Red wine's heart health chemical unlocked at last

FANCY receiving the heart protecting abilities of red wine without having to drink a glass every day? Soon you may be able to, thanks to the synthesis of chemicals derived from resveratrol, the molecule believed to give wine its protective powers. The chemicals have the potential to fight many diseases, including cancer.

Plants make a huge variety of chemicals, called polyphenols, from resveratrol to protect themselves against invaders, particularly fungi. But they only make tiny amounts of each chemical, making it extremely difficult for scientists to isolate and utilise them. The unstable nature of resveratrol has also hindered attempts at building new compounds from the chemical itself.

Scott Snyder at Columbia University in New York and his team have found a way around this: building polyphenols from compounds that resemble, but are subtly different to, resveratrol. These differences make the process much easier. Using these alternative starting materials, they have made dozens of natural polyphenols, including vaticanol C, which is known to kill cancer cells (Nature, DOI: 10.1038/nature10197).

"It's like a recipe book for the whole resveratrol family," says Snyder. "We've opened up a whole casket of nature's goodies."

Source New Scientist

How dense is a cell?

Combining an ancient principle with new technology, MIT researchers have devised a way to answer that question.

MIT researchers designed this tiny microfluidic chip that can measure the mass and density of single cells.

More than 2,000 years after Archimedes found a way to determine the density of a king’s crown by measuring its mass in two different fluids, MIT scientists have used the same principle to solve an equally vexing puzzle — how to measure the density of a single cell.

“Density is such a fundamental, basic property of everything,” says William Grover, a research associate in MIT’s Department of Biological Engineering. “Every cell in your body has a density, and if you can measure it accurately enough, it opens a whole new window on the biology of that cell.”

The new method, described in the Proceedings of the National Academy of Sciences the week of June 20, involves measuring the buoyant mass of each cell in two fluids of different densities. Just as measuring the crown’s density helped Archimedes determine whether it was made of pure gold, measuring cell density could allow researchers to gain biophysical insight into fundamental cellular processes such as adaptations for survival, and might also be useful for identifying diseased cells, according to the authors.

Grover and recent MIT PhD recipient Andrea Bryan are lead authors of the paper. Both work in the lab of Scott Manalis, a professor of biological engineering, member of the David H. Koch Institute for Integrative Cancer Research and senior author of the paper.

Going with the flow

Measuring the density of living cells is tricky because it requires a tool that can weigh cells in their native fluid environment, to keep them alive, and a method to measure each cell in two different fluids.

How the lab can determine the weight and density of individual cells.

In 2007, Manalis and his students developed the first technique to measure the buoyant mass of single living cells. Their device, known as a suspended microchannel resonator, pumps cells, in fluid, through a microchannel that runs across a tiny silicon cantilever, or diving-board structure. That cantilever vibrates within a vacuum; when a cell flows through the channel, the frequency of the cantilever’s vibration changes. The cell’s buoyant mass can be calculated from the change in frequency.

To adapt the system to measure density, the researchers needed to flow each cell through the channel twice, each time in a different fluid. A cell’s buoyant mass (its mass as it floats in fluid) depends on its absolute mass and volume, so by measuring two different buoyant masses for a cell, its mass, volume and density can be calculated.

The new device rapidly exchanges the fluids in the channel without harming the cell, and the entire measurement process for one cell takes as little as five seconds.

David Weitz, professor of physics at Harvard University, says the new technique is a clever way of measuring cell density, and opens up many new avenues of research. “The very interesting thing they show is that density seems to have a more sensitive change than some of the more standard measurements. Why is that? I don’t know. But the fact that I don’t know means it’s interesting,” he says.

Changes in density

The researchers tested their system with several types of cells, including red blood cells and leukemia cells. In the leukemia study, the researchers treated the cells with an antibiotic called staurosporine, then measured their density less than an hour later. Even in that short time, a change in density was already apparent. (The cells grew denser as they started to die.) The treated leukemia cells increased their density by only about 1 percent, a change that would be difficult to detect without a highly sensitive device such as this one. Because of that rapid response and sensitivity, this method could become a good way to screen potential cancer drugs.

“It was really easy, by the density measurement, to identify cells that had responded to the drug. If we had looked at mass alone, or volume alone, we never would have seen that effect,” Bryan says.

The researchers also demonstrated that malaria-infected red blood cells lose density as their infection progresses. This density loss was already known, but this is the first time it has been observed in single cells.

Being able to detect changes in red-blood-cell density could also offer a new way to test athletes who try to cheat by “doping” their blood — that is, by removing their own blood and storing it until just before their competition, when it is transfused back into the bloodstream. This boosts the number of red blood cells, potentially enhancing athletic performance.

Storing blood can alter the blood’s physical characteristics, and if those include changes in density, this technique may be able to detect blood doping, Grover says.

Researchers in Manalis’ lab are now investigating the densities of other types of cells, and are starting to work on measuring single cells as they grow over time — specifically cancer cells, which are characterized by uncontrolled growth.

“Understanding how density of individual cancer cells relates to malignant progression could provide fundamental insights into the underlying cellular processes, as well as lead to clinical strategies for treating patients in situations where molecular markers don’t yet exist or are difficult to measure due to limited sample volumes,” Manalis says.

Other authors on the paper are MIT research scientist Monica Diez-Silva; Subra Suresh, former dean of the MIT School of Engineering; and John Higgins of Massachusetts General Hospital and Harvard Medical School.

Source MIT

50-year search for calcium channel ends

Cell's power generator depends on long-sought protein.

Boston, MA (June 19, 2011)—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 will appear 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."

Source EurekaAlert!

Study Reveals Important Aspects of Signalling Across Cell Membranes in Plants

Plant receptors use different signalling method than do animal receptors.

