Sunday, January 30, 2005

At last!

Most of you probably wonder why the site hasn't been updated in about a week. No, I wasn't kidnapped by ninjas because I was working on a top secret project. The answer is simpler : I was preparing a surprise! I registered a domain, www.BiologyNews.net, got hosted at TotalChoiceHosting (I totally recommend these guys, incredible price, package, service!), moved all the content and the template of TheScientistBlog to Movable Type, optimized everything, installed a phpBB forum, coded some PHP scripts, etc.

TheScientistBlog won't be updated anymore, so update your bookmarks / rss feeds over to Biology News. Same quality news, updated daily by yours truly, with the occasional comment. If you were kind enough to link to TheScientistBlog, feel free to update your links. The new site loads much faster now (the pages are smaller; Blogger hid the extended post content, but it was still downloaded), news are Category sorted, I will be able to put up PHP scripts, including polls on the frontpage, comment security is better (I was getting spam in some posts, and blogger wasn't very comment deletion friendly) with typekey authentication, future posting... See you there!


Wednesday, January 26, 2005

Spleen may be source of versatile stem cells

year ago, Massachusetts General Hospital (MGH) researchers discovered that the spleen might be a source of adult stem cells that could regenerate the insulin-producing islets of the pancreas. In a follow-up to that unexpected finding, members of the same team now report that these potential adult stem cells produce a protein previously believed to be present only during the embryonic development of mammals.

The finding both supports the existence of these splenic stem cells and also suggests they may be able to produce an even greater variety of tissues. The report appears in the January 19 issue of SAGE KE, an online resource on the science of aging from the publishers of the journal Science.

"There may be a previously undiscovered pocket of primitive stem cells in the spleen that are important for healing several types of damage or injury," says Denise Faustman, MD, PhD, director of the MGH Immunobiology Laboratory and senior author of the SAGE KE report. "If so, these cells could have much broader therapeutic applications than suggested by our earlier work."

In 2001 Faustman's team found that a treatment designed to address the autoimmune reaction underlying type 1 diabetes actually cured the disease in diabetic mice. Late in 2003 they reported the mechanism behind the earlier discovery: cells from the spleens of donor mice - intended to train the diabetic animals' immune systems not to attack islet cells - were actually producing new islets. The result suggested that the adult spleen - previously regarded as playing a fairly minor role in regenerative medicine - might contain a population of potential islet stem cells.

In their pursuit of that finding, the MGH researchers investigated the possible presence of a protein called Hox11 in these cells. In mammals, Hox11 is a controller of key steps in embryonic development - including the formation of the spleen - but it was not known to be present in adults under normal circumstances. In some other animals, however, the protein has an intriguing function: when creatures like newts regenerate a lost limb or tail, production of Hox11 is radically increased.

As reported in their SAGE KE article, the MGH team did find that Hox11 was produced in the spleens of adult mice by the same cells that regenerated the islets in the earlier study. They also found that these cells did not produce a protein known to be associated with a cellular commitment to develop into a particular type of tissue. Without that commitment, the splenic cells may be able to differentiate into a wider variety of cells than can adult stem cells from bone marrow, which do not produce Hox11.

The researchers also note that the spleen develops from embryonic tissue that is known not only to generate precursors to many types of blood cells, a function shared by the bone marrow, but potentially to form such diverse organs as the small intestine, uterus, vascular system and lung. They theorize that a pocket of these uncommitted cells might remain in the spleen though adulthood. In addition to regeneration of islets, these cells might also produce bone cells - suggested by findings from other researchers - or potentially even cells of the nervous system, development of which depends on the correct production of Hox11.

"We know that if you have a major loss of blood, the spleen is turned on to supplement the bone marrow in replenishing your blood supply. We may find that the spleen kicks in to help with many more biological emergencies. What has been considered a practically unnecessary organ may actually provide critical healing cells," says Faustman, an associate professor of Medicine at Harvard Medical School.

She adds, "This data also shows the kind of payback that can come from studies of lower animals like newts and sponges. Combining the knowledge of Hox11's role in those animals with what we'd found about islet cell regeneration in mice helped us find this previously unknown example of normal, controlled Hox11 expression in an adult mammal."

Co-authors of the SAGE KE report are first author Shohta Kodama, MD, PhD, of the MGH Immunobiology Laboratory, and Miriam Davis, PhD, of George Washington University. The group's research is supported by grants from the Iacocca Foundation. Founder Lee Iacocca is also spearheading an effort to raise money for a clinical trial of the islet-regeneration technique in human patients. For more information about this project, go to http://www.joinleenow.org.

Massachusetts General Hospital, established in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of more than $400 million and major research centers in AIDS, cardiovascular research, cancer, cutaneous biology, medical imaging, neurodegenerative disorders, transplantation biology and photomedicine. In 1994, MGH and Brigham and Women's Hospital joined to form Partners HealthCare System, an integrated health care delivery system comprising the two academic medical centers, specialty and community hospitals, a network of physician groups, and nonacute and home health services.

Source : Massachusetts General Hospital


Genes In The Interferon System Important In Systemic Lupus Erythematosus

Two genes with very strong associations with the disease systemic lupus erythematosus (SLE) have been identified by a team of scientists headed by researchers at the Department of Medical Sciences at Uppsala University. The findings are being published today on the Web page of the highly prestigious American Journal of Human Genetics.

"These findings are probably the first genetic pieces of a huge 'interferon puzzle,' with whose help it will be possible to discover the mechanisms behind the disease SLE, and maybe other autoimmune diseases at the molecular level," says Professor Lars Rönnblom.

"It is remarkable that by studying only eleven of the some 200 genes that are seen as belonging to the interferon system, we were able to identify two genes with such clear connection to SLE," says Professor Ann-Christine Syvänen.

A few years ago Lars Rönnblom and Professor Gunnar Alm at the Swedish University of Agricultural Sciences were virtually the only researchers who claimed that the interferon system, which is involved in the body's defense against viruses, etc., was also behind the autoimmune disease SLE. Since then they have shown the importance of the interferon system in a number of works. This has led to the recognition of their hypothesis in the last year, and today it represents a white-hot field of research that has attracted a great deal of interest in the pharmaceutical industry. This picture has now been further reinforced by new findings­ - the result of multidisciplinary and international collaboration involving world experts on the interferon system, immunology, and the disease SLE, combined with world leaders in the technology for large-scale genetic analyses and statistics. The genetic and statistical analyses were performed by the doctoral student Snaevar Sigurdsson and Professor Ann-Christine Syvänen at the Center for Clinical Medical Research at Uppsala University.

The study, comprising nearly 2,000 individuals, shows that two genes in the interferon system are very strongly associated with the disease SLE. One of the genes codes for a so-called thyrosinkinase enzyme, whose function is to convey signals from interferon outside the cells to the cell nucleus.

"We found that genetic variants of thyrosinkase protect against SLE. It probably has an inhibited function that blocks the interferon effect. It is therefore possible to imagine the development of methods of treatment for SLE based on blocking the function of the thyrosinkinase enzyme," explains Lars Rönnblom.

The other gene codes for a transcription factor, which also plays an important role in regulating the interferon effect. Further functional analyses will be necessary to map the molecular mechanisms in detail.

Besides the Rheumatology Clinic at Uppsala Akademiska Hospital, hospitals in Umeå and Lund, in Sweden, and several hospitals in Finland as well as one hospital in Reykjavik provided the project with DNA samples and diagnostic information from SLE patients.

Source : Science Daily


Researchers discover way to make cells in the eye sensitive to light

Researchers have discovered a way to make light sensitive cells in the eye by switching on a single gene.

According to research published online today in Nature, the team from Imperial College London and the University of Manchester, have discovered that activating the melanopsin gene in the nerve cells causes them to become light responsive, or photoreceptive.

Using mouse cells, the researchers found that melanopsin could be used to make neurones light responsive. They found that as well as being sensitive to blue light, melanopsin uses light at different wavelengths to regenerate itself. In some forms of hereditary blindness photoreceptors are lost entirely, but the retinal ganglion cells, the cells which signal to the brain, remain intact. The researchers believe that by activating the melanopsin, these cells may gain the ability to sense and respond to light.

Professor Mark Hankins, from Imperial College London and Charing Cross Hospital, and one of the papers authors, comments: "It is quite remarkable that the activation of a single gene can create a functional photoreceptor. It is an important proof of principle that melanopsin can make non-light sensitive cells receptive to light, and although not a cure, could have applications in treating some forms of blindness."

Dr Rob Lucas, from the University of Manchester, and one of the paper's authors, adds: "The discovery that melanopsin is capable of making cells photosensitive has given us a unique opportunity to study the characteristics of this interesting protein. The textbook view of the eye is that it contains only two light sensing systems, the rods and cones. However, over the last few years it has become increasingly accepted that we have a third system, which uses melanopsin, that has lain undetected during decades of vigorous scientific investigation."

Professor Hankins and Dr Lucas were part of the team who previously discovered a new light detection system in the eye, totally independent of the rods and cones, which were thought to be the only systems providing sight. They measured light-induced pupillary constriction in genetically modified mice that lacked melanopsin. When the mice lacking melanopsin were exposed to low light, their pupillary response was the same as normal mice, but when they were exposed to bright light their pupil constriction was incomplete.

The researchers believe that while not a cure for blindness, the findings could lead to therapies for treating some forms of blindness, such as retinitis pigmentosa. Retinitis pigmentosa is a form of hereditary blindness where the rods and cones are destroyed, but the rest of the eye and retina remains intact. By switching on the melanopsin it could be possible to restore the eyes ability to respond to light.

