Researchers at Washington University School of Medicine in St. Louis have shed new light on a process that fixes breaks in the genetic material of the body’s cells. Their findings could lead to ways of enhancing chemotherapy drugs that destroy cancer cells by damaging their DNA.

An illustration of two proteins involved in DNA repair by artist Amy VanDonsel

Using yeast cells, the scientists studied protein molecules that have an important role in homologous recombination, which is one way that cells repair breaks in the DNA double helix. The process in yeast is similar to that in humans and other organisms.

Earlier research had established that a protein molecule named Srs2 regulates homologous recombination by counteracting the work of another protein, Rad51. Reporting in the July 10 issue of the journal Molecular Cell, the research team reveals the mechanism of how Srs2 removes Rad51 from DNA and thereby prevents it from making repairs to broken strands.

“Our findings may make it possible to uncover ways to augment the effect of DNA-damaging agents that are used for cancer chemotherapy,” says senior author Tom Ellenberger, D.V.M, Ph.D., the Raymond H. Wittcoff Professor and head of the Department of Biochemistry and Molecular Biophysics. “Many chemotherapeutic agents work by causing DNA damage in cancer cells, leading to their death, and tumors can become resistant to chemotherapy by using DNA repair mechanisms to keep the cells alive. Drugs that inhibit the DNA repair process could help increase the efficiency of chemotherapeutic agents.”

Ellenberger is also co-director of the Pharmacology Core at Siteman Cancer Center at Barnes-Jewish Hospital and Washington University. The facility aids in the development of anti-cancer agents.

Srs2 is a helicase molecule — a motor protein that’s able to walk or slide along a strand of DNA and remove other proteins from DNA or separate the two strands of the twisted double helix. For studies of Srs2, Ellenberger’s laboratory collaborated with Timothy Lohman, Ph.D., the Marvin A. Brennecke Professor of Biochemistry and Molecular Biophysics, a prominent expert in the biochemistry of motor proteins like Srs2.

Rad51’s job in the cell is to promote the exchange of sequences between two related DNA molecules, which can be used to repair breaks in DNA where both strands of the double helix are compromised. As a DNA matchmaker, Rad51 forms long filaments on DNA. Srs2 can remove these to prevent unwanted exchanges of DNA sequences. Without Srs2, cells lose their ability to maintain the normal structure of chromosomes, and DNA sequences become shuffled.

The biochemists found that Srs2 possesses a small arm that interacts with Rad51 and triggers a chemical reaction within the Rad51 protein causing it to fall off the DNA.

“Scientists had assumed that as Srs2 moved along the DNA strand, it just pushed off everything in its path,” says lead author Edwin Antony, Ph.D., a postdoctoral research associate in biochemistry and molecular biophysics. “This isn’t the case — we showed that Srs2 has a specialized structure that allows it to interact specifically with Rad51.”

This finding shows how a motor protein like Srs2 can perform the specialized task of remodeling a protein-DNA complex without interference by other similar helicases, he adds.

Because they now know more precisely the nature of this interaction between Srs2 and Rad51, the researchers can narrow their search for drugs that will block DNA repair by Rad51. This type of drug could make a lower dose of a DNA-damaging drug effective in treating cancer.

The research team is now trying to identify the Srs2 homologue in human cells and will study its structure in combination with Rad51. That will allow a more rational approach to understanding how cells cope with DNA damage and how some tumors evade cancer therapeutics, they say.

“In the long-term, my laboratory will look for drug-like molecules that influence this interaction,” Ellenberger says. “We are using the Chemical Genetics Screening Center here at the University (http://htc.wustl.edu). It has vast libraries of molecules that may have the activity we want. Edwin’s work on Srs2 and Rad51 will allow us to develop an assay to screen for agents that augment or supersede Srs2’s interference with DNA repair.”

Antony E, Tomko EJ, Xiao Q, Krejci L, Lohman TM, Ellenberger T. Srs2 disassembles Rad51 filaments by a protein-protein interaction triggering ATP turnover and dissociation of Rad51 from DNA. Molecular Cell. 2009;35(1):105-115.

Funding from the National Institutes of Health and the Young Scientist Program at Washington University supported this research.

Washington University School of Medicine’s 2,100 employed and volunteer faculty physicians also are the medical staff of Barnes-Jewish and St. Louis Children’s hospitals. The School of Medicine is one of the leading medical research, teaching and patient care institutions in the nation, currently ranked third in the nation by U.S. News & World Report. Through its affiliations with Barnes-Jewish and St. Louis Children’s hospitals, the School of Medicine is linked to BJC HealthCare.

Siteman Cancer Center is the only federally designated Comprehensive Cancer Center within a 240-mile radius of St. Louis. Siteman Cancer Center is composed of the combined cancer research and treatment programs of Barnes-Jewish Hospital and Washington University School of Medicine. Siteman has satellite locations in West County and St. Peters, in addition to its full-service facility at Washington University Medical Center on South Kingshighway.

