In a promising finding for the field of regenerative medicine, stem cell researchers at Children’s Hospital of Pittsburgh of UPMC have identified a source of adult stem cells found on the walls of blood vessels with the unlimited potential to differentiate into human tissues such as bone, cartilage and muscle.

The scientists, led by Bruno Péault, PhD, deputy director of the Stem Cell Research Center at Children’s Hospital, identified cells known as pericytes that are multipotent, meaning they have broad developmental potential. Pericytes are found on the walls of small blood vessels such as capillaries and microvessels throughout the body and have the potential to be extracted and grown into many types of tissues, according to the study.

“This finding marks the first direct evidence of the source of multipotent adult stem cells known as mesenchymal stem cells. We believe pericytes represent one of the most promising sources of multipotent stem cells that scientists have been searching for in the quest to make regenerative medicine possible,” Dr. Péault said. “The encouraging aspect of this source is that blood vessels are the one structure that all tissues in the human body have in common. These cells can be extracted easily and painlessly from convenient sources such as fat tissue, dental pulp, umbilical cord and placental tissue, then grown in culture to large numbers and, possibly, re-injected into the patient to heal a broken bone, a failing joint or an injured muscle.”

Results of the study are published in the September issue of the journal Cell Stem Cell.

In their laboratory in the John G. Rangos Sr. Research Center, researchers were able to identify pericytes in all human tissues they analyzed, including muscle, fat, pancreas, placenta and many other samples. Through purification in the lab, these pericytes could then be coaxed into becoming whatever type of tissue the scientists desired. For instance, the researchers took pericytes from the pancreas and then reinjected them into an injured muscle. The cells immediately began regenerating muscle tissue.

Source: Children’s Hospital of Pittsburgh

A new mouse model has provided some surprising insight into XLF, a molecule that helps to repair lethal DNA damage. The research, published by Cell Press in the September 5th issue of the journal Molecular Cell, suggests that although XLF shares many properties with well known DNA repair factors, certain cells of the immune system possess an unexpected compensatory mechanism that that can take over for nonfunctional XLF.

Genetic instability can lead to multiple problems, including cell death and many forms of cancer. Therefore, it is absolutely critical for cells to have both the means to constantly survey genes for damage and the mechanisms to repair broken DNA. Currently, there are six well characterized classical non-homologous end-joining (C-NHEJ) factors that repair double strand breaks (DSBs) in mammalian cells.

Lymphocytes, a type of immune cell, use a kind of genetic shuffling called variable, diversity, joining V(D)J recombination. This gene shuffling occurs during lymphocyte development and helps to produce diverse immune system cells that can recognize all sorts of different foreign substances, called antigens, that might pose a threat to the organism. Previous work in mice has shown that deficiency of C-NHEJ factors results in a severely compromised immune system, because of incomplete V(D)J recombination, along with increased sensitivity to cellular ionizing radiation (IR) and genomic instability.

Some recent studies have suggested that XLF may serve as an additional C-NHEJ factor. “We know that XLF mutations in humans lead to decreased numbers of lymphocytes and a somewhat less severe form of immunodeficiency,” says senior study author Dr. Frederick W. Alt from the Howard Hughes Medical Institute and Harvard Medical School. “While a role of XLF in C-NHEJ might explain lower than normal numbers of lymphocytes in human XLF-mutant patients, the reason for their relatively mildly impaired lymphocyte development is not clear.”

To examine XLF function, Dr. Alt and colleagues generated and characterized XLF-mutant mice. XLF-deficient mouse cells were IR sensitive, had substantial genomic instability and displayed major defects in the ability to repair DSBs. Surprisingly, however, mature lymphocyte numbers were only modestly decreased in the XLF-deficient mice and developing B cells exhibited nearly normal V(D)J recombination. This finding was in direct contrast to results seen in previously characterized C-NHEJ-deficient mice. Further, on a tumor suppressor p53-deficient background, XLF-deficient mice were not prone to lymphomas as were C-NHEJ-deficient mice, even though they were just as likely to develop non-immune cell tumors.