Every living plant cell and animal cell is surrounded by a membrane. These cellular membranes contain receptor molecules that serve as the cell's eyes and ears, and help it communicate with other cells and with the outside world.
The receptor molecules accomplish three basic things in the communication process: 1) recognize an outside signal, 2) transport that signal across the cell's membrane and 3) initiate the reading of the signal inside the cell and then initiate the cell's response to that signal. These steps are collectively known as transmembrane signaling.

 Click on image for larger version.

 Transmembrane signaling in a plant cell aided by a steroid.

Transmembrane signaling in animal cells has been significantly more studied and observed than that in plant cells. But now, with support from the National Science Foundation, researchers from Joanne Chory's laboratory at the Salk Institute have published new observations about transmembrane signaling in plants; their paper appears in the June 12, 2011, advanced online edition of Nature.
According to the study, transmembrane signaling mechanisms used by plants differ from those used by animals. Specifically, Michael Hothorn of the Salk Institute reports that a small steroid molecule on the outside of the plant cell assists in the transmembrane signaling process. By contrast, this sort of molecule and its receptor is generally located inside the nuclei of animal cells.

While studying transmembrane signaling in plants, Hothorn and colleagues observed the steroid, shown in yellow, attach to a membrane-bound receptor, shown in blue. This attachment enabled the steroid's counterpart--a co-receptor protein, shown in orange--to bind to the blue receptor. Once bound, the orange co-receptor and the blue receptor become glued together by the yellow steroid, allowing their intracellular domains to touch and initiate communication.
In the case observed by Hothorn, transmembrane signaling initiated plant growth.

Source National Science Fundation

How killer immune cells avoid killing themselves

After eight years of work, researchers have unearthed what has been a well-kept secret of our immune system's success. The findings published online on June 9th in Immunity, a Cell Press publication, offer an explanation for how specialized immune cells are able to kill infected or cancerous cells without killing themselves in the process.

The focus of the study is a molecule known as perforin, whose job it is to open up a pore in cells targeted for destruction. With that pore in place, proteases known as granzymes can enter target cells and destroy them.
Perforin is one of the most critical ingredients for a functional immune system. Without it, mice succumb to viral illness and lymphoma. Humans born without a working perforin gene develop an aggressive immunoregulatory disorder in the first few months of life and usually die unless treated with cytoxic drugs or a bone marrow transplant.

But perforin itself is an incredibly destructive molecule. "Perforin forms a massive pore," said Ilia Voskoboinik of the Peter MacCallum Cancer Centre in Australia. "It allows almost any protein to diffuse into a target cell. A few hundred molecules of perforin is sufficient to obliterate any cell."
When the immune cells known as cytotoxic lymphocytes (including cytotoxic T lymphocytes and natural killer cells) are activated, "they produce a massive amount of perforin, yet the cells are fine," Voskoboinik said. The question was: how do our immune cells manage such toxic cargo without endangering themselves?
Before perforin is released, the cells that produce it have to transport it from one part of the cell to another. That transport chain starts in a component of the cell known as the endoplasmic reticulum (ER). From there, it moves to the Golgi and into secretory granules where it is packaged together with granzymes. It is those secretory granules that ultimately fuse with the plasma membrane of the cytotoxic cell and allow its release into the junctions between the immune cell and the cell it aims to kill.

Scientists used to think perforin had an inhibitory domain within its structure that was only removed once they were safely stored in the secretory granules. (The acidic environment within secretory granules keeps perforin inactive until its release.) But Voskoboinik's team purified perforin and found that the protein was always active regardless of whether they had removed the supposed inhibitory domain or not.
"It seeded doubt about how perforin is inhibited," he says. "It was a puzzle. Perforin was fully functional but for some reason it couldn't kill the cell [in which it was synthesized]."
The real danger zone for the cell when it comes to perforin is the ER, Voskoboinik explained. Conditions there should be ideal for perforin to work, but something keeps it from doing so. The new study links that protection to a single amino acid at one end of the perforin protein. When that amino acid is substituted with another, perforin doesn't make it to the Golgi compartment, it builds up in the ER, and the cell dies.
"Perforin goes from zero to extremely high levels within 24 hours and it has everything it needs to be functional," Voskoboinik said. "The cell relies on a really efficient transport system to move perforin away from the danger zone and as a result the cell is absolutely protected."

The findings "close a chapter" in our understanding of the immune system that has existed in the field since perforin was discovered almost 25 years ago, Voskoboinik says. "It was one of those things that was out there on Olympus untouched. Everyone would just stare at it. That's what got us interested."

Source  EurekaAlert!

Malaria caught on camera breaking and entering cell

The video above captures the moment when a malaria parasite invades a human red blood cell - the first time the event has been caught in high resolution.
The Plasmodium parasite responsible for malaria is transmitted by the bite of infected mosquitoes, and is thought to kill almost 1 million people worldwide each year.

Jake Baum at the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia, and colleagues used transmission electron microscopy, immuno-fluorescence and 3D super-resolution microscopy to record thousands of high-definition images of separate invasion events, a process that takes less than 30 seconds.
To boost their chances of catching Plasmodium parasites in the act of attacking a red blood cell the team controlled the process using two drugs. The first - heparin - prevents parasites entering a new red blood cell, while the second - E64 - prevents their exit. Carefully timing the treatments meant "we knew we were going to get huge number of invasion events", says Baum.

The parasites produce a protein called the tight junction marker and use it to attach to and drill into red blood cells, says Baum. "At the beginning of invasion it's a dot, as the parasite enters the cell it becomes a beautiful circle, and then the marker is behind the parasite."
The images that the researchers generated show that invasion is not a well-ordered process, as we had thought, says Baum. "Initial attachment using the tight junction marker is the main switch, and then the parasite does everything at once." Simultaneously, it releases a vacuole to live in and switches on a motor complex allowing it to move within the cell.

