Notebook

Image provided by Genzyme Transgenics Corporation CLONING GOATS In contrast to cloning's popular image as a brave new way to mass-produce monsters, biologists have described cloning as a tool to ease other biotechnologies. Researchers from Louisiana State University in Baton Rouge, Genzyme Transgenic Corp. in Framingham, Mass., and Tufts University School of Medicine in North Grafton, Mass., have shown how cloning can dramatically increase the efficiency of creating goats that secrete valuable biopharmaceuticals in their milk (A. Baguisi et al., "Production of goats by somatic cell nuclear transfer," Nature Biotechnology, 17:456-61, May 1999). Traditional transgenics microinjects a gene of interest into a just-fertilized egg--an effort more often miss than hit. Transfer of an already-transgenic nucleus would ensure that the offspring are female and transgenic. So following in the hoofsteps of Dolly the sheep and cloned cattle George and Charlie, the researchers transferred nuclei from cultured fetal fibroblasts into enucleated oocytes. The fetus' father was transgenic for the human antithrombin III (AT III) gene, which encodes an anticoagulant. Three as-yet-unnamed goats resulted, and a battery of genetic tests proved their cloneworthiness. One she-goat, given appropriate hormonal stimulation, indeed produced human AT III in her milk. "The market for AT III is $200 million, and that amount can be produced by under 100 goats. Cloning is simply a tool to reproduce those animals as rapidly as possible. Another aspect is the health issue--we can get away from using serum-based therapeutic proteins," says Richard Denniston, an instructor in the department of animal science at the LSU Agricultural Center. AT III from serum is currently in clinical trials to be used in coronary bypass surgery, and Genzyme has several noncloned transgenic goats that produce the drug. Why goats? They are far less likely to develop scrapie than their ovine and bovine barnyard mates. --Ricki Lewis GUARDING THE GUARD Inhibiting the very enzyme that helps kill off Huntington's disease-afflicted cells can slow the sickness (V.O. Ona et al., "Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease," Nature, 399:263-7, May 20, 1999). Robert M. Friedlander of Brigham and Women's Hospital, Harvard Medical School, led a team of researchers to a mutant form caspase-1. They found that introducing the enzyme into transgenic mice carrying the human huntingtin gene delayed symptom onset and extended the animal's life up to 25 percent longer than in diseased mice without the mutant caspase. "We show how caspase-1 is not an all-or-none thing," comments Friedlander. He says the slow, chronic neurodegeneration in Huntington's disease may initiate a sublethal level of caspase activation, compared to stroke or other severe brain trauma, in which caspases busily cleave damaged proteins, resulting in massive apoptosis. Instead, Huntington's-afflicted cells go through an "ill phase" characterized by various abnormalities and dysfunction that only eventually progress to death. "So we can block what's making the cell sick, in a way," Friedlander notes. But available caspase inhibitors are too toxic for safe use in humans. "The next step is, in part, up to the pharmaceutical companies," he declares. "I know a lot of biotech companies are working very energetically to make a [safe] caspase inhibitor." --Steve Bunk NEW DIRECTIONS Although federal funding for medical research increased from $2 billion in 1960 to almost $16 billion this year, the inherently political process of appropriating funding is counterproductive to long-term planning. To find ways to cope with the process and boost research funding, the American Association for the Advancement of Science (AAAS) and the Funding First Program of the Mary Woodard Lasker Trust sponsored a workshop in February. The Burroughs Wellcome Fund cosponsored the workshop, which attracted about 30 experts in funding, policy, and research. The three groups released their report, "How to Fund Science: The Future of Medical Research," at a congressional briefing in Washington, D.C., May 25. "It is clear that medical research enjoys a healthy investment from the federal government," said Albert H. Teich, AAAS director of science and policy programs. "However, the scientific community needs to find ways to diversify the support base for medical research and the many other fields of science and engineering on which medical progress depends." The report recommends that the primary system of federal funding should continue as it is today, but three additional measures should be put into place: 1) Set up a secondary source of funding (a trust or reserve fund) as a supplement to annual appropriations. 2) Mandate public and private insurers to pay the cost of health care services for beneficiaries participating in federally supported clinical trials, except for beneficiaries who stand to gain a commercial profit from a patentable product linked to the trial. 3) Make permanent the research and experimentation tax credit and expand it to include clinical trials. Texts of presentations made at the workshop are available on the AAAS Web site at www.aaas.org/spp/dspp/cstc/fundscience.htm. --Larry Hand MOLECULAR LEGOS Proteins associate to form signal transduction complexes in a manner similar to Lego pieces snapping together to form a ship. Mingjie Zhang and colleagues of the Hong Kong University of Science and Technology have used nuclear magnetic resonance (NMR) studies to elucidate the mechanism by which PDZ domains localize channels, signaling enzymes, and adhesion molecules to sites of cell-cell contact such as synapses (M. Zhang et al., "Solution structure of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated peptide," Nature Structural Biology, 6:417-21, May 1999). PDZ domains (named after the first proteins in which this motif was found, PSD-95, Dlg, and ZO-1) contain a groove that binds the C-terminal residues of membrane-associated receptors or channels. The NMR structure of neuronal nitric oxide synthase (nNOS) PDZ domain in complex with the PDZ domain of target peptides suggests a new role for this motif as a versatile scaffold, which clusters multiple components. An approximately 25-residue C-terminal extension of the nNOS PDZ domain forms a "beta finger" that can insert into the binding site of the PDZ domain in either PSD-95 