By Gwen Ericson – Washington University in St. Louis, School of Medicine

WEST LAFAYETTE, Ind. – A new prostate cancer “homing device” could improve detection and allow for the first targeted treatment of the disease.

A new prostate cancer “homing device” could improve detection and allow for the first targeted treatment of the disease.

This image depicts transporter molecules carrying therapeutic drugs to PSMA targets on a prostate cancer cell. A Purdue research team designed a molecule that finds and penetrates prostate cancer cells and can transport drugs or imaging agents into the cell. (Image courtesy of Low laboratory)

A team of Purdue University researchers has synthesized a molecule that finds and penetrates prostate cancer cells and has created imaging agents and therapeutic drugs that can link to the molecule and be carried with it as cargo.

A radioimaging application used for body scans is expected to enter clinical trials this fall, and an optical imaging application used to measure prostate cancer cells in blood samples is already in clinical trials.

Philip Low, the Ralph C. Corley Distinguished Professor of Biochemistry who led the team, said a targeted treatment could be much more effective in treating cancer and would greatly reduce the harmful side effects associated with current treatments.

“Currently none of the drugs available to treat prostate cancer are targeted, which means they go everywhere in the body as opposed to only the tumor, and so are quite toxic for the patient,” said Low, who is a member of the Purdue Cancer Center. “By being able to target only the cancer cells, we could eliminate toxic side effects of treatments. In addition, the ability to target only the cancer cells can greatly improve imaging of the cancer to diagnose the disease, determine if it has spread or is responding to treatment.”

Prostate cancer is the most common cancer, other than skin cancers, and is the second leading cause of cancer death in American men, according to the American Cancer Society. It is estimated that about 192,280 new cases will be diagnosed and 27,360 men will die of prostate cancer in the United States this year.

The molecule Low’s team created attaches to prostate-specific membrane antigen, or PSMA, a protein that is found on the membrane of more than 90 percent of all prostate cancers. It also is found on the blood vessels of most solid tumors and could provide a way to cut off the tumor blood supply, Low said.

“A lot of new drugs are being designed to destroy the vasculature of solid tumors, and, if they could be linked to this new targeting molecule, we could have a two-pronged attack for prostate cancer,” he said. “We could not only kill the prostate cancer cells directly, we could also destroy the vasculature that feeds the tumors.”

There also is potential for the targeting molecule to be used to attack the vasculature of solid tumors of other types of cancers, Low said.

Two papers detailing the work of the Purdue team were published in the June 1 issue of Molecular Pharmaceutics. Endocyte Inc. funded the work.

The team’s animal study data shows an ability to eliminate human prostate cancer cells in mice with no evidence of collateral toxicity in normal tissue.

Sumith Kularatne, a graduate student in Purdue’s chemistry department and first author of both papers, compared the targeting molecule to a homing device.

“The molecule acts like a homing device for prostate cancer,” he said. “PSMA, which is found only on prostate cancer cells and tumor blood vessels, acts as the homing signal that the molecule targets. The molecule and its cargo go only to cancerous tissue, leaving healthy tissue unharmed.”

Philip Low, the Ralph C. Corley Distinguished Professor of Biochemistry at Purdue, and graduate student Sumith Kularatne, in foreground, examine the uptake of an imaging agent in prostate cancer cells. Low led a research team that designed a molecule to find and penetrate prostate cancer cells and has created imaging agents and therapeutic drugs that can link to the molecule and be carried with it as cargo. (Purdue University photo/Andrew Hancock)

Once the molecule reaches the PSMA protein, it binds to it. The molecule is designed with a specific shape that fits with the protein like a key to a lock, Kularatne said. The molecule and its cargo are then carried inside the cell with the protein as it goes through its normal cycle.

In 1995 Low developed a similar method to infiltrate cancer cells by attaching treatments to the vitamin folate, which many cancers rapidly consume. This method provided a “Trojan Horse” entry of large treatment molecules that otherwise would not be able to enter cancer cells.

Low was inspired to find a similar way to target prostate cancer, which does not have the same appetite for folate, he said.

A clinical trial of the radioimaging application is expected to begin at the Indiana University Medical Center in the fall through a collaboration between the Purdue Cancer Center and the Indiana University Cancer Center with additional support from Endocyte Inc.

A radioimaging agent linked to the targeting molecule will be injected into prostate cancer patients and pictures will be taken using a special camera that detects radioactivity. The pictures show where the cancer is present to help doctors determine if it has metastasized, or spread, to any other areas of the body. It also will help doctors decide on the best course of treatment, Low said.

There is currently only one radioimaging agent for prostate cancer approved by the Food and Drug Administration.

“The current imaging capabilities available for prostate cancer are very poor,” Low said. “The existing imaging agent is limited because of its large size, which is difficult to get into a solid tumor. Also it seeks out a target located inside the cancer cell and is only able to mark injured cells that are falling apart as opposed to actively growing cancer cells.”

The targeting molecule and radioimaging agent combination designed by Low’s group is more than 150 times smaller than the existing agent and has much easier penetration through a solid tumor to reach all of the cells inside, he said. It also has the advantage of targeting an area of PSMA exposed on the outside of cancer cells.