Although making cells in the eye responsive to light is not a cure for blindness, the team are collaborating with engineers from Imperial to develop a functional retinal prosthesis which would allow the information from the light responsive cells to be used by the brain to process images.

Source : Eurekalert


Quantum Dots Research Leads to New Knowledge about Protein Binding in Plants

UC Riverside researchers from the Departments of Chemical and Environmental Engineering, Mechanical Engineering and Botany and Plant Sciences have worked together to discover a way to utilize Quantum Dot bio-conjugates to uncover new knowledge about the binding of a protein at the growing pollen tube tip. This protein plays a critical role along with another protein (chemocyanin) in guiding sperm-laden pollen tubes to the eggs found in ovules.

Applying nanoparticles for imaging the protein localization revealed information that could not be observed previously by conventional imaging techniques. This study provides a new tool to botanical scientists by merging areas of materials science, chemistry and plant biology.

The findings are the result of an interdisciplinary research team including Sathyajith Ravindran of the Chemical and Environmental Engineering Department; Sunran Kim, Rebecca Martin and Elizabeth M. Lord of the Botany and Plant Sciences Department; and Cengiz S. Ozkan of the Mechanical Engineering Department at UC-Riverside.

The results of their collaborative research appeared in an article titled “Quantum Dots as Bio-labels for the Localization of a Small Plant Adhesion Protein” and published in the January 2005 issue of Nanotechnology, and is a featured article at http://nanotechweb.org. Journal Nanotechnology has an international readership among academic, government and corporate sectors, and is dedicated to coverage of all aspects of nanoscale science and technology from a multidisciplinary perspective.

Ozkan and his colleagues utilized cadmium selenide (CdSe) quantum dots coated with zinc sulphide as fluorescent probes. The particles had a diameter of 6.3 nm. The team terminated the quantum dots with carboxylic groups by reacting them with mercaptoacetic acid. Then they conjugated the quantum dots with the amine groups of stigma/stylar cysteine-rich adhesin (SCA) - a plant pollen tube adhesion protein. This labeled the protein molecules with fluorescent tags.

Quantum dots are much more resistant to photobleaching than conventional fluorescent markers and their small size make them ideal for biological imaging. The researchers then added the molecules to germinated lily pollen grains and examined them under a confocal microscope.

This is the very first time that Quantum Dots have been utilized for live imaging in plant systems. The study opens the door for the potential use of Quantum Dots in live imaging of plant cells and provides valuable understanding of the mechanism of interaction between the pollen tube and female tissue during reproduction.

“Integrating materials science, chemistry and plant biology to understand how and where specific proteins act on a pollen tube is one more step towards a better understanding of the fundamental processes involved, namely the network of the signaling process in plant reproduction,” said Ozkan. "A better understanding of the interaction of SCA with pollen tubes could help with successful plant breeding”.

Source : UCR News

Related : QuantumDot


Tuesday, January 25, 2005

University of Manchester makes made-to-measure skin and bones a reality using inkjet printers

Made-to-measure skin and bones, which could be used to treat burn victims or patients who have suffered severe disfigurements, may soon be a reality using inkjets which can print human cells.

Scientists at The University of Manchester have developed the breakthrough technology which will allow tailor-made tissues and bones to be grown, simply by inputting their dimensions into a computer.

Professor Brian Derby, Head of the Ink-Jet Printing of Human Cells Project research team, said: "It is difficult for a surgeon to reconstruct any complex disfiguring of the face using CT scans, but with this technology we are able to build a fragment which will fit exactly. We can place cells in any designed position in order to grow tissue or bone."

This breakthrough overcomes problems currently faced by scientists who are unable to grow large tissues and have limited control over the shape or size the tissue will grow to. It also allows more than one type of cell to be printed at once, which opens up the possibility of being able to create bone grafts.

"Using conventional methods, you are only able to grow tissues which are a few millimetres thick, which is fine for growing artificial skin, but if you wanted to grow cartilage, for instance, it would be impossible," Professor Derby says.

The key to the advance which Professor Derby and his team have made is the innovative way in which they are able to pre-determine the size and shape of the tissue or bone grown.

Using the printers, they are able create 3-dimensional structures, known as 'tissue scaffolds'. The shape of the scaffold determines the shape of the tissue as it grows. The structures are created by printing very thin layers of a material repeatedly on top of each other until the structure is built. Each layer is just 10 microns thick (1,000 layers equals 1cm in thickness).

This method allows larger tissues to be grown than previously possible. The reason for this is the way in which the cells are inserted into the structures.

Before being fed into the printer, the cells are suspended in a nutrient rich liquid not dissimilar to ink, which ensures their survival. The cells are then fed into the printer and seeded directly into the structure as it is built. This avoids any 'sticking to the surface' which is a major disadvantage of current methods that infuse the cells into the structure after it has been built.

"The problem is getting cells into the interior of these constructions as they naturally stick to the sides of whatever they are being inserted into. If they stick to the sides then this limits the number of cells which can grow into tissues, and the lack of penetration also limits their size. By using inkjet printing we are able to seed the cells into the construction as we build it, which means 'sticking' isn't a problem," says Professor Derby.

Professor Derby believes the potential for this technology is huge: "You could print the scaffolding to create an organ in a day," he says.

Source : University of Manchester


Enzyme, lost in most mammals, is shown to protect against UV-induced skin cancer

In a finding that broadens our insight into the cause of certain kinds of UV-induced skin cancer, researchers at Erasmus University Medical Center (Rotterdam, The Netherlands) have employed an evolutionarily ancient enzyme-repair system to identify the principal type of DNA damage responsible for the onset of skin-tumor development. The researchers' findings also suggest that this enzyme system may be useful in developing preventative therapies against skin cancer.

Ultraviolet light is a known source of damage to our DNA, but under normal conditions humans and other mammals are capable of removing UV-induced DNA damage by a DNA repair mechanism called nucleotide excision repair. Insufficient repair of UV-induced DNA damage, which for example may occur after excessive unprotected sunbathing, can lead to cellular death – recognized as sunburn of the skin – and may cause permanent changes in the DNA (mutations) that ultimately can result in the onset of skin cancer. Thus far it was not clear how the two major types of UV-induced DNA lesions – cyclobutane pyrimidine dimers (CPDs) and (6-4)photoproducts (6-4PPs) – contribute to the processes of cell death and cancer formation. Identifying the relative contributions of the two types of damage to tumor formation is critical for the development of therapies that could help prevent skin cancer. Moreover, CPDs and 6-4PPs have particular potential to cause lasting damage to mammalian cells because photolyases – a class of enzymes capable of efficiently repairing these lesions – have apparently been lost from placental mammals over the course of evolution.

Thus, most mammals, including humans, can only repair these lesions through a much less direct and elaborate process called nucleotide excision repair.

In the new work, Dr. Bert van der Horst and colleagues studied the effects of CPD and 6-4PP lesions by providing mice with transgenes encoding CPD and 6-4PP photolyase enzymes.

Although mice do not ordinarily produce these enzymes, which remove either CPD or 6-4PP lesions by using visible light as an energy source, expression of the transgenes allowed rapid photolyase-mediated repair of these lesions. The researchers found that transgenic mice bearing the CPD photolyase transgene, in contrast to mice bearing the 6-4PP photolyase transgene, showed superior resistance to the deleterious effects of UV irradiation. Not only could CPD photolyase transgenic animals withstand doses of UV light that cause severe sunburn in normal mice, but they also showed superior resistance to UV-induced skin cancer. This work clearly points to CPD lesions as the major intermediate in UV-induced cellular damage leading to non-melanoma skin cancer. Importantly, it also suggests that photolyases may be successfully employed as a genetic tool to combat UV-induced skin cancer.

Source : Eurekalert


Yellowstone microbes fueled by hydrogen, according to U. of Colorado study

Microbes living in the brilliantly colored hot springs of Yellowstone National Park use primarily hydrogen for fuel, a discovery University of Colorado at Boulder researchers say bodes well for life in extreme environments on other planets and could add to understanding of bacteria inside the human body.

A team of CU-Boulder biologists led by Professor Norman Pace, one of the world's leading experts on molecular evolution and microbiology, published their report "Hydrogen and bioenergetics in the Yellowstone geothermal system" this week in the online edition of the Proceedings of the National Academy of Sciences.

The team's findings, based on several years of research at the park, refute the popular idea that sulfur is the main source of energy for tiny organisms living in thermal features.

"It was a surprise to find hydrogen was the main energy source for microbes in the hot springs," Pace said. "This project is also interesting in the context of microbiology because it's one of the few times we've been able to study microbes to get information on an entire ecosystem. That's never before been possible."

The study was specifically designed to determine the main source of metabolic energy that drives microbial communities in park features with temperatures above 158 degrees Fahrenheit. Photosynthesis is not known to occur above that temperature.

A combination of three different clues led researchers to conclude that hydrogen was the main source of energy. Genetic analysis of the varieties of microbes living in the hot springs communities revealed that they all prefer hydrogen as an energy source. They also observed ubiquitous H2 in all the hot springs at concentrations sufficient for microbial bioenergetics.

Thermodynamic models based on field data confirmed that hydrogen metabolism was the most likely fuel source in these environments.

