stocks for clinical or experimental use can be easily made. A third benefit is that HSV is neurotropic, meaning that it preferentially targets neurons, especially sensory neurons.

Fourthly, unlike the murine leukemia virus, HSV can infect post-mitotic cells such as

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neurons43. As mentioned in Perez et al.43, another advantage is that HSV has a large cloning capacity, which allows the insertion of multiple genes of up to 30 kb42. For the purposes of downregulating transgene expression, HSV has the sixth benefit of entering a latent state where gene transcription is effectively restricted to a small region of the viral genome.

The human cytomegalovirus immediate early promoter (HCMV IEp) promoter can be used for dynamic transgene expression. Transgene expression under this promoter peaks within a few days of infection and then rapidly decreases, although low levels (1% or less of peak levels) of HCMV IEp driven expression may continue for up to 90 days. This may be enough time for the necessary regenerative effects to take place at the lesion site, since at 6 days post-injury, axons had already crossed the lesion site and entered the caudal host tissue in Li et al.30 Future Direction The spinal cord environment is extremely complex; it is a common notion that there will be no simple treatment, or “silver bullet” for spinal cord injury. A combinatorial approach will most likely be necessary to deal with the multitude of obstacles in the treatment of CNS damage. Although excellent cellular candidates overexpressing neurotrphic transgenes addresses the inhibitory growth environment of the CNS, and the failure of most adult neurons to regenerate after injury, another problem was developed in the process: the entrapment of regenerating axons within a high concentration of neurotrophic factors. This issue can be better understood by comparing continuous transgene expression and direct neurotrophin injection to dynamic transgene expression in SCI models.

References 1.

2.

3.

4.

5.

6.

Au, E., and Roskams, A.J. Olfactory ensheathing cells of the lamina propria in vivo and in vitro. Gliq. 41:224– 236 (2003).

Azizi, S.A., Stokes, D., Augelli, B.J., DiGirolamo, C., and Prockop, D.J. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats--similarities to astrocyte grafts. Proc. Natl.

Acad. Sci. U. S. A. 95:3908–3913 (1998).

Barnett, S.C., Alexander, C.L., Iwashita, Y, Gilson, J.M., Crowther, J., Clark, L., Dunn, L.T., Papanastassiou, V., Kennedy, P.G., and Franklin, R.J. Identification of a human olfactory ensheathing cell that can effect transplant-mediated remyelination of demyelinated CNS axons. Brain. 123:1581–1588 (1993).

Barnett, S.C., and Hutchins, A.M., and Noble, M. Purification of olfactory nerve ensheathing cells from the olfactory bulb. Dev. Biol. 155:337–350 (2000).

Bianco, J.I., Perry, C., Harkin, D.G., Mackay-Sim, A., and Feron, F. Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose. Glia. 45:111–123 (2004).

Bianco, P., Riminucci, M., Gronthos, S., and Robey, P.G. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells. 19:180–192

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7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

(2001).

Blesch, A., Conner, J., Pfeifer, A., Gasmi, M., Ramirez, A., Britton, W., Alfa, R., Verma, I., and Tuszynski, M.H. Regulated lentiviral NGF gene transfer controls rescue of medial septal cholinergic neurons. Mol Ther.

11:916–925 (2005).

Boyd, J.G., Jahed, A., McDonald, T.G., Krol, K.M., Van Eyk, J.E., Doucette, R., and Kawaja, M.D. Proteomic evaluation reveals that olfactory ensheathing cells but not Schwann cells express calponin. Glia. 53:434–440 (2006).

Brazelton, T.R., Rossi, F.M., Keshet, G.I., and Blau, H.M. From marrow to brain: expression of neuronal phenotypes in adult mice. Science. 290:1775–1779 (2000).

Cao, L., Liu, L., Chen, Z.Y., Wang, L.M., Ye, J.L., Qiu, H.Y., Lu, C.L,. and He, C. Olfactory ensheathing cells genetically modified to secrete GDNF to promote spinal cord repair. Brain. 127:535–549 (2004).

Chopp, M., Zhang, X.H., Li, Y., Wang, L., Chen, J., Lu, D., Lu, M., and Rosenblum, M. Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation. Neuroreport. 11:3001–3005 (2000).

Chuah, M.I., and Au, C. Cultures of ensheathing cells from neonatal rat olfactory bulbs. Brain Res. 601:213– 220 (1993).

Corti, S., Locatelli, F., Donadoni, C., Strazzer, S., Salani, S., Del Bo, R., Caccialanza, M., Bresolin, N., Scarlato, G., and Comi, G.P. Neuroectodermal and microglial differentiation of bone marrow cells in the mouse spinal cord and sensory ganglia. J. Neurosci. Res. 70:721– 733 (2002a).

Corti, S., Locatelli, F., Strazzer, S., Salani, S., Del Bo, R., Soligo, D., Bossolasco, P., Bresolin, N., Scarlato, G., and Comi, G.P. Modulated generation of neuronal cells from bone marrow by expansion and mobilization of circulating stem cells with in vivo cytokine treatment.

Exp. Neurol. 177:443–452 (2002b).

Deng, W., Obrocka, M., Fischer, I., and Prockop, D.J. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem. Biophys.

Res. Commun. 282:148–152 (2001).

Devon, R., and Doucette, R. Olfactory ensheathing cells myelinate dorsal root ganglion neurites. Brain Res. 589:175–179 (1992).

Digirolamo, C.M., Stokes, D., Colter, D., Phinney, D.G., Class, R., and Prockop, D.J. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate.

Br J Haematol. 107:275–281 (1999).

Favre, D., Blouin, V., Provost, N., Spisek, R., Porrot, F., Bohl, D., Marme, F., Cherel, Y., Salvetti, A., Hurtrel, B., Heard, J.M., Riviere, Y., and Moullier, P. Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycyclineregulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus. J Virol. 76:11605–11611 (2002).

Feron, F., Perry, C., Cochrane, J., Licina, P., Nowitzke, A., Urquhart, S., Geraghty, T., and Mackay-Sim, A. Autologous olfactory ensheathing cell transplantation in human spinal cord injury. Brain. 128:2951–2960 (2005).

Feron, F., Perry, C., McGrath, J.J., and Mackay-Sim, A. New techniques for biopsy and culture of human olfactory epithelial neurons. Arch Otolaryngol Head Neck Surg. 124:861–866 (1998).

Franceschini, I.A., and Barnett, S.C. Low-affinity NGFreceptor and E-N-CAM expression define two types of olfactory nerve ensheathing cells that share a common lineage. Dev. Biol. 173:327–343 (1996).

Glorioso, J.C., and Fink, D.J. Herpes vector-mediated gene transfer in treatment of diseases of the nervous system. Annu Rev Microbiol. 58:253–271 (2004).

Hendriks, W.T., Ruitenberg, M.J., Blits, B., Boer, G.J., and Verhaagen, J. Viral vector-mediated gene transfer of neurotrophins to promote regeneration of the injured spinal cord. Prog. Brain Res. 146:451–476 (2004).

Imaizumi, T., Lankford, K.L., and Kocsis, J.D. Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord. Brain Res. 854:70–78 (2000).

Jiang, Y., Henderson, D., Blackstad, M., Chen, A., Miller, R.F., and Verfaillie, C.M. Neuroectodermal differentiation from mouse multipotent adult progenitor cells.

Proc. Natl. Acad. Sci. U. S. A. 100 Suppl 1:11854– 11860 (2003).

Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lis-

2005–06 Volume 3

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

berg, A., Low, W.C., Largaespada, D.A., and Verfaillie, C.M. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 418:41–49 (2002).

Kopen, G.C., Prockop, D.J., and Phinney, D.G. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc. Natl. Acad.

Sci. U. S. A. 96:10711–10716 (1999).

Latta-Mahieu, M., Rolland, M., Caillet, C., Wang, M., Kennel, P., Mahfouz, I., Loquet, I., Dedieu, J.F., Mahfoudi, A., Trannoy, E., and Thuillier, V. Gene transfer of a chimeric trans-activator is immunogenic and results in short-lived transgene expression. Hum Gene Ther.

13:1611–1620 (2002).

Lendahl, U., Zimmerman, L.B., and McKay, R.D. CNS stem cells express a new class of intermediate filament protein. Cell. 60:585–595 (1990).

Li, Y., Field, P.M., and Raisman, G. Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells. Science. 277:2000–2002 (1997).