Already in clinical trials is an optical imaging application that involves attaching a fluorescent dye to the targeting molecule and mixing it with a patient’s blood sample. Circulating prostate cancer cells in the sample fluoresce and are easily measured to help in diagnosing patients with prostate cancer. Researchers also are investigating whether this could be used to evaluate a patient’s response to therapy, Low said.

Low’s research group modeled the targeting molecule after a naturally occurring molecule that strongly binds to PSMA, called DUPA. Several alterations were necessary to create a molecule that fit the needs of a homing device and delivery vehicle, Kularatne said. The team created an area on the molecule that would link to various imaging or therapeutic agents to bring them along as cargo and created a spacer that would stretch the molecule so that its cargo would not keep it from properly fitting into the binding site. The spacer also was designed to improve binding of the targeting molecule to PSMA.

In addition to Low and Kularatne, co-authors of the papers include Endocyte researchers Kevin Wang and Hari-Krishna R. Santhapuram, graduate student in medicinal chemistry Zhigang Zhou, graduate student in chemistry Jun Yang, and professor of medicinal chemistry and molecular pharmacology Carol B. Post.

Low is the chief science officer for Endocyte, a Purdue Research Park-based company that develops receptor-targeted therapeutics for the treatment of cancer and autoimmune diseases. Endocyte holds the license to many of Low’s drug-targeting technologies.

Papers:

Prostate-Specific Membrane Antigen Targeted Imaging and Therapy of Prostate Cancer Using a PSMA Inhibitor as a Homing Ligand
Sumith A. Kularatne, Kevin Wang, Hari-Krishna R. Santhapuram, and Philip S. Low

Design, Synthesis, and Preclinical Evaluation of Prostate-Specific Membrane Antigen Targeted ^99mTc-Radioimaging Agents
Sumith A. Kularatne, Zhigang Zhou, Jun Yang, Carol B. Post, and Philip S. Low

By Elizabeth K. Gardner – Purdue University

COLLEGE STATION – People smell them, thump them and eyeball their shape. But ultimately, it’s sweetness and a sense of healthy eating that lands a melon in a shopper’s cart.

Color melon flesh is full of nutrients. Plant breeders may develop even better varieties now that the melon genome with hundreds of DNA markers has been mapped . (Texas AgriLife Photo by Kathleen Phillips)

Plant breeders now have a better chance to pinpoint such traits for new varieties, because the melon genome with hundreds of DNA markers has been mapped by scientists with Texas AgriLife Research. That means tastier and healthier melons are likely for future summer picnics.

“This will help us anchor down some of the desirable genes to develop better melon varieties,” said Dr. Kevin Crosby, who completed the study with Drs. Soon O. Park and Hye Hwang. “We can identify specific genes for higher sugar content, disease resistance and even drought tolerance.”

The results are reported in the Journal of the American Society of Horticultural Sciences.

Melons are fleshy, edible cucurbits grown worldwide in a multitude of varieties. Not only are they economically important, the scientists noted, but they are a favorite among consumers internationally.

The average person in the U.S. eats about 25 pounds of melon every year, according to the Agricultural Marketing Resource Center at Iowa State University.

Scientists from France and Spain already had completed partial maps of segments of the melon DNA sequence. The Texas researchers connected those segments with new findings in their study to complete the entire melon genome map.

For the study, the Deltex ananas melon was crossed with a wild melon called TGR 1551. More than 100 of the offspring from that cross were grown in the AgriLife Research greenhouses at Weslaco, Crosby noted.

DNA was extracted from leaf tissue collected 21 days after planting. Results from these tests were integrated into partial maps created by other researchers.

Previous knowledge of melon DNA was like two sets of directions – one from Miami to Houston and the other from El Paso to Los Angeles. That would make one wonder how to get from Houston to El Paso. The study by Crosby’s group, in essence, devised the path from Miami to LA and all points between.

In addition to the complete map, the researchers located genetic markers linked to fruit sugars, ascorbic acid (vitamin C) and male sterility, which is useful for developing hybrid varieties.

The trio said the genetic map will be helpful for future studies in identifying fruit sweetness, quality, size, shape and resistance to disease.

By Kathleen Phillips – Texas A&M AgriLife

Baltimore, MD—Scientists working at the Carnegie Institution’s Department of Embryology, with colleagues, have overturned previous research that identified critical genes for making muscle stem cells. It turns out that the genes that make muscle stem cells in the embryo are surprisingly not needed in adult muscle stem cells to regenerate muscles after injury. The finding challenges the current course of research into muscular dystrophy, muscle injury, and regenerative medicine, which uses stem cells for healing tissues, and it favours using age-matched stem cells for therapy.  The study is published in the June 25 advance on-line edition of Nature.

