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Revved-up protein fights aging

An unlikely, decade-long journey that began with the discovery of a rapidly aging mouse has led scientists to a protein that seems to protect animals from cancer and other scourges of old age—with no apparent downsides. There are still lots of mysteries about the protein, called BubR1, but the work offers clues about how protecting chromosomes can enhance health. Cancer biologist Jan van Deursen at the Mayo Clinic in Rochester, Minnesota, and his colleagues were initially interested in studying a common feature of cancers, called aneuploidy. Aneuploid cells have too few or too many chromosomes. Nearly all cancer cells fall into this category, but it’s not clear whether aneuploidy actually causes cancer. Van Deursen, along with a then-graduate student, Darren Baker, engineered mice to produce less BubR1, a protein that helps cells segregate their chromosomes when they divide. When BubR1 is reduced, chromosomes can’t properly separate into identical daughter cells, leaving some daughters with the wrong number of chromosomes. Van Deursen, Baker, and their colleagues wanted to see whether these mice would develop cancer.

To their surprise, instead of tumor-filled mice, they wound up with animals that aged very quickly. “These mice were clearly very, very different than a normal mouse,” says Baker, who now studies the biology of aging at the Mayo Clinic. Last year, they reported that removing old cells—that is, cells with a genetic marker indicating senescence—from these mice could help them stay healthier longer. Adding intrigue is an extremely rare human condition caused by mutations in the BubR1 gene. Patients with the disease, mosaic variegated aneuploidy syndrome, age prematurely and have an elevated risk of cancer. Too little BubR1 seems to be bad news. Too much, on the other hand, might be a good thing. In work published today in Nature Cell Biology, the biologists report that genetically engineered mice that make extra BubR1 are less prone to cancer. For example, they found that when they exposed normal mice to a chemical that causes lung and skin tumors, all of them got cancer. But only 33% of those overexpressing BubR1 at high levels did. They also found that these animals developed fatal cancers much later than normal mice—after about 2 years, only 15% of the engineered mice had died of cancer, compared with roughly 40% of normal mice.

The animals that overexpressed BubR1 at high levels also lived 15% longer than controls, on average. And the mice looked veritably Olympian on a treadmill, running about twice as far—200 meters rather than 100 meters—as control animals. All of this left Baker, van Deursen, and their colleagues thinking that BuBR1’s life-extending effects aren’t due to only its ability to prevent cancer, although that’s not yet certain. A big question now is why having your chromosomes out of order might accelerate aging, says Wei Dai, a cell biologist at the New York University Langone Medical Center who’s based in Tuxedo, New York. Although aneuploidy seems less than desirable, studies haven’t been consistent about its effects on animals. “We found that when the aneuploidy level became low”—just like in van Deursen’s healthy mice—”you had more tumorigenesis,” not less, says Cristina Montagna, a molecular geneticist at Albert Einstein College of Medicine in the Bronx, New York. She and her colleague Jan Vijg are collaborating with van Deursen to study the brains of his BubR1 mice. One possibility is that both very low and very high aneuploidy can protect from cancer, perhaps because highly aneuploid cells are so damaged they don’t have the ability to quickly divide. Still, there’s hope that van Deursen’s group may have identified a new drug target to slow aging. “There are no negative consequences that he identified” to having more BubR1, says Paul Hasty, who studies aging and DNA repair at the University of Texas Health Science Center in San Antonio. “You need to figure out exactly what BubR1 is doing to achieve this desired effect,” he adds, but this could be the first step on a long path toward new treatments that delay aging—and possibly prevent cancer.

ScienceNow
January 8, 2013

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New insights into virus proteome: Unknown proteins of the herpesvirus discovered

The genome encodes the complete information needed by an organism, including that required for protein production. Viruses, which are up to a thousand times smaller than human cells, have considerably smaller genomes. Using a type of herpesvirus as a model system, the scientists of the Max Planck Institute (MPI) of Biochemistry in Martinsried near Munich and their collaboration partners at the University of California in San Francisco have shown that the genome of this virus contains much more information than previously assumed. The researchers identified several hundred novel proteins, many of which were surprisingly small. The results of the study have now been published in Science. More than 80 percent of the world’s population is infected with the herpesvirus, which can cause severe diseases in newborns and in persons with weakened immune system. Researchers had already sequenced the herpesvirus genome 20 years ago, thinking they could then predict all proteins that the virus produces (virus proteome). Now scientists from the research department of Matthias Mann, director at the MPI of Biochemistry, and their American colleagues have analyzed the information content of the genome more precisely.

To carry out their study, the scientists infected cells with herpesvirus and observed which proteins the virus produced inside the cell over a period of 72 hours. In order for proteins to be produced at all, the cell machinery must first make copies of the genetic material as intermediate products (RNA). While investigating the intermediate products of the herpesvirus, the American collaborators discovered many novel RNA molecules which were in large part surprisingly short. They also found that the organization of information required for protein production in the virus genome was far more complex than previously assumed. Annette Michalski, a scientist in the Department of Proteomics and Signal Transduction at the MPI of Biochemistry, was subsequently able to confirm directly the predicted viral proteins in the infected cell using mass spectrometry. This method enables an overview of the complete proteome of the virus-infected cell. The results of the American and German researchers provide detailed insight into the complex mechanisms used by the virus. “We showed that it’s not enough merely to know the virus genome to understand the biology of the herpesvirus,” Annette Michalski said. “What is important is to look at the products actually produced from the genome.” Even human genes may be much more complex than the genome sequence itself indicates, commented the researchers. Matthias Mann and his colleagues plan to investigate this question further in the coming years.

Science Daily
December 11, 2012

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Telomere lengths predict life expectancy in the wild, research shows

Researchers at the University of East Anglia have found that biological age and life expectancy can be predicted by measuring an individual’s DNA. They studied the length of chromosome caps — known as telomeres — in a 320-strong wild population of Seychelles Warblers on a small isolated island. Published Nov. 20 in Molecular Ecology, their research shows that individuals differ radically in how quickly their telomeres shorten with age, and that having shorter telomeres at any age is associated with an increased risk of death. Telomere length is a better indicator of future life-expectancy than actual age and may, therefore, be an indicator of biological age. The 20-year research project is the first of its kind to measure telomeres across the entire lifespan of individuals in a wild population. Telomeres are found at the end of chromosomes. They act as protective caps to stop genes close to the end of the chromosome degenerating — like the hard plastic ends of a boot lace. Lead researcher Dr David S Richardson said: “Over time these telomeres get broken down and become shorter. When they reach a critical short length they cause the cells they are in to stop functioning. This mechanism has evolved to prevent cells replicating out of control — becoming cancerous. However the flip side is that as these zombie cells build up in our organs it leads to their degeneration — aging — and consequently to health issues and eventually death.

“Telomeres help safeguard us from cancer but result in our aging.” Researchers studied the warbler population on Cousin Island. Blood samples were collected twice a year and telomere length analysed. “We wanted to understand what happens over an entire lifetime, so the Seychelles Warbler is an ideal research subject. They are naturally confined to an isolated tropical island, without any predators, so we can follow individuals throughout their lives, right through to old age. “We investigated whether, at any given age, their telomere lengths could predict imminent death. We found that short and rapidly shortening telomeres were a good indication that the bird would die within a year. “We also found that individuals with longer telomeres had longer life spans overall. “It used to be thought that telomere shortening occurred at a constant rate in individuals, and that telomere length could act as an internal clock to measure the chronological age of organisms in the wild. “However while telomeres do shorten with chronological age, the rate at which this happens differs between individuals of the same age. This is because individuals experience different amounts of biological stress due to the challenges and exertions they face in life. Telomere length can be used as a measure of the amount of damage an individual has accumulated over its life.

“We saw that telomere length is a better indicator of life expectancy than chronological age — so by measuring telomere length we have a way of estimating the biological age of an individual — how much of its life it has used up.” The research is important because while these ideas have been researched in the lab, they have never been tested in a wild environment. “It would be virtually impossible to do such a study in humans,” said Dr Richardson. “For one thing it would take a very long time to study a human lifespan. Also in humans we would normally, quite rightly, intervene in cases of disease, so it wouldn’t be a natural study. “We found that telomeres are linked to body condition and reflect the history of oxidative stress that has occurred within an individual’s lifetime. The healthier you are, or have been, the better telomeres you have. But it’s hard to know whether this is a consequence of being healthy, or a cause. “Oxidants attack telomeres. So things like smoking, eating foods that are bad for you, and putting your body through extreme physical or mental stress all have a shortening affect on telomeres. “All these stresses do damage to our bodies. You hear people saying ‘oh they look like they’ve had a hard life’. This is why. A shortened telomere shows an accumulation of damage life has done to you.” ‘Telomere length and dynamics predict mortality in a wild longitudinal study’ is authored by Dr David S Richardson and Emma Barrett from the University of East Anglia (UK), Terry Burke from the University of Sheffield (UK), and Jan Komdeur and Martijn Hammers from the University of Groningen (Netherlands).

Science Daily
December 11, 2012

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Closer to a cure? Chemists synthesize compound that flushes out latent HIV

A new collection of compounds, called “bryologs” — derived from a tiny marine organism — activate hidden reservoirs of the virus that currently make the disease nearly impossible to eradicate. Thanks to antiretrovirals, an AIDS diagnosis hasn’t been a death sentence for nearly two decades. But highly active antiretroviral therapy, or HAART, is also not a cure. Patients must adhere to a demandingly regular drug regimen that carries plenty of side effects. And while the therapy may be difficult to undergo in the United States, it is nearly impossible to scale to the AIDS crisis in the developing world. The problem with HAART is that it doesn’t address HIV’s so-called proviral reservoirs — dormant forms of the virus that lurk within T-cells and other cell types. Even after all of the body’s active HIV has been eliminated, a missed dose of antiretroviral drugs can allow the hibernating virus to emerge and ravage its host all over again. “It’s really a two-target problem,” said Stanford chemistry Professor Paul Wender, “and no one has successfully targeted the latent virus.” But Wender’s lab is getting closer, exciting many HIV patients hoping for a cure.

The lab has created a collection of “bryologs” designed after a naturally occurring, but difficult to obtain, molecule. The new compounds have been shown to activate latent HIV reservoirs with equal or greater potency than the original substance. The lab’s work may give doctors a practical way to flush out the dormant virus. The findings were published on July 15 in the journal Nature Chemistry. The first attempts to reactivate latent HIV were inspired by observations of Samoan healers. When ethnobotanists examined the bark of Samoa’s mamala tree, traditionally used by healers to treat hepatitis, they found a compound known as prostratin. Prostratin binds to and activates protein kinase C, an enzyme that forms part of the signaling pathway that reactivates latent viruses. The discovery sparked interest in the enzyme as a potential therapeutic target, especially as it was discovered that prostratin isn’t the only biomolecule to bind to the kinase. The bryozoan Bugula neritina – a mossy, colonial marine organism — produces a protein kinase C-activating compound that is many times more potent than prostratin. The molecule, named bryostatin , was deemed to hold promise as a treatment, not only for HIV but for cancer and Alzheimer’s disease as well.

The National Cancer Institute initiated a Phase II clinical trial for the compound in 2009 for the treatment of non-Hodgkin lymphoma. But the substance had a number of side effects and proved prohibitively difficult to produce. “It took 14 tons of bryozoans to make 18 grams of bryostatin,” said Wender. “They’ve stopped accrual in trials because, even if the trials worked, the compound cannot be currently supplied.” Patient enrollment was suspended until more accessible compounds came out of the Wender Group’s lab. Wender, who published the first practical synthesis of prostratin and its analogs in 2008, had set out to make a simpler, more effective synthetic analog of bryostatin. “We can copy the molecule,” he said, “or we can learn how it works and use that knowledge to create something that has never existed in nature and might be superior to it.” The seven resulting compounds, called bryologs, share two fundamental features with the original bryostatin: the recognition domain, which directly contacts protein kinase C, and the spacer domain, which allows the bryolog-protein kinase C complex to be inserted into the cell membrane.

