Labslink Research News

Researchers 'print' polymers that bend into 3-D shapes

Fri, 09 Mar 2012 17:22:34 PDT

Christian Santangelo, Ryan Hayward and colleagues at the University of Massachusetts Amherst recently employed photographic techniques and polymer science to develop a new technique for printing two-dimensional sheets of polymers that can fold into three-dimensional shapes when water is added. The technique may lead to wide ranging practical applications from medicine to robotics........> Full story

'Label-free' imaging tool tracks nanotubes in cells, blood for biomedical research

Mon, 05 Dec 2011 17:07:49 PDT

Researchers have demonstrated a new imaging tool for tracking structures called carbon nanotubes in living cells and the bloodstream, which could aid efforts to perfect their use in biomed.......> Full story

Researchers generate first complete 3-D structures of bacterial chromosome

Fri, 21 Oct 2011 18:20:53 PDT

A team of researchers at the University of Massachusetts Medical School, Harvard Medical School, Stanford University and the Prince Felipe Research Centre in Spain have deciphered the complete three-dimensional structure of the bacterium Caulobacter crescentus's chromosome. Analysis of the resulting structure —published this week in Molecular Cell — has revealed new insights into the function of genetic sequences responsible for the shape and structure of this genome. Scientists know that the three-dimensional shape of a cell's chromosome plays a role in how genetic sequences and genes are regulated. However, technical challenges have limited genome-wide analysis of a chromosome's architecture that would allow for simultaneous identification of the elements involved in shaping it and analysis of specific features of the structure. In this study, researchers used high-throughput chromatin interaction detection; next-generation DNA sequencing; computational modeling; and fluorescent microscopy to build the first 3D model of the architecture of the bacteria's chromosome and analyze the resulting structures. This new experimental approach revealed novel characteristics of a specific genetic sequence called the parS site, which helps to define the chromosome's shape. "What we've shown is that it's possible to combine molecular biology with 3D modeling technology to perform studies that tell us novel things about how genomes fold and identify the genetic sequences that are responsible." said Job Dekker, PhD, a pioneer in chromosome interaction detection technologies, professor of biochemistry & molecular pharmacology at the University of Massachusetts Medical School, and one of the authors on the study. Dekker and colleagues used "5C" technology to map more than 28,700 contact points in the Caulobacter crescentus's genome and used these contacts to approximate spatial distance in the folded chromosome. Plugged into a computational model, these contact points yielded a structural model of the bacterial chromosome which was strikingly beautiful: ellipsoidal in shape with arms helically arranged on either side. Marc A. Marti-Renom, PhD, a computational biologist who leads the Structural Genomics Laboratory at the Prince Felipe Research Center in Spain, and study author said "This work demonstrates that hybrid methods combining 5C maps with the Integrative Modeling Platform can produce genome-wide 3D models of unprecedented resolution, which for the first time allows for spatially pinpointing regulatory elements responsible of organizing the structure of a genome." The resulting 3D models of the Caulobacter crescentus genome, in conjunction with fluorescent microscopy, illustrate that the parS sequence, located in the pole of one arm of the chromosome, potentially served as an anchor for the genome and were instrumental in defining its overall structure. To unravel the role the parS site plays in the 3D organization of the chromosomal structure, Dekker and colleagues constructed mutant bacteria in which the parS site had been moved away from its normal position. Building 3D models of the shape of the mutated bacteria, they observed a change in the chromosome's structure; the entire genome had rotated clockwise. Changing the position of the parS site had resulted in a large-scale reorganization of the chromosome's shape that repositioned these sites at the cell's poles. Mark Umbarger, a post doctoral fellow at Harvard Medical School and study author notes, "Strikingly, we found that moving sequence elements which are no larger than 500 base pairs, led to a change in the conformation of all of the 4 million base-pairs of the chromosome!" "Our study is the first to test the effect of altering chromosome architecture. We were able to show that a very simple system, with a single anchor, can orient the whole chromosome inside of the cell." said Esteban Toro, PhD, one of the study authors and now a post doctoral fellow at the University of Pennsylvania. "These results suggest that the parS site in Caulobacter crescentus determines the orientation and global structure of the entire chromosome and are the only sequence elements that stably anchor the chromosome to the cell." The ability for scientists to perform structure function studies on chromosomes has the potential to yield powerful new insights into the biology of genomes. "When we began this project, most scientists were assessing the positions of a handful of genomic loci and attempting to derive general conclusions about genome structure. We were unhappy with this approach and sought to develop an integrated experimental approach to generate higher-resolution, and genome-wide insights," Umbarger said. "This isn't something we could have predicted from just looking at the DNA sequence," said Dekker. "This study illustrates how an investigation of 3D genomic structure can provide insights into how the complex relationships between genome sequence and structure can impact function. By studying genomic architecture we can potentially identify new classes of genomic sequences that are important in chromosome function and structure that we otherwise couldn't."

Manipulated gatekeeper: How viruses find their way into the cell nucleus

Mon, 03 Oct 2011 17:38:57 PDT

They have been around since the dawn of time and are a model of evolutionary success: viruses. Viruses are extremely adaptable but they have a problem: They cannot reproduce, so they smuggle their genes into suitable host cells. In the case of some viruses, the viral DNA has to enter the cell nucleus to reproduce. This has been known for almost 50 years. We know, for instance, that the adenovirus disassembles its protein shell in the first step. Just how the DNA is exposed and infiltrates the host cell, however, remained unclear despite decades of research. A research group headed by Urs Greber, a cell biologist at the University of Zurich, has now managed to clear up these points. As the scientists recently revealed in the journal Cell Host & Microbe, viruses use the cell's own mechanisms. The adenovirus latches onto a gatekeeper molecule, which sits on the nuclear pore complex in the nucleus envelope and controls the passage in and out of the nucleus. Another protein in the nuclear pore complex binds and activates a motor protein from the kinesin family, which regulates the transport of substances near the nucleus. Virus DNA uncoated with aid of host cell "The motor protein is in an active condition, can bind to micro-tubules and migrate along them," says Professor Greber, explaining his team's observations. And the docked virus uses precisely this situation for its purposes. The virus binds to the kinesin and uses the energy of the motor to disrupt its own shell, which exposes the virus DNA and prepares it for transport into the nucleus. The action of the activated motor has another effect, too: The nuclear pore ruptures and becomes markedly bigger, which enables the viral DNA to enter the cell nucleus more easily. Surprisingly, the cell repairs the defective nuclear pore so that the virus breach in the nucleus does not leave any visible damage in its wake. The viral DNA is smuggled into the nucleus practically without trace, where it can reproduce easily. The researchers used adenoviruses for their study. Adenoviruses cause, among other things, respiratory or epidemic ocular disease. Until recently, they were thought to be relatively harmless for healthy humans. However, the results of another research group recently demonstrated that a new kind of adenovirus triggered a dreaded zoonotic disease, meaning it was transmitted from an animal to humans before spreading from one person to another.

New cellular surprise may help scientists better understand human mitochondrial diseases

Tue, 06 Sep 2011 17:26:54 PDT

A surprising new discovery by the University of Colorado Boulder and the University of California, Davis regarding the division of tiny "power plants" within cells known as mitochondria has implications for better understanding a wide variety of human diseases and conditions due to mitochondrial defects. Led by CU-Boulder Assistant Professor Gia Voeltz and her team in collaboration with the UC-Davis team led by Professor Jodi Nunnari, the researchers analyzed factors that regulate the behavior of mitochondria, sausage-shaped organelles within cells that contain their own DNA and provide cells with the energy to move and divide. The dynamics of mitochondrion were intimately tied to another cell organelle known as the endoplasmic reticulum, which is a complex network of sacs and tubules that makes proteins and fats. Voeltz and her colleagues showed that the division of the mitochondria within cells is tied to the point or points where they are physically touching the endoplasmic reticulum in both yeast and mammalian cells. "This is the first time one cell organelle has been shown to shape another," said Voeltz of CU's molecular, cellular and developmental biology department. A paper on the study was published in the Sept. 2 issue of the journal Science. Co-authors on the study included CU-Boulder graduate student Jonathan Friedman, researcher Matthew West and senior Jared DiBenedetto and UC-Davis postdoctoral researcher Laura Lackner. Enclosed by membranes, mitochondria vary vastly in numbers per individual cells depending on the organism and tissue type, according to the researchers. While some single-cell organisms contain only a single mitochondrion, a human liver cell can contain up to 2,000 mitochondria and take up nearly one-quarter of the cell space. Since numerous human diseases are associated with mitochondrial dysfunction, it is important to understand how the division process is regulated, said Voeltz. Mitochondrial defects have been linked to a wide range of degenerative conditions and diseases, including diabetes, cardiovascular disease and stroke. "Our studies suggest the possibility that human mitochondrial diseases could result from disruption or excessive contact between the endoplasmic reticulum and the mitochondria." Previous work, including research in Nunnari's lab at UC-Davis, has shown that mitochondrial division is regulated by a protein known as "dynamine-related protein-1" that assembles into a noose-like ligature that tightens around individual mitochondrion, causing it to divide. The team found that several additional proteins linked to mitochondrial division also were found where the endoplasmic reticulum and mitochondria touched. "The new function for the endoplasmic reticulum expands and transforms our view of cell organization," said Nunnari, a professor and chair of molecular cell biology at UC-Davis. "It's a paradigm shift in cell biology."

New insight in how cells' powerhouse divides

Fri, 02 Sep 2011 18:44:19 PDT

New research from the University of California, Davis, and the University of Colorado at Boulder puts an unexpected twist on how mitochondria, the energy-generating structures within cells, divide. The work, which could have implications for a wide range of diseases and conditions, was published today (Sept. 2) in the journal Science........> Full story

The turn of the corkscrew: Structural analysis uncovers mechanisms of gene expression

Thu, 07 Jul 2011 17:49:51 PDT

The DNA in the cells of higher organisms is tightly wrapped around protein complexes called nucleosomes. This type of structural organization not only makes it possible to package the long DNA molecules in a highly compact form, it also provides the basis for the controlled expression of genetic information. Densely packed sections of the molecule are effectively in a repressed state, and genes located in these DNA segments cannot be transcribed.......> Full story

How muscle develops: A dance of cellular skeletons

Fri, 03 Jun 2011 18:48:13 PDT

Revealing another part of the story of muscle development, Johns Hopkins researchers have shown how the cytoskeleton from one muscle cell builds finger-like projections that invade into another muscle cell’s territory, eventually forcing the cells to combine. Such muscle cell fusion, the researchers say, is not only important for understanding normal muscle growth, but also muscle regeneration after injury or disease. The work, they believe, could further development of therapies for muscular dystrophy or age-related muscle wasting.......> Full story

New malaria protein structure upends theory of how cells grow and move

Mon, 30 May 2011 18:34:57 PDT

Researchers from the Walter and Eliza Hall Institute have overturned conventional wisdom on how cell movement across all species is controlled, solving the structure of a protein that cuts power to the cell 'motor'. The protein could be a potential drug target for future malaria and anti-cancer treatments. By studying the structure of actin-depolymerising factor 1 (ADF1), a key protein involved in controlling the movement of malaria parasites, the researchers have demonstrated that scientists' decades-long understanding of the relationship between protein structure and cell movement is flawed. Dr Jake Baum and Mr Wilson Wong from the institute's Infection and Immunity division and Dr Jacqui Gulbis from the Structural Biology division, in collaboration with Dr Dave Kovar from the University of Chicago, US, led the research, which appears in today's edition of the Proceedings of the National Academy of Sciences USA. Dr Baum said actin-depolymerising factors (ADFs) and their genetic regulators have long been known to be involved in controlling cell movement, including the movement of malaria parasites and movement of cancer cells through the body. Anti-cancer treatments that exploit this knowledge are under development. "ADFs help the cell to recycle actin, a protein which controls critical functions such as cell motility, muscle contraction, and cell division and signaling," Dr Baum said. "Actin has unusual properties, being able to spontaneously form polymers which are used by cells to engage internal molecular motors – much like a clutch does in the engine of your car. A suite of accessory proteins control how the clutch is engaged, including those that dismantle or 'cut' these polymers, such as ADF1. "For many years research in yeast, plants and humans has suggested that the ability of ADFs to dismantle actin polymers – effectively disengaging the clutch – required a small molecular 'finger' to break the actin in two," Dr Baum said. "However, when we looked at the malaria ADF1 protein, we were surprised to discover that it lacked this molecular 'finger', yet remarkably was still able to cut the polymers. We discovered that a previously overlooked part of the protein, effectively the 'knuckle' of the finger-like protrusion, was responsible for dismantling the actin; we then discovered this 'hidden' domain was present across all ADFs." Mr Wong said that the Australian Synchrotron was critical in providing the extraordinary detail that helped the team pinpoint the protein 'knuckle'. "This is the first time a 3D image of the ADF protein has been captured in such detail from any cell type," Mr Wong said. "Imaging the protein structure at such high resolution was critical in proving beyond question the segment of the protein responsible for cutting actin polymers. Obtaining that image would have been impossible without the synchrotron facilities." Dr Baum said the new knowledge will give researchers a much clearer understanding of one of the fundamental steps governing how cells across all species grow, divide and, importantly, move. "Knowing that this one small segment of the protein is singularly responsible for ADF1 function means that we need to focus on an entirely new target not only for developing anti-malarial treatments, but also other diseases where potential treatments target actin, such as anti-cancer therapeutics," Dr Baum said. "Malaria researchers are normally used to following insights from other biological systems; this is a case of the exception proving the rule: where the malaria parasite, being so unusual, reveals how all other ADFs across nature work." More than 250 million people contract malaria each year, and almost one million people, mostly children, die from the disease. The malaria parasite has developed resistance to most of the therapeutic agents available for treating the disease, so identifying novel ways of targeting the parasite is crucial. Dr Baum said that the discovery could lead to development of drugs entirely geared toward preventing malaria infection, without adverse effects on human cells. "One of the primary goals of the global fight against malaria is to develop novel drugs that prevent infection and transmission in all hosts, to break the malaria cycle," Dr Baum said. "There is a very real possibility that, in the future, drugs could be developed that 'jam' this molecular 'clutch', meaning the malaria parasite cannot move and continue to infect cells in any of its conventional hosts, which would be a huge breakthrough for the field."