The findings demonstrate that although the XLF-deficient mice share many characteristics associated with C-NHEJ-deficient mice, lymphocytes have a distinct developmental signature when it comes to XLF. “Together, our results implicate XLF as a C-NHEJ factor, but also indicate that developing mouse lymphocytes harbor cell type specific factors/pathways that compensate for absence of XLF function during V(D)J recombination,” explains Dr. Alt.

Source: Cell Press

Just one night without sleep can increase the amount of the chemical dopamine in the human brain, according to new imaging research in the August 20 issue of The Journal of Neuroscience. Because drugs that increase dopamine, like amphetamines, promote wakefulness, the findings offer a potential mechanism explaining how the brain helps people stay awake despite the urge to sleep. However, the study also shows that the increase in dopamine cannot compensate for the cognitive deficits caused by sleep deprivation.

“This is the first time that a study provides evidence that in the human brain, dopamine is involved in the adaptations that result from sleep deprivation,” said Nora Volkow, MD, director of the National Institute on Drug Abuse, who led the study.

Volkow and colleagues found that in healthy participants, sleep deprivation increased dopamine in two brain structures: the striatum, which is involved in motivation and reward, and the thalamus, which is involved in alertness. The researchers also found that the amount of dopamine in the brain correlated with feelings of fatigue and impaired performance on cognitive tasks.

“These findings suggest dopamine may increase after sleep deprivation as a compensatory response to the effects of increased sleep drive in the brain,” said David Dinges, PhD, at the University of Pennsylvania School of Medicine, an expert unaffiliated with the study. “The extent to which this occurs may differentiate how vulnerable people are to the neurobehavioral effects of sleep loss,” Dinges said.

The researchers studied 15 healthy participants who were either kept awake all night or allowed a good night’s sleep. Researchers tested the same participants in both conditions. On the morning of the study, participants rated how tired they were and did cognitive tasks testing visual attention and working memory.

The researchers used the imaging technique positron emission tomography to study the changes in the dopamine system that occur with sleep deprivation. Compared to well-rested participants, sleep-deprived participants showed reduced binding of a radiolabeled compound ([¹¹C]raclopride) that binds to dopamine receptors in the striatum and thalamus. Because raclopride competes with dopamine for the same receptors, decreased raclopride binding indicates increased levels of dopamine, according to the study authors.

Although decreases in raclopride binding could also indicate a reduction in the number of dopamine receptors, these findings are consistent with prior research implicating increased dopamine levels in wakefulness. For example, some stimulants that prevent sleep, like amphetamines, increase dopamine in the brain, and sleepiness is common in people with Parkinson’s disease, which kills dopamine neurons.

The rise in dopamine following sleep deprivation may promote wakefulness to compensate for sleep loss. “However, the concurrent decline in cognitive performance, which is associated with the dopamine increases, suggests that the adaptation is not sufficient to overcome the cognitive deterioration induced by sleep deprivation and may even contribute to it,” said study author Volkow.

Future research will examine the long-term effects of chronic sleep disturbances on dopamine brain circuits.

Source: Society for Neuroscience

The largest genetic analysis of its kind to date for bipolar disorder has implicated machinery involved in the balance of sodium and calcium in brain cells. Researchers supported in part by the National Institute of Mental Health, part of the National Institutes of Health, found an association between the disorder and variation in two genes that make components of channels that manage the flow of the elements into and out of cells, including neurons.

“A neuron’s excitability – whether it will fire – hinges on this delicate equilibrium,” explained Pamela Sklar, M.D., Ph.D., of Massachusetts General Hospital (MGH) and the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard, who led the research. “Finding statistically robust associations linked to two proteins that may be involved in regulating such ion channels – and that are also thought to be targets of drugs used to clinically to treat bipolar disorder – is astonishing.”

Although it’s not yet known if or how the suspect genetic variation might affect the balance machinery, the results point to the possibility that bipolar disorder might stem, at least in part, from malfunction of ion channels.

Sklar, Shaun Purcell, Ph.D., also of MGH and the Stanley Center, and Nick Craddock, M.D., Ph.D., of Cardiff University and the Wellcome Trust Case Control Consortiuum in the United Kingdom and a large group of international collaborators report on their findings online Aug. 17, 2008 in Nature Genetics.