"This work presents some interesting associated questions," said John Spear, lead author of the report. "Hydrogen is the most abundant element in the universe. If there is life elsewhere, it could be that hydrogen is its fuel," Spear said. "We've seen evidence of water on Mars, and we know that on Earth, hydrogen can be produced biogenetically by photosynthesis and fermentation or non-biogenetically by water reacting with iron-bearing rock. It's possible that non-biogenic processes produce hydrogen on Mars and that some microbial life form could be using that," he said.

There are many examples of bacteria living in extreme environments -- including the human body -- using hydrogen as fuel, according to Spear. "Recent studies have shown that Helicobacter pylori bacteria, which cause ulcers, live on hydrogen inside the stomach," said Spear. "Salmonella metabolizes hydrogen in the gut. It makes me wonder how many different kinds of microbes out there are metabolizing hydrogen in extreme environments."

Instead of relying on traditional techniques of microbiology that utilize cultures grown in the lab, the CU-Boulder team used methodology developed by Pace to genetically analyze the composition of the microbial community as it appeared in the field. "We didn't look at what grows in a culture dish, we looked at the RNA of samples directly from the field," Spear said.

"We've never before known what microbes were living in Yellowstone hot springs, and now we do," Pace said.

A novel suite of instruments was used to gather data, some of which had never before been collected. "No one had measured the concentration of hydrogen in the hot springs before because the technology didn't exist until about seven years ago. Now we can detect very low-level concentrations of hydrogen in water," Spear explained.

"We found lots of hydrogen in the hot springs -- an endless supply for bacteria," he said. Measurements of the amount of H2 in water were recorded in Yellowstone hot springs, streams and geothermal vents in different parts of the park and during different seasons. All of the environments had concentrations appropriate for energy metabolism.

The team used computer-generated thermodynamic models to find out if hydrogen was indeed the principle source of energy. "You can smell sulfide in the air at Yellowstone, and the accepted idea was that sulfur was the energy source for life in the hot springs," Spear said. Not so, according to the team's computer models built on field measurements of hydrogen, sulfide, dissolved oxygen concentration and other factors.

Spear said it was difficult to explore a microbial ecosystem. "We have a hard enough time explaining what's going on in a forest, for example, with all the interlacing systems. We can't even see a microbial system."

Sample extraction was a dangerous and delicate operation. In order to accurately analyze a hot spring's entire microbial community, Spear needed to collect only about as much material as a pencil eraser. Sediment samples were scooped into special sample vials and immediately frozen in liquid nitrogen canisters to preserve the microbial community.

In springs where there was no sediment, Spear collected samples of planktonic organisms by hanging a glass slide in the water and allowing the microbes to accumulate. "Bacteria are just like us. They like to be together, they like to be attached to a surface and they like to have their food - dissolved hydrogen, in this case -- brought to them."

Spear explained that the hot springs' colors are the result of interactions between minerals and the microbes living in the pools. Hotter water usually shows colors from minerals, and cooler water plays host to photosynthetic pigments.

"Based on what I've seen in this analysis, I think hydrogen probably drives a lot of life in a lot of environments," Spear said. "It's part speculation, but given the number and kinds of bacteria that are metabolizing hydrogen, it's probably a very old form of metabolism.

That's important because it tells us about the history of life on Earth," he said. "And if it works this way on Earth, it's likely to happen elsewhere. When you look up at the stars, there is a lot of hydrogen in the universe."

Source : University of Colorado at Boulder


Columbia research lifts major hurdle to gene therapy for cancer

Researchers at Columbia University Medical Center have discovered a way to overcome one of the major hurdles in gene therapy for cancer: its tendency to kill normal cells in the process of eradicating cancer cells.

In a new study published in the Jan. 25 issue of the Proceedings of the National Academy of Sciences (PNAS), the researchers demonstrated that the technique works by incorporating it into a specially designed virus. The virus eradicated prostate cancer cells in the lab and in animals while leaving normal cells unscathed.

Gene therapy based on the new technique should also be effective for a wide range of tumors - such as ovarian, breast, brain (glioma), skin (melanoma) and colon cancer - because the virus is constructed to exploit a characteristic of all solid cancers.

"What's exciting is we may now be able to design a therapy that will seek out and destroy only cancer cells," said the study's senior author, Paul B. Fisher, Ph.D., professor of clinical pathology and Michael and Stella Chernow Urological Cancer Research Scientist at Columbia University Medical Center. "We hope it will be particularly powerful in eradicating metastases that we can't see and that can't be eliminated by surgery or radiation. Gene therapy, especially for cancer, is really starting to make a comeback."

The virus's selectivity for cancer cells is based on two molecules called PEA-3 and AP-1 that, the researchers found, are usually abundant inside cancer cells. Both of the molecules flip a switch (called PEG) that turns on the production of a cancer-inhibiting protein uniquely in tumor cells.

The researchers say the PEG switch can be exploited to produce gene therapies that will only kill cancer cells even if the therapy enters normal cells.

As an example, the researchers constructed an adenovirus that carries the PEG switch and a toxic protein. The switch and the protein were connected to each other so that the deadly protein is only unleashed inside cancer cells when the switch is flipped on by PEA-3 or AP-1.

When added to a mix of normal and prostrate cancer cells, the virus entered both but only produced the toxic protein inside the cancer cells. All the prostrate cancer cells died while the normal cells were unaffected.

The same virus also selectively killed human cancer cells from melanoma and ovarian, breast, and glioma (brain) tumors.

Dr. Fisher's team is now altering the virus and developing additional viruses based on the PEG switch for use in clinical trials with patients. Other investigators associated with the PNAS study include Drs. Zao-zhong Su (research scientist), Devanand Sarkar (associate research scientist) and Luni Emdad (postdoctoral research scientist) in Dr. Fisher's group; Drs. Gregory J. Duigou (associate research scientist) and C. S. Hamish Young (professor) in the Department of Microbiology (Columbia University Medical Center); and Dr. Joy Ware (professor), Mr. Aaron Randolph (graduate student) and Dr. Kristoffer Valerie (professor) at Virginia Commonwealth University, Richmond, VA.

Source : Eurekalert


Novel technology detects human DNA mutations

Researchers at Nanosphere, Inc. today reported unprecedented benefits in the company's technology for the medical analysis of human DNA.

Nanosphere's nanoparticle-based technology allows for rapid, highly-sensitive and specific Single Nucleotide Polymorphism (SNP) genotyping, which is the direct detection of a particular gene and the extent to which it is normal or mutated. The technology, reported in the February 2005 (Volume 33, Number 2), issue of Nucleic Acids Research, allows detection of a SNP in an unknown genotype with a greater than 99 percent confidence threshold and can be used with human DNA obtained from samples as small as a drop of blood. Importantly, the technology eliminates the need for costly, time and labor intensive gene amplification or enzymatic interventions – two widespread methods currently used to perform such analyses.

"Nanosphere's new SNP analysis methodology for whole genomic human DNA is a powerful example of the versatility of our proprietary ClearReadTM nanoparticle technology," said William Moffitt, Nanosphere's President and CEO. "This study and the use of nanoparticles to dramatically increase sensitivity in our other proprietary applications -- such as bio-barcode for ultra sensitive detection of proteins -- demonstrate the broad applicability of nanotechnology and its potential to markedly advance the state-of-the-art in nucleic acid and proteomic research and diagnostics."

The analysis of whole human genomic DNA is extraordinarily complex as it requires sifting through the more than one billion base pairs of DNA to find a particular base pair of interest. Once that base pair is located, it is then necessary to determine if either of the bases is mutated (i.e., has SNPs). Nanosphere's technology can rapidly, easily, and accurately interrogate both bases in the pair to determine if they are homozygous (i.e., both are mutant or normal) or heterozygous (i.e., one is mutant, one is normal) – the most critical step in correlating the SNP with a disease or drug sensitivity.

To do so, Nanosphere scientists employ a two-step process called ClearReadTM technology. This method sandwiches a target DNA SNP segment between two oligonucleotide sequences to greatly increase detection specificity and sensitivity. One segment identifies any mutations in the DNA and the probe, a highly sensitive gold nanoparticle, creates a strong signal accurately indicating the presence of a specific target SNP. Proof of principle, reproducibility, and the robust, simple and rapid characteristics of this technology were demonstrated with unamplified DNA samples representing all possible forms of three genes implicated in hypercoagulation disorders.

Related : Nanosphere Inc. Technology

Source : Eurekalert


Contamination of Stem Cells - Continued

Following the "Current human embryonic stem cell lines contaminated UCSD/Salk team finds" story from two days ago, I spotted an interesting comment from the White House Gaggle :

"This is an issue that has been previously raised and discussed. We've known from the very beginning that the lines that were authorized for research had this particular trait…and the scientists at NIH are very well aware of it and remain confident that the stem cell lines that are available will provide us with the adequate supply to do the most basic research." The NIH had asked the FDA to specifically look at the issue and the FDA concluded that the same issue is presented by human feeder cells. "There is still much uncertainty about the promise of stem cell research. We are only at the stage of the beginning of basic research to understand the promise of embryonic stem cell research."


Monday, January 24, 2005

Priming embryonic stem cells to fulfill their promise

Bioengineering researchers at the University of California, San Diego have invented a process to help turn embryonic stem cells into the types of specialized cells being sought as possible treatments for dozens of human diseases and health conditions. Sangeeta Bhatia and Shu Chien, UCSD bioengineering professors, and Christopher J. Flaim, a bioengineering graduate student, described the cell-culture technique in a paper published in the February issue of Nature Methods, which became available online on Jan. 21.

