Friday, May 6, 2011

Researchers show heparan sulfate adjusts functions of growth factor proteins

When the human genome project produced a map of human genes, the number of genes in humans turned out to be relatively small, approximately the same number as in primitive nematode worms. The difference in complexity between human and primitive organisms results from the ways in which the functions of genes are elaborated, rather from just the number of genes. Boston University School of Medicine (BUSM) researchers are showing how heparan sulfate, a carbohydrate that is expressed on the surface of all human cells, adjusts the functions of growth factor proteins. These findings currently appear on-line in the Journal of Biological Chemistry.

Each cell responds to signals in the form of growth factor proteins that bind to cell surfaces. "The heparan sulfate on each cell helps the growth factor proteins connect with a that is necessary for the signaling to occur," explained Joseph Zaia, PhD, an associate professor of Biochemistry at BUSM. Cells can change the way they respond to growth factors by altering the structure of the heparan sulfate on their surfaces.

Under the direction of Zaia, researchers from BUSM's department of Biochemistry have produced a new picture of the structure of the heparan sulfate and how it interacts with growth factor proteins. These new results demonstrate that growth factors home into regions of the heparan sulfate chains known as non-reducing ends. "Such binding of growth factors to the non-reducing ends of heparan sulfate chains may be a general means whereby normal cell growth is maintained. Conversely, a breakdown in such signaling may contribute to abnormal cell growth," he added.

Provided by Boston University Medical Center

New evidence that caffeine is a healthful antioxidant in coffee


Scientists are reporting an in-depth analysis of how the caffeine in coffee, tea, and other foods seems to protect against conditions such as Alzheimer's disease and heart disease on the most fundamental levels. The report, which describes the chemistry behind caffeine's antioxidant effects, appears in ACS' Journal of Physical Chemistry B.

Annia Galano and Jorge Rafael León-Carmona describe evidence suggesting that coffee is one of the richest sources of healthful in the average person's diet. Some of the newest research points to caffeine (also present in tea, cocoa, and other foods) as the source of powerful antioxidant effects that may help protect people from Alzheimer's and other diseases. However, scientists know little about exactly how caffeine works in scavenging the so-called free radicals that have damaging effects in the body. And those few studies sometimes have reached contradictory conclusions.

In an effort to bolster scientific knowledge about caffeine, they present detailed theoretical calculations on caffeine's interactions with free radicals. Their theoretical conclusions show "excellent" consistency with the results that other scientists have report from animal and other experiments, bolstering the likelihood that caffeine is, indeed, a source of healthful antioxidant activity in coffee.

More information: “Is Caffeine a Good Scavenger of Oxygenated Free Radicals?” Journal of Physical Chemistry B. DOI: 10.1021/jp201383y

The reactions of caffeine (CAF) with different reactive oxygen species (ROS) have been studied using density functional theory. Five mechanisms of reaction have been considered, namely, radical adduct formation (RAF), hydrogen atom transfer (HAT), single electron transfer (SET), sequential electron proton transfer (SEPT), and proton coupled electron transfer (PCET). The SET, SEPT, and PCET mechanisms have been ruled out for the reactions of CAF with •OH, O2•-, ROO•, and RO• radicals. It was found that caffeine is inefficient for directly scavenging O2•- and •OOCH3 radicals and most likely other alkyl peroxyl radicals. The overall reactivity of CAF toward •OH was found to be diffusion-controlled, regardless of the polarity of the environment, supporting the excellent •OH scavenging activity of CAF. On the other hand, it is predicted to be a modest scavenger of •OCH3, and probably of other alkoxyl radicals, and a poor scavenger of HOO•. RAF has been identified as the main mechanism involved in the direct ROS scavenging activity of CAF. The excellent agreement with the available experimental data supports the reliability of the present calculations.

Provided by American Chemical Society (news : web)

Advanced instrument used to read cells' minds

Researchers at the Stanford University School of Medicine have taken a machine already in use for the measurement of impurities in semiconductors and used it to analyze immune cells in far more detail than has been possible before. The new technology lets scientists take simultaneous measurements of dozens of features located on and in cells, whereas the existing technology typically begins to encounter technical limitations at about a half-dozen.

The investigators were able not only to simultaneously categorize more immune cell types than ever before seen at once but, at the same time, to peer inside those and learn how various internal processes differed from one cell type to the next.

"We can tell not only what kind of cell it is, but essentially what it's thinking, what it's been doing, and what it may soon do or become," said Garry Nolan, PhD, professor of microbiology and immunology and the senior author of the study detailing the advance, to be published May 6 in Science.