Packaging process for genes discovered in new research

Thu, 19 May 2011 17:37:47 PDT

Scientists at Penn State University have achieved a major milestone in the attempt to assemble, in a test tube, entire chromosomes from their component parts. The achievement reveals the process a cell uses to package the basic building blocks of an organism's entire genetic code -- its genome. The evidence provided by early research with the new procedure overturns three previous theories of the genome-packaging process and opens the door to a new era of genome-wide biochemistry research. A paper describing the team's achievement will be published in the journal Science on 20 May 2011. The research was accomplished with the help of a new laboratory procedure developed by the team of scientists led by B. Franklin Pugh, the Willaman Chair in Molecular Biology at Penn State. The procedure allows scientists, for the first time, to do highly controlled biochemical experiments with all the components of an organism's genome. The team's research is designed to reveal the construction process for the chromosome -- the super-compressed marvel of molecular packaging that contains all an organism's DNA and associated proteins. "Our procedure starts with an entire genome of DNA from yeast cells that we propagate through bacteria, then purify, "Pugh said. "Next, we add equal parts of pure histones, the protein building blocks of chromosomes. Then we allow the assembly process to begin." The result was that short sections of the lanky string of gene-containing DNA became wound around a series of histone proteins, forming a line of knots called nucleosomes separated by unknotted sections of DNA. Although earlier studies in other labs had shown that histones and DNA alone could construct a series of nucleosome knots along the DNA string, the overall structure of this construction was not nearly as organized as it needed to be in order to look like chromatin inside of a cell -- the material that the cell remodels to form chromosomes. Pugh's team sought out the recipe that would produce the actual, highly organized structure of chromatin. "Just like baking a mixture of flour and water produces unleavened bread that lacks the texture of leavened bread, so too did the mixture of histones and DNA lack the texture of chromatin," explains Pugh. To provide "texture" to the histone-DNA mix, graduate student Christian Wippo added yeast extract, under the guidance of laboratory head Philipp Korber at the University of Munich, Germany, and co-investigator on the project. "But, like adding yeast to flour and water without the sugar, this was not enough," Pugh said. As Korber recounts, "Once we added ATP, 'the bread began to rise'." In other words, chromatin remodeling enzymes in the extract needed the energy from ATP to reposition the nucleosome knots along the DNA, thereby giving rise to the chromatin the texture that is seen inside of cells. "Chromatin-remodeling enzymes actively pack nucleosomes against barriers that sit at the beginning of every gene, and this process creates uniformly positioned nucleosome arrays," Pugh said. A critical part of the study that allowed the scientists to "see" the chromatin texture was developed by Graduate Student Zhenhai Zhang, under Pugh's direction. "Because there are more than 60,000 nucleosomes that comprise chromosomes in yeast cells, seeing patterns in this texture would be impossible without the computational pattern-searching algorithms developed by Zhang," Pugh said. Zhang explained, "Remarkably, when all genes were aligned, nucleosomes at the beginning of the genes also aligned, rather being randomly scattered about. Without the yeast extract and ATP, only nucleosome-free zones could be seen at the beginning of genes." This work is significant because it now allows scientists to experimentally probe the structure and function of chromosomes and their component genes in ways that simply were off limits before. "The cell protects chromosomes from the outside environment, including probing scientists," Pugh said. "We now have a way to study the components of the chromosome outside the protective confines of the cell." Because defects in chromatin organization lead to medical problems -- including certain cancers and developmental disorders -- more direct access to chromatin in its properly organized state is expected to help hasten the search for remedies to many human diseases.

Researchers get a first look at the mechanics of membrane proteins

Sun, 17 Apr 2011 20:46:33 PDT

In two new studies, researchers provide the first detailed view of the elaborate chemical and mechanical interactions that allow the ribosome – the cell's protein-building machinery – to insert a growing protein into the cellular membrane. The first study, in Nature Structural and Molecular Biology, gives an atom-by-atom snapshot of a pivotal stage in the insertion process: the moment just after the ribosome docks to a channel in the membrane and the newly forming protein winds its way into the membrane where it will reside. A collaboration between computational theoretical scientists at the University of Illinois and experimental scientists at University of Munich made this work possible. Using cryo-electron microscopy to image one moment in the insertion process, the researchers in Munich were able to get a rough picture of how the many individual players – the ribosome, membrane, membrane channel and newly forming protein – come together to get the job done. Each of these structures had been analyzed individually, but no previous studies had succeeded in imaging all of their interactions at once. "The computational methodology contributed by the Illinois group was crucial in interpreting the new cryo-EM reconstruction in terms of an atomic level structure, and testing the interpretation through simulation," said co-author Roland Beckmann at the University of Munich. "Our joint study is unique in so closely and successfully combining experimental and computational approaches." To image the ribosome's interaction with the membrane, Beckmann's team used small disks of membrane held together with belts of engineered lipoproteins. University of Illinois biochemistry professor Stephen Sligar developed and pioneered the use of these "nanodiscs." The Illinois team used the cryo-EM images as well as detailed structural information about the ribosome and other molecules to construct an atom-by-atom model of the whole system and "fit the proteins into the fuzzy images of the electron microscope," said University of Illinois physics and biophysics professor Klaus Schulten, who led this part of the analysis with postdoctoral researcher James Gumbart. "The ribosome with the membrane and the other components is a simulation of over 3 million atoms," Schulten said, a feat accomplished with powerful computers and "over 20 years of experience developing software for modeling biomolecules." (Schulten is principal investigator of the NIH-funded Resource for Macromolecular Modeling and Bioinformatics at Illinois, which supports the study of large molecular complexes in living cells, with a special focus on the proteins that mediate the exchange of materials and information across biological membranes.) This analysis found that regions of the membrane channel actually reach into the ribosome exit to help funnel the emerging protein into the channel. Depending on the type of protein being built, the channel will thread it all the way through the membrane to secrete it or, as in this case, open a "side door" that directs the growing protein into the interior of the membrane, Schulten said. The researchers also saw for the first time that the ribosome appears to interact directly with the membrane surface during this process. The researchers found that a signal sequence at the start of the growing protein threads through the channel and anchors itself in the membrane. Previous studies suggested that this signaling sequence "tells" the ribosome what kind of protein it is building, directing it to its ultimate destination inside or outside the cell. "This new work visualizes this process for the first time, giving researchers the first image of how nascent proteins actually get into membranes," Schulten said. "It's like going to Mars and being the first to look at Mars." In a second study, in the Proceedings of the National Academy of Sciences, Schulten, Gumbart and graduate student Christophe Chipot found that proteins get inserted into the membrane in two stages. First, the ribosome "pushes" the growing protein into the membrane channel, and then, in a second step, the protein enters the membrane. The original push, driven by the chemical energy that the ribosome harvests from other high-energy molecules in the cell, allows even highly charged proteins to pass easily into the oily, nonpolar environment of the membrane, the researchers found.

Exercise may prevent stress on telomeres, a measure of cell health

Mon, 04 Apr 2011 17:57:47 PDT

UCSF scientists are reporting several studies showing that psychological stress leads to shorter telomeres – the protective caps on the ends of chromosomes that are a measure of cell age and, thus, health. The findings also suggest that exercise may prevent this damage. The team focused on three groups: post-menopausal women who were the primary caregivers for a family member with dementia; young to middle-aged adults with post-traumatic stress disorder; and healthy, non-smoking women ages 50 to 65 years. They examined telomeres in leukocytes, or white blood cells, of the immune system, which defends the body against both infectious agents and cell damage. "Our findings suggest that traumatic and chronic stressful life events are associated with shortening of telomeres in cells of the immune system, but that physical activity may moderate this impact," said co-author Jue Lin, PhD, associate research biochemist in the laboratory of senior author and Nobel laureate Elizabeth Blackburn, PhD, professor of biochemistry and biophysics at UCSF. Lin presented the findings in a poster session on Monday, April 4, 2011, at the AACR 102nd Annual Meeting 2011. Telomeres are tiny units of DNA at the ends of chromosomes that protect and stabilize chromosomes. Every time a cell divides, some telomeres drop off. After a certain number of cell divisions, which varies depending on the cell type, the telomeres reach a critical length and the cell typically dies. Sometimes, however, the cells cease to divide and are subjected to genomic instability, promoting inflammation in the body. Scientists have known for more than a decade that the length of telomeres in immune system cells is a marker of cell aging. In recent years, they have discovered that shorter telomeres are associated with a broad range of aging-related diseases and are predictive of incidence and poor prognosis of cardiovascular disease and a variety of cancers. A 2004 study led by Blackburn and UCSF colleague Elissa Epel, PhD, associate professor of psychiatry, suggested that psychological stress may impact the length of telomeres in immune system cells. They reported that the perception of psychological stress in female caregivers of chronically sick children was related to shorter telomeres in lymphocytes, key cells of the immune system. This offered the first evidence that telomere maintenance potentially mediates the well documented detrimental effects of stress on health. (Proceedings of the National Academy of Sciences, Nov. 29, 2004) In the current research, one study, led by Epel, followed for two years 63 healthy postmenopausal women who were the primary caregivers for a family member with dementia. In an earlier analysis of 36 of these women, pessimism was associated with high levels of a pro-inflammatory protein often associated with aging and disease states, and with short telomeres. In a recent and separate analysis of the full group of women, an increase in perceived stress was related to an increase in the odds of having short telomeres only in the non-exercising women. Among those who exercised, perceived stress was unrelated to telomere length. In the current analysis of the larger group, it was revealed that an increase in perceived stress over the course of one year was associated with a decrease in telomere length during that time. A second study, led by Aoife O'Donovan, PhD, and Thomas Neylan, MD, UCSF professor of psychiatry at the San Francisco VA Medical Center, examined 43 people ages 20 to 50 with chronic post-traumatic stress disorder. They were compared to 47 age- and sex-matched individuals without PTSD.The results showed a relationship between PTSD and short telomere length. But even more interesting, said Lin, the finding showed that, in these adults, exposure to childhood trauma – at or before age 14 – also was associated with telomere shortening and accounted for the link between PTSD and telomeres. A third study, led by Eli Puterman, PhD, analyzed data from 251 healthy, non-smoking women ages 50-65 of varying activity levels. The findings showed that non-exercising women with histories of childhood abuse had shorter telomeres than those with no histories of abuse. But, in those women who exercised regularly, there was no link between childhood abuse and telomere length, after controlling for body mass index, income, education and age. "We saw a relationship between childhood trauma and short telomere length but the relationship seems to go away in people who exercise vigorously at least three times a week," Lin said.

Syracuse University research team shapes cell behavior research

Fri, 11 Mar 2011 03:44:23 PDT

A team led by James Henderson, assistant professor of biomedical and chemical engineering in Syracuse University’s L.C. Smith College of Engineering and Computer Science (LCS) and researcher in the Syracuse Biomaterials Institute, has used shape memory polymers to provide greater insight into how cells sense and respond to their physical environment.......> Full story

The connection between a cell's cytoskeleton and its surface receptors

Mon, 07 Mar 2011 03:17:54 PDT

New findings from researchers at Harvard Medical School in Boston and the Hospital for Sick Children in Toronto may shed light on the mechanisms that regulate the organization of receptors on the cell surface, a critical aspect of cell signaling not well understood at this time. The group reports on their use of the macrophage protein CD36, a clustering-responsive class B scavenger receptor, as a model for studying the processes governing receptor clustering and organization. The protein is involved in a number of cellular and physiological functions that range from lipid metabolism to immunity, but it is unknown how the CD36 protein is organized in the cell (as monomers or as oligomers) and how that organization leads to its biological functions. The researchers employed a combination of powerful tools: quantitative live-cell single-molecule imaging and biochemical/pharmacological approaches to study the dynamics, oligomerization and signaling of CD36 in primary human macrophages. The group reports that movement of CD36 in the macrophage plasma membrane is regulated by the sub-membranous actin meshwork and by microtubules, demonstrating that these cytoskeletal components might play a critical role in receptor function, in general. In terms of the impact of this research, lead researcher Khuloud Jaqaman says: "In the long run, establishing the relationship between receptor organization and cell signaling might aid in the development of drugs since receptors on the cell surface are the most accessible to pharmacological manipulation."

Atomic model of tropomyosin bound to actin

Wed, 16 Feb 2011 03:29:17 PDT

New research sheds light on the interaction between the semi-flexible protein tropomyosin and actin thin filaments. The study, published by Cell Press on February 15th in the Biophysical Journal, provides the first detailed atomic model of tropomyosin bound to actin and significantly advances the understanding of the dynamic relationship between these key cellular proteins. Tropomyosin is a long protein that associates with actin, a highly conserved thin filament protein found in organisms from yeast to humans. Actin, a major part of the cell's cytoskeleton, drives shape changes and cellular locomotion in many types of cells, and is part of the contractile apparatus in muscle cells. Tropomyosin binds to actin ad acts as a molecular barrier, essentially covering up active sites that are required for actin to interact with other proteins. In turn cellular signals can trigger additional regulatory proteins to move tropomyosin, dislodging the barrier in order to allow actin to associate with remodeling and motor proteins. "Previous studies examining tropomyosin in isolation suggested that it is a coiled coil that matches the shape of actin filaments and is arranged along their surface," explains senior study author, Dr. William Lehman from the Department of Physiology and Biophysics at Boston University School of Medicine. "However, a complete elucidation of tropomyosin-based regulatory mechanisms requires a complete representation of the atomic structure and mechanical properties of the tropomyosin molecule linked to its biological substrate." Building on previous findings that the association between tropomyosin and actin is an electrostatic attraction between oppositely charged amino acids, Dr. Lehman and colleagues explored thousands of combinations of different rotations and positions of tropomyosin to find the most favorable interaction between tropomyosin and actin. The researchers then used electron microscopy as a second approach to also reconstruct the interaction. The two methods yielded virtually identical solutions, "which is very gratifying", says Dr. Lehman. The authors discuss how the interaction between tropomyosin and actin is just weak enough that tropomyosin can be readily perturbed by regulatory proteins and act as a molecular switch to regulate actin interaction with other proteins. "The atomic model that we propose can serve as a reference location to characterize tropomyosin regulatory movements on actin thin filaments," concludes Dr. Lehman. "Moreover, the map of actin-tropomyosin provides a structural platform to assess mutations that influence actin-tropomyosin behavior and also to develop tropomyosin-mimicking peptide drugs designed to modulate actin-myosin or other interactions."

