RxPG News : Biotechnology

Carbon nanotubes can affect lung lining

Tue, 03 Nov 2009 23:05:36 PST

( from http://www.rxpgnews.com ) Carbon nanotubes which are used in everything from sports equipment to medical applications can affect the lining of the lungs, say researchers.

The long term effects, however, remain unclear.

The study was a collaboration between North Carolina State University -, The Hamner Institutes for Health Sciences, and the National Institute of Environmental Health Sciences.

Using mice in an animal model study, researchers set out to determine what happens when multi-walled carbon nanotubes are inhaled.

Specifically, researchers wanted to determine whether the nanotubes would be able to reach the pleura, which is the tissue that lines the outside of the lungs and is affected by exposure to certain types of asbestos fibres which cause cancer.

Researchers found that inhaled nanotubes do reach the pleura and cause health effects. Short-term studies described in the paper do not allow conclusions about long-term responses such as cancer.

The 'unique reaction' began within one day of inhalation of the nanotubes, when clusters of immune cells - began collecting on the surface of the pleura.

Localised fibrosis, or scarring on parts of the pleural surface that is also found with asbestos exposure, began two weeks after inhalation.

The study showed the immune response and fibrosis disappeared within three months of exposure. However, this study used only a single exposure to the nanotubes, says an NCSU release.

It remains unclear whether the pleura could recover from chronic, or repeated, exposures.

'More work needs to be done in that area and it is completely unknown at this point whether inhaled carbon nanotubes will prove to be carcinogenic in the lungs or in the pleural lining,' an NCSU release said.

These findings were published in Nature Nanotechnology.

Chicken egg whites - answer to three-dimensional cell culture systems

Tue, 07 Oct 2008 12:22:34 PST

( from http://www.rxpgnews.com ) More and more laboratories are seeking to develop three-dimensional cell culture systems that allow them to test their new techniques and drugs in a system that more closely mimics the way in which cells grow. However, a big sticking point is the cost of commercial media for growing such cultures.

Dr. Steffi Oesterreich, associate professor in the Lester and Sue Smith Breast Center at Baylor College of Medicine, and Dr. Benny A. Kaipparettu, a postdoctoral associate in her laboratory, found a solution – chicken egg whites. Their process has garnered attention in other laboratories around the world. A report on their technique appeared in a recent issue of the journal BioTechniques, which featured their article on its cover.

"It's important because the architecture of the cell is different in two dimensions compared to three," she said. "Understanding how the cell communicates, how protein work requires three dimensions."

For example, breast cells in the mammary gland form ducts through which milk flows when a woman breastfeeds.

"These are the same cells that cause cancer," said Oesterreich. "When you put these cells in the egg white preparation, it forms a structure like a duct. In the two-dimensional form, the cells cannot form a duct."

Only a three-dimensional culture allows cells to signal or send messages to one another as they would in a normal environment. Understanding cell signaling has become an increasingly important part of understanding how cells operate normally and what does wrong when they do not.

The use of a three-dimensional cell culture systems has become so important that the National Cancer Institute has launched a new Tumor Microenvironment Network focusing on studies of the cellular microenvironment – relying heavily on three-dimensional culture systems and encouraging initiatives to improve techniques.

Oesterreich and Kaipparettu in cooperation with others in their laboratory found that chicken eggs whites enabled them to grow both normal and tumors cells in three-dimensions.

"We have known for centuries that a baby chick can grow in three dimensions in an egg shell without any external support," said Kaipparettu. "Now we have found that Mother Nature has provided us a valuable tool for medical research. It gives an 'eggcellent' tool for researchers around the world to perform three-dimensional cellular research."

They are seeking a patent on the process, and hoping to find corporate partners.

Egg whites are a good tool because they are easy and cheap to obtain and they are transparent, allowing the researchers to see the cells under a microscope.

"It seemed a good idea and we thought we would try it," said Kaipparettu.

Nanoparticles hitchhike on red blood cells for drug delivery

Wed, 27 Jun 2007 03:59:37 PST

( from http://www.rxpgnews.com ) Researchers at the University of California, Santa Barbara have discovered that attaching polymeric nanoparticles to the surface of red blood cells dramatically increases the in vivo lifetime of the nanoparticles. The research, published in the July 07 issue of Experimental Biology and Medicine, could offer applications for the delivery of drugs and circulating bioreactors.

Polymeric nanoparticles are excellent carriers for delivering drugs. They protect drugs from degradation until they reach their target and provide sustained release of drugs. Polymeric nanoparticles, however, suffer from one major limitation: they are quickly removed from the blood, sometimes in minutes, rendering them ineffective in delivering drugs.

The research team, led by Samir Mitragotri, a professor of chemical engineering, and Elizabeth Chambers, a recent doctoral graduate, found that nanoparticles can be forced to remain in the circulation when attached to red blood cells. The particles eventually detach from the blood cells due to shear forces and cell-to-cell interactions, and are cleared from the system by the liver and spleen. Red blood cell circulation is not affected by attaching the nanoparticles.

"Attachment of polymeric nanoparticles to red blood cells combines the advantages of the long circulating lifetime of the red blood cell, and their abundance, with the robustness of polymeric nanoparticles," said Mitragotri. "Using red blood cells to extend the circulation time of the particles avoids the need to modify the surface chemistry of the entire particle, which offers the potential to attach chemicals to the exposed surface for targeting applications."

The researchers have learned that particles adhered to red blood cells can escape phagocytosis because red blood cells have a knack for evading macrophages. Nanoparticles aren't the first to be piggybacking on red blood cells; the strategy has already been adopted by certain bacteria, such as hemobartonella, that adhere to RBCs and can remain in circulation for several weeks.

The researchers say that it may be possible to keep the nanoparticles in circulation for a relatively long time, theoretically up to the circulation lifetime of a red blood cell - which is 120 days - if the binding between particles and the red blood cells is strengthened. The methodology is applicable to drugs that are effective while still attached to a red blood cell, although the researchers say that slow release from the red blood cell surface is also feasible.

Mitragotri says "this mode of prolonging particle circulation has significant implications in drug delivery, potentially leading to new treatments for a broad variety of conditions such as cancer, blood clots and heart disease". Dr. Steven R. Goodman, Editor-in-Chief of the journal, said "this study dealing with the attachment of nanoparticles to red blood cells may also have important implications for future treatment of hematologic disorders. This fusion of modern nanobioscience with cell biology and hematology is precisely the type of interdisciplinary study that the new Experimental Biology and Medicine is interested in publishing."

Gold Nanoparticle Molecular Ruler to Measure Smallest of Lifes Phenomena

Thu, 12 Oct 2006 13:22:37 PST

( from http://www.rxpgnews.com ) Scientists from the U.S. Department Energys Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California at Berkeley have developed a ruler made of gold nanoparticles and DNA that can measure the smallest of lifes phenomena, such as precisely where on a DNA strand a protein attaches itself.

The molecular ruler, detailed in the October premier issue of the journal Nature Nanotechnology, offers label-free and real-time measurement of a range of protein-DNA interactions at an extremely high resolution. As such, it promises to play a key role in the current push in biology to understand how genetic information flows from DNA to RNA to gene expression. Today, scientists involved in this research typically examine the final products of this chain of events by cataloging the expression levels of various genes and proteins.

The newly developed molecular ruler, however, can give scientists a much earlier glimpse into this process by measuring the initial protein-DNA binding interactions that unleash the flow of information which, in turn, sparks gene expression.

We can use the ruler to look at this process much more upstream. We can measure the beginning stages of DNA-binding activities, says Fanqing Frank Chen, a scientist in Berkeley Labs Life Sciences Division who was a member of the research team that, for the first time, used the molecular ruler to map protein-DNA interactions.

The existing techniques used to measure protein-DNA interactions involve labeling DNA and proteins with either radioactive or fluorescent compounds. But radioactive labels require tedious sample preparation and incur radiation-use restrictions, and fluorescent labels are short-lived and unable to measure complex protein-DNA interactions that measure more than 8 nanometers in length.

Our work promises to be a fast and convenient alternative for mapping DNA-protein interactions. We can measure precisely how a protein interacts with the information inscribed in the DNA and begins to regulate genetic information, says Chen. We can also measure large protein-DNA interactions that span up to 17 nanometers in length, and, in theory, span as much as 70 nanometers in length.

The molecular ruler was developed by a team of scientists that includes UC Berkeley Bioengineering Professor Luke Lee, UC Berkeley Ph.D. student Gang Liu, and Paul Alivisatos, Director of Berkeley Lab's Materials Sciences Division and an Associate Laboratory Director. Its composed of gold nanoparticles that are coated with a substance that makes the nanoparticles soluble. Next, about 100 double-stranded DNA segments are tethered to the gold nanoparticle in a configuration that resembles a many-legged spider.

The ruler works because of plasmon resonance, which is the collection of electrons that resonate in a metallic particle, in this case the gold-DNA conjugate. Plasmon resonance changes as a particle changes, leading to differences in scattering wavelength. For example, if the gold particles spidery DNA strands, which are 54 base pairs long, are shortened for whatever reason, then the gold-DNA particles scattering wavelengths also shift and this shift can be easily detected using spectroscopy. This method is so sensitive that scientists can use it to detect whether a DNA strand has been shortened by as little as one base pair in length, which opens the door for mapping the exact location of protein-DNA interactions.

These before-and-after images reveal how the gold nanoparticles change after DNA strands are added the nanoparticles. Chen and colleagues use these shifts in plasmon resonance to measure how proteins bind to DNA (Image: Berkeley Lab).

Chen and colleagues put the ruler to the test by using it to conduct DNA footprinting, a process in which scientists identify where on a DNA strand a particular protein attaches itself. DNA footprinting is most commonly performed on proteins that are thought to play a significant functional role, such as in regulating gene expression.

To conduct this genetic sleuthing, they developed a customized gold-DNA conjugate. As usual, they attached to each gold nanoparticle roughly 100 DNA strands that are 54 base pairs long. But among these base pairs they inserted a sequence of six base pairs that are specially tailored to bind to a model protein, in this case EcoRI(Q111). In other words, at the same location on each strand, they encoded the perfect home for an EcoRI(Q111) protein. They introduced this protein to the specially prepared gold-DNA conjugates, and allowed the protein to bind to the DNA strands.

Next, to map exactly where the protein attaches to the DNA, they introduced an enzyme called an exonuclease. This enzyme clamps onto the free end of the DNA strands, and chomps down each strand, removing base pair after base pair, until its blocked by the recently attached EcoRI(Q111) protein. Its like someone slurping down a spaghetti noodle, only to be stopped cold by a fly sitting on the noodle.

In this way, the gold particless DNA strands are shortened, with their newly sheared free ends marking the location of the protein. And this, in turn, allows the research team to zero in on the DNAs protein binding site. They already know the plasmonic scattering signature of the gold-DNA particle with all of its 54 base pairs. Now, they can then measure the plasmonic signature of the gold-DNA particle after its DNA has been trimmed. The difference between the two spectra correlates to the number of base pairs eliminated by the exonuclease.

The plasmon resonance wavelengths decrease by a certain number of nanometers, which translates to a certain number of DNA base pairs removed, says Chen.

By attaching DNA strands to gold nanoparticles, Berkeley Lab and UC Berkeley scientists have developed a ruler capable of measuring protein-DNA interactions (Image: Berkeley Lab).

This allows Chen and colleagues to measure how far the exonuclease travels down the DNA strand, which enables them to determine precisely where the protein binding site is located. The result is a quick and relatively cheap glimpse into the earliest stages of genetic activity.

We are monitoring the actual mechanism that causes genetic information to begin to flow, such as gene regulation, not the expression levels of genes and proteins, which are endpoint measurements adds Chen.

In addition to DNA footprinting, the molecular ruler can be used to monitor any enzyme that causes length changes in DNA, such as nucleases that cleave DNA strands in two. And the molecular rulers ability to measure changes in a single nanoparticle without the need for radioactive or fluorescent labeling makes it possible to perform high-throughput screening in a high-density microarray or a microfluidic device.

Tiny inhaled particles take easy route from nose to brain

Thu, 03 Aug 2006 17:28:37 PST

( from http://www.rxpgnews.com ) In a continuing effort to find out if the tiniest airborne particles pose a health risk, University of Rochester Medical Center scientists showed that when rats breathe in nano-sized materials they follow a rapid and efficient pathway from the nasal cavity to several regions of the brain, according to a study in the August issue of Environmental Health Perspectives.

Researchers also saw changes in gene expression that could signal inflammation and a cellular stress response, but they do not know yet if a buildup of ultrafine particles causes brain damage, said lead author Alison Elder, Ph.D., research assistant professor of Environmental Medicine.

The study tested manganese oxide ultrafine particles at a concentration typically inhaled by factory welders. The manganese oxide particles were the same size as manufactured nanoparticles, which are controversial and being diligently investigated because they are the key ingredient in a growing industry -- despite concerns about their safety.

Nanotechnology is a new wave of science that deals with particles engineered from many materials such as carbon, zinc and gold, which are less than 100 nanometers in diameter. The manipulation of these materials into bundles or rods helps in the manufacturing of smaller-than-ever electronics, optical and medical equipment. The sub-microscopic particles are also used in consumer products such as toothpaste, lotions and some sunscreens.

Some doctors and scientists are concerned about what happens at the cellular level after exposure to the ultrafine or nano-sized particles, and the University of Rochester is at the forefront of this type of environmental health research. In 2004 the Defense Department selected the University Medical Center to lead a five-year, $5.5 million investigation into whether the chemical characteristics of nanoparticles determine how they will interact with or cause harm to animal and human cells.

In the current study, the particles passed quickly through the rats' nostrils to the olfactory bulb, a region of the brain near the nasal cavity. They settled in the striatum, frontal cortex, cerebellum, and lungs.

After 12 days, the concentration of ultrafine particles in the olfactory bulb rose 3.5-fold and doubled in the lungs, the study found. Although the ultra-tiny particles did not cause obvious lung inflammation, several biomarkers of inflammation and stress response, such as tumor necrosis factor and macrophage inflammatory protein, increased significantly in the brain, according to gene and protein analyses.

"We suggest that despite differences between human and rodent olfactory systems, this pathway is likely to be operative in humans," the authors conclude.

DNA Amplification and Detection Made Simple

Wed, 12 Jul 2006 05:21:37 PST

( from http://www.rxpgnews.com ) Twenty-three years ago, a man musing about work while driving down a California highway revolutionized molecular biology when he envisioned a technique to make large numbers of copies of a piece of DNA rapidly and accurately. Known as the polymerase chain reaction, or PCR, Kary Mullis's technique involves separating the double strands of a DNA fragment into single-strand templates by heating it, attaching primers that initiate the copying process, using DNA polymerase to make a copy of each strand from free nucleotides floating around in the reaction mixture, detaching the primers, then repeating the cycle using the new and old strands as templates. Since its discovery in 1983, PCR has made possible a number of procedures we now take for granted, such as DNA fingerprinting of crime scenes, paternity testing, and DNA-based diagnosis of hereditary and infectious diseases.

As valuable as conventional PCR is, it has limits. Heat is required to separate the DNA and cooler temperatures are needed to bind the primer to the strands, so the reaction chamber must repeatedly cycle through hot and cold phases. As a result, the technique can only be performed in laboratories using sophisticated equipment.

Now Olaf Piepenburg, Niall Armes, and colleagues have come up with a new approach to DNA amplification that can be carried out at a constant temperature, using only a tiny amount of DNA, without elaborate equipment. Called recombinase polymerase amplification (RPA), the technique opens the door to dramatically extending the application of DNA amplification in fieldwork and in laboratories where PCR machines are not available.

RPA uses five main ingredients: a sample of the DNA to be amplified; a primerrecombinase complex, which initiates the copying process when it attaches to the template; nucleotides from which to form the new strands; a polymerase, which brings them together in the right order; and single-stranded DNA-binding proteins (SSBs), which help keep the original DNA from zipping back together while the new DNA is being made. The primerrecombinase complex is able to attach to the double-stranded DNA, eliminating the need to heat the mixture. After the complex is in place, it disassembles, allowing the DNA polymerase to begin synthesizing a new strand of DNA complementary to the template, while the SSBs attach to and stabilize the displaced strand. Under the right conditionsa precise milieu of process-regulating chemicalsthe process automatically repeats, resulting in an exponential increase in the DNA sample.

The researchers tested the sensitivity, specificity, and speed of RPA by using it to amplify three kinds of human DNA, as well as DNA from Bacillus subtilis. They found it to be rapid and accurate. However, they also noted that using RPA to detect the presence of a specific type of DNAfor example, that of a specific pathogenwas complicated by the fact that, at low or zero concentration, the primer also produced an artifact effect (akin to some similar PCR artifacts). To counteract this, the researchers developed a probe-based detection method that causes the sample to glow in the presence of the DNA being tested for, but not in the presence of primer alone.

To demonstrate the usefulness of the new system, the researchers used it to test for the presence of methicillin-resistant Staphylococcus aureus (MRSA), a disease-causing bacterium known as a superbug because it is unharmed by penicillin antibiotics. They found that RPA could detect fewer than ten copies of MRSA DNA. It could also determine the presence of three different genotypes of MRSA, and distinguish them from a methicillin-sensitive S. aureus strain.

How easy would it be to apply such a test in real-life situations? The researchers demonstrated one possible approach by encapsulating the entire process in a dipstick that could be used in the field to detect the presence of a pathogen.

As great as the potential of RPA is for making DNA amplification and detection easier and more broadly applicable, that's not its only value. The researchers noted that the reaction environment they developed also provides a framework for improving understanding of recombination and the application of DNA hybridization.

Solitons Could Power Artificial Muscles

Fri, 07 Jul 2006 18:14:37 PST

( from http://www.rxpgnews.com ) Scientists have discovered something new about exotic particles called solitons.

Since the 1980s, scientists have known that solitons can carry an electrical charge when traveling through certain organic polymers. A new study now suggests that solitons have intricate internal structures.

Scientists may one day use this information to put the particles to work in molecular electronics and artificial muscles, said Ju Li, assistant professor of materials science and engineering at Ohio State University.

Li explained that each soliton is made up of an electron surrounded by other particles called phonons. Just as a photon is a particle of light energy, a phonon is a particle of vibrational energy.

The new study suggests that the electron inside a soliton can attain different energy states, just like the electron in a hydrogen atom.

"While we know that such internal electronic structures exist in all atoms, this is the first time anyone has shown that such structures exist in a soliton," Li said.

The soliton's quantum mechanical properties -- including these newly discovered energy states -- are important because they affect how the particle carries a charge through organic materials such as conducting polymers at the molecular level.

"These extra electronic states will have an effect -- we just don't know right now if it will be for better or worse," he said.

Li and his longtime collaborators from MIT published their findings in a recent issue of the Proceedings of the National Academy of Sciences (PNAS).

The name "soliton" is short for "solitary wave." Though scientists often treat particles such as electrons as waves, soliton waves are different. Ordinary electron waves spread out and diminish over time, and soliton waves don't.

"It's like when you make a ripple in water -- it quickly spreads and disappears," Li said. "But a soliton is a strange kind of object. Once it is made, it maintains its character for a long time."

In fiber optics, normal light waves gradually flatten out; unless the signal is boosted periodically, it disappears. In contrast, solitonic light waves retain their structure and keep going without assistance. Some telecommunication companies have exploited that fact by using solitons to cheaply send signals over long distances.

Before solitons can be fully exploited in a wider range of applications, scientists must learn more about their basic properties, Li said. He's especially interested in how solitons carry a charge through conducting polymers, which consist of long, skinny chains of molecules.

The tiny chains are practically one-dimensional, and this calls some strange physics into play, Li said.

In their PNAS paper, Li and MIT colleagues Xi Lin, Clemens Först, and Sidney Yip describe a detailed calculation of what happens to solitons at a quantum-mechanical level as they travel along a chain of the organic polymer polyacetylene.

Their mathematical model builds upon a 1979 model called the Su-Schrieffer-Heeger (SSH) model. Alan Heeger, a University of California, Santa Barbara physicist who discovered solitons, won the Nobel Prize in 2000 for his pioneering work on conducting polymers.

Li said the new work extends the SSH model by including the full flexibility of the polymer chain, as well as interactions between electrons.

The finding will likely affect the development of molecular electronics -- devices built from individual molecules.

Because polymer chains tend to bend and twist as solitons pass through them, scientists have wondered whether solitons could be used to power artificial muscles for high-tech robots and devices to aid human mobility. Such muscles would be made of organic polymers, and flex in response to light or electrochemical stimulation.

"If fully understood, solitons may also be harnessed to drive molecular motors in nanotechnology," Li said.

Nanoparticles could deliver multi-drug therapy to tumors

Thu, 22 Jun 2006 17:07:37 PST

( from http://www.rxpgnews.com ) In the ongoing search for better ways to target anticancer drugs to kill tumors without making people sick, researchers find that nanoparticles called buckyballs might be used to significantly boost the payload of drugs carried by tumor-targeting antibodies.

In research due to appear in an upcoming issue of the journal Chemical Communications, scientists at Rice University and The University of Texas M. D. Anderson Cancer Center describe a method for creating a new class of anti-cancer compounds that contain both tumor-targeting antibodies and nanoparticles called buckyballs. Buckyballs are soccer ball-shaped molecules of pure carbon that can each be loaded with several molecules of anticancer drugs like Taxol®.

In the new research, the scientists found they could load as many as 40 buckyballs into a single skin-cancer antibody called ZME-018. Antibodies are large proteins created by the immune system to target and attack diseased or invading cells.

Previous work at M. D. Anderson has shown that ZME-018 can be used to deliver drugs directly into melanoma tumors, and work at Rice has shown that Taxol can be chemically attached to a buckyball.

"The idea that we can potentially carry more than one Taxol per buckyball is exciting, but the real advantage of fullerene immunotherapy over other targeted therapeutic agents is likely to be the buckyball's potential to carry multiple drug payloads, such as Taxol plus other chemotherapeutic drugs," said Rice's Lon Wilson, professor of chemistry. "Cancer cells can become drug resistant, and we hope to cut down on the possibility of their escaping treatment by attacking them with more than one kind of drug at a time."

Researchers have long dreamed of using antibodies like ZME-018 to better target chemotherapy drugs like Taxol, and M. D. Anderson's Michael G. Rosenblum, Ph.D., professor in the Department of Experimental Therapeutics and Chief of the Immunopharmacology and Targeted Therapy Laboratory, has conducted some of the pioneering work in this field.

"This is an exciting opportunity to apply novel materials such as fullerenes to generate targeted therapeutics with unique properties," Rosenblum said. "If successful, this could usher in a new class of agents for therapy not only for cancer, but for other diseases as well."

