Five subtypes of muscarinic acetylcholine receptors (mAChRs) are expressed throughout the body, where they exert diverse effects, such as smooth muscle contraction, glandular secretion, thermoregulation, and regulation of behavior, learning, and cognition.  MAChRs have been implicated in schizophrenia and Alzheimer’s disease (AD), making them attractive as candidate drug targets.  Several cholinergic agonists have shown promise for treating these conditions, but most of these drugs bind to the acetylcholine binding site—which is highly conserved across receptor subtypes—and therefore have undesirable side effects.  Because of this, drug developers have recently turned to allosteric agonists, which activate receptors by binding to subtype-specific domains outside the acetylcholine binding site.  Jones et al.  Report that one such agonist, which is highly specific for M1 mAChRs, produced effects in mice similar to effects of atypical antipsychotic drugs, without producing undesirable side effects.  Moreover, the drug regulated processing of amyloid precursor protein, suggesting that it may effectively treat AD.

Images of the brain’s fastest signals reveal an electromagnetic marker that predicts a patient’s response to a fast-acting antidepressant, researchers have discovered.

“Such biomarkers that identify who will benefit from a new class of antidepressants could someday minimize trial-and-error prescribing and speed delivery of care for what can be a life-threatening illness,” said Carlos Zarate, M.D., of the National Institute of Mental Health (NIMH), Mood and Anxiety Disorders Program.

In the new study at the National Institutes of Health in Bethesda, MD, depressed patients showed increasing activity in a mood-regulating hub near the front of the brain while viewing flashing frightful faces – the more the increase, the better their response to an experimental fast-acting medication called ketamine.  By contrast, healthy controls showed decreasing activity in this brain area under the same conditions.

Zarate, Giacomo Salvadore, M.D., Brian Cornwell, Ph.D., and NIMH colleagues report on their discovery online in Biological Psychiatry September 24, 2008.

Two years ago, Zarate and colleagues reported that ketamine, which targets the brain chemical glutamate, can lift depressions in just hours, instead of the weeks it takes conventional antidepressants, which work through the brain chemical serotonin.  Evidence suggests that glutamate likely acts closer to the source of the depression than serotonin, and is not dependant on slower mechanisms, such as the synthesis of new neurons.

Earlier imaging studies with conventional antidepressants had hinted that increased activity of the mood-regulating hub, called the anterior cingulate cortex (ACC), signals a better response.

To find out if ACC activity might also forecast response to glutamate-targeting medications, the NIMH researchers imaged the brain activity of 11 depressed patients and 11 healthy participants, using magnetoencephalography (MEG).  This imaging technology can non-invasively detect brain electromagnetic activity lasting only milliseconds – the speed of communications in neural circuits – whereas other functional brain imaging techniques can only capture activity that last seconds or minutes, and some involve radiation exposure.

This precise timing enabled the MEG scanner to capture the brain’s split-second responses to rapidly flashing pictures of fearful faces, a task known to activate the ACC.  While healthy participants’ ACC activity dropped off as they quickly habituated to the faces, patients’ ACC activity showed an opposite trend.  The more robust this increase, the more symptoms improved just four hours after a patient received a single infusion of ketamine.

“The ACC may be slow to respond, but not completely impaired, in patients who respond to ketamine,” explained Cornwell.

The lag in ACC activity could be a window into the dysfunctional workings of the glutamate-related circuitry targeted by the medication, the researchers suggest.  Ketamine’s side effects make it a poor candidate for becoming a practical antidepressant, but the new findings are helping to focus the search for new treatments that work through the same mechanism, they say.

Using computer models and live cell experiments, biomedical engineers at the Johns Hopkins University School of Medicine have discovered more than 100 human protein fragments that can slow or stop the growth of cells that make up new blood vessels.

Reporting online last week in the Proceedings of the National Academy of Sciences, the researchers say the findings could lead to developing treatments to fight diseases that depend on the growth of new blood vessels, including cancer, macular degeneration and rheumatoid arthritis.

“Before, there were only 40 known antiangiogenesis peptides,” says Aleksander Popel, Ph.D., a professor of biomedical engineering at Hopkins.  “Now, using a whole-genome, computer-based approach, we have identified more than 100 new ones, all of which can be further researched for their ability to fight the more than 30 known diseases affected by excessive blood vessel growth.”

To identify short protein fragments — peptides — that can block blood vessel growth, the team started by looking at 40 known peptides that have been studied and characterized by other experts in the field to stop blood vessel growth in animal models of disease.  Working under the assumption that the antivessel activity of these peptides can be attributed to similar features that are shared by a number of proteins, like the sequence of the peptide building blocks, the team first categorized the 40 known peptides by where they are located and what they look like.