This cross section of hind limb muscle tissue is from a mouse five days after injury. The uninjured cells are at top and stained red. The blue cells below are regenerating muscles cells. They were labeled with a blue stain and formed from muscle stem cells. Image courtesy Christoph Lepper

Previous studies have shown that two genes Pax3 and Pax7, are essential for making the embryonic and neonatal muscle stem cells in the mouse. Lead researcher Christoph Lepper, a predoctoral fellow in Carnegie’s Chen-Ming Fan’s lab and a Johns Hopkins student, for the first time looked at these two genes in promoting stem cells at varying stages of muscle growth in live mice after birth.

As Christoph explained: “The paired-box genes, Pax3 and Pax7 are involved in the development of the skeletal muscles. It is well established that both genes are needed to produce muscle stem cells in the embryo. A previous student, Alice Chen, studied how these genes are turned on in embryonic muscle stem cells (also published in Nature). I thought that if they are so important in the embryo, they must be important for adult muscle stem cells.  Using genetic tricks, I was able to suppress both genes in the adult muscle stem cells. I was totally surprised to find that the muscle stem cells are normal without them.”

The researchers then looked at whether the same was true upon injury, after which the repair process requires muscle stem cells to make new muscles. For this, they injured the leg muscles between the knee and ankle. They were again surprised that these muscle stem cells, without the two key embryonic muscle stem cell genes, could generate muscles as well as normal muscle stem cells. They even performed a second round of injury and found that the stem cells were still active.

The scientists then wondered when these genes become unnecessary for muscle stem cells to regenerate muscles. It turned out that these embryonic genes are important to muscle stem cell creation up to the first three weeks after birth. What makes the muscle stem cells different after three weeks? The scientist believe that these two embryonic muscle stem cell genes also tell the stem cells to become quiet as the organism matures. After that time is reached, they “hand over” their jobs to a different set of genes. The researchers suggest that since the adult muscle stem cells are only activated when injury occurs (by trauma or exercise), they use a new set of genes from those used during embryonic development, which proceeds without injury. The scientists are eager to find these adult muscle stem cell genes.

“We are just beginning to learn the basics of stem cell biology, and there are many surprises,” remarked Allan Spradling, director of Carnegie’s Department of Embryology. “This work illustrates the importance of carrying out basic research using animal models before rushing into the clinic with half-baked therapies.”

Listen to the scientist in his own words at this link: http://www.ciw.edu/publications_online/embedded_video/stem_cell_surprise.html

Carnegie Institution For Science

Vaseline, a known molecule from apples and a gene network encapsulated in algal gelatin are the components of a possible gene therapy which literally gets under the skin. This is what a research group in the Department of Biosystems (D-BSSE) in Basle managed to achieve.

New way to gene therapy: first implant a capsule with a particular gene under the skin, apply skin cream in order to stimulate the gene into action, which expresses an active principle which is able to escape from the capsule. (Image: P. Rüegg/ ETH Zürich)

“An apple a day keeps the doctor away”. This English proverb now has a new meaning. Marc Gitzinger from the research group of Martin Fussenegger, Professor of Biotechnology and Bioengineering Science in the Department of Biosystems (D-BSSE) in Basle, has developed a prototype for gene therapy through the skin. An important part in this is played by phloretin, an antioxidant found in apples which makes cell walls more permeable and is used in cosmetics as an anti-wrinkle agent. The researchers have presented their new therapeutic approach online in the current edition of PNAS.

Capsules and cream

The method of administration sounds very simple: first implant a capsule with a particular gene under the skin and then apply skin cream in order to stimulate the gene into action, which finally expresses an active principle which is able to escape from the capsule in a precise dose.

Fussenegger’s group has managed to do something which sounds like science fiction. The researchers have produced alginate capsules with living cells containing a specially designed genetic network. This network produces the protein SEAP. The capsules were implanted under the skin of test mice which were then coated with an ointment. This skin cream consists of commercial milk fat mixed with phloretin according to a particular formula.

And it worked. Phloretin penetrated the skin, the gel capsules and the cells contained within. As hoped for by the researchers, the antioxidant from the apples reduced the production of protein. With a large dose of phloretin in the cream, the production of SEAP could be stopped altogether.

“When developing the principle we had no particular clinical picture in mind”, emphasised the ETH professor. “We were concentrating on the route of administration through the skin”. A genetic network such as this can also be designed in such a way that when activated correctly, insulin or growth factors are produced. The researcher can imagine that certain metabolic diseases might be treatable by this method. The D-BSSE scientists have already had the method patented and hope that the pharmaceutical industry will be interested in further developing this principle.

Liver spared

This form of gene therapy has several advantages, stressed the ETH professor. It puts no strain on the liver because it has a very local action and phloretin is a molecule which can be found in everyday foodstuffs and undergoes rapid degradation in the body. Furthermore, the network can be precisely controlled and the therapy is well tolerated by the liver, adds Fussenegger. The disadvantage of orally administered therapeutic agents is that the liver, as the detoxifying organ, destroys most of the active agent before it reaches the target site.

Fussenegger is also convinced that implants are well accepted by the public. Implants can be stored in the body for a relatively long time and are easily removed after the end of therapy or in the event of complications.