Li, Y., Field, P.M., and Raisman, G. Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells. J. Neurosci. 18:10514– 10524 (1998).

Li, Y., and Raisman, G. Sprouts from cut corticospinal axons persist in the presence of astrocytic scarring in long-term lesions of the adult rat spinal cord. Exp. Neurol. 134:102–111 (1995).

Lu, J., Feron, F., Ho, S.M., Mackay-Sim, A., and Waite, P.M. Transplantation of nasal olfactory tissue promotes partial recovery in paraplegic adult rats. Brain Res. 889:344–357 (2001).

Lu, J., Feron, F., Mackay-Sim, A., and Waite, P.M. Olfactory ensheathing cells promote locomotor recovery after delayed transplantation into transected spinal cord.

Brain. 125:14–21 (2002).

Lu, P., Blesch, A., and Tuszynski, M.H. Induction of bone marrow stromal cells to neurons: differentiation, transdifferentiation, or artifact? J. Neurosci. Res. 77:174–191 (2004a).

Lu, P., Jones, L.L., and Tuszynski, M.H. BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp. Neurol. 191:344– 360 (2005).

Lu, P., Yang, H., Jones, L.L., Filbin, M.T., and Tuszynski, M.H. Combinatorial therapy with neurotrophins and cAMP promotes axonal regeneration beyond sites of spinal cord injury. J. Neurosci. 24:6402–6409 (2004b).

Mackay-Sim, A. Olfactory ensheathing cells and spinal cord repair. Keio J Med. 54:8–14 (2005).

Mezey, E., Chandross, K.J., Harta, G., Maki, R.A., and McKercher, S.R. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science. 290:1779–1782 (2000).

Nash, H.H., Borke, R.C., and Anders, J.J. New method of purification for establishing primary cultures of ensheathing cells from the adult olfactory bulb. Glia, 34:81–87 (2001).

Oudega, M., Moon, L.D., and de Almeida Leme, R.J. Schwann cells for spinal cord repair. Braz J Med Biol Res. 38:825–835 (2005).

Palmer, J.A., Branston, R.H., Lilley, C.E., Robinson, M.J., Groutsi, F., Smith, J., Latchman, D.S., and Coffin, R.S. Development and optimization of herpes simplex virus vectors for multiple long-term gene delivery to the peripheral nervous system. J Virol. 74:5604–5618 (2000).

Perez, M.C., Hunt, S.P., Coffin, R.S., and Palmer, J.A. Comparative analysis of genomic HSV vectors for gene delivery to motor neurons following peripheral inoculation in vivo. Gene Ther. 11:1023–1032 (2004).

Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R, Mosca, J.D, Moorman, M.A, Simonetti, D.W., Craig, S., and Marshak, D.R. Multilineage potential of adult human mesenchymal stem cells. Science.

284:143–147 (1999).

Plant, G.W., Christensen, C.L., Oudega, M., and Bunge, M.B. Delayed transplantation of olfactory ensheathing glia promotes sparing/regeneration of supraspinal axons in the contused adult rat spinal cord. J Neurotrauma. 20:1–16 (2003).

Plant, G.W., Currier, P.F., Cuervo, E.P., Bates, M.L., Pressman, Y., Bunge, M.B., and Wood, P.M. Purified adult ensheathing glia fail to myelinate axons under culture conditions that enable Schwann cells to form myelin. J. Neurosci. 22:6083–6091 (2002).

Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P.N., Bregman, B.S., and Filbin, M.T. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron. 34:895–903 (2002).

Ramon-Cueto, A., Cordero, M.I., Santos-Benito, F.F.,

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

and Avila, J. Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia. Neuron. 25:425–435 (2000).

Ramon-Cueto, A., and Nieto-Sampedro, M. Regeneration into the spinal cord of transected dorsal root axons is promoted by ensheathing glia transplants. Exp. Neurol. 127:232–244 (1994).

Ramon-Cueto, A., Plant, G.W., Avila, J., and Bunge, M.B. Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants. J. Neurosci. 18:3803– 3815 (1998).

Richter, M.W., Fletcher, P.A., Liu, J., Tetzlaff, W., and Roskams, A.J. Lamina propria and olfactory bulb ensheathing cells exhibit differential integration and migration and promote differential axon sprouting in the lesioned spinal cord. J. Neurosci. 25:10700–10711 (2005).

Rizek, P.N., and Kawaja, M.D. Cultures of rat olfactory ensheathing cells are contaminated with Schwann cells. Neuroreport. 17:459–462 (2006).

Ruitenberg, M.J., Levison, D.B., Lee, S.V., Verhaagen, J., Harvey, A.R., and Plant, G.W. NT-3 expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration. Brain. 128:839–853 (2005).

Ruitenberg, M.J., Plant, G.W., Christensen, C.L., Blits, B., Niclou, S.P., Harvey, A.R., Boer, G.J., and Verhaagen, J. Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord. Gene Ther. 9:135–146 (2002).

Sasaki, M., Black, J.A., Lankford, K.L., Tokuno, H.A., Waxman, S.G., and Kocsis, J.D. Molecular reconstruction of nodes of Ranvier after remyelination by transplanted olfactory ensheathing cells in the demyelinated spinal cord. J. Neurosci. 26:1803–1812 (2006).

Sasaki, M., Lankford, K.L., Zemedkun, M., and Kocsis, J.D. Identified olfactory ensheathing cells transplanted into the transected dorsal funiculus bridge the lesion and form myelin. J. Neurosci. 24:8485–8493 (2004).

Skinner, R.D., Houle, J.D., Reese, N.B., Berry, C.L., and Garcia-Rill, E. Effects of exercise and fetal spinal cord implants on the H-reflex in chronically spinalized adult rats. Brain Res. 729:127–131 (1996).

Steward, O., Sharp, K., Selvan, G., Hadden, A., Hofstadter, M., Au, E., and Roskams, J. A re-assessment of the consequences of delayed transplantation of olfactory lamina propria following complete spinal cord transection in rats. Exp. Neurol. (2006) Takami, T., Oudega, M., Bates, M.L., Wood, P.M., Kleitman, N., and Bunge, M.B. Schwann cell but not olfactory ensheathing glia transplants improve hindlimb locomotor performance in the moderately contused adult rat thoracic spinal cord. J. Neurosci. 22:6670– 6681 (2002).

Tuszynski, M.H. Growth-factor gene therapy for neurodegenerative disorders. Lancet Neurol. 1:51–57 (2002).

Tuszynski, M.H., Peterson, D.A., Ray, J., Baird, A., Nakahara, Y., and Gage, F.H. Fibroblasts genetically modified to produce nerve growth factor induce robust neuritic ingrowth after grafting to the spinal cord. Exp. Neurol. 126:1–14 (1994).

Van Damme, A., Vanden Driessche, T., Collen, D., and Chuah, M.K. Bone marrow stromal cells as targets for gene therapy. Curr Gene Ther. 2:195–209 (2002).

Weidner, N., Blesch, A., Grill, R.J., and Tuszynski, M.H. Nerve growth factor-hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expression of L1. J. Comp. Neurol. 413:495–506 (1999).

Weimann, J.M., Charlton, C.A., Brazelton, T.R., Hackman, R.C., and Blau, H.M. Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains. Proc. Natl. Acad. Sci. U. S. A. 100:2088–2093 (2003).

Whishaw, I.Q., O’Connor, W.T., and Dunnett, S.B. The contributions of motor cortex, nigrostriatal dopamine and caudate-putamen to skilled forelimb use in the rat.

Brain. 109:805–843 (1986).

Woodbury, D., Reynolds, K., and Black, I.B. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci. Res. 69:908-917 (2002).

Woodbury, D., Schwarz, E.J., Prockop, D.J., and Black, I.B. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61:364370 (2000).

Xu, X.M., Chen, A., Guenard, V., Kleitman, N., and Bunge, M.B. Bridging Schwann cell transplants pro-

UCSD Biological Sciences Saltman | Quarterly 45

69.

mote axonal regeneration from both the rostral and caudal stumps of transected adult rat spinal cord. J Neurocytol. 26:1–16 (1997).

Xu, X.M., Guenard, V., Kleitman, N., and Bunge, M.B. Axonal regeneration into Schwann cell-seeded guid-

70.

ance channels grafted into transected adult rat spinal cord. J. Comp. Neurol. 351:145–160 (1995).

Ying, Q.L., Nichols, J., Evans, E.P., and Smith, A.G. Changing potency by spontaneous fusion. Nature.