Having defined nine families, the researchers then used computer programs and compared the peptide families to all of the proteins encoded by the genome.  They found more than 120 peptides contained in 82 different proteins, many of which were not previously known to have any activity on blood vessel development.

“Computational methods only identify potential candidates,” says Popel.  “We next had to do the experiments on live cells to see if they had any real activity.  Of the 82 proteins we identified, most were not previously known to have any antiangiogenic activity.”

To test the activity of these candidate peptides, the researchers applied them to blood vessel cells growing in the lab and examined whether they had any effect on the growth, survival and movement of these cells.  To test growth and survival, they added different amounts of peptide to dishes containing roughly 2,000 cells and after three days, counted how many cells were still alive.

To test cell movement, they placed cells in double-chambered dishes and treated the cells with a growth factor known to encourage cells to move.  To some of the dishes they added the test peptides.  After 20 hours, they measured the number of cells that had crawled from one chamber to the other.  They then identified the protein receptors that the peptides bind to and were able to show in some cases that combinations of more than one peptide were better able to stop the cells than using single peptides.

“Basic, computational studies like this are critical to understanding normal blood vessel growth,” says Popel.  “A better understanding of normal growth gives us a better idea of what happens in disease.”

The next step, Popel says, is to test these peptides in animal models of human disease and to identify the diseases most appropriately treated by these newly identified peptide inhibitors.

Novel platform technologies and key advances in genomics are rapidly driving the development of molecular diagnostics, reports Genetic Engineering and Biotechnology News (GEN).  The payoff for successful molecular diagnostic products can be significant as Kalorama Information predicts that this market currently exceeds $3.2 billion worldwide and will reach $5.4 billion in four years, according to an article in the October 1 issue of GEN.

“Molecular diagnostic products are based on cutting-edge research in two of the most promising biotechnologies, genomics and proteomics.  These novel tests also utilize sophisticated analytical techniques such as microarrays and mass spectrometry,” notes John Sterling, Editor-in-Chief of GEN.  “Molecular diagnostics are particularly applicable to the early detection of cancer.”

Affymetrix and Illumina have both created array-based products that enable high-speed analysis of DNA, RNA, and proteins as tools for disease research, drug development, and molecular tests.  These gene-sequencing tools are being applied at an earlier stage.

Genetic tests can optimize drug therapy, and companion diagnostics are being touted as a method to better define a patient’s need or predict clinical outcome from a specific drug.  The FDA recently approved a HER-2 test from Invitrogen called Spot-Light that can be used to identify breast cancer patients who are candidates for treatment with Herceptin.  In addition, data was recently presented showing the importance of testing for the K-ras gene to assess the clinical benefit of Erbitux for metastatic colorectal cancer.

Of all the larger integrated healthcare companies, Roche has best executed the synergies of molecular diagnostics and biopharmaceuticals and is well positioned for the future with products in oncology and infectious disease.  Its genetic tests include CYP450 for drug metabolism studies and HER-2 for use with tamoxifen therapy.

Scientists have discovered a compound that could lead to new treatments for heart attacks as well as methods to protect hearts during open heart surgery and other situations in which blood flow to the heart is interrupted.In the process, the researchers uncovered cellular mechanisms that help explain how alcohol can protect against heart attack damage. In addition, they have uncovered a possible key to reducing chest pain and the heart attack damage among millions of people of East Asian descent who are genetically unable to respond to nitroglycerin and other cardiovascular treatments.

A research team of scientists at Stanford and Indiana universities schools of medicine reports in the Sept. 12 issue of the journal Science that by jump-starting a particular enzyme they were able to significantly reduce the amount of cell death caused by lack of blood flow to the heart.

The group, led by Daria Mochly-Rosen, Ph.D., professor of chemical and systems biology at Stanford, found that administering a compound called Alda-1 activated the enzyme, reducing heart muscle damage in experiments involving rats.

First, however, the researchers studied various mechanisms known to provide cardioprotection to heart muscle cells, including the use of ethanol, to better understand how those mechanisms worked. That work revealed a cellular signaling system that activated a particular enzyme called ALDH2.

“The idea was to find a small molecule that could bypass the signaling process and activate the enzyme directly,” said Thomas D. Hurley, Ph.D., professor of biochemistry and molecular biology and director of the Center for Structural Biology at the IU School of Medicine. Hurley’s research has included years of study of the ALDH2 enzyme.

