Structure formed by strep protein can trigger toxic shock

Infection with some strains of strep turn deadly when a protein found on their surface triggers a widespread inflammatory reaction.

In a report published April 7 in the journal Nature, researchers describe the precise architecture of a superstructure formed when the bacterial protein called M1 links with a host protein, fibrinogen, that is normally involved in clotting blood.

The proteins form scaffolds with M1 joints and fibrinogen struts that assemble into dense superstructures. Frontline immune cells called neutrophils mistake these thick networks for blood clots and overreact, releasing a chemical signal that can dilate vessels to the point where they leak, the team reports.

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M1 joints (red) and fibrinogen struts (blue) form a scaffold. Dense assemblies trigger a pathological response that can lead to toxic shock. Credit: Partho Ghosh lab

"We knew that M1 plus fibrinogen was inflammatory, but how was unknown. By determining the structure of this complex, we were able to identify the characteristics that lead to a sepsis response," said Partho Ghosh, Ph.D., professor of chemistry and biochemistry at the University of California, San Diego who studies the structure of virulence factors and led this project.

Ghosh and colleagues found that the density of the M1-fibrinogen structure was a critical characteristic. Looser structures or separate fibers formed by altered versions of M1 failed to trigger a pathological response.

"This research provides the first snapshot of the interaction between this key bacterial virulence factor and its human target at the atomic level," said Victor Nizet, M.D., professor of pediatrics and pharmacy and a co-author of the report.

Difficult to treat once they set in, the leaking blood vessels and organ failure of strep-induced toxic shock prove fatal for 30 percent of patients. Ghosh and Nizet have a long-standing collaboration aimed at designing treatments to counteract the toxic effects of strep protein.

More information: Streptococcal M1 protein constructs a pathological host fibrinogen network. DOI:10.1038/nature09967

Provided by University of California - San Diego (news : web)

OSU chemist developing solution to nerve agent exposure

Scientists are working to develop a new drug that will regenerate a critical enzyme in the human body that "ages" after a person is exposed to deadly chemical warfare agents.

Christopher Hadad, Ph.D., professor of chemistry at The Ohio State University (OSU), is leveraging Ohio Supercomputer Center (OSC) resources to help develop a more effective antidote to lethal chemicals called organophosphorus (OP) nerve agents.

"This project is a combination of synthetic and computational organic chemistry conducted through OSC at Ohio State, and biochemical studies conducted by colleagues at the U.S. Army Medical Research Institute of Chemical Defense at Aberdeen Proving Ground in Maryland," said Hadad.

OP nerve agents inhibit the ability of an enzyme called acetylcholinesterase (AChE) to turn off the messages being delivered by acetylcholine (ACh), a neurotransmitter, to activate various muscles, glands and organs throughout the body. After exposure to OP agents, AChE undergoes a series of reactions, culminating in an "aging" process that inhibits AChE from performing its critical biological function. Without the application of an effective antidote, neurosynaptic communication continues unabated, resulting in uncontrolled secretions from the mouth, eyes and nose, as well as severe muscle spasms, which, if untreated, result in death.

Conventional antidotes to OP nerve agents block the activity of the nerve agent by introducing oxime compounds, which have been the focus of a number of studies. These compounds attach to the phosphorus atom of the nerve agent, after the OP is bound to AChE, and then split it away from the AChE enzyme, allowing the AChE to engage with receptors and finally relax the tissues.

However, in some cases, the combined nerve agent/AChE molecule undergo a process called aging, in which groups of single-bonded carbon and hydrogen atoms called alkyl groups are removed from the molecule and a phosphonate residue is left behind in the AChE active site. Relatively unstudied in nerve agents, this process, called dealkylation, makes the nerve agent/AChE molecule unreceptive to oximes – an unfortunate situation, considering that certain nerve agents (e.g., soman) can undergo aging within minutes of exposure to AChE.

A docking simulation constructed at the Ohio Supercomputer Center by Ohio State Professor Christopher Hadad, Ph.D., illustrates binding in the active site of tabun-inhibited AChE. Credit: Hadad/OSU

Hadad's study is focused on the identification of compounds that would return an appropriate alkyl group to the aged nerve agent/AChE molecule, thus allowing treatment with oximes to provide for complete recovery. The project is investigating common OP nerve agents Tabun, VX, VR, Sarin, Soman, Cyclosarin and Paraoxon, all of which take on a similar molecular structure upon aging.