With this new approach, the scientists were further able to show the unexpected effects of a drug recently approved for treating certain leukemias — dasatinib — on biochemical activities taking place inside various types of cells, offering a possible explanation for some of dasatinib's side effects as well as suggesting potential new uses for the drug.

In the study, Nolan and his colleagues simultaneously monitored 34 different substances found inside and on the surface of different cell types produced in human bone marrow, the place where all immune and blood cells, as well as blood disorders such as leukemia, originate.

By measuring large numbers of cell features all at once with the — called mass cytometry — the team could capture subtle transitions between cell states in, essentially, a high-resolution snapshot of the entire blood-forming system, he said. Scientists normally think of the blood and as differentiating in a series of discrete steps. However, the authors showed that the transitions from one cell state to another are marked by gradually shifting levels of cell-surface markers and varying amounts and activation states of several intercellular molecules.

Mass cytometry builds on an established technology known as fluorescence-activated cell sorting, or FACS, which is in widespread use throughout the world. FACS was developed in the laboratory of Leonard Herzenberg, PhD, professor emeritus of genetics, under whose direction Nolan did his PhD work in the 1980s.

Both FACS and mass cytometry employ antibodies to specifically tag particular surface features on cells.

With traditional FACS, antibodies are designed to tag diverse cell features. Then the antibodies are affixed to differently fluorescent dyes that color-code these antibodies according to which cell feature they target. After being bathed in these antibody-dye preparations, cells are passed single-file through a tube and stimulated by laser pulses, which cause the dye molecules to give off bursts of light. Different wavelengths of light emitted by the dyes correspond to the cellular features the dyes have tagged. FACS technology, though over 30 years old, is a mainstay of immune studies, as well as cancer and vaccine research.

But researchers are eager to squeeze ever more information out of each cell they examine. This requires examining ever more cell features at once, and there are only so many colors in the rainbow. The ability of FACS to distinguish between any more than a half-dozen dyes is constrained by those dyes' overlapping fluorescence patterns.

Three years ago, Nolan was approached by Scott Tanner, a physical chemist now at the University of Toronto.

"He buttonholed me at a meeting," said Nolan, laughing. "I was trying to get away from him, but after he'd been talking for a few minutes I realized this was something I'd better start paying attention to. He clearly had something that, if true, was revolutionary in its potential."

Tanner's team was adapting for biological purposes an existing instrument that is typically used for gauging precise levels of added rare-earth in and for geological purposes. The new instrument, called a mass cytometer, promised to more than double the number of molecular features that could be measured simultaneously in each cell. Nolan, realizing that such an instrument could be used to learn much more about the immune system and cancer stem cells, was eager to bring his group's expertise to bear on its development. The Stanford team has worked in close collaboration with the new instrument's developers ever since.

Instead of dyes, mass cytometry joins rare-earth metals to antibodies, which in turn detect cellular features and processes. "The rare earths are a series of 17 elements, mostly at the bottom of periodic table, that nobody wanted to learn about in chemistry class, myself included," said Sean Bendall, PhD, a postdoctoral researcher in Nolan's lab. However, these elements turn out to particularly useful for biological applications, said Bendall, who shared first authorship of the Science paper with Erin Simonds, a graduate student in Nolan's lab.

"They're not all that rare in nature, but they're normally never found in the body," Bendall said. "If I looked at a sample of your blood and found some europium or ytterbium or neodymium in it, I'd say you were in deep trouble." So rare-earth elements stand out in a crowd.

What's more, these elements can be subdivided into as many as 100 variants with distinct atomic weights. Mass cytometry can easily detect those differences. "We need relatively few rare-earth atoms per cell for our instrument to see them," said Bendall.

In mass cytometry, cells are paraded one by one through a tube and sprayed into a tiny chamber in which they are heated to about 13,000 degrees Fahrenheit and vaporized into successive clouds of atomic nuclei and loose electrons. Next, the contents of each cloud that was once a cell are essentially flung against a wall with equal force. The lightest atoms arrive first, then the next-lightest and so forth. A detector counts the atoms as they land, and from this the instrument can determine their mass. The mass tallies how many copies of each metal-tagged antibody were stuck to the cell and, therefore, how many copies of each molecular feature were present on, or in, the cell in the first place.