Although the Alda-1 molecule reduced heart tissue damage in laboratory tests, years of work will be necessary to refine the compound into a version that would be potentially effective and safe for human use, Dr. Hurley said. That benefit could extend to about 40 percent of people of East Asian descent who carry a mutated form of the ALDH2 enzyme, which puts them at increased risk of cardiovascular damage.

Some of the most challenging obstacles limiting the reprogramming of mature human cells into stem cells may not seem quite as daunting in the near future.  Two independent research papers, published by Cell Press in the September 11th issue of the journal Cell Stem Cell, describe new tools that provide invaluable platforms for elucidating the molecular, genetic, and biochemical mechanisms associated with reprogramming.  The new findings also offer considerable hope toward making the reprogramming process more therapeutically relevant.

Although scientists have successfully reprogrammed mature human skin cells into induced pluripotent stem (iPS) cells by expressing a few key transcription factors, the conversion has been extremely inefficient.  “Little is known about the mechanisms by which reprogramming occurs, in part because of the low efficiency,” says senior study author Dr. Konrad Hochedlinger from the Harvard Stem Cell Institute.  In addition, the iPS cells created thus far have been generated with retroviruses and noninducible lentiviruses, both of which have major limitations that are not compatible with clinical applications.

The Hochedlinger group created a drug-inducible viral system to generate human iPS cells that were molecularly and functionally similar to human embryonic stem cells.  This method was unique in that it allowed the researchers to create iPS cells by using the drug doxycycline to control expression of the necessary factors that had been delivered to the cells with viruses.

The researchers then found that when doxycycline was removed and these “primary” iPS cells differentiated to mature cells, another exposure to the drug reactivated the genes required for reprogramming and induced generation of “secondary” iPS cells at a frequency that was far greater that the initial “primary” conversion.  The idea of generating these secondary cells was conceived in previous experiments with mice performed in the lab of Dr. Rudolf Jaenisch from the Massachusetts Institute of Technology.

“The secondary system will enable chemical and genetic screening efforts to identify key molecular constituents of reprogramming, as well as important obstacles in this process, and will ultimately lend itself as a powerful tool in the development and optimization methods to produce human iPS cells,” explains Dr. Hochedlinger.

In a separate paper, Dr. Jaenisch’s group reports on their success in deriving human secondary iPS cells using doxycycline-inducible transgenes.  “The drug-inducible system we describe represents a novel, predictable, and highly reproducible platform to study the kinetics of iPS cell generation,” says Dr. Jaenisch.  “Further, the genetic homogeneity of secondary cells makes chemical and genetic screening approaches to enhance reprogramming efficiency or to replace any of the original reprogramming factors feasible.”

Both research teams found that generation of secondary human iPS cells required less time than the initial reprogramming.  Interestingly, the time required to generate iPS cells varied among the types of skin cells that were used.  For instance, human fibroblasts required several weeks, while keratinocytes required only about 10 days.  “The fast kinetics of reprogramming observed for keratinocytes suggests that these cells would be useful for development and optimization of methods to reprogram cells by transient delivery of factors,” suggests Dr. Hochedlinger.

The combined results from both research groups represent a major advance toward more efficient strategies for reprogramming differentiated human cells into iPS cells.  The methods described here will not only provide critical insight into the reprogramming process, but also, because of the abbreviated time frame, may lead to the generation of cells that will be amenable for therapies, as reprogramming might be achievable without the prohibitive viruses or genetic modifications.

If your experiment doesn’t go the way you expect, take a closer look -something even more interesting may have happened.  That strategy has led scientists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory to discover a fundamental shift in an enzyme’s function that could help expand the toolbox for engineering biofuels and other plant-based oil products.  The results will be published online the week of September 8, 2008, in the Proceedings of the National Academy of Sciences.

The Brookhaven scientists were trying to understand the factors that affect where carbon-carbon double bonds are placed in fatty acids, the building blocks of oils and fats, when they are “desaturated” -that is, when a desaturase enzyme removes hydrogen from the carbon chain.

“Placing double bonds in different positions allows you to change the structure of the fatty acids to make products with different potential applications,” explained Brookhaven biochemist John Shanklin, who led the research.  The ultimate goal: engineering designer plant oils to be used as biofuels and/or raw materials to reduce the use of petroleum.

To try to change the position of a double bond, the Brookhaven team modified a desaturase enzyme, changing three of the 363 amino acids in its protein sequence.  But when they tested the modified enzyme and looked for the expected product with its altered double-bond position, it wasn’t there.