"Computational studies of the interaction of the alkylating compounds with AChE were used to provide insight for the design of selective reagents," Hadad explained. "Ligand-receptor docking, followed by molecular dynamics simulations of the interactions of alkylating compounds with aged OP-AChE, was carried out in conjunction with experimental studies to investigate the binding of alkylating compounds to AChE. These results were then used to suggest interactions that aided in the orientation of alkylating compounds for maximal efficacy."

Throughout the project, Hadad employed computational studies to guide the progress of each objective, as well as to rationalize the observed experimental results.

"Dr. Hadad's work on this project has made use of a range of the tools of electronic structure theory, molecular docking, molecular dynamics and hybrid quantum mechanical/molecular mechanical methods," said Ashok Krishnamurthy, interim co-executive director of OSC. "It was by design that OSC's flagship system, the Glenn IBM 1350 Opteron cluster, was developed to meet the needs of the bioscience research investigators, such as Dr. Hadad."

Provided by Ohio Supercomputer Center

Did clay mould life's origins?

An Oxford University scientist has taken our understanding of the origin of life a step further.

Professor Don Fraser from the Department of Earth Sciences has carried out neutron scattering experiments to try to find out more about the role of geochemistry in determining the origin of our amino acids – key building blocks of life on Earth – and specifically why the DNA-coded amino acids that make up our proteins are all left-handed.

There are two varieties of amino acids, known as left- or right-handed (referred to as S and R). They are mirror images of each other and both exist in nature, as shown for other substances by Louis Pasteur.

Biochemical processes in living organisms use left- and right-handed or ‘chiral’ receptors that template differently with these two forms. The olfactory receptors in our noses, for example, easily distinguish the distinct smells of the otherwise identical molecules (called carvones) of spearmint (R-carvone) and caraway (S-carvone).

Another example is the thalidomide tragedy that was related to the presence in the drug of both the mirror-image forms. One of these (S) was later understood to be harmful.

An important and outstanding mystery is why nature chooses only exclusively left-handed amino acids in forming proteins.

In the late 19th century, TH Huxley and Charles Darwin realised that life may have begun abiotically in a ‘warm little pond’ containing all the elements needed for life, ‘so that a protein compound was chemically formed ready to undergo still more complex changes,’ Darwin wrote.

Much later, in 1924, the Soviet scientist Alexander Oparin returned to the idea of spontaneous generation, suggesting that a ‘primeval soup’ of organic molecules, created by the action of sunlight in an oxygen-free environment, was the basis of all life.

Electric spark experiments subsequently carried out by Stanley Miller in model primeval atmospheres showed that amino acids form in lightning discharges. In contrast to biological systems, these show no preference for either mirror image form and are 50%:50% (racemic) mixtures.

Clay was suggested by the crystallographer John Bernal as a means of concentrating primitive biomolecules onto its surface so as to be available for further reactions. Clays again became the focus of studies more recently when James Ferris showed that they can act as catalysts for the formation of long strands of RNA, which with proteins and DNA are major compounds essential for the origin of life.

In a second paper, also published in Physical Chemistry Chemical Physics, Professor Fraser has extended these ideas to consider amino acids and to try to understand why all amino acids used to make proteins are left-handed.

With colleagues Professor Neal Skipper from UCL, Dr Martin Smalley from York University and Dr Chris Greenwell from Durham University he replaced the cations between the layers making up natural clay molecules with weakly bound organic cations, causing the clay layers to drift apart.
That created an extremely sensitive clay system with sufficient space to insert both left-handed and right-handed forms of the amino acid histidine between the layers.

"We found that the R- and S-histidine molecules interact differently with the clay surfaces. These clays are abiotically able to select for chirality – left- or right-handedness – as well as being implicated in the abiotic synthesis of RNA," Professor Fraser says. "Our experiments were the first to show that clay molecules could do that.

"We also found that the tiny interlayer space some 5nm wide was a very important dynamic region for studying prebiotic chemistry and that the reactions of simple chemicals there leads to the formation both of RNA oligomers and the selection of left-handed amino acids.’

Clays have also been shown by Jack Szostak and others to enable fatty acids to form primitive cells and, interestingly, clays show similar selective behaviour in space, as reported recently by the NASA scientists Glavin and Dworkin.