In the Science study, Nolan and his colleagues used the instrument to simultaneously monitor 13 separate molecular features on the surfaces of cells in samples taken from two healthy humans' bone marrow, and classified the cells into numerous distinct categories. The investigators simultaneously monitored activation states of 18 different intracellular protein targets. Protein activation levels give important clues about particular cellular decisions that have been or can be made, such as whether a cell is about to divide.

"As a prelude to looking at leukemic bone-marrow samples down the road, we wanted to first characterize the cells in normal bone marrow to see how their behaviors change as they mature," said Simonds.

The Nolan group perturbed cells by exposing them to various substances, including signaling molecules that sometimes circulate in our own blood, as well as foreign materials such as fragments of bacterial cell walls that are known to excite immune responses. "In essence," said Nolan, "we are interviewing or interrogating the cells, forcing them to reveal their inner thought processes." Some of these stimulatory tests were done in the presence of dasatinib, a drug used to treat chronic myelogenous leukemia and certain cases of acute lymphoblastic . Dasatinib is in clinical trials for several other indications, including some solid cancers.

When the Nolan group used a chemical, pervanadate, to "release the brakes" on a universal pro-cell-survival behavior, dasatinib blocked action in every cell type except one, the immune sentinels called dendritic cells. Simonds said this new finding demonstrates mass cytometry's capacity to ferret out tiny differences in cellular behavior that may help explain drugs' side effects as well as to indicate potential new uses for existing drugs.

The more measurements your tailor makes, the better the fit. It's the same with cell biology. "Our entire lab has already shifted from fluorescence-based measurements of cell features to this new MS-based method, because we get a much more complete picture," said Bendall.

Nolan has reported that he owns stock in the company Tanner created to develop and market the new system.

Provided by Stanford University Medical Center (news : web)

New route to map brain fat

Mapping the fat distribution of the healthy human brain is a key step in understanding neurological diseases, in general, and the neurodegeneration that accompanies Alzheimer's disease in particular. Antonio Veloso and colleagues, from the University of the Basque Country in Leioa, Spain, find a new technique to reveal the fat distribution of three different areas of the healthy human brain. Their work is published online in Springer's journal, Analytical & Bioanalytical Chemistry.

The human central nervous system has an abundance of lipid molecules - some are structural and energetic components of cells; others play a role in neurotransmission and are known as neurolipids. Mapping these neurolipids can increase neurologists' knowledge of the precise metabolic routes that produce them and where this production takes place. Knowing the fat composition of the healthy and the distribution of the different lipid species can give them clues about how neurodegenerative diseases develop.

The multi-disciplinary team used a combination of MALDI-TOF imaging mass spectrometry (a technique used to visualize the spatial distribution of compounds by their molecular mass) and functional autoradiography (an image recorded on a photographic film that shows the distribution of the activity induced by a drug) to scan healthy brain tissue slices. They mapped out, in detail, the lipid distribution of 43 types of lipids in three distinct areas of the human brain: the frontal cortex, hippocampus and striatum.* What is unique about their method, is its ability to identify the lipid species as well as locate them. Indeed, localization of lipid species is lost with the use of traditional techniques.

The authors conclude: "The application of imaging mass spectrometry to the localization of lipid species in the brain will be especially helpful to elucidate the specific functions of each type of lipid. Moreover, during the last few years the modulation of the signaling by neurolipids has been found to be implicated in neurodegenerative diseases. In this context, Alzheimer's disease is especially interesting since the familial mutation of some proteins that transport lipids, as the Apolipoprotein E, is a risk factor in this disease. The imaging technique is still in an early stage. It is expected that in the near future, new hardware developments will allow a precise determination of an increasing number of lipid species, aiming at producing a three-dimensional map of the lipid distribution in the brain."

More information: Veloso A et al (2011). Distribution of lipids in human brain. Analytical and Bioanalytical Chemistry; DOI:10.1007/s00216-011-4882-x

Provided by Springer

Organic chemistry: Amino acids made easy

Amino acids are the building blocks of proteins. There are 22 different amino acids and they can combine in a myriad ways to form a vast array of proteins. All amino acids except glycine are chiral molecules, meaning they exist in two mirror-image, or enantiomeric, forms—only one of which is naturally occurring. These unnatural enantiomers of amino acids are in great demand by the pharmaceutical industry as the raw materials for the production of a variety of drugs, including the antibiotic amoxycillin and the anti-nausea drug aprepitant (see image).