Kiaran Kirk at the Australian National University in Canberra says the "clever cell preparation and stunning microscopy" is a "tour de force".
The movie could have implications for the treatment of malaria too. Leann Tilley of La Trobe University in Melbourne, Australia, says the results confirm that interfering with the master switch would stop the parasites from entering red blood cells and "thereby stop disease".
Journal reference: Cell Host & Microbe, DOI: 10.1016/j.chom.2010.12.003

Source New Scientist

Scientists use super microscope to pinpoint body’s immunity 'switch'

Molecular mechanism driving the immune response identified for the first time.

Using the only microscope of its kind in Australia, medical scientists have been able for the first time to see the inner workings of T-cells, the front-line troops that alert our immune system to go on the defensive against germs and other invaders in our bloodstream.
The discovery overturns prevailing understanding, identifying the exact molecular 'switch' that spurs T-cells into action — a breakthrough that could lead to treatments for a range of conditions from auto-immune diseases to cancer.

The findings, by researchers at the University of New South Wales (UNSW), are reported this week in the high-impact journal Nature Immunology.
Studying a cell protein important in early immune response, the researchers led by Associate Professor Katharina Gaus from UNSW's Centre for Vascular Research at the Lowy Cancer Research Centre, used Australia's only microscope capable of super-resolution fluorescence microscopy to image the protein molecule-by-molecule to reveal the immunity 'switch'.
The technology is a major breakthrough for science, Dr Gaus said. Currently there are only half a dozen of the 'super' microscopes in use around the world.

"Previously you could see T-cells under a microscope but you couldn't see what their individual molecules were doing," Dr Gaus said.
Using the new microscope the scientists were able to image molecules as small as 10 nanometres. Dr Gaus said that what the team found overturns the existing understanding of T-cell activation.
"Previously it was thought that T-cell signalling was initiated at the cell surface in molecular clusters that formed around the activated receptor.

"In fact, what happens is that small membrane-enclosed sacks called vesicles inside the cell travel to the receptor, pick up the signal and then leave again," she said.
Dr Gaus said the discovery explained how the immune response could occur so quickly.
"There is this rolling amplification. The signalling station is like a docking port or an airport with vesicles like planes landing and taking off. The process allows a few receptors to activate a cell and then trigger the entire immune response," she said.
PhD candidate David Williamson, whose research formed the basis of the paper, said the discovery showed what could be achieved with the new generation of super-resolution fluorescence microscopes.
"In conventional microscopy, all the target molecules are lit up at once and individual molecules become lost amongst their neighbours – it's like trying to follow a conversation in a crowd where everyone is talking at once.

"With our microscope we can make the target molecules light up one at a time and precisely determine their location while their neighbours remain dark. This 'role call' of all the target molecules means we can then build a 'super resolution' image of the sample," he said.
The next step was to pinpoint other key proteins to get a complete picture of T-cell activity and to extend the microscope to capture 3-D images with the same unprecedented resolution.
"Being able to see the behaviour and function of individual molecules in a live cell is the equivalent of seeing atoms for the first time. It could change the whole concept of molecular and cell biology," Mr Williamson said.

Source  EurekaAlert!

Team solves decades-old molecular mystery linked to blood clotting

CHAMPAIGN, lll. — Blood clotting is a complicated business, particularly for those trying to understand how the body responds to injury. In a new study, researchers report that they are the first to describe in atomic detail a chemical interaction that is vital to blood clotting. This interaction – between a clotting factor and a cell membrane – has baffled scientists for decades.

Above is a movie of the supercomputer simulation of the blood clotting factor interacting with the membrane. The GLA domain of the clotting factor is depicted as a purple tube; individual GLA amino acids are yellow; tightly bound calcium ions are pink spheres; and the interacting phospholipids that make up the membrane are below.

The study appears online in the Journal of Biological Chemistry.
“For decades, people have known that blood-clotting proteins have to bind to a cell membrane in order for the clotting reaction to happen,” said University of Illinois biochemistry professor James Morrissey, who led the study with chemistry professor Chad Rienstra and biochemistry, biophysics and pharmacology professor Emad Tajkhorshid. “If you take clotting factors off the membrane, they’re thousands of times less active.”
The researchers combined laboratory detective work with supercomputer simulations and solid-state nuclear magnetic resonance (SSNMR) to get at the problem from every angle. They also made use of tiny rafts of lipid membranes called nanodiscs, using an approach developed at Illinois by biochemistry professor Stephen Sligar.

Previous studies had shown that each clotting factor contains a region, called the GLA domain, which interacts with specific lipids in cell membranes to start the cascade of chemical reactions that drive blood clotting.
One study, published in 2003 in the journal Nature Structural Biology, indicated that the GLA domain binds to a special phospholipid, phosphatidylserine (PS), which is embedded in the membrane. Other studies had shown that PS binds weakly to the clotting factor on its own, but in the presence of another phospholipid, phosphatidylethanolamine (PE), the interaction is much stronger.

Both PS and PE are abundant in the inner – but not the outer – leaflets of the double-layered membranes of cells. This keeps these lipids from coming into contact with clotting factors in the blood. But any injury that ruptures the cells brings PS and PE together with the clotting factors, initiating a chain of events that leads to blood clotting.
Researchers have developed many hypotheses to explain why clotting factors bind most readily to PS when PE is present. But none of these could fully explain the data.
In the new study, Morrissey’s lab engineered nanodiscs with high concentrations of PS and PE, and conducted functional tests to determine if they responded like normal membranes.
“We found that the nanodisc actually is very representative of what really happens in the cell in terms of the reaction of the lipids and the role that they play,” Morrissey said.