Embryonic stem cells are considered the blank-slate, raw material needed to repair or replace damaged or missing liver, nerve, muscle, and other tissues and organs. However, in order to fulfill their therapeutic promise, scientists believe that stem cells must first be coaxed to differentiate, or mature, into precursors of specialized cell types found in the body.

Embryonic stem cell differentiation is complex and far from fully understood. Scientists are focusing on four types of external inputs known to be involved in triggering the differentiation of stem cells: soluble growth factors, adjacent cells, mechanical forces, and extracellular matrix proteins that form the support structure of almost all tissues. Bhatia, Chien, and Flaim focused on just one - the extracellular matrix.

"We kept the other factors constant and developed a miniaturized technique to precisely vary extracellular matrix proteins as a way to identify which combinations were optimal in producing differentiated cells from stem cells," said Bhatia. She, Chien, and Flaim described in their paper a technique that enabled them to identify the precise mix of proteins that optimally prompted mouse embryonic stem cells to begin the differentiation process into liver cells. Bhatia, Chien, and Flaim designed the technique with other cell biologists in mind so that any of them could duplicate it with off-the-shelf chemicals and standardized laboratory machinery. "We think other researchers could easily use this technique with any other tissue in mouse, or human, or any other species," said Bhatia.

Scientists have identified about 100 proteins - including laminin, fibronectin, and several kinds of collagen - that function as the extracellular matrix, or scaffolding, of most mammalian tissues. Until now, there has been no practical way to evaluate the daunting number of possible combinations of these proteins, any one of which could form the optimal "niche" for a desired type of differentiated cell.

In their experiments, the UCSD researchers took advantage of the knowledge that the extracellular matrix in liver is comprised primarily of just five proteins. They applied spots of all 32 possible combinations of the five proteins as engineered niches onto the surface of gel-coated slides, and then added mouse embryonic stem cells to the niches. After the cells were allowed to grow, the researchers assayed their progression into liver cells. "We looked at all the combinations at once," said Bhatia. "Nobody has done this combinatorial approach before."

Bhatia, Chien, and Flaim reported that either collagen-1 or fibronectin had strongly positive effects on the differentiation of the stem cells they tested. Unexpectedly however, when both collagen-1 and fibronectin were combined in one niche, the liver cell differentiation rate was subtly inhibited. "You would not predict that from the customary cell biology experiments," said Bhatia. "By using this combinatorial technique we were surprised to find many interesting interactions, and we were able to tease out the effects of each protein, alone and in combination with others."

Cell biologists have not performed such combinatorial assays for other desired cell types because they have no practical way to do so. Bhatia, Chien, and Flaim seized on the unique ability of so-called DNA spotting machines to deliver tiny volumes of liquid, about one trillionth of a liter per spot. The spotting machines, which cost as little as $20,000, have become common fixtures at most research universities, but the innovation reported today in Nature Methods involved using such a machine to spot solutions of proteins rather than DNA. The UCSD researchers also refined other parameters so that the technique would be reproducible in other research laboratories.

"When we talked to our colleagues, it was clear that, whether it's cells in the liver, brain, or heart, there had been no practical way for researchers to find the optimal extracellular matrix needed to turn embryonic stem cells into cells with therapeutic potential," said Bhatia. "We think we've developed an enabling technology for stem cell research and other areas of cell biology in the sense that all of a sudden scientists can use inexpensive and widely available reagents and machinery to optimize the conditions needed to optimize embryonic stem cell differentiation."

Bhatia is planning further studies on generating liver cells from embryonic stem cells to make an artificial liver. She plans to seek funding to further her artificial liver research from the new California Institute for Regenerative Medicine. The institute was created after California voters in November approved Proposition 71, a measure that authorized the state to borrow $3 billion to fund stem cell research over the next 10 years.

"I'm really excited about the stem cell applications of our new technology," said Bhatia. "We feel that this extracellular matrix part of the stem cell niche has been understudied. If we can now take what we've learned, add combinations of growth factors, and even add other cells to embryonic stem cells, we may be able for the first time to study all the dimensions of the niches required to very specifically control embryonic stem cell differentiation."

Source : Eurekalert


New, automated tool successfully classifies and relates proteins in unprecedented way

For the first time, researchers have automatically grouped fluorescently tagged proteins from high-resolution images of cells. This technical feat opens a new way to identify disease proteins and drug targets by helping to show which proteins cluster together inside a cell.

The approach, developed by Carnegie Mellon University, outperforms existing visual methods to localize proteins inside cells, says Professor Robert F. Murphy, whose report, "Data Mining in Genomics and Proteomics," appears in an upcoming special issue of the Journal of Biomedicine and Biotechnology.

"Our approach really enables the new field of location proteomics, which describes and relates the location of proteins within cells," said Murphy, a professor of biological sciences, machine learning, and biomedical engineering. "This work should provide a more thorough understanding of cellular processes that underlie disease."

Using this approach to spot a protein cluster could help scientists identify a common protein structure that enables those proteins to gather in one part of the cell, according to Murphy. Getting this information is critical to foil a disease like cancer, where you might want to identify and disable part of a tumor cell's machinery needed to process proteins for cancer growth.

"Our tool represents a step forward because it is based on standardized features and not on features chosen by the human eye, which is unreliable. By automating the clustering of proteins inside cell images, we also can study thousands of images fast, objectively and without error," Murphy said.

Murphy's tool has two key components. One is a set of subcellular location features (SLFs) that describe a protein's location in a cell image. SLFs measure both simple and complex aspects of proteins, such as shape, texture, edge qualities and contrast against background features. Like fingerprints, a protein's SLFs act as a unique set of identifiers. Using a set of established SLFs, Murphy then developed a computational strategy for automatically clustering, or grouping, proteins based on SLF similarities and differences. For his study, Murphy used images of randomly chosen, fluorescently labeled proteins. These proteins were produced inside living cells using a technology called CD tagging, which was developed by Jonathan Jarvik and Peter Berget, both associate professors of biological sciences at Carnegie Mellon. The computational analyses were carried out together with Xiang Chen, a graduate student in the Merck Computational Biology and Chemistry program.

Chen and Murphy found that the new tool outperformed existing methods of identifying overlapping proteins within cells, such as simple visual categorization of their locations.

"Our tool outperformed clustering based on the terms developed by the Gene Ontology Consortium, the best previous way of describing protein location. We found that the Gene Ontology terms were too limited to describe the many complex location patterns we found. Of course, the other drawback of term-based approaches is that they have to be assigned manually by database curators, and this is often not consistent between different curators," said Murphy.

Murphy and his colleagues are currently amassing more protein image data using CD Tagging so that they can refine their approach further. They are also working on ways to "train" a general system that will work for different cell types.

Source : Carnegie Mellon

Related : Journal of Biomedecine and Biotechnology article


Lack of enzyme turns fat cells into fat burners

Lack of the enzyme, acetyl CoA carboxylase 2 or ACC2, appears to turn the adipose or fat cells of mice into fat burners, explaining in part why the animals can eat more and weigh less than their normal counterparts, said Baylor College of Medicine researchers.

The report that appears online today in the Proceedings of the National Academy of Sciences.

"We studied the fat cells in these mice bred to lack ACC2," said Dr. Salih Wakil, chair of the BCM department of biochemistry and molecular biology. "We found that the adipose in the mutant mice are now oxidizing fat, hydrolyzing (breaking down using water) fat, and passing it on to the heart and muscle because there is an increase in oxidation of fat in those organs. It also starts oxidizing glucose. In other words, the adipose tissue is becoming a little more oxidative and less involved in the synthesis and storage of fat. We feel this contributes to the status of the animal."

In prior studies, Wakil and his colleagues have demonstrated the effect ACC2 has on mice. Mice bred to lack the enzyme can eat a high fat, high carbohydrate diet without gaining weight, while their normal counterparts become obese and develop type 2 diabetes.

"This adds another tissue or organ that helps out in the process of energy maintenance," said Wakil. "ACC2 is potentially a key enzyme in the regulation of weight, obesity, and related problems."

Wakil and his colleagues studied the oxidation of fatty acid and glucose in cultures of fat cells isolated from both normal and mutant mice that lacked ACC2. When the mice were fed a normal diet, fatty acid oxidation was 80 percent higher in the fat cells of the mice lacking ACC2 when compared to normal mice. When they were fed a high fat, high carbohydrate diet for four to five months, the ACC2-deficient mice had a 25 percent higher rate of fatty acid oxidation and twofold higher rate of glucose oxidation than the normal mice.

Others who participated in the research included Drs. WonKeun Oh, Lutfi Abu-Elheiga, Parichher Kordari, Zeiwei Gu, Tattym Shaikenov, Subrahmanyam S. Chirala. The work was supported in part by by the Clayton Foundation for Research and the National Institutes of Health.

Source : Eurekalert

Related : Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets, PNAS


Sunday, January 23, 2005

Emory Study Tests Bone Marrow Stem Cells to Improve Circulation in Legs

Physicians at Emory University School of Medicine are conducting a clinical trial using stem cells generated within the bone marrow to grow new blood vessels that could improve circulation in patients with blockages in the arteries of their legs -- a condition called peripheral vascular disease (PVD). Individuals with PVD have decreased blood flow to the muscles of the legs, especially during exercise, which causes pain, aching, cramping or fatigue in the muscles of their legs when they walk. This condition also is called "intermittent claudication".