One method that is widely used to produce generally is Strecker synthesis—a chemical reaction devised by the nineteenth-century German chemist Adolph Strecker that combines an aldehyde, ammonia and hydrogen cyanide to produce an aminonitrile that can be easily converted into an amino acid. Unfortunately, the original Strecker synthesis can only produce a mixture of the enantiomeric forms of an amino acid. For this reason, many chemists have taken an interest in the development of enantioselective, or asymmetric, catalytic reactions—reactions that use a catalyst to selectively increase the formation of a particular enantiomer.

Some catalytic enantioselective variations of Strecker synthesis have already been reported, but there are problems. Many require the use of expensive sources of cyanide—typically a compound called trimethylsilylcyanide—and very low temperatures, which can be difficult to achieve on an industrial scale.

Abdul Majeed Seayad at the A*STAR Institute of Chemical and Engineering Sciences and co-workers have now developed an asymmetric Strecker protocol that uses hydrogen cyanide as the cyanide source and which proceeds at room temperature. The new methodology still requires the use of trimethylsilylcyanide, which the researchers found to be essential to achieving an enantioselective reaction, but only a relatively small catalytic amount is required and it is regenerated in the reaction by the addition of cheaper hydrogen cyanide. Seayad and his co-workers showed that they can use their conditions to produce a variety of unnatural amino acids.

As with most methodology developments, there is room for improvement with further research. “So far we’ve tackled only amino acids with aromatic side-chains,” explains Seayad. “We would like to develop the process to produce amino acids with other side chains. is inexpensive but it is extremely toxic and special equipment and training are needed to handle it. We are exploring ways in which we might generate it in the reaction, which would be much safer.”

More information: Ramalingam, B. et al. A remarkable titanium-catalyzed asymmetric Strecker reaction using hydrogen cyanide at room temperature. Advanced Synthesis and Catalysis 352, 2153–2158 (2010). … sc.201000462

Provided by Agency for Science, Technology and Research (A*STAR)

New woes for silicones in cosmetics and personal care products

At a time when cosmetics, shampoos, skin creams, and other personal care products already are going green — with manufacturers switching to plant-derived extracts and other natural ingredients — government regulators in Canada are adding to the woes of the silicone-based ingredients long used in these products. That's the topic of an article in the current edition of Chemical & Engineering News (C&EN), ACS' weekly newsmagazine.

C&EN Senior Correspondent Marc S. Reisch points out that manufacturers have used silicones for decades in an array of personal care products. Antiperspirants and underarm deodorants account for about half the entire U.S. personal care market for silicones. Manufacturers voluntarily stopped using one type of silicone ingredient in over the last decade. Now government regulators in Canada are proposing regulations limiting use of another widely used type of silicone ingredient. They cite concerns that the might built up in the environment and harm wildlife.

The article notes that some manufacturers, despite the concerns, are sticking with the traditional ingredients, termed cyclic methylsiloxanes. Others are using the concerns as a basis for jumping on the natural ingredient bandwagon and reformulating their products with other silicones or as "silicone-free."

More information: “Storm Over Silicone” is available at

Provided by American Chemical Society (news : web)

Tests show new biosensor can guide environmental clean-ups

Tests of a new antibody-based "biosensor" developed by researchers at the Virginia Institute of Marine Science show that it can detect marine pollutants like oil much faster and more cheaply than current technologies. The device is small and sturdy enough to be used from a boat.

Testing of the biosensor in the Elizabeth River and Yorktown Creek, which both drain into lower Chesapeake Bay, shows that the instrument can process samples in less than 10 minutes, detect pollutants at levels as low as just a few parts per billion, and do so at a cost of just pennies per sample. Current technology requires hours of lab work, with a per-sample cost of up to $1,000.

"Our biosensor combines the power of the immune system with the sensitivity of cutting-edge electronics," says Dr. Mike Unger of VIMS. "It holds great promise for real-time detection and monitoring of oil spills and other releases of contaminants into the marine environment."

The biosensor was developed and tested by Unger, fellow VIMS professor Steve Kaattari, and their doctoral student Candace Spier, with assistance from marine scientist George Vadas. The team's report of field tests with the sensor appears in this month's issue of .

The instrument was developed in conjunction with Sapidyne Instruments, Inc., with funding from the state of Virginia, the Office of Naval Research, and the Cooperative Institute for Coastal and Estuarine Environmental Technology, a partnership between NOAA and the University of New Hampshire.

The tests in the Elizabeth River took place during clean up of a site contaminated by (PAHs), byproducts of decades of industrial use of creosote to treat marine pilings. The U.S. considers PAHs highly toxic and lists 17 as suspected carcinogens.