The researchers tested the new compounds’ ability to reactivate viral reservoirs in J-Lat cell lines, which contain latent HIV and begin to fluoresce when they express the virus. In the J-Lat line, bryologs induced virus in as many or more cells than bryostatin at a variety of concentrations, and ranged from 25 to 1,000 times more potent than prostratin. The compounds showed no toxic effects. Bryolog testing remains in the early stages — the researchers are currently conducting in vivo studies in animal models. But practical bryostatin substitutes may be the first step toward true HIV-eradication therapy.

Science Daily
August 7, 2012

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Differences between human twins at birth highlight importance of intrauterine environment

Your genes determine much about you, but environment can have a strong influence on your genes even before birth, with consequences that can last a lifetime. In a study published online in Genome Research, researchers have for the first time shown that the environment experienced in the womb defines the newborn epigenetic profile, the chemical modifications to DNA we are born with, that could have implications for disease risk later in life. Epigenetic tagging of genes by a chemical modification called DNA methylation is known to affect gene activity, playing a role in normal development, aging, and also in diseases such as diabetes, heart disease, and cancer. Studies conducted in animals have shown that the environment shapes the epigenetic profile across the genome, called the epigenome, particularly in the womb. An understanding of how the intrauterine environment molds the human epigenome could provide critical information about disease risk to help manage health throughout life.

Twin pairs, both monozygotic (identical) and dizygotic (fraternal), are ideal for epigenetic study because they share the same mother but have their own umbilical cord and amniotic sac, and in the case of identical twins, also share the same genetic make-up. Previous studies have shown that methylation can vary significantly at a single gene across multiple tissues of identical twins, but it is important to know what the DNA methylation landscape looks like across the genome. In this report, an international team of researchers has for the first time analyzed genome-scale DNA methylation profiles of umbilical cord tissue, cord blood, and placenta of newborn identical and fraternal twin pairs to estimate how genes, the shared environment that their mother provides and the potentially different intrauterine environments experienced by each twin contribute to the epigenome. The group found that even in identical twins, there are widespread differences in the epigenetic profile of twins at birth. “This must be due to events that happened to one twin and not the other,” said Dr. Jeffrey Craig of the Murdoch Childrens Research Institute (MCRI) in Australia and a senior author of the report. Craig added that although twins share a womb, the influence of specific tissues like the placenta and umbilical cord can be different for each fetus, and likely affects the epigenetic profile.

Interestingly, the team found that methylated genes closely associated with birth weight in their cohort are genes known to play roles in growth, metabolism, and cardiovascular disease, lending further support to a known link between low birth weight and risk for diseases such as diabetes and heart disease. The authors explained that their findings suggest the unique environmental experiences in the womb may have a more profound effect on epigenetic factors that influence health throughout life than previously thought. Furthermore, an understanding of the epigenetic profile at birth could be a particularly powerful tool for managing future health. “This has potential to identify and track disease risk early in life, said Dr. Richard Saffery of the MCRI and a co-senior author of the study, “or even to modify risk through specific environmental or dietary interventions.”

Science Daily
August 7, 2012

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Computer-designed proteins programmed to disarm a variety of flu viruses

Computer-designed proteins are under construction to fight the flu. Researchers are demonstrating that proteins found in nature, but that do not normally bind the flu, can be engineered to act as broad-spectrum antiviral agents against a variety of flu virus strains, including H1N1 pandemic influenza. “One of these engineered proteins has a flu-fighting potency that rivals that of several human monoclonal antibodies,” said Dr. David Baker, professor of biochemistry at the University of Washington, in a report in Nature Biotechnology. Baker’s research team is making major inroads in optimizing the function of computer-designed influenza inhibitors. These proteins are constructed via computer modeling to fit exquisitely into a specific nano-sized target on flu viruses. By binding the target region like a key into a lock, they keep the virus from changing shape, a tactic that the virus uses to infect living cells. The research efforts, akin to docking a space station but on a molecular level, are made possible by computers that can describe the landscapes of forces involved on the submicroscopic scale.

Baker heads the new Institute for Protein Design Center at the University of Washington. Biochemists, computer scientists, engineers and medical specialists at the center are engineering novel proteins with new functions for specific purposes in medicine, environmental protection and other fields. Proteins underlie all normal activities and structures of living cells, and also regulate disease actions of pathogens like viruses. Abnormal protein formation and interactions are also implicated in many inherited and later-life chronic disorders. Because influenza is a serious worldwide public health concern due to its genetic shifts and drifts that periodically become more virulent, the flu is one of the key interests of the Institutes for Protein Design and its collaborators in the United States and abroad. Researchers are trying to meet the urgent need for better therapeutics to protect against this very adaptable and extremely infective virus. Vaccines for new strains of influenza take months to develop, test and manufacture, and are not helpful for those already sick. The long response time for vaccine creation and distribution is unnerving when a more deadly strain suddenly emerges and spreads quickly. The speed of transmission is accelerated by the lack of widespread immunity in the general population to the latest form of the virus.

Flu trackers refer to strains by their H and N subtypes. H stands for hemagglutinins, which are the molecules on the flu virus that enable it to invade the cells of respiratory passages. The virus’s hemagglutinin molecules attach to the surface of cells lining the respiratory tract. When the cell tries to engulf the virus, it makes the mistake of drawing it into a more acidic location. The drop in pH changes the shape of the viral hemagglutinin, thereby allowing the virus to fuse to the cell and open an entry for the virus’ RNA to come in and start making fresh viruses. It is hypothesized that the Baker Lab protein inhibits this shape change by binding the hemagglutinin in a very specific orientation and thus keeps the virus from invading cells. Baker and his team wanted to create antivirals that could react against a wide variety of H subtypes, as this versatility could lead to a comprehensive therapy for influenza. Specifically, viruses that have hemagglutinins of the H2 subtype are responsible for the deadly pandemic of 1957 and continued to circulate until 1968. People born after that date haven’t been exposed to H2 viruses. The recent avian flu has a new version of H1 hemagglutinin. Data suggests that Baker’s proteins bind to all types of the Group I Hemagglutinin, a group that includes not just H1 but the pandemic H2 and avian H5 strains.

Recognizing the importance of new flu therapies to national and international security, the Defense Advanced Research Projects Agency and the Defense Threat Reduction Agency funded this work, along with the National Institutes of Health’s National Institute for Allergy and Infectious Diseases. The researchers also used the Advanced Photon Source at Argonne National Laboratories in Illinois, with support from the Department of Energy, Basic Energy Sciences. The methods developed for the influenza inhibitor protein design, Baker said, could be “a powerful route to inhibitors or binders for any surface patch on any desired target of interest.” For example, if a new disease pathogen arises, scientists could figure out how it interacts with human cells or other hosts on a molecular level. Scientists could then use protein interface design to generate a diversity of small proteins that they predict would block the pathogen’s interaction surface. Genes for large numbers of the most promising, computer-designed proteins could be tested using yeast cells. After further molecular chemistry studies to find the best binding among those proteins, those could be re-programmed in the lab to undergo mutations, and all the mutated forms could be stored in a “library” for an in-depth analysis of their amino acids, molecular architecture and energy bonds. Advanced technologies would allow the scientists to quickly thumb through the library to pick out those tiny proteins that clung to the pathogen surface target with pinpoint accuracy. The finalists would be selected from this pool for excelling at stopping the pathogen from attaching to, entering and infecting human or animal cells.

The use of deep sequencing, the same technology now used to sequence human genomes cheaply, was especially crucial in creating detailed maps relating sequencing to function. These maps were used to reprogram the design to achieve a more precise interaction between the inhibitor protein and the virus molecule. It also enabled the scientists, they said, “to leapfrog over bottlenecks” to improve the activity of the binder. They were able to see how small contributions from many tiny changes in the protein, too difficult to spot individually, could together create a binder with better attachment strength. “We anticipate that our approach combining computational design followed by comprehensive energy landscape mapping,” Baker said, “will be widely useful in generating high-affinity and high-specificity binders to a broad range of targets for use in therapeutics and diagnostics.”

eBioNews
June 26, 2012

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Long-lived rodents have high levels of brain-protecting factor

The typical naked mole rat lives 25 to 30 years, during which it shows little decline in activity, bone health, reproductive capacity and cognitive ability. What is the secret to this East African rodent’s long, healthy life? Scientists from the United States and Israel found a clue. From infancy to old age, naked mole rats are blessed with large amounts of a protein essential for normal brain function. “Naked mole rats have the highest level of a growth factor called NRG-1 in the cerebellum. Its levels are sustained throughout their life, from development through adulthood,” said Yael Edrey, doctoral student at The University of Texas Health Science Center San Antonio’s Barshop Institute for Longevity and Aging Studies. The Barshop Institute has the largest colony of naked mole rats in the U.S. — 2,000 rodents scampering around a network of tubes and cages in humid conditions that mimic their natural underground habitat. Edrey is the lead author of research that compared lifelong NRG-1 levels across seven species of rodents, from mice and guinea pigs to blind mole rats and Damaraland mole rats. NRG-1 levels were monitored in naked mole rats at different ages ranging from 1 day to 26 years. The other six rodent species have maximum life spans of three to 19 years. The cerebellum coordinates movements and maintains bodily equilibrium. The research team hypothesized that long-lived species would maintain higher levels of NRG-1 in this region of the brain, with simultaneous healthy activity levels. Among each of the species, the longest-lived members exhibited the highest lifelong levels of NRG-1. The naked mole rat had the most robust and enduring supply. “In both mice and in humans, NRG-1 levels go down with age,” Edrey said. Researchers have documented various characteristics of naked mole rat physiology, revealing the integrity of proteins in the liver, kidney and muscle. This is the first set of data evaluating species’ differences in a key factor involved in maintaining the integrity of the rodent’s brain.

“The strong correlation between this protective brain factor and maximum life span highlights a new focus for aging research, further supporting earlier findings that it is not the amount of oxidative damage an organism encounters that determines species life span but rather that the protective mechanisms may be more important,” said senior author Rochelle Buffenstein, Ph.D., professor of physiology and cellular and structural biology at the Barshop Institute. She is Edrey’s research mentor. The finding, while not directly applicable to humans, has many implications for NRG-1’s role in maintaining neuron integrity. Naked mole rats are burrowing rodents with a distinctive appearance — hairless with wrinkled pinkish skin, tiny eyes and protruding front teeth. Their native habitat is the Horn of Africa. The rodent’s capacity to resist cancer and maintain protein integrity in the face of oxidative damage makes it an ideal animal model for aging and biomedical research.

Science Daily
May 29, 2012

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California BSE prion comes with a different twist

Reports of ‘mad cow’ disease in the United States erupted in the news this week after the US Department of Agriculture (USDA) confirmed that the remains of a California dairy cow had tested positive for bovine spongiform encephalopathy (BSE). This marks the fourth case of BSE identified in the US, and the first case in six years. In spongiform encephalopathy diseases, abnormally folded prion proteins accumulate in the brain, causing other proteins to deform as well. BSE has proved to be unusually adept at jumping between species; humans exposed to BSE can develop its human counterpart: Creutzfeldt-Jakob disease (CJD). In a statement released on 24 April, Karen Ross, Secretary of the California Department of Food and Agriculture said, “The detection of BSE shows that the surveillance program in place in California and around the country is working.” Food safety advocates such as Yonkers, New York, -based Consumers Union say it’s a warning sign that surveillance is inadequate and needs to be stepped up. Ross’s statement also makes a point of noting a key feature of this particular case: The infected cow carried what is known as ‘L-type’ BSE, a version of the disease that has not been detected before in the US and has so far not been associated with transmission through animal feed. As the policy debate over testing rumbles on, here is a short guide to what is known and not known about this rare strain and its unexpected appearance.