The Emory team, led by cardiologist Arshed A. Quyyumi, MD, and cardiology fellow Veerappan Subramaniyam, MD, is using colony stimulating factors (growth factors), to prod the bone marrow to release a type of stem cells called endothelial progenitor cells, which are used by the body to form new blood vessels or to repair damaged ones.

Decreased blood flow in the legs is caused by the blockage or narrowing of the arteries due to build-up of cholesterol. Normally, with exercise, the blood vessels dilate (get bigger), but clogged blood vessels constrict during exercise. In some individuals the vascular system corrects the problem on its own either by forming new blood vessels, called "collaterals," that bypass the blockages, or by repairing the diseased blood vessels. This repair process results in improved circulation even during exercise. Some people are not able to repair their own vessels, however, and physicians don't completely understand the reasons why.

Recent studies show that when muscles do not receive enough blood, the body makes growth factors that stimulate the bone marrow to release stem cells that "home" to the muscle that is not getting enough blood. These stem cells include endothelial progenitor cells (EPCs), which is the type of cell needed to make new blood vessels and to repair damaged ones.

Patients in the clinical trial will be given an injection of either a growth factor called GM-CSF (granulocyte-macrophage colony stimulating factor) or placebo (sterile salt water) three times a week for two weeks. The level of EPCs in the volunteers' blood will be measured before, during and after administration of the drug or placebo. The study is randomized and blinded, which means that volunteers will not know whether they are receiving the study drug or the placebo.

The goal of the study is to determine if and how much GM-CSF will increase the number of circulating EPCs in patients with peripheral vascular disease. Another goal is to find out whether or not increasing the number of circulating EPCs results in improved blood flow to the leg, improved blood vessel function and improvement of patients' symptoms.

Currently, GM-CSF is approved by the FDA for several uses, including in cancer patients to increase the number of white blood cells to fight infection after chemotherapy; in healthy individuals serving as bone marrow donors to stimulate the bone marrow to release stem cells; and in patients who have had a bone marrow transplant to increase the number of white blood cells. It is still considered experimental, however, for use to increase the level of EPCs in patients with peripheral vascular disease.

The investigators are seeking patients in whom prior treatments, including surgery or angioplasty, have been unsuccessful, or patients for whom those treatments are not options. To find out more about the study and eligibility, call 404-712-0170.

Source : Woodruff Health Sciences Center


Controversial drug shown to act on brain protein to cut alcohol use

Medication against nicotine addiction is nowadays readily available. However, a similar and equally dangerous addiction, alcoholism, can't yet be controlled by drugs. Or can it be? Researchers from the University of California in San Diego identified a natural compound able to block alcohol addiction in rodents. We can only hope that anti-alcoholism patchs or gum will be available in a close future; it would help fix a problem that we've been struggling with for ages.

A naturally occurring hallucinogen advocated by some clinicians as a potent anti-addiction drug has been rigorously studied for the first time, confirming its ability to block alcohol craving in rodents, and clarifying how it works in the brain. The new research findings about the drug Ibogaine open the way for development of other drugs to reverse addiction without Ibogaine's side effects, potentially adding to the small arsenal of drugs that effectively combat addiction.

Derived from a West African shrub, Ibogaine has been championed for years by a cadre of clinicians and drug treatment advocates impressed with its ability to reverse withdrawal symptoms and craving for alcohol and various drugs of abuse. It has been used outside of the U.S. to treat addiction by American and other clinicians. But its side effects, including hallucinations, which made it popular in the 1960s drug culture, and evidence of toxicity to certain nerve cells in rodent studies have discouraged careful studies of its clinical potential against drug and alcohol addiction. The FDA has not approved use of Ibogaine in the U.S.

Scientists at UCSF's Ernest Gallo Clinic and Research Center have now shown definitively in experiments with mice and rats that Ibogaine does reduce alcohol consumption, and they have determined that it does so by increasing the level of a brain protein known as glial cell line-derived neurotrophic factor, or GDNF. In a separate study, they demonstrated that GDNF by itself decreases alcohol consumption.

The research is being published in the January 19 issue of The Journal of Neuroscience.

"By identifying the brain protein that Ibogaine regulates to reduce alcohol consumption in rats, we have established a link between GDNF and reversal of addiction -- knowledge of a molecular mechanism that should allow development of a new class of drugs to treat addiction without Ibogaine's side effects," said Dorit Ron, PhD, UCSF associate professor of neurology and also principal investigator at the Gallo Center. Ron is co-senior author of the paper with Patricia Janak, PhD, UCSF assistant professor of neurology and also principal investigator at the Gallo Center.

In their research, the scientists first carried out classic behavioral studies showing that Ibogaine reduced alcohol consumption. They induced the rats to consume alcohol in daily drinking sessions and then demonstrated that their drinking declined precipitously when they received Ibogaine. The drug was administered either by injection or directly into the same brain region where GDNF levels were shown to increase.

The research also showed that Ibogaine was quite effective in preventing relapse, or "falling off the wagon" -- the vulnerability of recovered alcoholics or addicts to return to uncontrolled drinking or drug use when exposed to the drug of abuse months or even years after breaking the habit.

In this analysis, the researchers provided alcohol to rats until they had become "experienced" daily drinkers. They then withheld alcohol for two weeks, which normally leads to greatly increased drinking when when alcohol is again available. When they administered Ibogaine, they found that the heightened craving and consumption was significantly reduced.

"The discovery that Ibogaine reduced binge drinking after a period of abstinence was an exciting finding for us because this is the type of behavior in alcoholics for which very few effective drugs exist," Janak said.

The scientists confirmed in a cell model that Ibogaine stimulated GDNF activity. Finally, they showed that a known inhibitor of GDNF blocked Ibogaine's ability to decrease alcohol craving in the rats, suggesting a direct link between Ibogaine's desirable actions and GDNF.

"If we can alter the GDNF pathway, we may well have a new treatment against alcohol and drug addiction without the unwanted side effects of Ibogaine," Ron said.

Colleagues in the research and coauthors on the paper are postdoctoral fellows Dao-Yao He, PhD, Nancy N.H. McGough, PhHD; Ajay Ravindranathan, PhD; Jerome Jeanblanc, PhD; Marian Logrip, BA, UCSF neurology graduate student; and Khanhky Phamluong, BA, research associate, all at the Gallo Center.

The research is supported by funds provided by the State of California through UCSF for medical research on alcohol and substance abuse, and by the Department of Defense.

Source : University of California, San Francisco


Current human embryonic stem cell lines contaminated UCSD/Salk team finds

Currently available lines of human embryonic stem cells have been contaminated with a non-human molecule that compromises their potential therapeutic use in human subjects, according to research by investigators at the University of California, San Diego (UCSD) School of Medicine and the Salk Institute in La Jolla, California.

In a study published online January 23, 2005 in the journal Nature Medicine, the researchers found that human embryonic stem cells, including those currently approved for study under federal funding in the U.S., contain a non-human, cell-surface sialic acid called N-glycolylneuraminic acid (Neu5Gc), even though human cells are genetically unable to make it. In a related paper published November 29, 2004 by the Journal of Biological Chemistry (JBC), the Varki group has also discovered the exact cellular mechanism by which this occurs.

In studies with one of the federally approved human embryonic stem cell lines, the investigators determined that the Neu5Gc is incorporated by the stem cells when they are grown or derived from laboratory cultures that contain animal sources of the non-human Neu5Gc molecule. All traditional culture-dish methods used to grow all human embryonic stem cells include animal-derived materials, including connective tissue cells (so-called "feeder layers") from mice and fetal calf serum.

"The human embryonic stem cells remained contaminated by Neu5Gc even when grown in special culture conditions with commercially available serum replacements, apparently because these are also derived from animal products," said both papers' senior author Ajit Varki, M.D., UCSD professor of medicine and cellular & molecular medicine, and co-director of the UCSD Glycobiology Research and Training Center.

The research in Nature Medicine was done with human embryonic stem cells grown in the laboratory of Fred Gage, Ph.D., professor, Laboratory of Genetics, the Salk Institute, La Jolla, California, an adjunct professor of neurosciences at UCSD, and an author on the Nature Medicine paper.

Previously, the Varki lab found in 1998 that humans are uniquely different from other mammals studied in that people do not express Neu5Gc*. In a 2003 study**, the UCSD researchers found that humans have naturally occurring antibodies that are directed against Neu5Gc. In the current Nature Medicine paper, the scientists found that the human embryonic stem cells contaminated with Neu5Gc became, effectively, like animal cells, being attacked by human antibodies, and thus rendering them useless as a potential therapeutic tool in humans.

"It's an important safety issue because this opens up the idea that metabolic transfer of glycans is occurring between cells," said Gage. "Also, components of the growth medium have the capacity to change the immunological characteristics of the human ES cells. More research is needed to understand the optimal conditions for preparing human cells for therapeutic application."

"We considered that one partial solution to the problem was to use human serum in the growth medium," Varki said. When the team grew the cells in heat-inactivated human serum specially selected for low concentrations of anti-Neu5Gc antibodies, the immune response was significantly reduced, but not completely eliminated.

In their experiments, the researchers used recently developed probes to detect the presence of Neu5Gc on the cell surface of human embryonic stem cells that had been grown in traditional culture conditions. The scientists further confirmed the presence of Neu5Gc with a process called electrospray mass spectrometry. The percentage of total sialic acids present as Neu5Gc in the embryonic stem cells varied from 2.5 to 10.5 percent. In human embryonic stem cells that had been allowed to differentiate into embryoid bodies (EB), which is the first step in preparing them for potential use in humans, the percentage of total sialic acids present still ranged from 5 to 17 percent.