416:545–548 (2002).

71.

Hofstetter, C.P., Schwarz, E.J., Hess, D., Widenfalk, J., El Manira, A., Prockop, D.J., and Olson, L. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc. Natl. Acad. Sci. U.S.A. 99:2199-2204 (2002).

Ethidium Bromide Exposure Has Teratogenic Effects on Xenopus Embryos Jeffrey Cantle

Roger Revelle College, Senior, Animal Physiology and Neuroscience Major (Article Accepted Spring 2006)

Xenopus blastula stage embryos were treated with ethidium bromide in order to determine if the compound behaves as a teratogen—an agent that can disrupt development or cause malformations in an embryo. It was hypothesized that, due to the DNA and RNA synthesis-inhibiting properties of ethidium bromide, it would decrease the head-to-tail length of embryos, as well as produce gross malformations of the major organ systems. Cellular localization of ethidium bromide was expected in the nucleus of affected cells because of the DNA intercalating nature of the compound. Ethidium bromide was found to reduce the overall length of Nieuwkoop and Faber stage 45 embryos from 10.54mm to 5.90mm, in addition to producing abnormal eyes, bent tail and spine, and enlarged gut in many of the embryos treated. Tumors were also prevalent in embryos treated with higher concentrations of ethidium bromide. This led to the classification of ethidium bromide as teratogenic to normal Xenopus development, with implications for re-thinking safe handling and discarding of ethidium bromide from the laboratory.

Introduction Ethidium bromide (EB), 3,8-Diamino-5ethyl-6-phenylphenanthridinium bromide is commonly used in the laboratory for the visualization of DNA in electrophoresis gels.

What allows for the visualization of the DNA bands in the electrophoresis gels is the tendency of EB to intercalate in DNA, coupled with the compound’s ability to fluoresce under ultraviolet (UV) light1,2. The same ability of EB to intercalate in DNA gives EB toxic and mutagenic properties3. A search of the Sigma-Aldrich material safety data sheets indicates that EB is known to be mutagenic and toxic in multiple species, but states only that EB may pose harm to developing organisms.

The mutagenic potential of EB is fairly well known, but because its effects as a teratogen remain poorly understood, the effects of EB were studied on Xenopus embryos.

Xenopus laevis, the African clawed frog, is a model organism frequently used in developmental biology laboratories. They are frequently used in teratogen experiments as a way to screen chemicals and environmental factors for damaging effects on embryos. In addition to being easily attainable and relatively simple to work with in the lab, recently there has been much attention given to the decline in frog populations world wide.

While Xenopus itself has been implicated in the decline of some native species, frogs and

amphibians in general remain used by ecologists as indicators of ecosystem health.

Ethidium bromide is classified as a DNA and RNA synthesis inhibitor, thus EB exposure is likely to result in malformations typical of DNA and RNA synthesis inhibitors, such as reduced head-to-tail length and gross malformations to all major organ systems4. The concentrations of ethidium bromide used for testing were the lower limits of the range between the concentration that kills half of the embryos (LC50) and the amount that causes the first noticeable abnormalities as determined by Courchesne and Bantle. These values were found to be 5.0x10-2mg/mL and 1.0x10-3mg/mL, respectively5. Initially, this entire range was of interest; however, the focus of the study was on the teratogenic effects and not the toxic effects, so experimental concentrations centered on the lower portion of the range to keep embryonic death minimized.

By minimizing the toxic effects of ethidium bromide, it was possible to determine whether or not EB is teratogenic to Xenopus development. It was hypothesized that EB would inhibit embryonic growth (measured head-to-tail) and produce gross morphogenic and organogenic abnormalities. It was also hypothesized that, due to the ability of EB to fluoresce under UV light, the cellular location of ethidium bromide in

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embryonic cells could be determined using UV microscopy.

Methods Healthy appearing mid- to fine-cell blastula stage Xenopus embryos (Nieuwkoop and Faber stages 8 and 9) were de-jellied using 2.0% cisteine (pH=8), then sorted for use in the study. In the first batch, 30 embryos were used for each of five groups, but because of the difficulty of measuring and characterizing 150 embryos, 10 per group were used for subsequent batches. As discussed above, EB concentrations at the lower range of the Courchesne study were used (Table 1).

Solutions were made using an EB stock of 0.625mg/mL and 10% modified Ringer’s (MR) solution. Group E was 10% MR with no EB, and was used to establish the baselines of mortality and length of embryo, in addition to establishing normal morphogenesis. Ethidium bromide is known to degrade when exposed to light, so solutions were kept covered after preparation. To prevent degradation of the EB during the course of the experiment, the embryos were covered to prevent the entry of light. Approximately 12mL of solution were used per 60mm Petri dish that contained the embryos for the course of the study. Embryos remained in the same solution (with no changes) until the control group (E group) reached stage 45, at

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which time the embryos were removed to 10% MR solution with no EB. Embryos were observed at different times during the course of the treatment for abnormalities and mortality, but final measurements were taken when the control group was in Nieuwkoop and Faber (N&F) stage 45. At least one embryo was removed from the groups and fixed in 2% paraformaldehyde for later photographing. These embryos were not included in the length measurements, but are included here as qualitative examples of the changes induced by ethidium bromide exposure. Embryos were observed and measured with a dissecting microscope and a calibrated ocular micrometer.

For visualizing individual cells and areas of embryo under UV light, a Nikon compound microscope with a UV lamp was used.

This allowed for the viewing of any Figure 1. Representative embryonic development. Common embryo developmental abnormalities included localization of ethidium bromide, as deformed eyes, enlarged gut, bent spine/tail, and tumors. the EB fluoresces under UV light.

For images requiring less magnificaindividuals decreased as the concentration no countable tumors. tion, whole embryos were placed in a depres- of ethidium bromide decreased, as did the As seen in Figure 1, the effects of sion slide. For higher magnifications, tissue severity of the gut enlargement. There was ethidium bromide varied widely. Many of samples were taken from specific embryos no noticeable gut enlargement in any of the the embryos exposed to ethidium bromide, and viewed with a microscope slide and cover 10% MR control embryos (group E). Bend- and especially those are higher concentraslip. This allowed for higher magnifications ing of the spine/tail was also a fairly recogniz- tions, showed malformations of the eye, gut, than the depression slide would allow due to able trait, as it caused the embryo to swim in and tail/spine. It is not known whether the its larger height. circles in extreme cases. In moderate cases, hypothesized random intercalation of ethidResults Morphology Embryos were monitored for developmental abnormalities daily throughout development, and several malformations were found to be more common than others, including eye malformations, enlarged gut, permanent bending of the tail, and tumors (Figure 1).

Although qualitative, the following data trends were observed. Eye malformations included small and oddly shaped eyes, as well as missing lenses. These malformations were fewer in number and less severe in the lower EB concentrations and were non-existent in the control solution. Enlarged gut was the most common developmental abnormality found, as it occurred in almost all of the D group embryos. The number of affected

the bending was recognized after several start/stop swimming cycles: if the bend was in the same place as previously found after several bursts of swimming, it was taken to be due to an anatomical feature rather than chance. Again, the severity of this developmental abnormality was found to be higher in the embryos exposed to higher concentrations of EB, while no embryos in the control group (E) were found to have bent tails. Tumors proved to be harder to classify, as they were often nearly clear, small, and difficult to distinguish from normal structures. Tumors were also found more frequently on embryos exposed to higher concentrations of ethidium bromide (with the most occurring in the D group); both group A (0.001mg/mL EB), and the negative control group E had

ium bromide is actually specific for certain genes or regions of the genome, but it is of interest that these defects occurred more commonly than others. Molecular biology techniques such as a DNA microarray chip experiment could be used to assay the differences between the normal gene expression and the gene expression of the treated embryos. These experiments assay the expression of known genes, and analysis of several treated embryos could determine with great accuracy whether certain genes are being specifically knocked out, or whether the eye, gut, and tail/spine problems are due to nonspecific inhibition of DNA and RNA synthesis.

Sample Group

A

B

C

D

E

[EB] (mg/mL)

0.001

0.003

0.01

0.03

0.0 (Control)

Table 1. Ethidium bromide concentrations used.

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Length and Mortality of Embryos 12

10

Sample Group

8

Average Length (mm) Number of Dead Embryos

6

4

2

0 Control

0.001 mg/mL

0.003 mg/mL

0.01 mg/mL

0.03 mg/mL

Figure 2. Length and mortality of embryos. The average length of the embryos at N&F stage 45 is shown in blue, while the number of dead embryos from the same group is shown in maroon. It should be noted that increasing EB concentrations did not correlate with increased mortality at this range of concentrations.