Then Tajkhorshid’s lab used advanced modeling and simulation methods to position every atom in the system and simulated the molecular interactions on a supercomputer. The simulations indicated that one PS molecule was linking directly to the GLA domain of the clotting factor via an amino acid (serine) on its head-group (the non-oily region of a phospholipid that orients toward the membrane surface).
More surprisingly, the simulations indicated that six other phospholipids also were drawing close to the GLA domain. These lipids, however, were bending their head-groups out of the way so that their phosphates, which are negatively charged, could interact with positively charged calcium ions associated with the GLA domain. (Watch a movie of the simulation.)
“The simulations were a breakthrough for us,” Morrissey said. “They provided a detailed view of how things might come together during membrane binding of coagulation factors. But these predictions had to be tested experimentally.”

Rienstra’s lab then analyzed the samples using SSNMR, a technique that allows researchers to precisely measure the distances and angles between individual atoms in large molecules or groups of interacting molecules. His group found that one of every six or seven PS molecules was binding directly to the clotting factor, providing strong experimental support for the model derived from the simulations.
“That turned out to be a key insight that we contributed to this study,” Rienstra said.
The team reasoned that if the PE head-groups were simply bending out of the way, then any phospholipid with a sufficiently small head-group should work as well as PE in the presence of PS. This also explained why only one PS molecule was actually binding to a GLA domain. The other phospholipids nearby were also interacting with the clotting factor, but more weakly.
The finding explained another mystery that had long daunted researchers. A different type of membrane lipid, phosphatidylcholine (PC), which has a very large head-group and is most abundant on the outer surface of cells, was known to block any association between the membrane and the clotting factor, even in the presence of PS.

Follow-up experiments showed that any phospholipid but PC enhanced the binding of PS to the GLA domain. This led to the “ABC” hypothesis: when PS is present, the GLA domain will interact with “Anything But Choline.”
“This is the first real insight at an atomic level of how most of the blood-clotting proteins interact with membranes, an interaction that’s known to be essential to blood clotting,” Morrissey said. The findings offer new targets for the development of drugs to regulate blood clotting, he said.
Morrissey and Tajkhorshid have their primary appointments in the U. of I. College of Medicine. Tajkhorshid also is an affiliate of the Beckman Institute at Illinois.
The National Heart, Lung and Blood Institute and the National Institute for General Medical Sciences provided funding for this study.

Source University of Illinois

MDC Researchers Discover Key Molecule for Stem Cell Pluripotency

Researchers of the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch have discovered what enables embryonic stem cells to differentiate into diverse cell types and thus to be pluripotent. This pluripotency depends on a specific molecule – E-cadherin – hitherto primarily known for its role in mediating cell-cell adhesion as a kind of “intracellular glue”. If E-cadherin is absent, the stem cells lose their pluripotency. The molecule also plays a crucial role in the reprogramming of somatic cells (body cells) into pluripotent stem cells (EMBO Reports, advance online publication 27 May 2011; doi:10.1038/embor.2011.88)*. 

Dr. Daniel Besser, Prof. Walter Birchmeier and Torben Redmer from the MDC, a member of the Helmholtz Association, used mouse embryonic fibroblasts (MEFs) in their stem cell experiments. In a first step they showed that the pluripotency of these stem cells is directly associated with the cell-adhesion molecule E-cadherin. If E-cadherin is absent, the stem cells lose their pluripotency.

In a second step the researchers investigated what happens when somatic cells that normally neither have E-cadherin nor are pluripotent are reprogrammed into a pluripotent stem cell state. In this reprogramming technique, somatic cells are converted into induced pluripotent stem cells (iPSCs). This new technique may help researchers avoid the controversies that come with the use of human embryos to produce human embryonic stem cells for research purposes.

The MDC researchers found that in contrast to the original cells, the new pluripotent cells derived from mouse connective tissue contained E-cadherin. “Thus, we have double proof that E-cadherin is directly associated with stem-cell pluripotency. E-Cadherin is necessary for maintaining pluripotent stem cells and also for inducing the pluripotent state in the reprogramming of somatic cells,” Dr. Besser said. “If E-cadherin is absent, somatic cells cannot be reprogrammed into viable pluripotent cells.” In addition, E-Cadherin can replace OCT 4, one of the signaling molecules until now considered indispensable for reprogramming.

Next, the MDC researchers want to find out to what extent E-cadherin also regulates human embryonic stem cells. “Understanding the molecular relationships is essential for using human somatic cells to develop stem cell therapy for diseases such as heart attack, Alzheimer’s or Parkinson’s disease or diabetes,” Dr. Besser said.

Source MDC

Researchers model genome copying-collating steps during cell division

Researchers from Virginia Tech and Oxford University have proposed a novel molecular mechanism for the living cell's remarkable ability to detect the alignment of replicated chromosomes on the mitotic spindle in the final phase of the cell division cycle. This checkpoint mechanism prevents mistakes in the cell division process that could damage dividing cells and the organism they inhabit.

John Tyson, University Distinguished Professor of Biological Sciences in the College of Science at Virginia Tech, and Bela Novak, professor of integrative systems biology at Oxford University, have been using mathematical models for many years to study the checkpoints that regulate irreversible progression through the cell cycle. Their latest modeling effort, on the chromosome alignment checkpoint, is published in the online early edition of the Proceedings of the National Academy of Sciences (PNAS) the week of May 23 in the article, "System-level feedbacks make the anaphase switch irreversible," with coauthors Enuo He and Orsolya Kapuy of the Centre for Integrative Systems Biology at the University of Oxford; Raquel A. Oliveira of the Department of Biochemistry, University of Oxford; and Frank Uhlmann of the Chromosome Segregation Laboratory, Cancer Research UK, London.

The article provides theoretical and experimental evidence that bistability of the checkpoint machinery ensures irreversibility of the metaphase-anaphase transition.