After brain tissue samples turned up inconclusive results in California, scientists at the USDA’s Veterinary Research Laboratories in Ames, Iowa ran two additional biochemical tests and got positive results for BSE. The second technique, western blotting, separates proteins based on their molecular weight to create a pattern on a gel. L-type prions have low weights, and another atypical strain, H-type prions have high weights compared to classic (C) strains. This sample produced a clear L-type pattern, said Mark Hall, head of the pathology, parasitology, and entomology section at Ames. In addition to weighing less, L-type strains also produce different lesion profiles in the brain. Both H and L types are extremely rare and found in older cattle, with only around 30 reported cases each worldwide. Perhaps this contributes to the fact that we don’t know much about them. “Right now, the full pathogenesis is not well characterized,” says Hall of L-type. Unlike their classical counterparts, “past studies and current diagnostic tests to detect ruminant DNA and RNA in feed haven’t found atypical variants,” says Jim Cullor, a veterinarian at the University of California at Davis.

“There is some evidence in primates and transgenic mice that it seems to spread faster – meaning it maybe more virulent – but we don’t know how representative these models are of the disease in humans,” says Linda Detwiler, a clinical professor at Mississippi State University’s College of Veterinary Medicine. Whether or not L-type could jump species without direct injection into the brain remains unclear, but so far it can transmit to transgenic humanized, ovinized (modified to genetically mimic sheep), bovinized, and normal mice, as well as macaques in the lab. “We do know that L-type can transmit from one cow to another through injection in the brain,” says Detwiler. “Long term studies are beginning to look at whether or not it’s capable of transmission orally through feed, but we don’t have the data yet.” Whether these strains have been around for a long time or recently developed is still unknown. “No one knows the origin at this point,” says Detwiler. “It might be sporadic, similar to sporadic types of Creutzfeldt-Jakob disease in humans. It might be some kind of genetic mutation, or it could be a modification of classical BSE or TSE.” In a 2008 study, out of all known cases of atypical BSE, only one contained a genetic mutation. So, a mutant cause can’t be ruled out and could arise in countries that have never seen BSE. In humans, sporadic cases of CJD – those that aren’t caused by transmission from cattle – occur at random in humans without a clear cause.

The USDA bans cow material in cow feed and “specific risk material” – meaning bone, brain, and other organs where the disease persists – from all livestock feed. “The L-type’s infectivity alone reinforces the need for regulations,” says Detwiler. “It’s really important to monitor any emerging disease around the world so that we can change our policies if we need to.” Some feel that the regulations aren’t enough. Michael Hansen, a senior staff scientist with Consumer’s Union, notes that only .13% of the 30 million cattle that go to slaughter annually in the US are tested. “I would argue that’s not enough,” says Hansen. “The feed ban is not a firewall.” The USDA has sent samples to official World Animal Health (OIE) reference labs in Canada and Great Britain. An on-site farm investigation may reveal more about the source and potential extent of infection. Whether or not the case produces any astounding revelations in the coming weeks, it has already underscored the many general unknowns that researchers face when it comes to L-type. “It’s important to look at whether it can transmit orally into to cattle or other species that consume feed, and to figure out where infection occurs in case we need to look for different high risk material,” says Detwiler. “With continued research and epidemiology studies, we’ll get more clues.”

Nature
May 15, 2012

Original web page at Nature

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Sirtuin protein linked to longevity in mammals

At last, a member of the celebrated sirtuin family of proteins has been shown to extend lifespan in mammals — although it’s not the one that has received the most attention and financial investment. Sirtuin genes and the proteins they encode have intrigued many researchers who study ageing ever since they were first linked to longevity in yeast. Results published in Nature suggest that the overexpression of one gene, called sirtuin 6 (SIRT6), can lengthen lifespan in male mice by as much as 15.8%. Male mice with boosted levels of the sirtuin protein SIRT6 could live longer. For years, another member of the family, SIRT1, has hogged much of the spotlight because it is the mammalian member of the sirtuin clan most closely related to the longevity-linked yeast gene. Some researchers speculated that SIRT1 may also boost lifespan in mammals, and that it was the target of resveratrol, a compound found in red wine that had been linked to a variety of health benefits. Sirtuin fervour reached its height in 2008, when the London-based drug company GlaxoSmithKline paid US$720 million for a biotechnology company that was initially focused on finding SIRT1-activating compounds as possible treatments for type 2 diabetes. But since then, results suggesting that SIRT1 affects lifespan in the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans have been questioned. And no effect of SIRT1 on longevity in mammals has been reported, although its expression is associated with a healthier metabolism in mice fed a high-fat diet.

Amid the excitement about SIRT1, it was in part the relative obscurity of SIRT6 that drew molecular biologist Haim Cohen of Bar-Ilan University in Ramat-Gan, Israel, to study the gene. “People were mostly interested in SIRT1,” he says. “So I thought it might be better for us as a new lab to work on something that is less crowded.” In 2006, researchers had reported that mice lacking SIRT6 seemed to age more quickly. The mice were small and sickly, had a reduced capacity to repair damaged DNA, and died a month after birth. Cohen and his colleagues decided to find out what would happen if mice expressed higher levels of the SIRT6 protein than normal. They found that longevity in female mice was unaffected by the excess protein, but that the median lifespan of male mice rose by 14.5% in one line of their transgenic mice and 9.9% in another. Another measure of longevity, maximum lifespan (generally more valued by researchers into ageing because it is less likely to be affected by other factors such as changes in infant mortality), rose by 15.8% in the first line of mice, and 13.1% in the second, although the latter increase was not statistically significant. Furthermore, in the transgenic mice, levels of proteins involved in the ‘insulin-like growth factor 1’ pathway, which has been previously linked to longevity, were also affected by SIRT6 expression.

The results are interesting, and the magnitude of lifespan extension is impressive, says Richard Miller, who studies ageing at the University of Michigan in Ann Arbor. But the work must be interpreted with care, he adds. “It’s a good bet that each of the sirtuins does something interesting,” says Miller. “But the case for whether any one of them is important to ageing and longevity in mammals is somewhat weak and circumstantial.” The strain of mice used in the study is particularly prone to tumours, especially in males, says Miller. It’s possible, then, that the longer lifespans could be the result of an anti-cancer effect of SIRT6 rather than a direct effect on ageing. Cohen acknowledges that this is a possibility, but notes that statistical analyses found no evidence that differences in tumour rates were contributing to the longevity effects of SIRT6. David Lombard, a sirtuin researcher at the University of Michigan agrees with Miller, saying that it is important for researchers to directly address whether SIRT6 affects several of the conditions associated with ageing, such as cataract formation and declines in memory and mobility. Since the initial work with SIRT6-deficient mice was published, he notes, researchers have found that much of what initially seemed to be an accelerated rate of age-related degeneration may in fact be attributable to metabolic defects that cause extremely low blood-sugar levels.

And why does SIRT6 affect males and females differently? Cohen’s lab is trying to piece that together, but for now he can only offer speculation. He notes that in the strain of mice his team used, females live about 15% longer than males and that overexpression of SIRT6 simply allowed the males to catch up to the females. Perhaps, then, SIRT6 is mimicking effects already seen in the females of this strain of mice. In this context, Rafael de Cabo, who studies ageing at the National Institute on Ageing in Baltimore, Maryland, notes that the expression of some proteins in the transgenic mice producing excess SIRT6 matched the expression of those proteins in normal, control female mice. The new focus on SIRT6 does not mean that the other sirtuins have been left by the wayside, says Miller. Researchers are beginning to look at the effects of SIRT1 when expressed in specific tissues, and work on the other members of the family is continuing apace. “People are just beginning to come to grips with the fact that there are seven sirtuins and each may do different things,” says Miller. “The quicker people stop thinking in terms of ‘it’s either gold or tin’ and start addressing the nuances of sirtuin function, the better.”

Nature
March 6, 2012

Original web page at Nature

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Discovery of extremely long-lived proteins may provide insight into cell aging and neurodegenerative diseases

One of the big mysteries in biology is why cells age. Now scientists at the Salk Institute for Biological Studies report that they have discovered a weakness in a component of brain cells that may explain how the aging process occurs in the brain. The scientists discovered that certain proteins, called extremely long-lived proteins (ELLPs), which are found on the surface of the nucleus of neurons, have a remarkably long lifespan. While the lifespan of most proteins totals two days or less, the Salk Institute researchers identified ELLPs in the rat brain that were as old as the organism, a finding they reported February 3 in Science. The Salk scientists are the first to discover an essential intracellular machine whose components include proteins of this age. Their results suggest the proteins last an entire lifetime, without being replaced. ELLPs make up the transport channels on the surface of the nucleus; gates that control what materials enter and exit. Their long lifespan might be an advantage if not for the wear-and-tear that these proteins experience over time. Unlike other proteins in the body, ELLPs are not replaced when they incur aberrant chemical modifications and other damage.

Damage to the ELLPs weakens the ability of the three-dimensional transport channels that are composed of these proteins to safeguard the cell’s nucleus from toxins, says Martin Hetzer, a professor in Salk’s Molecular and Cell Biology Laboratory, who headed the research. These toxins may alter the cell’s DNA and thereby the activity of genes, resulting in cellular aging. Funded by the Ellison Medical Foundation and the Glenn Foundation for Medical Research, Hetzer’s research group is the only lab in the world that is investigating the role of these transport channels, called the nuclear pore complex (NPC), in the aging process. Previous studies have revealed that alterations in gene expression underlie the aging process. But, until the Hetzer lab’s discovery that mammals’ NPCs possess an Achilles’ heel that allows DNA-damaging toxins to enter the nucleus, the scientific community has had few solid clues about how these gene alterations occur. “The fundamental defining feature of aging is an overall decline in the functional capacity of various organs such as the heart and the brain,” says Hetzer. “This decline results from deterioration of the homeostasis, or internal stability, within the constituent cells of those organs. Recent research in several laboratories has linked breakdown of protein homeostasis to declining cell function.”

The results that Hetzer and his team just report suggest that declining neuron function may originate in ELLPs that deteriorate as a result of damage over time. “Most cells, but not neurons, combat functional deterioration of their protein components through the process of protein turnover, in which the potentially impaired parts of the proteins are replaced with new functional copies,” says Hetzer. “Our results also suggest that nuclear pore deterioration might be a general aging mechanism leading to age-related defects in nuclear function, such as the loss of youthful gene expression programs,” he adds. The findings may prove relevant to understanding the molecular origins of aging and such neurodegenerative disorders as Alzheimer’s disease and Parkinson’s disease.

Science Daily
February 21, 2012

Original web page at Science Daily

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One molecule for muscle growth and insulin sensitivity

Two independent studies in the Nov.11 issue of the journal Cell, a Cell Press publication, suggest a common way to pump up muscles and prevent diabetes. The key is a molecule required for fine-tuning metabolism by selectively and subtly modifying core metabolic programs. Researchers show that loss of this molecule specifically in muscle produces mice with more fat-burning muscle and greater exercise capacity. “We turned mice into super-marathon mice,” said Johan Auwerx of École Polytechnique Fédérale de Lausanne. “They had more stamina and more endurance.” Another group of researchers show that loss of the same molecule specifically in fat cells produces mice that become more insulin sensitive even as they grow fatter. Despite being fat, the mice are a lot like animals on diabetes drugs known collectively as TZDs (thiazolidinediones) minus the side effects of water retention and heart disease. “They were more glucose tolerant, even though they were more obese,” said Jerrold Olefsky of the University of California, San Diego. “They were less insulin resistant and had less systemic inflammation — all features common to TZD treatment.”

This molecule is called NCoR. It integrates complex signaling pathways, adjusting specific metabolic programs in a manner similar to a dimmer switch, Auwerx explains. The particular genes it acts on apparently vary with cell type. These studies are surprising because earlier work had shown that complete loss of NCoR early in development is fatal. Scientists, including Auwerx and Olefsky, had anticipated that NCoR in specific adult cells would have broader effects than it does. What these findings suggest is that limiting the levels or activity of NCoR could improve human metabolism for the better. It might be possible to produce drugs that specifically target NCoR activity only in one tissue or another. Olefsky’s work suggests that fat is the prime target for improving insulin sensitivity since the mice with changes only in adipose experience systemic improvements. “There is no doubt where this begins,” Olefsky says, “and with this adipocyte [NCoR] knockout you get systemic insulin sensitivity; the liver and muscle gets better too.” The fact that NCoR deficiency comes with benefits in two totally different contexts makes such a treatment strategy that much more compelling, Auwerx said. “At the end of the day, it’s doing something good for metabolism,” Olefsky said.