They could have moved on and made different amino acid changes to accomplish the initial goal.  But Brookhaven research associate Edward Whittle was determined to figure out what was going on with the unusual result.  “The substrate, or starting material, had been used up, so something was being produced -substrates can’t just disappear,” Whittle said.  “If it wasn’t the product we were looking for, what was it?”

Whittle’s detective work uncovered a remarkable discovery.  Instead of producing a shift in double-bond position, the enzyme modification had yielded three completely new products -two variations of a hydroxylated product called an allylic alcohol and a fatty acid containing two double bonds.  “This was a profound shift in enzyme function,” noted Shanklin, who has been working with modified enzymes for 15 years.  “Usually you make changes very gradually, getting a few percent of a new product mixed with the original product.  This was more like throwing a switch, making the change in function close to complete.”

The discovery is also notable because the starting enzyme, like other soluble (membrane-independent) desaturases, can ordinarily perform only its one specified reaction -desaturation.  This is unlike desaturase enzymes that reside within the cell membrane, which appear to be more versatile, performing a range of reactions.  The soluble and membrane enzymes, however, do share one key feature: both perform reactions that require the production of a highly reactive form of oxygen.

“Since both classes of enzymes produce activated oxygen, in theory the soluble enzymes, like their membrane counterparts, should be able to perform a variety of reactions as well,” Shanklin said.  “Our work demonstrates that this is indeed the case.  Making small changes to the enzyme’s amino acid sequence has unlocked the soluble desaturase’s potential to facilitate a wider range of chemistry than has been seen before,” Shanklin said.

The challenge is to figure out how these structural changes to the enzyme lead to the observed changes in reaction chemistry.  Computer-generated models combining the known structure of the starting enzyme in conjunction with its new substrates are helping the scientists understand how the enzyme works.  The next step is to obtain real 3-D crystal structures of enzyme-substrate complexes, using the National Synchrotron Light Source at Brookhaven Lab, to see how they match up with the predictions.

Analyzing the structures of soluble enzymes is much simpler than obtaining structures for membrane enzymes.  So, in effect, this work is a fast-track approach for correlating structure with function, which should help scientists gain general mechanistic insights relevant to both classes of enzymes.  “Understanding how nature has figured out how to do this very difficult chemistry, and how to control that chemistry,” Shanklin said, “would be extremely satisfying from a purely scientific perspective.  But applying this knowledge could have benefits for us all.”

“Right now, the materials we use -the plastics, foams, nylons -have been limited by the structures of petroleum-based chemical feedstocks.  But if we understand how to engineer designer desaturase-like plant enzymes, we can tailor-make feedstocks with optimal properties, instead of relying on the properties of preexisting raw materials,” said Shanklin.  “We’d no longer have to say, ‘this is what we have, so this is what we can make.’  Instead, we could make the best feedstock for a particular application by designing the raw materials that will yield it.”

A well-nursed prejudice in scholarly communication is that researchers avoid journalists and are disappointed with the coverage when they do have contact with the media.  A current study in the specialist journal Science shows the opposite to be true: more than half of the researchers questioned described their contact with journalists as predominantly good.  Four out of ten found coverage in the public-sector beneficial to their career.  The idea of the “ivory tower of science” can therefore no longer be upheld.

“The second prejudice we need to dispense with is that German researchers tend to find dealing with journalists more difficult and are less motivated to report on their research in the public sphere than their colleagues in the USA”, said head of the study Prof.  Hans Peter Peters from Forschungszentrum Jülich, a member of the Helmholtz Association.  The number of interactions with the media was similarly high in all of the countries investigated.  More than two thirds of the researchers had contact with the media over a period of three years.  Their experience in all of the countries was also positive.  “The main reason for the similarity in this pattern can be seen in the social need for a public legitimation of science.”

The fact that media presence and management positions clearly go hand-in-hand also backs up this point.  “Being a leading researcher now requires a readiness to liaise with the mass media”, explained Peters.  This can be construed from the clear correlation between the number of contacts with the media and those holding management positions.  “In other words, it is not left up to the discretion of each scientist as to whether they want to forge links with the media”, explained Peters.  “In certain positions and situations, it is expected of them.  Subjective attitudes only play a secondary role.”

The study which has now been published is the first comprehensive international survey of scientists in the world on this topic.  It was conducted by Forschungszentrum Jülich and partners in France, the United Kingdom, Japan, and the USA.  The sample consisted of around 1,350 biomedical researchers from the five largest science nations who produced at least two pertinent publications in their field between 2002 and 2004.  For reasons of comparability, all of those interviewed were selected from two clearly defined fields of research – epidemiology and stem-cell research.