Scientists develop method to identify fleetingly ordered protein structures

Wed, 09 Feb 2011 03:17:27 PDT

A team of scientists from The Scripps Research Institute and the University of California, San Diego (UCSD) have developed a novel technique to observe previously unknown details of how folded structures are formed from an intrinsically disordered protein. The insights could help scientists to better understand the mechanism of plaque formation in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. The results of the study, which has broad implications for the field, were recently published in an advanced, online issue of the journal Nature Methods. The new technique allows previously unheard-of rapid detection—in less than 0.001 seconds—of transiently folded single-molecule structures from a class of often-amorphous molecules known as "intrinsically disordered proteins." The method also permits new types of observations of short-lived protein complexes. "This exciting new technique allowed us to visualize multiple short-lived folded states," said Scripps Research Associate Professor Ashok Deniz, Ph.D., who led the study with UCSD Professor Alex Groisman, Ph.D., and Yann Gambin, Ph.D., of Scripps Research. "Further, better understanding of complexity during folding may offer more ways to regulate this interesting class of proteins." The specific protein examined in the study was -synuclein, which is highly concentrated in neural tissue. The protein has been implicated in Parkinson's and Alzheimer's diseases, as it is found in high concentrations in aggregates from the brains of patients with these conditions. Mixing It Up Unlike typical proteins in the cell, intrinsically disordered proteins such as -synuclein do not adopt a stable globular form in isolation. Rather, intrinsically disordered proteins are like a messy, unfolded string of yarn, whereas typical globular proteins more closely resemble yarn neatly knit into complicated and functional shapes like that of a glove. Studying intrinsically disordered proteins has been a challenge. Known techniques to determine protein structures are often designed for ordered proteins, and detection of transient shapes in structurally heterogenous proteins such as -synuclein has been difficult. To remedy this situation, the Deniz and Groisman labs set out to devise advanced technologies to shed light on this novel class of proteins. The new experimental technique described in the Nature Methods paper successfully combines and improves upon two established experimental methods: single-molecule Förster Resonance Energy Transfer (smFRET) and microfludic mixing. smFRET detection allows for the observation of very small distances (in the range of one billionth of a meter), and can reveal changes in the molecular structure in real time. The method works by transfer of energy between single fluorescent dye molecules used as tags on a protein. One dye (donor) absorbs light and can emit red-shifted fluorescent light, whereas the other dye (acceptor) can receive the energy from the donor and emit even more red-shifted light. The relative amount of light emitted by the two dyes depends on the distance between them, and hence can be used as a molecular ruler to measure distances in proteins. Microfluidic mixing in high-speed laminar flows has been used previously to rapidly initiate protein-folding reactions, but most observations have been made on a bulk rather than single-molecule level. Detecting Protein Folding in a Chip The key innovation of the research was to combine rapid mixing in a high-speed flow with single-molecule detection in a slow flow by abruptly decelerating the flow between mixing and detection regions. The protein started in an aqueous solution and was mixed with a substance known as sodium dodecyl sulfate (SDS), which is normally used to unfold proteins but also facilitates the folding of -synuclein due to its special interactions with amphipathic environments. This combination of rapid mixing and detection enabled the discovery of short-lived protein states previously invisible to researchers. The microfluidic mixing itself was performed inside a small chip housing several hollow channels (or tunnels). The main channel is like a freeway upon which the protein travels. This channel connects at junction points to other inlets (on-ramps to the freeway) or outlets (off-ramps from the freeway). The two inlets were used to funnel buffer and SDS into the central stream, effectively focusing the central protein stream into a narrow, fast-moving lane, and allowing a rapid switch into a solution containing SDS. Further along the channel at another junction, two outlets forced most of the "traffic" to exit. As a result, the speed of the remaining central part of the stream, or central lane, abruptly decreased. Protein molecules in this slower, focused stream of protein were then detected by smFRET. The rapid slowing was a critical new element in the method, providing just enough time for scientists to examine individual slower-moving proteins as they passed by the detector. A movie of the proteins' changing shapes could be recorded over time. At the inlet (on-ramp), -synuclein was introduced to negatively charged SDS, and -synuclein began to fold. Combined with the rapid mixing, the fluorescence from the dye tags—which had been placed far apart on -synuclein—revealed previously unknown details of transiently folded structures of -synuclein, observed in the sub-millisecond timeframe. Dynamic Conditions Prior to this work, the equilibrium state of -synuclein in the SDS-containing solution was known to be an extended helix (like the coil of a phone cord) called the F state. This ordered structure exists in the presence of the negatively charged biological membrane or SDS. "So the question was: 'Do we go directly from the disordered protein to that F state?'" said Deniz. "And the answer from our experiments was, 'No.' We visit an intermediate structure, which has a similar FRET efficiency to what was previously observed to be a helix-kink-helix (I state), like a coil with a kink that bends the coil into a U-shape instead of a straight coil. Surprisingly, even this initial transition is complex, and provides us views of how the protein shape changes soon after binding to its partner molecules. What this means is that, as conditions in the cell are dynamic, these new states might give us many more points of regulation of -synuclein." Next in the lab's research, Deniz plans to examine questions including: "Do different -synuclein structures aggregate differently, and how do they couple to function?" "What triggers the aggregation?" "What exactly are the roles of aggregates?" and "What kinds of structures will be detected for -synuclein interacting with other protein, lipid, and small-molecule partners?" In addition, Deniz believes the developed microfluidic method will improve scientists' understanding of complexity in many other biological and health-related molecules.

A protein reinforces memory and prevents forgetfulness

Thu, 03 Feb 2011 03:13:09 PDT

An international research study has shown that, in animal models, type 2 insulin (IGF-II) growth factor reinforces memory and prevents forgetfulness. The results of the work, developed at the Mount Sinai Hospital Medical School in New York, in which Ana García-Osta, researcher at the Centro de Investigación Médica Aplicada (CIMA) of the University of Navarra took part, was published in the latest issue of Nature. The article describes the role played by IGF-II in the processes of consolidation of long-term memory. “Through microarray studies (gene identification) we see that the gene that codes this protein increases in the brain of rats exposed to a learning session. On administering IGF-II locally into the hypocampus (the part of the brain where memory is acquired and consolidated) of these animals, we observe that the animal undergoes reinforcement of memory and prevention of forgetfulness”, explained the CIMA researcher........> Full story

New microscopy method opens window on previously unseen cell features

Tue, 25 Jan 2011 03:29:58 PDT

Despite the sophistication and range of contemporary microscopy techniques, many important biological phenomena still elude the precision of even the most sensitive tools. The need for refined imaging methods for fundamental research and biomedical applications related to the study of disease remains acute. Nongjian (N.J.) Tao and his colleagues at the Biodesign Institute at Arizona State University have pioneered a new technique capable of peering into single cells and even intracellular processes with unprecedented clarity. The method, known as electrochemical impedance microscopy (EIM) may be used to explore subtle features of profound importance for basic and applied research, including cell adhesion, cell death (or apoptosis) and electroporation—a process that can be used to introduce DNA or drugs into cells........> Full story

How the hat fits: Structural biology study reveals shape of epigenetic enzyme complex

Fri, 21 Jan 2011 03:32:16 PDT

To understand the emerging science of epigenetics—a field that describes how genes may be regulated without altering the underlying DNA itself—scientists are deciphering the many ways in which enzymes act on the proteins surrounding DNA within cells. One type of these enzymes, proteins known as histone acetyltransferases (HATs), act on DNA by modifying DNA-bound proteins called histones. This act of modification, called acetlyation, can dictate how histones interact with DNA and other proteins affecting processes such as DNA replication, transcription (reading the gene), and repair. In the February 9 issue of the journal Structure, available online, researchers at The Wistar Institute are the first to describe the complete atomic structure formed by a yeast HAT, known as Rtt109, and one of its associated proteins. Their findings demonstrate how a particular histone acetylation event works, a crucial step to understanding epigenetics and the related processes that underlie both health and disease. According to the study's senior author, Ronen Marmorstein, Ph.D., professor and program leader of Wistar's Gene Expression and Regulation Program, two copies of Rtt109 bind to two copies of a "chaperone" protein to form a ring. "The ring fits atop a histone much like a halo, and we find that the type of chaperone dictates exactly how the enzyme affects the histone by determining the exact position of acetylation," said Marmorstein. "The structure represents a nice model system for the regulation of protein acetylation, and teaches us something new about the biology of this enzyme, Rtt109." The act of acetylation adds an "acetyl group," a small chemical structure, to a lysine—one of the amino acids that make up a given protein. Altering one lysine could change the shape of a protein, such as a histone, in a subtle way, perhaps redirecting how it functions. Rtt109, the researchers say, acetylates any of three specific lysines on histones, and exactly which of the histone lysines are modified is determined by which chaperone escorts Rtt109 into place. Since histones are such crucial DNA-associated proteins, altering a single lysine in a single part of the structure can have profound effects on the "behavior" of that histone, such as exposing a particular set of genes to be read, for example. In the paper, Marmorstein and his colleagues show how Rtt109 associates with a particular chaperone called Vps75. Rtt109 also associates with another chaperone, Asf1, which has been shown to enable the Rtt109 to modify lysines in a different spot on a given histone, creating a different effect in how that histone interacts with DNA and in turn changing the cell's biological properties. Their study is the first to show that two Rtt109 enzymes pair up with two Vps75 chaperones to form a ring. The laboratory created crystals of the protein complex and used a technique called X-ray crystallography to "see" the structure of the complex by analyzing the patterns formed when X-rays bounce off the crystals. They used the powerful X-ray source at the Argonne National Laboratory's Advanced Photon Source, which enabled the team to determine the structure of the protein complex at the atomic scale—at a resolution of 2.8 angstroms (2.8 billionths of a meter), which is smaller than the distance between individual rungs on the DNA ladder. Since the Marmorstein laboratory began its work on HATs over a decade ago, several large-scale studies have shown that acetylation occurs to over 2000 proteins, not just histones. According to Marmorstein, it appears there is an entire web of communication going on within cells directly attributable to protein acetylation, another level of complexity in an already-complex field. "We have seen many different proteins over several different pathways become affected by acetylation, which can alter the processes of RNA metabolism, cell cycle control, cancer, and a number of different aspects of life. It looks like protein acetylation has much broader biological implications than initially appreciated," said Marmorstein. "In many ways, it seems a lot like what we have seen in recent years with protein kinases and cell signaling," said Marmorstein. "What we're learning is that these HATs, and possibly other protein acetyltransferases, are regulated in much the same way. They have these profound effects within cells, but it is still very new to science. How it works is a big black box that we intend to decipher."

With chemical modification, stable RNA nanoparticles go 3-D

Fri, 21 Jan 2011 03:24:30 PDT

For years, RNA has seemed an elusive tool in nanotechnology research—easily manipulated into a variety of structures, yet susceptible to quick destruction when confronted with a commonly found enzyme.

"The enzyme RNase cuts RNA randomly into small pieces, very efficiently and within minutes,” explains Peixuan Guo, PhD, Dane and Mary Louise Miller Endowed Chair and professor of biomedical engineering at the University of Cincinnati (UC). "Moreover, RNase is present everywhere, making the preparation of RNA in a lab extremely difficult.”

But by replacing a chemical group in the macromolecule, Guo says he and fellow researchers have found a way to bypass RNase and create stable three-dimensional configurations of RNA, greatly expanding the possibilities for RNA in nanotechnology (the engineering of functional systems at the molecular scale)........> Full story

Scientists reveal complete structure of HIV's outer shell

Thu, 20 Jan 2011 03:19:14 PDT

A team of scientists at The Scripps Research Institute and the University of Virginia has determined the structure of the protein package that delivers the genetic material of the human immunodeficiency virus (HIV) to human cells. The work is the culmination of studies carried out over the last decade looking at different portions of the cone-shaped container, or the capsid. The final piece of the puzzle, described in an article published in Nature on January 20, 2011, details the structure of the two ends of the cone. "This paper is a real milestone for research from our group," says the study's senior author Mark Yeager, M.D., Ph.D., a Scripps Research professor and staff cardiologist and chair of the Molecular Physiology and Biological Physics Department at The University of Virginia School of Medicine. A detailed description of the complete HIV capsid will provide a roadmap for developing drugs that can disrupt its formation and thus prevent infection by HIV. Assembling the Package HIV binds to receptors on human cells and then delivers the capsid inside them. Once inside a cell, the capsid comes apart, releasing its precious cargo—the virus's genetic material. HIV then sabotages the cell machinery to make many copies of its genes and proteins. As new viruses are made, the genetic material is packaged into spherical immature capsids that HIV uses to escape from the infected cell. But before these newly released viruses can infect other cells, the immature capsid undergoes a dramatic rearrangement to form the mature, cone-shaped shell. If formation of the mature capsid is disrupted, the virus is no longer infectious. Thus, new drugs targeting capsid formation could provide valuable additions to the arsenal of existing drugs against HIV. A "Floppy" Bridge To develop drugs that disrupt capsid formation, however, scientists first need to know precisely how it is formed. One technology researchers use to obtain detailed structures of biological molecules is X-ray crystallography. This technique requires growing crystals of a molecule and then bombarding the crystals with X-rays to determine the positions of all the atoms. But unlike the cone-shaped capsids of other viruses, such as the poliovirus, which have a rigid, symmetrical structure that obediently assembles into crystals, the HIV capsid is flexible and can adopt slightly different shapes. Part of the reason for this flexibility is the protein that makes up the HIV capsid, the CA protein, consists of two ends held together by a "floppy" bridge. In the capsid, each CA protein joins hands with other CA proteins, forming groups of five or six proteins. The main body of the capsid contains about 250 of the six-fold units or hexamers. Each end of the cone is then closed off by either five or seven smaller five-fold units or pentamers. "It is impossible to grow crystals of the entire HIV capsid," says Yeager. As a result, his team used a "divide and conquer approach." Divide and Conquer Working with husband-and-wife team Owen Pornillos and Barbie Ganser-Pornillos, investigators in his lab, Yeager partitioned the HIV capsid into smaller components, then determined their respective structures. Yeager's group started by focusing on the structure of the CA hexamer. A breakthrough came in a 2007, when the group viewed the CA hexamers with a powerful electron microscope. Guided by information from that structure, in 2009 the team managed to trick the CA hexamers into forming crystals. The researchers were then able to determine the particles' structures at 2-Angstrom resolution (one Angstrom equals one ten-billionth of a meter). Having cracked the atomic structure of the hexamer, the investigators turned their attention to the more elusive pentamers. Next Came the Pentamer In this latest study, Yeager, Pornillos, and Ganser-Pornillos used techniques similar to those they had applied to the hexamers to obtain the crystal structures of the CA pentamers. The new structure reveals that five CA proteins link hands at one end, called the N-terminal domain (NTD), to form a circle. The opposite ends of the CA proteins, called C-terminal domain (CTD), form a floppy belt around this central core. Then, CTD links to CTD to connect adjacent pentamers. The structure reveals flexibility and mobility both between the central core and belt within each pentamer and at the CTD-CTD interfaces of adjacent pentamers. The CTD subunits can rotate relative NTDs. "As a result, each ring can adopt slightly different angles relative to its adjacent rings," says Pornillos, first author of the paper. The structure of the pentamers is remarkably similar to that of the hexamers, notes Pornillos, with one important difference. Because pentamers are smaller than hexamers, the amino acids, the building blocks of proteins, at the center of the pentamer ring are closer together than in the hexamer. Many amino acids have positive or negative charges. When two amino acids with the same charge are close together they tend to push each other away. One amino acid in the CA protein, called arginine, with a positive charge, sits smack in the middle of both the hexamer and pentamer ring. Because in the pentamer the arginines are packed much closer together, they repel one another, making the pentamer a less stable structure than the hexamer. This may explain why there are many more hexamers in the mature HIV capsid compared to pentamers. The only place where pentamers are likely to form is at the capsids' ends, where the linked CA proteins have to bend dramatically to close off the capsid—a feat the pentamer is more apt to perform. "Arginine is the critical switch between hexamer and pentamer formation," says Yeager. "We can finally explain why the CA protein would make one or the other." An Atomic Model of the HIV Capsid Having solved the atomic structures of both CA hexamers and pentamers, Yeager and colleagues for the first time were able to build a complete atomic model of the mature HIV capsid. The researchers now plan to further refine the model using sophisticated computer programs to determine the stability of the structure in different regions and to identify possible "weak" points they can target using newly designed drugs. They will also begin studying the structure of the immature capsid to determine how this version of the capsid transitions to the mature form—a step in the virus lifecycle that has remained mysterious. "We don't have the full story yet, but we have volume one," says Yeager.