Science Daily
February 7, 2012

Original web page at Science Daily

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Septin proteins take bacterial prisoners

Cellular proteins called septins might play an important part in the human body’s ability to fight off bacterial infections, according to a study. Septins are found in many organisms, and are best known for building scaffolding to provide structural support during cell division and to rope off parts of the cell. However, most studies of septins, or guanosine-5′-triphosphate (GTP) binding proteins, have been confined to yeast cells. The latest research in human cells suggests that septins build ‘cages’ around bacterial pathogens, immobilizing the harmful microbes and preventing them from invading other healthy cells. This cellular defence system could help researchers to create therapies for dysentery and other illnesses, the researchers say. “This is a new way for cells to control an infection,” says Pascale Cossart, a cell biologist at the Pasteur Institute in Paris, who presented the findings in a poster session at the annual meeting of the American Society for Cell Biology in Denver, Colorado.

The researchers discovered the caging behaviour with Shigella, a bacterium that causes sometimes lethal diarrhoea in humans and other primates. To propagate from cell to cell, Shigella bacteria develop actin-polymer ‘tails’, which propel the microbes around and allow them to force their way into neighbouring host cells. To counterattack, human cells produce a cell-signalling protein called TNF-α. The researchers found that when TNF-α is present, thick bundles of septin filaments encircle the microbes. This, in turn, interferes with tail formation and stops Shigella in its tracks. Microbes that become trapped in septin cages are broken down in a stage of the cell’s life cycle called autophagy. “Autophagy is more efficient because of the septin cage, and the septin cage does not occur if you do not have the autophagy,” says Cossart.

Nature
January 10, 2012

Original web page at Nature

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Novel prion protein in BSE-affected cattle

Bovine spongiform encephalopathy (BSE) is a feed-borne prion disease that affects mainly cattle but also other ruminants, felids, and humans. Currently, 3 types of BSE have been distinguished by Western immunoblot on the basis of the signature of the proteinase K–resistant fragment of the pathologic prion protein (PrPres): the classic type of BSE (C-BSE) and 2 so-called atypical types of BSE with higher or lower molecular masses of PrPres (H-BSE and L-BSE, respectively). C-BSE is transmitted to cattle by ingestion of contaminated meat-and-bone meal, a feed supplement produced from animal carcasses and by-products. H-BSE and L-BSE have been identified by active disease surveillance, and incidence in aged cattle is low; but little is known about their epidemiology, pathobiology, and zoonotic potential. We describe 2 recent cases of BSE in aged cattle in Switzerland in which a PrPres phenotype distinct from those of C-, L- and H-BSE was unexpectedly displayed.

In April 2011, an 8-year-old cow (cow 1) died of accidental injury, with no apparent precedent clinical signs, on a farm in the canton of St. Gallen, Switzerland. In the context of active surveillance for BSE, the medulla oblongata was tested and found to be BSE positive by using the PrioStrip test (Prionics AG, Schlieren, Switzerland), a lateral-flow immunochromatographic assay for detection of PrPres. One month later, another cow (cow 2), 15 years of age, in the canton of Berne, Switzerland, was slaughtered because of a hind limb fracture. Information on this animal’s health status before death was unavailable. Statutory testing of the medulla oblongata gave a BSE-positive result by using the Prionics Check Western, a rapid Western blot technique. Medulla oblongata samples from the 2 animals were forwarded to the National Reference Laboratory for confirmatory testing. In accordance with the guidelines of the World Organisation for Animal Health, BSE was confirmed for each animal by positive test results in independent, approved screening tests, of which 1 must be a Western blot.

Because the tissues were severely autolyzed, target structures for the diagnosis of BSE could not be identified, and histopathologic and immunohistochemical results were inconclusive. The Prionics Western blot detected a similar 3-band PrPres glycoprofile with molecular masses of roughly 16, 20, and 25 kDa for each animal, lower than equivalent PrP protein bands detected in animals with C-BSE. Sequencing of the open reading frame of the PRNP gene of cow 2 (which was unsuccessful for cow 1) indicated that the encoded protein was identical to the common bovine PrP amino acid sequence (as translated from GenBank accession no. AJ298878) and therefore was not likely to account for the differences observed by Western blot testing. We next investigated which region of the prion protein was present in these aberrant PrPres fragments by probing with a panel of antibodies in the Western blot that bind to different regions of the prion protein. PrPres in cows 1 and 2 was readily detected by antibodies Sha31, 94B4, and JB10. By contrast, antibody 9A2, which maps to the PrPres N terminus, bound only to PrPres in samples from animals with C-, L- and H- BSE but not in samples from cows 1 and 2. The molecular masses of the PrPres moieties from the 2 cows were also clearly distinct from those from controls with L- and H-BSE. For samples from animals with H-BSE, enzymatic deglycosylation demonstrated PrPres subtypes, 1 and 2, the latter being a C-terminal PrPres fragment of 12–14 kDa. To investigate whether the novel PrPres type corresponds to PrPres subtype 2, we compared samples from cow 2 with those from the H-BSE control by Western blot. The PrPres type from the 2 cows reported here and PrPres subtype 2 from the H-BSE control were indeed distinct.

We report a novel PrPres signature in 2 cows with BSE diagnoses determined according to established criteria. Combining Western blot analysis with an epitope mapping strategy, we ascertained that these animals displayed an N terminally truncated PrPres different from currently classified BSE prions. The interpretation of these findings remains difficult because neuropathologic and systematic clinical data for the 2 cases are not available. Moreover, the tissue samples were autolyzed, and the question of whether this affected the PrPres molecular signature is of concern. Nonetheless, our findings raise the possibility that these cattle were affected by a prion disease not previously encountered and distinct from the known types of BSE. To confirm this possibility and to assess a potential effect on disease control and public health, in vivo transmission studies using transgenic mouse models and cattle are ongoing. Until results of these studies are available, molecular diagnostic techniques should be used so that such cases are not missed.

Emerging Infectious Diseases
December 13, 2011

Original web page at Emerging Infectious Diseases

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Diseased hearts to heal themselves in future

Oncostatin M regulates the reversion of heart muscle cells into precursor cells and is vitally important for the self-healing powers of the heart. Cellular reversion processes arise in diseases of the heart muscle, for example myocardial infarction and cardiomyopathy, which limit the fatal consequences for the organ. Scientists from the Max Planck Institute for Heart and Lung Research in Bad Nauheim and the Schüchtermann Klinik in Bad Rothenfelde have identified a protein which fulfils a central task in this reversion process by stimulating the regression of individual heart muscle cells into their precursor cells. They now plan to improve the self-healing powers of the heart with the help of this protein. In order to regenerate damaged heart muscle as caused by a heart attack, for example, the damaged muscle cells must be replaced by new ones. The number of cells to be replaced may be considerable, depending on the extent of the damage caused. Simpler vertebrates like the salamander adopt a strategy whereby surviving healthy heart muscle cells regress into an embryonic state. This process, which is known as dedifferentiation, produces cells which contain a series of stem cell markers and re-attain their cell division activity. Thus, new cells are produced which convert, in turn, into heart muscle cells. The cardiac function is then restored through the remodelling of the muscle tissue.

An optimised repair mechanism of this kind does not exist in humans. Although heart stem cells were discovered some time ago, exactly how and to what extent they play a role in cardiac repair is a matter of dispute. It has only been known for a few years that processes comparable to those found in the salamander even exist in mammals. Thomas Braun’s research group at the Max Planck Institute for Heart and Lung Research in Bad Nauheim has now discovered the molecule responsible for controlling this dedifferentiation of heart muscle cells in mammals. The scientists initially noticed the high concentration of oncostatin M in tissue samples from the hearts of patients suffering from myocardial infarction. It was already known that this protein is responsible for the dedifferentiation of different cell types, among other things. The researchers therefore treated cultivated heart muscle cells with oncostatin M in the laboratory and were then able to trace the regression of the cells live under the microscope: “Based on certain changes in the cells, we were able to see that almost all heart muscle cells had been dedifferentiated within six days of treatment with oncostatin M,” explains Braun. “We were also able to demonstrate the presence of various stem cell markers in the cells. This should be understood as an indicator that these cells had been switched to a repair mode.”

Using a mouse infarct model, the Max Planck researchers succeeded in demonstrating that oncostatin M actually does stimulate the repair of damaged heart muscle tissue as presumed. One of the two test groups had been modified genetically in advance to ensure that the oncostatin M could not have any effect in these animals. “The difference between the two groups was astonishing. Whereas in the group in which oncostatin M could take effect almost all animals were still alive after four weeks, 40 percent of the genetically modified mice had died from the effects of the infarction,” says Braun. The reason for this was that oncostatin M ensured clearly quantifiable better cardiac function in the unmodified animals. The scientists in Bad Nauheim would now like to find a way of using oncostatin M in treatment. The aim is to strengthen the self-healing powers of the damaged heart muscle and to enable the restoration of cardiac function for the first time. The downside, however, is that oncostatin M was also observed to be counterproductive and exacerbated the damage in an experiment on a chronically diseased heart. “We believe that oncostatin M has considerable potential for efficiently healing damaged heart muscle tissue. What we now need is to be able to pinpoint the precise window of application to prevent any possible negative effects,” says Braun.

Science Daily
November 29, 2011

Original web page at Science Daily

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Warning on neural technique

Chemical marker that is commonly used to identify newly generated cells in the brain may be distorting the results of studies of neurogenesis, according to research published today in The Journal of Neuroscience. Bromodeoxyuridine (BrdU) is a synthetic analogue of thymidine, one of the building blocks of DNA. When injected into animals, it becomes incorporated into newly synthesized DNA, enabling researchers to identify cells that are dividing. The technique has been used routinely for decades to identify replicating cells, and particularly to investigate neurogenesis, the generation of neurons in the developing brain. It is also widely used in studies of neurogenesis in the adult mammalian brain. “BrdU has contributed enormously to understanding the timing and pattern of neurogenesis,” says Pasko Rakic, a neurobiologist at Yale University School of Medicine in New Haven, Connecticut, who performed the work with colleague Alvaro Duque.

Rakic and Duque injected seven rhesus monkeys with BrdU and seven others with tritiated thymidine, another DNA marker, at various stages of embryonic development. Previous studies have shown that the two markers produce similar results in rodents. The researchers used monkeys because these animals have larger brains that develop more slowly, enabling cellular events to be analysed in greater detail. The authors examined the animals’ brains at between two-and-a-half and three months of age, and found differences in the number and distribution of labelled cells between the two groups, with BrdU-labelled cells dispersed more widely than those labelled with tritiated thymidine. These differences occur, the researchers say, because the structure of BrdU differs markedly from that of thymidine. This may result in random mutations that have unpredictable effects on the cells being studied. “Many neuroscientists who use BrdU may not be familiar with its side effects on DNA structure and function and perhaps do not know the full potential of its toxicity,” says Rakic. Furthermore, BrdU also labels cells that are dying or repairing their DNA. “BrdU is not a specific marker for new cells,” Rakic says. “The caveat is that all dividing cells are labelled, but not all labelled cells are dividing.” In studies of embryonic development, when much cell division is occurring, these factors are not hugely problematic.

They could, however, distort the data in studies using BrdU to quantify cell proliferation in postnatal and adult brain tissue. “This is a scary thought for scientists studying neurogenesis,” says Jason Snyder, a neurobiologist at the National Institutes of Health’s Neuroplasticity Unit in Bethesda, Maryland, who uses the method to investigate adult neurogenesis in rodents. “It is fortunate that the differences are not observed in rodents,” he adds, “because the majority of neurogenesis studies are performed using rats and mice. But BrdU could still have more subtle side effects in rodents, and this study reminds us to be aware of this possibility.” Despite these limitations, BrdU is still the most reliable and cheapest method for studying the generation, movement and ultimate fate of neurons. But, says Henry Kennedy, director of research at the Stem-cell and Brain Research Institute in Bron, part of France’s national biomedical agency INSERM, the study “comes as a much needed reminder of the limitations of these markers”.