This new genetic network is a typical example of progress in synthetic biology. Researchers use known and well-characterised biological components to construct artificial networks which in turn are able to produce gene products such as specific proteins. Researchers can also use certain components to make biological switches which in turn allow such systems to be switched on or off.

Reference:

Gitzinger M, Kemmer C, El-Baba MD, Weber W, Fussenegger M. Controlling Transgene Expression in Subcutaneous Implants Using a Skin Lotion Containing the Apple Metabolite Phloretin. PNAS, online publication 22 June 200. doi:10.1073/pnas.0901501106

By Peter Rüegg – ETH Zürich

EMBL researchers provide the as yet closest look at the structure of immature HIV.

Lattice maps for immature HIV particles. The 3D computer reconstruction shows the immature Gag lattice of HIV that matures to form the protein shell of the infecious virus. Maps are shown in perspective such that hexamers on the rear surface of the particle appear smaller. The side of the particle toward the viewer lacks ordered Gag. (John Briggs/EMBL)

Scientists at the European Molecular Biology Laboratory (EMBL) and the University Clinic Heidelberg, Germany, have produced a three-dimensional reconstruction of HIV (Human Immunodeficiency Virus), which shows the structure of the immature form of the virus at unprecedented detail. Immature HIV is a precursor of the infectious virus, which can cause AIDS. The study, published in the 22-26 June online edition of PNAS, describes how the protein coat that packages the virus’ genetic material assembles in human cells. Drugs that block this assembly process and prevent the virus from maturing into its infectious form are considered a promising therapeutic approach. HIV consists of an RNA molecule that carries the genetic information of the virus and is surrounded by protective protein and membrane layers. During infection the virus deposits its genetic material into a human cell where it reprogrammes the host cell machinery to generate many copies of the viral genome and initiates the production of a viral protein called Gag. In the immature virus, many copies of Gag interact to form a roughly spherical lattice that encloses the virus’ genetic material.The virus then leaves the cell with the help of proteins of the host and infects new cells.

A simplified representation of HIV’s lifecycle. For a detailed description please refer to page 2. (John Briggs/EMBL)

Using a method called cryoelectron tomography researchers in the groups of John Briggs at EMBL and Hans-Georg Kräusslich at the University Clinic Heidelberg generated the as yet highest resolution 3D computer reconstruction images of the immature Gag lattice. The results suggest a simple model of HIV formation in human cells: multiple Gag proteins interact to form a hexameric lattice that grows with an inherent curvature and that incorporates new proteins stochastically. Several further steps in which Gag is cleaved by an enzyme are necessary to transform this immature lattice into its mature, infectious form.

Briggs and his team are now working on producing an even higher resolution structure of the protein lattice to gain a more detailed understanding of the virus’ assembly and maturation processes, which may eventually help to find weak points that could be targeted by drugs.

Cryoelectron tomography is a technique with which a sample is instantly frozen in its natural state and then examined with an electron microscope. Images are taken from different directions and assembled into an accurate 3D reconstruction by a computer.

Source Article: Briggs, J.A.G. et al. Structure and assembly of immature HIV. PNAS online, 22 June 2009

European Molecular Biology Laboratory (EMBL)

A discovery by a team of Canadian and American researchers could provide new ways to fight HIV-AIDS. According to a new study published in Nature Medicine, HIV-AIDS could be treated through a combination of targeted chemotherapy and current Highly Active Retroviral (HAART) treatments. This radical new therapy would make it possible to destroy both the viruses circulating in the body as well as those playing hide-and-seek in immune system cells.

(Université de Montréal)

The study was led by Dr. Rafick-Pierre Sékaly, of the Université de Montréal. Dr. Jean-Pierre Routy of the Research Institute of the McGill University Health Centre (RI-MUHC) and scientists from the National Institutes of Health (NIH) and the University of Minnesota in the United States also collaborated on the investigation. To date, anti-AIDS treatments have been stymied by “HIV reservoirs” – immune system cells where the virus hides and where existing HAART treatments cannot reach. The researchers successfully identified the cells where HIV hides and the “stealth” mechanisms that allow the virus to escape existing treatments. This breakthrough opens the way towards innovative therapies that are completely different from current approaches.

“Our results argue in favour of a strategy similar to the one used against leukemia, which is targeted chemotherapy, associated with a targeted immune treatment. This would make it possible to destroy the cells containing a virus, while giving the immune system time to regenerate with healthy cells,” says Dr. Rafick-Pierre Sékaly, a professor at the Université de Montréal, researcher at the Centre Hospitalier de Université de Montréal (CHUM), director of INSERM 743 and scientific director of the Vaccine and Gene Therapy Institute of Florida.

“For the first time, this study proves that the HIV reservoirs are not due to a lack of potency of the antiretroviral drugs, but to the virus hiding inside two different types of long life CD4 memory immune cells,” explains Dr. Jean-Pierre Routy, a hematologist with the MUHC, researcher in infection and immunity at the RI-MUHC and professor of hematology at McGill University. “There are several types of HIV reservoirs, each necessitating a different treatment to eliminate them.”