Varki and his team noted that many efforts have been made during the last few years to try to eliminate any animal-derived culture components in human stem cell culture. However, many of the specialized media used for growth and differentiation still contain materials from animal sources and are hence contaminated with Neu5Gc.

In addition to using human serum, the researchers suggested the possibility of using what are called "feeder cells" from mice with a human-like defect in Neu5Gc production. They noted that they have recently produced such a mouse. Another possibility being attempted by groups in other parts of the world is to use human embryo-derived connective tissue cells as the feeder layer in the culture.

A further solution might be a short-term culture in heat-inactivated serum from the actual patient who is going to receive the therapy, the scientists said. However, it may still prove difficult to completely eliminate the Neu5Gc, because is has become metabolically incorporated into the currently available, federally-funded human embryonic stem cell lines.

"With this discovery, the preexisting general concern about using animal products for deriving human embryonic stem cells has become more specific, being defined in molecular terms," Varki said.

"Such issues will, of course, become irrelevant if complete elimination of Neu5Gc can be achieved by deriving new human embryonic stem cells that have never been exposed to Neu5Gc-containing animal products of any kind," the researchers said in the Nature Medicine paper, noting that none of the suggested approaches guarantees the complete elimination of Neu5Gc from existing cultures. "Therefore, it would seem best to start over again with newly derived human embryonic stem cells that have never been exposed to any animal products, and ideally, only ever exposed to serum from the intended transplant recipient."

"However, such an approach could not be pursued under existing rules for the use of federal grant dollars," Varki said.

The first author of the Nature Medicine study is Maria J. Martin, Ph.D., a post doctoral researcher in Varki's lab at UCSD. An important additional author is Gage's post doctoral fellow Alysson Muotri, Ph.D. The study was funded by the National Institute of General Medical Sciences at the National Institutes of Health, the Lookout Fund, and by the G. Harold and Leila Y. Mathers Charitable Foundation of New York.

In addition to Varki, authors of the related study in JBC included Muriel Bardor, Ph.D. and Dzung Nguyen, Ph.D., post-doctoral fellows, and Sandra Diaz, a research associate. They determined that Neu5Gc gets into human cells by being engulfed in fluid droplets and then moved to the cytoplasm of the cell by a "pump" called the lysosomal sialic acid transporter.

Varki noted that this pathway is an unusual and previously unknown one that may also be relevant to the entry of other small molecules into cells. In addition, the JBC study showed how Neu5Gc attached to dietary proteins from animals could be incorporated into cells lining the stomach and colon, organs where consumption of red meat has been associated with risk of cancer.

"Knowing the mechanism that this molecule uses to get into human cells may give us clues to possible solutions to the problems that it may cause in various situations," Varki said.

Source : Eurekalert


Drug That 'Tags' Decision-making Areas Of The Brain May Aid Battle Against Nicotine Addiction, Alzheimer's And Other Disorders

Along with aiding efforts to study addicted smokers, a new drug that attaches only to areas of the brain that have been implicated in nicotine addiction may help studies of people battling other disorders such as Alzheimer’s disease and schizophrenia.

Developed by UC Irvine Transdisciplinary Tobacco Use Research Center scientists, the new drug – Nifrolidine – is a selective binding agent that identifies specific areas of the brain responsible for decision-making, learning and memory. Lead researcher Jogeshwar Mukherjee, UCI associate professor of psychiatry and human behavior, developed Nifrolidine to measure a subtype of nicotine receptors in the living brain by using an imaging technique, positron emission tomography, more commonly known as PET scans. After proving the drug’s effectiveness, Mukherjee believes the drug will have implications for other conditions, as well.

Study results appear in the January issue of the Journal of Nuclear Medicine.

“Nifrolidine is suited to provide reliable, quantitative information of these receptors and may therefore be very useful for future human brain imaging studies of nicotine addiction and other clinical conditions in which these brain regions have been implicated,” Mukherjee said.

He found in animal tests that Nifrolidine binds to receptors in the temporal and frontal cortex, areas that are responsible for learning and memory as well as reasoning, planning, problem solving and emotion. According to Mukherjee, patients with Alzheimer’s disease have been known to have a 30 percent to 50 percent loss of these receptors “If there is a gradual loss of these receptors over time, Nifrolidine could be a potential marker for early diagnosis of Alzheimer’s disease,” Mukherjee said.

Scientists have known that nicotine’s action on these receptors elicits dopamine in various brain regions implicated in nicotine addiction and other disorders. Nicotine acts by opening specific membrane proteins, called nicotinic receptors, and changing the electrical properties of the cell.

The human brain coordinates billions of neurons to mediate complex behaviors such as an infant learning to recognize his or her parents, a senior citizen learning to play piano, or the process of addiction following repetitive exposure to specific drugs. These behaviors all result from the formation of new connections between individual neurons or modifications of existing connections in the brain.

“Imaging of nicotine receptors also gives us the potential to study why some people are more addicted to nicotine than others,” Mukherjee said.

Additional information : Chattopadhyay S, Xue B, Collins D, Pichika R, Bagnera R, Leslie FM, Christian BT, Shi B, Narayanan TK, Potkin SG, Mukherjee J.
Synthesis and Evaluation of Nicotine {alpha}4{beta}2 Receptor Radioligand, 5-(3'-18F-Fluoropropyl)-3-(2-(S)-Pyrrolidinylmethoxy)Pyridine, in Rodents and PET in Nonhuman Primate. Journal of Nuclear Medicine. 2005 Jan; 46(1): 130-140.

Source : University of California, Irvine


Saturday, January 22, 2005

Mouse brain cells rapidly recover after Alzheimer's plaques are cleared

Brain cells in a mouse model of Alzheimer's disease have surprised scientists with their ability to recuperate after the disorder's characteristic brain plaques are removed.

Researchers at Washington University School of Medicine in St. Louis injected mice with an antibody for a key component of brain plaques, the amyloid beta (Abeta) peptide. In areas of the brain where antibodies cleared plaques, many of the swellings previously observed on nerve cell branches rapidly disappeared.

"These swellings represent structural damage that seemed to be well established and stable, but clearing out the plaques often led to rapid recovery of normal structure over a few days," says senior author David H. Holtzman, M.D., the Charlotte and Paul Hagemann Professor and head of the Department of Neurology. "This provides confirmation of the potential benefits of plaque-clearing treatments and also gets us rethinking our theories on how plaques cause nerve cell damage."

Prior to the experiment, Holtzman and some other scientists had regarded plaque damage to nerve cells as a fait accompli--something that the plaques only needed to inflict on nerve cells once. According to Holtzman, the new results suggest that plaques might not just cause damage but also somehow actively maintain it.

The study, will appear in the Feb. 5 issue of the Journal of Clinical Investigation.

Lead author Robert Brendza, Ph.D., research instructor, began the experiment with one key question: how did clearance of brain plaques, made possible by the development of Abeta antibodies, affect the progression of Alzheimer's disease? Through collaborations with researchers at other institutions, he had acquired several key techniques and technologies that allowed him to closely track changes in live brain cells in mice with an Alzheimer's-like condition.

The mice he used for the study had two mutations. One, utilized by scientists at Eli Lilly, causes amyloid plaques to build up, creating the Alzheimer's-like condition. The second, developed by scientists at Washington University, causes some of the mouse brain cells to produce a dye that allowed Brendza to obtain detailed images of nerve cell branches.

To correlate brain cell changes with plaque development, Brendza injected another dye, developed by scientists at the University of Pittsburgh, that temporarily sticks to amyloid. He showed that as the plaques appeared, nearby branches of nerve cells developed bumps and swellings.

"If you look under the electron microscope at these swellings, they are filled with abnormal amounts of different types of cellular parts known as organelles," Holtzman explains. "Normally any given segment of a nerve cell branch would have only very small amounts of these organelles."

Nerve cells move organelles along their branches as a part of their regular function. Holtzman suspects that this transport breaks down in the mice, leading to pileups that become swellings. Scientists have previously demonstrated that such swellings make it difficult or impossible for nerve-cell branches to send signals.

After showing that the swellings were mostly stable in number and size over the course of three to seven days, Brendza injected Abeta antibodies directly onto the surface of the mouse brains. In the region of the injection, the antibodies cleared the plaques, confirming earlier research results. Then Brendza closely monitored the swellings for three days.

"We thought that clearing the plaques would halt the progression of the damage--stop the development of new swellings," says Brendza. "But what we saw was much more striking: in just three days, there were 20 to 25 percent reductions in the number or size of the existing swellings."

The nerve cells' rapid ability to regain normal structure has Holtzman and Brendza wondering if the nerve cells are constantly trying to restore their normal structure. If so, that recuperative effort must somehow be countered on an ongoing basis by the effects of the plaques.

More research is needed to determine if similar effects will occur in humans. Abeta antibodies are currently being considered for use in Alzheimer's patients in clinical trials.

In the mice, the largest swellings were least likely to heal. Brendza plans to look into whether additional treatment can prompt their recovery.

Holtzman and Brendza plan to continue using the mouse model to study disease treatments and the cellular abnormalities caused by their Alzheimer's-like condition.

"For example, we'd like to know what's going wrong in the nerve cell branches that get these swellings," Holtzman says. "Is it really a cellular transport problem, or do the swellings result from the plaques' effects on nearby support cells? Or is it something else?"