Amino acids contained in the meteorites Murchison and Orgueil show enrichment in S-amino acids and this correlates with the amount of intrinsic hydrous clay present in these primitive meteorites that are 4.55 billion years old. ‘The amino acid studied – isovaline – cannot be a contaminant as it is not found in terrestrial living systems,’ Professor Fraser explains. ‘We are thus building an increasingly detailed picture of the steps that lead to the origin of life.

"We are continuing our research next month on the new NIMROD instrument at the ISIS neutron source near Oxford. This will involve amino acids enriched in deuterium, an isotope of hydrogen, and will provide a detailed atomic picture of the way amino acids interact with the layers of clay for the first time.

"In the long term, this work could have significant applications not only for our understanding of the origin of life, but also in medicine as the design of new mineral surfaces that aid the production of chiral drugs would be of great benefit to the pharmaceutical industry."

Provided by Oxford University (news : web)

Capturing the fugitive... in art

Enlarge
Winslow Homer (1836-1910) For to Be a Farmer's Boy 1887 (Gift of Mrs George T. Langhorne in memory of Edward Carson Waller, AIC 1963.760). This image had long puzzled scholars due to the seemingly unfinished and flat sky in a highly finished work. Credit: ? The Art Institute of Chicago
What do Winslow Homer's For to Be a Farmer's Boy (1887) and Vincent van Gogh's The Bedroom (1889) have in common?
First, they are both displayed at the Art Institute of Chicago (AIC). Homer's painting represents a high point in the career of America's premiere watercolorist, while Van Gogh's painting is perhaps one of most recognizable paintings in the world. However, they also share a key physical trait.
"These breathtaking artworks are both painted with colorants that are sensitive to light, or, as we say in museums, they are 'fugitive,' meaning they quickly vanish if exposed to too much light," says Francesca Casadio, A.W. Mellon senior conservation scientist at the AIC. "Fading can dramatically change the color balance of fragile works of art and go so far as to obfuscate, in part, the artist's intended effect."
In For to Be a Farmer's Boy, the sky is starkly blank--gone are the vibrant colors that Homer is known to have used for his evocative renditions of skies and seas. Yet, through research funded by the National Science Foundation's (NSF) Chemistry and Materials Research in Cultural Heritage Science (CHS) program, a new story is being revealed about Homer's painting.


Laboratory replicas of watercolor brushstrokes containing cochineal (Carmine Naccarat) and madder lakes before (below) and after (above) fading for 20 months in a sun-exposed window. Credit: ? Kristi Dahm, The Art Institute of Chicago
After painstakingly peering through binocular microscopes, art conservators working behind the scenes at AIC discovered some microscopic particles of colored pigments trapped in the artwork's paper fibers. Precise identification of such pigments was key to "recognizing the implicit emotional, narrative and symbolic content" of the artist's work, according to AIC curator Martha Tedeschi.
Effectively identifying the red "lake" pigments that Homer used is difficult using conventional analytical techniques. For example, Raman spectroscopy, normally used to fingerprint artists' palettes, is strongly affected by the overwhelming fluorescence of natural colorants.
Nanotechnology comes to the rescue
Now, art historians and art conservators have one more tool in their arsenal to preserve our cultural treasures: Surface Enhanced Raman Spectroscopy (SERS). Although this technique has been around for almost 30 years, only recently has SERS fully realized its potential, thanks to the nanotechnology boom.

SERS is an ideal technique for art analysis--it is highly sensitive and can detect vanishingly small amounts of organic pigments that have long eluded identification by other approaches. Yet, only a handful of research groups are working on this application.
"Imagine a child in a sandbox with toys. The toys are the molecules--we want to study them, but they are hidden under the sand and you cannot see them," explains Richard P. Van Duyne, the Charles E. and Emma H. Morrison Professor of Chemistry at Northwestern University, who is best known for the discovery of SERS. "Our technique gets rid of the sand so that you can see the toys and identify what they are."