Nicolas Chomont and Rafick-Pierre Sékaly of the Université de Montréal with McGill University’s Jean-Pierre Routy. (Université de Montréal)

Indeed, once the virus is hidden in these reservoir cells, it becomes dependent on them: if the cell lives, the virus lives, but if the cell dies, so does the virus. As such, destroying these immune cells will allow for the elimination of the resilient or hidden parts of the virus. Existing HAART treatments destroy the viruses circulating in the body, yet cannot reach those hidden in reservoir cells.

“We now have brand-new options to fight HIV,” concludes Nicolas Chomont, a postdoctoral intern at the Université de Montréal’s Department of Microbiology and Immunology and one of the co-authors of this study. “The combination of fundamental and clinical approaches led to amazing results that allow us to elucidate another mystery of this virus of a thousand faces.”

These new therapeutic options will require many more years of research before they are validated and become a reality for patients. However, this study represents an invaluable work plan that will provide a map for many laboratories around the world.

Partners in research
This study was funded by the American Foundation for AIDS Research (amfAR), the National Institutes of Health, the Canadian Institutes of Health Research and the FRSQ-AIDS and Infectious Diseases Network.

About the study
The study, “HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation,” published in Nature Medicine, was coauthored by Rafick-Pierre Sékaly, Elias K. Haddad, Nicolas Chomont, Mohamed El Far, Petronela Ancuta, Lydie Trautmann, Francesco A. Procopio, Bader Yassine-Diab and Geneviève Boucher of the Université de Montréal and Centre Hospitalier de Université de Montréal (CHUM), Jean-Pierre Routy, Mohamed-Rachid Boulassel and Georges Ghattas of the McGill University Health Centre (MUHC) and McGill University, Brenna J. Hill, Daniel C. Douek and Jason M. Brenchley of the National Institutes of Health, U.S.A., and Timothy W. Schacker of the University of Minnesota, U.S.A.

On the web
About the Université de Montréal’s Faculty of Medicine
About the Research Centre of the Centre Hospitalier de Université de Montréal
About the Research Institute of the McGill University Health Centre
About McGill University
About INSERM
About Vaccine and Gene Therapy Institute of Florida

Université de Montréal

Inhibitor molecules mimic interaction; could become drugs to block deadly poison.

UPTON, NY — New structures of a botulism toxin interacting with a mimic of the nerve-cell protein it destroys suggest new ways to block this often-fatal interaction. Indeed, the mimic molecules have such high affinity for the toxin and bind to it so tightly that they themselves could possibly serve as anti-toxin drugs with further modification, the researchers said.

The catalytic domain of Clostridium botulinum neurotoxin type F (represented as a molecular surface, gray) bound to an inhibitor molecule (colored ribbon) designed to mimic the nerve-cell protein the toxin cleaves. The mimic protein interacts with the toxin at several exosites (purple-, brown-, and green-shaded areas) in addition to the active site (red) that performs the cleaving action, suggesting that blocking these interactions could thwart the toxins deadly action. (Brookhaven National Laboratory)

The atomic-resolution structures were made at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory in collaboration with scientists from the U.S. Army Medical Research Institute for Infectious Diseases, and will be published online on June 21, 2009, in Nature Structural & Molecular Biology.

Botulism toxins are among the deadliest known poisons. Minute quantities in improperly canned goods can cause a fatal form of food poisoning. Recently, some forms have been used in medical settings to smooth facial wrinkles and to quell bladder spasms to stem urinary leakage. But fear of their use as a bioterror weapon has made the toxins notorious — and the push for developing antitoxin drugs or vaccines a high priority.

The toxins come in seven distinct varieties, but all work the same way: One portion of the toxin binds to a nerve-cell membrane; another portion moves a smaller “catalytic domain” into the cell; then this catalytic domain binds to and cleaves a nerve-cell protein, making it impossible for the nerve cell to “fire,” or send signals. The result is paralysis — and often, death.

“This study looked specifically at how the catalytic domain of one type of neurotoxin, neurotoxin F, recognizes and binds to its target nerve-cell protein to perform this final, paralyzing step,” said Brookhaven Lab biologist Subramanyam Swaminathan, who led the research team.

The team first synthesized two different mimics of the target nerve-cell protein. They then allowed each to bind to the catalytic domain of the toxin, and analyzed the structures using high-intensity x-rays at Brookhaven’s National Synchrotron Light Source (NSLS). Analyzing how the x-rays bounce off the structure allows scientists to reconstruct extremely high-resolution, 3-D images showing the positions and relative orientations of the atoms making up the proteins.

“Our structures reveal that portions of the toxin that are distant from the ‘active site’ that cleaves the nerve-cell protein are crucial to the toxin’s ability to bind to and destroy this protein,” Swaminathan said. Biochemical analysis confirmed the existence and importance of these “exosites,” further validating the crystal structures.

“Because these exosites play such an essential role in the toxin’s ability to bind to and cleave the nerve-cell protein, they could serve as additional targets for the development of drugs designed to interfere with the toxin’s deadly action,” Swaminathan said.