Source : Washington University School of Medicine


Key molecule in plant photo-protection identified

Another important piece to the photosynthesis puzzle is now in place. Researchers with the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California at Berkeley have identified one of the key molecules that help protect plants from oxidation damage as the result of absorbing too much light.

The researchers determined that when chlorophyll molecules in green plants take in more solar energy than they are able to immediately use, molecules of zeaxanthin, a member of the carotenoid family of pigment molecules, carry away the excess energy.

This study was led by Graham Fleming, director of Berkeley Lab's Physical Biosciences Division and a chemistry professor with UC Berkeley, and Kris Niyogi, who also holds joint appointments with Berkeley Lab and UC Berkeley. Its results are reported in the January 21, 2005 issue of the journal Science. Co-authoring the paper with Fleming and Niyogi were Nancy Holt, plus Donatas Zigmantas, Leonas Valkunas and Xiao-Ping Li.

Through photosynthesis, green plants are able to harvest energy from sunlight and convert it to chemical energy at an energy transfer efficiency rate of approximately 97 percent. If scientists can create artificial versions of photosynthesis, the dream of solar power as a clean, efficient and sustainable source of energy for humanity could be realized.

A potential pitfall for any sunlight-harvesting system is that if the system becomes overloaded with absorbed energy, it will likely suffer some form of damage. Plants solve this problem on a daily basis with a photo-protective mechanism called feedback de-excitation quenching. Excess energy, detected by changes in pH levels (the feedback mechanism), is safely dissipated from one molecular system to another, where it can then be routed down relatively harmless chemical reaction pathways.

Said Fleming, "This defense mechanism is so sensitive to changing light conditions, it will even respond to the passing of clouds overhead. It is one of Nature's supreme examples of nanoscale engineering."

The light harvesting system of plants consists of two protein complexes, Photosystem I and Photosystem II. Each complex features antennae made up of chlorophyll and carotenoid molecules that gain extra "excitation" energy when they capture photons. This excitation energy is funneled through a series of molecules into a reaction center where it is converted to chemical energy. Scientists have long suspected that the photo-protective mechanism involved carotenoids in Photosystem II, but, until now, the details were unknown.

Said Holt, "While it takes from 10 to 15 minutes for a plant's feedback de-excitation quenching mechanism to maximize, the individual steps in the quenching process occur on picosecond and even femtosecond time-scales (a femtosecond is one millionth of a billionth of a second). To identify these steps, we needed the ultrafast spectroscopic capabilities that have only recently become available."

The Berkeley researchers used femtosecond spectroscopic techniques to follow the movement of absorbed excitation energy in the thylakoid membranes of spinach leaves, which are large and proficient at quenching excess solar energy. They found that intense exposure to light triggers the formation of zeaxanthin molecules which are able to interact with the excited chlorophyll molecules. During this interaction, energy is dissipated via a charge exchange mechanism in which the zeaxanthin gives up an electron to the chlorophyll. The charge exchange brings the chlorophyll's energy back down to its ground state and turns the zeaxanthin into a radical cation which, unlike an excited chlorophyll molecule, is a non-oxidizing agent.

To confirm that zeaxanthin was indeed the key player in the energy quenching, and not some other intermediate, the Berkeley researchers conducted similar tests on special mutant strains of Arabidopsis thaliana, a weed that serves as a model organism for plant studies. These mutant strains were genetically engineered to either over express or not express at all the gene, psbS, which codes for an eponymous protein that is essential for the quenching process (most likely by binding zeaxanthin to chlorophyll).

"Our work with the mutant strains of Arabidopsis thaliana clearly showed that formation of zeaxanthin and its charge exchange with chlorophyll were responsible for the energy quenching we measured," said Niyogi. "We were surprised to find that the mechanism behind this energy quenching was a charge exchange, as earlier studies had indicated the mechanism was an energy transfer."

Fleming credits calculations performed on the supercomputers at the National Energy Research Scientific Computing Center (NERSC), under the leadership of Martin Head-Gordon, as an important factor in his group's determination that the mechanism behind energy quenching was an electron charge exchange. NERSC is a U.S. Department of Energy national user facility hosted by Berkeley Lab. Head-Gordon is a UC Berkeley faculty chemist with Berkeley Lab's Chemical Sciences Division.

"The success of this project depended on several different areas of science, from the greenhouse to the supercomputer," Fleming said. "It demonstrates that to understand extremely complex chemical systems, like photosynthesis, it is essential to combine state-of-the-art expertise in multiple scientific disciplines."

There are still many pieces of the photosynthesis puzzle that have yet to be placed for scientists to have a clear picture of the process. Fleming likens the on-going research effort to the popular board game, Clue.

"You have to figure out something like it was Colonel Mustard in the library with the lead pipe," he says. "When we began this project, we didn't know who did it, how they did it, or where they did it. Now we know who did it and how, but we don't know where. That's next!"

Source : Eurekalert


Anti-bacterial additive widespread in U.S. waterways

Many rivers and streams in the United States are believed to contain a toxic antimicrobial chemical whose environmental fate was never thoroughly scrutinized despite large scale production and usage for almost half a century, according to an analysis conducted by researchers at the Johns Hopkins Bloomberg School of Public Health. The chemical, triclocarban, has been widely used for decades in hand soaps and other cleaning products, but rarely was monitored for or detected in the environment. The new findings suggest that triclocarban contamination is greatly underreported. The study is published in the current online edition of Environmental Science & Technology, a peer-reviewed journal of the American Chemical Society.

"We've been using triclocarban for almost half a century at rates approaching 1 million pounds per year, but we have essentially no idea of what exactly happens to the compound after we flush it down the drain," said the study's lead author, Rolf U. Halden, PhD, PE, assistant professor in the School's Department of Environmental Health Sciences and founding member of its Center for Water and Health.

The nationwide assessment of triclocarban contamination is based in part on an analysis of water samples collected from rivers in and around Baltimore, Md., as well as from local water filtration and wastewater treatment plants. From the samples, Dr. Halden and his summer research intern, Daniel H. Paull, now a graduate student in the Chemistry department at Johns Hopkins University, observed the occurrence of triclocarban in the environment correlated strongly with that of triclosan, another commonly used antimicrobial chemical that has been studied in much greater detail because it is more easily detectable.

Using an empirical model and published data on the environmental occurrence of triclosan, the researchers predicted triclocarban concentrations for 85 U.S. streams. The study results suggest that the antimicrobial contaminant is present in 60 percent of the U.S. water resources investigated, thereby making it the fifth most frequent contaminant among 96 pharmaceuticals, personal care products and organic wastewater contaminants evaluated.

To determine the validity of the analysis, the researchers compared their predicted nationwide levels of contamination to experimentally measured concentrations in the Greater Baltimore region, and found no statistically significant differences. The results also show that the levels of triclocarban in water resources nationwide are much higher than previously thought.

In surface water from the Baltimore region, the researchers detected triclocarban at concentrations of up to 6.75 micrograms per liter (parts-per-billion). This maximum concentration was 28 fold higher than previously reported levels, which are currently used by the U.S. Environmental Protection Agency for evaluation of the ecological and human health risks of triclocarban.

"Along with its chemical cousin triclosan, the antimicrobial compound triclocarban should be added to the list of polychlorinated organic compounds that deserve our attention due to unfavorable environmental characteristics, which include long term persistence and potential bioaccumulation. Triclocarban, for example, has an estimated half life of 1.5 years in aquatic sediments. Do the potential benefits of antimicrobial products outweigh their known environmental and human health risks? This is a scientifically complex question consumers, knowingly or unknowingly, answer to everyday in the checkout line of the grocery store," said Dr. Halden.

Source : Eurekalert


Timing is everything: First step in protein building revealed

Timing is everything, it seems, even in science. A team led by Johns Hopkins scientists has unraveled the first step in translating genetic information in order to build a protein, only to find that it's not one step but two.

In a series of experiments, the scientists found that when yeast's protein-building machinery recognizes the starting line for a gene's instructions, it first alters its structure and then releases a factor known as eIF1, a step necessary to let it continue reading the assembly instructions. Even though yeast are the most primitive relatives of humans, the protein-building machinery, or ribosomes, of each are quite similar.

"The idea is to really know at the molecular level how life is put together," says Jon Lorsch, Ph.D., professor of biophysics and biophysical chemistry, one of the departments in Johns Hopkins' Institute for Basic Biomedical Sciences. "We see disease largely as an incorrect timing event -- the wrong thing happening at the wrong time, or the lack of the right thing."

As a result, Lorsch studies the timing of how the ribosome complex itself assembles and how other factors come and go as it translates genetic information to build proteins, the workhorses of cells. If the ribosome doesn't start in the right place along a gene's instructions, it will make the wrong protein, which can kill the cell or lead to disease.

"The ribosome is the end stage of gene expression, and gene expression keeps us alive and causes disease," says Lorsch. "If we can better understand how the ribosome works, perhaps we can harness it to help us fix disease."

Already, scientists knew that without eIF1, the ribosome can start reading the gene's RNA instructions at places other than a particular three-block piece of RNA known as the "start codon." And excessive amounts of eIF1 are associated with cardiac hypertrophy, or an enlarged heart.

While eIF1's role in cardiac hypertrophy remains a mystery, the new discovery reveals exactly how eIF1 regulates the ribosome's activity. The research team has demonstrated that eIF1's mere presence on the yeast ribosome prevents the machinery from getting started. Only after its release from the complex can the ribosome start making proteins.