CSHL study finds that 2 non-coding RNAs trigger formation of a nuclear subcompartment

Mon, 20 Dec 2010 03:23:20 PDT

The nucleus of a cell, which houses the cell's DNA, is also home to many structures that are not bound by a membrane but nevertheless exist as distinct compartments. A team of Cold Spring Harbor Laboratory (CSHL) scientists has discovered that the formation of one of these nuclear subcompartments, called paraspeckles, is triggered by a pair of RNA molecules, which also maintain its structural integrity. As reported in a study published online ahead of print on December 19 in Nature Cell Biology, the scientists discovered this unique structure-building role for the RNAs by keeping a close watch on them from the moment they come into existence within a cell's nucleus. The scientists' visual surveillance revealed that when the genes for these RNAs are switched on, and the RNAs are made, they recruit other RNA and protein components and serve as a scaffolding platform upon which these components assemble to form paraspeckles. The two RNAs described in the study, named MENε and MENβ, are "non-coding" RNAs —a type of RNA that does not serve as a code or template for the synthesis of cellular proteins. The genes that give rise to these non-coding RNAs are now thought to make up most of the human genome, in contrast to the genes that produce protein-coding RNAs, which account for approximately 2% of the human genome. "We've known for several years that much of the other 98% of the genome doesn't encode for useless RNA," explains CSHL's Professor David L. Spector, who led the current study. "Various types of non-coding RNAs have been found that regulate the activity of protein-coding genes and cellular physiology in different ways. Our results reveal a new and intriguing function for a non-coding RNA—the ability to trigger the assembly and maintenance of a nuclear body." The nuclear bodies in question—the paraspeckles—are believed to serve as nuclear storage depots for RNAs that are ready to be coded, or translated, into proteins but are retained in the cell nucleus. Paraspeckles are thought to release this RNA cache into the cell's cytoplasm—the site of protein synthesis—under certain physiological conditions, such as cellular stress. Spector estimates that storing pre-made protein-coding RNA within the paraspeckles and releasing them as needed allows the cell to respond faster than if it had to make the RNA from scratch. Previous experiments by Spector's team and two other groups indicated that MENε and MENβ RNAs were the critical elements for paraspeckle formation. "What wasn't clear was how the paraspeckles actually form and the dynamics of how the non-coding MEN RNAs help organize and maintain its structure," says Spector. To address this question, the team developed an innovative approach—spearheaded by CSHL postdoctoral fellow Yuntao (Steve) Mao and graduate student Hongjae Sunwoo—to peer into living cells and capture the real-time dynamics of the interactions among the set of molecules known to be involved in paraspeckle formation. The scientists engineered cells in which each of these players—the MENε/β genes, the newly formed MEN RNAs, and the various paraspeckle protein components—each carried a different colored fluorescent tag. The cells were also genetically manipulated such that the MEN genes could be switched on by exposing the cells to a drug. The resulting movies shot by the Spector team, showed that within five minutes of switching on the MENε/β gene, individual paraspeckle proteins arrived and assembled at the sites of MEN RNA transcription. As the RNA transcripts accumulated, the fully functional paraspeckles enlarged in tandem and eventually broke away to cluster around the transcription sites. "Our experiments show that it is the act of MEN RNA transcription alone that triggers paraspeckle formation and sustains them," says Spector. In the absence of transcriptional activity—such as during cell division or when the scientists added drugs that block RNA transcription or specifically switched off the MEN genes—the newly formed paraspeckles fell apart. This dependency on RNA transcription seems to be unique, as other nuclear compartments such as Cajal bodies can form when one of their components is simply tethered to a site on the genome, which in turn causes other components to coalesce around it. In contrast, says Spector, "Paraspeckles seem to follow a different assembly model in which MEN non-coding RNAs serve as seeding molecules that are driven by transcription to recruit the other components."

Tools used to decipher 'histone code' may be faulty

Fri, 17 Dec 2010 03:48:35 PDT

The function of histones -- the proteins that enable yards of DNA to be crammed into a single cell -- depends on a number of chemical tags adorning their exterior. This sophisticated chemical syntax for packaging DNA into tight little coils or unraveling it again -- called the "histone code" -- is the latest frontier for researchers bent on understanding how genetics encodes life. But recent research from the University of North Carolina at Chapel Hill has found a number of issues with histone antibodies, the main tools used to decipher this code, suggesting they may need more rigorous testing. "When I have presented our findings at major meetings, the reactions of my peers have been shock and awe across the board," said senior study author Brian Strahl, PhD, associate professor of biochemistry and biophysics at UNC. "Hundreds and hundreds of researchers around the world use them and assume they are accurate. Yet we have found that they need to be used with caution." Strahl is a member of the UNC Lineberger Comprehensive Cancer Center. The results of the study, which appears online December 16, 2010, in the journal Current Biology, also found that the proteins that interpret the histone instructions are affected not just by the specific chemical tag they land on but also by other tags in the neighborhood. The "Histone Code" was first proposed almost ten years ago by Strahl and epigenetics researcher C. David Allis, who was his postdoctoral advisor at the time. In a review article published in the journal Nature that has since been cited over 3000 times, Strahl and Allis suggested a model of how histones and their posttranslational modifications may function in chromatin. Histones are the protein spools around which strands of DNA are wrapped to form a package called chromatin. Depending on the modifications or tags decorating the histones, DNA is either closed up tightly within this package or lies open so that its genes can be read. Strahl and Allis hypothesized that distinct combinations of histone modifications work together to form a code, akin to the classic genetic code in which distinct combinations of nucleotides make an amino acid. These histone modifications – chemical changes like phosphorylation, acetylation and methylation -- generate a language that is interpreted through the ability to recruit the proteins that modulate chromatin. "But this histone code is way more complicated, because there are over a 100 different histone modifications, and they are working in a three-dimensional space that is very difficult to visualize," said Strahl. "We can't say that this mark or this combination of modifications will always mean a certain thing. But what I think we can say is that multiple modifications can help tip the balance of one chromatin state to another, making the underlying DNA more or less accessible to the protein machinery." In order to uncover what some of those codes might be, the researchers started generating chunks of histone proteins, each engineered to contain various combinations of modifications. In a completely new approach to the histone code, Strahl and his colleagues printed these modified chunks or peptides onto glass slides, generating peptide arrays akin to DNA arrays. When they tested widely used commercial antibodies that were directed against specific modifications on histones, like methylysine or methylarginine, they found the antibodies didn't always recognize the site they were supposed to, sometimes even binding to off-targets better than their intended target. The results fit nicely with a study published recently in Nature Structural Biology by Jason Lieb, Ph.D., a professor of biology at UNC and a Lineberger Center member. Lieb used older approaches like immunofluorescence, CHIP and Western blots to show that many commercial antibodies were not performing as they should. An additional finding of the study by Strahl and colleagues was that antibodies, as well as the proteins that naturally bind chromatin, were greatly affected by neighboring modifications. "This result gives further support to the idea of the histone code, in that the ability of a protein to bind to histones may depend on a particular modification landscape and not just one single modification" said Strahl. "The presence of an acetylation site nearby could impact the binding of a protein at its intended phosphorylation site. So altogether these modifications generate a landscape that is vitally important in how proteins read the histone code."

New microscope reveals ultrastructure of cells

Mon, 22 Nov 2010 03:26:52 PDT

For the first time, there is no need to chemically fix, stain or cut cells in order to study them. Instead, whole living cells are fast-frozen and studied in their natural environment. The new method delivers an immediate 3-D image, thereby closing a gap between conventional microscopic techniques. The new microscope delivers a high-resolution 3-D image of the entire cell in one step. This is an advantage over electron microscopy, in which a 3-D image is assembled out of many thin sections. This can take up to weeks for just one cell. Also, the cell need not be labelled with dyes, unlike in fluorescence microscopy, where only the labelled structures become visible. The new X-ray microscope instead exploits the natural contrast between organic material and water to form an image of all cell structures. Dr. Gerd Schneider and his microscopy team at the Institute for Soft Matter and Functional Materials have published their development in Nature Methods (DOI:10.1038/nmeth.1533). With the high resolution achieved by their microscope, the researchers, in cooperation with colleagues of the National Cancer Institute in the USA, have reconstructed mouse adenocarcinoma cells in three dimensions. The smallest of details were visible: the double membrane of the cell nucleus, nuclear pores in the nuclear envelope, membrane channels in the nucleus, numerous invaginations of the inner mitochondrial membrane and inclusions in cell organelles such as lysosomes. Such insights will be crucial for shedding light on inner-cellular processes: such as how viruses or nanoparticles penetrate into cells or into the nucleus, for example. This is the first time the so-called ultrastructure of cells has been imaged with X-rays to such precision, down to 30 nanometres. Ten nanometres are about one ten-thousandth of the width of a human hair. Ultrastructure is the detailed structure of a biological specimen that is too small to be seen with an optical microscope. Researchers achieved this high 3-D resolution by illuminating the minute structures of the frozen-hydrated object with partially coherent light. This light is generated by BESSY II, the synchrotron source at HZB. Partial coherence is the property of two waves whose relative phase undergoes random fluctuations which are not, however, sufficient to make the wave completely incoherent. Illumination with partial coherent light generates significantly higher contrast for small object details compared to incoherent illumination. Combining this approach with a high-resolution lens, the researchers were able to visualize the ultrastructures of cells at hitherto unattained contrast. The new X-ray microscope also allows for more space around the sample, which leads to a better spatial view. This space has always been greatly limited by the setup for the sample illumination. The required monochromatic X-ray light was created using a radial grid and then, from this light, a diaphragm would select the desired range of wavelengths. The diaphragm had to be placed so close to the sample that there was almost no space to turn the sample around. The researchers modified this setup: Monochromatic light is collected by a new type of condenser which directly illuminates the object, and the diaphragm is no longer needed. This allows the sample to be turned by up to 158 degrees and observed in three dimensions. These developments provide a new tool in structural biology for the better understanding of the cell structure.

Scientists at IRB Barcelona discover a new protein critical for mitochondria

Wed, 03 Nov 2010 03:31:23 PDT

A study by the team headed by Lluís Ribas de Pouplana, ICREA professor at the Institute for Research in Biomedicine (IRB Barcelona), has been chosen as “Paper of the week” in the December issue of the Journal of Biological Chemistry, which is already available online. The article describes the discovery of a new protein in the fly Drosophila melanogaster (fruit fly) that is crucial for mitochondria. The removal of SLIMP in these flies leads to aberrant mitochondria and loss of metabolic capacity, thus causing death. The study, whose first author is Tanit Guitart, a PhD student in Ribas’ lab, has been recognised as “Paper of the week” award because of the “significance and global relevance” of the research performed. Furthermore, the editors have included it among the best studies that have appeared in the journal this year. Of the 6600 articles published, only between 50 and 100 receive the distinction of “Article of the week”.......> Full story

Structural genomics accelerates protein structure determination

Thu, 28 Oct 2010 03:22:24 PDT

Proteins are molecular machines that transport substances, catalyze chemical reactions, pump ions, and identify signaling substances. They are chains of amino acids and the individual amino acid sequence is known for many of them. However, the functions a protein can carry out inside the cell are determined by the three-dimensional spatial structure of the protein. Establishing this so-called tertiary structure presents a great challenge to scientists. There is, thus, a lot of catching up to be done in structure analysis. To push progress, the National Institute of General Medical Sciences (NIGMS) of the USA National Institutes of Health (NIH) has invested over 500 million dollars in this field over the last ten years as part of the Protein Structure Initiative with the hope of making significant progress in medicine and biological research. Informatics professor Burkhard Rost and Marco Punta, Carl von Linde Junior Fellow at the Institute for Advanced Study (IAS) of the TU München, are involved in this large-scale project. They are affiliated with the New York Consortium on Membrane Protein Structure (NYCOMPS), which is among nine funded membrane research centers. The NYCOMPS scientists put a special emphasis on membrane proteins. That is because they play a key role in pharmacological research. When a pharmaceutical agent enters the cell, it normally interacts first with membrane proteins. Knowing the protein structure is essential to understanding this interaction at the molecular level. However, in the case of these very important membrane proteins, experimentally deciphering the tertiary structure is particularly difficult. For example the recombinant production of many membrane proteins is a major challenge and purification and crystallization are also difficult steps. The result: although around 25 percent of all proteins are membrane proteins, they account for less than one percent of the total number of proteins with known structures. Membrane protein structures are thus underrepresented 25-fold. Given their medical relevance, they should be much better known. Since the experimental analysis of a membrane protein can take up to several years, the NYCOMPS scientists applied a bioinformatics strategy, the so-called homology modeling. The basic assumption of this strategy is that proteins with common evolutionary predecessors resemble each other in their amino acid sequences, as well as in their three-dimensional structure. If the structure of one of the related proteins can be determined experimentally, the remaining ones can be predicted. In the case of the bacterial membrane protein TehA they could bring all pieces of the puzzle together. “In a screening procedure we searched for TeHA-related membrane proteins by comparing tens of thousands of amino acid sequences. Using a multistage selection process we chose 43 proteins from 38 different organisms,” says TUM computational biologist Marco Punta. Scientists at Columbia University now succeeded in experimentally determining the tertiary structure of the membrane protein TehA of the bacterium Haemophilus influenzae using X-ray crystallography. With a resolution of 0.12 nanometers (1.2 Ångstrøm), this structure is among the best crystal structures ever obtained for a membrane protein. Furthermore, the experiment harbored a surprise: The TehA membrane protein has a hitherto entirely unknown fold. After getting to know the “TehA family,” the scientists at Columbia University succeeded in deriving the structures of the individual proteins. In particular, they modeled the structure of the plant membrane protein SLAC1. Comparing this to the protein structure of TehA derived experimentally, they could build a structural model for SLAC1 – entirely without experimentation, using nothing but bioinformatics methods. “Using this procedure we aim to have a high structure determination throughput rate. determining more protein structures in a shorter time – that was our goal, in particular for the membrane proteins. The results at hand show that this strategy can work for membrane proteins, too,” says Burkhard Rost. Ultimately, the three-dimensional structures are determined to identify the function of the proteins using mutagenesis tests. Although the membrane proteins TehA and SLAC1 are only distantly related – the overlap of the amino acid sequence is only 19 percent – the predicted tertiary structure of SLAC1 was so good that a new hypothesis on the function of the SLAC1 membrane protein could be put forward. SLAC1 is found in the stomata of the plant Arabidopsis thaliana. Stomata control the exchange of water vapor and carbon dioxide between the plant and its environment. This is very important in photosynthesis. The membrane protein SLAC1 plays a role in this process, as well, as part of the anion channel: It influences the turgor pressure – the pressure of cell fluid on the cell wall – and thus the gas exchange of the plant cell as a reaction to environmental influences such as aridity and high carbon dioxide concentration. SLAC1 anion channels are entirely novel in structure and, apparently, in the mechanism for ion conductance. The SLAC1 pore has a relatively uniform diameter, but in the middle a Phenylalanine-group blocks the way. The results suggest that this amino acid is turned away when the ion channel is activated through binding of a triggering protein.