Diffusible molecules of the Slit family inhibit midline crossing by axons and neurons that express Robo receptors. For example, migrating inferior olive (IO) neurons extend a leading process across the midline, but the somata stop upon reaching the floor plate, which expresses Slits; the leading process forms the axon. Robo3 knock-out prevents midline crossing by the leading process, suggesting that Robo3 may interfere with repulsive signaling by other Slit–Robo pairs. To test this hypothesis, Di Meglio et al. knocked out Slits and Robos individually and in combination. As expected, IO somata crossed the midline in Slit1/2 and Robo1/2 knock-outs, confirming that these proteins normally repel neurons. Unexpectedly, however, axons failed to cross the midline in Robo1/2/3 triple knock-outs, indicating that Robo3 actively promotes crossing, rather than simply interfering with Robo1/2 signaling. In addition, the patterning of IO subnuclei was disrupted in knock-outs, suggesting an additional role for Slits and Robos.

Soon after an individual becomes infected with HIV the virus infects cells in the brain and spinal cord (the central nervous system [CNS]).  Although this causes no immediate problems, during the late-stages of disease it can cause dementia and encephalitis (acute inflammation of the brain that can cause death).  Monkeys infected with a relative of HIV (SIV) also sometimes develop CNS damage and provide a good model of CNS disease in individuals infected with HIV.  Insight into the mechanisms of CNS damage in SIV-infected monkeys has now been provided by a team of researchers at The Scripps Research Institute, La Jolla, who developed an approach to identify molecular changes in the fluid bathing the CNS (the CSF).  The researchers, who were led by Howard Fox and Gary Siuzdak, hope that similar approaches could be used to provide new information about other neurodegenerative and neuropsychiatric disorders.

In the study, an approach known as global metabolomics was used to assess the levels of molecules known as metabolites in the CSF before and after SIV-induced encephalitis was manifest.  The level of a number of metabolites, including some known as fatty acids and phospholipids, was observed to increase during infection.  Consistent with this, a protein known to be important in the generation of fatty acids was found to be increased in the brain of monkeys with SIV-induced encephalitis.  Further studies will be required to determine the precise role of the increased level of each metabolite, but it should be noted that many of them are known to induce receptor signaling and thereby might be able to further modulate CNS function.

A new mouse model for the study of Inclusion Body Myositis (IBM), a type of muscular dystrophy, has been developed by Dr. Ze’ev Ronai and a worldwide team of researchers.  The protein RNF5 is over-produced in the mice, resulting in extensive muscle damage similar to that seen in IBM patients.  The IBM mouse model will allow researchers to further study the mechanisms underlying development of the disease, as well as test potential new therapies.

A group of scientists at Yale have now provided a glimpse of the ancient mechanism that helped diversify our genomes; it illuminated a relationship between gene processing in humans and the most primitive organisms by creating the first crystal structure of a crucial self-splicing region of RNA.

The genes of higher organisms code for production of proteins through intermediary RNA molecules. But, after transcription from the DNA, these RNAs must be cut into pieces and patched together before they are ready for translation into protein. Stretches of the RNA sequence that code for protein are kept, and the intervening sequences, or introns, are spliced out of the transcript.

This work, published in Science, highlights a 16-year quest by Anna Marie Pyle, the William Edward Gilbert Professor of Molecular Biophysics & Biochemistry at Yale, and her research team into the nature of “group II” introns, a particular type of intron within gene transcripts that catalyzes its own removal during the maturation of RNA.

Group II introns are found throughout nature, in all forms of living organisms. Although much has been learned about their structure and how they work through biochemical and computational analysis, until now there have been no high-resolution crystal structures available. The resulting images have provided both confirmation of the earlier work and new information on the three-dimensional structure of RNA and the mechanism of splicing.

“One of the most exciting aspects of this work was that we did not need to do anything disruptive to these molecules to prepare them for structural analysis,” said Pyle. “The molecules showed us their structure, their active site and their activity — all in a natural state. We were even able to visualize their associated ions.”

According to Pyle, the crystal structure revealed some unexpected features — showing two sections that were most implicated as key elements of the active site and strengthening a theory that the process of splicing in humans “shares a close evolutionary heritage” with ancient forms of bacteria.

Looking to future applications of the work, Pyle said, “Group II introns hold promise in the future as agents of gene therapy. A free intron is an infectious element that is special because it targets DNA sites very specifically. We hope that further knowledge of these structures may lead to the development of new genetic tools and therapeutics.”

Citation: Science 320 , 77-82 (April 4, 2008). [DOI: 10.1126/science.1153803]