While it's possible to attach drug molecules directly to antibodies, Wilson said scientists haven't been able to attach more than a handful of drug molecules to an antibody without significantly changing its targeting ability. That happens, in large part, because the chemical bonds that are used to attach the drugs -- strong, covalent bonds -- tend to block the targeting centers on the antibody's surface. If an antibody is modified with too many covalent bonds, the chemical changes will destroy its ability to recognize the cancer it was intended to attack.

Wilson said the team from Rice and M. D. Anderson had planned to overcome this limitation by attaching multiple molecules of Taxol to each buckyball, which would then be covalently connected to the antibodies. To the team's surprise, many more buckyballs than expected attached themselves to the antibody. Moreover, no covalent bonds were required, so the increased payload did not significantly change the targeting ability of the antibody.

Wilson said certain binding sites on the antibody are hydrophobic (water repelling), and the team believes that these hydrophobic sites attract the hydrophobic buckyballs in large numbers so multiple drugs can be loaded into a single antibody in a spontaneous manner to give the antibody-drug agent more "bang for the buck."

"The use of these nanomaterials solves some intractable problems in targeted therapy and additionally demonstrates the increasing value of the team science approach bridging different disciplines to uniquely address existing problems," Rosenblum said.

Nanotechnology can identify disease at early cellular level

Tue, 25 Apr 2006 21:12:37 PST

( from http://www.rxpgnews.com ) Nanotechnology may one day help physicians detect the very earliest stages of serious diseases like cancer, a new study suggests.

It would do so by improving the quality of images produced by one of the most common diagnostic tools used in doctors' offices the ultrasound machine.

In laboratory experiments on mice, scientists found that nano-sized particles injected into the animals improved the resulting images. This study is one of the first reports showing that ultrasound can detect these tiny particles when they are inside the body, said Thomas Rosol, a study co-author and dean of the college of veterinary medicine at Ohio State University.

Given their tiny size, nobody thought it would be possible for ultrasound to detect nanoparticles, he said.

It turns out that not only can ultrasound waves sense nanoparticles, but the particles can brighten the resulting image. One day, those bright spots may indicate that a few cells in the area may be on the verge of mutating and growing out of control.

The long-term goal is to use this technology to improve our ability to identify very early cancers and other diseases, said Jun Liu, a study co-author and an assistant professor of biomedical engineering at Ohio State University. We ultimately want to identify disease at its cellular level, at its very earliest stage.

The researchers injected a solution of silica nanoparticles into the tail vein of each mouse. They then anesthetized the animals and placed them on their backs on a warm imaging table.

Rosol said that Liu and her team are working on creating biodegradable nanoparticles. For the purposes of this study, however, the researchers wanted to use a hard substance, silica, to see if their idea would work. The strongest ultrasound signals are those produced by sound waves bounce off a hard surface. While not biodegradable, the nanoparticles used in the study were biologically inert.

The researchers took ultrasound images of the animals' livers every five minutes for 90 minutes after the injection. The nanoparticles had accumulated in the animals' livers. Another future step for this work is to label nanoparticles with a molecular road map of sorts, which would direct the particles to go to specific locations in the body.

The liver takes up foreign substances in the body, so it's not surprising that that's where we saw the particles, Rosol said. But we ultimately want to be able to make these particles to go to the mammary glands or other tissues we're interested in.

The ultrasound images grew brighter over the 90-minute period. The researchers compared these images to those from a group of control mice injected with a saline solution. There was no change in ultrasound image brightness in the control mice after that injection.

While this research is still in its infancy, Rosol and his colleagues foresee a day when nanotechnology can alert a physician to the beginnings of cancer or heart disease, perhaps in a woman who has a family history of breast cancer:

Her doctor could inject the breast with nanoparticles and the resulting ultrasound image would alert the doctor to any suspicious areas in the tissue, even at the cellular level, Rosol said.

The hope is that combining ultrasound and nanotechnology may provide a definitive diagnosis in lieu of an invasive procedure like a biopsy.

These nanoparticles may make it possible for physicians to screen for tumors very quickly, and perhaps lessen the need for a biopsy in many cases, Liu said.

Nanoparticles are smaller than any cell in the human body, so they may pass through the walls of the leaky blood vessels, or capillaries, of tumor tissue and actually infiltrate the tumor.

Until now, nobody knew what these particles would do in the blood, Rosol said. But they made it into the liver.

And despite their miniscule size, nanoparticles are still big enough to carry a payload of medicine, Rosol said.

That the particles made it into the liver suggests that they could be used to deliver toxic chemotherapeutic drugs that would act locally on a tissue, at the site of a tumor, and not have such a pronounced affect on the rest of the body, Rosol said. The problem with chemotherapy is that the drug affects the whole body, causing a host of problems such as hair loss, diarrhea and anemia.

Light-sensitive particles change chemistry at the flick of a switch

Mon, 27 Mar 2006 16:36:37 PST

( from http://www.rxpgnews.com ) A light-sensitive, self-assembled monolayer that provides unique control over particle interactions has been developed by scientists at the University of Illinois at Urbana-Champaign. Particles coated with the monolayer change their surface charge and chemistry upon exposure to ultraviolet light.

"Tailoring interactions between particles allows us to design colloidal fluids, gels and crystals for use as ceramic, photonic and pharmaceutical materials," said Jeffrey Moore, a William H. and Janet Lycan Professor of Chemistry and a researcher at the Frederick Seitz Materials Research Laboratory and at the Beckman Institute for Advanced Science and Technology. "We are assembling a toolkit of molecules that can be incorporated as monolayers on particles to achieve desired effects."

Light-induced modification of colloidal interactions provides an 'extra handle' for tailoring system behavior, said Jennifer Lewis, the Thurnauer Professor of Materials Science and Engineering and interim director of the Frederick Seitz Materials Research Laboratory.

"The monolayer is designed so that light triggers the cleavage of a specific chemical bond, thereby exposing an underlying functional group of interest," said Lewis, who also is a professor of chemical and biomolecular engineering and a researcher at the Beckman Institute.

Moore and Lewis first demonstrated the technique in a paper published in the Sept. 30, 2005, issue of the Journal of the American Chemical Society. In that work, the surface charge and, thus, the electrostatic interactions between photosensitive silica microspheres, were modified by exposure to ultraviolet light.

In recent work, the researchers documented the gel-to-fluid transition in binary mixtures that initially were oppositely charged. "Exposure to ultraviolet light rendered all of the particles negative and converted the system into a colloidal fluid that settled to form a dense sediment," said Moore, who will present the team's findings at the national meeting of the American Chemical Society, to be held in Atlanta, March 26-30.

"These light-responsive systems will enable novel assembly routes for creating colloidal structures in a variety of materials," Lewis said. "We are currently investigating the ability to locally photo-pattern such assemblies in three dimensions without requiring multiple processing steps."

Light-sensitive colloidal particles could also be used to "tune" the elastic properties, viscous response and microstructure of gel-based inks used in the direct-write assembly of complex, three-dimensional structures formed by robotic deposition.

The Moore group is developing multiple wavelength-specific triggers that would allow different wavelengths of light to induce changes sequentially.

DNA Fragments for Making Tomatoes Taste Better Identified

Mon, 27 Mar 2006 04:18:37 PST

( from http://www.rxpgnews.com ) Tomatoes are a major nutrient for humans. In 2004, 120,000 tonnes of tomatoes were harvested worldwide - and every year this number increases. Numerous medical studies have shown the health value of tomatoes. Lycopen, the pigment that makes tomatoes red, can for example prevent heart disease. Tomatoes furthermore contain a lot of vitamins C and E, indispensable for human nourishment. But after centuries of cultivation for shape, colour, and other useful qualities, our cultured tomatoes today are of small genetic diversity, in comparison with wild types. This has affected the taste and health value of the fruits.

To cultivate tomato strains with particular characteristics, researchers have to increase the genetic diversity of cultured tomatoes. This can be done either by cross-breeding them with wild tomatoes, or changing their genetic make-up technologically. Scientists from the Max Planck Institute for Molecular Plant Physiology in Golm, and their Israeli colleagues at Hebrew University in Jerusalem, chose the second option. They investigated strains of tomatoes created from the crossing of cultured and wild types. Their goal was to identify the biochemical composition of fruits and determine which factors control their development. The German-Israeli research team used a method of analysis developed at the Max Planck Institute for Molecular Plant Physiology. The technique - a combination of mass spectrometry and gas chromatography - analyzes the composition of biological samples. It can be used to quickly and simultaneously look into a fruits amino acids, organic acids, sugar and vitamins.

Dr. Alisdair Fernie, head of the Institutes "Central Metabolism" research group, discovered that there were 880 variations in the content composition of descendants produced by crossing cultured tomatoes and wild tomatoes. "On one hand, we measured higher amounts of essential amino acids and vitamins, on the other hand the fruits showed an altered combination of various sugars and organic acids," Fernie says. These contents have a great influence on the taste of tomatoes.

A) tomato plants and B) tomato fruits of the Solarum Lycopersicum complex, which are easily cross-bred with each other. Various wild tomatoes - (I) S. chmielewskii, (II) S. habrochaites, (IV) S. pimpinellifolium, (V) S. neorickii, (VI) S. pennellii - are all excellent for hybridisation with the cultured tomato (III) S. lycoperisicum. Image Courtesy: Max Planck Institute of Molecular Plant Physiology

The scientists used molecular biological methods to identify parts of the tomato genomes responsible for biochemical changes. The researchers findings could make it possible in the future to cross-breed wild tomatoes with cultured tomatoes in a targeted way to make them more nutritious.

'Custom' nanoparticles could improve cancer diagnosis and treatment

Mon, 27 Mar 2006 01:34:37 PST

( from http://www.rxpgnews.com ) Researchers have developed "custom" nanoparticles that show promise of providing a more targeted and effective delivery of anticancer drugs than conventional medications or any of the earlier attempts to fight cancer with nanoparticles. Designed at the molecular level to attack specific types of cancer without affecting healthy cells, the nanoparticles also have the potential to reduce side effects associated with chemotherapy, the researchers say. Their study was described today at the 231st national meeting of the American Chemical Society, the worlds largest scientific society.

The particles, considered the next generation of cancer therapeutics, are the most uniform, shape-specific drug delivery particles developed to date, according to researchers at the University of North Carolina (UNC) in Chapel Hill. Other potential benefits of the tiny uniform particles include enhanced imaging of cancer cells for improved diagnosis and use as delivery vehicles for gene therapy agents, they say.

To date, the UNC researchers have produced a variety of custom nanoparticles from biocompatible organic materials using techniques they adapted from processes used by the electronics industry to make transistors. In cell studies, they have shown that the uniform nanoparticles can attach to specific cell targets, release important chemotherapy drugs inside cells, and hold MRI contrast agents. Animal studies began recently and human studies are anticipated, the researchers say.

"I think this will transform the way one detects and treats disease," says study leader Joseph DeSimone, Ph.D., a chemistry professor at UNC and director of the schools Institute for Advanced Materials, Nanoscience and Technology. He has co-founded a company, Liquidia Technologies, to develop and produce the nanoparticles.

Researchers have been experimenting with nanoparticles as drug delivery vehicles for years but have had only limited success in cell and animal studies, DeSimone says. Each carrier has drawbacks with regard to stability in the bloodstream or ability to be directed toward a specific cancer site. In addition, there has been no general method available that allows precise control of the particles size, shape and composition, which are considered key features for the success of targeted drug delivery, he says.

Now, DeSimone and his associates at UNC have developed a new fabrication technique that allows, for the first time, unprecedented control over the structure and function of drug delivery nanoparticles. Called PRINT (Particle Replication In Non-wetting Templates), the technique is similar to injection molding and uses principles borrowed from the electronics industry for transistor fabrication, they say. The technique was first detailed last June in the online version of the Journal of the American Chemical Society.

The manufacturing process starts with a silicon wafer that is etched with the shape and size of the desired nanoparticle, resulting in a template. Next, nonstick liquid fluoropolymers are poured into the template and cured to form a fixed mold. The finished mold is then injected with organic materials that can contain imaging agents, anticancer drugs, DNA (for gene therapy) and other materials, depending on the intended function, DeSimone says. The new manufacturing technique uses gentler processing methods that are less likely to harm important organic components than traditional nanoparticle manufacturing techniques, he adds.

The resulting nanoparticles can be as small as 20 nanometers, or thousands of times smaller than the width of a single human hair. The shapes of the particles can also be made to mimic the shapes of objects found in nature like red blood cells or virus particles, DeSimone says.

Human albumin from tobacco plants

Sat, 25 Mar 2006 15:34:37 PST

( from http://www.rxpgnews.com ) Human serum albumin (HSA) is the intravenous protein most commonly used in the world for therapeutic ends. It is employed to stabilise blood volume and to avoid risk of a heart attack, its administration in operating theatres being almost a daily occurrence. It is used for haemorrhages, burns, surgical operations or when the patient shows symptoms of malnutrition or dehydration, chronic infections and renal or liver illnesses. The annual consumption in Spain is about 10 tons but, at a worldwide level, the demand exceeds 500 tons.

Agricultural engineer, Alicia Fernández San Millán, has developed a novel technique in Spain - plastidial transformation, in order to produce, in a recombinant form, human albumin from tobacco plants. According to her PhD thesis, plastidial transformation is an economically viable alternative, as it enables increasing the levels of HSA by between 10 and a 100 times, compared to levels obtained by nuclear transformation.

The title of the PhD is: Production of human serum albumin in tobacco plants by means of plastidial transformation. It should be added that this novel technique, fruit of Ms Fernández San Milláns PhD, has been patented at a world level and there is already a company interested in marketing it.

Commercial albumin is currently extracted from blood, but the lack of sufficient reserves to cover all worldwide needs has instigated researchers to look for new formulae to multiply this protein. One of the methods most used has been the obtention of HSA from yeasts and mammal cells. However, their high market-place costs have meant that these methods are not competitive. While the price at the pharmacy of albumin produced using plasma is 4 euros per gram, that obtained from yeasts or mammal cells costs between 300 and 4,000 euros per gram. Another option worked on over recent years has been the production of albumin from vegetables, always using nuclear transformation.

The novelty in this research arises from the method of obtention of the HSA. The plastidial system enables the extraction of great quantities of albumin. With nuclear transformation, the maximum level obtained is 0.5% of the total soluble protein of the plant, while application of the plastidial system multiplies this percentage by fourteen (to 7%), reaching an average of 0.9 milligrams of HSA per gram of fresh leaf weight.

The key is the place where the gene in question is deposited. With the nuclear transformation method, it integrates into the DNA of the cell nucleus of the leaf and, thus, can only manage a small number of copies of the gene. With the plastidial system, on the other hand, the gene is introduced into the chloroplast, where photosynthesis takes place and where the genomes can multiply up to 10,000 times.

A property highly valued by the experts has to be added to these positive results: the production of albumin from plants using this technique does not involve the escape of genes through pollen transmission given that, with most crops under cultivation, the genome of the plastids is inherited maternally.

The tobacco plant is very easy to handle genetically and also it is great generator of biomass. The authoress of the thesis says that up to 100 tons of biomass per hectare can be obtained in optimum growth conditions. Given that the protein is produced in the chloroplasts, the more the leaf biomass we have, the more albumin we can get.

To date all the trials undertaken with tobacco plants have been with laboratory varieties. The aim is to do tests with commercial varieties. Laboratory plants are very small and, as a result, the quantity of albumin extracted is not sufficient. However, the commercial varieties of tobacco are some 30 times more productive in terms of biomass.

Despite the advantages demonstrated by the experts, there is still a long way to go. Involving, as it does, a protein that is intravenously injected into patients, it has to be thoroughly purified to eliminate any kind of contaminant. Moreover, it is necessary to assure that the protein obtained has an identical structure to the human one to guarantee that its functioning will be 100%.

A new metal detector to study human disease

Wed, 22 Mar 2006 08:06:37 PST

( from http://www.rxpgnews.com ) Zinc may be a familiar dietary supplement to millions of health-conscious people, but it remains a mystery metal to scientists who study zincs role in Alzheimers disease, stroke and other health problems.

They are just beginning to fathom how the body keeps levels of zinc under the precise control that spells the difference between health and disease.

Researchers now have developed a biochemical metal detector to help crack the mystery. It is a biosensor that has yielded the first measurements of the tiny amounts of zinc ordinarily present inside living cells.

It was conducted by Rebecca A. Bozym and Richard B. Thompson, Ph.D. of the department of biochemistry and molecular biology, University of Maryland School of Medicine, Baltimore, and Andrea K. Stoddard and Carol A. Fierke, Ph.D. of the Department of Chemistry, University of Michigan, Ann Arbor.

The question of how much zinc is available in a cell has emerged at the forefront of chemical biology, Amy R. Barrios, Ph.D., of the University of Southern California, Los Angeles, wrote in an accompanying Point of View in ACS Chemical Biology.

Barrios described the new research as a critical step forward, and predicted many more exciting breakthroughs in measuring levels of metals in human cells.

Just 2-3 grams of zinc (the weight of a penny coin) exist in the entire human body. The metal is a key building block in enzymes and other substances involved in functioning of the nervous system, the immune response, and the reproductive system.

We believe this new technique can help us understand how zinc is involved in plaque formation in Alzheimers disease, how prolonged seizures or stroke kill brain cells, and how the cell normally allocates zinc to different proteins, said Thompson.

Thompson explained that almost all zinc inside cells is incorporated into proteins, where it plays many vital roles, such as helping to read the genetic code of DNA.

We know that if there is much zinc in the cell that is not attached to protein or otherwise encapsulated so-called free zinc the cell is stressed or may be undergoing programmed cell death. This has been observed in animal models of epilepsy and stroke.

In the past, scientists could only measure the relatively high levels of zinc in sick cells. The new sensing technology can measure very low free zinc concentrations in healthy cells.

The technique uses a special protein molecule that has been re-engineered to report when zinc becomes stuck to it as a change in luminescence that can be seen in the microscope. This protein (originally found in blood cells) is very selective, recognizing tiny levels of free zinc even in the presence of the million-fold higher levels of other metals present in cells, such as calcium or magnesium.

Because proper zinc levels are so important in health and disease, scientists have been seeking ways of measuring zinc inside and outside of cells for more than a decade.

This is an important discovery, said Sarah B. Tegen, Ph.D., managing editor of ACS Chemical Biology. We need to know how the body controls levels of zinc inside cells. Too much zinc can kill nerve cells. With too little, nerve cells will not work properly.

Now we have a metal detector, technology that can measure tiny amounts of zinc in living cells. Understanding how zinc is stored and released in different cells throughout the body may help us understand some of the nerve damage that occurs during a stroke and other nerve injuries.

Crucial breakthrough in pectin biosynthesis

Wed, 22 Mar 2006 07:54:37 PST

( from http://www.rxpgnews.com ) Most people know pectin as a common household gelling agent in making jams and jellies, but its uses are vast. It has anticancer properties, for instance, and may have a role in important biological functions including plant growth and development and defense against disease.

Despite the importance of pectin as a major component in the primary walls of plants, scientists have known relatively little about how this family of complex polysaccharides is made. Especially perplexing has been how the synthesis of the three different classes of pectic polysaccharides is coordinated to produce the pectin matrix in cell walls.

Now, in what has been described as a crucial breakthrough in pectin biosynthesis, a team of researchers at the University of Georgia has discovered a gene that encodes one of the proteins responsible for pectin synthesis. This is a powerful new molecular tool that could help researchers understandand potentially manipulatepectins.

The result, which arose from the use of biochemistry and bioinformatics to discover gene sequences, could be genetically altered pectins that might dramatically improve plant species ability to fight disease and new pectins that could be specifically targeted to fight cancers in humans. (It has also been demonstrated that pectin lowers serum cholesterol levels.) While not the breakthrough that will allow immediate manipulation of total pectin biosynthesis, it is, one of the researchers involved said, the first word of the Rosetta Stone that will show us the blueprint for pectin biosynthesis.

The project, led by Debra Mohnen of the UGA department of biochemistry and molecular biology and the Complex Carbohydrate Research Center, was just published in the Proceedings of the National Academy of Sciences (PNAS). Other authors of the paper include Jason Sterling, Melani Atmodjo, Sarah Inwood, V.S. Kumar Kolli, Heather Quigley and Michael Hahn.

Numerous studies show that pectins contribute to the physical and biochemical properties of the plant cell wall, said Mohnen. We know they are required for normal plant growth and development, but to really understand pectin function, we need to identify and be able to manipulate the biosynthetic enzymes and corresponding genes.

The breakthrough came in the identification, for the first time, of a gene sequence encoding a pectin biosynthetic enzyme, which the team named galacturonosyltransferase-1 or GAUT1. The researchers discovered the gene and the protein it encodes while searching the genetic map database for a common laboratory plant in the mustard family, Arabidopsis thaliana.

The identification of GAUT1 as a galacturonosyltransferase that synthesizes pectin, a family of complex polysaccharides present in the cell walls of all land plants, means that the ability to manipulate pectin synthesis and thereby improve pectins plant-helping or cancer-fighting properties is now on the horizon.

We knew that the enzymes we were looking for were membrane-bound, so we took advantage of our understanding of the enzymes biochemistry to identify the genes, said Mohnen.

While this first step may well be a crucial one in elucidating pectin biosynthetic genes, Hahn said the research is still at the bottom rung of the ladder, but the team has for the first time genetic tools that should help to identify multiple genes encoding enzymes involved in pectin biosynthesis.

Pectic polymers appear to have multiple roles in growth, development and disease resistance, and the new tools will open new areas of inquiry for researchers.

We could, for instance, modify a pectic structure to get a specific biological effect, said Mohnen. The ability to modify pectin synthesis could have huge ramifications.

In an accompanying commentary on the research, to be published later in PNAS, Antony Bacic of the Australian Centre for Plant Functional Genomics at the University of Melbourne said the description of the biosynthetic processes involved in the synthesis of the non-cellulosic and pectic polysaccharides of the cell wall has, until the 21st century, been slow to unfold.

He points out the large number of pectin uses, from food production to cancer prevention and treatment, and notes that more information on the process of manipulating the quality and quantity of wall polysaccharides is crucial and badly needed.

The work [described in the UGA paper] represents a significant advance, as it is the first functional identification of an Arabidopsis pectin homogalacturonan galacturonosyltransferase [GAUT1] using biochemical and functional genomic approaches.

While questions remain regarding pectin biosynthesis, the identification of GAUT1 and the GAUT1-related gene family provides the molecular tools to begin to break through the bottleneck of our understanding of pectin synthesis, Bacic said.