Breakthrough: With a chaperone, copper breaks through

Tue, 19 Oct 2010 03:20:47 PDT

Information on proteins is critical for understanding how cells function in health and disease. But while regular proteins are easy to extract and study, it is far more difficult to gather information about membrane proteins, which are responsible for exchanging elements essential to our health, like copper, between a cell and its surrounding tissues.

Now Prof. Nir Ben-Tal and his graduate students Maya Schushan and Yariv Barkan of Tel Aviv University's Department of Biochemistry and Molecular Biology have investigated how a type of membrane protein transfers essential copper ions throughout the body. This mechanism, Schushan says, could also be responsible for how the body absorbs Cisplatin, a common chemotherapy drug used to fight cancer. In the future, this new knowledge may allow scientists to improve the way the drug is transferred throughout the body, she continues.

Their breakthrough discovery was detailed in a recent issue of PNAS (Proceedings of the National Academy of Sciences).

Cellular gatekeepers and chaperones

Most proteins are water soluble, which allows for easy treatment and study. But membrane proteins reside in the greasy membrane that surrounds a cell. If researchers attempt to study them with normal technology of solubilization in water, they are destroyed — and can't be studied.

Copper, which is absorbed into the body through a membrane protein, is necessary to the healthy functioning of the human body. A deficiency can give rise to disease, while loss of regulation is toxic. Therefore, the cell handles copper ions with special care. One chaperone molecule delivers the copper ion to an "entrance gate" outside the cell; another chaperone then picks it up and carries it to various destinations inside the cell.

The researchers suggest that this delicate system is maintained by passing one copper ion at a time by the copper transporter, allowing for maximum control of the copper ions. "This way, there is no risk of bringing several copper ions into the protein at the same time, which ultimately prevents harmful chemical reactions between the ions and the abundant chemical reagents within the cell," explains Prof. Ben-Tal. Once the ion has passed through the transporter into the cell, the transporter is ready to receive another copper ion if necessary.

Improving cancer drugs — and more

The mechanism which transfers copper throughout the body may also be responsible for the transfer of the common chemotherapy drug Cisplatin. By studying how copper is transferred throughout the body, researchers may also gain a better understanding of how this medication and others are transferred into the cell.

With this information, says Prof. Ben-Tal, scientists could improve the transfer of the drug throughout the body, or develop a more effective chemotherapy drug. And that's not the only pharmaceutical dependent on the functioning of membrane proteins. "Sixty percent of drugs target membrane proteins," he explains, "so it's critical to learn how they function." This work was done in collaboration with Prof. Turkan Haliloglu from Bogazici University, Istanbul.

Scientists find signals that make cell nucleus blow up like a balloon

Fri, 15 Oct 2010 03:22:18 PDT

Size matters when it comes to the nucleus of a cell, and now scientists have discovered the signals that control how big the nucleus gets. Nuclear size varies not only among different species, but also in different types of cells in the same species and at different times during development. In addition, cancer cells are known to develop larger nuclei as they become more malignant. Screening for cervical cancer, for example, involves looking for grossly distorted nuclei in cervical cells collected during a Pap smear. "Pathologists look at nuclear size in cancer cells for staging different cancers, but nobody knows what is behind this," said Rebecca Heald, professor of molecular and cell biology at the University of California, Berkeley. As a result, she and post-doctoral fellow Daniel L. Levy explored why the nuclei of two species of African clawed frog are so different in size and discovered that the large nucleus of Xenopus laevis sucks in more material while growing than does the small nucleus of Xenopus tropicalis. The two researchers tracked down the proteins streaming into the nucleus and discovered that they were importing structural material used to build the web of lamin proteins that shores up the inside of the nuclear shell. The faster the import of lamin and other structural proteins, the faster it is added to the underside of the nuclear envelope, inflating it like a balloon. In addition, they found that another protein sits like a plug at the entrances to the nuclear envelope – the nuclear pore complexes – to slow the importation of large proteins. Together, these two different proteins – the importing protein, importin-alpha, and the gatekeeper, ntf2 – account for the difference in size between the nuclei of the two frogs. "The different levels of these two factors is sufficient to account for our nuclear size differences," Levy said. "There was a lot more importin-alpha and a lot less ntf2 in Xenopus laevis, and we found out that we can convert Xenopus tropicalis into laevis just by adding excess importin and partially getting rid of ntf2 in egg extracts," Heald said. "We thought this could be really complicated, but it isn't. "Now that we understand some of the mechanisms that regulate nuclear size, we can try to decrease nuclear size in cancer cells ands ask, does the cancer cell care? Maybe it will and maybe it won't." Levy and Heald report their findings in the Oct. 15 issue of the journal Cell. Heald has long been interested in what regulates the size of a cell's internal structures – in particular, the nucleus and the spindles that pull chromosomes apart during cell division. In certain amphibians, the larger the animal, the larger the genome and the larger the cells and nuclei. For mammals, however, that is not necessarily the case. Nevertheless, when mammalian cells become aneuploid – that is, they no longer have two copies of each chromosome – they often grow a larger nucleus. Aneuploidy is associated with cancer. In 2006, Heald was awarded a five-year, $2.5 million Pioneer Award from the National Institutes of Health to pursue research on cell organelle size, work deemed too "risky" for regular NIH funding. To explore these questions, she works in test tubes filled with the guts of hundreds of frog eggs. The guts consist of cell-free cytoplasm extracted from the cellular envelopes by centrifuging. By adding new proteins or antibodies that block existing proteins, she can explore the regulation of many activities inside the cell. For these experiments, she and Levy extended the technique from X. laevis to the smaller X. tropicalis, whose eggs have about one-fifth the volume. Part of the size difference is due to the fact that X. laevis is tetraploid – it has four copies of nearly every chromosome – whereas X. tropicalis is diploid, with two copies of each chromosome. Working with extracts allowed the researchers to find importation proteins that differed in concentration between the two species and thereby track down the mechanism of nuclear inflation. Specifically, X. laevis has three times more importin-alpha than does X. tropicalis, while tropicalis has four times more ntf2 than does laevis. By adjusting levels of these proteins in X. tropicalis egg extracts, they were able to make this species' nuclei balloon up to the size of X. laevis nuclei. "We can now ask physiologically what happens when you change nuclear size," Heald said. "If you make the nucleus bigger, does it become more cancer-like? How related are these two phenomena, cancer and nuclear size, which are associated but with no real causal effect." Heald and her colleagues are also tracking down the proteins that control the size of the spindles in X. laevis and X. tropicalis. Malfunctioning of spindles can lead to improper segregation of chromosomes during cell duplication, which results in aneuploidy and cancer.

First observation of the folding of a nucleic acid

Wed, 22 Sep 2010 03:18:58 PDT

The prediction of the structure and function of biological macromolecules (i.e., the machinery of life) is of foremost importance in the field of structural biology. Since the elucidation of the three-dimensional structure of DNA (the molecule that carries all genetic information) by Watson and Crick, scientists have strived to decipher the hidden code that determines the evolution of the spatial arrangement of these molecules towards their functional native state. Attempts to follow these structural transitions experimentally and with atomic resolution are hampered by the ultra-fast nature of the folding process. To date, the characterization of these processes by pure computational means has also been very challenging, since monitoring the folding of nucleic acid with realistic methods requires years of computing time in a regular PC. The researchers Modesto Orozco, Life Sciences Director of the Barcelona Supercomputing Center and responsible of the Molecular Modelling and Bioinformatics group at the Institute for Research in Biomedicine (IRB Barcelona), and Guillem Portella, postdoctoral researcher of his group, have been able to describe for the first time the folding process of a small DNA hairpin in water and with atomic resolution. The study reveals that, unexpectedly, the folding process appears as a competition between different fast folding and slow folding routes, and that microscopic details determine in a random fashion which route is explored. This investigation, conducted by means of molecular dynamics techniques, has been possible thanks to the enormous computing power of the MareNostrum supercomputer. The present study has far-reaching implications not only because it represents a new milestone in the theoretical study of nucleic acids' folding, but also it is of great importance for the design of new therapeutic strategies based on oligonucleotides. This is the case, e.g., for RNA interference based treatments, which will help to fight complex diseases such as cancer.

Misfolded neural proteins linked to autism disorders

Sat, 11 Sep 2010 03:48:54 PDT

An international team of scientists, led by researchers at the University of California, San Diego, has identified misfolding and other molecular anomalies in a key brain protein associated with autism spectrum disorders. Palmer Taylor, associate vice chancellor for Health Sciences at UC San Diego and dean of the Skaggs School of Pharmacy and Pharmaceutical Sciences, and colleagues report in the September 10 issue of the Journal of Biological Chemistry that misfolding of a protein called neuroligin-3, due to gene mutations, results in trafficking deficiencies that may lead to abnormal communications between neurons. Genetic misfolding of neuroligins is thought to prevent normal formation and function of neuronal synapses. The gene mutation has been documented in patients with autism. "It makes sense that there's a connection," said Taylor. "The neuroligins are involved in maintaining neuronal synapses and their malfunction is likely to affect a neurodevelopmental disease." Neuroligins are post-synaptic proteins that help glue together neurons at synapses by connecting with pre-synaptic protein partners called neurexins. They are part of a larger family of alpha-beta-hydrolase fold proteins that includes many molecules with diverse catalytic, adhesion and secretory functions. Using live neurons in culture, the researchers found that different mutations caused different degrees of misfolding of the protein structure, which translated into trafficking deficiencies of varying severity regardless of alpha-beta-hydrolase protein type, yet resulted in distinctly different congenital disorders in the endocrine or nervous systems. Both neuroligins and the autism mutations are relatively new to science. The former were characterized 15 years ago, the latter discovered just seven years ago. Taylor said identifying and describing the misfolded protein link advances understanding of the complex causes of certain autisms, including the influences of genes versus environment, and perhaps offers a new target for potential drug therapies. "If the mutation is identified early, it might be possible to rescue affected neurons before abnormal synaptic connections are established" said co-author Davide Comoletti, a research scientist at the Skaggs School of Pharmacy. "But much work remains. We may be able to find a treatment to fix a cell in culture, but to rescue function in vivo may not be feasible with the same strategy."

Scientists of Helmholtz Zentrum Muenchen and TU Muenchen elucidate structure details of protein Sam68

Sat, 11 Sep 2010 03:41:04 PDT

Using NMR spectroscopy, Professor Michael Sattler and his team elucidated the spatial structure of the Qua1 region of Sam68, which is responsible for the dimerization of the protein. In collaboration with the research group of Professor Ruth Brack-Werner of the Institute of Virology, the authors showed that this region is essential for the biological function of Sam68. The image reveals an unusual spatial structure, in which two helices of respectively one Qua1 region (green and blue) interact with each other and mediate the dimerization of Qua1 and thus of Sam68.

Sam68 belongs to the family of STAR proteins which carry out important tasks in the signal-regulated processing of genetic information and its translation into protein. Among others, Sam68 regulates specific processes linked to the cell cycle and apoptosis and plays a key role in the pathogenesis of cancer........> Full story

Brown-led research divines structure for class of proteins

Thu, 09 Sep 2010 03:26:48 PDT

Most proteins are shapely. But about one-third of them lack a definitive form, at least that scientists can readily observe. These intrinsically disordered proteins (IDPs) perform a host of important biological functions, from muscle contraction to other neuronal actions. Yet despite their importance, "We don't know much about them," said Wolfgang Peti, associate professor of medical science and chemistry. "No one really worried about them." Now, Peti, joined by researchers at the University of Toronto and at Brookhaven National Laboratory in New York, has discovered the structure of three IDPs — spinophilin, I-2, and DARPP-32. Besides getting a handle on each protein's shape, the scientists present for the first time how these IDPs exist on their own (referred to as "free form") and what shape they assume when they latch on to protein phosphatase 1, known as "folding upon binding." The findings are reported in the journal Structure. Determining the IDPs' shape is important, Peti explained, because it gives molecular biologists insight into what happens when IDPs fold and regulate proteins, such as PP1, which must occur for biological instructions to be passed along. "What we see is some amino acids don't have to change much, and some have to change a lot," Peti, a corresponding author on the paper, said. "That may be a signature how that (binding) interaction happens." For two years, the researchers used a variety of techniques to ascertain each IDP's structure. With I-2, which instructs cells to divide, they used nuclear magnetic resonance spectroscopy to create ensemble calculations for the protein in its free and PP1-bound form. They confirmed I-2's binding interaction with PP1 (known as the PP1:I-2 complex) with the help of small-angle X-ray scattering measurements at the National Synchrotron Light Source, located at the Brookhaven lab. The researchers did the same thing to determine the structure of spinophilin and DARPP-32 in their free-form state and to gain insights into their shapes when they bind with PP1. "It's analogous to putting a sack cloth over a person," Peti explained. "You can't see the details, but you can get the overall shape. This is really a new way to create a structure model for highly dynamic complexes."

