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Reprinted with permission from Nature CKI produces complete secondary dorsal axes. Synthetic mRNA encoding CKI, mutant CKI (K > R), or Xwnt-8 was injected at the eight-cell stage into one ventral vegetal blastomere. Embryos injected with CKI or Xwnt-8, but not inactive CKI (K > R), developed complete secondary dorsal axes. CANCER CLUE The rush of discoveries over the past two years concerning the Wnt signaling pathway--known to be crucial to normal development and altered in human melanoma and colon cancers--has uncovered an as-yet unnamed complex of proteins within the cell that appear to function together as a machine. Wnt signals regulate this developmental machinery by binding with membrane receptors to enter the cell and influence gene expression from the nucleus. Now another component in this convoluted signaling cascade has been established and its likely position identified (J.M. Peters et al., "Casein kinase I transduces Wnt signals," Nature, 401:245-50, Sept. 23, 1999). Senior author Jonathan M. Graff, an assistant professor in the Center for Developmental Biology at the University of Texas Southwestern Medical Center, notes that the paper describes the function of the casein kinase I (CKI) but not its mechanism. "We think that somehow, CKI [has an impact on] that protein machinery," he says. In a series of experiments with worms and frogs, his group showed that CKI probably is located upstream of a protein called GSK-3 that is part of the machinery and that must be turned off to activate the pathway. But CKI does not phosphorylate GSK-3. Instead, it increases phosphorylation of the protein just upstream from it, [called] dishevelled, which also is thought to be part of the complex. CKI binds to dishevelled somewhere within a fragment of 80 amino acids. Could this fragment be a pathogenic key? "We're certainly working on that," Graff says. "Absolutely." --Steve Bunk RESERVOIR JOB In a review paper that may be more forward-challenging than backward-looking, Anthony S. Fauci exhorted AIDS researchers to find a way around the hidden pools that harbor minute amounts of the virus (T. Chun, A.S. Fauci, "Latent reservoirs of HIV: obstacles to the eradication of the virus," Proceedings of the National Academy of Sciences, 96:10958-61, Sept. 28, 1999). Attacking those pools directly with the much-vaunted highly active antiretroviral treatment (HAART)--combination therapy that includes at least one protease inhibitor--has its limits, wrote the National Institute of Allergy and Infectious Diseases (NIAID) director. Even when HAART reduces the virus to nondetectable levels, it persists in hard-to-reach tissues, such as the brain, gut-associated lymph tissues, and bone marrow. "We don't have access to all the organs in the body," comments coauthor Tae-Wook Chun, a staff fellow with NIAID. "I don't think eradicating [the virus] is going to be easy." Instead, Fauci and Chun recommend that AIDS researchers look for additional approaches--particularly finding ways to marshal the body's immune response to keep the disease at bay, rather than destroy every trace of HIV. Without a new strategy, HAART may wear out its welcome. "More people are experiencing side effects. It would be nice to control the viral replications without HAART," Chun concludes. --Paul Smaglik FAT TRANSFER Fatty acids--once thought to flow unaided from the gut into the small intestine--are actually shepherded across the cell membrane by fatty acid transport protein 4 (FATP4), report a group of scientists from the Whitehead Institute and Millennium Pharmaceuticals Inc. The Cambridge, Mass.-based team discovered the novel protein expressed at high levels in the small intestine's villi, tiny, finger-shaped projections that absorb nutrients (A. Stahl et al., "Identification of the major intestinal fatty acid transport protein," Molecular Cell, 4:299-308, September 1999). While the fatty acids do indeed diffuse freely across the cell membrane, they cross that cellular threshold much more quickly in the presence of FATP4. Whitehead researcher Harvey F. Lodish, the study's senior author, and colleagues discovered the first FATP five years ago in mice, but those molecules weren't anywhere near the digestive tract (J.E. Schaffer, H.F. Lodish, "Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein," Cell, 79:427-36, 1994). The growing databases resulting from the Human Genome Project led the group to a human homologue, then to more members of the FATP family. The Millennium collaborators then expressed the protein products one at a time. Those expression experiments led to the discovery that FATP4 is concentrated in a location key to dietary uptake. Further experiments using antisense technology to inhibit the gene's expression led to decreased fatty acid uptake and cemented the protein's function. "There may be other proteins that may interact with this one in other cells that may enhance its activity," Lodish notes. "We're not claiming this is the only protein that's involved in fatty acid uptake. There may be accessory proteins that help." Jean E. Schaffer , assistant professor of medicine at Washington University in St. Louis, agrees. "How all those proteins contribute so far is unknown." Understanding FATP4's function may lead to new anti-obesity drugs. --Paul Smaglik CRYSTAL CLEAR Scientists are finally seeing the four-way DNA junction proposed by Robin Holliday in 1964. The Holliday junction as it is now known is a model that explains recombination of genes and DNA repair events. By 1988 David Lilley, professor of biochemistry at the University of Dundee, United Kingdom, solved the structure in solution, but it took another 10 years to solve the actual crystal structure. "People have tried to design their molecules cleverly, but none of it has worked. The curious thing is, two crystal structures [have been determined] in