“Faced with little agreement among previous studies searching for the genomic hot spots in bipolar disorder, these researchers pooled their data for maximal statistical power and unearthed surprising results,” said NIMH Director Thomas R. Insel, M.D. “Improved understanding of these abnormalities could lead to new hope for the millions of Americans affected by bipolar disorder.”

In the first such genome-wide association study for bipolar disorder, NIMH researchers last fall reported the strongest signal associated with the illness in a gene that makes an enzyme involved the action of the anti-manic medication lithium. However, other chromosomal locations were most strongly associated with the disorder in two subsequent studies.

Since bipolar disorder is thought to involve many different gene variants, each exerting relatively small effects, researchers need large samples to detect relatively weak signals of illness association.

To boost their odds, Sklar and colleagues pooled data from the latter two previously published and one new study of their own. They also added additional samples from the STEP-BD study and Scottish and Irish families, and controls from the NIMH Genetics Repository. After examining about 1.8 million sites of genetic variation in 10,596 people – including 4,387 with bipolar disorder – the researchers found the two genes showing the strongest association among 14 disorder-associated chromosomal regions.

Variation in a gene called Ankyrin 3 (ANK3) showed the strongest association with bipolar disorder. The ANK3 protein is strategically located in the first part of neuronal extensions called axons and is part of the cellular machinery that decides whether a neuron will fire. Co-authors of the paper had shown last year in mouse brain that lithium, the most common medication for preventing bipolar disorder episodes, reduces expression of ANK3.

Variation in a calcium channel gene found in the brain showed the second strongest association with bipolar disorder. This CACNA1C protein similarly regulates the influx and outflow of calcium and is the site of interaction for a hypertension medication that has also been used in the treatment of bipolar disorder.

Source: NIH/National Institute of Mental Health

In a first, scientists from Weill Cornell Medical College and Columbia University Medical Center have described the specifics of how brain cells process antidepressant drugs, cocaine and amphetamines. These novel findings could prove useful in the development of more targeted medication therapies for a host of psychiatric diseases, most notably in the area of addiction.

Their breakthrough research, featured as the cover story in a recent issue of Molecular Cell, describes the precise molecular and biochemical structure of drug targets known as neurotransmitter-sodium symporters (NSSs), and how cells use them to enable neural signaling in the brain. A second study, published in the latest issue of Nature Neuroscience, pinpoints where the drug molecules bind in the neurotransmitter transporter — their target in the human nervous system.

“These findings are so clear and detailed at the level of molecular behavior that they will be most valuable to developing more effective therapies for mood disorders and neurologic and psychiatric diseases, and to direct effective treatments for drug addiction to cocaine and amphetamines,” says co-lead author Dr. Harel Weinstein, Chairman and Maxwell M. Upson Professor of Physiology and Biophysics, and director of the Institute for Computational Biomedicine at Weill Cornell Medical College. “This research may also open the door to the development of new therapies for dopamine-neurotransmitter disorders such as Parkinson’s disease, schizophrenia, and anxiety and depression.”

To make their observations, the research team led by Dr. Jonathan Javitch, senior author of the Molecular Cell study and contributing author to the Nature Neuroscience study, and professor of Psychiatry and Pharmacology in the Center for Molecular Recognition at Columbia University Medical Center, stabilized different structural states of the neurotransmitter-sodium-symporter molecule that relate to steps in its function. This allowed the team to study how substrates and inhibitors affect the transition between these different states, and thus to understand the way in which its function is accomplished.

“Crystallography had allowed the identification of only one structural form of the molecule, but our experiments and computations were able to identify how this form changes and thereby add an understanding of the functional role of the different forms that the molecule must adopt to accomplish transport activity,” says Dr. Javitch.

The main surprise was the realization that two binding sites on the transporter molecule need to be filled simultaneously and cooperate in order for transport to be driven across the cell membrane. For these studies, the scientists used the crystal structure of a bacterial transporter that is very similar to human neurotransmitter transporters. They performed computer simulations to reveal the path of the transported molecules into cells. Laboratory experimentation was used to test the computational predictions and validate the researchers’ inferences.

Together, these procedures revealed a finely-tuned process in which two sodium ions bind and stabilize the transporter molecule for the correct positioning of the two messenger molecules — one deep in the center of the protein, and the other closer to the entrance. Like a key engaging a lock mechanism, this second binding causes changes in the transporter throughout the structure, allowing one of the two sodium molecules to move inward, and then release the deeply bound messenger and its sodium partner into the cell.