Length and mortality Upon reaching stage N&F stage 45, the head-to-tail length of all non-fixed embryos was recorded. Figure 2 shows the average head-to-tail length of the embryos at stage 45 decreases steadily as the treatment concentration of ethidium bromide increases.

Although it was hoped that there would be a dose-response relationship between the concentration of EB administered and the headto-tail length, that kind of relationship was not observed. That is not to say that there is not a strong relationship between EB concentration and head-to-tail length, however.

Group A embryos averaged 8.57mm in length at an EB concentration of 0.001mg/mL, while group C embryos averaged 8.02mm at 0.01mg/mL: a 0.55mm decrease in length with a ten-fold increase in concentration. In contrast between group B (0.003mg/mL) and group D (0.03mg/mL) the head-to-tail length decreased from 8.45mm to 5.90mm.

Again, this is a ten-fold increase in concentration, but it resulted in 2.55mm of loss in length of the embryos. This is not a strictly linear dose-response relationship, but there is a strong correlation between the concentra-

tion of EB administered and the decrease in length of the embryos. It should be noted that the highest concentration of EB that embryos were exposed to decreased the average embryo length from 10.54mm in the control group E to 5.90mm in the group D (0.03mg/mL) concentration. This is a significant decrease in the overall length of the embryos.

Mortality can be seen in Figure 2 as a raw number of embryos that were found dead from each sample group. There is no well-defined trend in the mortality data.

Death was determined by either the lack of a beating heart or the continued absence of development for greater than 24 hours.

Only one embryo died in the control group E, as did one embryo in the group with the highest EB concentration (group D). Most deaths were observed in group C, although it is not known why this group suffered the highest mortality rates.

Tissue and cellular specificity Figure 3 shows both the ease of viewing individual cells after treatment with ethidium bromide, and the possible cellular localiza-

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tion of EB. Under visible light conditions at 400X magnification, melanocytes can be discerned from the background, and a cell can be made out in the center of the field.

Under UV illumination, regions of this cell begin to fluoresce as the intercalated EB begins to release photons in the visible spectrum. There are several brightly fluorescing spots within the cell, and it is likely that the brightest one is the nucleus, as it contains the highest concentration of nucleic acids in the cell. Other bright regions may include areas of high concentrations of mitochondria (as they contain their own DNA) or cellular RNA. To confirm that the nucleus is the part of the cell that is fluorescing the strongest, a clear image of the nucleus under visible light illumination would be required, after which illumination with UV light only could confirm the location of the brightest fluorescence to the nucleus. This would be stronger evidence of the cellular location of ethidium bromide in the cell, but based upon the hypothesized mechanism of intercalation of EB into nucleic acids, it is likely that this conclusion is accurate.

Viewing of embryonic tissue sam-

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ples under UV light allowed for the observation of any tissue specificity of the ethidium bromide treatment. As viewed, no tissue specificity could be found; indeed it appeared that nearly every tissue sample had some cells fluorescing from ethidium bromide treatment. The exception to this was any cell that was heavily pigmented, such as melanocytes and most of the eye (the lens still fluoresced).

The broad range of tissues that are affected by the ethidium bromide treatment is likely due to the very early stage of the embryos at the initial time of treatment. The late blastula stage is likely to be early enough in development that most cells could absorb EB before they move inside the embryo during gastrulation—resulting in a thoroughly treated embryo.

Discussion The head-to-tail length of the embryos was used as the major quantitative criterion for determination of teratogenicity, while the severity of changes in morphogenesis and organogenesis were qualitatively recorded.

Ethidium bromide was found to reduce the head-to-tail length of the embryos from the normal 10.54mm in the 0.00mg/mL EB control group E to 5.90mm in the 0.03mg/ mL EB treated group D. Changes in morphology were very common and were present in most embryos treated with ethidium bromide. The severity of abnormalities increased as the concentration of EB given to the sample group increased. In addition, compound microscopy with UV light illumination showed that the cellular localization of EB in the embryos appeared to be in the nucleus and that there did not appear to be specific tissue localization.

Morphology There is no standard way to classify the level of morphological damage sustained by ethidium bromide, such as the Index of Axis Deficiency7. An index of morphological changes was too difficult to establish because of the varying locations of the changes. Owing to this difficulty, the data on morphological changes due to EB exposure is highly qualitative. This resulted from an initial plan to count the number of abnormal embryos.

This plan was quickly changed when it became apparent that almost all EB treated embryos developed some kind of morphological abnormality, and the head-to-tail length became the chief quantitative data on ethidium bromide’s effects on embryonic development.

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Length and mortality What is clear from this data is that ethidium bromide is not toxic at the concentrations tested. This is an important consideration for this study as the goal is to classify ethidium bromide not as being toxic, but rather as a teratogen. As toxicity at this concentration is not pronounced, ethidium bromide can be classified as a teratogen. Mutagenesis was not tested, as DNA sequencing was not performed and embryos were never allowed to develop into reproducing adults.

Tissue and cellular specificity The techniques performed to image the tissue samples were crude, and improvements to the techniques could yield better imaging.

This said, there was still localized EB fluorescence in the nuclei of cells from the tissue. Future studies focusing on later stages of embryonic growth could yield more tissue specificity as there could be more treatment of certain cell layers than others.

Figure 3. Xenopus embryo cell under visible spectra, and then UV illumination. In the top image, under visible light illumination, a cell can barely be made out as a darker-colored region against the light-blue background. In the bottom image, the same cell’s cell boundaries are clearly defined and regions in the cell are fluorescing. The larger dark spots are melanocytes.

Broad implications Current laboratory practices vary by institution as to the correct handling of ethidium bromide waste, with many institutions allowing used gels to be discarded in normal garbage instead of being treated as toxic waste.

In this study, ethidium bromide was found to be quite teratogenic to Xenopus embryos, in addition to the more common classification of EB in the scientific literature as both a toxin and a mutagen. Sigma-Aldrich recommends using ethidium bromide at a concentration of 10µg/mL in DNA electrophoresis gels. This is a much higher concentration than used in even the highest concentration that embryos were treated to in this study. If a university were to discard a thousand gels averaging 100mL and 10µg/mL, it would be equivalent to one gram of EB discarded.

Even though ethidium bromide breaks down from exposure to light, this large an amount of EB could pose a significant threat if not

disposed of correctly.

Frogs and other amphibians are used by ecologists as indicator species for the health of ecosystems. Frogs live in the interface between air and water and, as a result, are exposed to the hazards of each. In past decades, and for unknown reasons there has been a decline in frog populations world wide. Many factors may contribute to this decline, including increased predation, decreased habitat, introduction of more competitive species, and environmental toxins7.

At this time, it is impossible do determine for certain which factors are responsible, but more attention is being shifted to the role that frogs play because changes in the health of frog populations can mark potential problems faced by other populations—including human. Although there is not good enough reason yet to believe that environmental toxins are the cause of the decline in frog populations, and by no means is ethidium bromide implicated as one of the major players in this debate, it has been shown that EB exposure

UCSD Biological Sciences Saltman | Quarterly 49

can severely affect Xenopus embryos.

More care should be taken in the treatment of ethidium bromide waste. As seen in Xenopus, it has the potential to cause great and lasting harm to embryos that it comes in contact with. With newer, safer DNA marking dyes on the market, such as SYBR Safe DNA Gel Stain, expense is the only issue preventing the safe staining of DNA gels in the laboratory8.

Acknowledgements I would like to thank Kathy French, Krista

Todd, Dennis Hickey, and Albert Kim for their help and support.

References 1.

2.

3.

4.

Benesova-Minarikova L., Fantova L., and Minarik, M. Multicapillary electrophoresis of unlabeled DNA fragment,s with high-sensitive laser-induced fluorescence detection by counter-current migration of intercalation dye. Electrophoresis 26(21):4064-9 (2005).

Vardevanian P.O., Antonian A.P., Davtian A.G., Arakelian A.V., Minasian S.G., and Tavadian, L.A. [Study of complexes of ethidium bromide with DNA by differential pulse voltammetry]. Biofizika 50(2):371-3 (2005).

(Translated from Russian) Smith, S. Personal Communication (2005).