Nature
November 1, 2011

Original web page at Nature

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Protein reveals secret of stem cell pluripotency

A protein that helps maintain mouse stem cell pluripotency has been identified by researchers at the RIKEN Omics Science Center. The finding, published in the August issue of Stem Cells, points the way to advances in regenerative medicine and more effective culturing techniques for human pluripotent stem cells. Through their capacity to differentiate into any other type of cell, embryonic stem cells (ES cells) and induced-pluripotent stem cells (iPS cells) promise a new era of cell-based treatments for a wide range of conditions and diseases. Cultivating such cells, however, commonly relies on the use of so-called “feeder” cells to maintain pluripotency in cell culture conditions. Feeder cells keep stem cells in their undifferentiated state by releasing nutrients into the culture medium, but they have the potential to introduce contamination which, in humans, can lead to serious health risks. Previous research has shown that mouse pluripotent stem cells can be cultured without feeder cells through the addition of a cytokine called Leukemia Inhibitory Factor (LIF) to the culture media (“feeder-free” culture). LIF is secreted by mouse feeder cells and activates signal pathways reinforcing a stem cell regulatory network. The researchers discovered early in their investigation, however, that the amount of LIF secreted from feeder cells is much less than the amount needed to maintain pluripotency in feeder-free conditions. This points to other, as-of-yet unknown contributing factors.

To clarify these factors, the research group analyzed differences in gene expression between mouse iPS cells cultured on feeder cells and those cultured in feeder-free (LIF treated) conditions. Their results revealed 17 genes whose expression level is higher in feeder conditions. To test for possible effects on pluripotency, they then selected 7 chemokines (small proteins secreted by cells) from among these candidates and overexpressed them in iPS cells grown in feeder-free conditions. They found that one chemokine in particular, CC chemokine ligand 2 (CCL2), enhances the expression of key pluripotent genes via activation of a well-known signal pathway known as Jak/Stat3. While CCL2 is known for its role in recruiting certain cells to sites of infection or inflammation, the current research is the first to demonstrate that it also helps maintain iPS cell pluripotency. The findings also offer broader insights applicable to the cultivation of human iPS/ES cells, setting the groundwork for advances in regenerative medicine.

Science Daily
October 4, 2011

Original web page at Science Daily

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Scientists reengineer antibiotic to overcome dangerous antibiotic-resistant bacteria

A team of scientists from The Scripps Research Institute has successfully reengineered an important antibiotic to kill the deadliest antibiotic-resistant bacteria. The compound could one day be used clinically to treat patients with life-threatening and highly resistant bacterial infections. The results were published in an advanced online issue of the Journal of the American Chemical Society. “These results have true clinical significance and chart a path forward for the development of next generation antibiotics for the treatment of the most serious resistant bacterial infections,” said Dale L. Boger, who is Richard and Alice Cramer Professor of Chemistry at The Scripps Research Institute and senior author of the new study. “The result could not be predicted. It really required the preparation of the molecule and the establishment of its properties.” The compound synthesized is an analogue of the well-known commercial antibiotic vancomycin. The new analogue was prepared in an elegant total synthesis, a momentous achievement from a synthetic chemistry point of view. “In addition to the elegantly designed synthesis,” said Jian Xie, postdoctoral fellow in Boger’s group and first author on the publication, “I am exceedingly gratified that our results could have the potential to be a great service to mankind.”

Vancomycin is an antibiotic of last resort, which is used only after treatment with other antibiotics has failed. Clinically, it is used to treat patients that are either infected with the virulent methicillin-resistant Staphylococcus aureus (MRSA), individuals on dialysis, or those allergic to beta-lactam antibiotics (penicillin, cephalosporins). The drug was first used clinically in the 1950s, and the first vancomycin-resistant bacterial strains were isolated in the 1980s. Vancomycin normally works by grabbing hold of and sequestering the bacterial cell-wall making machinery, a peptidoglycan (carbohydrate and peptide containing molecule). Only Gram-positive bacteria have a cell wall, which is a membrane on the cell’s outer surface. The antibiotic binds so tightly to the peptidoglycan that the bacteria can no longer use the machinery to make their cell wall and thus die. Unfortunately, bacteria have found a way to alter the peptidoglycan in such a way that the antibiotic can no longer grab hold. Think of it as trying to hold a ball without any fingers. Biochemically the bacteria express a mutant form of the peptidoglycan in which properties of a key atom used in the recognition process are changed. This simply means where there once was something attractive there is now something repulsive. Chemically, the bacteria replace an amide (carbonyl, RC=O linked to an amine) with an ester (a carbonyl, RC=O linked to an oxygen, O).

This one atom change changes the entire game and renders vancomycin ineffective. Until now. Like magnets, molecular interactions can be attractive (oppositely charged) or repulsive (identically charged). What chemists in the Boger lab have done is made this key interaction no longer repulsive, but attractive. So now the new vancomycin analogue can grab hold of the mutant peptidoglycan, and again prevent the bacteria from making the cell wall and killing the resistant bacteria. But what is so remarkable about the design is that the redesigned antibiotic maintains its ability to bind the wild type peptidoglycan as well. Changing the properties of a key amide at the core of the natural products structure required a new synthetic strategy that only the most talented chemists could achieve in the lab. The preparation of the entire structure took a great deal of time and a fresh approach. The new compound has an amidine (an iminium, RC=NH+ linked to a nitrogen, N) instead of an amide at a key position buried in the interior of the natural product. However, to install such a functional group, the chemical properties of the amide carbonyl were not useful, as the natural product has seven of them. Instead, the group relied on the chemical properties of sulfur (S), oxygen’s downstairs neighbor in the periodic table, to install the desired nitrogen. To do this, a second analogue was prepared in which the key amide was chemically altered to a thioamide. “The thioamide allowed us to make any modification at the residue 4 amide that we would like to make, such as the amidine, but we could also make the methylene analogue,” said Boger citing work published in another paper (B. Crowley and D. L. Boger, J. Am. Chem. Soc. 128: 2885-2892). “And there are other modifications that we are making at the present time that we haven’t disclosed. We are just getting to that work.”

The most fundamental finding in the synthesis was that the installation of the amidine could be done in the last step, as a single-step conversion, on the fully unprotected thioamide analogue. Providing an elegant and novel approach to the analogue, which contrasts other published multistep procedures. This chemical behavior was, as Boger said, “an astonishing result as there are no protecting groups and it is a single step reaction… in the end it was the simplest and most straightforward way to prepare the amidine.” Although it is still at its early stages and there is much work ahead. Currently, the only route known to make the new antibiotic is the one published by Boger and his co-workers, which presently provides laboratory amounts of the compound. So Professor Boger now looks forward and will continue to investigate the “host of alternative approaches” for the preparation of the molecule “such as reengineered organisms to produce the material or semi-synthetic approaches to the analogue. That is going to be part of the next stage of the work.”

Science Daily
September 6, 2011

Original web page at Science Daily

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New understanding of biomarkers could lead to earlier diagnosis of fatal diseases

A new research paper sheds light on the way antibodies distinguish between different but closely related ‘biomarkers’ — proteins which reveal information about the condition of the human body. This new understanding could enable pharmaceutical companies to develop new technologies for quickly diagnosing and treating fatal diseases. All diseases have proteins, or concentrations of proteins, specifically linked to them called biomarkers. Identifying these can prove a powerful diagnostic tool. These biomarkers are detected by immunoassays — a test which mixes a substance (e.g. blood, urine) with antibodies, which bind to the protein if it is present. The antibodies can then be measured to identify the level of the biomarker, which in turn indicates the presence and extent of an illness. Antibodies bind with high specificity to one protein molecule or a limited group of molecules (e.g. hormones), which is why we can use antibodies to test for specific biomarkers. Problems arise when they bind to groups of similar hormones that are associated with normal bodily changes. This leads to false positives and therefore unreliable information. New research, carried out by the National Physical Laboratory (NPL), the University of Edinburgh and industrial partners from the UK (Mologic ltd), US (IBM’s Watson Research Center) and the Netherlands (Pepscan Presto BV), changes this. The research shows how different proteins are made up, and therefore how they can be identified reliably.

The highly sought solution is ‘intelligent selection’ of antibody-specific interaction sites on hormones that can differ from similar sites of other hormones by just one molecule. The research focused on hCG (human chorionic gonadotropin), a hormone produced during pregnancy. A subunit of hCG — hCGβ — is secreted by some cancers, meaning detection can give early warning of tumors. hCG is very similar to other reproductive hormones, known as LH and FSH, which are always present in the body. Detecting hCG can be confused with these other hormones, leading to unreliable results. The immunoassay antibodies bind to a tiny part of the hormone called an epitope. Hormones are made up of thousands of ‘building blocks’, with epitopes making up less than 10 of these blocks. The difference between hormones can be as little as one of these epitope blocks. The research team took a variety of precise measurements of the hCG hormone. The team showed how very subtle, atomic level characteristics define the antibody selectivity in closely related epitopes of different proteins. They identified that specific antibodies are highly selective in immunoassays and can distinguish between hCGβ and closely related LH fragments. Understanding these structural differences explains the observed selectivity in the full hormones. Armed with this knowledge, scientists can develop intelligent epitope selection to achieve the required assay performance. This means reliable tests can be developed to identify the presence of different hormones — in this case the presence of hCGβ which indicates cancer, as opposed to LH, which is always present.

The advances described in this research will enable development of further immunoassays to identify other biomarkers from similar groups. Pharmaceutical companies could use this to develop new technologies for diagnostics and clinical disease treatments, for example tests for tumour as part of routine screenings. Max Ryadnov, Principal Research Scientist at the National Physical Laboratory, says “This work answers one of the big questions in distinguishing biomarkers which are critical for identifying and treating serious diseases. We hope this breakthrough will underpin the development of a range of new diagnostic techniques and treatment.” Prof Paul Davis, Chief Scientific Officer of Mologic ltd — a UK diagnostic company that initiated the study, said: “It was a great collaborative effort, and it stands as a fine example of what can be achieved when motivated scientists work together openly across boundaries.”

Science Daily
August 9, 2011

Original web page at Science Daily

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New method used to detect 20 drugs in cow, goat and human milk

A Spanish-Moroccan research team has developed a method that makes it possible to simultaneously detect 20 pharmaceutical products in cow, goat and human milk. The samples of the three types of milk studied showed that they all contain anti-inflammatories, although the largest number of drugs was found in whole cows’ milk. Up to 20 kinds of antibiotics, anti-inflammatories, antiseptics, lipid regulators, beta-blockers and hormones can be detected simultaneously in various kinds of milk, thanks to a new method developed by researchers at the universities of Jaén and Córdoba in Spain and the Abdelmalek Essaadi University in Morocco. “We used this methodology to analyse 20 samples of cows’ milk (fresh, whole, semi-skimmed, skimmed and powdered), goats’ milk (whole and semi-skimmed) and breast milk from human volunteers, and we found that the drug content differs according to the type of milk,” Evaristo Ballesteros, a researcher at the University of Jaén and the study director, said.

The highest number of pharmacological substances was found in whole cows’ milk, particularly niflumic acid, mefenamic acid and ketoprofen (three anti-inflammatory drugs) and the hormone 17-beta-estradiol. Niflumic acid was also found in goats’ milk, along with flunixin. The human milk analysed, meanwhile, also contained anti-inflammatory drugs (such as ibuprofen and naproxen), as well as the antiseptic triclosan and some hormones, such as 17-alfa-ethinyl estradiol, 17-beta-estradiol and estrone. The researchers acknowledge that the results of the study, published in the Journal of Agricultural and Food Chemistry, cannot be extrapolated to all kinds of milk in general due to the small number of samples analysed, but they say it does confirm the validity of the method. The technique uses a “system of continuous extraction of substances in solid phase” and classifies them using “gas chromatography-mass spectrometry.” “The validation results clearly show that this method is the most sensitive and one of the most selective described to date in the scientific literature,” explains Ballesteros. “It is also highly precise and exact, with short analysis times (around 30 minutes).” The scientists believe the new methodology will help to provide a more effective way of determining the presence of these kinds of contaminants in milk or other products. Food quality control laboratories could use this new tool to detect these drugs before they enter the food chain. “This would raise consumers’ awareness and give them the knowledge that food, aside from its good organoleptic properties and good value, is also harmless, pure, genuine, beneficial to health and free of toxic residues,” the researcher concludes.