In the bacterial transporter studied, antidepressant molecules bind in the outer one of two sites, and stop the transport mechanism, leaving the messenger molecule outside the cell.

The second team of researchers, involving a collaboration of the Weinstein and Javitch labs with colleagues in Denmark (the labs of Ulrik Gether and Claus Loland), found that in the human dopamine transporter cocaine binds in the deep site, unlike the antidepressant binding in the bacterial transporter. Therefore, the researchers conclude that anti-cocaine therapy will be more complicated, because interfering with cocaine binding also means interference with the binding of natural messengers.

“This finding might steer anti-cocaine therapy in a completely new direction,” says Dr. Weinstein.

Molecular understanding at this level of structural and dynamic detail is rare in the world of drug development, the authors note. Only about 15 percent of all drugs have a known molecular method-of-action, even though the effects of these drugs within the body — after very stringent and controlled laboratory testing — are well understood pharmacologically.

Source: New York- Presbyterian Hospital/Weill Cornell Medical Center/Weill Cornell Medical College

In recent years, a class of small molecules known as microRNA have been found to play an important role in regulating gene products in most animal and plant species. A new study now indicates that microRNA may influence the development of alcohol tolerance, a hallmark of alcohol abuse and dependence. Researchers supported by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) report the findings in the July 31 issue of the journal Neuron.

“This is an intriguing contribution to efforts aimed at identifying the molecular bases of alcohol tolerance,” noted NIAAA Director Ting-Kai Li, MD.

Tolerance is the decrease in sensitivity to alcohol that develops with repeated exposures to alcohol over time. Individuals who develop high tolerance (low sensitivity) to alcohol are at increased risk for becoming alcohol dependent. Thus, an important research objective has been to identify the adaptations within individual molecules that underlie tolerance.

In previous experiments, Steven N. Treistman, PhD, Professor of Psychiatry at the University of Massachusetts Medical School (UMMS), and colleagues at the university’s Brudnick Neurospychiatric Research Institute (BNRI), determined that a brain cell membrane structure known as the BK channel develops tolerance to alcohol, particularly in the supraoptic nucleus and the striatum, two brain regions important in alcohol’s effects. In both regions, alcohol tolerance was manifested as decreased alcohol sensitivity and reduced BK channel density. Previous studies have also shown that there are numerous variants of the BK channel gene.

In the current study, researchers led by Dr. Treistman, who is the director of the BNRI, examined whether microRNA might be involved in the alcohol tolerance observed in the BK channel.

In test tube experiments, the researchers showed that the amount of a specific microRNA molecule known as miR-9 increases in brain cells within minutes of exposure to alcohol. They also found that miR-9 blocks the expression of BK gene variants that contain a specific binding site for the molecule, while sparing those that lack a miR-9 binding site. Remarkably, the BK gene variants were destroyed exhibited high alcohol sensitivity, while those that remained showed significantly lower sensitivity, consistent with the development of tolerance.

“This represents a novel and elegant mechanism by which neurons are able to adapt to alcohol,” said Treistman. “Moreover, since adaptation, or tolerance, to the drug likely contributes to alcohol abuse, our findings identify a potential molecular target for therapeutic intervention.” Treistman credited his colleagues, especially Andrzej Z. Pietrzykowski, MD, PhD, research assistant professor of psychiatry, for their contributions to this important work.

A widely published expert on the molecular basis of addiction—in particular, the changes in the brain that occur as a function of drug exposure, which may make an individual prone to substance abuse and the compulsive behavior associated with drug addiction—Dr. Treistman noted that the microRNA process observed in this study may represent a general mechanism of neuronal adaptation to alcohol, with miR-9 playing a pivotal role in a complex regulatory network.

“This study demonstrates for the first time that alcohol exposure can cause rapid changes in microRNA levels, altering gene expression and perhaps behavior,” said Antonio Noronha, PhD, director of NIAAA’s Division of Neuroscience and Behavior. “In future studies, it will be interesting to determine if similar microRNA-based regulatory mechanisms influence alcohol problems in human populations.”