the past several months, and both were obtained completely by accident," says Lilley. Gerald Joyce , professor of biochemistry at The Scripps Research Institute, was trying to crystallize a DNA enzyme complex with an RNA substrate when it dimerized, forming a four-way junction of two DNA strands and two RNA strands (J. Nowakowski et al., "Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10-23 DNA enzyme," Nature Structural Biology, 6:151-6, February 1999). Recently Miquel Coll, from the Institut de Biologia Molecular de Barcelona, Spain, crystallized a true Holliday junction when studying a DNA duplex containing central consecutive adenine-guanine mismatches (M. Ortiz-Lombardia et al., "Crystal structure of a DNA Holliday junction," Nature Structural Biology, 6:913-7, October 1999). The structures found by both groups support the Holliday junction model formed over the years. Joyce comments, "We were stunned by how closely [our structure] matches up with the proposals." Technically, the molecule from the Joyce lab is not a Holliday junction, since it is formed of RNA and DNA strands. Crystal structures of DNA junctions alone and junctions with the enzymes that catalyze reactions are snapshots in a chain of molecular events. "What one would really like, of course, is to order the snapshots along the pathway of recombination. It's not realistic to think we will catch it in the act of completely swapping its strands, but we can begin to see what that pathway looks like," explains Joyce. The crystal structures may also have an impact on engineering DNA "machines" for nanotechnology research. --Nadia S. Halim THE SAME, OR 'NO' DIFFERENCE? Perhaps the great divide in the sexes boils down to the activity of a few lousy molecules. Researchers at Johns Hopkins University (JHU) recently documented the rather schizophrenic activity of the infamous chemical messenger nitric oxide (NO), a small molecule well known for its roles as both neurotransmitter and vasodilator. According to studies done at JHU, NO, like the neurotransmitter seratonin, seems to have different functions in male versus female mice. Work done in 1995 by Randy Nelson , a professor of psychology and neuroscience at JHU, suggested that a strain of male mice with defective copy of the gene that codes for neuronal nitric oxide synthase (nNOS) behaved unusually aggressively (R.J. Nelson et al., "Behavioral abnormalities in male mice lacking neuronal nitric-oxide synthase," Nature, 378:383-6, 1995). A September 1999 study done on the same line of mice, but with females, came up with different results (S.C. Gammie and R.J. Nelson, "Maternal aggression is reduced in neuronal nitric oxide synthase-deficient mice," Journal of Neuroscience, 19:8027-35, Sept. 15, 1999). Investigators found that the NO-less females were actually less aggressive; compared to controls, they spent only one-fifteenth of the time attacking strange males. An evolutionary explanation is unclear, but Nelson speculates that NO may act like a brake in male mice because aggression is needed more frequently. NO's effects in females, however, could be stimulatory because they need only be aggressive around their pups. Confusing matters, a third study by the team in the October 1 Journal of Neuroscience suggests that mice lacking endothelial NOS (eNOS), an NOS released in blood vessels exclusively and involved in increasing blood flow, demonstrate low levels of aggression. "It may have to do with regulation of blood flow," says JHU researcher Stephen Gammie , a psychology postdoctoral fellow. "[Lack of eNOS] might generally slow down a brain's functioning." The JHU team has just submitted a paper on the effect of eNOS in female mice. Future studies will attempt to locate NO's site of activation. Says Gammie, "The definitive experiments are still in the works." --Eugene Russo PROMISING VACCINE There has been a lot of noise in the cancer vaccine area, but few vaccines make it to advanced clinical testing. A vaccine for B-cell (white blood cells that produce antibodies) lymphoma is beating the odds and moving into a large-scale Phase III clinical trial after promising Phase II results. Researchers from the National Cancer Institute (NCI) report that 18 of 20 patients who were vaccinated against this blood-cell tumor remain in complete remission an average of four years after vaccine therapy began (M. Bendandi et al., "Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma," Nature Medicine, 5:1171-7, October 1999). To create the vaccine, researchers fused tumor cells from individual patients to antibody-producing mouse cells that synthesize large quantities of tumor proteins. Then the protein of interest, a receptor molecule on the outer surface of the cancerous B cells, is isolated from the mixture. Essentially the vaccine presents the tumor protein as a sign that the immune system can recognize; any cell bearing the sign will be destroyed. "This is a highly purified, well-defined protein that is exquisitely specific for the tumor cell," says Larry W. Kwak , principal investigator in NCI's division of clinical sciences. The nature of the vaccine and the clinical design may be keys to the trial's success. The vaccine was administered after chemotherapy, which reduces 95 percent of the initial tumor burden. Kwak adds, "We saw an increase in the number of CD8+ T cells and CD4+ T cells, which supports our central hypothesis that this arm of the immune response is paramount [in this approach]." Though the vaccine induced antibodies against the tumor in more than 50 percent of the patients, the study indicates that antibodies were not required for the observed molecular remissions. One potential advantage of the vaccination approach is that activation of a patient's immune system may be effective months or years after administration. --Nadia S. Halim

Notebook

Oct 10, 1999

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