The most important goal of the cell division cycle is to make a new copy of each of the cell's DNA molecules and then to separate these identical molecules, called sister chromatids, to the two new cells so that each cell gets one and only one copy of each DNA molecule. "Think of it like copying the pages of a book," said Tyson, "where the two copies of each page are stuck together when they come out of the copy machine, and then putting the copies through a collating machine that pulls apart the identical pages, placing one copy in a stack to the right and the other copy in a stack to the left. The DNA synthesis phase of the cell cycle, called S phase, is the copy machine, and mitosis, called M phase, is the collating machine," he said.

"The copying and collating machines must be carefully monitored by the cell, because mistakes in replicating the DNA molecules or partitioning the sister chromatids to the new cells can be fatal. It is the job of molecular 'surveillance mechanisms' to look for mistakes and correct them," said Tyson. "If a mistake is found, then further progress through the cell division cycle must be blocked until the problem can be corrected."
These block-points are called checkpoints. Once a cell has passed one of these checkpoints, it may not back up to an earlier phase of the cell division cycle; it must proceed to the next phase. In this sense, the checkpoint transitions are said to be irreversible.

In 1993, Tyson and Novak proposed that the transition into mitosis (the G2/M transition) is irreversible because it is controlled by a molecular toggle switch.

"A mechanical toggle switch, like an old-fashioned light switch, has two states: off and on. To turn the light on, the lever must be pushed up, above the mid-point, before the switch flips on," said Tyson. "Once the light is turned on, it stays on; the transition is irreversible in the sense that this same switch will not mistakenly turn the light off of its own accord. To turn the light off, the lever must be pushed down, below the mid-point, before the switch flips off. When the lever is in the central position, the lights may be either on or off, depending on which direction the lever is moving. In the central position, the switch is 'bistable'. The irreversible transition points (where the switch flips) lie above and below the central, bistable area."

This connection between bistability and irreversible transitions extends to the tiny molecular switches in the cell division cycle, as proposed by Novak and Tyson in 1993. In 2003, their prediction was confirmed experimentally by Jill Sible, associate professor of biological sciences at Virginia Tech, and her research group, and by Jim Ferrell's group at Stanford University. Since then bistability and irreversibility have been confirmed at two other cell cycle transitions, but the 'metaphase-anaphase transition' remained a puzzle.
Tyson explained that metaphase is the critical step in the collating machine, when the glued-together pages (the sister chromatids) are all lined up in the central zone (the metaphase plate) with one page attached by a string (microtubule) coming from the left side of the cell and the other page attached by a string coming from the right side of the cell. "As the cell leaves metaphase and enters anaphase, the glue is dissolved and the pages are pulled apart by the strings to the stacks on the left and right. In this fashion, each stack (each daughter nucleus) gets one and only one copy of each page (each chromatid)."

There is a surveillance mechanism for chromosome alignment that makes sure that none of the glue is dissolved until every one of the glued-together sister-chromatid pairs are properly aligned on the metaphase plate, Tyson said. "Human cells have 46 pairs of sister chromatids. Even if 45 of the 46 pairs are properly aligned, the cell may not pass the metaphase checkpoint. But as soon as the 46th pair comes into alignment, the checkpoint is rapidly lifted, the glue is dissolved, and the cell moves on to the next stage of the division process. The transition is irreversible in the sense that once the glue is dissolved and the pages are separated, the cell cannot easily return to the pre-metaphase stage. It must go on to divide and start again at the beginning of a new cell cycle."

The theoreticians attribute bistability of the metaphase checkpoint and irreversibility of the metaphase-anaphase transition to two positive feedback loops in the molecular interactions that comprise the surveillance mechanism. The experimentalists (Oliveira and Uhlmann) have shown that, if one of these feedback loops is broken by mutations, then bistability is lost and the transition becomes reversible. The nature of the second feedback loop is still controversial and the subject of ongoing experimental studies.

"Understanding these control mechanisms is important," said Tyson, "because mistakes in partitioning chromosomes at anaphase are the root cause of many human maladies, like birth defects and cancer."

Source  EurekaAlert!

The dance of the cells: A minuet or a mosh?

Boston, MA – The physical forces that guide how cells migrate—how they manage to get from place to place in a coordinated fashion inside the living body— are poorly understood. Scientists at the Harvard School of Public Health (HSPH) and the Institute for Bioengineering of Catalonia (IBEC) have, for the first time, devised a way to measure these forces during collective cellular migration. Their surprising conclusion is that the cells fight it out, each pushing and pulling on its neighbors in a chaotic dance, yet together moving cooperatively toward their intended direction.

The study appears May 22, 2011, in an advance online edition of Nature Materials.
Until now it was known that cells could follow gradients of soluble chemical cues, called morphogens, which help to direct tissue development, or they could follow physical cues, such as adhesion to their surroundings. Fundamental studies of these and other mechanisms of cellular migration have focused on dissecting cell behavior into ever smaller increments, trying to get to the molecular roots of how migration occurs. In contrast, the HSPH team worked at a higher level—the group level—and focused upon the forces that cells exert upon their immediate neighbors, to begin to resolve the riddle of collective cellular migration.

Collective cellular migrations are necessary for multicellular life; for example, in order for cells to form the embryo, cells must move collectively. Or in the healing of a wound, cells must migrate collectively to fill the wound gap. But the migration process is also dangerous in situations such as cancer, when malignant cells, or clumps of cells, can migrate to distant sites to invade other tissues or form new tumors. Understanding how and why collective cellular migration happens may lead to ways to control or interrupt diseases that involve abnormal cell migration.

The laboratories of Jeffrey Fredberg, professor of bioengineering and physiology at HSPH, and his colleague Xavier Trepat, a researcher at IBEC, are the only ones in the world that can now measure the forces within and between complex cellular groups. "We're beginning for the first time to see the forces and understand how they work when cells behave in large groups," said Trepat.

To do this, the researchers invented a measurement technology called Monolayer Stress Microscopy, which allows them to visualize the minute mechanical forces exerted at the junctions where individual cells are connected. Their studies led to discovery of a new phenomenon, which they named "plithotaxis," a term derived from Greek "plithos" suggestive of throng, swarm or crowd.