Source: University of Massachusetts Medical School

University of Florida College of Pharmacy researchers have discovered a marine compound off the coast of Key Largo that inhibits cancer cell growth in laboratory tests, a finding they hope will fuel the development of new drugs to better battle the disease.

The UF-patented compound, largazole, is derived from cyanobacteria that grow on coral reefs. Researchers, who described results from early studies today (Aug. 7) at an international natural products scientific meeting in Athens, Greece, say it is one of the most promising they’ve found since the college’s marine natural products laboratory was established three years ago.

An initial set of papers in the Journal of the American Chemical Society also has garnered the attention of other scientists, and the lab is racing to complete additional research. The molecule’s natural chemical structure and ability to inhibit cancer cell growth were first described in the journal in February and the laboratory synthesis and description of the molecular basis for its anticancer activity appeared July 2.

“It’s exciting because we’ve found a compound in nature that may one day surpass a currently marketed drug or could become the structural template for rationally designed drugs with improved selectivity,” said Hendrik Luesch, Ph.D., an assistant professor in UF’s department of medicinal chemistry and the study’s principal investigator.

Largazole, discovered and named by Luesch for its Florida location and structural features, seeks out a family of enzymes called histone deacetylase, or HDAC. Overactivity of certain HDACs has been associated with several cancers such as prostate and colon tumors, and inhibiting HDACs can activate tumor-suppressor genes that have been silenced in these cancers.

Although scientists have been probing the depths of the ocean for marine products since the early 1960s, many pharmaceutical companies lost interest before researchers could deliver useful compounds because natural products were considered too costly and time-consuming to research and develop.

Many common medications, from pain relievers to cholesterol-reducing statins, stem from natural products that grow on the earth, but there is literally an ocean of compounds yet to be discovered in our seas. Only 14 marine natural products developed are in clinical trials today, Luesch said, and one drug recently approved in Europe is the first-ever marine-derived anticancer agent.

“Marine study is in its infancy,” said William Fenical, Ph.D., a distinguished professor of oceanography and pharmaceutical sciences at the University of California, San Diego. “The ocean is a genetically distinct environment and the single, most diverse source of new molecules to be discovered.”

The history of pharmacy traces its roots back thousands of years to plants growing on Earth’s continents, used by ancient civilizations for medicinal purposes, Fenical added. Yet only in the past 30 years have scientists begun to explore the organisms in Earth’s oceans, he said. Fewer than 30 labs exist worldwide and research dollars have only become available in the past 15 years.

HDACs are already targeted by a drug approved for cutaneous T-cell lymphoma manufactured by the global pharmaceutical company Merck & Co. Inc. However, UF’s compound does not inhibit all HDACs equally, meaning a largazole-based drug might result in improved therapies and fewer side effects, Luesch said.

Since 2006, Luesch and his team of researchers have screened cyanobacteria provided by collaborator Valerie Paul, Ph.D., head scientist at the Smithsonian Marine Station in Fort Pierce. They check the samples for toxic activity against cancer cells and last year encountered one exceptionally potent extract — the one that ultimately yielded largazole.

To conduct further biological testing on the compound, Luesch and his team have been collaborating with Jiyong Hong, an assistant professor in the department of chemistry at Duke University, to replicate its natural structure and its actions in the laboratory.

Luesch said that within the next few months he plans to study whether largazole reduces or prevents tumor growth in mice.

Luesch has several other antitumor natural products from Atlantic and Pacific cyanobacteria in the pipeline.

“We have only scratched the surface of the chemical diversity in the ocean,” Luesch said. “The opportunities for marine drug discovery are spectacular.”

Source: University of Florida

If you’re a fan of habañero salsa or like to order Thai food spiced to five stars, you owe a lot to bugs, both the crawling kind and ones you can see only with a microscope. New research shows they are the ones responsible for the heat in chili peppers.

The spiciness is a defense mechanism that some peppers develop to suppress a microbial fungus that invades through punctures made in the outer skin by insects. The fungus, from a large genus called Fusarium, destroys the plant’s seeds before they can be eaten by birds and widely distributed.

“For these wild chilies the biggest danger to the seed comes before dispersal, when a large number are killed by this fungus,” said Joshua Tewksbury, a University of Washington assistant professor of biology. “Both the fungus and the birds eat chilies, but the fungus never disperses seeds – it just kills them.”