Enzyme computer could live in human body

Sat, 25 Feb 2006 09:59:37 PST

( from http://www.rxpgnews.com ) Israeli researchers have invented a molecular computer that uses enzymes to perform calculations and could eventually be implanted into the human body and monitor the release of drugs.

Itamar Willner, who built the computer with colleagues at the Hebrew University of Jerusalem in Israel, said the enzyme-powered computers could have significant pharmaceutical and biomedical applications and could, in the future, be implanted into humans.

The researchers used two enzymes, glucose dehydrogenase (GDH) and horseradish peroxidase (HRP), to trigger two interconnected chemical reactions, the online edition of New Scientist reported.

According to the report, enzymes are already widely used to assist calculations.

Molecular computers have the potential to surpass the speed and power of existing silicon computers because they can perform many calculations simultaneously and pack a vast number of components into a tiny space.

Willner said his computer would eventually be incorporated into bio-sensing equipment to help with intelligent drug delivery according to the needs of the patient.

"This is basically a computer that could be integrated with the human body," Willner said. "We feel you could implant an enzyme computer into the body and use it to calculate an entire metabolic pathway."

Using biologically compatible materials to fabricate a nanoshuttle

Tue, 24 Jan 2006 15:50:37 PST

( from http://www.rxpgnews.com ) Researchers at The University of Texas M. D. Anderson Cancer Center report that they have created a way for viral and gold particles to "directly assemble" and potentially seek out and treat disease where it resides in the body.

Their study, published in the online early edition of The Proceedings of the National Academy of Sciences (PNAS) the week of Jan. 23 - 27, 2006, shows the use of biologically compatible materials to fabricate a "nanoshuttle" - thousands of times smaller than a human hair - which can be harnessed to viral particles to precisely home to disease wherever it hides.

Once there, the nanoshuttle can perform a variety of functions. The study defines how assembled particles of gold - a metal that is not rejected by the body - could possibly be "tuned" to destroy tissue or emit signals that can be detected by imaging devices. The system also can be adapted to form a flexible scaffold that can carry drugs, genes or even cradle restorative stem cells.

"Gold is a perfect metal to perform these different functions, and scientists have been trying to find a way to target such particles to specific organs or tissues, but it has been extremely difficult," says the co-leader of the study, Renata Pasqualini, Ph.D., professor of medicine and cancer biology. "Instead of taking the usual approach by using a synthetic molecule or polymer, we have found a way to mix a 'genetically programmable' nanoparticle with a biologically compatible metal that together target specific locations in the body."

For example, these nanoplatforms could potentially locate damaged areas on arteries that have been caused by heart disease, and then deliver stem cells to the site that can build new blood vessel tissue. To treat cancer, they also may be able to locate specific tumors by using an array of imaging techniques. The tumors could then be treated by either heating the gold particles with laser light and/or using the nanoparticles to selectively deliver a drug to destroy the cancer.

"Gold nanoshells and laser light have been tested in pre-clinical models previously, but it has been difficult to accurately target the therapy," says Wadih Arap, M.D., professor of medicine and cancer biology, co-leader on the study.

These nanoplatforms and scaffolds have not as yet been tested in vivo, but this study is the first to show how, in a laboratory, gold and phage (viruses that infect only bacteria) can combine and build a matrix that can support stem cells.

The disease-finding capability of these scaffolds is due to the specially engineered virus that displays a peptide that matches a protein receptor "zip code" on the tissue of interest. This homing technique was pioneered by the lead authors on the current study, Pasqualini and Arap. Their previous work revealed that the human vascular system contains unique molecular addresses, depending on the site of an organ or tissue, and that blood vessels also acquire abnormal signatures on diseased organs. They were the first to attach such unique vascular "zip codes" to phage, engineering them in such a way that these viral particles would go to these target addresses.

This advance was only made possible, Pasqualini says, because she and Arap invited chemist Glauco Souza, Ph.D., the paper's first author, to work on the problem.

"This was truly a multidisciplinary approach, and it brings together something chemists, physicists and biologists have been trying to do, separately and unsuccessfully, for a long time," Souza says.

"The beauty of this approach is that the phage can already be screened and selected to either target a certain cell type in the body, or home to certain tissues," Pasqualini says.

During their preliminary work, Souza discovered that certain properties of the capsid (the outer shell of the phage virus) would allow it to spontaneously assemble with gold particles.

"So if you can assemble gold particles onto the phage and incorporate a 'signature' molecule like imidazole, you immediately have an entity that is both a sensor, because it binds to a specific molecular signature, and a reporter, because it picks up specific properties of the gold which can be measured in a number of ways," Souza says.

The team also found that by manipulating solution conditions, the network of scaffolds would form a "hydrogel," a bio-inorganic environment capable of sustaining and nurturing stem cells. This biological matrix can potentially be used in two ways, according to Pasqualini and Arap. First, it could be used to grow needed tissue in a laboratory, which could then be delivered to patients. Alternatively, the matrix could be directly injected so that it can implant at the site of injury. There, the stem cells could potentially morph into tissue needed to internally repair the wound, the researchers say.

"This is our vision of the future, and, of course, it all needs to be further studied and translated into real clinical applications," Arap says. "But we can now think in those terms because of this pioneering work that merges the fields of vascular targeting and nanotechnology."

Buckyballs Deform DNA - Surprising Simulation Findings

Wed, 07 Dec 2005 19:21:38 PST

( from http://www.rxpgnews.com ) Soccer-ball-shaped "buckyballs" are the most famous players on the nanoscale field, presenting tantalizing prospects of revolutionizing medicine and the computer industry. Since their discovery in 1985, engineers and scientists have been exploring the properties of these molecules for a wide range of applications and innovations.

But could these microscopic spheres represent a potential environmental hazard?

A new study published in December 2005 in Biophysical Journal raises a red flag regarding the safety of buckyballs when dissolved in water. It reports the results of a detailed computer simulation that finds buckyballs bind to the spirals in DNA molecules in an aqueous environment, causing the DNA to deform, potentially interfering with its biological functions and possibly causing long-term negative side effects in people and other living organisms.

The research, conducted at Vanderbilt by chemical engineers Peter T. Cummings and Alberto Striolo (now a faculty member at the University of Oklahoma), along with Oak Ridge National Laboratory scientist Xiongce Zhao, employed molecular dynamics simulations to investigate the question of whether buckyballs would bind to DNA and, if so, might inflict any lasting damage. "Safe is a difficult word to define, since few substances that can be ingested into the human body are completely safe," points out Cummings, who is the John R. Hall Professor of Chemical Engineering and director of the Nanomaterials Theory Institute at Oak Ridge National Laboratory.

"Even common table salt, if eaten in sufficient quantity, is lethal. What we are doing is looking at the mechanisms of interaction between buckyballs and DNA; we don't know yet what actually happens in the body," he says.

Surprising findings

Despite the caveat, Cummings suggests that his research reveals a potentially serious problem: "Buckyballs have a potentially adverse effect on the structure, stability and biological functions of DNA molecules."

The findings came as something of a surprise, despite earlier studies that have shown buckyballs to be toxic to cells unless coated and to be able to find their way into the brains of fish. Before these cautionary discoveries, researchers thought that the combination of buckyballs' dislike of water and their affinity for each other would cause them to clump together and sink to the bottom of a pool, lake, stream or other aqueous environment. As a result, researchers thought they should not cause a significant environmental problem.

Cummings' team found that, depending on the form the DNA takes, the 60-carbon-atom (C60) buckyball molecule can lodge in the end of a DNA molecule and break apart important hydrogen bonds within the double helix. They can also stick to the minor grooves on the outside of DNA, causing the DNA molecule to bend significantly to one side. Damage to the DNA molecule is even more pronounced when the molecule is split into two helices, as it does when cells are dividing or when the genes are being accessed to produce proteins needed by the cell.

"The binding energy between DNA and buckyballs is quite strong," Cummings says. "We found that the energies were comparable to the binding energies of a drug to receptors in cells."

It turns out that buckyballs have a stronger affinity for DNA than they do for themselves. "This research shows that if buckyballs can get into the nucleus, they can bind to DNA," Cummings says. "If the DNA is damaged, it can be inhibited from self-repairing."

Computer simulations

The computer simulations showed that buckyballs make first contact with the DNA molecule after one to two nanoseconds. Once the C60 molecules bind with the DNA, they remained stable for the duration of the simulation.

Researchers tested the most common forms of DNA, the "A" and "B" forms. The "B" form is the most common form. In a stronger saline solution, or when alcohol is added, the DNA structure can change to the "A" form. A third, rarer form, "Z," occurs in high concentrations of alcohol or salt and was not tested.

The researchers found that buckyballs docked on the minor groove of "A" DNA, bending the molecule and deforming the stacking angles of the base pairs in contact with it. The simulations also showed that buckyballs can penetrate the free end of "A" form DNA and permanently break the hydrogen bonds between the end base pair of nucleotides.

As expected, the buckyballs bound most strongly to single helix DNA, causing the most deformation and damage. While buckyballs did bind to "B" form double-strand DNA, the binding did not affect the overall shape of the DNA molecule. More research needed

What the researchers don't know is whether these worrisome binding events will take place in the body. "Earlier studies have shown both that buckyballs can migrate into bodily tissues and can penetrate cell membranes," Cummings says. "We don't know whether they can penetrate a cell nucleus and reach the DNA stored there. What this study shows is that if the buckyballs can get into the nucleus they could cause real problems. What are needed now are experimental and theoretical studies to demonstrate whether they can actually get there. Because the toxicity of nanomaterials like buckyballs is not well known at this point, they are regarded in the laboratory as potentially very hazardous, and treated accordingly."

ATP Hydrolysis is Required to Reset the ATP-binding Cassette Dimer

Sun, 04 Dec 2005 09:47:38 PST

( from http://www.rxpgnews.com ) Scientists have a tough time visualizing the tiny hatchways that allow nutrients to pass into our cells, but a group of Purdue University biologists may have found the next best thing: a glimpse into the workings of the "motor" that opens and closes them.

A research team led by Jue Chen has clarified the connection between these minuscule gates which are called membrane transport proteins and the steps by which they use a cell's energy to permit or deny materials entry into the interior of the cell from the outside world.

In what the team perceives to be a three-step process, cells feed chemical energy to a tiny machine called an ABC protein, which is the part of the membrane protein that connects it to the interior of the cell. These ABC proteins use the energy to bend the membrane protein into its open and closed positions, allowing the cell both to bring in nutrients and to flush out waste.

"We think we have a better handle on a process fundamental to life in creatures from bacteria to humans," said Chen, who is an assistant professor of biology in Purdue's College of Science. "This is the first time the entire cycle has been visualized, and this could enhance our understanding of how the process of metabolism unfolds."

The team's paper appears in this week's issue of Proceedings of the National Academy of Sciences. Chen's group also includes her Purdue colleagues Gang Lu and James M. Westbrooks, as well as Amy L. Davidson, who recently relocated to Purdue from the Baylor College of Medicine. The team used X-ray crystallography and other advanced imaging techniques to obtain a clear picture of the ABC protein, a method which has only had limited success in revealing secrets of the membrane proteins themselves.

Membrane proteins in cells have been likened to spacecraft airlocks, which ensure that only the astronauts gain entry and no air is lost. Where spacecraft have metal walls, cells have membranes that surround their inner protoplasm, and their airlock proteins are highly complex individual molecules that allow nutrients to enter cells and waste products to leave them.

Of the thousands of membrane proteins that exist, scientists only know the structure of a few dozen. They are of great interest to biologists because, as the regulators of intercellular commerce, they essentially permit metabolism and, thus, life itself to continue. However, while most proteins dissolve in water and can be easily crystallized and examined, membrane proteins dissolve only in fatty substances, making it difficult to isolate them for study.

"If we had a better understanding of this class of proteins, we might know more about how our bodies use and transfer energy," Chen said. "It's an unfortunate gap in our knowledge of how living things work. But in this study, we looked at a protein that is a bit of a hybrid: one part of it is fat-soluble, and the other is water-soluble."

Because the entire membrane protein would not submit to crystallization, Chen's team focused their efforts on the ATP-binding cassette proteins, or ABC proteins for short, that connect the membrane proteins with the cell's interior. This portion of the protein is of the more study-friendly, water-soluble variety, and also plays a critical role in cellular commerce: It is the motor that drives a membrane protein's motion.

"We isolated the ABC proteins from an E. coli bacterium, which is a very common research subject," Chen said. "Different as these single-celled organisms are, their ABC proteins are structurally very similar to those in human cells, so studying them could help our knowledge of our own metabolism."

ABC proteins function like tiny tweezers and are powered by ATP, a chemical that animal cells use for energy. When ATP causes the tweezers to squeeze shut, the membrane proteins open to reveal a small cavity that can hold a nutrient or other substance the cell requires from the outside. Once the nutrient is in place, the cell uses water to break down the ATP, signaling the "tweezers" to relax, closing the membrane protein gate and capturing the nutrient. Lastly, the membrane protein releases the nutrient into the cell's interior.

"The ABC protein is like the inner door of the airlock; that's what we were able to see in operation in this study," Chen said. "If you opened both it and the membrane protein simultaneously, nothing would stop the interior of the cell from getting sucked out."

Chen admits that the team is not yet certain that the description of the process is complete, though it does seem compelling based on what science already knows about the workings of membrane proteins.

"We need to look closer at our information and try to find out more," Davidson said. "We will be applying several tests to our data in the near future to determine if our image of these proteins accurately describes their behavior."

This graphic illustrates the process by which a membrane protein opens and closes, as envisioned by Jue Chen's research team at Purdue University. ABC proteins, which are the inner portion of a membrane protein, function like tiny tweezers and are powered by ATP, a chemical that animal cells use for energy transport. When the tweezers squeeze shut, the outer section of the membrane protein opens to reveal a small cavity that can hold a nutrient or other substance the cell requires from the outside. Once the nutrient is there, the cell uses water to signal the "tweezers" to relax, closing the membrane protein gate and capturing the nutrient. Lastly, the membrane protein releases the nutrient into the cell's interior. (Purdue graphic/Chen labs)

Chen said the work might have long-term payoffs in the fight against cancer, though it was too soon to make more than general statements as to how.

"Many cancer cells are resistant to anticancer drugs because the ABC proteins are overabundant and get too good at pumping the drugs out before they can work," she said. "Future therapies might exploit what we are finding out about these proteins' operation. It's too soon to talk about specific therapies, but because there are so many kinds of cancer out there, every piece of knowledge helps."

First comprehensive map of the proteins and kinase signaling network

Thu, 01 Dec 2005 05:36:38 PST

( from http://www.rxpgnews.com ) A team of scientists at Yale University has completed the first comprehensive map of the proteins and kinase signaling network that controls how cells of higher organisms operate, according to a report this week in the journal Nature.

The study is a breakthrough in understanding mechanisms of how proteins operate in different cell types under the control of master regulator molecules called protein kinases. Although protein kinases are already important targets of cancer drugs including Gleevec and Herceptin, until recently, it has been difficult to identify the proteins regulated by the kinases.

Led by Michael Snyder, Lewis B Cullman Professor of Molecular, Cellular and Developmental Biology, these researchers focused on the expression and relationship between proteins of the yeast cell "proteome," or the proteins that are active in a cell.

Protein kinases act as regulator switches and modify their target proteins by adding a phosphate group to them. This process, called "phosphorylation," results in altered activity of the phosphorylated protein. It is estimated that 30% of all proteins are regulated by this process.

Using technology developed in Snyder's laboratory, graduate students Jason Ptacek and Geeta Devgan used proteome microarrays to assay the thousands of different proteins in a yeast cell for targets of the protein kinases. The 82 unique kinases, representing the majority of master regulators in the yeast cell, were tested separately with the microarrays to determine which proteins were modified by each kinase.

From the wealth of information generated by these experiments Snyder's team constructed a complex map of the regulatory networks governing the functions and activities of the kinases in the yeast cell. The map shows several distinct patterns.

"It was a little like having all the pieces of an airplane separated out, and not knowing how those pieces function together to create an airplane and make it fly," said Snyder. "We wanted to know how the tens of thousands of proteins coordinate to carry out complex processes such as growth, cell division and formation of complex cell types such as brain cells and intestinal cells."

Over the past several years, a large volume of information on genes in organisms as diverse as man, mouse, baker's yeast and viruses has been generated. While genomic DNA is the blueprint, the encoded proteins are the products that carry out the complex biological functions of cells. Although scientists can predict from the DNA what proteins are in the proteome of an organism, this study opens the door to seeing how they are coordinated to work together.

"This insight into the regulation and integration of biological networks has broad applications for basic science and clinical research," said Snyder. "Biological networks determine the development and function of organisms from the single-celled yeast to man; aberrations in those networks signal disease."

Biological networks are typically conserved between species, meaning that often the same type of protein carries out the same type of function, whether it is in a yeast cell or a human cell. According to Snyder, these findings in yeast are of immediate use for understanding both human development from the fertilized egg to full grown organism, and for drug discovery targeting human diseases.

Magnetic probe successfully tracks implanted cells

Mon, 21 Nov 2005 20:12:38 PST

( from http://www.rxpgnews.com ) By using MRI to detect magnetic probes of tiny iron oxide particles, an international research team for the first time has successfully tracked immune-stimulating cells implanted into cancer patients for treatment purposes.

"In four of the eight patients, MRI revealed that the implanted cells weren't where they needed to be to be effective for treatment," says Jeff Bulte, Ph.D., an associate professor of radiology at Hopkins' Institute for Cell Engineering who developed methods to optimally label cells with the clinically approved iron oxide particles.

This new application of the probes -- already clinically approved for MRI scanning of the liver -- could dramatically improve efforts to test and use cellular therapies such as vaccines to treat cancer or prevent its recurrence or stem cells to repair damaged organs, say the researchers.

Bulte and a team of Dutch researchers used MRI and a magnetic probe approved by both European and U.S. agencies to locate therapeutic cells injected into eight melanoma patients.

"Our results show that the MRI-based technique was more accurate than tracking the cells using radioactivity and that ultrasound failed to accurately guide injection of the cells into lymph nodes in half of the patients," says Bulte, an author on the report, which appears in the November issue of Nature Biotechnology.

The cells used in the current study, so-called dendritic cells, are the immune system's own "most wanted posters" because they take up and display foreign proteins that tell the immune system's fighters what cells to look for and destroy.

Since the mid-1990s, clinical trials have been testing dendritic cells to see whether they can stimulate the immune system to kill cancer cells. In these trials, dendritic cells from patients are exposed to proteins from the patients' cancer cells and then returned to the patients.

However, some of the clinical trials of such "cancer vaccines" have been disappointing, with some patients responding very well but others not at all. A critical issue behind each patient's success on the treatment, however, is whether the cells get to the lymph nodes, where the immune system's fighters are normally "trained" by dendritic cells. Until now, there's been no accurate way to know where the cells end up.

It's thought, but not proven, that the best way to get the cells where they need to be is to inject them directly into the lymph nodes that drain the area containing a tumor. Currently, doctors use ultrasound to guide the needle, and dendritic cells carrying a radioactive tag are sometimes used to try to double-check the cells' final resting place.

However, in this study, the Dutch team discovered that using MRI and iron oxide particles was able to track the cells' location much more accurately than the radioactive tracking method and provided anatomic detail simultaneously -- structural detail not possible by tracking radioactivity.

"On the MR images, we can see the lymph nodes, and we can see the magnetically labeled dendritic cells, and we can tell very clearly whether they are in the same place," says the study's first author, Jolanda de Vries, an assistant professor at the Nijmegen Center for the Molecular Life Sciences (NCMLS) of the Radboud University Nijmegen Medical Center in The Netherlands. "The cells can't get from the fat into the lymph nodes by themselves, so injecting them properly is very important."

Bulte says he, Dara Kraitchman, Ph.D., D.V.M., and colleagues at Hopkins are already testing magnetically labeled stem cells with MRI-compatible injection systems to allow MRI guidance of injection in large animals.

The current clinical trial builds on Bulte's earlier work tracking magnetically labeled cells in animals. Four years ago, he and colleagues reported that stem cells containing so-called magnetodendrimers could be followed by MRI.

But to advance to clinical trials, the research team switched from the experimental magnetic tags to formulations of iron oxide already approved for clinical use in Europe (as Endorem) and the United States (as Feridex). Because immature dendritic cells naturally take up materials around them, they simply absorbed, or ingested, the iron oxide particles when exposed to them in the lab. The magnetically labeled, cancer-primed cells were then returned to the patients, all of whom had stage III melanoma.

"Although dendritic cell therapy is used in clinical trials to treat patients with melanoma, in this study we wanted to see whether the magnetically labeled cells could be tracked by MRI, to study their migratory behavior in more detail," says Carl Figdor, principal investigator of the study, of the NCMLS. "We were very pleased that they showed up clearly. With the anatomic information from the MRI, we could see precisely where they were -- inside or outside of the lymph nodes."

New Microscope Tracks Functioning Protein at Atomic Level

Mon, 14 Nov 2005 01:48:38 PST

( from http://www.rxpgnews.com ) A Stanford University research team has designed the first microscope sensitive enough to track the real-time motion of a single protein down to the level of its individual atoms. Writing in the Nov. 13 online issue of the journal Nature, the Stanford researchers explain how the new instrument allowed them to settle long-standing scientific debates about the way genes are copied from DNA--a biochemical process that's essential to life.

In a second paper published in the Nov. 8 online issue of the journal Physical Review Letters, the scientists offer a detailed description of their novel device, an advanced version of the "optical trap," which uses infrared light to trap and control the forces on a functional protein, allowing researchers to monitor the molecule's every move in real time.

"In the Nature experiment, we carried out the highest-resolution measurement ever made of an individual protein," says Steven Block, professor of applied physics and of biological sciences. "We obtained measurements accurate to one angstrom, or one-tenth of a nanometer. That's a distance equivalent to the diameter of a single hydrogen atom, and about 10 times finer than any previous such measurement."

Block co-authored the papers in Nature and Physical Review Letters with three current members of his Stanford Lab--graduate students Elio Abbondanzieri and William Greenleaf and postdoctoral fellow Michael Woodside--together with former graduate student Joshua Shaevitz, now at the University of California-Berkeley, and longtime collaborator Robert Landick at the University of Wisconsin.

Central dogma

In the Nature study, Block and his colleagues tackled a fundamental principal of biology known as the central dogma, which states that in living organisms, genetic information flows from DNA to RNA to proteins.