"If you studied a cell in isolation, you'd never be able to understand the behavior of a cell in a crowd," said Dhananjay Tambe, the first author and a research fellow at HSPH. Instead, the researchers studied groups of cells living in a single thin layer—a monolayer—and precisely measured the forces each cell was experiencing as it was navigating within the group. The findings surprised them.

"We thought that as cells are moving—say, to close a wound—that the underlying forces would be synchronized and smoothly changing so as to vary coherently across the crowd of cells, as in a minuet," said co-first author Corey Hardin, a research fellow at Massachusetts General Hospital. "Instead, we found the forces to vary tremendously, occurring in huge peaks and valleys across the monolayer. So the forces are not smooth and orderly at all; they are more like those in a 'mosh pit'—organized chaos with pushing and pulling in all directions at once, but collectively giving rise to motion in a given direction," he said.

"This new finding has the potential to alter, in a fundamental way, our understanding of mechano-biology and its role in the basic processes that underlie the function of monolayers in health and disease," said Fredberg. He also predicted the new report would be interesting for both physicists and biologists, and might even spur new research collaborations between the two disciplines.

The study findings should provide a better understanding of cell migration as it occurs in embryonic development—how the human body gets put together soon after fertilization, say the researchers. The findings may also help to explain how cancer cells migrate in the deadly process called metastasis.

Source EurekaAlert!

Dynamics of crucial protein 'switch' revealed

Cell signaling networks tied to diabetes and cancer.

Researchers at the University of Texas Medical Branch at Galveston and the University of California-San Diego School of Medicine have published a study that offers a new understanding of a protein critical to physiological processes involved in major diseases such as diabetes and cancer. This work could help scientists design drugs to battle these disorders.

The article was deemed a "Paper of the Week" by and will be on the cover of the Journal of Biological Chemistry. It is scheduled for publication May 20 and now available online.
"This study applied a powerful protein structural analysis approach to investigate how a chemical signal called cAMP turns on one of its protein switches, Epac2," said principal investigator Xiaodong Cheng, professor in the Department of Pharmacology and Toxicology and member of the Sealy Center for Structural Biology and Molecular Biophysics at UTMB.

The cAMP molecule controls many physiological processes, ranging from learning and memory in the brain and contractility and relaxation in the heart to insulin secretion in the pancreas. cAMP exerts its action in cells by binding to and switching on specific receptor proteins, which, when activated by cAMP, turn on additional signaling pathways.
Errors in cell signaling are responsible for diseases such as diabetes, cancer and heart failure. Understanding cAMP-mediated cell signaling, in which Epac2 is a major player, likely will facilitate the development of new therapeutic strategies specifically targeting the cAMP-Epac2 signaling components, according to the researchers.

The project involved an ongoing collaboration between Cheng's research group at UTMB, experts in the study of cAMP signaling, and UCSD professor of medicine Virgil Woods Jr. and colleagues at UCSD, pioneers in the development and application of hydrogen/deuterium exchange mass spectrometry (DXMS) technology. Compared with other protein-analysis techniques, DXMS is especially good at studying the structural motion of proteins.

Using this novel approach, the investigators were able to reveal, in fine detail, that cAMP interacts with its two known binding sites on Epac2 in a sequential fashion and that binding of cAMP changes the shape of the protein in a very specific way – switching on its activity by exposing further signaling interaction sites on Epac2.
"DXMS analysis has proved to be an amazingly powerful approach, alone or in combination with other techniques, in figuring out how proteins work as molecular machines, changing their shapes – or morphing – in the normal course of their function," said Woods. "This will be of great use in the identification and development of therapeutic drugs that target these protein motions."

Source EurekaAlert!

Fundamental question on how life started solved

German and US researchers calculate a carbon nucleus of crucial importance

The researchers published their results in the coming issue of the scientific journal Physical Review Letters.

"Attempts to calculate the Hoyle state have been unsuccessful since 1954," said Professor Dr. Ulf-G. Meißner (Helmholtz-Institut für Strahlen- und Kernphysik der Universität Bonn). "But now, we have done it!" The Hoyle state is an energy-rich form of the carbon nucleus. It is the mountain pass over which all roads from one valley to the next lead: From the three nuclei of helium gas to the much larger carbon nucleus. This fusion reaction takes place in the hot interior of heavy stars. If the Hoyle state did not exist, only very little carbon or other higher elements such as oxygen, nitrogen and iron could have formed. Without this type of carbon nucleus, life probably also would not have been possible.

The search for the "slave transmitter"

The Hoyle state had been verified by experiments as early as 1954, but calculating it always failed. For this form of carbon consists of only three, very loosely linked helium nuclei - more of a cloudy diffuse carbon nucleus. And it does not occur individually, only together with other forms of carbon. "This is as if you wanted to analyze a radio signal whose main transmitter and several slave transmitters are interfering with each other," explained Prof. Dr. Evgeny Epelbaum (Institute of Theoretical Physics II at Ruhr-Universität Bochum). The main transmitter is the stable carbon nucleus from which humans - among others - are made. "But we are interested in one of the unstable, energy-rich carbon nuclei; so we have to separate the weaker radio transmitter somehow from the dominant signal by means of a noise filter."

What made this possible was a new, improved calculating approach the researchers used that allowed calculating the forces between several nuclear particles more precisely than ever. And in JUGENE, the supercomputer at Forschungszentrum Jülich, a suitable tool was found. It took JUGENE almost a week of calculating. The results matched the experimental data so well that the researchers can be certain that they have indeed calculated the Hoyle state.

More about how the Universe came into existence

"Now we can analyze this exciting and essential form of the carbon nucleus in every detail," explained Prof. Meißner. "We will determine how big it is, and what its structure is. And it also means that we can now take a very close look at the entire chain of how elements are formed."