Stanford-developed app shows 2-D structure of thousands of RNA molecules

Thu, 02 Sep 2010 03:27:37 PDT

"There's an app for that." To a cadre of scientists, the familiar phrase will soon mean they can enter a specific RNA from baker's yeast into their iPhone and see a depiction of its two-dimensional structure - thanks to a new technology developed by scientists at Stanford University. The application is cool, but it's just window dressing for the real advance: For the first time, it's possible to experimentally capture a global snapshot of the conformation of thousands of RNA molecules in a cell. The finding is important because this scrappy little sister of DNA has recently been shown to be much more complex than previously thought. "We used to think of RNA as just a long, floppy string that delivers instructions from DNA to the protein-assembly points in the cell," said associate professor of dermatology Howard Chang, MD, PhD. "But now we're learning that often the molecule's structure - and not just its sequence of nucleotide letters - determines its function. So we set out to develop a method that can map the structure of all the RNA in a cell." Chang, who was selected last year as a Howard Hughes Medical Institute Early Career Scientist, and Eran Segal, PhD, of the Weizmann Institute of Science in Israel, are the senior authors of the research, which will be published Sept. 2 in Nature. Michael Kertesz, PhD, previously at the Weizmann Institute and currently a postdoctoral scholar in Stanford's Department of Bioengineering, and Stanford graduate student Yue Wan are co-first authors. For years, RNA was known only for its role in shuttling information in the form of nucleotide sequences from the DNA in a cell's nucleus to the protein factories in the cytoplasm. Now we know that RNAs control many aspects of gene regulation and function. In comparison to DNA - a relatively inflexible, double strand of paired nucleotides that spiral around one another in a helix formation - RNA is a veritable circus contortionist. It can fold back on itself to form stem and loop structures, and these structures can bind to one another in pseudoknots, which can twist around and ... well, you get the idea. Until now, the only way to know what shape a particular RNA molecule preferred was to conduct a laborious series of experiments focused on just that molecule. But the effort was necessary to fully understand what it might be doing in the cell. The researchers capitalized on the recent development of deep-sequencing techniques that allow scientists to simultaneously sequence millions of nucleotide fragments for their analysis. They treated the pool of more than 3,000 protein-encoding RNA molecules from Saccharomyces cerevisiae, also known as baker's yeast, with structure-specific enzymes (one cleaves only single-stranded nucleotides at specific sequences and while another cleaves only double-stranded, or paired, RNA sequences). They then sequenced the fragments and pieced together the structure of each RNA molecule in a process they call "parallel analysis of RNA structure" or PARS. "It's now possible to look at RNA structure much more quickly and comprehensively," said Chang. "Now we can see patterns that were not previously evident, and begin to categorize RNAs by structure rather than sequence." Some of the patterns they identified were surprising. The researchers found that regions of RNA that encode specific instructions for protein tend to have more secondary structure than do other regions, and that it is possible to identify the beginning, middle and end of an RNA transcript simply by analyzing its structure. Finally, they found that RNA molecules that had similar functions often have similar structures - perhaps to better direct them to specific locations within the cell. The researchers tested their technique on baker's yeast because it is a well-studied organism with a relatively limited number of RNA molecules in action at any one time (about 3,000 vs. 10,000 in humans). But they plan to tackle other organisms soon, and to expand their analysis to include regulatory RNAs that don't carry protein-building instructions. "There's so much more information to be discovered," said Chang. "This is just a snapshot of RNAs in isolation. But we can leverage this information for biological insight into how RNA structures may change under different conditions. There are levels of complexity that we're only just beginning to understand." The researchers are also developing a searchable website (http://genie.weizmann.ac.il/pubs/PARS10/index.html) with their data. And then there's that iPhone application. "Now you can use your phone to look up structures and pull up RNA sequences," said Chang.

Opening the gate to the cell's recycling center

Thu, 15 Jul 2010 03:23:25 PDT

In cells, as in cities, disposing of garbage and recycling anything that can be reused is an essential service. In both city and cell, health problems can arise when the process breaks down.

New research by University of Michigan cell biologist Haoxing Xu and colleagues reveals key details about how the cell's garbage dump and recycling center, the lysosome, functions. These insights, which may lead to better understanding of conditions such as amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) and Charcot-Marie-Tooth (CMT) disease, suggest new avenues of treatment for these and other diseases that cause nerves and muscles to malfunction........> Full story

Nanotubes pass acid test

Thu, 15 Jul 2010 03:14:07 PDT

Rice University scientists have found the "ultimate" solvent for all kinds of carbon nanotubes (CNTs), a breakthrough that brings the creation of a highly conductive quantum nanowire ever closer. Nanotubes have the frustrating habit of bundling, making them less useful than when they're separated in a solution. Rice scientists led by Matteo Pasquali, a professor in chemical and biomolecular engineering and in chemistry, have been trying to untangle them for years as they look for scalable methods to make exceptionally strong, ultralight, highly conductive materials that could revolutionize power distribution, such as the armchair quantum wire. The armchair quantum wire -- a macroscopic cable of well-aligned metallic nanotubes -- was envisioned by the late Richard Smalley, a Rice chemist who shared the Nobel Prize for his part in discovering the the family of molecules that includes the carbon nanotube. Rice is celebrating the 25th anniversary of that discovery this year. Pasquali, primary author Nicholas Parra-Vasquez and their colleagues reported this month in the online journal ACS Nano that chlorosulfonic acid can dissolve half-millimeter-long nanotubes in solution, a critical step in spinning fibers from ultralong nanotubes. Current methods to dissolve carbon nanotubes, which include surrounding the tubes with soap-like surfactants, doping them with alkali metals or attaching small chemical groups to the sidewalls, disperse nanotubes at relatively low concentrations. These techniques are not ideal for fiber spinning because they damage the properties of the nanotubes, either by attaching small molecules to their surfaces or by shortening them. A few years ago, the Rice researchers discovered that chlorosulfonic acid, a "superacid," adds positive charges to the surface of the nanotubes without damaging them. This causes the nanotubes to spontaneously separate from each other in their natural bundled form. This method is ideal for making nanotube solutions for fiber spinning because it produces fluid dopes that closely resemble those used in industrial spinning of high-performance fibers. Until recently, the researchers thought this dissolution method would be effective only for short single-walled nanotubes. In the new paper, the Rice team reported that the acid dissolution method also works with any type of carbon nanotube, irrespective of length and type, as long as the nanotubes are relatively free of defects. Parra-Vasquez described the process as "very easy." "Just adding the nanotubes to chlorosulfonic acid results in dissolution, without even mixing," he said. While earlier research had focused on single-walled carbon nanotubes, the team discovered chlorosulfonic acid is also adept at dissolving multiwalled nanotubes (MWNTs). "There are many processes that make multiwalled nanotubes at a cheaper cost, and there's a lot of research with them," said Parra-Vasquez, who earned his Rice doctorate last year. "We hope this will open up new areas of research." They also observed for the first time that long SWNTs dispersed by superacid form liquid crystals. "We already knew that with shorter nanotubes, the liquid-crystalline phase is very different from traditional liquid crystals, so liquid crystals formed from ultralong nanotubes should be interesting to study," he said. Parra-Vasquez, now a postdoctoral researcher at Centre de Physique Moleculaire Optique et Hertzienne, Universite' de Bordeaux, Talence, France, came to Rice in 2002 for graduate studies with Pasquali and Smalley. Study co-author Micah Green, assistant professor of chemical engineering at Texas Tech and a former postdoctoral fellow in Pasquali's research group, said working with long nanotubes is key to attaining exceptional properties in fibers because both the mechanical and electrical properties depend on the length of the constituent nanotubes. Pasquali said that using long nanotubes in the fibers should improve their properties on the order of one to two magnitudes, and that similar enhanced properties are also expected in thin films of carbon nanotubes being investigated for flexible electronics applications. An immediate goal for researchers, Parra-Vasquez said, will be to find "large quantities of ultralong single-walled nanotubes with low defects -- and then making that fiber we have been dreaming of making since I arrived at Rice, a dream that Rick Smalley had and that we have all shared since."

Case Western Reserve University-led team takes high resolution photo of a K+ channel

Wed, 14 Jul 2010 03:20:30 PDT

Using chemical labeling and mass spectrometry-based techniques, Mark Chance, PhD, director of the Case Western Reserve University School of Medicine Center for Proteomics and Bioinformatics and professor of physiology and biophysics; Sayan Gupta, PhD, instructor at the Case Center for Proteomics and Bioinformatics; and a research team from the University of Oxford, for the first time, were able to take a high resolution picture of the open state of a K+ channel, allowing them to comparatively analyze gating mechanisms important to heart function and nerve signaling – in addition these techniques have already permitted Case Western investigators to gain a deeper understanding of G-protein coupled receptors (GPCRs). GPCRs, the targets of more than half of all pharmaceutical drugs, are important signaling molecules and help regulate proper channel function. With a new window on the open state of the channel, researchers have an opportunity to better understand the effectiveness of drugs used to treat and prevent cardiovascular diseases. "The discoveries made by Mark [Chance] and his team have the potential to revolutionize modern medicine as we know it," said Daniel Simon, MD, Chief of Cardiovascular Medicine at University Hospitals Case Medical Center, director of the Case Cardiovascular Center and the Herman K. Hellerstein Professor of Cardiovascular Research at Case Western Reserve School of Medicine. "With an advanced understanding of GPCRs and ion channels comes a better understanding of how modern medicine works, which would theoretically increase pharmaceutical efficiency and enhance the quality of countless cardiovascular-related drugs." In the study, published in the July edition and featured on the cover of the July issue of Structure, Dr. Chance and his research team discovered the details of the paths by which ions flow through the inwardly-rectifying potassium channel KirBac3.1.The results of this study provide support for a newly proposed gating mechanism of the K+ channel, which if perfected, could offer scientists a new method of probing other important membrane proteins and ion channels that could lead to advancements in the field of cardiovascular medicine. Cardiovascular complications such as stroke and coronary heart disease remain the number one cause of death in all Americans*. NIBIB Grant for $1.1M to Continue Structural Protein Footprinting Research To further support his research efforts, Dr. Chance and the Case Western Reserve Center for Proteomics and Bioinformatics received a four-year grant totaling $1.1 million from the National Institute of Biomedical Imaging and Bioengineering (NIBIB). The grant work will be performed in collaboration with Krzysztof Palczewski, PhD, chair of the Department of Pharmacology at Case Western Reserve University, and Dr. Gupta, instructor at the Case Center for Synchrotron Biosciences (CSB) in New York. Dr. Chance is also the director of the CSB, which provides unique facilities to carry out protein footprinting experiments on GPCRs and their complexes. "Determining the structure of membrane proteins like GPCRs is particularly difficult, while understanding how drugs function to turn them on and off is even harder, but very important to treating major diseases like depression, heart failure, and diabetes," Dr. Chance said of his research. "Our techniques have provided novel insight into addressing these questions and this funding from the NIH will accelerate these studies." With this funding, Dr. Chance and co-investigators will continue their development of new biotechnology methods to study GPCRs. The aim of their research is as follows:

To improve their mass spectrometry based structural imaging technology 1,000-fold in an effort to better understand how GCPRs are activated and signal information to protein receptors
To develop a novel oxygen-18 based water labeling technique to examine the locations and dynamics of structural waters and the exchange properties of bulk water in multiple biological states of interest
To improve detection efficiency in current protein footprinting experiments in hopes of enhancing the number of amino acids routinely detected
To develop an algorithmic formula in relation to current footprinting data to determine the outlines of different protein structures

Rice program takes on protein puzzle

Thu, 08 Jul 2010 03:21:46 PDT

All proteins self-assemble in a fraction of the blink of an eye, but it can take a long time to mimic the process. And there has been no guarantee of success, even with the most powerful computers – until now. Rice University researchers have come up with a computer program to accurately simulate protein folding dramatically faster than previous methods. It will allow scientists to peer deeper into the roots of diseases caused by proteins that fold incorrectly. Authors Cheng Zhang and Jianpeng Ma describe their simulation of three short proteins with the new technique in the cover story of the current Journal of Chemical Physics. Ma is a professor in the Department of Bioengineering at Rice University and the Department of Biochemistry and Molecular Biology at Baylor College of Medicine. Zhang is an applied physics graduate student at Rice. "Protein folding is regarded as one of the biggest unsolved problems in biophysics," Ma said. "This is a technically challenging task, and many groups around the world have been competing for years to make the process faster and more accurate." Understanding the intricacies of protein folding is a crucial step in deciphering the genetic code that serves as the operating system of all living things. Correctly folded proteins perform many roles: as enzymes vital to metabolism; structural elements in bone, muscle and cell scaffolding; mechanisms in cell signaling and immune response and much more. But protein misfolding is a critical factor in many diseases, including Alzheimer's, cystic fibrosis, emphysema and various cancers. Proteins start as amino acid molecules floating in a cell. Following DNA blueprints, the molecules are strung together like beads on a necklace, called a polypeptide. Every polypeptide of a given sequence will fold precisely the same way into the shape, called the native state, that determines its function. Like a river finding the shortest route to the sea, proteins always find their way to their native states in an instant. How that happens is one of life's great mysteries. "The question is how nature finds this final folded state so quickly," Ma said. Zhang and Ma reached unprecedented accuracy and speed in simulating the folding of three relatively short but well-understood proteins -- trpzip2, trp-cage and the villin headpiece -- in the presence of water molecules, which Ma described as the best way to simulate physiological conditions. Though the proteins assemble themselves in nature almost instantly, the Rice team's algorithm took weeks to run the simulation. Still, that was far faster than others have achieved. "And for trpzip and villin, nobody has reached this level of accuracy in the native state under similar simulation conditions -- that is, in the presence of water, which is the most stringent condition," Ma said. The researchers employed two novel strategies, continuously variable temperature and single-copy simulation. "In the process of simulation, called sampling, the computer has to search through many, many possible structures of the protein chain to find the lowest-energy solution," Ma said. "A polypeptide chain en route to its native state encounters many energy barriers, much like when one navigates through a rugged mountain landscape. "Speeding up the process of crossing those barriers is the key to finding the true global minimum (energy state)," he said. "In our simulation, temperature is a variable that goes continuously up and down. When the temperature is higher, proteins can overcome energy barriers faster. It's equivalent to speeding up the motion of atoms." Ma said the previous state of the art was to run multiple copies of a simulation in parallel on many computers -- an intensive and expensive approach. "The single-copy approach uses only one simulation, essentially, to find the native state of the protein. This is a major plus, because anyone with reasonable computing power can run this method." Even so, it takes computational muscle to simulate a biological task that the body's cells accomplish as a matter of course. Zhang and Ma found it in Rice's supercomputer cluster, the Shared University Grid at Rice, aka SUG@R. "We can't overstate the significance of state-of-the-art computing facilities, as well as excellent service from Rice's Research Computing Support Group," Ma said. His group is continuing its work on Rice's newest supercomputer cluster, BlueBioU, for longer polypeptide sequences. "These supercomputer resources will continue to make Rice one of the leading institutions in the field of protein folding and computational biophysics," Ma said. The National Institutes of Health, the National Science Foundation, the Welch Foundation, the Welch Chemistry and Biology Collaborative Grant from the John S. Dunn Gulf Coast Consortium for Chemical Genomics and the Rice Faculty Initiatives Fund supported the research.