The process begins with DNA, the famous double helix, which stores genetic data. DNA is often compared to a twisted ladder consisting of two strands connected by molecular rungs called "bases," which are known by the abbreviations A, T, G and C. Lengthier DNA sequences code for genes, which contain explicit instructions for building a specific protein.

A typical DNA ladder carries thousands of genes that encode thousands of proteins, which keep the organism alive and functioning. A single misplaced letter in gene's DNA sequence--a G substituted for a T, for example--can produce a defective protein that may cause a serious disease.

Transcription

The Block team focused on a crucial step in the central dogma, a process known as "transcription," where each gene is copied from DNA onto RNA. Transcription begins when an enzyme called RNA polymerase (RNAP) latches onto the DNA ladder and pulls a small section apart lengthwise. The RNAP enzyme then builds a new, complementary strand of RNA by chemically copying each base in one of the exposed DNA strands. RNAP continues moving down the DNA strand until the gene is fully copied.

For the Nature experiment, Block and his colleagues used DNA and RNAP extracted from E. coli bacteria, which is remarkably similar to RNAP in more complex organisms, including humans. "RNAP is one of the most important enzymes in nature," Block says. "Without it there would be no RNA messages, no proteins and no life."

Inchworms and scrunching

Exactly how transcription works at the molecular level has been intensely debated among scientists.

"People for years have known that RNA is made one base at a time," Block says. "But that has left open the question of whether the RNAP enzyme actually climbs up the DNA ladder one rung at a time, or does it move instead in chunks--for example, does it add three bases of RNA, then jump along and add another three bases." The latter process, called discontinuous elongation, is like reading a book, he explains: "When you read, you don't advance your eyes one letter at a time. You 'chunk': You read it in pieces."

Two basic hypotheses have been proposed for discontinuous elongation:

* Ihe inchworm model, in which RNAP moves along DNA like an inchworm, with the front end of the enzyme always ahead of the rear.
* The scrunching model, whereby RNAP pulls in ("scrunches") a loop of DNA, copies each base in the loop, then grabs another loop farther up the ladder.

Determining which model is correct has been a difficult challenge, because until now, no instrument was sensitive enough to track each microscopic step taken by RNAP along DNA during transcription. That's because conventional optical traps can't measure anything smaller than about 10 angstroms (1 nanometer). However, each base in the DNA ladder--A, T, G or C--is only separated by about 3.4 angstroms. "My lab has been working very hard for the better part of a decade to break the nanometer barrier and attain angstrom-level resolution," Block says.

Light and motion

To achieve that goal, the Block team had to overcome two inherent problems with conventional force clamps: fluctuating signals and bending light waves.

"When you shine a laser through the air, the light beam wiggles around a bit, for the same reason that stars twinkle in the sky," Block explains. "But we want to use that beam to measure the position of something to within the size of an atom, so if the beam moves just 1 angstrom, that's the end of the story. We took all the optics external to the microscope, enclosed them in a sealed box and replaced the air with helium gas, which has a refractive index that's 10 times closer to that of a vacuum than air. So you get, roughly speaking, 10 times less twinkling and an instrument with angstrom-level stability."

In addition to stabilizing the light, the researchers also had to improve the method for detecting force and displacement. Optical force clamps use tiny forces from an infrared laser beam to trap DNA and other molecules. In a conventional force clamp experiment, microscopic beads are attached near the opposite ends of a long DNA molecule--an arrangement that resembles a weight lifter's dumbbell. A single RNAP enzyme attached to the surface of one bead then moves along the DNA and churns out a complementary strand of RNA, drawing the ends of the dumbbell closer together as it advances. The two beads that form the dumbbell are usually held near the center two separate optical traps. But graduate student William Greenleaf discovered that if one of the two beads in the dumbbell was placed near the outer edge of its trap, the force on it would remain constant, allowing angstrom-level measurements to be made quickly and efficiently.

"That's just what you want--a clamp that allows RNAP to move with impunity, but the force itself doesn't change," Block says. "Normally the bead is inside the trap in the center, but right at the edge of the trap we have this magical property where the force is constant."

Unlike conventional instruments, the new force clamp requires no time-consuming computer computations to correct for competing forces. "This new technique is entirely passive, like a thermos that just sits there and keeps something cool," Block says. "All we have to do is shine light on the system and everything takes care of itself. As a result, we were finally able to resolve the minuscule, 3.4-angstrom steps taken by E. coli RNAP as it transcribes a bacterial gene."

Settling the debates

With these innovations in place, the research team appears to have settled some of the fundamental arguments over DNA-RNA transcription. "Quite simply, our experiment rules out both discontinuous-location models," Block says. "Neither the inchworm nor the scrunching model is consistent with our data, and the idea that some have held all along--that RNAP climbs the DNA ladder one base pair at a time--is probably the right answer."

The Stanford group also weighed in on another controversy concerning the actual mechanism that allows RNAP to advance. "RNAP is a molecular motor that starts at one end of the DNA and walks down to the other end," Block explains. "It gets its energy from the chemical reaction that occurs when it copies A, T, G or C. It's as if a machine that lays down asphalt could somehow be powered by the asphalt itself."

Scientists have come up with two different models to explain what drives this molecular motor:

* The power stroke model, in which pent up energy thrusts the enzyme forward--like a loaded spring that's periodically released.
* The Brownian (or thermal) ratchet model, whereby random thermal energy causes the RNAP enzyme to jiggle back and forth. Each incoming DNA base then locks the enzyme into the forward position so that it cannot jiggle backwards. "It would be as if you were repeatedly bouncing off a wall, and every time you happened to bounce a bit farther away, somebody came in and moved the wall up behind you, so you couldn't bounce so far back. You'd wind up drifting forwards, even though your own motion was mostly random," Block explains.

In the Nature study, Block and his colleagues concluded that the Brownian ratchet model is probably correct for RNAP, even though several other motor proteins are believed to move instead by the power stroke mechanism. "We've certainly come down hard in favor of the Brownian ratchet camp and against the power stroke camp," Block says. "But does that mean all power stroke models have been ruled out and that all Brownian ratchet models are acceptable? No."

Molecular folding

The Block team also applied the new force clamp technology to one the hottest fields in biomedical research--molecular folding. For a protein to function properly, it has to fold into a specific, intricate three-dimensional shape. Diseases such as Alzheimer's, Mad Cow and Parkinson's may result when proteins do not fold into their correct 3-D conformation. Medical researchers are trying to solve the mystery of how proteins fold in hopes of some day curing these and other diseases.

In the experiment published in Physical Review Letters, the Block group addressed certain aspects of the general folding problem on a simpler scale by focusing on single DNA hairpins--folded structures that can form when a single strand of DNA pairs with itself instead of with the opposite strand. "Hairpins are wonderful models," Block says. "By keeping the force constant, we were able to measure the folding and unfolding transitions of a single DNA hairpin at the angstrom scale. In the future, this may help us understand and predict what shape a more complex linear protein will assume in three-dimensional space."

Major advance

The development of an ultra-stable optical trapping system with angstrom resolution is "a major advance," says Charles Yanofsky, the Morris Herzstein Professor of Biological Sciences at Stanford and a pioneer of modern molecular genetics. The new device is like "adding movies to stills in understanding enzyme action," he says.

"This technical achievement will no doubt lead to new information about the molecular machinery that carries out basic cellular processes, particularly those related to replication, transcription and translation," adds Catherine Lewis, a program director in biophysics at the National Institute of General Medical Sciences (NIGMS).

"If I look in my crystal ball and see where this is going, I think this blows open the field of single-molecule biophysics," Block says. "We have achieved a resolution for a single molecule comparable to what a crystallographer typically achieves in a millimeter-sized crystal, which has 1,000 trillion molecules in it. Not only are we doing all this with one molecule at one-angstrom resolution, we're doing it in real time while the molecule is moving at room temperature in an aqueous solution."

Block notes that it took "years of careful instrument development, sponsored by the National Institutes of Health, and the construction of a special laboratory built by Stanford University to make this possible, along with the simply outstanding efforts of some incredibly bright and hard-working graduate students and postdocs here at Stanford. I am especially proud of this work."

Selenium Speeds Enzymatic Reactions

Tue, 08 Nov 2005 17:43:38 PST

( from http://www.rxpgnews.com ) At the heart of every reaction of every cell lies an enzyme, a protein catalyst. At its active siteâa special pocket on its surfaceâit binds reactants (substrates) and rearranges their chemical bonds, before releasing them as useful products. Rearranging some bonds may require help from certain chemical elements that are present in trace amounts. Many enzymes place these elements at the center of their active sites to do the most critical job.

Selenium is one such element. In large quantities, selenium is toxic, but, in trace amounts, it is absolutely essential for life in many organisms, including humans. Selenium is present in proteins in the form of selenocysteine, a rare amino acid that helps promote antioxidant reactions. These selenocysteine-containing proteins are called selenoproteins. One important selenoprotein is the enzyme methionine-R-sulfoxide reductase (MsrB) 1, whose job is to repair proteins injured by oxidative damage, caused by sunlight, toxic chemicals, or a variety of other insults.

In mammals, there are two other forms of MsrB, which also can efficiently perform this task, but use the abundant amino acid cysteine instead of selenocysteine. So why do cells go to the trouble and metabolic expense of acquiring selenium from the environment? In this issue, Hwa-Young Kim and Vadim Gladyshev explore the details of active-site chemistry of these three related enzymes, and show that the selenoprotein form employs a different catalytic mechanism.

The authors began by identifying three key amino acids in the active site of the cysteine-containing forms, which did not occur in the selenoprotein MsrB1. When any of these amino acids were mutated, the activity of the cysteine-containing enzymes was greatly diminished. This result indicates that these amino acids likely play a role at the active site, a supposition supported by previous work on related enzymes in bacteria.

Kim and Gladyshev next systematically mutated MsrB1 to include one, two, or all three of these amino acids, and discovered that inclusion of one or any combination of them diminished activity of the selenocysteine-containing enzyme. This suggested that while these amino acids support the mechanism of the cysteine-containing forms, they interfere with the mechanism of the selenoprotein. Not surprisingly, when the selenium was removed from MsrB1, the enzyme was significantly impaired. But when the three amino acids were added to this crippled enzyme, they restored some of the diminished activity, probably by carrying out the same mechanism they do in the cysteine-containing enzymes.

The authors then inserted a selenium atom into each of the cysteine-containing enzymes, in the same spot in the active site where it sits in MsrB1. They found that the initial activity of each enzyme was increased over 100-fold, indicating the inherent capacity of selenium to promote catalytic activity. These souped-up enzymes were unable to complete the reaction, however, because they lacked other features of MsrB1's active site. Further scrutiny of the enzymes revealed these critical features, and inserting them allowed the artificial selenoproteins to carry out the entire reaction.

The authors suggest the explanation for these findings relates to a difference in the catalytic mechanism of selenocysteine- and cysteine-containing enzymes. The substrate for both enzyme types, methionine-R-sulfoxide, is found within oxidized proteins. The job of both enzymes is to reduce this compound back to the amino acid methionine. Both do so by accepting an oxygen atom from the sulfoxide.

In the presence of selenium, the oxygen temporarily binds to the selenium. The selenium's electrons then shift to bond with a sulfur on a neighboring cysteine amino acid, kicking out the oxygen as part of a water molecule. Finally, the selenium-sulfur bond is broken and the enzyme is restored to its original state by the intervention of thioredoxin, a ubiquitous cell molecule whose job is to undo just such temporary linkages in a wide variety of enzymes.

Without selenium, the oxygen binds directly to sulfur, and thioredoxin intervenes to form the water and restore the sulfur. This reaction occurs in fewer steps, but is slower. The authors propose that the evolution of selenium-containing MsrB1 from cysteine-containing forms was likely favored by the higher rate of reaction it offered, although this trend is likely limited by the requirement for changes in other portions of the enzyme to accommodate the trace element. The authors suggest that selenium provides inherent catalytic advantages to certain types of enzymatic reactions, even though utilization of these advantages is sometimes tricky. If so, manipulation of related enzymes by insertion of selenium may increase their catalytic efficiency, perhaps much above that designed by nature. This may offer advantages for some biotechnology and biomedical applications that depend on antioxidants. âRichard Robinson

Exploring Dynamic Personalities of Proteins

Thu, 03 Nov 2005 16:23:38 PST

( from http://www.rxpgnews.com ) A Brandeis University study published in Nature this week advances fundamental understanding of the dynamic personalities of proteins and proposes that these enzymes are much more mobile, or plastic, than previously thought. The research, based on nuclear magnetic resonance (NMR) experiments, may shed new light on how to improve rational drug design through docking to dynamic targets.

For the first time ever, the study linked both the low-energy as well as the much rarer high-energy state of enzymes to their function, said lead author Brandeis biophysicist Dorothee Kern, who is also an investigator at the Howard Hughes Medical Institute.

This is important because drugs seek to bind, or dock, to target enzymes in the infrequent high-energy state. Kern believes the study brings scientists a step closer to a new area of research that seeks to elucidate the structures of enzymes in high-energy states that can be ultimately used for rational drug design.

"This research shifts the paradigm of how we thought proteins work. The traditional view is that proteins are not terribly dynamic when they do not perform their function, and that they become dynamic only during catalysis, their active state. What we have learned now is that there is no resting state, that even in the absence of substrates, before catalysis, defined motions of many atoms is an intrinsic property of these enzymes," explained Kern.

"Much like a rousing basketball game in which all the players continuously but strategically move with or without the ball nature has evolved these biomolecules so that they are constantly moving in highly-defined directions conducive to their function with or without the substrate," explained Kern, who played for the East German National basketball team before the Berlin Wall fell in 1989 and later professional basketball for united Germany.

The research involved NMR studies of the enzyme cyclophilin A, a highly conserved protein found in all organisms from yeast to the human body, and which is involved in HIV replication in humans. Elucidating the role that cyclophilin A plays in the body would be a major step toward creating drugs that impede its virulence, without interfering with normal cellular function.

Kern summed up: "The fundamental principal of life is that molecules constantly change over time that is the definition of dynamics."

Biotech failed to meet promises

Fri, 14 Oct 2005 21:42:38 PST

( from http://www.rxpgnews.com ) Promises of cheaper and better drugs using biotechnologies have not been met, say researchers in this weeks BMJ. They assessed biotech products approved by the European Medicine Evaluation Agency between 1995 and 2003.

Of 61 products licensed for therapeutic use, only 15 were for diseases without effective treatment, more effective than existing treatment, or active in patients resistant to current treatment.

A further 22 offered limited advantages over existing products, and 24 were copycat drugs, many of which have failed to offer new options for patients and provide no cost advantage, say the authors.

Furthermore, evaluation of these substances was not always based on rigorous methodological criteria, suggesting that commercial priorities come before the sound development of drugs in the interest of patients.

The promises of biotechnology substances to be more effective and less toxic than conventional drugs have been only partially fulfilled, they add. Many of the substances produced so far are analogues of existing drugs and have contributed little to innovation in medicine.

Nevertheless, biotechnology has made it possible to make available drugs that would otherwise be impossible to obtain in large amounts or research tools that are useful for discovering new drugs. Let us hope that in future biotechnology will better live up to its promises.

Understanding how voltage-gated ion channels operate

Wed, 12 Oct 2005 04:55:38 PST

( from http://www.rxpgnews.com ) One of the biggest mysteries in molecular biology is exactly how ion channels tiny protein pores through which molecules such as calcium and potassium flow in and out of cells operate. Such channels can be extremely important; members of the voltage-gated ion channel family are crucial to generating electrical pulses in the brain and heart, carrying signals in nerves and muscles. When channel function goes awry, the resulting diseases known as channelopathies, including epilepsy, a number of cardiomyopathies and cystic fibrosis can be devastating.

Ion channels are also controversial, with two competing theories of how they open and close. Now, scientists at Jefferson Medical College, reporting October 6, 2005 in the journal Neuron, have detailed a part of this intricate process, providing evidence to support one of the theories. A better understanding of how these channels work is key to developing new drugs to treat ion channel-based disorders.

According to Richard Horn, Ph.D., professor of physiology at Jefferson Medical College of Thomas Jefferson University in Philadelphia, voltage-gated ion channels are large proteins with a pore that pierces the cell membrane. They open and close in response to voltage changes across the cell membrane, and the channels determine when and which ions are permitted to cross a cell membrane.

In the conventional theory, when an electrical impulse called an action potential travels along a nerve, the cell membrane charge changes. The inside of the cell (normally electrically negative), becomes more positive. In turn, the voltage sensor, a positively charged transmembrane segment called S4, moves towards the outside of the cell through a small molecular gasket called a gating pore. This movement somehow causes the ion channel to open, releasing positively charged ions to flow across the cell membrane. After the action potential is over, the cell's inside becomes negative again, and the membrane returns to its normal resting state.

The more recent and controversial theory proposed by Nobel laureate Roderick MacKinnon of Rockefeller University holds that a kind of molecular paddle comprised of the S4 segment and part of the S3 segment moves through the cell membrane, carrying S4's positive charges with it across the lipid. As in the conventional theory, the S4 movement controls the channel's opening and closing. The two theories differ in part because the paddle must move its positive charges all the way across the cell membrane. The conventional theory says that charges move a short distance through the gating pore.

In the current work, Dr. Horn and colleague Christopher Ahern, Ph.D., a research assistant in the Department of Physiology at Jefferson Medical College, showed that the field through which the voltage sensor's charges moved is very short, lending support to the conventional model.

"Using a molecular tape measure with a very fine resolution 1.24 Angstroms we tethered charges to the voltage sensor," Dr. Horn explains. "When the tether is too long, the voltage sensor can't pull it through the electric field," meaning the electric field is highly focused.

"This is another nail in the coffin of the paddle model," he says, "because the thickness of the electric field is much smaller than predicted by that model. The measurement is unambiguous in terms of the relationship between length of the tether and how much charge gets pulled through the electric field.

Next, the researchers are tackling the relationship between S4's movement and the gates that open and close the channels.

Call for funding boost for robotics research in US

Tue, 20 Sep 2005 22:03:38 PST

( from http://www.rxpgnews.com ) When it comes to developing robots for use in biology and medicine, no country is currently a match for the United States . But that situation could change within the next few years, according to a new report.Unless the government boosts funding for robotics research, the United States the world leader for research and manufacturing of robotic systems for tasks such as surgery and DNA sequencing will likely have to start relying on technology from other countries, said Yuan F. Zheng, professor of electrical and computer engineering at Ohio State.

Zheng is lead author of the chapter on robotics for biological and medical applications. We cannot say there's a systematic theory for robotics in biological and medical applications in this country, because the scientists who do the work come from so many different disciplines, Zheng said.Most American scientists who develop biomedical robotics belong to one academic department on a university campus, and perform interdisciplinary research that crosses over into other departments. Some are engineers who know a little biology; others are biologists who know a little engineering. If more universities had such programs, the United States could grow the culture it needs to sustain its role as a robotics leader, Zheng and his colleagues suggest. Universities will need more funding to establish these programs, Zheng said. While the United States has reduced its support of robotics research in recent years, other countries are boosting resources in this area, Zheng noted. I remember a few years ago when American research accounted for 80 percent of papers presented at robotics conferences. To compile the report, Zheng and his coauthors surveyed major foreign universities that had a robotics program for biology or medicine. They visited Japan , Korea , and countries in Western Europe .

The report details applications of robotics for biology and medicine that are under development worldwide. Doctors routinely use remote-controlled robotics to operate in constrained spaces, such as inside the heart, brain, spinal cord, throat and knee.

Biochemistry's future with quantum physics

Mon, 19 Sep 2005 12:19:38 PST

( from http://www.rxpgnews.com ) Using powerful supercomputers to analyze the interplay of the dozens of electrons that whirl in clouds about these molecules, a team of physicists led by Purdue's Jorge H. Rodriguez has found that the quantum property of electrons called "spin" needs to be considered to obtain a complete and fundamental picture of how many biochemical reactions take place. In particular, a class of metal-based proteins that includes hemoglobin and chlorophyll, and their reactions in plants and animals, can be better understood with the technique.

Not only will this discovery sharpen our basic knowledge of biology, Rodriguez said, but it also could help scientists with a number of practical problems such as selecting the best potential new drug compounds from a vast group of candidates, a process that can cost pharmaceutical companies years of work and millions of dollars.

"Whereas we have had to be satisfied with observing the chemistry in living things and describing it afterward without complete understanding, we are developing computational tools that can predict what will happen between molecules before they meet in the test tube," said Rodriguez, who is an assistant professor of physics in Purdue's College of Science. "Not only does this research open up a new field of science that reveals how metalloproteins and their constituent particles interact, but the quantum theory behind it also should allow us to model and predict these behaviors accurately with computer simulation alone. It is an example of how much can be accomplished with interdisciplinary science."

Rodriguez is pioneering a new field he calls "quantum biochemistry" a field that involves both biochemistry and particle physics, which are often cited among the more formidable subjects science students tackle. Ordinarily, the two disciplines share little common ground. Although biochemistry deals with interactions among the complex molecules that our bodies use for the fundamental processes of life, these microscopically small molecules are nonetheless gargantuan entities in comparison with the tinier subatomic particles such as protons and electrons that physicists study.

"Despite these differences, there is one point of overlap between chemistry and physics that has interested me, and that is in the elementary particles that whirl about these molecules the electrons," Rodriguez said. "Physicists have long known that, according to the laws of quantum mechanics, there are some chemical reactions in our bodies that are 'forbidden' such as hemoglobin's binding oxygen in our lungs when we breathe. But they do happen nonetheless. So, because these reactions involve electron spin, we decided to take a closer look at them."

Charge is a familiar property of an electron, but it is not the only one. Electrons also have another quantum property called spin, and though they are all negatively charged, they can spin in one of two opposing directions up or down.

"Nature loves balance, and you see evidence of it in both charge and spin," Rodriguez said. "For example, electrons of opposite spin like to pair up with each other as they fly around the nucleus. This allows their spins to balance one another, just as positive and negative charges do between protons and electrons. Even when you have hundreds of electrons forming an immense cloud around a complex molecule, you still see balance in both charge and spin; we call this balance 'conservation,' and it's something we count on in both chemistry and physics to help us understand these tiny objects.

"But sometimes the electrons in metalloproteins seem to be playing a trick on us. As we see with hemoglobin, nature appears to be conserving electronic charge while sacrificing this conservation in spin."

Hemoglobin's active center contains iron, one of the so-called transition metals. These metals are noted for the way several of their electrons can fly around the nucleus unpaired.

When a red blood cell encounters oxygen in our lungs, its hemoglobin is able to grasp some of the oxygen with some of these unpaired electrons, carrying it to the rest of our body. But in the process, the cumulative spin of the system changes in a way that is not conserved, which to a physicist looks as strange as a ball hitting the water without making a splash.