Courchesne, C.L., and Bantle, J.A. Analysis of the activity of DNA, RNA, and protein synthesis inhibitors on Xenopus embryo development. Teratog. Carcinog. Mutagen.

5.

6.

7.

8.

5(3):177-93 (1985).

Courchesne, C.L., Dawson, D.A., and Bantle, J.A. Detection of inhibitors of protein and nucleic acid synthesis using oocytes of Xenopus laevis microinjected with the herpes thymidine kinase gene. Chem. Biol. Interact.

60(1):13-30 (1986).

Kao, K.R., and Elinson, R.P. The entire mesodermal mantle behaves as Spemann’s organizer in dorsoanterior enhanced Xenopus laevis embryos. Dev. Biol. 127, 64-77 (1988).

Hayes, M.P., and Jennings, M.R. Decline of ranid frog species in western North America: Are bullfrogs (Rana catesbeiana) responsible? Journal of Herpetology.

10(4): 490-509 (1986).

Huang Q., and Fu W.L. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clin. Chem. Lab. Med. 43(8):841-2 (2005).

PIK3CA Mutations Are Rare in Neuroblastoma: Development and Use of a Highly Sensitive dHPLC Assay for the Mutational Analysis of PIK3CA

Youngjin Kim*, Alice L. Yu, and Mitchell B. Diccianni

*Roger Revelle College, Senior, General Biology Major, UCSD Medical Center, Department of Pediatrics Hematology/Oncology

(Article Accepted Spring 2006)

Activating mutations of the PIK3CA gene have been found at a high frequency in many human cancers. Since neuronal-derived tumors of the brain have been shown to harbor PIK3CA mutations, and deletion and mutation of the specific inhibitor of PIK3CA are rare in neuroblastoma, we considered the possibility of PIK3CA alterations in this tumor type. Using denaturing High Performance Liquid Chromatography (dHPLC), we have analyzed 23 neuroblastoma cell lines and 101 primary neuroblastoma samples for PIK3CA mutations in exons 9 and 20 where greater than 75% of the reported mutations have been localized. No neuroblastoma cell lines were found to have PIK3CA mutations. However, one stage III primary neuroblastoma sample was found to have a heterozygous mutation at bp3236, which resulted in a leucine to proline amino acid change at codon 1079. This sample exhibited N-myc amplification but was otherwise unremarkable in comparison with the rest of the neuroblastoma samples analyzed. In summary, we have developed a highly sensitive, rapid, and cost-effective dHPLC technique to screen for mutations in PIK3CA exons 9 and 20. With regard to neuroblastoma, PIK3CA mutation is an uncommon event that may only rarely be involved in the pathology of this disease.

Introduction Neuroblastoma is a malignant tumor comprised of undifferentiated neuroectodermal cells derived from the neural crest. It is the most common extracranial solid tumor of childhood, accounting for about 10% of all pediatric cancers. Though the etiology of neuroblastoma is unknown, a number of genetic alterations have been associated with the disease, including deletions of chromosome 1p or 11q, unbalanced gain of 17q, and amplification of N-myc (MYCN), each of which has adverse prognostic significance for neuroblastoma.1 Neuroblastoma can broadly be divided into two categories based on biological

and molecular characteristics and prognosis.

Advanced stage disease (stages III and IV) accounts for about 60% of neuroblastoma patients and is commonly associated with metastases in bone or bone marrow and amplification of the MYCN proto-oncogene.

The prognosis for recovery of stage III patients with MYCN amplification and stage IV patients is less than 50% in spite of intensive multimodality treatment including bone marrow transplantation.2 In contrast to the poor prognosis of stages III and IV neuroblastoma is the favorable prognosis observed for patients with localized disease (stages I and II) and stage IVS disease. Stages I and II neuroblastoma patients without MYCN

50 Saltman | Quarterly UCSD Biological Sciences

amplification respond favorably to surgery and/or chemotherapy, with eradication of the disease obtained in greater than 90% of the patients. Stage IVS tumors are particularly unique; patients survive with little or no cytotoxic therapy due to the spontaneous regression of the disease or, occasionally, the differentiation of the tumor into benign ganglioneuroma.

The Akt pathway is a multifaceted pathway of positive regulators such as PIK3CA and negative regulators such as PTEN, that act through the kinase activation of Akt to regulate cell growth and differentiation.

Loss of cell cycle regulation is often attributed to deregulation of Akt, whose activa-

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tion is due to alterations in regulatory genes such as PTEN or PIK3CA.3 The PTEN gene is located on chromosome 10q23, a region of frequent heterozygous deletions in many cancers.4 Consistent with PTEN tumor suppressor activity, deletions and mutations of the PTEN gene have been found in many cancers.4 Reports of 10q alterations in neuroblastoma led to the hypothesis that PTEN may also be involved in neuroblastoma.5 However, mutations, deletions, and transcriptional inactivation of PTEN have only been rarely found in neuroblastoma,6, 7 suggesting the non-involvement of PTEN in this disease.

Recently, the presence of activating mutations of PIK3CA has been reported in numerous cancers.8,9 Several considerations led us to consider PIK3CA alterations in neuroblastoma as well. Of particular interest to us was the observation that there existed a high frequency of PIK3CA mutations in neuronal tissue.8 We also noted that many neuroblastoma cell lines contain the active form of Akt.10,11 LY294002, a specific inhibitor of PIK3CA, also impaire retinoic acid (RA)-induced down-regulation of members of the Inhibitor of Differentiation (ID) family of proteins in a PIK3-dependent manner,12 and has been hypothesized to be involved in neuroblastoma pathogenesis.13 These evidences support a role for the PI3K/Akt signaling pathway in the regulation of neuronal cell survival, with the possibility that deregulation of PIK3CA through mutation could hold an oncogenic role in neuroblastoma cell growth.

In order to screen for mutations of PIK3CA in neuroblastoma, we developed a highly sensitive dHPLC technique to analyze exons 9 and 20 where greater than 75% of the mutations have been reported. Our technique was sensitive enough to detect a heterozygous PIK3CA mutation at one part in 20, or a homozygous mutation present in a sample containing only 2.5% tumor cells.

Using this technique, we analyzed 23 neuroblastoma cell lines and 101 primary neuroblastoma patient samples at all stages. Our investigations identified only a single primary neuroblastoma sample harboring a PIK3CA alteration. Confirmation of this alteration by direct sequence analysis revealed it to be a heterozygous mutation at bp3236, which results in a Leu ‡ Pro amino acid change at codon 1079 of exon 20. We concluded that mutations of PIK3CA are very rare in neuroblastoma and that alterations of this gene likely do not play a role in neuroblastoma pathogenesis.

2005–06 Volume 3

Materials and Methods Cell lines and primary sample accrual The 23 cell lines were obtained from various sources and the primary neuroblastoma samples were obtained from the COG Tumor Bank or the Cooperative Human Tissue Network (CHTN) and consisted of 46 favorable stage [I (N=16), II (N=19), and IVS (N=11)] and 55 unfavorable stage [III (N=14) and IV (N=41)] tumor samples, classified according to the International Neuroblastoma Staging System.14 Samples are fully encoded to protect patient confidentiality and conform to HIPAA standards and are utilized under a University of California, San Diego-approved IRB protocol (#041429). Tumor cell content was determined to be 80% or more in all samples, as reported by analysis of a tissue section by the institution submitting the sample or by a CHTN or COG tumor bank pathologist.

RNA and DNA preparation Primary neuroblastoma tumors were crushed over dry ice, lysed in Trizol® (Invitrogen, Carlsbad, CA) and RNA extracted according to the manufacturers’ instructions. DNA was subsequently extracted from the organic phase of the RNA-extracted Trizol samples as described.15 DNA from cell lines, harvested at approximately 60-80% confluence, was extracted by lysis of the cells in 320 mM sucrose, 10 mM Tris-HCl (pH 7.8), 5 mM MgCl2, and 1% Triton X-100 at 4oC, digestion of protein with protease K, extraction with phenol-chloroform, spooling of the DNA on a glass rod, and resuspension in 10 mM Tris-HCl, 1 mM EDTA (pH 8.0) buffer.