The biosensor allowed the researchers to quantify PAH concentrations while the Elizabeth River remediation was taking place, gaining on-site knowledge about water quality surrounding the remediation site. Spier says the test was "the first use of an antibody-based biosensor to guide sampling efforts through near real-time evaluation of environmental contamination."

In the Yorktown Creek study, the researchers used the biosensor to track the runoff of PAHs from roadways and soils during a rainstorm.

Biosensor development

Kaattari says "Our basic idea was to fuse two different kinds of technologies—monoclonal antibodies and electronic sensors—in order to detect contaminants."

Antibodies are proteins produced by the immune system of humans and other mammals. They are particularly well suited for detecting contaminants because they have, as Kaattari puts it, an "almost an infinite power to recognize the 3-dimensional shape of any molecule."

Mammals produce antibodies that recognize and bind with large organic molecules such as proteins or with viruses. The VIMS team took this process one step further, linking proteins to PAHs and other contaminants, then exposing mice to these paired compounds in a manner very similar to a regular vaccination.

"Just like you get vaccinated against the flu, we in essence are vaccinating our mice against contaminants," says Kaattari. "The mouse's lymphatic system then produces antibodies to PAHs, TNT, tributyl tin [TBT, the active ingredient in anti-fouling paints for boats], or other compounds."

Once a mouse has produced an antibody to a particular contaminant, the VIMS team applies standard clinical techniques to produce "monoclonal antibodies" in sufficiently large quantities for use in a biosensor.

"This technology allows you to immortalize a lymphocyte that produces only a very specific antibody," says Kaattari. "You grow the lymphocytes in culture and can produce large quantities of antibodies within a couple of weeks. You can preserve the antibody-producing lymphocyte forever, which means you don't have to go to a new animal every time you need to produce new antibodies."

From antibody to electrical signal

The team's next step was to develop a sensor that can recognize when an antibody binds with a contaminant and translate that recognition into an electrical signal. The Sapidyne® sensor used by the VIMS team works via what Kaattari calls a "fluorescence-inhibitory, spectroscopic kind of assay."

In the sensor used on the Elizabeth River and Yorktown Creek, antibodies designed to recognize a specific class of PAHs were joined with a dye that glows when exposed to fluorescent light. The intensity of that light is in turn recorded as a voltage. The sensor also houses tiny plastic beads that are coated with what Spier calls a "PAH surrogate"—a PAH derivative that retains the shape that the antibody recognizes as a PAH molecule.

When water samples with low PAH levels are added to the sensor chamber (which is already flooded with a solution of anti-PAH antibodies), the antibodies have little to bind with and are thus free to attach to the surrogate-coated beads, providing a strong fluorescent glow and electric signal. In water samples with high PAH concentrations, on the other hand, a large fraction of the antibodies bind with the environmental contaminants. That leaves fewer to attach to the surrogate-coated beads, which consequently provides a fainter glow and a weaker electric signal.

During the Elizabeth River study, the biosensor measured PAH concentrations that ranged from 0.3 to 3.2 parts per billion, with higher PAH levels closer to the dredge site. In Yorktown Creek, the biosensor showed that PAH levels in runoff peaked 1 to 2 hours after the rain started, with peak concentration of 4.4 parts per billion.

Comparison of the biosensor's field readings with later readings from a mass spectrometer at VIMS showed that the biosensor is just as accurate as the more expensive, slower, and laboratory-bound machine.

A valuable field tool

Spier says "Using the biosensor allowed us to quickly survey an area of almost 22 acres around the Elizabeth River dredge, and to provide information about the size and intensity of the contaminant plume to engineers monitoring the dredging from shore. If our results had shown elevated concentrations, they could have halted dredging and put remedial actions in place."

Unger adds "measuring data in real-time also allowed us to guide the collection of large-volume water samples right from the boat. We used these samples for later analysis of specific PAH compounds in the lab. This saved time, effort, and money by keeping us from having to analyze samples that might contain PAHs at levels below our detection limit."

"Biosensors have their constraints and optimal operating conditions," says Kaattari, "but their promise far outweighs any limitations. The primary advantages of our biosensor are its sensitivity, speed, and portability. These instruments are sure to have a myriad of uses in future environmental monitoring and management."

One promising use of the biosensor is for early detection and tracking of oil spills. "If biosensors were placed near an oil facility and there was a spill, we would know immediately," says Kaattari. "And because we could see concentrations increasing or decreasing in a certain pattern, we could also monitor the dispersal over real time."

Provided by Virginia Institute of Marine Science