In future, this may even allow answering philosophical questions using science. For decades, the Hoyle state was a prime example for the theory that natural constants must have precisely their experimentally determined values, and not any different ones, since otherwise we would not be here to observe the Universe (the anthropic principle). "For the Hoyle state this means that it must have exactly the amount of energy it has, or else, we would not exist," said Prof. Meißner. "Now we can calculate whether - in a changed world with other parameters - the Hoyle state would indeed have a different energy when comparing the mass of three helium nuclei." If this is so, this would confirm the anthropic principle.

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The study was jointly conducted by the University of Bonn, Ruhr-Universität Bochum, North Carolina State University, and Forschungszentrum Jülich.

Source EurekaAlert!

Harvey Fineberg: Are we ready for neo-evolution?

Medical ethicist Harvey Fineberg shows us three paths forward for the ever-evolving human species: to stop evolving completely, to evolve naturally -- or to control the next steps of human evolution, using genetic modification, to make ourselves smarter, faster, better. Neo-evolution is within our grasp. What will we do with it?

Osama bin Laden: how DNA identified his body

Achieving a satisfactory identification of the world's most wanted man is not, it turns out, a simple matter. According to the US government, the body of Osama bin Laden, killed during a raid on a compound in Abbottabad, Pakistan, on 2 May, was identified by comparison to photographs, confirmation from one of his wives at the compound, facial-recognition software, and – the gold standard for identification – DNA analysis.

John Brennan, Assistant to the President for Homeland Security and Counterterrorism, said the DNA evidence provided a match with "99.9 per cent confidence". That would require the comparison of DNA from the body with that of people known to be related to bin Laden. Bin Laden had no full siblings, but more than 50 half-siblings and up to 24 children.

Using DNA from many half-siblings could produce a DNA match of greater than 90 per cent confidence, but it would be difficult to get as high as 99.9 per cent without a closer relative says Rhonda Roby, a forensic geneticist at the University of North Texas Health Science Center, Fort Worth. Roby, who led the team using DNA evidence to identify the remains of people killed in the 9/11 attacks in 2001, says that the statistical analysis based on DNA from half-siblings is more complex and less reliable than analysis based on DNA from a closer relative like a parent or child.

Reports indicate that one of bin Laden's sons was also killed in the raid, possibly 20-year-old Hamza bin Laden. Roby says DNA from a son and several half-siblings could confirm Osama's identity with 99.9 per cent accuracy. 

Full paternity trio?

If, however, the government was able to obtain DNA from bin Laden's body, his son and also that son's biological mother – who might have been at the compound during the raid, it could perform DNA profiling with a "full paternity trio", assuring 99.9 per cent accuracy, Roby says.

The provenance of the DNA used in the comparison is unknown, as is the nature of the test itself. However, it is likely that forensic scientists analysing bin Laden's DNA used the polymerase chain reaction to amplify between 13 and 16 key regions called short tandem repeats (STRs), then compared the pattern of these with the same STRs from that of his relatives.

The agency responsible for conducting the analysis is also unconfirmed: media reports have named both FBI and the CIA. Several sources suggested that DNA was subpoenaed from the body of a half-sister of bin Laden, when she reportedly died at Massachusetts General Hospital in Boston, but the hospital was unable to confirm the report. "There is no evidence she was even here," says spokesperson Sue McGreevey.

FBI special agent Greg Comcowich would not confirm or deny any investigative steps the FBI has taken. When asked whether the agency would release any further information on the DNA analysis, he replied: "Not that I'm aware of."

Source  New Scientist

New protein regulates water in the brain to control inflammation

New research in the FASEB Journal suggests that aquaporin-4, a water transporting protein, plays a role in brain inflammation via a mechanism involving astrocyte swelling and cytokine release. 


A new protein, called aquaporin-4, is making waves and found to play a key role in brain inflammation, or encephalitis. This discovery is important as the first to identify a role for this protein in inflammation, opening doors for the development of new drugs that treat brain inflammation and other conditions at the cellular level rather than just treating the symptoms. This discovery was published in the May 2011 issue of The FASEB Journal (http://www.faseb.org).

"Our study establishes a novel role for a water channel, aquaporin-4, in neuroinflammation, as well as a cell-level mechanism," said Alan S. Verkman, M.D., Ph.D., a senior researcher involved in the work from the Department of Medicine and the Department of Physiology at the University of California, San Francisco. "Our data suggest that inhibition or down-regulation of aquaporin-4 expression in brain and spinal cord may offer a new therapeutic option in diseases such as multiple sclerosis, neuromyelitis optica and other conditions associated with neuroinflammation."

Scientists compared normal mice and mice without genes for producing aquaporin-4 using a model of brain inflammation. These experiments showed significantly reduced brain inflammation in the mice that did not produce aquaporin-4. Researchers then systematically investigated the various possible causes of this reduced neuroinflammation and surprisingly found that aquaporin-4 deletion causes the brain to be less susceptible to inflammation, involving differences in astrocyte reaction to stress. The involvement of aquaporin-4 in brain inflammation provides a new determinant and better understanding of how the brain responses to inflammatory stresses. This suggests that using drugs or other agents that target this protein may be effective for treating a variety of conditions associated with brain or spinal cord inflammation.
"This a new lead in our efforts to stem inflammation in the brain," said Gerald Weissmann, M.D., Editor-in-Chief of The FASEB Journal. "The importance of water movement in and out of cells cannot be understated, and this paper helps to clarify what has otherwise been a muddy view of aquaporins."

Source  EurekaAlert!

