Nuclear Protein Unexpectedly Limits Mammalian Cell Life Span

A gene found in mice limits the life span of cells, according to a recent study by HMS researchers. The findings are surprising because the presence of the same version of the gene in invertebrates seems to extend cell life. The study appears in the July 2005 Cell Metabolism.

The gene produces SIRT1, a nuclear protein involved in cellular senescence, a process thought to represent cell aging. According to lead researcher Frederick Alt, the Charles A. Janeway professor of pediatrics at Children’s Hospital Boston, SIRT1 suppresses cellular longevity in mice rather than promoting it. Previous studies in the field showed that activation of Sir2 proteins in nonmammalian organisms could increase life span. This led to speculation that the removal of mammalian SIRT1 would cause the cells to senesce sooner. Alt and his colleagues tested this hypothesis on mouse embryonic fibroblasts, employed as a model system for cell aging, and found that SIRT1 loss had the opposite effect. “Unlike wild-type cells that only undergo a limited number of divisions before they reach senescence, SIRT1-deficient cells continued to grow on and on,” Alt said.

The researchers also found that when the SIRT1-deficient cells were exposed to chronic oxidative stress, the cells continued to show enhanced proliferative ability. Because oxidative stress can lead to DNA damage, it is possible that SIRT1 limits cell proliferation to ensure that harmful mutations are not propagated. The team found, however, that while the cells lacking SIRT1 continued to divide, DNA damage did not increase. It will be important to determine whether SIRT1 inactivation similarly extends the life span of human cells, and if so, whether this is accompanied by increased genomic instability.

Results from similar studies on SIRT1 and its homologs have led to speculation that this gene could be manipulated in humans to defer aging. And while Alt and his lab are hopeful their mouse study results will be useful, they caution that the pathway in humans could be very different.

Another insight of the new study is the potential use for SIRT1-deficient cells in research. “Many labs and companies have developed a wide range of modulators of SIRT1,” Alt said. “I think our findings offer one interesting possibility for their use, in that inhibiting SIRT1 might enable the production of large numbers of cells that are relatively normal.”

—Rachel Patzer

Bone Marrow Transplantation Restores Oogenesis in Mice

Ovaries of sterilized adult female mice rapidly generate hundreds of oocytes when given a bone marrow transplant from normal female mice, according to a recent study from researchers at Massachusetts General Hospital and HMS. The work is a follow-up to their 2004 findings that challenged the central dogma of reproductive biology: that females of most mammalian species produce only a finite stockpile of oocytes because oogenesis halts during fetal development. The researchers hope their study, which appears in the July 29 Cell, will ultimately lead to better treatment and management of female infertility and menopause.

In the earlier study, the scientists used molecular markers to demonstrate the existence of an unidentified pool of germline stem cells in mouse ovaries. The current study aimed to identify the source of these stem cells. The existence of an embryonic germ cell marker in the blood vessel–rich portion of the ovaries led the team to hypothesize that the bone marrow could be responsible for the production of germ cells. “We found that every germ cell marker we could think of was expressed in the bone marrow of adult female mice,” said senior author Jonathan Tilly, director of the Vincent Center for Reproductive Biology at MGH and HMS associate professor of obstetrics, gynecology and reproductive biology. “Everyone had missed finding female germline stem cells because they are not in the ovaries, where everyone would have looked for them.”

The scientists conducted a series of experiments to verify the function of the markers in the bone marrow. Two mouse groups, one consisting of normal mice treated with chemotherapy drugs to destroy the ovaries and the other of genetically sterile females, were used as models. Some of each group received transplants of bone marrow from normal females. Two months later, the mice that did not receive transplants lacked oocytes, and the ovaries of the mice that received a transplant looked identical to the normal mice since oocyte production was restored.

In the future, to bolster their controversial findings, the scientists hope to show that the mice can produce offspring after transplants of bone marrow or peripheral blood. They also plan to investigate the specific signal the ovary sends to the bone marrow.

—Rachel Patzer

Antibiotic Probe Spotlights Bacterial Defenses

HMS researchers have developed a tool that could help solve one of the oldest questions in biology—how bacteria assemble their hardy cell wall. Previous methods have not yielded precise information about when and where various components of the wall are made. Using an antibiotic probe that attacks components just as they are emerging onto the cell surface, Suzanne Walker and her colleagues were able to directly observe early cell wall growth in living bacteria. Their approach, which was presented on Aug. 31 at the American Chemical Society national meeting in Washington, D.C., could be used to better understand, and possibly enhance, how antibiotics actually work.