Carbon nanotubes form ultrasensitive biosensor to detect proteins

Mon, 28 Jun 2010 03:18:06 PDT

A cluster of carbon nanotubes coated with a thin layer of protein-recognizing polymer form a biosensor capable of using electrochemical signals to detect minute amounts of proteins, which could provide a crucial new diagnostic tool for the detection of a range of illnesses, a team of Boston College researchers report in the journal Nature Nanotechnology. The nanotube biosensor proved capable of detecting human ferritin, the primary iron-storing protein of cells, and E7 oncoprotein derived from human papillomavirus. Further tests using calmodulin showed the sensor could discriminate between varieties of the protein that take different shapes, according to the multi-disciplinary team of biologists, chemists and physicists. Molecular imprinting techniques have shown that polymer structures can be used in the development of sensors capable of recognizing certain organic compounds, but recognizing proteins has presented a difficult set of challenges. The BC team used arrays of wire-like nanotubes – approximately one 300th the size of a human hair – coated with a non-conducting polymer coating capable of recognizing proteins with subpicogram per liter sensitivity. Central to the function of the sensor are imprints of the protein molecules within the non-conducting polymer coating. Because the imprints reduce the thickness of the coating, these regions of the polymer register a lower level of impedance than the rest of the polymer insulator when contacted by the charges inherent to the proteins and an ionized saline solution. When a protein molecule drops into its mirror image, it fills the void in the insulator, allowing the nanotubes to register a corresponding change in impedance, signaling the presence of the protein, according to co-author Dong Cai, an associate research professor of Biology at BC. The detection can be read in real time, instead of after days or weeks of laboratory analysis, meaning the nanotube molecular imprinting technique could pave the way for biosensors capable of detecting human papillomavirus or other viruses weeks sooner than available diagnostic techniques currently allow. As opposed to searching for the HPV antibody or cell-mediated immine responses after initial infection, the nanotube sensor can track the HPV protein directly. In addition, no chemical marker is required by the lebel-free electrochemical detection methods. "In the case of some diseases, no one can be sure why someone is ill," said Cai. "All that may be known is that it might be a virus. At that time, the patient may not have detectable serum antibodies. So at a time when it is critical to obtain a diagnosis, there may not be any traces of the virus. You've basically lost your chance. Now we can detect surface proteins of the virus itself through molecular imprinting and do the analysis."

Insight into cells could lead to new approach to medicines

Tue, 22 Jun 2010 03:11:24 PDT

A surprising discovery about the complex make-up of our cells could lead to the development of new types of medicines, a study suggests. Scientists studying interactions between cell proteins – which enable the cells in our bodies to function – have shown that proteins communicate not by a series of simple one-to-one communications, but by a complex network of chemical messages. The findings suggest that medicines would be more effective if they were designed differently. Drugs could have a greater effect on cell function by targeting groups of proteins working together, rather than individual proteins. Results were obtained by studying yeast, which has many corresponding proteins in human cells. Researchers, including scientists from the University of Edinburgh, used advanced technology to identify hundreds of different proteins, and then used statistical analysis to identify the more important links between them, mapping almost 2000 connections in all. Scientists expected to find simple links between individual proteins but were surprised to find that proteins were inter-connected in a complex web. Dr Victor Neduva, of the University of Edinburgh, who took part in the study, said: "Our studies have revealed an intricate network of proteins within cells that is much more complex than we previously thought. This suggests that drugs should be more complex to treat illnesses effectively. Professor Mike Tyers, who led the study, said: "Medicines could work better if they targeted networks of proteins rather than sole proteins associated with particular illnesses."

Raising the bar for biomolecular modeling

Tue, 15 Jun 2010 03:18:26 PDT

Researchers at the University of Calgary found that amino acid residues form a type of barrier to help in the process of electron transfer between proteins. "This raises the bar for biomolecular modeling," says Dennis Salahub, U of C co-author of a paper published today in the prestigious journal Proceedings of the National Academy of Sciences (PNAS). "At a fundamental level, it is by far the most detailed insight that has been obtained for the dynamic role of water in this kind of electron transfer, or for that matter any biochemical reaction." Electron transfer between proteins is the cornerstone of biological energy transfer. Every life-form uses this process to convert food or sunlight into chemical energy. The interdisciplinary team of researchers found that the electron travels over a bridge made of a water molecule, while residues on one of the proteins form a sort of 'molecular breakwater' to keep other water molecules away while the electron travels across the bridge. "You don't want too many (water molecules around the bridge) because it gets too crowded and they're all bumping into each other and you can't get one to fit at just the right position and the right angle (for the bridge) for any length of time," says PhD student and co-author Nathan Babcock. "It's like being on a crowded subway where you can't get comfortable." In artificial mutations with a faulty breakwater, the water bridge is disrupted and the rate of electron transfer is markedly reduced, he says. Using the CHARMM molecular simulation computer program, the research team examined a 40 nanosecond period of electronic coupling of the proteins methylamine dehydrogenase and amicyanin from the bacterium Paracoccus denitrificans. "This is fundamental research but you can imagine how studies like this can be applied to various genetically modified organisms, and if you can gain control over some, you can use it to either speed up or slow down a particular reaction," says Salahub. He says the work was made possible with the collaboration of two of the U of C's interdisciplinary research institutes; the Institute for Biocomplexity and Informatics (IBI) and the Institute for Quantum Information Science (IQIS). Babcock, whose background is in quantum information theory, was pleased to do research at the union of these two disciplines. "When you think of quantum mechanics, usually you're thinking solid state semi conductors, atoms trapped with lasers, etc. It's usually cold laboratory stuff, not warm globby biological stuff," says the PhD student. "I think the union of biology and quantum mechanics is very, very exciting."

Nuclear pores call on different assembly mechanisms at different cell cycle stages

Fri, 11 Jun 2010 03:34:03 PDT

Nuclear pores are the primary gatekeepers mediating communication between a cell's nucleus and its cytoplasm. Recently these large multiprotein transport channels have also been shown to play an essential role in developmental gene regulation. Despite the critical role in nuclear function, however, nuclear pore complexes remain somewhat shadowy figures, with many details about their formation shrouded in mystery. Now a team of investigators from the Salk Institute for Biological Studies has illuminated key differences in the mechanisms behind nuclear pores formed at two distinct stages in the cell cycle. Their findings, to be published in the June 12 issue of Cell, may provide insights into conditions such as cancer, developmental defects, and sudden cardiac arrest. Nuclear pores, which are built from 30 different proteins, assemble during interphase, the period when the nucleus expands and replicates its DNA, and following mitosis, when the nuclear membrane reforms around the segregated chromosomes to create two identical nuclei. But, explains Martin Hetzer, Ph.D., Hearst Endowment associate professor in Salk's Molecular and Cell Biology Laboratory, who led the study, there has been a longstanding question about whether assembly pathways at the distinct cell cycle stages use different or similar mechanisms. "Interphase assembly is different from post-mitotic assembly in that the nuclear membrane is fully formed around chromatin," he says, "whereas post-mitotic assembly occurs into the reforming nuclear membrane. So the topology of the nuclear membrane is very different during these two cell cycle stages." While some aspects of post-mitotic assembly were known, almost nothing was understood about how assembly of the pores occurs during interphase, when the cell doubles the number of nuclear pores to provide sufficient levels of NPC components for the two daughter cells. A parallel process takes place during differentiation of an oocyte, when millions of nuclear pore components are integrated into the nuclear membrane of the egg cell, so any findings about interphase assembly could also be relevant to embryonic development. "We were able to show for the first time that there are two distinct mechanisms behind how these large protein complexes assemble to accommodate cell cycle-dependent differences in nuclear membrane topology," says Hetzer. The team identified a key difference in how the Nup107/160 complex, which is essential for NPC formation, is targeted to new assembly sites in the NE. Surprisingly, one of the complex members, Nup133, is directed to the pore assembly site via a completely novel mechanism that involves sensing of the nuclear membrane's curvature. "The sensor was identified in a bioinformatics screen, and it was not known whether it was really functional in vivo," says co-first author Christine Doucet, Ph.D., a postdoctoral fellow in Hetzer's lab. "But we thought it would fit in with the topology of the nuclear membrane and the sites of the new nuclear pore complexes because they are highly curved. So if the sensor was playing a role in assembly, it was a really neat way to coordinate the assembly of all the components at the right position and the right time." The second difference the group discovered is that in post-mitotic assembly, but not during interphase, a protein called ELYS played a key role in directing the NUP107/160 complex, which is critical to the formation of pores, to the assembly sites. In contrast, the transmembrane Nup POM121, is specifically required for interphase assembly.POM121 is the earliest known protein at pore assembly sites yet how it is directed there is still under investigation. "We knew both proteins were essential for pore assembly in different ways, but we didn't know how," says co-first author Jessica Talamas, also a postdoctoral fellow in Hetzer's lab. "There was a discrepancy in the literature about POM121, so we were trying to figure out what was going on. It was one of those lightbulb moments, we were looking at the data and realized that POM121 was only required for interphase assembly, and then everything just made sense." Because these processes are at work in every cell that divides, the study is especially germane to one of the big questions in the field: how the number of nuclear pores is regulated. It's a question with multiple ramifications. Nuclear pore numbers are misregulated in cancer cells, for example, so the findings have applications in cancer research. In addition, because neurons require a large number of nuclear pores, evidence is mounting that defects in nuclear pore assembly are linked to developmental defects in the central nervous system. Assembly defects during development have also been implicated in conditions such as sudden cardiac arrest. "In establishing differences between the two assembly pathways, the findings have provided the first glimpse of a mechanistic understanding," Hetzer says.

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This study was supported by a grant from the National Institutes of Health. About the Salk Institute for Biological Studies: The Salk Institute for Biological Studies is one of the world's preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused both on discovery and on mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer's, diabetes and infectious diseases by studying neuroscience, genetics, cell and plant biology, and related disciplines. Faculty achievements have been recognized with numerous honors, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent nonprofit organization and architectural landmark.

Researchers capture first images of sub-nano pore structures

Wed, 09 Jun 2010 03:41:02 PDT

Moore's law marches on: In the quest for faster and cheaper computers, scientists have imaged pore structures in insulation material at sub-nanometer scale for the first time. Understanding these structures could substantially enhance computer performance and power usage of integrated circuits, say Semiconductor Research Corporation (SRC) and Cornell University scientists. To help maintain the ever-increasing power and performance benefits of semiconductors – like the speed and memory trend described in Moore's law – the industry has introduced very porous, low-dielectric constant materials to replace silicon dioxide as the insulator between nano-scaled copper wires. This has sped up the electrical signals sent along these copper wires inside a computer chip, and at the same time reduced power consumption. "Knowing how many of the molecule-sized voids in the carefully-engineered Swiss cheese survive in an actual device will greatly affect future designs of integrated circuits," said David Muller, Cornell University professor of applied and engineering physics, and co-director of Kavli Institute for Nanoscale Science at Cornell. "The techniques we developed look deeply, as well as in and around the structures, to give a much clearer picture so complex processing and integration issues can be addressed." The scientists understand that the detailed structure and connectivity of these nanopores have profound control on the mechanical strength, chemical stability and reliability of these dielectrics. With today's announcement, researches now have a nearly atomic understanding of the three-dimensional pore structures of low-k materials required to solve these problems. Welcome to the atomic world: SRC and Cornell researchers were able to devise a method to obtain 3-D images of the pores using electron tomography, leverages imaging advances used for CT scans and MRIs in the medical field, says Scott List, director of interconnect and packaging sciences at SRC, at Research Triangle Park, N.C. "Sophisticated software extracts 3-D images from a series of 2-D images taken at multiple angles. A 2-D picture is worth a thousand words, but a 3-D image at near atomic resolution gives the semiconductor industry new insights into scaling low-k materials for several additional technology nodes."