Several baffling puzzles in protein molecular structure solved with new method

Determining the molecular configuration of proteins is important in nanotechology, drug design, disease research and many other fields

The structures of many protein molecules remain unsolved even after experts apply an extensive array of approaches. An international collaboration has led to a new, high-performance method that rapidly determined the structure of protein molecules in several cases where previous methods had failed.
The usefulness of the new method is reported May 1 in Nature advanced online publication. The lead authors are Dr. Frank DiMaio of the University of Washington (UW) in Seattle and Dr. Thomas C. Terwilliger of Los Alamos National Laboratory in New Mexico. The senior author is Dr. David Baker, of the UW Department of Biochemistry.

This is Dr. David Baker with a model of a molecule in his biochemistry lab at the University of Washington in Seattle.

A protein's molecular structure shapes its functions. In biomedical and health research, for example, scientists are interested in the molecular structure of specific proteins for many reasons, a few of which are:

1. To design drugs that selectively target, at the molecular level, particular biochemical reactions in the body
2. To understand abnormal human proteins in disease, such as those found in cancer and neurodegenerative disorders like Alzheimer's, and how these abnormal proteins cause malfunctions
3. To learn the shape and function of virus particles and how they act to cause infections
4. To see how the chains of amino acids, decoded from the DNA in genes, fold and twist into normally or abnormally shaped protein molecules
5. To design new proteins not found in the natural world, such as enzymes to speed up a slow biochemical reaction
6. To find ways to replace malfunctioning molecular parts of proteins that are critical to health
7. To devise nano-scale tools, such as molecular motors

"The important new method described this week in Nature highlights the value of computational modeling in helping scientists to determine the structures and functions of molecules that are difficult to study using current techniques," said Dr. Peter Preusch, who oversees Baker's research grant and other structural biology grants at the National Institutes of Health (NIH). "Expanding the repertoire of known protein structures -- a key goal of the NIH Protein Structure Initiative, which helped fund the research – will be of great benefit to scientists striving to design new therapeutic agents to treat disease."
The methods devised by the group overcome some of the limitations of X-ray crystallography in determining the molecular structure of a protein. X-ray crystallography obtains information about the positions of atoms, chemical bonds, the density of electrons and other arrangements within a protein molecule.
The information is gleaned by striking protein crystals with X-ray beams. The beams bounce off in several directions.

Measuring the angles and intensities of these diffracted beams enables scientists to produce a 3-dimensional image of electron density. However, information about the molecular structure can be lost in taking the measurements, due to restraints posed by physics.
Scientists attempt to sidestep this problem by comparing the crystallography results to previously solved protein structures that resemble the unknown structure. The technique to "fill in the blanks" is called molecular replacement.

Molecular replacement has its own limitations in interpreting the electron density maps produced by X-ray crystallography, according to the authors of the paper. Techniques such as automatic chain tracing often follow the comparative model more closely than the actual structure of the protein under question. These mistakes lead to failure to obtain an accurate configuration of the molecule.
The researchers showed that this limitation can be substantially reduced by combining computer algorithms for protein structure modeling with those for determining structure via X-ray crystallography.
Several years ago, University of Washington researchers and their colleagues developed a structure prediction method called Rosetta. This program takes a chain of amino acids – protein-building blocks strung all in a row -- and searches for the lowest energy conformation possible from folding, twisting and packing the chain into a three-dimensional (3-D) molecule.

The researchers found that even very poor electron density maps from molecular replacement solutions could be useful. These maps could guide Rosetta structural prediction searches that are based on energy optimization. By taking these energy-optimized predicted models, and looking for consistency with the electron density data contained in the X-ray crystallography, new maps are generated. The new maps are then subjected to automatic chain tracing to produce 3-D models of the protein molecular structure. The models are checked with a sophisticated monitoring technique to see if any are successful.
To test the performance of their new integrated method, the researchers looked at 13 sets of X-ray crystallography data on molecules whose structures could not be solved by expert crystallographers. These structures remained unsolved even after the application of an extensive array of other approaches. The new integrated method was able to yield high resolution structures for 8 of these 13 highly challenging models.
"The results show that structural prediction methods such as Rosetta can be even more powerful when combined with X-ray crystallography data," the researchers noted. They added that the integrated approach probably outperforms others because it provides physical chemistry and protein structural information that can guide the massive sampling of candidate configurations. This information eliminates most conformations that are not physically possible.

Our procedures, the authors noted, required considerable computation, as up to several thousand Rosetta model predictions are generated for each structure. The researchers have developed automated procedures that potentially could narrow down the possibilities and lessen the number of times a model is rebuilt to make corrections. This automation could reduce computing time.
Through Baker's laboratory, many members of the general public contribute their unused home computer time to help in the effort to obtain structural models of proteins that are biologically and medically significant. The scientific discovery game is called "Fold It." (http://fold.it/portal/)

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Other authors of the paper appearing this week in Nature are Dr. Randy J. Read of the University of Cambridge, United Kingdom; Dr. Alexander Wlodawer of the National Cancer Institute; Drs. Gustav Oberdorfer and Ulrike Wagner of University of Graz, Austria; Dr. Eugene Valkov of the University of Cambridge; Drs. Assaf Alon and Deborah Fass of the Weizmann Institute of Science in Rehovot, Israel; Drs. Herbert L. Axelrod and Debanu Das of the SLAC National Accelerator Laboratory in Menlo Park, Calif.; Dr. Sergey M. Vorobiev of Columbia University in New York; and Dr. Hideo Iwai of the University of Helsinki, Finland.
The research was funded by the National Institute of General Medical Sciences and the National Center for Research Resources at the National Institutes of Health, the Howard Hughes Medical Institute, the Israel Science Foundation, DK Molecular Enzymology, Austrian Science Fund, the Center for Cancer Research at the National Cancer Institute, the Academy of Finland, and the U.S. Department of Energy's Office of Science, Biological and Environmental Research. The Joint Center for Structural Genomics, which is supported by the NIH's Protein Structure Initiative, contributed to the protein production and structural work.

Source EurekaAlert!