or * -syntrophin. The C-terminal extension and the peptide-binding groove are located at opposite ends of the nNOS PDZ domain, indicating that the two modes of activity can be independent of each other. Binding of a C-terminal peptide and binding of another PDZ domain could, in principle, occur simultaneously. The authors suggest that the extended nNOS PDZ domain may be a bifunctional adapter that can link various signaling components or signaling proteins and the cytoskeleton. Zhang comments, "The data presented in the paper provide a novel approach [to rational drug design] aimed at uncoupling the interaction between nNOS PDZ domain and PSD95." This type of nNOS inactivation would be valuable in controlling nitric oxide generation in cases of stroke, Huntington's disease, and muscular dystrophy. Independent NMR studies supporting this work were published in Science (B.J. Hillier et al., "Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex," Science, 284:812-5, April 30, 1999). --Nadia S. Halim SAVING SPACE, WITH MOUSE SPERM Mice make terrific models for many human diseases, and amassing new mutants isn't a problem--but housing animals is. A way to solve the space problem while easing shipment of strains is to use sperm, according to investigators at the Institute of Mammalian Genetics of the GSF Research Center for Environment and Health in Neuherberg, Germany (S. Marschall, M. Hrabé de Angelis, "Cryopreservation of mouse spermatozoa--double your mouse space," Trends in Genetics, 15:128-30, April 1999). Colonies could be nurtured in as little as a few weeks from a single sperm donor, says Martin Hrabé de Angelis, head of the genome project at GSF. Alternative reproductive technologies replace old-fashioned breeding. "[In vitro fertilization] is a well-established standard technique used worldwide--it is no problem for mouse houses with several users. And although artificial insemination is still under development, new methods look very promising and might be the way for single users and centers in the future," he says. The world's major mouse facilities are gearing up to collect and distribute sperm on a large scale. Centers include GSF, the Induced Mutant Resource at the Jackson Laboratory in Bar Harbor, Maine, the Mammalian Genetics Unit of the MRC in Harwell, U.K., the European Mouse Mutant Archive in Monterotondo, and a planned Japanese Mouse Mutant Archive. "All centers use it or are ... establishing the method. Smaller labs are following--I know that by looking at the addresses of our sperm-freezing course participants. The courses are always fully booked," Hrabé de Angelis adds. --Ricki Lewis SYNTHETIC CANCER VACCINE A carbohydrate cancer vaccine may be able to trigger the body's immune response to attack wandering cancer cells before they develop into metastases. This is the goal of Samuel Danishefsky and his coworkers at Memorial Sloan-Kettering Cancer Center (R. Ragupathi et al., "A fully synthetic globo H carbohydrate vaccine induces a focused humoral response in prostate cancer patients: a proof of principle," Angewandte Chemie International Edition, 38:563-6, 1999). Molecules with a protein and a carbohydrate component, known as glycoproteins, often provide a distinguishing mark on the surface of cancer cells. Danishefsky and colleagues have synthesized one such carbohydrate component called globo H and have used it in clinical trials (S.F. Slovin et al., "Carbohydrate vaccines in cancer: immunogenicity of a fully synthetic globo H hexasaccharide conjugate in man," Proceedings of the National Academy of Sciences, 96:5710-5, May 11, 1999). The carbohydrate molecule is made up of six sugar units and is found in nature on the coating of breast and prostate cancer cells. The synthetic carbohydrate was conjugated to a protein carrier, keyhole limpet hemocyanin (KLH), and administered as a vaccine to 20 patients with prostate cancer who relapsed after surgery or radiation therapy. The vaccine was established as safe and capable of inducing specific antibody response to globo H. More importantly, the immune response to the vaccine based on synthetic globo H-protein conjugate also recognized naturally occurring tumor-derived glycoprotein. Danishefsky comments, "[These results] represent a feasibility demonstration. The next move will be a large clinical trial to get statistically significant data." --Nadia S. Halim DEMETHYLATION DETECTION DNA methylation is a fundamental biological phenomenon that silences gene expression by attaching a methyl group to a specific nucleotide sequence. The role the process plays in cell development and tumorigenesis is therefore enormous. Not surprisingly, its reverse process, demethylation, is also critical to scientists' understanding of the life and death of a cell. But, although known to occur during development, demethylation was thought to be unachievable in developed cells; methylation was considered irreversible. A recent paper from scientists at McGill University in Montreal, however, suggests otherwise (S. Ramchandani et al., "DNA methylation is a reversible biological signal," Proceedings of the National Academy of Sciences [PNAS], 96:6107-12, May 25, 1999). The main problem with achieving demethylation: breaking the strong chemical bond between methyl groups and the carbon molecules of the nucleotide sequence. The authors of this study have shown that an enzyme, which they isolated from human lung cancer cells, is capable of breaking that bond and of removing specific products of the DNA methylation reaction. According to senior author Moshe Szyf, an associate professor in McGill's department of pharmacology, researchers had already "documented the fingerprints" of such an enzyme, but had not isolated it. "The problem is that methylation happens early in development," explains Szyf. "It's very hard to do biochemistry with very few cells." National Institute of Child Health and Human Development investigator Alan Wolffe, lead author of an accompanying PNAS commentary on DNA demethylation (A.P. Wolffe et al., "DNA demethylation," PNAS, 96:5894-6, May 25, 1999), cautions, however, that the precise activity and role of this enzyme "remains to be seen." Such an enzyme would be a potentially excellent target for cancer therapy; recent studies indicate that methylation has a causal role in tumor suppressor activity. --Eugene Russo

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