Science Daily
July 26, 2011

Original web page at Science Daily

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Researchers identify mechanism that seems to protect brain from aging

Researchers from the Universities of Bonn and Mainz have discovered a mechanism that seems to protect the brain from aging. In experiments with mice, they switched off the cannabinoid-1 receptor. As a consequence, the animals showed signs of degeneration — as seen in people with dementia — much faster. Humans are getting older and older, and the number of people with dementia is increasing. The factors controlling degeneration of the brain are still mostly unknown. However, researchers assume that factors such as stress, accumulation of toxic waste products as well as inflammation accelerate aging. But, vice versa, there are also mechanisms that can — like a bodyguard — protect the brain from degenerating, or repair defective structures. Researchers from the Universities of Bonn and Mainz have now discovered a hitherto unknown function of the cannabinoid-1 receptor (CB1). A receptor is a protein that can bind to other substances, triggering a chain of signals. Cannabinoids such as THC — the active agent in cannabis sativa — and endocannabinoids formed by the body bind to the CB1 receptors. The existence of this receptor is also the reason for the intoxicating effect of hashish and marijuana.

Not only does the CB1 receptor have an addictive potential, but it also plays a role in the degeneration of the brain. “If we switch off the receptor using gene technology, mouse brains age much faster,” said Önder Albayram, principal author of the publication and a doctoral student on the team of Professor Dr. Andreas Zimmer from the Institut für Molekulare Psychiatrie at the University of Bonn. “This means that the CB1 signal system has a protective effect for nerve cells.” The researchers studied mice in different age categories — young six week old animals, middle-aged ones at five months, and those of an advanced age at 12 months. The animals had to master various tasks — first, they had to find a submerged platform in the pool. Once the mice knew its location, the platform was moved, and the animals had to find it again. This was how the researchers tested how well the rodents learned and remembered. The animals in which the CB1 receptor had been switched off (the knock-out mice) clearly differed from their kind. “The knock-out mice showed clearly diminished learning and memory capacity,” said Privatdozent Dr. Andras Bilkei-Gorzo from Professor Zimmer’s team, who led the study. So, animals that did not have the receptor were less successful in their search for the platform. “In addition, they showed a clear loss of nerve cells in the hippocampus,” he explained further. This part of the brain is the central area for forming and storing information. In addition, the researchers found inflammation processes in the brain. As the mice advanced in age, the degenerative processes became increasingly noticeable.

The animals with the intact CB1 receptor, to the contrary, did clearly better with regard to their learning and memory capabilities, as well as the health of their nerve cells. “The root cause of aging is one of the secrets of life,” commented Albayram. This study has begun to open the door to solving this enigma. The processes in the mouse brains have a surprising number of parallels with age-related changes in human brains. So, the endocannabinoid system may also present a protective mechanism in the aging of the human brain. The principal author cautioned, “This will require additional research.” The scientists would like to better understand the mechanism by which CB1 receptors protect the brain from inflammation processes. And based on these signal chains, it might then be possible to develop substances for new therapies.

Science Daily
July 26, 2011

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Fifty-year search for calcium channel ends: Cell’s power generator depends on long-sought protein

Mitochondria, those battery-pack organelles that fuel the energy of almost every living cell, have an insatiable appetite for calcium. Whether in a dish or a living organism, the mitochondria of most organisms eagerly absorb this chemical compound. Because calcium levels link to many essential biological processes — not to mention conditions such as neurological disease and diabetes — scientists have been working for half a century to identify the molecular pathway that enables these processes. After decades of failed effort that relied on classic biochemistry and membrane protein purification, Vamsi Mootha, HMS associate professor of systems biology, and colleagues have discovered, through a combination of digital database mining and laboratory assays, the linchpin protein that drives mitochondria’s calcium machinery. “This channel has been studied extensively using physiology and biophysics, yet its molecular identity has remained elusive,” said Mootha, who also has appointments at Massachusetts General Hospital and at Broad Institute. “But thanks to the Human Genome Project, freely downloadable genomic databases, and a few tricks — we were able to get to the bottom of it.” These findings appeared online June 19 in Nature.

The results build on work from Vamsi and his group over the past decade. In 2008, he and his team published a near-comprehensive protein inventory, or proteome, of human and mouse mitochondria. This inventory, called MitoCarta, consisted of just over 1,000 proteins, most of which had no known function. In a September 2010 paper, Mootha’s group described using the MitoCarta inventory to identify the first protein specifically required for mitochondrial calcium uptake. Their strategy was simple. They knew that mitochondria from humans and Trypanosomes (a parasitical organism), but not baker’s yeast, are capable of absorbing large amounts of calcium. By simply overlapping the mitochondrial protein profiles of these three organisms, the group could spotlight roughly 50 proteins out of the 1,000 that might be involved with calcium channeling. They found that one protein, which they dubbed MICU1, is essential for calcium uptake. “That was an significant advance for the field,” says Mootha. “We showed that MICU1 was required for calcium uptake, but because it did not span the membrane, we doubted it was the central component of the channel. But what it provided us with was live bait to then go and find the bigger fish.”

Traditionally, researchers used standard laboratory methods for such a fishing exhibition, such as attaching biochemical hooks to the protein, casting it into the cell’s cytoplasm, then reeling it back in the hope that another, related protein will have bitten. But MICU1’s function as a regulator of a membrane channel made this technically prohibitive. Instead, graduate student Joshua Baughman and postdoctoral researcher Fabiana Perocchi went fishing in publicly available genomic databases. With MICU1 as their point of reference, they scoured those databases that measure whole genome RNA and protein expression, as well as an additional database containing genomic information for 500 species, and looked for proteins whose activity profile mirrored MICU1’s. A single anonymous protein with no known function stood out. The researchers named it MCU, short for “mitochondrial calcium uniporter.” To confirm that MCU is central to mitochondria’s calcium absorption, the team collaborated with Alnylam Pharmaceuticals, a company that leverages a laboratory tool called RNAi in order to selectively knock out genes in both cells and live animals. Using one of the company’s platforms, the researchers deactivated MCU in the livers of mice. While the mice displayed no immediate reaction, the mitochondria in their liver tissue lost the capacity to absorb calcium.

This basic science finding may prove relevant in certain human diseases. “We’ve known for decades now that neurons in the brains of people suffering from neurodegenerative disease are often marked by mitochondrial calcium overload,” said Mootha, an expert on rare mitochondrial diseases who sees patients at Massachusetts General Hospital when he’s not in the lab. “We also know that the secretion of many hormones, like insulin, are triggered by calcium spikes in the cell’s cytoplasm. By clearing cytosolic calcium, mitochondria can shape these signals. Scientists studying the nexus of energy metabolism and cellular signaling will be particularly interested in MICU1 and MCU. It’s still very early, but they could prove to be valuable drug targets for a variety of diseases — ranging from ischemic injury and neurodegeneration to diabetes.”

Science Daily
July 26, 2011

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The smell of a meat-eater

A chemical found in the urine of carnivores such as bobcats could shed light on the control of instinctive behaviour. If you are a small animal, it is useful to know whether there is anything around that might want to eat you. Stephen Liberles from Harvard Medical School in Cambridge, Massachusetts, and his colleagues have analysed urine samples from a variety of zoo inhabitants, including lions and bears, and discovered how rodents can use smell to do just that. In a research published today in the Proceedings of the National Academy of Science, the team identifies a chemical found in high concentrations in the urine of carnivores that makes mice and rats run for cover. Chemicals have already been identified that allow prey to recognize a known predator. But this is the first example of a generic clue that allows an animal to detect any potential predator, irrespective of whether the two species have ever come into contact. The researchers started by analysing an engimatic group of olfactory receptors discovered in 2001 called trace amine-associated receptors (TAARs). They are found in most vertebrates, in varying numbers. Mice, for example, have 15, rats 17 and humans have just 6. Very little is known about what chemicals bind to them. “A giraffe had to be trained to urinate in a cup.”

Liberles and his colleagues found that one member of the receptor family, TAAR4, is strongly activated by bobcat urine, which is sold online and used by gardeners to keep rodents and rabbits away. They managed to extract the molecule responsible for activating the receptor, called 2-phenylethylamine. They then wondered whether the molecule was specific to the bobcat. But the urine from other animals cannot always be bought as easily. “Also, commercial products may be contaminated, whereas we wanted to be sure we were studying only natural substances,” says David Ferrero, a graduate student in Liberles’s lab and first author of the study. So the researchers collected urine samples from a range of sources, including zoos in New England and South Dakota. Their collection covered 38 species from predators such as lions, snow leopards and servals to herbivores including cows, giraffes and zebra. They also tested humans, cats and various rodents.

The operation was not trivial. A giraffe had to be trained to urinate in a cup, and Ferrero had a nose-to-nose encounter with an uncooperative jaguar when the animal jumped against the bars as he approached its cage. Carnivores had by far the greatest concentration of 2-phenylethylamine in their urine, with the highest levels in lion, serval and tiger. Levels in the herbivores’ urine were up to 3,000 times lower. The chemical might be a by-product of digesting meat proteins, although the researchers have yet to confirm this idea. Liberles and his team double-checked the role of 2-phenylethylamine by placing a few drops of it – on its own, or within lion urine – in a cage. They found that mice and rats stayed away from that part of the cage. But when they used an enzyme to remove the chemical from lion urine, the drops no longer caused any reaction. “The role of TAAR receptors is still a bit of a mystery,” says Anna Menini, a physiologist at the International School for Advanced Studies in Trieste, Italy, and president-elect of the European Chemoreception Research Organization in Paris. “Here we have the first convincing evidence that they might control instinctive behaviour.”

She adds that the study questions a dogma in olfactory studies: that the olfactory receptors that trigger instinctive responses are found only in the vomeronasal organ, a part of the olfactory system that humans have lost. TAARs are in the olfactory epithelium — specialized tissue on the roof of the nasal cavity — which humans have, although they do not have an active gene for TAAR4 itself. The researchers are still missing the smoking gun for proving that TAAR4 directly controls the animal’s behaviour: a mouse in which this receptor has been knocked out should be fearless when faced with a carnivore’s urine. Liberles says he is working on this, as well as studying what brain circuits are activated by the receptor. “That is the big black box in neuroscience” he says. “We know a lot about perception and we can observe behaviour, but we need to find the circuits in the brain that bridge the two. TAAR4 offers a way to do that.”

Nature
July 12, 2011

Original web page at Nature

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New clues about aging: Genetic splicing mechanism triggers both premature aging syndrome and normal cellular aging

National Institutes of Health researchers have identified a new pathway that sets the clock for programmed aging in normal cells. The study provides insights about the interaction between a toxic protein called progerin and telomeres, which cap the ends of chromosomes like aglets, the plastic tips that bind the ends of shoelaces. The study by researchers from the National Human Genome Research Institute (NHGRI) appears online in the Journal of Clinical Investigation. Telomeres wear away during cell division. When they degrade sufficiently, the cell stops dividing and dies. The researchers have found that short or dysfunctional telomeres activate production of progerin, which is associated with age-related cell damage. As the telomeres shorten, the cell produces more progerin. Progerin is a mutated version of a normal cellular protein called lamin A, which is encoded by the normal LMNA gene. Lamin A helps to maintain the normal structure of a cell’s nucleus, the cellular repository of genetic information.

In 2003, NHGRI researchers discovered that a mutation in LMNA causes the rare premature aging condition, progeria, formally known as known as Hutchinson-Gilford progeria syndrome. Progeria is an extremely rare disease in which children experience symptoms normally associated with advanced age, including hair loss, diminished subcutaneous fat, premature atherosclerosis and skeletal abnormalities. These children typically die from cardiovascular complications in their teens. “Connecting this rare disease phenomenon and normal aging is bearing fruit in an important way,” said NIH Director Francis S. Collins, M.D., Ph.D., a senior author of the current paper. “This study highlights that valuable biological insights are gained by studying rare genetic disorders such as progeria. Our sense from the start was that progeria had a lot to teach us about the normal aging process and clues about more general biochemical and molecular mechanisms.” Collins led the earlier discovery of the gene mutation responsible for progeria and subsequent advances at NIH in understanding the biochemical and molecular underpinnings of the disease.