Fruits use sugars and lipids to attract consumers such as birds that will scatter the seeds. But insects and fungi enjoy sugars and lipids too, and in tandem they can be fatal to a pepper’s progeny.

However, the researchers found that the pungency, or heat, in hot chilies acts as a unique defense mechanism. The pungency comes from capsaicinoids, the same chemicals that protect them from fungal attack by dramatically slowing microbial growth.

“Capsaicin doesn’t stop the dispersal of seeds because birds don’t sense the pain and so they continue to eat peppers, but the fungus that kills pepper seeds is quite sensitive to this chemical,” said Tewksbury, lead author of a paper documenting the research.

“Having such a specific defense, one that doesn’t harm reproduction or dispersal, is what makes chemistry so valuable to the plant, and I think it is a great example of the power of natural selection.”

The paper is published the week of Aug. 11 in the online Proceedings of the National Academy of Sciences. Co-authors are Karen Reagan, Noelle Machnicki, Tomás Carlo, and David Haak of the University of Washington; Alejandra Lorena Calderón Peñaloza of Universidad Autonoma Gabriel Rene Moreno in Bolivia; and Douglas Levey of the University of Florida. The work was funded by the National Science Foundation and the National Geographic Society.

The scientists collected chilies from seven different populations of the same pepper species spread across 1,000 square miles in Bolivia. In each population, they randomly selected peppers and counted scars on the outer skin from insect foraging. The damage was caused by hemipteran insects – insects such as seed bugs (similar to aphids and leaf hoppers) that have sucking mouth parts arranged into a beaklike structure that can pierce the skin of a fruit.

The researchers found that not all of the plants produce capsaicinoids, so that in the same population fruit on one plant could be hotter than a jalapeño while fruit from other plants might be as mild as a bell pepper. But there was a much-higher frequency of pungent plants in areas with larger populations of hemipteran insects that attack the chilies and leave them more vulnerable to fungus.

The scientists also found that hot plants got even hotter, with higher levels of capsaicinoids, in areas where fungal attacks were common. But in areas with few insects and less danger of fungal attack, most of the plants lacked heat entirely. In those areas, chilies from the plants that did produce capsaicinoids had a lot less kick because they only produced about half the capsaicinoids as the plants did in areas where fungal attack was common.

Using chemical substances as a defense is not unique to peppers. Tomatoes, for example, are loaded with substances that give their unripened fruit a decidedly unpleasant taste, allowing the seeds a chance to mature and be dispersed. But unlike peppers, tomatoes and most other fruits lose their chemical defenses when the fruit ripens. That is a necessary step, scientists believe, because otherwise the fruit would not be consumed by birds and other animals that disperse the seed. The problem with that strategy, Tewksbury said, is that it leaves the fruit exposed to fungal attack.

“By contrast, peppers increase their chemical defense levels, or their heat, as they ripen. This is a very different model and peppers can get away with it because birds don’t sense pain when they eat capsaicin,” Tewksbury said. “I think a lot of plants would love to come up with this way of stopping fungal growth without inhibiting dispersers. It’s just very hard to do.”

The fact that chilies have capsaicin could be the reason humans started eating the peppers in the first place, he said. Chili peppers and corn are among the earliest domesticated crops in the New World.

“Before there was refrigeration, it was probably adaptive to eat chilies, particularly in the tropics,” Tewksbury said. “Back then, if you lived in a warm and humid climate, eating could be downright dangerous because virtually everything was packed with microbes, many of them harmful. People probably added chilies to their stews because spicy stews were less likely to kill them.”

All chilies originated in South America, and wild chilies now grow from central South America to the southwestern United States. Explorers carried the plants back to Europe, but they were not widely used there. From Europe, chilies made their way to Asia and Africa, where they have become a common ingredient in nearly every tropical cuisine.

“In the north, any adaptive benefit to using eating chilies would be much smaller than at the equator because microbial infection of food is less common and it’s easier to keep food cold. Maybe that’s why food in the north can be so boring,” Tewksbury said.

“Along the equator, without access to refrigeration, you could be dead pretty quickly unless you can find a way to protect yourself against the microbes you ingest every day.”

Source: University of Washington