A digital recreation of Homer's For to Be a Farmer's Boy, simulating the appearance of the sky at sunset before fading, matched to the original, faded image. Credit: ? Kristi Dahm, Loren McDonald, The Art Institute of Chicago
By using a colloidal suspension of silver nanoparticles as a "performance enhancing drug," researchers, for the first time, can identify natural organic colorants on a single grain of pigment otherwise invisible to the naked eye.
SERS analysis
Indeed, only a handful of pigment particles were available from the Homer watercolor. Compared to reference 19th century watercolor pigments available at AIC, these colorants were identified as Indian purple (cochineal precipitated with copper sulfate) and madder purple, two natural dyestuffs derived from an insect and vegetable-root sources, respectively.
The results indicate that in Homer's For to Be a Farmer's Boy, the "empty" sky once depicted a vibrant autumn sunset, with organic purples and reds, in addition to inorganic reds and yellows. Although these results are promising, there is still a need to improve the identification of pigments from severely faded paintings. This is because pigment particles are normally applied in a complex medium, which increases the fluorescence background of the spectra, much like adding more sand to the sandbox covers up the toys (molecules) below.
In order to reduce the fluorescence, research professor Nilam Shah, also of Northwestern, will be developing ad hoc-tailored nanoparticles optimized to resonate with infrared lasers, which are less damaging to the artworks, and more universal. These next-generation nanoparticles hold promise as tools to unlock information on dyes, pigments and binding media, as present in Van Gogh's The Bedroom.
Capturing the fugitive
Typically, researchers who use SERS for materials identification search an unknown compound against a database of references, much like matching fingerprints of known criminals to forensic evidence collected on the crime scene.
Thanks to the theoretical expertise of George Schatz, Morrison Professor of Chemistry at Northwestern, this painstaking database search will no longer be a limiting factor. In fact, researchers will be able to calculate from theory not only the expected SERS spectrum of unknown fugitive dyes, but also the tell-tale signs of dyes disappearing after prolonged exposure to light.
Taking into account the pigment identification by SERS and nuances of shade and tone that are typical of Homer's paint handling, art conservators proposed a digital re-creation of the Homer watercolor. By shining laser light on particles buried in the artwork, SERS investigators have now unearthed the materials evidence that allows viewers to truly experience the hues of Homer's faded sunsets for the first time in modern times.

Provided by National Science Foundation (news : web)

Nano fit-ness: Helping enzymes stay active and keep in shape

Proteins are critically important to life and the human body. They are also among the most complex molecules in nature, and there is much we still don't know or understand about them.

One key challenge is the stability of enzymes, a particular type of protein that speeds up, or catalyzes, chemical reactions. Taken out of their natural environment in the cell or body, enzymes can quickly lose their shape and denature. Everyday examples of enzymes denaturing include milk going sour, or eggs turning solid when boiled.

Rensselaer Polytechnic Institute Professor Marc-Olivier Coppens has developed a new technique for boosting the stability of enzymes, making them useful under a much broader range of conditions. Coppens confined lysozyme and other enzymes inside carefully engineered nanoscale holes, or nanopores. Instead of denaturing, these embedded enzymes mostly retained their 3-D structure and exhibited a significant increase in activity.

"Normally, when you put an enzyme on a surface, its activity goes down. But in this study, we discovered that when we put enzymes in nanopores – a highly controlled environment – the enzymatic activity goes up dramatically," said Coppens, a professor in the Department of Chemical and Biological Engineering at Rensselaer. "The enzymatic activity turns out to be very dependent on the local environment. This is very exciting."

Results of the study are detailed in the paper, "Effects of surface curvature and surface chemistry on the structure and activity of proteins adsorbed in nanopores," published last month by the journal Physical Chemistry Chemical Physics.

Researchers at Rensselaer and elsewhere have made important discoveries by wrapping enzymes and other proteins around nanomaterials. While this immobilizes the enzyme and often results in high stability and novel properties, the enzyme's activity decreases as it loses its natural 3-D structure.

Coppens took a different approach, and inserted enzymes inside nanopores. Measuring only 3-4 nanometers (nm) in size, the enzyme lysozyme fits snugly into a nanoporous material with well-controlled pore size between 5 nm and 12 nm. Confined to this compact space, the enzymes have a much harder time unfolding or wiggling around, Coppens said.

The discovery raises many questions and opens up entirely new possibilities related to biology, chemistry, medicine, and nanoengineering, Coppens said. He envisions this technology could be adapted to better control nanoscale environments, as well as increase the activity and selectivity of different enzymes. Looking forward, Coppens and colleagues will employ molecular simulations, multiscale modeling methods, and physical experiments to better understand the fundamental mechanics of confining enzymes inside nanopores.

More information: The paper may be viewed online at: http://dx.doi.org/10.1039/C0CP02273J

Provided by Rensselaer Polytechnic Institute (news : web)

Chemical engineers design molecular probe to study disease

 

Chemical engineers at UC Santa Barbara expect that their new process to create molecular probes may eventually result in the development of new drugs to treat cancer and other illnesses.