Primers, PCR conditions, and sequencing A 258-bp fragment of exon 9 was amplified with primers X9FF and X9RR, while a 420-bp fragment of exon 20 from PIK3CA, containing the previously reported region of frequent mutation, were PCR-amplified with primers X20F2 and X20R2. PCR was performed in a 50-μl volume containing 50 ng genomic DNA, 2 mM MgSO4, 140 μM dNTPs, 0.3 μM of each primer, and 2.5 U Platinum Hi Fidelity DNA taq polymerase (Invitrogen). The initial denaturing step at 94°C for 2 min was followed by 35 cycles consisting of 30 sec at 94°C, 30 sec at 55°C, and 45 sec at 68°C. The final extension step at 72°C was 2 min. PCR was performed in a GeneAmp 2400 PCR System (Applied Biosystems). For direct sequence analysis, PCR products were purified on a Qiagen QiaQuick column and sequenced at the UCSD’s

Moores Cancer Center shared sequencing resource with internal primers X9F and X9RC for exon 9 and X20F and X20R for exon 20.

X9FF: X9RR: X20F2: X20R2: X9F: X9RC: X20F: X20R:

5’-CCAGAGGGGAAAAATATGACA-3’ 5’-TGCTGAGATCAGCCAAATTC-3’; 5’-GACCTGAAGGTATTAACATCATTTGC-3 5’-ATTCCTATGCAATCGGTCTTTG-3’ 5’-GATTGGTTCTTTCCTGTCTCTG-3’ 5’-CCACAAATATCAATTTACAACCATTG-3’ 5’-TTGCATACATTCGAAAGACC-3’ 5’-GGGGATTTTTGTTTTGTTTTG-3’

dHPLC analysis Denaturing high-performance liquid chromatography (dHPLC) was performed using the WAVE® DNA Fragment Analysis System (Model 3500 HT; Transgenomic), controlled by Navigator software. To enhance heteroduplex formation, PCR products were denatured at 95°C for 5 minutes, followed by gradual reannealing to 25°C at -0.1°C/ sec. Initially, for each PCR product, the melting behavior of the wild-type sequence was visualized using the WAVEMAKER™ software (Transgenomic) algorithm. The elution for each PCR product was performed at a temperature corresponding to 80-90% α-helical fraction for each melting domain.

After a few test trials with the positive control samples for each exon, the optimal temperature for mutation detection for 258-bp exon 9 was determined to be 59°C, and 58°C for 420-bp exon 20. Sample volumes of 10 μl (optimal quantity of the loaded PCR product corresponded to a peak of A260 ~4-12 mV) were loaded on a DNASep® (Transgenomic) cartridge and eluted within 4.5 min at a flow rate of 0.9 ml/min using a linear acetonitrile gradient 48.3 – 62.3% buffer B (0.1 M triethylammonium acetate [TEAA]; 25% acetonitrile) for exon 9 PCR products and 50.8 – 68.4% buffer B for exon 20 PCR products. Eluted DNA fragments were detected by the system’s ultraviolet detector at 260 nm. Regeneration of the column was achieved by washing with 100% buffer D (75% acetonitrile) for 30 sec followed by an equilibration time of 2 min.

Results To establish dHPLC as a viable approach to mutation screening of PIK3CA mutations, we utilized DNA from the MCF7 breast cancer cell line, which harbors a PIK3CA mutation in exon 9, and the LS174 colon cancer cell line, which has a PIK3CA mutation in exon 20. As shown in Figure 1A, a distinct two peak elution profile is observed in the DNA from the MCF7 cell line, whereas a single peak elution profile is observed from

UCSD Biological Sciences Saltman | Quarterly 51

the DNA of a non-tumor control sample.

Similarly, when DNA from the LS174 cell line was amplified for PIK3CA exon 20 and resolved by dHPLC, a semi-broad, shoulder containing elution profile is observed in comparison to a single, sharp peak elution profile from the DNA of a non-tumor control sample (Figure 1C). Sequence analysis of both of these samples confirmed the reported exon 9 (bp1633 G‡A, E545K) and exon 20 (bp3140 A‡G, H1047R) mutations (Figures 1 B and D, respectively).

To establish the sensitivity of our dHPLC assay, we applied a pooling strategy where DNA from the MCF7 cell line was serial diluted with DNA from a wild-type sample. As the MCF7 cell line contains a heterozygous polymorphism, our control (MCF7 DNA only) value represents a 50% dilution of mutation/wild-type sequence.

As seen in Figure 2, our dHPLC procedure could successfully distinguish the presence of a mutation in a sample in a 1:20 dilution (5%) of MCF7 DNA, representing the ability to detect a homozygous mutation present in a sample containing as few as 2.5% tumor cells.

Having established a highly sensitive and reproducible technique for the detection of PIK3CA exons 9 and 20 gene alterations, we screened DNA from 23 neuroblastoma cell lines and 101 primary neuroblastoma samples. None of the neuroblastoma cell lines harbored mutations in either of these exons. Next, we analyzed PIK3CA status in the 101 primary neuroblastoma

samples available from both favorable and unfavorable outcomes. No mutations were detected in any of the 46 favorable outcome neuroblastoma samples for either exons 9 or 20. Of the 55 unfavorable outcome tumor samples, only one stage III sample exhibited a dHPLC profile that varied from the profile of the wild-type DNA for exon 20 and was analogous to the profile observed for the LS174 cell line (Figure 3A). Sequence analysis of this sample revealed a heterozygous T‡C mutation at bp3236 (Figure 3B), resulting in a Leu to Pro substitution at codon 1079. According to our research, this mutation has not previously been reported. No aberrant dHPLC profiles for exon 9 were observed in any of these unfavorable outcome tumor samples.

Discussion The genotype of PIK3CA has become an important consideration in assessing the aggressiveness of some tumor types and a potential target of therapeutic intervention, thus necessitating the need for a sensitive, rapid, and high throughput approach to the screening of this gene. Numerous DNA screening techniques have been developed for the detection of gene mutations. We ourselves have used single stranded conformation polymorphism (SSCP) to screen for mutations.16 SSCP is simple and inexpensive to use, but depending on the location and substitution genotype of the mutation, can fail to detect some mutations. Direct sequencing is by far the most unequivocal and unambiguous

Figure 1. dHPLC analysis of PIK3CA exon 9 (A) from MCF7 DNA and exon 20 (C) from LS174 DNA show distinct elution profiles versus DNA from normal cells, suggesting the presence of mutations, which were confirmed by direct sequence analysis (B and D, MCF7 exon 9 and LS174 exon 20, respectively).

52 Saltman | Quarterly UCSD Biological Sciences

method of mutation detection, however, direct sequencing requires the use of a high quality polymerase during the amplification stage, the need to purify each amplicon, and the requirement for at least two sequencing experiments per amplicon (forward and reverse), resulting in a procedure in which accuracy comes at the cost of time and money.

This procedure becomes especially cost prohibitive when mutations are infrequent. In comparison, dHPLC can screen for multiple DNA variants in a high throughput mode and with a superior detection rate to SSCP, approaching accuracy rates equivalent to that seen by direct sequencing at a fraction of the cost and time, while requiring minimal postPCR handling before a sample is ready for analysis. These properties of dHPLC offers considerable advantages for mutation detection compared to other methods.

Until recently, deregulation of the PIK3/Akt pathway has been largely the consequence of inactivation of the PTEN protein, whose negative regulatory activity is lost through deletion or mutation in many cancer types resulting in activation of PIK3CA and phosphorylation of Akt. Akt activation

by PIK3CA leads to a cascade of responses, from cell growth and proliferation to survival and motility, that drive tumor progression.3 The presence of an activated Akt in some neuroblastoma suggests deregulation of this pathway at some level.10,17 It had been widely hypothesized that, with the identification of cytogenetic alterations at the PTEN locus in neuroblastoma, that PTEN involvement in this disease would be inevitable.5 However, mutation of PTEN is infrequent in neuroblastoma, with no PTEN deletions detected in 12 neuroblastoma cell lines investigated, and just 2 deletions detected out of 41 primary neuroblastoma samples investigated.7 We have used dHPLC to screen PIK3CA in neuroblastoma and found only one mutation, an unremarkable stage III primary sample, out of 105 neuroblastoma samples and 23 cell lines investigated. Thus, as with PTEN, alterations of PIK3CA are uncommon in neuroblastoma and not a primary mechanism of Akt pathway deregulation in this disease.