In a 2007 study, NIH researchers showed that normal cells of healthy people can produce a small amount of progerin, the toxic protein, even when they do not carry the mutation. The more cell divisions the cell underwent, the shorter the telomeres and the greater the production of progerin. But a mystery remained: What was triggering the production of the toxic progerin protein? The current study shows that the mutation that causes progeria strongly activates the splicing of lamin A to produce the toxic progerin protein, leading to all of the features of premature aging suffered by children with this disease. But modifications in the splicing of LMNA are also at play in the presence of the normal gene. The research suggests that the shortening of telomeres during normal cell division in individuals with normal LMNA genes somehow alters the way a normal cell processes genetic information when turning it into a protein, a process called RNA splicing. To build proteins, RNA is transcribed from genetic instructions embedded in DNA. RNA does not carry all of the linear information embedded in the ribbon of DNA; rather, the cell splices together segments of genetic information called exons that contain the code for building proteins, and removes the intervening letters of unused genetic information called introns. This mechanism appears to be altered by telomere shortening, and affects protein production for multiple proteins that are important for cytoskeleton integrity. Most importantly, this alteration in RNA splicing affects the processing of the LMNA messenger RNA, leading to an accumulation of the toxic progerin protein.

Cells age as part of the normal cell cycle process called senescence, which progressively advances through a limited number of divisions in the cell lifetime. “Telomere shortening during cellular senescence plays a causative role in activating progerin production and leads to extensive change in alternative splicing in multiple other genes,” said lead author Kan Cao, Ph.D., an assistant professor of cell biology and molecular genetics at the University of Maryland, College Park. Telomerase is an enzyme that can extend the structure of telomeres so that cells continue to maintain the ability to divide. The study supplied support for the telomere-progerin link, showing that cells that have a perpetual supply of telomerase, known as immortalized cells, produce very little progerin RNA. Most cells of this kind are cancer cells, which do not reach a normal cell cycle end point, and instead replicate out of control.

The researchers also conducted laboratory tests on normal cells from healthy individuals using biochemical markers to indicate the occurrence of progerin-generating RNA splicing in cells. The cell donors ranged in age from 10 to 92 years. Regardless of age, cells that passed through many cell cycles had progressively higher progerin production. Normal cells that produce higher concentrations of progerin also displayed shortened and dysfunctional telomeres, the tell-tale indication of many cell divisions. In addition to their focus on progerin, the researchers conducted the first systematic analysis across the genome of alternative splicing during cellular aging, considering which other protein products are affected by jumbled instructions as RNA molecules assemble proteins through splicing. Using laboratory techniques that analyze the order of chemical units of RNA, called nucleotides, the researchers found that splicing is altered by short telomeres, affecting lamin A and a number of other genes, including those that encode proteins that play a role in the structure of the cell. The researchers suggest that the combination of telomere fraying and loss with progerin production together induces cell aging. This finding lends insights into how progerin may participate in the normal aging process.

Science Daily
June 28, 2011

Original web page at Science Daily

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Similarities cause protein misfolding

A large number of illnesses stem from misfolded proteins, molecules composed of amino acids. Researchers at the University of Zurich have now studied protein misfolding using a special spectroscopic technique. Misfolding, as they report in Nature, is more frequent if the sequence of the amino acids in the neighboring protein domains is very similar. Proteins are the main molecular machines in our bodies. They perform a wide range of functions, from digesting and processing nutrients, converting energy and aiding cell structure to transmitting signals in cells and the whole body. In order to perform these highly specific functions, proteins have to adopt a well-defined, three-dimensional structure. Remarkably, in most cases they find this structure unaided once they have been formed out of their individual building blocks, amino acids, as a long chain molecule in the cell.

However, the process of protein folding can also go wrong, which means the proteins affected are no longer able to perform their function. In some cases, this can even have much more serious consequences if these misfolded proteins clump and trigger neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. In the course of evolution, a crucial factor in the development of proteins has thus been to avoid such “misfolding processes.” However, this is no easy task since the same molecular interactions that stabilize the correct structure of the individual proteins can also bring about interactions between protein molecules, causing them to misfold. Using a special spectroscopic method called single-molecule fluorescence, researchers from the Universities of Zurich and Cambridge have now studied the circumstances under which misfolding occurs. The team headed by Prof. Benjamin Schuler from the University of Zurich studied sections, or “domains,” of the largest protein in our bodies, titin, which helps the stability and elasticity of the muscle fibers. It is assumed that individual titindomains can unfold while the muscle is heavily exerted to avoid damaging the muscle tissue. When the muscle relaxes again, however, there is a danger that these unfolded domains might fold incorrectly. There is also a similar risk for other multidomain proteins.

For their study, the researchers attached small dye molecules as probes in the protein. “Using our laser-spectroscopic method we were able to determine distances on a molecular scale, i.e. down to a few millionths of a millimeter, through the energy transfer between the probes,” explains Prof. Schuler. This enabled the structures of correctly and misfolded proteins to be distinguished and thus the proportion of misfolding determined. “The study of different titin domains in our experiments revealed that the probability of misfolding increases if neighboring domains are very similar in the sequence of their amino acids,” says Prof. Schuler. This is apparently the reason why neighboring domains in proteins have a limited degree of similarity. “This seems to be a key evolutionary strategy to avoid protein misfolding and thus guarantee their maximum functionality,” says Schuler.

Science Daily
June 15, 2011

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Molecular discrimination of sheep bovine spongiform encephalopathy from scrapie

Sheep CH1641-like transmissible spongiform encephalopathy isolates have shown molecular similarities to bovine spongiform encephalopathy (BSE) isolates. We report that the prion protein PrPSc from sheep BSE is extremely resistant to denaturation. This feature, combined with the N-terminal PrPSc cleavage, allowed differentiation of classical scrapie, including CH1641-like, from natural goat BSE and experimental sheep BSE. Prion diseases, or transmissible spongiform encephalopathies (TSEs), are neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE) in cattle. TSEs are characterized by accumulation of an abnormal isoform of the host-encoded prion protein (PrPC), termed PrPSc.

A novel human prion disease, variant CJD, was reported in 1995 and postulated to be caused by eating beef infected with BSE. Biologic and molecular analyses provided evidence that the same agent was involved in BSE and variant CJD Evidence of sheep and goat susceptibility to BSE and discovery of natural BSE infections in 2 goats prompted the European Commission to increase the search for BSE infections in small ruminants. Although the BSE agent can be recognized by biologic strain typing in conventional mice, large-scale testing of small ruminants required molecular tests able to discriminate BSE from the most common TSEs of small ruminants. Molecular criteria used to discriminate BSE from scrapie are based on the low molecular weight of proteinase K–treated PrPSc (PrPres), a high proportion of the diglycosylated PrPSc, and poor or absent binding with antibodies directed at N-terminal epitopes. This last characteristic was fundamental in developing the discriminatory methods currently approved for surveillance in Europe.

The experimental scrapie isolate CH1641 reportedly shares molecular features with experimental sheep BSE, although lack of transmissibility of CH1641 to conventional mice in comparison to successful transmission of BSE provided evidence that CH1641 and BSE are caused by distinct prion agents. A few natural isolates have been described in sheep, showing molecular and biologic similarities to CH1641, and were named CH1641-like. Subtle pathologic differences were exploited to distinguish these CH1641-like isolates from BSE by immunohistochemical and biochemical analyses by glycoform profiling. However, routine testing by using discriminatory Western blot (WB) methods does not easily distinguish CH1641 and CH1641-like isolates from BSE. We report 2 new CH1641-like isolates; analyze the conformational stability of CH1641-like isolates, BSE, and classical scrapie; and show that a reliable molecular differentiation of these 3 TSE sources is possible by an improved discriminatory WB method.

Emerging Infectious Diseases
April 19, 2011

Original web page at Emerging Infectious Diseases

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New biomarker for Creutzfeldt-Jakob disease identified

Neena Singh, MD, PhD and colleagues at Case Western Reserve University School of Medicine have identified the first disease-specific biomarker for sporadic Creutzfeldt-Jakob disease (sCJD), a universally fatal, degenerative brain disease for which there is no cure. sCJD is one of the causes of dementia and typically leads to death within a year of disease onset. The finding, published in the March 9th issue of PLoS ONE, a scientific journal produced by the Public Library of Science, provides a basis for developing a test to diagnosis sCJD while patients are still alive. Presently, the only definitive diagnostic test for the disease requires brain tissue be obtained by biopsy or after death. In their study, Dr. Singh, associate professor of pathology at the School of Medicine, and her team found that levels of the iron-transport protein transferrin (Tf) are significantly decreased in the cerebrospinal fluid (CSF) of patients with sCJD well before the end stage of the disease, potentially allowing for earlier diagnosis.

“The decrease in Tf is significant enough to distinguish sCJD from dementia of non-CJD origin with an accuracy of 80 percent,” Dr. Singh says. “When combined with the currently used non-disease-specific biomarker T-tau, the diagnostic accuracy increases to 86 percent. This suggests that the two biomarkers represent separate disease processes, and complement each other as diagnostic biomarkers.” A decrease in the levels of CSF Tf reflects the imbalance of brain’s iron metabolism that is associated with sCJD. Being a part of the sCJD disease process, CSF Tf is likely to be a more precise indicator of sCJD than the current tests, Dr. Singh explains. “CSF Tf is the first biomarker that is related to the underlying pathology in sCJD brains,” Dr. Singh explains. Presently, sCJD is diagnosed by testing for elevated levels of the proteins 14-3-3 and T-tau in the CSF of cases suspected of the disease. Since these biomarkers are elevated in several other diseases besides sCJD, the incidence of false positive results is high. Replacement of 14-3-3 with Tf increases the specificity of the test significantly, providing a superior biomarker combination for the diagnosis of sCJD.

The ability to accurately diagnose patients while they are still living is critical to prevent inadvertent spread of sCJD to healthy individuals, to reduce the misdiagnosis of potentially treatable causes of dementia, and to eventually develop potential therapies for sCJD, according to Dr. Singh. As a part of their study, Dr. Singh and her team estimated levels of Tf in the CSF collected up to 24 months before death from confirmed cases of sCJD (n=99) and dementia of non-CJD origin (n=74). They found that levels of Tf were decreased significantly in sCJD cases compared to dementia of non-CJD origin. Further testing revealed that measurement of CSF Tf alone identified sCJD with a sensitivity of 85 percent, specificity of 72 percent, and accuracy of 80 percent. When combined with the surrogate biomarker T-tau, the CSF Tf and T-tau combination identified sCJD with an improved specificity of 87 percent and accuracy of 86 percent according to the research.

In addition to providing improved diagnostic accuracy, Dr. Singh notes that CSF Tf has several other advantages. It is resistant to degradation by enzymes, ensuring consistent results even in poorly preserved CSF samples; Tf-β2, the brain specific isoform of Tf is equally efficient in identifying sCJD and is likely to provide accurate results even from samples that are accidentally contaminated with blood during the collection process; and, Tf is abundant in the CSF relative to the currently used biomarkers 14-3-3 and T-tau, allowing accurate diagnosis from a small sample volume. Moving forward, researchers will work to establish a user-friendly, quantitative test for CSF Tf to provide a quick and uniform method of diagnosis for sCJD. They will also continue testing CSF samples from sCJD and other forms of human and animal prion disorders to establish the earliest time point in the disease course when this test becomes positive.