"This chemistry is vital for life, but physicists wonder how it can happen," Rodriguez said. "The charge between the electrons in the bonded oxygen and hemoglobin is balanced in the end, which makes sense to chemists. But the electronic spin of the entire system is not conserved, making a physicist frown at what appears to be a formally forbidden process. Of course, we needed to learn more about nature at the microscopic level."

As many of these supposedly forbidden reactions involve biomolecules centered upon transition metals, which can flip back and forth between different spin states under certain conditions, Rodriguez theorized that it was this variability in spin state that was influencing the rate of these reactions. To explore whether this effect, which Rodriguez calls spin-dependent reactivity, was indeed the decisive factor, the team is modeling the reaction rates with a supercomputer, the only tool capable of keeping track of the motion of so many particles at once.

"Supercomputers have allowed us to check our models against our understanding of spin's effect on a reaction, and our models have been closely checked by experiment," Rodriguez said. "The results suggest that our understanding of electron behavior is sufficient to create virtual models of molecules that we can then 'react' with one another in simulations that accurately predict what will happen when they meet in the physical world."

Rodriguez said the approach, though still in its nascent stages, could provide insight into far more biologically important molecules when it is further developed.

"We are at the point where we have developed computational tools to analyze the spin-dependent processes of biomolecules and have applied them to a few important test cases," he said. "But our methods are based on approaches that are valid for any molecular system. Therefore, hundreds more metalloproteins that are of great scientific and practical interest may be studied in the future with the methods we have developed."

For example, Rodriguez is planning to study the manganese involved in photosynthesis to understand how water is broken down to produce molecular oxygen. But for now, he is happy that the four years of work his team has put into the project have produced such encouraging results.

"We are creating a new field that attempts to understand biochemical processes at the most fundamental level that of quantum mechanics," he said. "It could be the most important step toward making biochemistry a predictive science rather than a descriptive one."

Jeffrey Long, a professor of chemistry at the University of California at Berkeley, commented on Rodriguez's work. "Rodriguez has come up with an elegant means of evaluating excited-state electronic structures," he said. "It lends insight to the detailed mechanisms of poorly understood transformations in inorganic complexes."

First comprehensive study of human hair on nanometer level

Fri, 09 Sep 2005 17:40:38 PST

( from http://www.rxpgnews.com ) Ohio State University researchers have just completed the first comprehensive study of human hair on the nanometer level.

Special equipment enabled Bharat Bhushan and his colleagues to get an unprecedented close-up look at a rogue's gallery of bad hair days from chemically overprocessed locks to curls kinked up by humidity.

Ultimately, the same techniques could be used to improve lipstick, nail polish and other beauty products, said Bhushan , Ohio Eminent Scholar and the Howard D. Winbigler Professor of mechanical engineering at Ohio State .

His specialty is nanotribology the measurement of very small things, such as the friction between moving parts in microelectronics.

At first, hair seemed like an unlikely study subject, he said. Then he was invited to give a lecture to scientists at Procter & Gamble Co.

It turns out that, for hair, friction is a major issue, he said. Everyday activities like washing, drying, combing and brushing all cause hairs to rub against objects and against each other, he explained. Over time, the friction causes wear and tear two processes that he and his colleagues are very familiar with, though they're normally studying the wear between tiny motors and gears.

We realized that beauty care was an emerging area for us and we should dive in, Bhushan said.

He consulted for the company until P&G became an industrial partner in his laboratory, supplying him with samples of healthy and damaged hair. The Ohio State engineers examined hairs under an atomic force microscope (AFM), a tool that let them scratch the surface of hairs and probe inside the hair shaft with a very tiny needle. They published their results in the journal Ultramicroscopy, in a paper now available on the Web.

Among their findings: hair conditioners typically do not evenly cover the entire hair shaft.

P&G recently developed a new formula with additives to make the conditioner coat the hair evenly. In tests, Bhushan found that the new conditioner did coat hair more evenly.

Meanwhile, they examined healthy and damaged hairs under an electron microscope and an AFM, and simulated everyday wear and tear by rubbing hairs together and against polyurethane film to simulate skin.

We didn't know what we were looking for, Bhushan said. People know a lot about hair, but nobody has used an AFM to really study the structure of hair. So we already knew some things, but otherwise we didn't know what to expect.

Under the electron microscope, individual hairs looked like tree trunks, wrapped in layers of cuticle that resembled bark. In healthy hair, the cuticle edges lay flat against the hair shaft, but as hair gets damaged from chemical treatments or wear and tear, the cuticle edges begin to peel away from the shaft. That much was already known.

The researchers simulated what happens when damaged hair is exposed to humidity; the hairs plump up, and the cuticles stick out even further, leading to frizz. More frizz meant more friction a fact confirmed by the AFM as researchers dragged a tiny needle across the surface.

Conditioner tends to stick to the cuticle edges, and can make the hair sticky on the nanometer scale. The researchers determined that by poking the hair shaft with the needle, and measuring the force required to pull it away.

They also probed inside hairs to measure the hardness of different layers of the shaft. Hair has a very complex structure, Bhushan said, and these first ultra-precise measurements of interior structure could one day lead to new products that treat hair from the inside.

In the future, he thinks his AFM techniques could be used to develop wear-resistant nail polishes and lipsticks.

Artificial intelligence to help intensive care doctors

Sun, 04 Sep 2005 09:26:38 PST

( from http://www.rxpgnews.com ) A team of systems engineers from the University of Sheffield is developing an intelligent computer system which imitates a doctor's brain to make treatment decisions for intensive care patients. The system will take some of the workload from emergency medical teams by monitoring patients' vital signs and then evaluating and administering the right amounts of different drugs needed - a job usually carried out by specialist medical doctors.

The team, led by Professor Mahdi Mahfouf in the University of Sheffield's Department of Automatic Control and Systems Engineering, is pioneering the intelligent decision-support system which, in effect, duplicates the decision making processes of specialist medical doctors in Intensive Care Units (ITU).

The system models all the possible interactions between different drugs and patients' bodies, and then makes intelligent decisions about the best way to treat patients during heart bypass operations, and post-operatively in the ITU. This unique system can decide on the types and quantities of drugs to give to patients in a matter of seconds. This will help doctors provide effective treatment for patients, whilst allowing them to concentrate on as many other important tasks as possible.

Professor Mahdi Mahfouf of the University of Sheffield explains that it is the system's ability to learn, adapt, and make informed decisions which is unique: "This new system not only monitors and treats critical patients, but it can also learn from the experiences of medical staff, who can override the machine at any time. If overridden, the system assimilates the doctor's input and uses the new information to make decisions about similar cases in the future.

"This system is not intended to replace the work that doctors do in intensive care units. However it will provide them with invaluable assistance by evaluating the complex interactions of different drugs which are needed to treat patients and protect them against the danger of septic shock."

A Global View of DNA-Packing Proteins Cracks the Histone Code

Wed, 31 Aug 2005 02:11:38 PST

( from http://www.rxpgnews.com ) In one of biology's most impressive engineering feats, specialized proteins package some six-and-a-half feet of human DNA into a nucleus that averages just 5 microns (0.0001969 inches) in diameter. In the first of a series of supercondensing steps, DNA winds around proteins called histones, which together form a complex called the nucleosome. Histones package DNA into repetitive coils, which not only provide genomic structure but also help regulate gene expression. These tasks are mediated in part by chemical modifications to histone proteinsmost commonly to histone tails, long, unstructured chains of amino acids that protrude from nucleosomes. Different chemical modifications are associated with different functional effects. Acetylation, which adds an acetyl group to an amino acid on the histone tail, has been linked to both gene activation and silencing, depending on which amino acid is modified. Methylation (addition of a methyl group to the histone tail) has also been linked to gene activation and repression, although the chemical effects of methylation differ dramatically from those of acetylation.

Even in yeast, amino acid modifications in the histone tails can number in the tens and twenties. Given the number of possible permutations of modification types and amino acids, the question arose, might different combinations of histone modifications produce discrete outcomes? The notion that a sequence or combination of specific modifications on histone tails acts as a signal to other proteins and produces distinct biological effects was advanced as the histone code hypothesis in 2000.

Progress in deciphering the vocabulary, mechanics, and function of the histone code has been hindered by the coarse resolution of available tools. Nucleosomes typically cover about 146 base pairs, but existing technology could only average over 500 to 1,000 base pairs at a timeconfounding the effects of single nucleosomes. In a new study, Oliver Rando and colleagues take advantage of the high resolution afforded by their custom-made microarray, which has a resolution of 20 base pairs. Working with the budding yeast Saccharomyces cerevisiae, the scientists examined 12 different histone modifications in individual nucleosomes and found only a small number of distinct combinations with few discrete histone modification patterns. The concurrent modifications fall into two categories: one set targets a transcriptional start site but is the same no matter what the level of transcription, while the other occurs throughout gene coding regions and is linked to transcription. Importantly, the only modifications that appear to correlate with transcription occur over transcribed regions, as though they were the consequence, rather than the cause, of transcription.

Why might histone tails exhibit so many modifications if they form only two independent categories? It's possible that histone-modifying enzymes may work best in groups and so the marks that recruit themacetyl and methyl groupsalso come in groups. Another possible explanation relates to how histone modifications signal transcription enzymes that a particular gene requires more or less transcription. When the positively charged amino acid lysine acquires an acetyl group, it loses its charge, and chargecharge interactions play a major role in many interactions between proteins and other molecules. Multiple lysine acetylations on the histone tail may thereby aid certain chemical reactions necessary for transcription in a continuous way; having multiple levels of acetylation, for example, may allow the cell to tune proteinprotein interactions, and thus gene expression, up and down, rather than simply turn it on or off.

Rando and colleagues propose that the histone modifications associated with transcription may facilitate rather than trigger gene expression, perhaps by clearing a path for the transcription machinery or attracting proteins needed for the job. The authors are careful to point out, however, that histone modifications may also play some role in initiating gene expression, but that any transcription pattern would likely be obscured, or erased, as transcription occurs. While future studies will help determine which role proves more common, these results suggest that histone modifications are facilitators rather than activators and that the histone code is more a transcription footprint than a starting signal. Liza Gross

DNA buckyballs for drug delivery created

Mon, 29 Aug 2005 22:35:38 PST

( from http://www.rxpgnews.com ) DNA isn't just for storing genetic codes any more. Since DNA can polymerize -- linking many molecules together into larger structures -- scientists have been using it as a nanoscale building material, constructing geometric shapes and even working mechanical devices.

Now Cornell University researchers have made DNA buckyballs -- tiny geodesic spheres that could be used for drug delivery and as containers for chemical reactions.

The term "buckyballs" has been used up to now for tiny spherical assemblies of carbon atoms known as Buckminsterfullerenes or just fullerenes. Under the right conditions, carbon atoms can link up into hexagons and pentagons, which in turn assemble into spherical shapes (technically truncated icosahedrons) resembling the geodesic domes designed by the architect-engineer Buckminster Fuller. Instead of carbon, the Cornell researchers are making buckyballs out of a specially prepared, branched DNA-polystyrene hybrid. The hybrid molecules spontaneously self-assemble into hollow balls about 400 nanometers (nm) in diameter. The DNA/polystyrene "rods" forming the structure are each about 15 nm long. (While still on the nanoscale, the DNA spheres are much larger than carbon buckyballs, which are typically around 7 nm in diameter.)

About 70 percent of the volume of the DNA buckyball is hollow, and the open spaces in the structure allow water to enter. Dan Luo, Cornell assistant professor of biological and environmental engineering in whose lab the DNA structures were made, suggests that drugs could be encapsulated in buckyballs to be carried into cells, where natural enzymes would break down the DNA, releasing the drug. They might also be used as cages to study chemical reactions on the nanoscale, he says.

The nanoscale, hollow buckyballs are also the first structures assembled from "dendrimerlike DNA." If three strands of artificial DNA are created such that portions of each strand are complementary to portions of another, the three strands will bind to each other over the complementary portions, creating a Y-shaped molecule. By joining several Y's in the same way, Luo's research group created molecules with several arms, a sort of tree shape (dendri- means tree in Greek). Then they attached polystyrene molecules to the dendrimerlike DNA forming a hybrid molecule called an amphiphile -- a molecule that both likes and hates water. DNA is hydrophillic -- attracted to water -- while polystyrene is hydrophobic -- water repels it.

The researchers expected the amphiphiles to assemble in water into some sort of solid structure arranged so that DNA would have a maximum interaction with water and polystyrene would avoid water as much as possible. Other researchers have used other amphiphiles to make spheres, rods and other solids. The hollow buckyballs were an intriguing and serendipitous surprise. A model suggests that one buckyball consists of about 19,000 amphiphiles, with their water-loving DNA mostly on the outside of the rods that form the structure. How these tens of thousands of molecules were able to self-organize to form such an intricate and complex structure is still an open question, the researchers say. They are seeking collaborators to solve the puzzle.

A scanning electron microscope photo of a self-assembled DNA buckyball.

Luo and Ph.D. graduate students Soong Ho Um, Sang Yeon Kwon and Jong Bum Lee described DNA buckyballs in an invited talk titled "Self-assembly of nanobuckyballs from dendrimer-like-DNA-polystyrene amphiphiles" Sunday, Aug. 28, at the 2005 annual meeting of the American Chemical Society in Washington, D.C. They reminded the audience that although the geometry of solid truncated icosahedrons was first described by Archimedes on paper more than 2,000 years ago, the skeletal, hollow-faced version of buckyballs had not been envisioned until Leonardo da Vinci's illustrations in 1494.

Luo added that DNA buckyballs may turn out to have unusual electronic, photonic and mechanical properties, and that because DNA is easily labeled and manipulated, his research group's work offers a way to study in detail the self-assembly process -- a process very important to the future development of nanotechnology.

Method to predict protein separation behavior directly from protein structure

Sat, 20 Aug 2005 16:37:38 PST

( from http://www.rxpgnews.com ) Applying math and computers to the drug discovery process, researchers at Rensselaer Polytechnic Institute have developed a method to predict protein separation behavior directly from protein structure. This new multi-scale protein modeling approach may reduce the time it takes to bring pharmaceuticals to market and may have significant implications for an array of biotechnology applications, including bioprocessing, drug discovery, and proteomics, the study of protein structure and function.

Predictive modeling is a new approach to drug discovery that takes information from lab analysis and concentrates it in predictive models that may be evaluated on a computer, said Curt M. Breneman, professor of chemistry and chemical biology at Rensselaer.

The ability to predict the separation behavior of a particular protein directly from its structure has considerable implications for biotechnology processes, said Steven Cramer, professor of chemical and biological engineering at Rensselaer. The research results thus far indicate that this modeling approach can be used to determine protein behavior for use in bioseparation applications, such as the protein purification methods used in drug discovery. This could potentially reduce the development time required to bring biopharmaceuticals to market.

The modeling technique is based on methods previously developed by Brenemans group for rapidly predicting the efficacy and side effects of small drug-like molecules. The newly developed model successfully predicted the amount of a protein that binds to a material under a range of conditions by using molecular information obtained from the protein structure. These predicted adsorption isotherm parameters then replicated experimental results by predicting the actual separation profile of proteins in chromatographic columns. Chromatography techniques are used to identify and purify molecules, in this case, particular proteins.

We intend to test the model against more complicated protein structures as part of its further development, said Breneman. The outcome of this work will yield fundamental information about the complex relationship between a proteins structural features and its chemical binding properties, and also aid in evaluating its potential biomedical applications.

The research findings are reported in the Aug. 16 issue of Proceedings of the National Academy of Sciences in a paper titled A Priori Prediction of Adsorption Isotherm Parameters and Chromatographic Behavior in Ion-Exchange Systems.

In addition to Breneman and Cramer, the collaborative research team includes Asif Ladiwala and Kaushal Rege, who both recently earned doctorates in chemical and biological engineering at Rensselaer. The work was supported by the National Science Foundation and GE Healthcare.

The researchers computational model uses a combination of molecular-level quantitative structure-property relationship models with macroscopic steric mass action isotherm models and support vector machine regression computations.

Gene silencing technique can theoretically cure any disease

Thu, 11 Aug 2005 17:28:38 PST

( from http://www.rxpgnews.com ) A new technique aimed at directly controlling the expression of genes by turning them on or off at the DNA level could lead to drugs for the treatment or cure of many diseases, say researchers at UT Southwestern Medical Center.

"Virtually every disease starts at the level of malfunctioning gene expression, or viral or bacterial gene expression," said Dr. David Corey, professor of pharmacology and biochemistry. "This is an approach that could theoretically produce a drug for the treatment or cure of almost any disease."

In two papers appearing in the online edition of the journal Nature Chemical Biology, Dr. Corey and his colleagues describe how they efficiently shut down gene expression in cultured cells by blocking the ability of chromosomal DNA to be copied into RNA and made into proteins. The studies, which Dr. Corey said represent the most significant findings thus far in his career, are the most definitive to date showing that chromosomal DNA is accessible to and can be controlled by synthetic and natural molecules.

"With this information, one could easily turn on or off gene expression, as well as think about ways to correct genetic disease by changing mutant gene sequences back to normal," Dr. Corey said. "Those types of things now look a lot more feasible."

Genes are segments of DNA housed in the chromosomes in the nucleus of every cell. Genes carry instructions for making proteins, which in turn carry out all of life's functions. Faulty or mutated genes lead to malfunctioning proteins, which cause disease.

The information in a gene is not directly converted into proteins, but first is copied by special enzymes into many copies of messenger RNA, which then move out of the nucleus and into the body of the cell, where they go on to create a protein.

Current techniques for turning genes on or off focus on controlling the messenger RNA once it's already produced. But blocking all the copies of messenger RNA before they can make a protein within a cell is akin to using a bucket to catch all the streams of water coming out of a yard sprinkler before they can hit the ground.

While that's certainly possible, a more efficient way to staunch the streams of water would be to turn off the faucet. By targeting the chromosomal DNA directly, that's just what Dr. Corey and his colleagues accomplished.

The researchers targeted chromosomal DNA in two ways. First, they developed a synthetic molecule called a peptide nucleic acid, or PNA, which physically binds to DNA and blocks enzymes from copying, or transcribing, the DNA into messenger RNA.

More importantly, the researchers also employed RNA itself as a silencing agent. Previous work by other scientists had shown that RNA might be able to target chromosomal DNA, so once Dr. Corey and his team saw that PNAs were working, they decided to try RNA as well.

"The RNA is more important because it may reflect the body's own natural mechanism for controlling gene expression, while the PNAs are synthetic," Dr. Corey said.

"The experiments worked beautifully," he said. "It's hard to believe that this strategy would work so well if nature wasn't doing it already."

The researchers designed their RNA to match up with and target specific genes. "It's possible that the body is making the RNAs that we are using, and that will be an exciting topic for further research, to determine whether the human body or viruses and bacteria make RNA sequences like this to control gene expression," Dr. Corey said.

So far, the researchers have inhibited the expression of nine different genes in cancer cell cultures. Dr. Corey said it's not clear whether the RNA is actually binding to the DNA itself, as the PNAs do, but it's clear the effects are occurring at the DNA level.

New role for CIB1 protein as fundamental inhibitor of cell movement

Sun, 07 Aug 2005 14:34:38 PST

( from http://www.rxpgnews.com ) Scientists from the University of North Carolina at Chapel Hill School of Medicine and the UNC Lineberger Comprehensive Cancer Center have identified a protein that may inhibit cellular movement, or migration.
The protein, CIB1, or calcium and integrin-binding protein 1, was originally discovered at UNC in 1997 as a blood platelet protein that may play a role in clotting.

Cell migration belongs to the most rudimentary of cellular functions that allow processes such as fetal development, new blood vessel formation and wound healing to occur in humans. Increased tumor cell migration also is one of the hallmarks of highly aggressive, rapidly spreading cancer tumors.

The study appears in the August issue of The Journal of Cell Biology.

The study indicates that CIB1 inhibits cell migration by binding to and activating a protein called PAK1, or p21-activated kinase, in cancer cells. When CIB1 activates PAK1, this kinase then inhibits cell migration by adding a phosphate group to a host of other proteins in the cell.

Thus, the study suggests that CIB1 may be a likely target for new drug development aimed at decreasing tumor metastasis, or spread, throughout the body.

"I was ecstatic to see these results and to discover that it also regulates the fundamental process of cell migration," said Dr. Tina Leisner, associate professor of pharmacology at UNC and the study's lead author. "CIB1 plays a prominent role in the activation of PAK1 and potentially may be another important player in the regulation of this kinase," she added.

The other activators of PAK1 include relatives of the notorious Ras family of tumor promoters, the GTPases Rac and Cdc42. CIB1 activation of PAK1, however, is different from these GTPases.

"CIB1 activates PAK1 before Rac and Cdc42," said Dr. Leslie V. Parise, UNC professor of pharmacology, member of UNC Lineberger and the study's senior author.

"The time course of PAK1 activation never synched up with the time course of Rac and Cdc42 activation; now we know why it was probably CIB1 that was activating PAK1 and not the Ras relatives."

In illustrating the role that CIB1 plays in cell migration and PAK1 activation, the authors used a new method known as RNAi or RNA interference to knock down or reduce CIB1 expression in various cell lines. Cells with less CIB1 had less PAK1 activation and migrated faster. The authors also showed that the more CIB1 these cells had, the less likely they were to move.

The key to understanding CIB1's multifunctional role in humans is that the protein has a relative that behaves in a very similar multifunctional fashion: calmodulin. This was one of the first regulatory proteins ever discovered.

"CIB1 is very similar to the protein calmodulin, which binds to a host of other proteins and regulates numerous cell functions, the fact that CIB1 and calmodulin are so similar could suggest that CIB1 may play multiple roles in multiple cell types."

"Our study of CIB1 is still very much in its early days, but its role in migration is already very clear," Parise said.

Structure of membrane protein NhaA revealed

Sat, 06 Aug 2005 11:46:38 PST

( from http://www.rxpgnews.com ) The structure of the membrane protein NhaA has been revealed by researchers at the Hebrew University of Jerusalem and the Max Planck Institute of Germany.

Membrane protein research is at the forefront of modern biological study, with great potential consequences for development of new medicinal treatments and genetic engineering of plants.

The research on NhaA has been carried out by Etana Padan, the Adelina and Massimo DellaPergola Professor of Life Sciences, with Dr. Rimon Avraham, both of the Silberman Institute of Life Sciences at the Hebrew University, and Prof. Hartmut Michel, Nobel prize winner for chemistry in 1988, of the Max Planck for biophysics in Frankfurt, Germany. Their work, described in a recent edition of the journal Nature, was supported by a grant from the German-Israel Binational Science Foundation;

Proteins such as NhaA are found in the membranes of every living cell, from bacteria and up to humans. Until now, the structure of fewer than 50 cell membrane proteins have been discovered, as opposed to 30,000 soluble proteins.