Preventing cells from getting the kinks out of DNA

Tue, 25 May 2010 04:04:38 PDT

Many standard antibiotics and anti-cancer drugs block the enzymes that snip the kinks and knots out of DNA – DNA tangles are lethal to cells – but the drugs are increasingly encountering resistant bacteria and tumors. A new discovery by University of California, Berkeley, biochemists could pave the way for new research into how to re-design these drugs to make them more effective poisons for cancer cells and harmful bacteria. "The development of the anti-bacterial and anti-tumor agents that target these enzymes thus far has been done entirely in the absence of any visualization of how these drugs actually interact with the protein itself. And they have done remarkably well," said James Berger, UC Berkeley professor of molecular and cell biology. "But we have increasing problems of resistance to these drugs. Being able to see how these drugs can interact with the enzyme and DNA is going to be critical to developing the next generation of therapeutics that can be used to overcome these resistance problems." Berger and colleagues at Emerald BioStructues of Bainbridge Island, Wash., and Vanderbilt University in Nashville, Tenn., report their new findings in a paper to be printed in the journal Nature and made available last week as an advance online publication at http://www.nature.com. The tangles in DNA, like those in a string of holiday lights, are a result of packing some six feet of DNA into a cell nucleus so small that it is invisible to the naked eye. Every time a cell divides, it has to unpack, duplicate and repack its DNA, generating about a million tangles among the newly-copied chromosomes in the process. As Berger has shown in previous work, enzymes called topoisomerases home in on the sharp turns in a knot and then progressively snip the DNA, unloop it, and restitch it flawlessly. If, however, the enzyme slips up, that one snip can turn into a potentially mutagenic or cell-killing DNA break. While the protein structure of these topoisomerases is known, the details of the chemical reactions that take place between the enzyme and DNA, and their reaction with the drugs that bind both, remain a mystery, Berger said. In fact, one of the main puzzles is why antibiotics like ciprofloxacin (Cipro) and anti-cancer drugs like etoposide, which vary widely in structure, have the same effect: jamming the enzyme and causing a break in the double-stranded DNA helix. Berger and his colleagues found a way to obtain a picture that shows the interaction of the protein bound to DNA. The next step is to do the same for a drug bound to the protein/DNA complex, getting an image of exactly how these drugs interfere with the knot elimination machinery. "The technique we used to trap this complex so that we could actually crystallize it and image it we think now gives us a handle on how to go after drug-bound complexes of human topoisomerases that have long eluded the field," said Berger, who also is a staff scientist at Lawrence Berkeley National Laboratory (LBNL). The scientists' new picture of the enzyme bound to DNA also turned up something totally unexpected. Most enzymes that bind DNA to snip or stitch it together use two metal ions – typically two magnesium ions – to catalyze the reaction. Berger found that type II topoisomerases, which target double-stranded DNA, make use of only one of their two magnesium ions and instead use the amino acid arginine as their second catalytic center. The second magnesium merely provides structural integrity to the protein. "We stumbled upon a new kind of cleavage mechanism for DNA, an example of a protein that uses a completely new approach for the same mechanism," Berger said. "It speaks to the evolutionary plasticity and adaptability of nature that continuously amazes us with finding new ways to carry out reactions that it needs to perform." Berger now plans to use his trick to trap the enzyme on a short segment of DNA, allowing him to collect enough to crystallize and analyze in an X-ray beam from LBNL's Advanced Light Source, to trap both drug and enzyme on DNA. Once crystallized and imaged, he will have the first full picture of a topoisomerase interacting the way it does in a real cancer cell or microbe.

Systems biology helps to understand hematopoiesis

Sat, 22 May 2010 06:13:24 PDT

Our body reacts to blood loss by stimulating the production of red blood cells (erythrocytes). The cells of the hematopoietic (blood-forming) system in the bone marrow do so upon receipt of a signal by a hormone called erythropoietin, or Epo for short. This hormone is produced mainly by the kidney that increases the Epo level by up to a thousand-fold as a response to falling oxygen saturation of the blood. The hematopoietic cells receive the Epo signal through Epo receptors on their surface. How do the blood progenitor cells that carry only few receptor molecules manage to react adequately to a high rise in the Epo level and to always provide the required amount of red blood cells? "If too much of the hormone floods too few receptor molecules, we would expect the saturation point to be reached soon. This would mean that the hematopoietic cell can no longer respond to a further increase in the hormone level," says Dr. Ursula Klingmüller of DKFZ.......> Full story

First-ever high-resolution observations of DNA unfolding

Fri, 21 May 2010 04:29:26 PDT

The separation of the two DNA strands occurs in millionths of a second. Consequently, it is extremely difficult to study this phenomenon experimentally and researchers must rely on computational simulations. After four years of fine-tuning an effective physical model and massive use of the supercomputer Mare Nostrum, researchers at IRB Barcelona and the Barcelona Supercomputing Center (BSC) have managed to produce the first realistic simulation of DNA opening at high resolution. The scientists Modesto Orozco, group leader of the Molecular Modelling and Bioinformatics Group at IRB Barcelona, Full Professor of Biochemistry and Molecular Biology at the University of Barcelona and director of the Life Sciences Dept. at the BSC, and Alberto Pérez, “Juan de la Cierva” researcher at BSC, currently at the University of California, San Francisco, (U.S.) publish their findings in a leading international chemistry journal, Angewandte Chemie. Alberto Pérez explains that “many of the functions of DNA come about when its two strands separate, when, for example, it has to replicate during cell division or in repair processes. With this study, we propose a mechanism for this process, which in turn, will lead to new experiments for its final corroboration”.......> Full story

Surprising infection inducing mechanism found in bacteria

Wed, 19 May 2010 03:27:42 PDT

A research appearing in Nature, with the participation of doctors Susana Campoy and Jordi Barbé from the Department of Genetics and Microbiology at UAB, demonstrates that bacteria have a surprising mechanism to transfer virulent genes causing infections. The research describes an unprecedented evolutionary adaptation and could contribute to finding new ways of treating and preventing bacterial infections. Pathogenic genes are responsible for making bacteria capable of causing diseases. These genes cause bacteria to produce specific types of toxins and determine whether or not a disease will later develop in an individual. These virulent genes can be passed from one bacteria to another if the genome segments containing them, known as pathogenicity islands, are transferred from one to another. A team of researchers from Universitat Autònoma de Barcelona, together with members of the CSIC Institute for Agrobiotechnology, Public University of Navarre, Virginia Commonwealth University, and New York University Medical Center, coordinated by the Valencian Institute for Agronomic Research (IVIA) and CEU-Cardenal Herrera University, have studied the mechanisms producing virulence in staphylococcus bacteria and causing the Toxic Shock Syndrome, a rare but potentially fatal illness in 50% of the cases........> Full story

Hammerhead shark study shows cascade of evolution affected size, head shape

Wed, 19 May 2010 03:18:29 PDT

The ancestor of all hammerhead sharks probably appeared abruptly in Earth's oceans about 20 million years ago and was as big as some contemporary hammerheads, according to a new study led by the University of Colorado at Boulder. But once the hammerhead evolved, it underwent divergent evolution in different directions, with some species becoming larger, some smaller, and the distinctive hammer-like head of the fish changing in size and shape, said CU-Boulder Professor Andrew Martin of the ecology and evolutionary biology department. Sporting wide, flattened heads known as cephalofoils with eyeballs bulging at each end, hammerhead sharks are among the most recognizable fish in the world. The bizarre creatures range in length from about 3 feet up to 18 feet and cruise warm waters around the world, Martin said. In the new study, scientists focused on the DNA of eight species of hammerhead sharks to build family "gene trees" going back thousands to millions of generations. In addition to showing that small hammerheads evolved from a large ancestor, the team showed that the "signature" cephalofoils of hammerheads underwent divergent evolution in different lineages over time, likely due to selective environmental pressures, said Martin. The team used both mitochondrial DNA passed from mother to offspring and nuclear DNA -- which is commonly used in forensic identification -- to track gene mutations. The researchers targeted four mitochondrial genes and three nuclear genes, which they amplified and sequenced for the study. "These techniques allowed us to see the whole organism evolving through time," Martin said. "Our study indicates the big hammerheads probably evolved into smaller hammerheads, and that smaller hammerheads evolved independently twice." A paper on the subject was published in this month's issue of Molecular Phylogenetics and Evolution. Led by former CU-Boulder ecology and evolutionary biology undergraduate student Douglas Lim, co-authors included Martin and University of South Florida researchers Philip Motta and Kyle Mara. Lim is currently a student at the University of Colorado School of Medicine. The National Science Foundation funded the study. The researchers sampled hammerheads from across the globe -- including the waters of the southeast United States now under siege by the Gulf oil spill -- as well as Australia, Panama, Hawaii, Trinidad and South Africa. Most of the hammerhead DNA was obtained at local markets, where the peddling of sharks and other fish is common practice. The team sequenced the DNA of the sharks, constructing a "phylogenetic" tree that shows how all of the species are related and when each species originated, said Martin. The hammerhead ancestor probably lived in the Miocene epoch about 20 million years ago. The team found that two divergent lineages of small sharks about 3 to 4 feet long originated independently at separate times in the past. One of the species, the winghead shark, now lives in the warm waters north of Australia and the other, the bonnethead shark, inhabits the Caribbean and tropical eastern Pacific Ocean. One reason for the "incredible shrinking shark" over the eons may be the process of neoteny -- the ability of some adult sharks to retain juvenile traits -- or their ability to achieve sexual maturity at earlier ages, Martin said. "As the sharks became smaller, they may have begun investing more energy into reproductive activities instead of growth." While the cephalofoils appear to provide "lift" to large hammerheads as they cruise through the water -- much like the wing of an airplane -- smaller hammerheads don't appear to gain an advantage in lift, but may gain other attributes. "It looks like they sacrifice locomotion advantages for prey detection and visualization," he said. Another advantage hammerheads may gain from larger cephalofoils is an increased number of electrical sensors in their flattened noses and heads that can detect extremely weak electrical emissions from molecules associated with potential prey. "Hammerheads appear to be able to triangulate on their prey, which is remarkable," said Martin. Small sharks are highly variable in the size and shape of their cephalofoils, said Martin. The winghead shark, for example, has a laterally expanded head that is about half the size of its roughly 4-foot body length. At the other end of the spectrum is the bonnethead shark, about 3 feet long but which has the smallest cephalofoil of all hammerhead species -- a protrusion that resembles the head of a shovel, Martin said. Martin said that hammerheads are an ideal biological study subject in part because of some important similarities to humans. Both have slow growth rates, mature late in life, give live birth and have relatively few offspring. While hammerheads may have a dozen or more pups, other oceanic fish regularly lay millions of eggs. Hammerheads generally live for about 30 years, he said. While hammerhead sharks appear intimidating, attacks on humans are extremely rare, said Martin. Hammerheads have relatively small mouths facing downward that are used to grab food like fish, shellfish, shrimp, squid, octopuses and stingrays. "If you see a hammerhead, I'd say grab your camera and jump into the water," said Martin. "Hammerheads are special fish, and there is nothing that remotely resembles them anywhere on the planet," said Martin. Unfortunately, hammerheads -- like most shark species -- are on the decline. In addition to being overfished, sharks often are the victims of a technique known as finning, in which fishermen catch them, cut off their fins for use in delicacy soups, and return them to the water to die, Martin said. Shark meat also is used for fertilizer and to make pet food. There currently are 233 shark species on the International Union for the Conservation of Nature's "Red List of Threatened Species," and 12 shark species are classified as critically endangered. A study led by Virginia Tech showed the great hammerhead, scalloped hammerhead and smooth hammerhead species declined by an average of 90 percent from 1981 to 2005. "Their situation is generally pretty dire," Martin said. A 2005 study by Martin and his colleagues on scalloped hammerheads indicated females tend to breed in the specific ocean regions where they were born, while males tended to move around more widely. A previous study by Martin's team also showed that male great white sharks roam Earth's oceans much more widely than females, a finding with implications for future conservation strategies for the storied and threatened fish.

Biologists discover an extra layer of protection for bacterial spores

Fri, 07 May 2010 04:15:31 PDT

Bacterial spores, the most resistant organisms on earth, carry an extra coating of protection previously undetected, a team of microbiologists reports in the latest issue of the journal Current Biology. Their findings offer additional insight into why spores of the bacteria that cause botulism, tetanus, and anthrax survive methods to eradicate them. The study was conducted by researchers at New York University's Center for Genomics and Systems Biology, Loyola (Ill.) University's Medical Center, and Princeton University's Department of Molecular Biology. The researchers studied the spores of a non-pathogenic bacterium, Bacillus subtilis, which is commonly found in soil. Although non-pathogenic, B. subtilis spores exhibit many of the same structural features of the spore-forming pathogens. In this study, the scientists examined the proteins that comprise spores' protective layers. Previous research has shown that 70 different proteins make up these layers. Less understood is how these proteins interact to form the spores' protective coats. To do this, the researchers examined coat formation of both normal and mutant spores. In the latter case, they removed genes for selected coat proteins, allowing them to determine which proteins were necessary in—and extraneous to—the formation of the spores' coats. To observe proteins' behavior in living cells, the researchers fused the genes encoding the spores' coat proteins to a marker, a Green Fluorescent Protein (GFP). This procedure allowed them to monitor how the proteins localized to form spores' protective coats. A combination of fluorescence microscopy experiments and high-resolution image analysis enabled the researchers to overcome a theoretical limitation of light microscopy, pinpoint the location of the spores' coat proteins with a high degree of precision, and build a map of the spore coat. These experiments suggested the existence of a new outermost layer of the spore coat. They were then able to confirm the existence of this new layer using electron microscopy. The researchers named this coat layer, located on the spores' outer surface, the "spore crust." While it has not yet been confirmed, it is possible that the spore crust is a common feature of all spore-forming bacteria, such as the botulism, tetanus, and anthrax pathogens.

NIH study confirms location of stem cells near cartilage-rich regions in bones

Tue, 27 Apr 2010 03:21:08 PDT

Working with mice, a team of researchers has pinpointed the location of bone generating stem cells in the spine, at the ends of shins, and in other bones. The team also has identified factors that control the stem cells' growth. The research was conducted at the National Institutes of Health and other institutions. "Identifying the location of bone stem cells and some of the genetic triggers that control their growth is an important step forward," said Alan E. Guttmacher, M.D., acting director of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the NIH institute where much of the research took place. "Now, researchers can explore ways to harness these cells so that ultimately they might be used to repair damaged or malformed bone. Also, studies of this stem cell population could yield insight into the formation of bone tumors." Researchers have long known that stem cells from bone marrow give rise to bone cells and to red and white blood cells. The current study is the first to identify the location of bone stem cells in the adult mouse skeleton. The researchers refer to the newly identified cells as bone stromal cells. "Stroma" is a term used to describe a supportive or connective structure in biological tissue. The term distinguishes the cells from hematopoietic stem cells, which give rise to blood cells, and which are found in bone marrow.......> Full story

New Method for Producing Proteins Critical to Medical Research

Sun, 04 Apr 2010 04:48:13 PDT

Scientists at the University of Delaware have developed a new method for producing proteins critical to research on cancer, Alzheimer's, and other diseases. Developed by Zhihao Zhuang, UD assistant professor of chemistry and biochemistry, and his research group, the chemical method yields hundredsfold more ubiquitylated proteins than current approaches. Such proteins may hold the key to revealing such mysteries as how cancer cells gain resistance to cancer drugs. The advance is reported in the April issue of Nature Chemical Biology, the leading journal in the field of chemical biology. Zhuang's co-authors include graduate students Junjun Chen and Jialiang Wang and postdoctoral fellow Yongxing Ai, all from UD, and Lajos Haracska, a researcher in the Institute of Genetics at the Hungarian Academy of Sciences. Ubiquitin is a small protein, the basis of Nobel Prize-winning research in 2004, which deemed the molecule the “kiss of death” for its role in tagging damaged or unneeded proteins for the cell's waste disposal in the constant process of protein generation and degradation. In recent years, the non-proteolytic functions of ubiquitin in diverse cellular processes, including protein trafficking, immune response, and DNA damage tolerance, have been discovered at a rapid pace, and it has become clear that ubiquitin plays far-broader roles in cell biology.......> Full story