Despite the absence of significant rates of PTEN or PIK3CA mutations in neuroblastoma, the deregulatory role of these genes in Akt activation in some neuroblastoma remains a possibility. Gene amplification of PIK3CA has been reported in several cancers including ovarian, cervical, squamous cell, and glioblastomas.18 In an analysis of more than 100 thyroid cancer samples, only

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tion is believed to result in the amplification of genes associated with the retinoid pathway and there is no evidence suggesting that the chromosomal duplication seen in this patient would extend as far as the PIK3CA locus at 3q26.22,23 In addition, duplication at 3q is a recognized medical Figure 2. Using MCF7 DNA and PIK3CA exon 9 as an example, our established syndrome not necdHPLC assay could detect a heterozygous mutation in a DNA sample diluted 20fold with normal DNA, representing the ability to detect a heterozygous mutation essarily associated present in a population containing as few as 5% tumor cells, or a homozygous with neuroblasmutation in a sample population containing as few as 2.5% tumor cells. toma.24 Thus, the amplification of one PIK3CA mutation was found.19 Howev3q26 and PIK3CA in neuroblastoma is uner, in this same sample population, PIK3CA common and rarely likely to be a significant gene amplification was found to be as high as 24% in some thyroid cancer subtypes, and contributor to neuroblastoma pathogenesis.

In summary, we sought to underwas found to be amplified in 5 of 7 thyroid stand the mechanism by which Akt is activatcancer cell lines investigated. The PIK3CA ed in some neuroblastomas and to contribute gene is located on chromosome 3q26.3. to the existing knowledge of the PIK3CA Comparative genomic hybridization (CGH) gene mutation in the most frequent solid studies of neuroblastoma do not identify this tumor of childhood by investigating somatic locus as a region of common alteration in 5 mutation of PIK3CA in a series of neuroneuroblastoma; however, CGH and other blastoma cell lines and tumor tissue samples cytogenetic techniques may not be able to from various prognostic stages. We develdetect small regions of amplification. Sevoped a dHPLC technique that may find utileral studies have identified chromosomal ality as a rapid and highly sensitive technique terations at 3q in neuroblastoma; Maier and 20 for screening tumor samples for PIK3CA Beck report gene duplication at 3q21-27, mutations.

Furthermore, we have found that a region encompassing the PIK3CA locus 20 PIK3CA is rarely mutated in neuroblastoma, at 3q26. Another report of chromosomal amplification at 3q in neuroblastoma was re- supporting the hypothesis that the involveported by Qureshi et al.21, who report a mi- ment of PIK3CA mutation in human cancer croscopic neuroblastoma in a fetus that har- pathogenesis may be different according to bored a translocation and gene duplication at the types of cancers. We conclude that dis3q21.21 However, the chromosomal duplica- ruption of the PIK3CA gene is not required for the major pathway leading to malignant

transformation of human neuroblastomas.

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Figure 3. PIK3CA exon 9 and exon 20 were screened from 23 neuroblastoma cell lines and 101 primary neuroblastoma samples. A single stage III sample exhibited an aberrant dHPLC elution profile (A). The presence of a mutation in this sample wasconfirmed as a heterozygous T◊C base change atbp3236 (L1079P) (B).

2005–06 Volume 3

Brodeur, G.M. Significance of intratumoral genetic heterogeneity in neuroblastomas. Med. Pediatr. Oncol. 38(2): 112-3 (2002).

Castel, V., and Canete A. A comparison of current neuroblastoma chemotherapeutics. Expert Opin. Pharmacother. 5(1): 71-80 (2004).

Vivanco, I., and Sawyers, C.L. The phosphatidylinositol 3Kinase AKT pathway in human cancer. Nat. Rev. Cancer.

2(7): 489-501 (2002).

Sansal, I., and Sellers, W.R. The biology and clinical relevance of the PTEN tumor suppressor pathway. J. Clin.

Oncol. 22(14): 2954-63 (2004).

Altura, R.A., Maris, J.M., Li, H., Boyett, J.M., Brodeur, G.M., and Look, A.T. Novel regions of chromosomal loss in familial neuroblastoma by comparative genomic hybridization. Genes Chromosomes Cancer 19(3): 176-84 (1997).

Moritake, H., Horii, Y., Kuroda, H., and Sugimoto, T. Analysis of PTEN/MMAC1 alteration in neuroblastoma. Cancer Genet. Cytogenet. 125(2): 151-5 (2001).

Munoz, J., Lazcoz, P., Inda, M.M., Nistal, M., Pestana, A., and Encio, I.J., et al. Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines. Int. J. Cancer. 109(5): 673-9 (2004).

Samuels, Y., Wang, Z., Bardelli, A., Silliman, N., Ptak, J., and Szabo, S., et al. High frequency of mutations of the PIK3CA gene in human cancers. Science. 304(5670): 554 (2004).

Kang, S., Bader, A.G., and Vogt, P.K. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc. Natl. Acad. Sci. U S A. 102(3): 802-7 (2005).

Kim, S., Kang, J., Qiao, J., Thomas, R.P., Evers, B.M., and Chung, D.H. Phosphatidylinositol 3-kinase inhibition down-regulates survivin and facilitates TRAIL-mediated apoptosis in neuroblastomas. J. Pediatr. Surg. 39(4): 51621 (2004).

Seo, J.H., Ahn, Y., Lee, S.R., Yeol Y.C., and Chung, H.K. The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway. Mol. Biol. Cell. 16(1): 348-57 (2005).

Lopez-Carballo, G., Moreno, L., Masia, S., Perez, P., and Barettino, D. Activation of the phosphatidylinositol 3-kinase/Akt signaling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J. Biol. Chem. 277(28): 25297-304 (2002).

Lasorella, A., Noseda, M., Beyna, M., Yokota, Y., and Iavarone, A. Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature. 407(6804): 592-8 (2002).

Brodeur, G.M., Pritchard, J., Berthold, F., Carlsen, N.L., Castel, V., and Castelberry R.P., et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J. Clin. Oncol. 11(8): 1466-77 (1993).

Chomczynski, P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques. 15(3): 532-4, 36-7 (1993).

Diccianni, M.B., Yu, J., Hsiao, M., Mukherjee, S., Shao, L.E., and Yu, A.L. Clinical significance of p53 mutations in relapsed T-cell acute lymphoblastic leukemia. Blood. 84(9): 3105-12 (1994).

Beierle, E.A., Nagaram, A., Dai, W., Iyengar, M., and Chen, M.K. VEGF-mediated survivin expression in neuroblastoma cells. J. Surg. Res. 127(1): 21-8 (2005).

Osaki, M., Oshimura, M., and Ito, H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis.

9(6): 667-76 (2004).

19. Wu, G., Mambo, E., Guo, Z., Hu, S., Huang, X., and Gollin, S.M., et al. Uncommon mutation, but common amplifications, of the PIK3CA gene in thyroid tumors. J. Clin.

Endocrinol. Metab. 90(8): 4688-93 (2005).

20. Maier, B., and Beck, J.D. Dup 3(q) syndrome and neuroblastoma. Eur. J. Pediatr. 151(9): 715-6 (1992).

21. Qureshi, F., Jacques, S.M., Johnson, M.P., Reichler, A., and Evans, M.I. Microscopic neuroblastoma in a fetus with a de novo unbalanced translocation 3;10. Am. J. Med. Genet. 53(1): 24-8 (1994).

22. Goodman, A.B. Disruption of genes in the retinoid cascade may explain the microscopic neuroblastoma in a fetus with de novo unbalanced translocation (3;10)(q21;q26). Am. J. Med. Genet. 56(1): 123. (1995) 23. Satge, D., Moore, S.W., Stiller, C.A., Niggli, F.K., Pritchard-Jones, K., and Bown, N., et al. Abnormal constitutional karyotypes in patients with neuroblastoma: a report of four new cases and review of 47 others in the literature. Cancer. Genet. Cytogenet. 147(2): 89-98 (2003).

24. Steinbach, P., Adkins, W.N., Jr., Caspar, H., Dumars, K.W., Gebauer, J., and Gilbert, E.F., et al. The dup(3q) syndrome: report of eight cases and review of the literature Am. J. Med. Genet. 10(2): 159-77 (1981).

UCSD Biological Sciences Saltman | Quarterly 53

Saltman | Quarterly Acknowledgements The staff of Saltman Quarterly would like to acknowledge the following individuals for their support. Each person has contributed to establishing SQ as a valuable educational tool for undergraduate life sciences students at UCSD. Dean Eduardo Macagno - Without your support, SQ would not be here today. You have contributed not only financial resources of the Dean’s Office but also your time and personal effort into building awareness of and support for SQ among UCSD faculty and administrators. Assistant Dean Barbra Blake - Thank you for your crucial role in establishing SQ and your direction and guidance throughout the year. Staff Advisor Patricia Walsh - Although SQ is a student-run publication, Pat is the one who makes sure the issue comes together and often keeps our heads on our shoulders.