"The location of the proteins in the cell membranes presents tremendous difficulties in research," said Prof. Padan. "Unlike the majority of those proteins which are soluble in water, the membrane proteins are soluble only in fats or in the presence of detergents."

The cell membrane is the crossroads of busy, two-way "traffic" through which materials and impulses travel into and out of the cell. The fatty cell membrane is impenetrable to most of these materials and signals; and it is therefore the proteins within the membranes that are responsible for the communication between the cell and its environment. Indeed, more than 60 percent of the medicines in use today are directed at the cell membrane proteins. Since the cell membrane proteins are exposed, in part, to areas extending outside the cells, the medicines are able to reach them without entering the cell itself.

In Prof. Padan's laboratory, the researchers succeeded in isolating the gene that encodes NhaA in bacteria and in producing a large quantity of the protein in its active state. This achievement paved the way for determining the structure of the protein, providing an essential insight into its mechanism of activity and regulation. NhaA protects the volume of the cell and its internal, normative state in terms of its salinity and acidity.

The deciphering of the NhaA protein's structure was done utilizing three-dimensional crystals of the protein which diffract x-rays. The work of analyzing the diffraction was done using the powerful electron accelerators in Grenoble, France, and Zurich, Switzerland.

"In this way we were able to reveal the wonderful architecture of the membrane protein, which was unknown before," said Prof. Padan. "In the center of the protein we found a wide funnel which extends into the cell. The funnel narrows and ends at the point at which it binds with the sodium or the hydrogen deep within the cell membrane. Near that point two chains of the protein unite into a unique structure."

The researchers believe that this unique structure is the basis for the activity of the protein. The protein operates as a kind of pump, utilizing energy which it receives from processes taking place within the cell. The protein structure thus acts as a kind of molecular motor. This "motor" is connected to the area found at the mouth of the funnel that apparently conveys signals to "modulate" the motor according to the acidity within the cell. The result is that the protein's activity is controlled in accordance with the needs of the cell in relation to its acidic and basic levels.

Molecule that inhibits regrowth of spinal nerve cells

Thu, 14 Jul 2005 23:04:38 PST

( from http://www.rxpgnews.com ) A molecule that helps the body's motor nerve cells grow along proper paths during embryonic development also plays a major role in inhibiting spinal-cord neurons from regenerating after injury, researchers at UT Southwestern Medical Center have found.

In cultured cells, the researchers found that a component of myelin - a substance that normally insulates and stabilizes long nerve fibers in adult vertebrates - chemically blocks the ability of nerve cells to grow through myelin that is released when the spinal cord is damaged.

While other myelin components also block nerve growth, a component called ephrin-B3 inhibits such activity as well or better than that of other known blocking agents combined, UT Southwestern researchers report in an upcoming issue of the Proceedings of the National Academy of Sciences.

"I believe that to the extent that overcoming myelin-based inhibition is going to provide some sort of functional recovery for spinal cord injury patients, understanding ephrins is a major step forward," said Dr. Luis Parada, senior author on the paper and director of the Center for Developmental Biology and the Kent Waldrep Center for Basic Research on Nerve Growth and Regeneration at UT Southwestern.

A mixture of molecules and proteins, myelin insulates nerve fibers and impedes them from having contact with other nerve cells. After a spinal-cord injury, myelin is released into the tissues. Not only does myelin encourage the growth of scars - called glial scars - which physically block nerve cells from regrowing in the damaged area, but components of myelin also chemically prevent nerve cells from regrowing there as well.

Considerable research has been done in the past 10 years to identify elements in myelin that chemically inhibit the regeneration of nerve cells, Dr. Parada said. Three individual components - the molecules Nogo, MAG and OMgp - have been shown to do so in isolation.

Developmental biologists at UT Southwestern have been studying how ephrin-B3 helps control how and where nerve fibers grow during early development. They previously showed that the molecule throws up "fences" that repel developing nerves and guide them along the pathways to their appropriate connections to muscles.

In 2002 Dr. Mark Henkemeyer, associate professor in the Center for Developmental Biology and of cell biology and one of the authors of the PNAS study, found that such a "fence" is erected specifically down the middle of the cortical spinal tract, which is damaged during spinal-cord injury.

In the current study, Dr. Parada and his colleagues asked: What is this molecule, whose normal function is to be repellent during embryonic development, doing in the mature system?

"To our surprise, we found that ephrin-B3, which normally is present as a 'wall' down the middle of adult spinal cords, also is found in very high levels in adult myelin," Dr. Parada said.

The researchers knew from previous work that ephrin-B3 interacts with receptors on neurons in the cortical spinal cord. So, in the lab, led by the study's lead author Dr. M. Douglas Benson, a postdoctoral research fellow, they cultured neurons together with isolated ephrin-B3 and confirmed that the molecule activated the neuron's receptors. They then cultured normal myelin together with the neurons and got the same results.

However, when they cultured neurons with myelin from which the ephrin-B3 had been removed, the receptors were not activated. The findings suggest that there is much more to be learned about myelin-based inhibition, Dr. Parada said.

"We firmly believe that ephrin-B3 is an important, functional, relevant component of myelin, although there may be other elements that are left to be discovered," he said.

Dr. Parada added that several factors must be overcome before spinal-cord regeneration and recovery from injury can occur in a meaningful way for patients.

"We have to figure out how to dissolve the glial scars or impede their formation," he said. "We also need to get mature neurons to be better at growing, similar to the way they do during embryonic development. And finally, we have to remove myelin-based inhibition. If and when we achieve those three things, then we'll have robust regeneration of injured nerves."

Aggresome plays a role in thiopurine metabolism

Sun, 10 Jul 2005 16:00:38 PST

( from http://www.rxpgnews.com ) Mayo Clinic researchers have discovered an inherited structural mechanism that can make drugs for some diseases toxic for some patients. The mechanism decreases a protein and in turn causes certain individuals to metabolize thiopurine drugs differently. Thiopurine therapies are used to treat patients with childhood leukemia, autoimmune diseases and organ transplants. The Mayo researchers say their finding advances the field of pharmacogenomics, which tailors medicine to a patient's personal genetic makeup.

In the current issue of the Proceedings of the National Academy of Sciences, (http://www.pnas.org/cgi/content/abstract/102/26/9394) Mayo researchers report that under certain genetic conditions, key proteins are not formed properly -- they are "misfolded." When misfolding happens, the quality-control process in the cell detects the misfolded proteins and tags them for immediate destruction or quarantines them in a "cellular trash can" known as an aggresome (last syllable rhymes with "foam"). Whether destroyed or aggregated into the aggresome, the effect is the same: the patient's body suffers a protein deficit that disrupts the enzyme that metabolizes thiopurine.

"Our finding is surprising because the aggresome is a new kind of mechanism to study to explain this. It's quite different from what we were thinking even a few years ago," says Liewei Wang, M.D., Ph.D., lead Mayo researcher in the study. "People are still debating what its function really is, but it appears to play a role here by receiving misfolded proteins."

Significance of the Research

"Nobody has shown before that the aggresome plays a role in thiopurine metabolism, and it's a significant contribution," says Richard Weinshilboum, M.D., the Mayo Clinic researcher who first described the genetically variable response to thiopurine drugs over 20 years ago. "From a clinical point of view, the genetic test we developed at Mayo to predict response to thiopurine drugs has been invaluable to pharmacogenomic medicine -- and now this finding is taking us in promising new directions because we believe our findings can be generalized to apply to many instances in the field."

The finding helps explain what goes wrong under certain genetic conditions -- and suggests mechanisms which might help predict which genetic changes could alter the effect of drugs. Prior efforts to explain the mystery of thiopurine metabolism had focused on biochemical mechanisms -- not changes in protein levels.

Background

Researchers have known for decades that 1 in 300 patients of Caucasian European genetic background has two copies of the variant gene -- specifically, a switch in 2 out of 245 amino acids -- that results in the absence of the protein needed to properly metabolize thiopurine drugs. In patients with the genetic defect, instead of helping heal, a standard dose of thiopurine drugs can cause fatal bone marrow destruction. Though Mayo Clinic researchers described this genetically variable response and the danger it presents over 20 years ago, no one had been able to explain the cellular mechanism behind it.

Simple Peptides Stabilize Mighty Membrane Proteins

Wed, 22 Jun 2005 13:04:38 PST

( from http://www.rxpgnews.com ) Cell membranes are largely made of proteins, and membrane proteins account for about a third of all genes. Despite their importance, they are devilishly hard to isolate and stabilize, and therefore are hard to study. The problem lies in their structure: membrane proteins have at least one hydrophobic domain, composed of a stretch of water-repelling amino acids, which holds the protein snugly in the lipid membrane. Purifying such a protein in an aqueous medium makes the hydrophobic parts aggregate, destroying the proteins delicate three-dimensional structure and often disrupting its function. The alternative is to extract the protein with a detergent, a two-headed Janus molecule with both hydrophobic and hydrophilic ends. The protein remains surrounded by the hydrophobic ends, while water clusters at the hydrophilic ends, easing the protein out of the membrane and into solution, where it can be studied.

To date, though, relatively few complex membrane proteins have been successfully purified with available detergents. In this issue, Shuguang Zhang and colleagues show that a simple amino acidbased detergent can successfully stabilize the dauntingly large protein complex photosystem I (PS-I), an integral part of the photosynthetic machinery.

The molecule they made, abbreviated A6K, links six units of the hydrophobic amino acid alanine to one of the hydrophilic amino acid lysine. The authors used it to stabilize PS-I and then attached the detergentprotein complex to a glass slide, allowed it to dry, and examined the stability of PS-I by testing its fluorescence. Intact PS-I emits red light with a characteristic peak wavelength; as it degrades, this peak subsides and is replaced by another, bluer peak. Even the two best standard detergents did poorly at maintaining the red peak. In contrast, the spectrum after A6K extraction was almost a perfect match for the normal one, indicating the complex was largely intact after drying. Furthermore, the complex appeared to remain stable for up to three weeks on the glass slide.

The potential applications of this work are severalfold. PS-I itself remains to be fully characterized, and this stabilization technique offers new means to explore its properties. In addition, an isolated and stabilized form of PS-I may hold some promise as an alternative energy source, since it generates an electric current in sunlight. Perhaps most importantly, the full potential of such simple amino acidbased detergents has only begun to be explored. It is likely that either this one, or others like it, can be used to isolate and stabilize hundreds of other membrane proteins, allowing them to be studied in detail for the first time.

Nano-particle Research to Benefit Inhaler Users

Sun, 01 May 2005 21:09:38 PST

( from http://www.rxpgnews.com ) Patients suffering from conditions as diverse as asthma and diabetes could benefit from research at Cardiff University to improve the effectiveness of drugs taken through spray inhalers.

Scientists in the Welsh School of Pharmacy are working on new nano-particle drug formulations for inhalers, and enhancers to improve the effectiveness of proteins, such as insulin, delivered to the lung.

"Drugs delivered through inhalers are usually either in a suspension (as particles dispersed in liquid), or in a solution (when the drug is dissolved in the liquid)," explained Dr James Birchall. "However, there are problems with both methods - a suspension can lead to sediment in the inhaler and less of the drug reaching the target area of the lung, while solutions present problems in dissolving the drug in the inhaler propellant liquid and can make the drug itself less stable."

The Cardiff team's approach is to prepare the drug in nano-particle form ensuring the correct dosage reaches the lung and the drug retains its stability, and providing the possibility of slowing the release of the drug in the lung for longer therapeutic effect.

This could lead to the possibility of more drugs being administered effectively by inhaler, rather than by tablet or injection.

Meanwhile, the team is also developing a process which uses a naturally occurring substance to enhance the absorption of insulin. Initial studies suggest insulin is absorbed three to four times more effectively by this process.

Now Dr Birchall and his colleague Dr Glyn Taylor of The Pulmonary Research Group aim to combine the two innovations to prolong and maximise the absorption effect.

"These two technologies could make a huge improvement in the effectiveness of spray inhalers for users suffering from a wide range of illnesses and conditions," said Dr Birchall.

Engineer turns Bacteria into Living Computers

Thu, 28 Apr 2005 00:12:38 PST

( from http://www.rxpgnews.com ) In a step toward making living cells function as if they were tiny computers, engineers at Princeton have programmed bacteria to communicate with each other and produce color-coded patterns.

The feat, accomplished in a biology lab within the Department of Electrical Engineering, represents an important proof-of-principle in an emerging field known as "synthetic biology," which aims to harness living cells as workhorses that detect hazards, build structures or repair tissues and organs within the body.

"We are really moving beyond the ability to program individual cells to programming a large collection -- millions or billions -- of cells to do interesting things," said Ron Weiss, an assistant professor of electrical engineering and molecular biology.

Collaborating with researchers at the California Institute of Technology, Weiss and graduate student Subhayu Basu programmed E. coli bacteria to emit red or green fluorescent light in response to a signal emitted from another set of E. coli. In one experiment, the cells glowed green when they sensed a higher concentration of the signal chemical and red when they sensed a lower concentration. In a Petri dish, they formed a bull's-eye pattern -- a green circle inside a red one -- surrounding the sender cells.

In addition to demonstrating that the genetic programming techniques work, this sensing system could be useful for the detection of chemicals or organisms in laboratory tests. "The bull's-eye could tell you: This is where the anthrax is," said Weiss.

The researchers published their results in the April 28 issue of Nature. In addition to Weiss and Basu, authors of the paper are postdoctoral researcher Yoram Gerchman at Princeton and professor of chemical engineering Frances Arnold and graduate student Cynthia Collins at Caltech. It was funded by a grant from the U.S. Defense Advanced Research Projects Agency.

In previous work, including a paper published March 8 in the Proceedings of the National Academy of Sciences along with Sara Hooshangi and Stephan Thiberge, Weiss showed the feasibility of inserting engineered pieces of DNA into cells to make them behave in the same manner as digital circuits. The cells, for example, could be made to perform basic mathematical logic and produce crisp, reliable readouts that are more commonly associated with silicon chips than biological organisms. The new paper applies similar techniques to a large population of cells.

"Here we're showing an integrated package where the cells have an ability to send messages and other cells have the ability to act on these messages," said Weiss.

The creation of patterns, such as the bull's-eye effect, is a key step in one of Weiss' eventual goals, which is to have the cells secrete materials that build physical devices such as antennas or transmitters in places that are hard for humans to reach. Programmed cells also could be used to control the repair or construction of tissues within the body, possibly guiding stem cells to the locations where they are needed for the growth of new nerve or bone cells in a process Weiss called "programmed tissue engineering."

Even the early step of creating patterns in a Petri dish, however, may be useful as a tool for other scientists, particularly developmental biologists who are trying to understand how the cells of an embryo arrange themselves into patterns that become the various body parts of a mature organism. In fruit fly embryos, for example, the first cells are thought to differentiate into the head, abdomen and other parts based on the concentration of chemical signals that are emitted from the ends of the embryo.

In addition to conducting laboratory experiments, Weiss and colleagues are creating computer models of their engineered systems, which allow them to study how small modifications would affect the ultimate behavior of the organisms. So far, said Weiss, the experimental results have matched the computer models fairly closely, but the goal is to have a mathematically exact description of how each component works.

"One of the nice things about synthetic biology is that because we built the network from scratch, we should be able to model all the important details," he said. At some point in the future, he said, scientists will be able to choose a behavior they want from cells, and a computer program will create a genetic circuit to accomplish the task. "Then we can do an experiment to see if the community of cells is behaving as we desire. That is going to have a tremendous number of applications."

Structural Insights into a Porphyrin-Binding Protein

Wed, 27 Apr 2005 02:26:38 PST

( from http://www.rxpgnews.com ) Eukaryotic cells have an organizational problem. The specialized proteins found in cellular organelles (structures with specific functions such as energy production) are mostly encoded within the nucleus. To build and maintain a cell that works efficiently under all conditions, each type of organelle needs to be able to send signals to the nucleus to say Send more protein X or hold back on enzyme Y. Think of it as the cellular version of grocery store clerks restocking orders to the warehouse.

In plant cells, the chloroplasts (the photosynthetic organelles that convert light excitation energy into chemical energy) send signals to the nucleus to control the expression of the genes that encode chloroplast-localized proteins such as the enzymes that fix carbon dioxide, make chlorophyll, or perform photosynthesis. The accumulation of the chlorophyll precursor Mg-protoporphyrin IX provides one of these signals. A protein called GUN4 both enhances the activity of Mg-chelatase, the enzyme that makes Mg-protoporphyrin IX, and plays a role in the chloroplast-to-nucleus signaling activity of Mg-protoporphyrin IX in Arabidopsis, a well-studied plant.

To discover how GUN4 has these effects, Mark Verdecia in Joseph Noels laboratory and Rob Larkin, formally in Joanne Chorys laboratory, determined the crystal structure of the GUN4 equivalent in the cyanobacteria Synechocystis. Cyanobacteria are the evolutionary ancestors of chloroplasts, so whatever GUN4 does in these cells is likely to be important in plant cells. The researchers crystallographic studies, together with nuclear magnetic resonance and other studies, indicate that the porphyrin-binding region of Synechocystis GUN4 has a unique three-dimensional shape that resembles a cupped hand, the inner concave surface of which is highly hydrophobic. Because of this tendency to repel water, the researchers call this region the greasy palm of the cupped hand.

This structure suggests how GUN4 is involved in the chloroplast-to-nucleus signaling activity of Mg-protoporphyrin. By wrapping Mg-protoporphyrin IX in its cupped, greasy palm, the GUN4 structure provides a novel vehicle for binding Mg-protoporphyrin IX and may be involved in transporting signals from the chloroplast to the nucleus. In addition, the structure also suggests that GUN4 may be involved in photoprotection. Although light drives photosynthesis, which is essential to green plants, light has a downsideporphyrins combine with the oxygen released during photosynthesis to generate reactive oxygen species, which are generally damaging to the cell. GUN4, by cocooning Mg-protoporphyrin IX in its protective hand, may provide a way to safely move porphyrin around the chloroplast without exposing it to oxygen. Finally, the detailed structural and functional studies described by Noel and colleagues explain how GUN4 enhances the activity of Mg-chelatase. GUN4 binds Mg-protoporphyrin IX, the product of the chelatase, much better than protoporphyrin IX and so will tend to enhance the enzymatic reaction by removing its product.

Domain protecting proteins from degradation identified

Mon, 18 Apr 2005 21:36:38 PST

( from http://www.rxpgnews.com ) The first stabilizing signal that protects a protein from degradation has been identified.

Dr Nico Dantumas research group at Karolinska Institutet has identified the first cellular stabilization signal, which protects a protein from being degraded.

Rad23 is a protein that is important for DNA repair and protein degradation and for both functions, it has to bind to the proteasome (see below). The new findings show that a motif in the tail of Rad23, the so called UBA domain, protects it from degradation. Interestingly, it has been described that Rad23 is itself linked to the proteasome but it is not degraded by the proteasome. Because of this stabilization signal, Rad23 is the perfect shuttle to bring things to the proteasome since it can escape every time and be reused over and over again. UBA domains from several other proteins can also protect proteins from degradation showing that it is a general phenomenon.

The ubiquitin/proteasome system:
Proteins that have to be degraded in the cell are labelled through the linkage of a chain of many ubiquitins, which is a small abundant protein. This chain of ubiquitins binds to the proteasome, which is a large barrel shaped protease complex. The proteasome starts to unravel the protein that has to be degraded and transports it into the belly of the proteasome, where the protein is chopped in small pieces. It is well established that proteins, that have to be labelled with ubiquitin and next degraded by the proteasome, are recognized by the presence of degradation signals, which are recognition motives within proteins that triggers enzymes to link a ubiquitin chain to this protein.

At the molecular level, the predator is the prey

Thu, 14 Apr 2005 16:00:38 PST

( from http://www.rxpgnews.com ) An evolutionary arms race between predatory garter snakes and their newt quarry is turning out to be something of an illusion. At the molecular level, another battle rages. And in this second, miniature realm, it's the newt who's the aggressor.

Biologists at Indiana University Bloomington, Utah State University and the University of Utah present evidence in this week's Nature that a toxin produced by the rough skinned newt, Taricha granulosa, has forced several evolutionary changes in the garter snake Thamnophis sirtalis or, more specifically, in the snake nerve cell protein that endures the toxin's attacks.

Embedded in the surface of garter snake nerve cells is tsNa(V)1.4, a tube-shaped protein that allows sodium ions to flow into the cell. When nerve cells' ability to move sodium in and out is hampered, paralysis and death can result. Tetrodotoxin (TTX), a powerful paralytic poison concentrated in the newts' skin, can bind to garter snake nerve cell channels and prevent sodium ions from flowing freely.

"These channels are absolutely fundamental to every aspect of nerve and muscle function and are highly specific gateways for sodium ions," said IUB biologist Edmund Brodie III, one of the paper's coauthors. "If the channels change too much or in the wrong way, they can't perform their basic, everyday functions. Sodium channel genes in different vertebrates are virtually identical to each other, but not in these snakes. We're finding a molecular arms race is driving rapid and repeated changes in the gene within this group of beasts."

For TTX to bind successfully to the sodium channel, the toxin needs something to bind to. At this moment in the garter snake's evolutionary history, TTX infiltrates a hole on tsNa(V)1.4's surface, binding to an aromatic amino acid and causing enough of a change in the sodium channel's shape to impair its function. Three of the four Pacific Northwest snake populations the scientists examined have evolved some degree of resistance to TTX by making this aromatic amino acid harder for TTX to grasp -- or by removing it altogether.

One-thirtieth of the TTX normally found in a T. granulosa newt is enough to kill the average human being. The only organisms on Earth that can eat T. granulosa newts and survive are some T. sirtalis garter snakes. TTX is a defensive compound found in some puffer fish, octopuses and primitive chordates. It is used in low concentrations to treat morphine and heroin addicts. It's also the "zombie" drug used by Haitian voodoo ritualists.

Despite its action at the molecular level, the evolution of TTX in some organisms is viewed by ecologists as a defense mechanism. In the case of T. granulosa newts and T. sirtalis garter snakes, the interaction has gone far beyond that simple fangs-off arrangement, evolving into a lethal contest of toxification/detoxification one-upsmanship.