The scientists are also exploring the possibility that the inhibitor molecules they used in this study as mimics for the nerve-cell protein could themselves serve as anti-toxin drugs.

“These inhibitors are attractive candidates for anti-botulinum drug development,” Swaminathan said. “To do so, we’d need to find a way for the inhibitor to reach the toxin inside nerve cells.” One possibility would be to add a transmembrane sequence or some other means of intracellular transport to the inhibitor molecule.

Work on all seven variations of the toxin is essential to understanding common mechanisms that may aid in the design of drugs that work across several different types, or ideally, broadly against all seven. Swaminathan’s group has studied six of the seven varieties. (See related links, below.)

Brookhaven biologists Subramanyam Swaminathan (left) and Rakhi Agarwal. (Brookhaven National Laboratory)

“The mere existence of a vaccine or anti-toxin drugs would help mitigate the extreme fear of a bioterror attack,” Swaminathan said.

Understanding the detailed structures of the toxins and how they interact with their target proteins could also lead to advances in the ways they can be used safely in a medical setting.

This research was funded by grants from the Defense Threat Reduction Agency/Joint Science and Technology Office for Chemical and Biological Defense, U.S. Department of Defense. Data for this study were measured at beamline X29 and X12C of the NSLS, which is supported by the offices of Biological and Environmental Research and of Basic Energy Sciences within DOE’s Office of Science.

Related Links:

• Scientists Reveal Structure of New Botulism Nerve Toxin Subtype, December 22, 2008

• Scientists Determine Drug Target for the Most Potent Botulinum Neurotoxin, April 29, 2008

• Botulinum Toxin Structure Offers Clues for Vaccines/Treatments, May 10, 2004

• Scientists Decipher Structure of Toxin Responsible for Botulism, July 31, 2000

Brookhaven National Laboratory

MANHATTAN — Owners of exotic animals like reptiles and birds need to be aware of illnesses that can affect both their pet and humans, according to a Kansas State University veterinarian.

Source: Kansas State University

Gary West, assistant professor of zoological medicine in K-State’s College of Veterinary Medicine, said exotic animals can make fun and interesting pets, but there are many health factors to consider before owning one.

West said common exotic pets are ferrets, rabbits, guinea pigs, reptiles and birds. He recommended several of these animals as good pets, including cockatiels, guinea pigs, rabbits, bearded dragons, corn snakes, blue-tongued skinks, some species of tarantulas, freshwater tropical fish and some tortoises.

Owning an exotic pet is different from owning pets like dogs and cats. Some exotic pets have specialized needs, such as lizards that require an ultraviolet light for basking, live insects for food or other whole prey items, West said.

“Many of our diets and health care are very advanced for dogs, which have been domesticated for a long time,” he said. “Many exotic pets are non-domestic, and although many advances have been made, there are still things we are learning about them.”

West said there are fad exotic animals every few years that can be challenging to own, such as a kinkajou. West said this South American animal is related to the raccoon and typically does not make a good pet because of special environmental requirements and that it can bite.

He said all exotic animals have specific needs and requirements, and it is important that the pet owner become educated about the animal before purchasing it. He said the best way to keep an exotic pet healthy is to know what it requires to stay healthy and thrive.

Exotic animals can carry diseases, West said. Reptiles are commonly known to be at risk of carrying salmonella, and there have been reports of other diseases, including chlamydia, in pet birds. Rabies also is a concern for mammals if they go outdoors.

However, unlike dogs and cats, exotic pets like reptiles can carry diseases like salmonella without getting sick — but that puts other animals and humans at risk.

West said after handling an exotic pet, people should wash their hands immediately. He also said reptiles should not be allowed to roam free, they should not be allowed in the kitchen or around people who are eating, and owners should disinfect surfaces where reptiles have been. Additionally, the U.S. Centers for Disease Control and Prevention recommends that reptiles not be kept in homes with children younger than 5 years or with immunocompromised people, he said.

Source: Kansas State University

West said typically there are no signs or symptoms to tell if an exotic animal is a carrier of salmonella or other diseases, though the animal could infect other pets. To keep other pets from getting the diseases, West said the same principles for humans apply. Other animals should not come into direct contact with the exotic pet, for mutual benefit, and the animals should keep away from each other’s food and water bowls.

Some exotic animals do not like much handling, noise or strangers, and agitating the animal puts the owner at risk for bites and scratches. Bite wounds can become infected with bacteria from the pet’s mouth and should be examined by a doctor. Additionally, injuries are fairly common in small pets, so owners should be careful when the animal is around larger animals like dogs or around young children, West said.

“We see many cases where the family cat or dog injures or bites the exotic pet, which are often can be life-threatening wounds,” he said.

He said owners should not impulse-buy pets, especially exotic animals. People also should buy from a good source who is knowledgeable about husbandry and care, and the source should also be able to recommend good products and guarantee that the animal is healthy.

West also said to see if the source is selling species that make good pets. He said there are many animals that should not be pets, such as monkeys. Primates make bad pets for several reasons, he said, including because they can carry diseases that are transmissible to humans.