"No one had any idea when eIF1 was released from the ribosome, or that its release might serve an important purpose, so this was a completely unexpected result," says graduate student David Maag, first author of the paper.

"It's impossible to know for sure whether eIF1 is released completely in living creatures, but in our laboratory experiments that is clearly the case," adds Lorsch. "Even if it isn't released completely in intact cells, our results would indicate that it must be very loosely associated for translation [protein building] to begin."

To monitor what was happening to eIF1, the researchers tagged it and a related part of the ribosome with different fluorescent chemicals. When two fluorescently labeled molecules are near one another, the fluorescent chemicals subtly interact, which changes the color or wavelength of light that is given off. If the distance between the fluorescent molecules changes, the color of the emitted light changes as well.

The researchers successfully used this phenomenon, known as fluorescence resonance energy transfer or FRET, to monitor the relationship between eIF1 and its relative as the ribosome complex assembled and after RNA was added to the mix.

"We weren't even sure the two fluorescent molecules would be close enough together to create a FRET signal at all," says Maag. "We were very pleased just to be able to monitor it, and then we were surprised and pleased by what we saw next."

They had expected -- or at least hoped -- to see a shift in the color of light once the RNA was mixed in. Instead, they saw two shifts in the color given off. First, there was a slight shift, indicating a small change in the distance between eIF1 and its relative, and then a much larger shift, indicating a much bigger separation.

To prove eIF1 was being released from the ribosome complex, the researchers examined how fast the pieces of the ribosome come together, and how long it takes them to fall apart under various circumstances. Their results support the idea that two separate steps take place once the instruction's starting point is found: first a structural change in the ribosome complex, and then release of eIF1.

Lorsch's goal is know the five "Ws" and one "H" that affect timing of all of the ribosome's pieces and activities. But unraveling every what, when, where, why, who and how is no small task -- roughly 27 bits like eIF1 play a role at one point or another. To tackle the problem, Lorsch and his colleagues move between "timing" studies of the ribosome's molecular comings and goings, and genetic studies that create mutant ribosome parts, which likely affect ribosome function -- and change its timing.

Source : Johns Hopkins Medical Institution


UW's Rosetta software to unlock secrets of many human proteins

University of Washington TechTransfer recently licensed software that will give scientists a huge advantage in the fight against disease.

The software, known as Rosetta, predicts how proteins fold, information that is highly valuable to biological and biomedical researchers.

UW Tech Transfer's Digital Ventures licensed Rosetta software without charge to the Institute for Systems Biology (ISB), a non-profit research organization. The institute has partnered with IBM and United Devices, an Austin-based company, to create the Human Proteome Folding Project, a global effort to determine the structures of the approximately 60 percent of human proteins with no known function.

"How proteins fold determines how they are structured," said Lars Malmstroem of the UW laboratory that developed the program, "And how they are structured is related to their function in the body."

Because there is an astronomical number of possible conformations for a given protein, collecting the data would take many thousands of lifetimes to complete with conventional computers, said Dr. Richard Bonneau, one of the researchers.

But by summoning the computing power of millions of volunteers around the world, he said, the task will be completed in less than a year.

IBM's World Community Grid, which was built using grid technology developed by United Devices, will enable millions of people to volunteer their personal computers to run Rosetta during periods of computer downtime.

The information will be entered into a publicly accessible database, which scientists can then use to conduct research into new drugs and treatments.

Rosetta works by virtually folding protein sequences into thousands of possible shapes, based on certain protein folding "rules" known by scientists. These rules are summarized in the program and are termed the "Rosetta score." The program tries a great many conformations and returns those with the lowest Rosetta scores; these conformations come closest to the actual shape of the protein.

Rosetta was developed in the laboratory of UW Professor David Baker by a large team of scientists and students. Former post-doctoral fellow Richard Bonneau, who is now with the ISB, is the technical lead for the project. Rosetta software is available for licensing at: http://depts.washington.edu/ventures/UW_Technology/Licensing/.

Source : University of Washington


Wednesday, January 19, 2005

Researchers find how protein allows insects to detect and respond to pheromones

How do insects smell? Badly, according to a new study, if they lack a certain kind of protein critical to their ability to detect and interpret pheromones – the insect equivalent of "smelling."

Researchers at UT Southwestern Medical Center have discovered how a protein, called an olfactory binding protein, links incoming pheromone signals and specific nerve cells in an insect's brain, which in turn translate those signals. Pheromones are chemical signals given off by animals that, when detected by others of the same species, mediate a variety of behaviors, such as feeding, mating and colonizing.

The findings not only shed light on insect behavior, but also suggest that olfactory binding proteins may be new targets for synthetic chemicals that could trick insects like mosquitoes into traps or could function as repellents, said Dr. Dean Smith, associate professor of pharmacology at UT Southwestern and senior author on the study. Humans give off signals that attract mosquitoes, the insect responsible for spreading malaria, which kills up to 3 million people each year.

The research, appearing in the Jan. 20 issue of the journal Neuron, is the first to directly link pheromone-induced behavior with the activity of olfactory binding proteins, or OBPs.

The nerve cells, or neurons, in insects responsible for picking up on pheromone signals have been studied for decades, as have pheromones themselves. But the biochemical mechanism by which pheromones and other odorants selectively activate those sensory neurons is poorly understood.

"We've known about OBPs for 20 years, but until now their function and significance was unclear," said Dr. Smith, who works in the Center for Basic Neuroscience. Olfactory binding proteins are produced by non-neuronal cells and are secreted into the fluid bathing the dendrites, or nerve endings, of olfactory neurons.

Dr. Smith's research group found that an OBP in fruit flies called LUSH is required for olfactory neurons to smell the pheromone 11-cis vaccenyl acetate, or VA. Mutant flies lacking the gene that codes for the LUSH protein are unable to detect the VA pheromone and do not display the behavior associated with that pheromone, which normally signals the flies to aggregate in groups.

When the VA pheromone contacts a tiny hair on a fly's antenna, it binds with the LUSH protein. Once bound, the LUSH protein changes its shape so it can fit into a receptor on the surface of a specific olfactory neuron inside the hair, which sends the appropriate behavioral signal to the bug.

"Without LUSH as a bridge, this pheromone can't get its signal to the neuron and the fly doesn't behave normally," Dr. Smith said. His research group reinstated the correct behavior in the mutant flies by injecting them with the missing lush gene.

In the absence of the pheromone, the researchers found that LUSH still binds to the olfactory neuron, sparking the neuron to fire a small electrical signal called "spontaneous activity." With the pheromone present, and bound to LUSH, the neuron exhibits a large burst of normal electrical activity. In mutants lacking LUSH, however, they found a 400-fold reduction in spontaneous activity, indicating that LUSH is necessary for the neuron to function properly.

"This reduction in spontaneous activity was a surprising finding," Dr. Smith said. "Our results indicate that LUSH, and not the pheromone, is what directly activates the chemosensory neurons. It is likely that OBPs in other insects also work this way, although the pheromones are different in different species. We think that OBPs might be new targets for insect control and repellents." Other studies have also linked OBPs to insect behavior. A 2002 fire ant study suggested a role for OBPs in worker ants' ability to recognize queens and regulate the number of queens in a colony.

The new UT Southwestern findings represent "a major breakthrough in our understanding of what role olfactory binding proteins play in insect pheromone detection," Drs. Leslie B. Vosshall and Marcus C. Stensmyr of The Rockefeller University wrote in a preview article in the same issue of Neuron.

Source : University of Texas Southwestern Medical Center at Dallas


Novel antiviral technology inhibits RSV infection in mice

A novel antiviral treatment combining nanoparticle and gene silencing technologies thwarts attacks of respiratory syncytial virus (RSV) -- a virus associated with severe bronchitis and asthma, an animal study by University of South Florida researchers found. The study was reported in the January 2005 issue of the journal Nature Medicine.

RSV infects lung cells and can be life-threatening in very young children, the elderly and those with compromised immune systems. No vaccine or widespread antiviral treatment is available for the infection.

Researchers at USF's Joy McCann Culverhouse Airway Disease Research Center, working with scientists from the Moffitt Cancer Center and TransGenex Nanobiotech Inc., used a revolutionary new technology known as RNA interference, or gene silencing, to knock out one of the key proteins needed for RSV to multiply in the lungs. The virus harnesses this protein, known as NS1, to block the body's own antiviral response, which would normally kill RSV before it could gain a foothold.

"This is an exciting advance in the fight against respiratory syncytial virus infection," said Shyam S. Mohapatra, PhD, principal investigator of the study and director of basic research at the USF Division of Allergy and Immunology. "We found that RNA interference targeting a virus's NS1 gene can be administered in the form of a nasal drop or spray. The treatment keeps the host's natural antiviral shield intact and attenuates virus reproduction, providing substantial protection from severe infections over days to weeks."

Dr. Mohapatra and his team developed nose drops containing vectors capable of producing small fragments of RNA (siRNA). These fragments were encapsulated within chitosan nanoparticles -- miniscule naturally-occurring, biodegradable particles that stick to mucous-producing cells lining the lungs. The RNA produced is specifically designed to suppress the protein NS1. Without NS1, the host antiviral defense remains intact and the virus cannot reproduce.

Mice treated intranasally with the gene-silencing nanoparticles, before and after infection with RSV, showed significantly lower levels of virus in the lung and less airway inflammation and hyper-reactivity than untreated mice.

Source : Eurekalert