"One might think that this sort of change in the sodium channel would be too costly to the snakes," said Utah State University biologist Shana Geffeney, who conducted the gene expression experiments. "What will be interesting in the future is to understand if there is a balance between the costs of the changes in the channel pore structure on channel function and the benefits of changes in TTX binding."

The evolution of new traits often happens one of two ways, either by altering existing genes or by changing patterns and amounts of expression. The current Nature report shows that snakes' ability to detoxify TTX involves changes in the sodium channel gene.

"That is generally the story as it is developing," Brodie said. "Ecological arms races that go on between predator and prey are really driven at the molecular level. We have no evidence, nor reason to believe, that TTX is changing too, but rather that the toxin responds in quantity. Pour on more toxin, change the snake's sodium channel. Add more toxin, force further changes in the channel."

Simplicity of Histone Code Revealed

Wed, 30 Mar 2005 06:44:38 PST

( from http://www.rxpgnews.com ) Histones, which package DNA in eukaryotes, play an important role in gene regulation. According to the histone code hypothesis, covalent posttranslational modification of histone tails, in this case, acetylation, influences gene regulation by altering transcriptional output.

Michael Dion et al. tested this hypothesis by creating mutant yeast strains in which the four lysines (K) in the histone H4 tail of budding yeast, lysines 5, 8, 12, and 16, were replaced with arginine (R) in all 16 possible combinations. The replacement of lysine with arginine mimics the unacetylated lysine. The team analyzed the gene expression with DNA microarrays and showed that only the K16R substitution changed gene expression of a unique set of genes. Of the 125 genes whose expression differed, 67 showed a >2-fold change.

The authors suggest that this result is consistent with a transcription mechanism involving a K16-binding protein. Mutations in the other three lysines did not uniquely affect gene expression. Instead, the total number of these K-to-R mutations caused incremental genome-wide shifts in expression. The authors conclude that the four H4 lysines produce a total of 8 transcription states rather than the possible 16, suggesting that the histone code is simpler than expected.

Dynamic, Artificial Cells and Vesicles

Wed, 30 Mar 2005 06:44:38 PST

( from http://www.rxpgnews.com ) Scott Long et al. have constructed synthetic cells comprised of lipid bilayer membranes surrounding a two-phase, aqueous polymer solution that can be reversibly converted to a single phase. An understanding of compartmentalization in cell function has been hampered by the lack of an experimental model system.

Using a previously developed method, the authors constructed artificial cells by encapsulating a poly(ethylene glycol) (PEG)/dextran aqueous two-phase system (ATPS) within a giant lipid vesicle. The researchers tagged each macromolecule, PEG, dextran, and lipid, with an individual fluorescent marker to monitor transitions by confocal microscopy.

The two encapsulated phases were found to contain different polymer concentrations, and the authors were able to create microcompartments within the synthetic cells by introducing molecules with varying affinities for PEG or dextran. Fluorescently tagged streptavidin accumulated preferentially in the PEG-rich phase of a vesicle containing biotinylated PEG, whereas SBA, a carbohydrate-binding lectin, partitioned into the dextran-rich phase of the vesicles.

Through osmotically driven dehydration, Long et al. were able to partition single-stranded DNA oligonucleotides into the dextran-rich phase of an ATPS-containing vesicle.

Bmpr1a and Bmpr1b Essential for Cartilage Formation

Wed, 30 Mar 2005 06:44:38 PST

( from http://www.rxpgnews.com ) Byeong Yoon et al. report that bone morphogenetic protein (BMP) signaling is essential for the generation of chondrocytes in vivo.

During development, chondrocytes proliferate and form the cartilage that acts as the template for bone formation. Previous studies have demonstrated that BMPs can promote differentiation of chondrocytes in vitro.

To clarify the in vivo role of BMP signaling during chondrocyte development, Yoon et al. generated mice lacking the BMP receptors Bmpr1a and Bmpr1b in chondrocytes. The researchers showed that mice deficient in either Bmpr1a or Bmpr1b were able to form cartilage elements and had few skeletal defects, but Bmpr1a/Bmpr1b double mutants developed severe generalized chondrodysplasia, including delayed bone formation and shortened long bones.

Cartilage condensations in double mutants were reduced in size because of decreased proliferation and increased apoptosis. The few cartilage condensations that developed were arrested in the prechondrocytic stage and never formed an organized growth plate. In addition, the mutant cartilage lacked Sox9, a transcription factor involved in early chondrocyte differentiation, as well as the downstream targets of Sox9, L-Sox5, and Sox6.

The authors conclude that Bmpr1a and Bmpr1b are functionally redundant during early chondrogenesis, and that BMP signaling is required for chondrocyte proliferation, survival, and differentiation in vivo.

Triterpenoids Protect Against Oxidative and Electrophile Stress

Wed, 30 Mar 2005 06:44:38 PST

( from http://www.rxpgnews.com ) Albena Dinkova-Kostova et al. report that synthetic triterpenoid (TP) analogs of oleanolic acid can activate the phase 2 response, which protects cells against electrophile and oxidant toxicities and blocks the inflammatory response to IFN-γ.

The specific mechanisms of the antiinflammatory and anticarcinogenic effects of these TPs remain unknown. To investigate these mechanisms, the authors exposed varying mouse and human cells to these TP analogs and detected an increase in the activity of NQO1, a chemoprotective enzyme, and a decrease in the synthesis of IFN-γ-evoked, proinflammatory iNOS and COX-2 enzymes.

Potencies of TP analogs for both responses closely correlated over a wide range of concentrations. The most effective analogs had activated Michael acceptor groups in rings A and C, at a critical distance from each other. In addition, TP-225, the most potent TP, protected human retinal epithelial cells against photooxidative damage by UV-A light.

Cells that lacked Nrf2, the principal phase 2 transcription factor, and/or Keap1, the sensor for inducers and a repressor of Nrf2, displayed diminished ability to respond to the TP analogs.

Triterpenoids Protect Against Oxidative and Electrophile Stress

Tue, 29 Mar 2005 17:49:38 PST

( from http://www.rxpgnews.com ) Albena Dinkova-Kostova et al. report that synthetic triterpenoid (TP) analogs of oleanolic acid can activate the phase 2 response, which protects cells against electrophile and oxidant toxicities and blocks the inflammatory response to IFN-γ.

The specific mechanisms of the antiinflammatory and anticarcinogenic effects of these TPs remain unknown. To investigate these mechanisms, the authors exposed varying mouse and human cells to these TP analogs and detected an increase in the activity of NQO1, a chemoprotective enzyme, and a decrease in the synthesis of IFN-γ-evoked, proinflammatory iNOS and COX-2 enzymes.

Potencies of TP analogs for both responses closely correlated over a wide range of concentrations. The most effective analogs had activated Michael acceptor groups in rings A and C, at a critical distance from each other.

In addition, TP-225, the most potent TP, protected human retinal epithelial cells against photooxidative damage by UV-A light. Cells that lacked Nrf2, the principal phase 2 transcription factor, and/or Keap1, the sensor for inducers and a repressor of Nrf2, displayed diminished ability to respond to the TP analogs.

Unique Mutant Leptin Protein Developed

Fri, 18 Mar 2005 22:26:38 PST

( from http://www.rxpgnews.com ) A unique technique for neutralizing the action of the leptin protein in humans and animals thereby providing a means for controlling and better understanding of leptin function, including its role in unwanted cell growth -- has been developed by researchers at the Hebrew University of Jerusalem.

Leptin was discovered ten years ago and has attracted attention first because of its involvement in control of appetite and later by its effect on growth, puberty, digestion and immunological processes. Leptin can also have negative consequences, such as, for example, enhancing the spread of tumorous growths.

In his laboratory at the Hebrew University's Faculty of Agricultural Food and Environmental Quality Sciences in Rehovot, Arieh Gertler, the Karl Bach Professor of Agricultural Biochemistry, along with his students, has developed a technique for genetically engineering mutations of the leptin protein. Prof. Gertler has been assisted in this work by graduate students Dana Gonen-Berger and Leonora Niv-Spector and research assistant Gili Benyehuda.

In experimental work carried out cooperatively with researchers at the Agronomic Research Institute of France and the University of Paris VI, the scientists have developed a model showing which amino acids in leptin are responsible for activating leptin receptors in living cells. By replacing these amino acids with others, they were able to create a leptin variant that could bind with cell receptors, but would be unable to activate them, thereby providing a unique, novel research tool. In this way, the mutated leptin, with the substituted amino acids, acts as an "antagonist," competing with the normal leptin for the "attention" of the cell receptors to which both leptins are attracted. The result is a "standoff" situation in which the normal leptin is neutralized.

Since leptin is involved in several cell functions, the development of this mutated "antagonistic leptin" could have significant consequences not only for better understanding of leptin action in animals but also on halting undesirable leptin effects in humans, such as undesired cell proliferation in cancer, or in controlling other pathological phenomena in which leptin is a factor.

Thus far, the researchers have succeeded in creating antagonists of human, sheep, rat and mouse leptins.

A company, Protein Laboratories Rehovot (PLR), that was formed by Prof. Gertler and the Hebrew University's Yissum Research Development Company 18 months ago, was given the license to produce and market the mutated leptin products. Further development is currently being pursued towards testing whether leptin antagonists are capable of anti-cancer activity. This work is being pursued in cooperation with Prof. Nira Ben-Jonathan of the University of Cincinnati in the U.S., with the assistance of Prof. Gertler's graduate student, Gila Ben Avraham.

Gene Therapy to Restore Hair Cells in Deaf

Tue, 01 Mar 2005 17:50:38 PST

( from http://www.rxpgnews.com ) Researchers supported by the National Institute on Deafness and Other Communication Disorders successfully used gene therapy to grow new hair cells and restore hearing in deaf guinea pigs. This is the first time researchers have been able to restore structural and functional levels to auditory hair cells in live adult mammals.

Scientists at the University of Michigan's Medical School inserted a gene called Atoh1, a key regulator of hair cell development into non-sensory epithelial cells that were in the inner ears of adult guinea pigs whose original hair cells were destroyed by exposure to ototoxic drugs. An adenoviral vector was used to deliver the Atoh1 gene into the left ears of ten guinea pigs whose hair cells had been destroyed; deaf guinea pigs that were not administered the gene served as matched controls, and the animals right ears served as an additional control.

Eight weeks after treatment they found new hair cells in the ears treated with Atoh, and auditory testing confirmed that the generation of hair cells coincided with restoration of auditory threshold levels.

The researchers caution that restoring auditory threshold levels is not the same as restoring normal hearing and it will be several years before Atoh1 gene therapy will be ready to test in humans. This study is an important advance in hearing research, and the findings bring scientists one step further in the search for new ways to treat hearing loss that affects approximately 28 million Americans.

Wnt10b signalling protein protects against bone loss from aging or lack of estrogen

Tue, 22 Feb 2005 18:16:38 PST

( from http://www.rxpgnews.com ) Leaping tall buildings in a single bound may be out of the question, but the genetically engineered "supermice" in Ormond MacDougald's laboratory at the University of Michigan Medical School are definitely stronger than average. With bone mass up to four times greater than ordinary mice, these research animals could hold the secret to new drugs for preventing or treating osteoporosis and other human diseases.
The secret appears to be a secreted signaling protein called Wnt10b. Known to inhibit the development of adipose tissue in mice, Wnt10b also stimulates the growth of bone cells, according to a new study that will be published February 21 in the Online Early Edition of the Proceedings of the National Academy of Sciences.

"High levels of Wnt10b expression in bone marrow directly increased bone mass and density in our experimental mice," says Ormond A. MacDougald, Ph.D., associate professor of molecular and integrative physiology in the U-M Medical School. "This is the first identification of a specific signaling protein in the Wnt family that regulates bone formation."

Wnt10b is one of a family of 19 related proteins. Wnts (pronounced "wints") regulate the complex changes that take place as an embryo develops. One step in this process determines the fate of primitive cells called mesenchymal stem cells.

"In bone marrow, mesenchymal stem cells have the potential to become either fat cells called adipocytes or bone-forming cells called osteoblasts," MacDougald says. "In adult animals, including humans, there's a reciprocal relationship between bone and marrow fat. Our research indicates that Wnt10b's signal blocks the fat cell pathway and stimulates the osteoblast pathway, which means less fat and more bone."

To study the effect of Wnt10b gene expression on tissue development, MacDougald's research team created an artificial sequence of DNA called a transgene linking Wnt10b to the FABP4 promoter, which is expressed in fatty tissue and in bone marrow. U-M scientists injected the transgene DNA into fertilized mouse eggs, and then bred mice that inherited the new gene to create the transgenic animals used in their research.

Kurt D. Hankenson, D.V.M., Ph.D., a U-M assistant professor of orthopedic surgery and laboratory animal medicine, and Christina N. Bennett, a U-M graduate student and first author of the PNAS paper, used a technology called micro-computerized tomography to scan femur (leg) bones from mice that inherited the FABP4-Wnt10b gene combination and compare them to scans from normal mice.

Bennett and Hankenson discovered that femurs from the transgenic mice had almost four times as much bone, and were mechanically stronger than femurs from control mice. (Note to editors: An image showing the femur scan comparison is available.)

"It was a very exciting moment the first time we saw scans showing increased bone mass in transgenic mice," Bennett says. "Visually, we don't see any abnormal side-effects in bone from the transgenic mice. Its development and morphology appear to be completely normal."

Loss of bone often develops with aging, but Wnt10b transgenic mice maintained their high levels of bone mass up to the ripe old age of 23 months, when the study was concluded.

Estrogen deficiency in females is another common cause of bone loss. When U-M scientists removed ovaries from normal mice in the study, they developed reduced bone mineral density and bone volume. But the Wnt10b females showed no bone loss after their ovaries were removed. "Because the transgenic mice have more trabecular bone, or bone within the marrow cavity, to begin with, they are doubly protected from the usual loss of bone density due to estrogen deficiency," MacDougald adds.

To confirm that Wnt10b was the key to increased bone formation, Bennett and Hankenson scanned bones from a strain of laboratory mice that didn't have a gene for Wnt10b. Lacking the ability to produce Wnt10b protein in bone marrow cells, these mice had 30 percent lower bone volume and bone mineral density than normal mice.

Using PCR analysis of Wnt10b-expressing cells in bone marrow, MacDougald found high levels of collagen and alkaline phosphatase, and expression of transcription factors that turn on genes involved in bone formation.

Bennett discovered another important clue when she found that Wnt10b expression shuts down activity of a gene called PPAR-gamma, which is required for the development of adipocytes or fat cells. "It suggests that Wnt10b's role may be to block PPAR-gamma, shifting development from the adipocyte pathway to the osteoblast pathway," she says.

In future research, MacDougald hopes to unravel the molecular mechanism for Wnt10b's bone-building effect. "It's not only an important scientific question, it's important to the understanding and potential treatment of osteoporosis and other human diseases," he says. "Right now, there is a need for drugs on the market to stimulate new bone formation. Being able to activate Wnt signaling in bone marrow and osteoblasts might help prevent the loss of bone associated with aging or menopause."

The research was funded by the National Institutes of Health, the U-M Diabetes Research and Training Center, the U-M Core Center for Musculoskeletal Disorders, and the Nathan Shock Mutant and Transgenic Rodent Core. Fellowships to Christina Bennett were from the Tissue Engineering and Regeneration Training Grant and the American Physiological Society Porter Fellowship. Kenneth Longo was supported by a mentor-based postdoctoral fellowship from the American Diabetes Association.

The experimental mice used in the study were produced in the U-M's Transgenic Animal Model Core facility. The University of Michigan has filed for patent protection on the Wnt10b transgenic mouse.

Additional collaborators on the study include Kenneth A. Longo, Ph.D., a former research fellow in MacDougald's lab who is now a postdoctoral fellow in the U-M School of Dentistry; Wendy S. Wright, research associate; Larry J. Suva, Ph.D., Center for Orthopaedic Research, University of Arkansas for Medical Sciences; and Timothy F. Lane, Ph.D., Jonsson Comprehensive Cancer Center, University of California, Los Angeles, who developed the Wnt10b knock-out mouse.

MacDougald and his research team published a paper in the August 2004 issue of the Journal of Biological Chemistry, which showed that Wnt10b over-expression in adipocytes produced mice with 50 percent less body fat and fewer fat cells.

Grant Announced for Latest Software to Produce Blueprint of Designer Drug to Fight Influenza

Tue, 22 Feb 2005 08:14:38 PST

( from http://www.rxpgnews.com ) Chemists are shortly to begin using state-of-the-art computer technology to tackle deadly diseases such as influenza.

Researchers at the University of Bath have won a £261,000 grant to use the latest software to produce a blueprint of a designer drug that could stop influenza and some other diseases from replicating in humans.

The announcement of the grant comes at a time when fears are rising that an influenza outbreak developing from Asian chickens could kill thousands of people.

Professor Ian Williams, of the Department of Chemistry, will begin work in April on a project that could help pharmaceutical companies develop a better drug that could be taken by people coming down with flu to stop the disease developing.

The drug would work by being chemically very similar to part of the protective coating around the cells in our throats that the flu virus first attacks when a person becomes infected. The flu virus would be deceived into attacking the drug, called an inhibitor, instead of the cells.

The three-year project will be largely carried out by examining the behaviour of atoms of the influenza virus which attack cells, and atoms of the throat cells that are attacked. By using advanced software to model the way these atoms behave in highly complex interactions, the atomic structure of a suitable drug can be worked out.

Using computer modelling in this way can be of great assistance in drug design. Normally drugs are produced by trial and error in a process that can take many years.

Professor Williams and his colleague Dr Gus Ruggiero will use part of the grant from the Biotechnology and Biological Sciences Research Council to buy computers with a combined power many times that of the most advanced desktop machines.

This will be the first time the software, developed in Germany, will have been used in Britain. It will allow accurate modelling of the behaviour of tens of thousands of atoms, many times more sophisticated than previous work.

Developing a blueprint for a new way of fighting influenza is a very important task," said Professor Williams.

We often think of flu as just a nasty illness which puts us in bed for a few days. But some outbreaks can cause death on a large scale the world-wide outbreak in 1918 killed more people than the First World War itself. We may now be facing another flu outbreak, this time originating from chickens in Asia.

If we are successful, we will have taken important steps in finding a new way of fighting influenza and other diseases. It will then be for the pharmaceutical companies to take our blueprint and turn it into a drug.

Professor Williams said that his work is a more sophisticated development from similar modelling which produced two anti-influenza drugs, Relenza and Tamiflu, whose effectiveness is limited.

He and Dr Ruggiero will study sialidases, enzymes used by the flu virus to snip off a special type of sugar, sialic acid, from the throat cell, allowing the virus to enter the cell and reproduce.

The new drug would be chemically similar to the sialic acid, but would act to inhibit the sialidase. This would hinder the virus from entering the cells, and from leaving them should they gain entry, thereby controlling the spread of the infection.

Because of similarities between the enzymes used by different viruses and bacteria, a similar approach may also be useful in fighting other diseases such as the South American sleeping sickness, Chagas Disease.

Dead bone graft can be converted to living tissue by gene therapy

Sat, 19 Feb 2005 16:57:38 PST

( from http://www.rxpgnews.com ) Researchers have created a way to transform the dead bone of a transplanted skeletal graft into living tissue in an experiment involving mice. The advance, which uses gene therapy to stimulate the body into treating the foreign splint as living bone, is a promising development for the thousands of cancer and trauma patients each year who suffer with fragile and failing bone grafts.

The procedure, designed by a team led by Edward M. Schwarz, Ph.D., associate professor of orthopedics and of microbiology and immunology at the University of Rochester Medical Center, is intended to eventually aid people with various cancers or injuries whose treatment involves the replacement of large sections of bone. Cancers such as osteosarcoma, one of the most common types of bone cancers, or tumors that occur adjacent to bones, often must be treated by removing the diseased section of bone and replacing it with the only alternative available a donated section of comparable bone from a cadaver. The new splint of bone is then literally screwed into place, giving the patient most of the strength and support of the original bone. Bone, unlike any other tissue in the human body, can still perform one of its functions, structural support, even if all its cells are completely dead. A serious problem arises, however, when the bone wears over time.

"Everyday activities cause microscopic fractures in our bones," explains Schwarz. "Those fractures are normal and healthy, and our bones re-knit them constantly. But when the bone is dead, there is no healing, and those tiny fractures begin to accumulate until finally, perhaps in 10 years, the implanted section collapses, and more drastic surgery becomes necessary."

To make the transplanted bone more robust, Schwarz looked into the activity of the genes and proteins that govern its health. His team replaced sections of bones in dozens of mice, using both healthy and dead segments, then scanned the surrounding inflammatory tissue for differences in the levels of the active genes. He discovered that the genes that create two key proteins in living bone, called RANKL and VEGF, were barely expressed around the dead bone. He then modified a harmless virus to carry these genes, devised a method of freeze-drying a paste containing the virus so it could be easily handled, and painted it directly onto a bone graft during surgery.

Numerous tests in mice confirmed that the virus permeated the inflammatory tissue around the dead bone and turned on the genes. The mouse body then began to treat the implanted bone as if it were its own tissue instead of a foreign object, which would normally trigger the body to wrap the "invader" in scar tissue.

"That recognition is the key," says Schwarz. "It's at that point that the body actually begins changing the dead, foreign bone splint, into the body's own, whole, living bone."

Such a transformation can occur because mammals use their skeletons for both support and as a kind of "calcium bank." If calcium, which is necessary for such important functions as maintaining the brain and heart, runs low, cells called osteoclasts dig out the calcium from our bones. This is why doctors encourage post-menopausal women to take calcium supplements, so that the body doesn't raid their bones for the calcium it needs. The process works both ways, fortunately, as another set of cells, called osteoblasts, rebuilds the bone when the body has excess calcium. In an average year, a healthy person may remove and rebuild 10 percent of his or her bone structure.

This process of teardown and rebuilding is triggered in the dead bone when Schwarz paints it with the genetically modified virus. New blood vessels begin to grow around and into the bone splint, stripping it down in times when the body needs the calcium, and rebuilding it when calcium levels rise. The bone that is rebuilt is now fully the patient's own, as if the dead bone were a house being renovated by replacing a single brick at a time without tearing the whole structure apart.

Schwarz's studies with mice showed their dead splints were quickly converted to new, healthy bone. He projects that the bone would be completely converted in just a year, and that a human bone might be completely converted in as little as five years. The Musculoskeletal Transplant Foundation has been trying for two decades to conquer the issues complicating bone transplants, and the group has pledged to continue supporting Schwarz's research. Schwarz hopes to begin early human trials with the procedure soon.

"This technology looks like it will have a dramatic effect on success rates for cancer patients who would otherwise be facing choices as drastic as amputation," says Arthur A. Gertzman, executive vice president for research and development at the Musculoskeletal Transplant Foundation.