Dean Eduardo Macagno

Faculty Advisory Committee - Drs. Lisa Boulanger, Milton Saier, Laurie Smith, and Christopher Wills. Thank you not only for reviewing the student research manuscripts submitted to SQ but also for sitting down with each of the authors to discuss your comments and suggestions, and for developing and conducting the first annual manuscript review workshop for student reviewers. Thank you for sharing your time, knowledge, experience and inspiration with SQ staff and authors.

Dr. Maarten Chrispeels, director of the San Diego Center for Molecular Biology - for your guest faculty review of a research manuscript and for stepping up and helping out at the last minute.

Drs. Milton Saier and Nigel Crawford for agreeing to be interviewed for Saltman Quarterly and for sharing their insights on science education and the process of discovery.

Mrs. Paul Saltman for your dedication to undergraduate science education at UCSD and for your help in building communication channels between faculty and undergraduates.

The faculty of the UCSD Division of Biological Sciences for your support of SQ and for inspiring us in the classroom and the laboratory with your love of science.

Our predecessors: Marika Orlov, Greg Emmanuel, & Louis Nguyen, editors of SQ, Volume 1; Cara Cast and Caroline Lindsay, editors of SQ, Volume 2, as well as all of the staff members and writers for each of these issues of SQ for laying the foundation on which we, your successors, continue to build.

Inaugural Faculty Advisory Committee (from left): Dr. Laurie Smith (Cell & Developmental Biology Section), Dr. Lisa Boulanger (Neurobiology Section), and Dr. Christopher Wills (Ecology, Behavior, Evolution Section). Not pictured: Dr. Milton Saier (Molecular Biology Section) (see page 13).

Saltman Quarterly Student Review Board 2005–06 Katherine Banares Senior, Marshall College, Human Biology Major

Mark Chen Senior, Warren College, Animal Physiology & Neuroscience Major

Prema Hampapur Sophomore, Muir College, Biochemistry & Cell Biology Major

Youngjin Kim Senior, Revelle College, General Biology Major

Kendra Bettis Senior, Warren College, General Biology Major

Kevin Day Senior, Marshall College, Biochemistry & Cell Biology Major

Cindy Huynh Senior, Marshall College, Biochemistry & Cell Biology Major

Eun Kyung (Joanne) Lee Junior, Warren College, Biochemistry & Cell Biology Major

Linda Boettger Senior, Revelle College, General Biology Major

Fernanda Delgado Sophomore, Muir College, Molecular Biology Major

Melanie Kho Senior, Muir College, Biochemistry & Cell Biology Major

Hyuma Leland Junior, Muir College, Molecular Biology Major

Iris Chen Junior, Revelle College, Human Biology Major

James Duguid Junior, Muir College, Microbiology Major

David Kim Senior, Revelle College, Molecular Biology Major

Nick Lind Senior, Warren College, Biochemistry & Cell Biology Major

54 Saltman | Quarterly UCSD Biological Sciences

Volume 3 2005–06

Saltman | Quarterly Staff 2005-06 Kyle Kuchinsky

Nicole Gabrielle Gomez

Editor-in-Chief

Junior, Sixth College, Biochemistry & Cell Biology / Psychology Major Last year I served as the SQ production editor and on the review board. I am interested in the interface between biology and psychology. I’d like to correlate biochemical events to psychological conditions. I plan to attend medical school to become a psychiatrist or neurologist.

Ann Cai

Managing Editor

Senior, Muir College, Molecular Biology / Music Major In prior years, I served as publicity chair for SQ, on the student review board, and on the Poster Session committee for 2003-04. I will be in the joint Harvard Medical School-MIT M.D. program next year, and hope to become a physician-scientist. I have been a two-time recipient of the Chancellor’s Research Scholarship for my work in the Partho Ghosh Lab on adenine-directed mutagenesis in bordetella bacteriophage; I am completing my senior honors thesis this year. Outside of my scientific endeavors, I love dancing, traveling and enjoying music.

Eric Chan

Features Editor

Senior, Marshall College, Political Science / History Major (former Biology Minor) I’m headed off to Washington D.C. to attend the George Washington University’s Elliott School of International Affairs for my M.A. in international relations.

I’m interested in pursuing work as a political analyst and in biological weapons (non)proliferation. I’m also interested in sushi, California girls and the sun (all of which will be sadly lacking on the East Coast). I served as features editor in 2004-05 and on the student review board for Volumes 1 & 2.

Lauren Ashley Miller Junior, Revelle College, Molecular Biology Major

Ji Woong (John) Suk Senior, Revelle College, Biochemistry & Cell Biology Major

Sara Paul Senior, Warren College, General Biology Major

Koh Tanimoto Senior, Muir College, Animal Physiology & Neuroscience Major

Ronnie Pezeshk Senior, Warren College, Human Biology Major

Kevin Tran Sophomore, Marshall College, Molecular Biology Major

Erica Sanford Senior, Warren College, Human Biology Major

2005–06 Volume 3

Research Editor/Review Board Manager

Senior, Warren College, General Biology Major I am participating in the UCSD Medical School post-baccalaureate program before applying to medical school. I volunteer at the UCSD student-run free clinics and shadow at the Hillcrest Medical Center.

What can I say ... I love science and want to “fix” people! I was also an author in last year’s Saltman Quarterly, served on the review board and as a co-chair of the 2004-05 SQ Poster Session.

Reeti Desai

Technical Editor

Senior, Revelle College, Molecular Biology Major I became involved with SQ during its firstyear as a member of the review board and as a co-chair of the SQ poster session in 2004-05. I am an avid reader and love traveling. I spent the first quarter of this academic year in Europe and made some great memories. I plan to stay in San Diego after I graduate. I am interested in a career in research and will soon apply to graduate school to pursue an advanced degree in biology.

Maximus Mu-Liang Chen Production Editor

Junior, Marshall College, Molecular Biology Major I’m involved in the LGBT community at UCSD in addition to the biology community with BSSA. I want to be a faculty member of a medical school someday and to teach, conduct research and practice medicine at the same time. I’m going for an M.D. program after a year abroad (summer in Boston, fall in England, spring in China). I enjoy dance and martial arts. Last year I served on the SQ student review board.

Shruti Jayakumar Publicity Chair

Sophomore, Roosevelt College, Human Biology Major I plan on going to medical school and becoming an ophthalmologist. Last year I was on the SQ student review board.

Grace Wang Webmaster

Junior, Muir College, Microbiology Major I plan to study immunology in graduate school then go into intellectual property (IP) law. I was involved with SQ last year as a student review board member.

UCSD Biological Sciences Saltman | Quarterly 55

Get Involved with S|Q !

The Saltman Quarterly Undergraduate Research Journal (SQ) is a forum for undergraduates at UCSD to present their life science research. A peer- and faculty-reviewed publication run by students with guidance from a faculty committee and Division staff, SQ provides young researchers with an opportunity to publish and review research articles. The journal is named after the late Dr. Paul D. Saltman to recognize and honor his commitment to undergraduates in the classroom and in the laboratory. His dedication continues to impact students today.

If you are interested in becoming involved with SQ there are many opportunities available.

In addition to staff positions, students can become review board members. They serve the critical role of peer review. In addition, they learn about writing and publishing research, critical skills for anyone whatever career path a student chooses to follow. Two other benefits of serving on the SQ review board are the opportunity to participate in a faculty-led scientific manuscript review workshop each fall and the opportunity to step into an SQ staff position in following years. All SQ staff positions are open to any UCSD undergraduate pursuing a major or minor in the life sciences.

If you have already conducted research, are currently working on a research project, or are planning to do research in the life sciences, you can submit your review article or completed research manuscript to SQ. Submissions are accepted fourth week of each quarter. See the website for specific dates and submission guidelines. SQ also publishes

editorials, interviews, and other feature articles about anything of scientific interest to undergraduates in the life sciences. It offers undergraduates considering a career in science writing experience and writing credits. For more information, visit the SQ website at: www.sq.ucsd.edu, or contact the current editor-in-chief at: sq@biomail.ucsd.edu.

Saltman | Quarterly http://sq.ucsd.edu / sq@biomail.ucsd.edu Saltman Quarterly Division of Biological Sciences University of California, San Diego 9500 Gilman Drive La Jolla, CA 92093 - 0376 (858) 534-3112