Science Daily
March 22, 2011

Original web page at Science Daily

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Molecular typing of protease-resistant prion protein in transmissible spongiform encephalopathies of small ruminants, France, 2002–2009

The agent that causes bovine spongiform encephalopathy (BSE) may be infecting small ruminants, which could have serious implications for human health. To distinguish BSE from scrapie and to examine the molecular characteristics of the protease-resistant prion protein (PrPres), we used a specifically designed Western blot method to test isolates from 648 sheep and 53 goats. During 2002–2009, classical non-Nor98 transmissible spongiform encephalopathy had been confirmed among ≈1.7 million small ruminants in France. Five sheep and 2 goats that showed a PrPres pattern consistent with BSE, or with the CH1641 experimental scrapie source, were identified. Later, bioassays confirmed infection by the BSE agent in 1 of the 2 goats. Western blot testing of the 6 other isolates showed an additional C-terminally cleaved PrPres product, with an unglycosylated band at ≈14 kDa, similar to that found in the CH1641 experimental scrapie isolate and different from the BSE isolate. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases that include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. TSEs are characterized by accumulation in the brain of a disease-associated isoform (PrPd) of a host-encoded cellular prion protein (PrPc). PrPd, in comparison with the normal prion protein PrPc, clearly differs in secondary and tertiary structures and in biochemical characteristics. Proteinase K (PK) digestion destroys PrPc, but in PrPd it generates a protease-resistant fragment known as PrPres. Most TSE diagnostic methods (e.g., ELISA and Western blot tests) are based on detection of PrPres.

The transmissible agent involved in BSE in cattle is known to cause prion diseases in other species under natural conditions. BSE can also be experimentally transmitted to sheep and goats, including after oral challenge to test for transmission. Because BSE-contaminated meat and bone meal may have been fed to small ruminants, BSE may have been transmitted to sheep or goats. Also, the Scientific Steering Committee of the European Commission has hypothesized that the BSE agent might have originated from a scrapie agent in sheep or goats and that these animals may represent a reservoir. In view of these data, the European Commission defined a strategy to investigate the possible presence of BSE in sheep and goats under natural conditions.

The standard for strain typing TSE agents is based on analysis of the phenotypic characteristics of the disease after transmission in laboratory rodents. Biological characterization of the BSE agent in inbred wild-type mice appeared to be reliable, because it showed uniform features in mice. However, this approach is time-consuming and costly. The identification of uniform molecular features of PrPres by Western blot in human variant CJD paved the way to a similar approach for detecting possible BSE in small ruminants. The molecular criteria defined in these studies included electrophoretic mobilities, glycosylation characteristics, and immunolabeling with different monoclonal antibodies. The last criteria enabled mapping of the protease cleavage site of the PrP protein fragment obtained after PK digestion. More recently, the identification of additional C-terminal PrPres products may contribute to discrimination of the different types of CJD or of different scrapie and BSE sources. Discriminant molecular features of the prion protein can also be investigated by immunohistochemical analysis or ELISA. In all of these studies, it was assumed that the strain information was closely associated with the structural features of PrPd.

The Western blot method enabled discrimination of experimental BSE in sheep from most scrapie-affected animals. Nevertheless, discrimination was more difficult with the CH1641 experimental scrapie isolate, which otherwise clearly differs from BSE by its absence of transmissibility to wild-type mice. Similar molecular features to those of CH1641 have been described in a few natural scrapie cases in France and in the United Kingdom. We describe the molecular findings obtained for a large series of TSE infections in France identified in small ruminants by active surveillance during 2002–2009 and for CH1641-like isolates in sheep and in 1 goat.

Emerging Infectious Diseases
January 24, 2011

Original web page at Emerging Infectious Diseases

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Protein-based biomarkers in blood serum could classify individuals with Alzheimer’s disease

An initial analysis suggests that biomarkers in blood serum can be combined with clinical information to accurately classify patients with Alzheimer’s disease, according to a report in the September issue of Archives of Neurology, one of the JAMA/Archives journals. “There is clearly a need for reliable and valid diagnostic and prognostic biomarkers of Alzheimer’s disease, and in recent years, there has been an explosive increase of effort aimed at identifying such markers,” the authors write as background information in the article. “It has been previously argued that, because of significant advantages, the ideal biomarkers would be gleaned from peripheral blood.” Identifying biomarkers in the blood has several advantages over other methods of classifying patients with Alzheimer’s disease, including detecting biomarkers in the cerebrospinal fluid and neuroimaging. Blood can be collected at any clinic or in-home visit and most patients will agree to the process, whereas not all facilities can conduct lumbar punctures to obtain cerebrospinal fluid. Older patients may not consent to lumbar puncture and may not be able to undergo neuroimaging because of pacemakers or other health issues.

Sid E. O’Bryant, Ph.D., of Texas Tech University Health Sciences Center, Lubbock, and colleagues in the Texas Alzheimer’s Research Consortium analyzed proteins in the serum of 197 patients diagnosed with Alzheimer’s disease and 203 controls without Alzheimer’s disease. Statistical analyses were used to create a biomarker risk score, which included levels of a number of protein biomarkers, including fibrinogen (a clotting protein), interleukin-10 (associated with the immune system) and C-reactive protein (an inflammatory marker). The final biomarker risk score correctly identified 80 percent of the individuals with Alzheimer’s disease and accurately excluded 91 percent of the individuals without Alzheimer’s disease. When other factors were also considered — age, sex, education and whether an individual had the APOE gene, which is associated with risk for Alzheimer’s disease — the score correctly identified 94 percent of the individuals with Alzheimer’s disease and accurately classified 84 percent of participants who did not have the disease.

“In addition to offering more accessible, rapid and cost- and time-effective methods for assessment, biomarkers (or panels of biomarkers) also hold great potential for the identification of endophenotypes within Alzheimer’s disease populations that are associated with particular disease mechanisms,” the authors write. In the current study, “a disproportionate number of inflammatory and vascular markers were weighted most heavily in the analyses.” The findings provide support for the existence of an inflammatory subtype of Alzheimer’s disease, they note. “The identification of blood-based biomarker profiles with good diagnostic accuracy would have a profound impact worldwide and requires further validation,” the authors conclude. “Additionally, the identification of pathway-specific endophenotypes among patients with Alzheimer’s disease would likewise have implications for targeted therapeutics as well as understanding differential progression among diagnosed cases. With the rapidly evolving technology and the analytic techniques available, Alzheimer’s disease researchers now have the tools to simultaneously analyze exponentially more information from a host of modalities, which is likely going to be necessary to understand this very complex disease.”

Science Daily
September 28, 2010

Original web page at Science Daily

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How fish oil fights inflammation

Omega-3 fatty acids, a main component of fish oil, have a reputation as potent anti-inflammatory agents. Now researchers think they know how the acids block this immune response. They’ve also found that omega-3s can help fight diabetes in obese mice, pointing the way to potential therapies in humans. To understand how omega-3s curb inflammation, Jerrold Olefsky, an endocrinologist at the University of California, San Diego, and his colleagues trawled through the data on a family of proteins called G protein-coupled receptors, which can bind to a number of different fatty acids. One of these receptors—GPR120—”jumped right out,” Olefsky says. Olefsky’s group found it on immune cells involved in inflammation, as well as in mature fat cells, and they noted that it seemed to bind to omega-3s. To confirm the link, the team doused GPR120-containing mouse immune cells with omega-3 fatty acids. That “shut down almost all of the inflammatory pathways,” Olefsky says. “It was a very powerful effect.”

The researchers also genetically modified mice to lack the GPR120 receptor. They then fed the mutant mice and normal mice a high-fat diet. Both groups became obese and developed a mouse form of diabetes. Scientists have long suspected a link between inflammation and obesity-linked diabetes; and indeed, when Olefsky’s team supplemented the fatty diet with a hefty helping of omega-3 fatty acids—enough to double the concentration of omega-3 in the mice’s blood—the normal mice experienced a reduction in their diabetic symptoms. The rodents remained obese, but they regained some sensitivity to insulin, meaning they didn’t need as much insulin to take up glucose and burn it to produce energy. In fact, the supplemented diet worked as well to combat insulin resistance as the common diabetes drug Avandia, the team reports in the 3 September issue of Cell. The mutant mice remained diabetic regardless of how many omega-3s they consumed, highlighting the importance of the GPR120 receptor. “The results are preliminary but exciting,” says Nader Moniri, a pharmacologist at Mercer University in Atlanta. “For the first time we’re linking inflammation to GPR120.” Moniri points out that the GPR120 receptor also shows up in intestinal cells, where it appears to regulate a hormone that prompts the pancreas to release insulin. That means there are two routes by which GPR120 could influence diabetes, which makes it a very nice drug target, he says.

Olefsky suggests that the GPR120 receptor is the main way by which omega-3s control inflammation, but he acknowledges that other mechanisms may exist. For example, digestion breaks down some omega-3s into shorter fatty acids. Some evidence suggests that these may also influence inflammation, though not through GPR120, he notes. Olefsky also won’t go so far as to recommend that anyone take fish oil pills to stave off inflammation or diabetes. “We’ve never worked with people on this,” he says, “so we have no idea how much omega-3 fatty acids a person would have to take.”

ScienceNow
September 14, 2010

Original web page at ScienceNow

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Merlin protein found to control liver stem cells, prevent tumor development

A protein known to be involved in a rare hereditary cancer syndrome may have a role in the regulation of liver stem cells and the development of liver cancer. In the August 15 issue of Genes & Development, a Massachusetts General Hospital (MGH) research team describes finding that the protein called merlin, encoded by the NF2 (neurofibromatosis type 2) gene, controls the activity of adult stem cells that give rise to the two major types of liver cells. “We found that mutation of the NF2 tumor suppressor gene in the mouse liver led to a dramatic overproliferation of liver stem cells – the cells that contribute to the liver’s remarkable ability to regenerate,” says Andrea McClatchey, PhD, of the MGH Center for Cancer Research, who led the study. “These mice go on to develop the two forms of liver cancer that are most common in humans, suggesting that liver stem cells may be the cells of origin of these tumors.”

The liver has a rare ability to regenerate and replace damaged or missing tissue. If one lobe is removed for transplantation, the rest of the donor’s organ will return to its previous size and the transplanted lobe will grow to match the needs of the recipient. This regeneration usually involves proliferation of the most characteristic liver cells, called hepatocytes, and of bile duct cells; but if that growth is blocked or those cells are damaged, a population of less-differentiated progenitor cells will start to expand. These liver stem cells have been identified in rodents, and potential equivalents found but not confirmed in humans. Previous research also indicated that liver stem cells may be the source of some tumors in animals, and suggested that the tumor suppressor gene NF2 may help prevent tumor development. Originally discovered through its involvement in the rare genetic disorder neurofibromatosis type 2, the NF2 gene codes for merlin, a protein known to suppress the activity of a number of cellular receptors. One of these is the epidermal growth factor receptor (EGFR), and oversignaling by that protein is known to lead to several types of cancer. The current study was designed to investigate the role of NF2 and merlin in the fetal and adult mouse liver, including possible involvement with tumor development.

The researchers found that infant mice lacking functioning NF2 in their livers developed dramatic overgrowth of liver stem cells, to the point of crowding out hepatocytes. Mice that did not die from a lack of functioning liver cells soon developed the two major types of liver cancer, and the fact that stem cell overgrowth preceded tumor development strongly suggested that the undifferentiated progenitors were the source of the tumors. Blocking the expression of NF2 in the livers of adult mice had minimal effect on the animals unless a portion of the liver was surgically removed, setting off the regeneration process and leading to the same stem cell overproliferation and tumor development. McClatchey explains that the study’s findings provide new information about liver stem cells and how their proliferation is controlled; identifies a new animal model for liver cancer, the lack of which has seriously impeded understanding the disease; and suggests that liver tumors may originate from liver stem cells and that excess EGFR signaling leads to liver tumor development. “These results are consistent with our previous studies showing that merlin helps to regulate EGFR activity at the cell membrane,” she says. “We also showed that merlin’s role in cell-to-cell communication is essential for cells to stop growing when they fill the appropriate space. Since liver progenitors need to be poised to regenerate in case of injury, they may be particularly sensitive to the loss of merlin’s regulatory function.” McClatchey is an associate professor of Pathology at Harvard Medical School.

EurekAlert! Medicine
August 31, 2010

Original web page at EurekAlert! Medicine