Other scientists have attempted to invoke a similar reaction in dead bone by infusing tissue-growing proteins directly into the bone. This has proven successful for the small bone transplants used in spinal fusion, but not for larger grafts, because the proteins' effectiveness wear off in just hours. By contrast, the gene therapy method triggers the tissue surrounding the graft to produce the proteins continuously for up to three weeks, long enough for the body to trigger the perpetual bone remodeling response. Stem cells have also seen success in this area, but Schwarz says their appeal has waned because their handling keeping them alive and ready to use is far harder than Schwarz's viral paste, which can be stored at room temperature and doesn't interfere with the normal grafting surgery.

"We're very excited about the prospects of this technology," says Schwarz. "Our ultimate goal is to apply this to one of the Holy Grails of orthopedics cartilage repair. Unlike bone, damaged cartilage, even in a healthy person, can't re-grow or repair itself.

"It's a steeper challenge that would require additional technology such as light-activated gene therapy (LAGT) to site-specifically target the genes to the edge of the damaged cartilage during arthroscopic surgery, but we're looking to use the same idea of triggering the cartilage to remake itself."

A Biotech Chip will Now Allow Rapid Screening of Drug Toxicity

Sat, 12 Feb 2005 10:26:38 PST

( from http://www.rxpgnews.com )

Researchers at the University of California, Berkeley, and Rensselaer Polytechnic Institute have created a biotech chip that mimics the metabolic reactions in the human liver, allowing rapid screening of potential drugs to identify those activated by the liver and to weed out those made toxic.

"The MetaChip would allow testing a backlog of compounds for toxicity earlier in the drug discovery process faster and more efficiently and help remove a current bottleneck in the drug discovery process," said Douglas S. Clark, professor of chemical engineering at UC Berkeley.

The MetaChip, short for metabolizing enzyme toxicology assay chip, was developed by Clark and colleague Jonathan S. Dordick, the Howard P. Isermann '42 Professor of Chemical and Biological Engineering at Rensselaer. The chip used in the current study was produced by the biotech company Solidus Biosciences, Inc., a startup they founded in Troy, New York, with funding assistance from the National Institutes of Health.

"The MetaChip offers a new approach in the identification of pharmacologically safe and effective lead drug compounds for advancement to the preclinical phase of drug development," said Dordick. "The research results thus far indicate that this technique could be incorporated into an effective process for toxicity analysis at early stages in drug discovery."

The liver is the body's detox station, degrading chemicals and often, in the case of drugs, activating them to become effective elsewhere in the body. Clark and Dordick took several of the liver's major detoxification enzymes, called cytochrome P450 enzymes, and put them on a chip in order to create liver metabolites of drug candidates and rapidly test them for toxicity against specific types of cells.

"Many compounds taken as drugs are not active until they are metabolized by enzymes in the liver," Clark explained. "The MetaChip products correspond to those generated in the liver, but then they can be screened against many different cell types.

In their new study, Clark and Dordick tested liver metabolites against breast cancer cells as a model system to find metabolites that damage or kill the cells.

The study was published by Clark, Dordick and their colleagues in the Online Early Edition of the Proceedings of the National Academy of Sciences. The paper is printed in the Jan. 25 issue.

Development of the MetaChip is part of a collaborative research project funded by the NIH to find more efficient ways to synthesize and identify compounds that merit further development as possible new drugs. According to Clark, while drug companies have found rapid means of generating new drug candidates, they have yet to come up with a way to rapidly screen these candidates for toxicity.

"There are high-throughput methods of generating new compounds, but few if any high-throughput methods for toxicity analysis, forcing chemists to select compounds for drug development based on limited information about their toxicological properties," he said. "This technology fills that gap. It enables basic human metabolism to be carried out on a chip and the products of that metabolism can be screened for toxicity using the same chip platform."

Current tox screening involves cultured liver cells and even slivers of liver, but these tend to give inconsistent results and contain low levels of the P450 enzymes responsible for the initial clearance of drugs from the body and the activation of prodrugs, the researchers said. P450 enzymes are iron-containing proteins that oxidize chemicals, often making them more water-soluble so that potentially harmful substances can be eliminated more easily from the body. The antihistamine loratidine is one example of a prodrug that must be activated by liver enzymes to be effective, the researchers pointed out in their paper. On the other hand, the common pain reliever acetaminophen is converted by the liver into a toxic chemical that can damage the liver.

The MetaChip contains recombinant P450 enzymes encapsulated in a sol-gel that immobilizes them on a glass slide, so that many drug candidates can be tested simultaneously. The team plans to merge the current MetaChip with a complementary chip on which live cells are growing to enable seamless testing of the drug metabolites against an array of different cell types from the body. This will identify organ-specific drug toxicity and possible adverse drug interactions.

"Our research will expand to include other cell types, compounds, and human enzymes responsible for drug metabolism, including the cytochrome P450s," Clark said. "The outcome of this work may facilitate the elimination of toxic drug candidates much earlier in the drug development process, thereby allowing research efforts to concentrate on more promising and less toxic candidates."

New protein tagging and detection system based on a process for "splitting" a green fluorescent protein

Tue, 04 Jan 2005 15:08:38 PST

( from http://www.rxpgnews.com ) University of California scientists working at Los Alamos National Laboratory have developed a new protein tagging and detection system based on a process for "splitting" a green fluorescent protein. Unlike current protein detection methods, the method works both in living cells and in the test tube and can be used to quantify proteins down to 0.1 picomole, or one billionth of a gram of a typical protein molecule. Because the method can be used to detect protein aggregation within the living organisms, it will be useful for high-throughput studies of protein structure and protein production and for studying diseases, like Alzheimer's, that are associated with protein misfolding and aggregation.

In research published recently in the online version of the scientific journal Nature Biotechnology, Los Alamos scientists Stéphanie Cabantous, Tom Terwilliger and Geoff Waldo describe a method for engineering soluble, self-associating fragments of green fluorescent proteins that can be used to tag or detect soluble and insoluble proteins in living cells or cell lysates without changing protein solubility.

According to team member Geoff Waldo, "we think this discovery will have a major impact in the field of protein biotechnology and work related to deciphering the structure and function of proteins. I like to think of it as an enabling technology, a toolbox, if you will, for protein researchers, that could help them close the gap between sequencing the DNA of the human genome and determining the structures and functions of the encoded proteins."

The new system is based on the Rapid Protein Folding Assay (RPFA) method developed several years ago by Waldo, which used green fluorescence to signal protein folding. That method worked by fusing a protein's DNA to the DNA for green fluorescent proteins (GFP). The hybrid protein created by this linking then had the characteristics of both the GFP and the protein being assayed. If the protein being produced, or expressed, folds correctly, then the attached GFP also will fold correctly as it too is expressed. If the protein being expressed does not fold correctly, then the GFP also will not fold correctly and not fluoresce green. After scientists discovered that the GFP had some drawbacks, they developed the new system, which uses GFP fragments instead.

The split green fluorescent protein research resulted from Laboratory scientists efforts to develop a practical method for engineering protein folding and solubility as part of the National Institutes of Health (NIH) Protein Structure Initiative, a large-scale effort to determine the structures of thousands of protein molecules. These protein structures can be used in the design of new therapeutics and to deepen our understanding of how cells work.

Mouse brain tumors mimic those in human genetic disorder

Wed, 29 Dec 2004 05:32:38 PST

( from http://www.rxpgnews.com ) A recently developed mouse model of brain tumors common in the genetic disorder neurofibromatosis 1 (NF1) successfully mimics the human condition and provides unique insight into tumor development, diagnosis and treatment, according to researchers at Washington University School of Medicine in St. Louis.

After validating their animal model, the team made two important discoveries: New blood vessels and immune system cells may be essential to the initial formation of tumors and therefore may be promising drug targets; and brain images often used to determine the need for treatment may not actually be diagnostically informative.

"These mice develop brain tumors with many of the same features as those seen in children with NF1, and studying those tumors has helped us understand the cellular events involved in NF1 brain tumor development," says principal investigator David H. Gutmann, M.D., Ph.D., the Donald O. Schnuck Family Professor of Neurology.

The study appears online and will be published in the January 2005 issue of the journal Annals of Neurology.

NF1 is one of the most common neurological disorders caused by a single gene mutation. The disorder can lead to a variety of complications including brain cancer.

To supplement their clinical research, Gutmann's team developed a mouse model in which the animals, like humans with the disease, have one abnormal copy of the gene for NF1 in every cell in their body, while specific support cells in the brain called astrocytes have two abnormal copies of this same gene.

Their latest paper shows that brain tumor formation in these mice has several of the same distinguishing clinical characteristics as tumor development in children with NF1.

First, the mice developed tumors along the optic nerve and optic chiasm, which transmit visual information from the eye to the brain. This type of tumor, called an optic pathway glioma, is the most common tumor in children with NF1.

Second, the time course of tumor development was similar to that seen in humans. Unlike most tumors, optic pathway gliomas associated with NF1 typically stop growing after a few years. Moreover, they almost always occur in children -- these tumors generally start growing in children younger than 5 years old and usually do not progress after age 10. A similar pattern occurred in the mice: The optic nerve and chiasm were enlarged and astrocytes along the optic pathway began multiplying and growing when the animals were around three weeks old, developing into optic pathway gliomas by two months of age. After that time-period, which is roughly equivalent to teenage years in humans, the cells slowed down to the same growth speed as astrocytes in control mice.

"The fact that cell growth is dramatically reduced after a few months in mice and after a few years in humans tells us there may be growth signals that are produced early in life, which are critical for tumor formation and expansion," Gutmann explains.

Optic pathway gliomas in humans are typically surrounded by blood vessels and microglia, which are immune system cells in the brain. But it was unclear whether the development of blood vessels and recruitment of microglia helped trigger tumor formation or if they appeared only after the tumor was fully developed. The researchers found that by three weeks of age, the mutant mice had about four times the number of small blood vessels in the optic nerves and chiasm as control mice. Similarly, microglia were also found in the nerve and chiasm of mutant mice prior to tumor formation.

"In our judgment, the fact that recruitment of new blood vessels and infiltration of immune system cells occurs before actual tumor formation suggests that these events are important for the development of tumors," Gutmann says. "These findings raise the possibility that targeted therapies for NF1 brain tumors may involve agents that prevent the supply of growth promoting factors provided by new blood vessels and microglia."

Next, the researchers used the mouse model to investigate a clinical concern. Physicians rely on several tests to determine whether a child with an optic pathway glioma should undergo treatment for the tumor, including the tumor's size and the patient's clinical symptoms. But often those tests aren't sufficiently informative, so experts also examine pictures of the patient's brain taken with magnetic resonance imaging (MRI). To capture such brain images, physicians inject a contrast dye into a patient's bloodstream and look for accumulation of dye around the tumor. Though dye accumulation may be a sign of tumor progression, it is unclear whether that is always the case, particularly in optic pathway gliomas associated with NF1.

Results from this latest study suggest that the two are not necessarily correlated. Gutmann's team found that optic pathway gliomas lit up just as brightly in 2-month-old mice as in 8-month-old mice, despite the fact that the tumors were actively growing only in the younger mice.

"If this finding is also true in humans, this strongly argues that contrast enhancement on MRI alone is not a reliable test of tumor progression," Gutmann says. "If we rely on contrast enhancement in children with NF1 optic pathway gliomas, we may be treating kids who don't need to be treated. Using this mouse model, we hope to continue to hone in on more accurate diagnostic, prognostic and treatment approaches."

Electro osmotic Mixing in Microchannels

Thu, 23 Dec 2004 22:53:38 PST

( from http://www.rxpgnews.com ) By alternating the flow of fluid through tiny plastic pipes, a team of mechanical engineers at New Jersey Institute of Technology (NJIT) has discovered a new and speedier way to mix liquids, which in turn will someday produce better and safer medications.

"Everybody looks at creating turbulence in three dimensions to mix liquids," said team leader Nadine Aubry, PhD, Jacobus distinguished professor and chair of the mechanical engineering department at NJIT. "We traded space for time, which is a much simpler way to handle this problem when space is at a premium."

A paper by Aubry and her team, "Electro osmotic Mixing in Microchannels," published in the Nov. 29, 2004 issue of Lab on a Chip, showed that mixing could be accomplished by changing the flow rates by simply varying the voltage applied to the electrodes that commonly pump the fluid through a micro-channel. This publication follows other journal articles about similar research using other types of pumping: the Aug. 15, 2004 issue of Analytical Chemistry as well as the May 19, 2003 issue of Lab on a Chip.

More recognition for Aubry's work has come from professional colleagues, who appointed her last month vice chair of the U.S. National Committee for Theoretical and Applied Mechanics. The Committee serves as a national forum for discussions on research, technology and education of mechanics, as well as represents the U.S. in international scientific activities related to mechanics.

"Normally when two pipes in a micro-scale chemical reactor meet, the two liquids fail to mix," said Aubry. But by switching the flow many times per second, the scientists were able to create - in just a second - a pseudo-turbulent flow that completely blended the two liquids. To demonstrate the method, Aubry used a "T" channel intersection whose segments were 200 microns wide by 120 microns deep about twice the circumference of a human hair.

The method caused the interface between the two liquids to stretch, fold, and sweep through, allowing the liquids to mix quickly after traveling only two millimeters down the channel. Aubry expects the new methods to have many useful applications, especially in the pharmaceutical industry.

"The process will be useful in the preliminary phases of drug discovery," she said, "where reagents need to be well-mixed to produce purer test drugs with fewer unwanted by-products." Her process will also help engineers design smaller, more sensitive detectors for nerve gases and pollutants. And on the domestic front, inexpensive lab-on-a-chip devices could be used to make sensors that will detect rotting food in kitchen refrigerators.

Aubry is the co-director of NJIT's Keck Laboratory, a biotechnology lab whose world-class technology can help identify and manipulate bacteria, viruses and cancer cells. She received her bachelor's degree in mechanical engineering from the National Polytechnic Institute in Grenoble, France, her master's degree in mechanical engineering from the Scientific University of Grenoble and her doctorate in mechanical and aerospace engineering from Cornell University, N.Y.

She has served as a member of the National Aeronautics and Space Engineering Board's Air Force Office of Scientific Research (AFOSR) Panel and as a member of the National Research Council Panel for the NASA Administrator's Fellowship Program. She is a recipient of the Presidential Young Investigator Award from the National Science Foundation and the Ralph R. Teetor Award from the Society of Automotive Engineers.

Aubry is looking forward to continuing her micro-fluidic investigations. "Fluid mechanics has always been at the forefront of engineering and science," she said. "The prominent role that it now plays in emerging areas such as nanotechnology and biomedicine makes it a particularly exciting field."

Research unlocks potential for new medications, vaccines and diagnostics

Thu, 23 Dec 2004 22:38:38 PST

( from http://www.rxpgnews.com ) Devices the size of a pager now have greater capabilities than computers that once occupied an entire room. Similar advances are being made in the emerging field of synthetic biology at the University of Houston, now allowing researchers to inexpensively program the chemical synthesis of entire genes on a single microchip.

Xiaolian Gao, a professor in the department of biology and biochemistry at UH, works at the leading edge of this field. Her recent findings on how to mass produce multiple genes on a single chip are described in a paper titled "Accurate multiplex gene synthesis from programmable DNA microchips," appearing in the current issue of Nature, the weekly scientific journal for biological and physical sciences research.

"Synthetic genes are like a box of Lego building blocks," Gao said. "Their organization is very complex, even in simple organisms. By making programmed synthesis of genes economical, we can provide more efficient tools to aid the efforts of researchers to understand the molecular mechanisms that regulate biological systems. There are many potential biochemical and biomedical applications."

Most immediately, examples include understanding the regulation of gene function. Down the road, these efforts will improve health care, medicine and the environment at a fundamental level.

Using current methods, programmed synthesis of a typical gene costs thousands of dollars. Thus, the prospect of creating the most primitive of living organisms, which requires synthesis of several thousand genes, would be prohibitive, costing millions of dollars and years of time. The system developed by Gao and her partners employs digital technology similar to that used in making computer chips and thereby reduces cost and time factors drastically. Gao's group estimates that the new technology will be about one hundred times more cost- and time-efficient than current technologies.

With this discovery, Gao and her colleagues have developed a technology with the potential to make complete functioning organisms that can produce energy, neutralize toxins and make drugs and artificial genes that could eventually be used in gene therapy procedures. Gene therapy is a promising approach to the treatment of genetic disorders, debilitating neurological diseases such as Parkinson's and endocrine disorders such as diabetes. This technology may therefore yield profound benefits for human health and quality of life.

"The technology developed by Dr. Gao and her collaborators has the potential to make research that many of us could only dream about both plausible and cost effective," said Stuart Dryer, chair of the department of biology and biochemistry at UH. "In my own research on neurological diseases, we've often wished we could rapidly synthesize many variations of large naturally occurring genes. The costs of current technology have prevented us from doing this, but Dr. Gao's research will break down that barrier."

This technology offers tremendous potential benefits, as synthetic genes could allow for development and production of safer, less toxic proteins that are currently used in disease treatment. It also could allow for production of large molecules that do not occur naturally, but that are needed for new generations of vaccines and therapeutic agents, including vaccines for HIV and other viral diseases. This technology also will facilitate development of new medications through the creation of humanized yet synthetic antibodies that could be especially useful in detection and treatment of infectious organisms that could be used by terrorists.

Gao's co-authors include Erdogan Gulari and Xiaochuan Zhou from the University of Michigan and George Church of Harvard University. Gao, Gulari and Zhou are partners in Atactic Technologies, a company that produces and markets products for life sciences research. Atactic Technologies currently holds the license to this breakthrough technology, called picoarray gene synthesis. UH and the University of Michigan are co-holders of the patents to these DNA microchip technologies.

Prior to coming to UH in 1992, Gao was a senior investigator at Glaxo Research Laboratory and received her postdoctoral training at Columbia University, her doctorate from Rutgers University and bachelor of science from the Beijing Institute of Chemical Technology. She is an expert in nucleic acid chemistry, biomolecular nuclear magnetic resonance technology, structural biological chemistry and combinatorial chemistry. Research in her lab involves the interface of chemistry and biological sciences. Holding patents in biochip technologies, her current focus is to understand the relationships of function and structure of complex genomes of humans and other species. Gao's research has been funded by the National Institutes of Health, the Welsh Foundation, the Texas Higher Education Coordinating Board, the National Foundation for Cancer Research, the Merck Genomic Research Institute and the Defense Advanced Research Projects Agency.

Meet the 'tadpole': a new breed of DNA-protein hybrid

Thu, 23 Dec 2004 22:34:38 PST

( from http://www.rxpgnews.com ) Researchers hungry for an adaptable system for the high-sensitivity quantification of biomolecules may have found the answer in tadpoles?

Considering the sensitivity and versatility of Polymerase Chain Reaction (PCR)-based applications, biologists can perhaps be forgiven for becoming a bit spoiled, and demanding the same ease and level of sensitivity for the detection of non-nucleic acid-based molecules. However, a powerful new tool described in the January issue of Nature Methods promises just such sensitivity for the detection of a versatile range of targets, including proteins and small-molecule compounds.

Meet the 'tadpole': a new breed of DNA-protein hybrid developed by Ian Burbulis, Roger Brent, and their colleagues at the Molecular Sciences Institute (Berkeley, CA). The tadpole 'head' consists of a protein element, such as an antibody, with targeted affinity for a specific molecule, while the 'tail' is a unique DNA tag that enables PCR-based quantification. The two halves are synthesized separately, then neatly joined via a naturally occurring splicing reaction to generate the ready-to-use tadpole.

After adding the tadpoles to a sample, the amount of tag sequence amplified by PCR reveals the quantity of tadpole-bound target with surprising accuracy. Burbulis et al. began with a tadpole targeted against the vitamin biotin, and found that they could detect as few as 600 molecules with 95% confidence, and that their tadpoles offered a limit of detection 109 lower than ELISA, a commonly used antibody-based diagnostic assay. They achieved equally sensitive detection using different kinds of 'head' molecules to quantify targets such as anthrax toxin, consistently surpassing the limits of ELISA.

In an accompanying News & Views feature, Stanford researcher Garry Nolan assesses the tadpole, and concludes that the simplicity, adaptability, and sensitivity of this approach "make this an appealing system for researchers wanting a standardized, high-throughput, and accurate detection system for... just about anything."

Leukotrienes are produced by the enzyme 5-lipoxygenase (5-LO). In the new study, Colin Funk and his colleagues show that cells expressing 5-LO are an important component of aortic aneurysms in mice. They also found that reducing 5-LO expression markedly attenuates the formation of aneurysms. This effect seems to depend on the regulation of inflammatory proteins: the leukotriene LTD4 stimulates the production of proinflammatory molecules, and the absence of 5-LO correlates with their reduced expression.

These data link 5-LO pathway to inflammation of the arterial wall and to pathogenesis of aortic aneurysms, identifying a potential therapeutic target for pharmacological intervention.

Carbon nanotubes yield a new class of biological sensors useful for diabetics

Tue, 14 Dec 2004 18:25:38 PST

( from http://www.rxpgnews.com ) Nanotechnology researchers at the University of Illinois in Urbana-Champaign have demonstrated a tiny, implantable detector that could one day allow diabetics to monitor their glucose levels continuously-without ever having to draw a blood sample.

The work, which is the first application of a whole new class of biological sensors, was funded by the National Science Foundation (NSF) and announced December 12 in the online edition of the journal Nature Materials.

Principal investigator Michael Strano, a professor of chemical and biomolecular engineering at Illinois, explains that the new sensors are based on single-walled carbon nanotubes: cylindrical molecules whose sides are formed from a lattice of carbon atoms. The idea is to exploit the nanotubes' ability to fluoresce, or glow, when illuminated by certain wavelengths of infrared light-"a region of the spectrum where human tissue and biological fluids are particularly transparent," says Strano.

To make a sensor, Strano and his collaborators first coat the nanotubes with a "molecular sheath": a one-molecule-thick layer of compounds that react strongly with a particular chemical-in this case, glucose. The mix of compounds is chosen so that the reaction also changes the nanotubes' fluorescent response. Then the researchers load the coated nanotubes into a needle-thin capillary tube that can safely be implanted into the body. The capillary keeps the nanotubes from directly touching living cells but still allows glucose to enter.

The Illinois researchers tested their glucose sensor by inserting it into a human tissue sample. Then they illuminated the sample with an infrared laser and verified that the strength of the fluorescence from the buried sensor was directly related to the glucose concentrations in the tissue.

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