“One of the things this could do is help reveal what happens when you treat bacteria with antibiotics that are cell wall active,” said Walker, HMS professor of microbiology and molecular genetics.

Bacteria are among the oldest and most successful organisms on the planet. Their success is due in large part to their peptidoglycan-containing cell wall, which serves to keep intruders out and also to prevent the intense internal osmotic pressure from causing the cell to explode. Peptidoglycans originate inside the cell as disaccharide building blocks that are converted on the surface of the cell into a cross-linked polymer.

In an effort to understand when and where this conversion occurs, some researchers have used metabolic labeling, but this approach has not provided sufficient temporal and spatial resolution. Others have used a fluorescent derivative of the antibiotic vancomycin to visualize new peptidoglycans synthesis. “That intrigued us, but vancomycin does not have the right specificity to probe the initiation sites of new peptidoglycans,” Walker said. A chemist by training, she and her colleagues selected another antibiotic, ramoplanin, which binds where the beginning of the peptidoglycans chain is made. The probes not only bound to the nascent chains in living bacteria, but when made fluorescent, could be followed in a way that promises to yield answers to the critical when and where questions.

Walker and her colleagues are currently using the ramoplanin-based probes to explore the assembly of the cell wall. “Having a chemical background is really useful for addressing certain types of biological questions,” she said.

—Misia Landau

Technique Set to Develop New Antibiotics of Last Resort

In U.S. hospitals, the growing frequency of pathogenic bacteria that are resistant to vancomycin, the drug of last resort against deadly infections, has become a serious public health threat.

Vancomycin was discovered nearly 50 years ago in soil-dwelling bacteria, which produce the compound to kill other bacteria. Now, researchers at Harvard University have harnessed the bacteria’s own enzymatic manufacturing process to make and test vancomycin variations that can kill bacteria in a new way.

The method will speed up research about how the drugs work and how they may overcome vancomycin resistance, said Catherine Leimkuhler, a graduate student in the Harvard Chemistry and Chemical Biology Department, who presented her findings on Aug. 28 at the American Chemical Society national meeting in Washington, D.C. The new technique is the latest advance in a long-standing collaboration between the labs of Daniel Kahne, professor of biological chemistry and molecular pharmacology at HMS and professor of chemistry at Harvard University, and Christopher T. Walsh, the Hamilton Kuhn professor of biological chemistry and molecular pharmacology at HMS. Ultimately, the researchers hope to learn how to design new antibiotics with activity against both sensitive and resistant bacterial strains.

Vancomycin works against gram-positive bacteria, which include Staphylococcus. These bugs must continually build and repair a protective wall of carbohydrate mesh around their outer membranes as they divide and grow. Specifically, the antibiotic binds to and interferes with a building block in the final stage of assembling the wall. Without intact cell walls to support them, the bacteria break apart and die. Unfortunately, a handful of genes that the vancomycin-making bacteria use to detect and defend themselves from their own deadly compound have been passed along to pathogenic bacteria, many of which can now repel the antibiotic.

Taking a cue from Mary Poppins, the fictional nanny who advised taking a spoonful of sugar with every dose of medicine, Kahne and Walsh speculated that modified antibiotics were still able to kill vancomycin-resistant bacteria because of variations in a sugar group that decorates these related antibiotics.

After researchers tinkered with vancomycin’s sugar group, the remodeled antibiotic was able to kill resistant bacteria. The sugar group seems unrelated to the original antibiotic activity, which happens elsewhere on the antibiotic molecule. The researchers proposed that the derivative worked in a new way, targeting the machinery that puts together the building blocks in the final stage of constructing the bacterial cell wall.

To test this idea and experiment with other variations, Kahne, Walsh, and their colleagues developed a recipe to tweak the sugar groups on vancomycin. In the latest work, Leimkuhler has shown that a simplified enzymatic process from the vancomycin-producing bacteria will enable researchers to quickly make and test many variations, leading to an understanding of how the different sugar flavors overcome bacterial resistance.

—Carol Cruzan Morton

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