“Wildlife do not make good pets, and it is illegal and irresponsible to take a baby animal from the wild and raise it or make a pet out of it,” West said. “You are not helping it.”

West said owning a pet can be good for children as a way to learn patience and responsibility, and all pets can be great companions and even stress relievers.

“Watching your fish or learning more about these fascinating creatures can be great hobbies for children and adults,” he said. “Observing and learning about their behavior can be very interesting and help foster an interest and love for animals and wildlife.”

By Kristin Hodges – Kansas State University

Banning or restricting the use of certain types of fishing gear could help the world’s coral reefs and their fish populations survive the onslaughts of climate change.

Credit: ARC Centre of Excellence

An international team of scientists led by Dr Josh Cinner of the ARC Centre of Excellence for Coral Reef Studies at James Cook University has proposed that bans on fishing gear – like spear guns, fish traps, beach seine nets, and gill nets – could aid in the recovery of reefs and fish populations hard hit by coral bleaching events.

Around the world corals have been dying at alarming rates, due to unusually warm water events resulting from global warming.

Research carried out in Kenya and Papua New Guinea has shown that certain types of gear are more damaging to corals, to coral-dependent fish and to the key species of fish that are needed to help reefs recover from bleaching or storm damage.

“This is creating a double jeopardy for both the corals and certain types of reef fish. They are already on the edge because of the overfishing– and the additional impact caused by a bleaching even can push them over” Dr Cinner explains. The result can be an accelerated decline of the reef, its fish populations – and their ability to sustain local people.

“From an ecological perspective, the best response to bleaching is to close reefs to fishing entirely.  But that is not feasible everywhere and is a particularly hard sell among the impoverished fishers in developing countries” says co-author Dr. Tim McClanahan of the Wildlife Conservation Society. “In areas where fishery closures are impractical, managers don’t have many options and haven’t been able to do much but watch the reef die and often not recover.”

“Selective gear restrictions offer reef managers and fishers alike some middle ground, reducing pressure on the reef and its fish while it is in the recovery phase, while also providing fishers with some options for their livelihood” Dr Cinner says.  This middle way is also more likely to be taken up by fishers.  “In other research we’ve found that fishers themselves prefer gear restrictions to total closures, because most fishers use several types of gear so they can still earn a living when the use of one sort of gear is banned. They are more likely to comply.”

The team investigated the effects of five main types of gear on different types of fish: spear guns, traps, hook and line, beach seine nets and gill nets.

They found that spear guns were the most damaging of all – to corals themselves, to susceptible fish species and to the fish needed to help reefs recover, such as parrot, surgeon and trigger fish, which keep seaweeds and urchins in check while the coral re-grows.

“Spear guns target a high proportion of species that help maintain the resilience of coral reefs, but also can result in a surprising amount of damage to the corals themselves.  When a fish is shot with a spear gun, it often hides in the reef, so some fishermen break the corals in their attempts to get it.” Dr Cinner says.

But in developing countries, spear guns can be the fishing tool most used by the poorest fishers because they are cheap to make and the yield can be high, so they are an important source of income for poor fishers.

Credit: ARC Centre of Excellence

“You can’t simply impose an arbitrary ban on their use – you need to consider issues like compensation, other fishing options, or alternative livelihoods for the affected fishers,” says co-author Dr. Shaun Wilson of the Western Australian Department of Environment and Conservation. “One key issue may be educating fishers about the importance of reef habitat and the species that help to maintain reef quality – and the need to be selective in what they shoot. This would mean fishers could still use this cheap and effective fishing tool without necessarily damaging habitat and reef resilience.”

Fish traps also targeted both the most susceptible reef fish and the ones most involved in reef recovery. Beach seine nets didn’t target as many key fish species as gill nets, traps, or spear guns, but were damaging both to corals directly and took large amounts of juvenile fish.

“Where people really depend on reef resources, it may not be possible to permanently ban all of these types of gear.   By creating temporary bans for specific types of gear following a coral bleaching event, reef managers could ease pressure on the reef and its fish population for a time when corals ecosystems are most sensitive without causing undue hardship to the human populations that depend on it.” Dr Cinner says

“Of course, where the conditions are right, managers and fishers don’t have to wait for a bleaching event- preventative gear bans are a good idea, particularly in areas that are highly susceptible to the impacts of bleaching,” says co-author Dr Nick Graham. “And our new research provides managers with some ideas about the trade-offs involved in banning certain gear.”

Dr Cinner says that temporary bans or imposing permanent restrictions on the use of various types of gear can apply to virtually any coral reef management – whether in the developing world or in developed countries such as on Australia’s Great Barrier Reef.

“In principle, it can be used anywhere. It offers both communities and reef managers much greater flexibility.  Around the world, communities are increasingly making their own decisions about how to protect their reefs and they could impose voluntary bans on certain gears.

The article Gear-based fisheries management as a potential adaptive response to climate change and coral mortality, by Cinner J et al. appears in the latest issue of the Journal of Applied Ecology.

ARC Centre of Excellence for Coral Reef Studies
James Cook University