Their work, reported in the journal Chemistry & Biology, published by Cell Press, describes a new strategy to build molecular probes to visualize, measure, and learn about the activities of enzymes, called proteases, on the surface of cancer cells.

Patrick Daugherty, senior author and professor of chemical engineering at UCSB, explained that the probes are effective at understanding proteases involved in tumor metastasis.

"Tumor metastasis is widely regarded as the cause of death for cancer patients," said Daugherty. "It's not usually the primary tumor that causes death. Metastasis is mediated by proteases, like the one we are studying here. These proteases can enable tumor cells to separate and degrade surrounding tissue, and then migrate to sites distant from the primary tumor. The tumor doesn't just fall apart. There are many events that must occur for a tumor to release cancerous cells into the blood stream that can circulate and end up in other tissues such as liver or bone."

The probes allowed the researchers, for the first time, to measure directly the activity of a protease involved in metastasis. They did this by adding their probe into a dish of tumor cells. They then measured the activity of this protease that breaks down collagen –– the single most abundant protein (by mass) in the human body.

"We have immediate plans to use similar probes to effectively distinguish metastatic HER2 positive tumors, one of the most commonly used biomarkers of breast cancer," said Daugherty. "A significant fraction of patients have HER2 positive tumors but we don't know which of those tumors is going to metastasize yet. But our ability to make these probes can allow us to identify which of those HER2 positive tumors have the ability to break down that surrounding tissue, to detach from the primary tumor, and to establish a separate tumor somewhere else in the body."

The authors designed the molecular probe to be recognized by a single protease rather than by the many proteases that are present in human tissues. That is half of the probe. The other half of the probe involves an optical technique used to measure activity. This approach relies upon the use of two engineered fluorescent proteins, derived from marine organisms, that absorb and emit light in a process called FRET, or Forster resonance energy transfer.

To prepare the probes, the researchers introduced a gene that encodes the probe into the bacteria E. coli. Then they produced and purified significant quantities of the probe. All of the information needed for the probe is encoded by a DNA sequence. The probes are easy and inexpensive to produce, as well as easily shared with other researchers.

In addition to studying cancer, similarly constructed probes have ramifications for studying Alzheimer's disease, arthritis and connective tissue diseases, bacterial infections, viruses, and many other diseases.

"The fact that you can generalize the concept, and the way you make these probes, to many systems, makes it attractive," said Daugherty. "We happen to study the activity of this protease and a certain type of tumor cells that are derived from cancer patients. But you could apply this to hundreds of molecules and really develop a working understanding of how groups of proteases function together in cell biology."

In individuals with rheumatoid arthritis, for example, there is increased production of proteases, including the one studied by Daugherty's team. This protease mediates collagen breakdown and joint destruction. "If you've got an enzyme that can chew up collagen and you've got lots of collagen in your joints, then you would expect that you would see more rapid degradation of the joint by those proteases," said Daugherty.

Daugherty's research group has created approximately 25 probes analogous to the one presented in the paper. They are building a panel of about 100 probes and will use this panel to characterize how different proteases function. This investigation could lead to new drug therapies for a variety of diseases.

Provided by University of California - Santa Barbara (news : web)

Tungsten may not be the best shot for making 'green' bullets

With efforts underway to ban lead-based ammunition as a potential health and environmental hazard, scientists are reporting new evidence that a prime alternative material for bullets — tungsten — may not be a good substitute The report, which found that tungsten accumulates in major structures of the immune system in animals, appears in ACS' journal Chemical Research in Toxicology.

Jose Centeno and colleagues explain that tungsten alloys have been introduced as a replacement for lead in bullets and other munitions. It resulted from concern that lead from spent ammunition could harm wildlife when it dissolves into water in the soil, streams, and lakes. Scientists thought that tungsten was relatively non-toxic, and a "green" replacement for lead. Recent studies suggested otherwise, and with small amounts of tungsten also used in some artificial hips and knees, Centeno's group decided to gather further information on tungsten.

They added small amounts of a tungsten compound to the drinking water of laboratory mice, used as surrogates for people in such research, and examined the organs and tissues to see exactly where tungsten ended up. The highest concentrations of tungsten were in the spleen, one of the main components of the immune system, and the bones, the center or "marrow" of which is the initial source of all the cells of the immune system. Further research, they say, will be needed to determine what effects, if any, tungsten may have on functioning of the immune system.

Provided by American Chemical Society (news : web)