Tag: Biochemistry

Viruses are able to redirect the functioning of cells in order to infect them. Inspired by their mode of action, scientists from the CNRS and Université de Strasbourg have designed a “chemical virus” that can cross the double lipid layer that surrounds cells, and then disintegrate in the intracellular medium in order to release active compounds. To achieve this, the team used two polymers they had designed, which notably can self-assemble or dissociate, depending on the conditions. This work, the result of collaborative efforts by chemists, biologists and biophysicists, is published in the 1st September issue of Angewandte Chemie International Edition.

Biotechnological advances have offered access to a wealth of compounds with therapeutic potential. Many of these compounds are only active inside human cells but remain unusable because the lipid membrane surrounding these cells is a barrier they cannot cross. The challenge is therefore to find transfer solutions that can cross this barrier.

By imitating the ability of viruses to penetrate into cells, chemists in the Laboratoire de Conception et Application de Molécules Bioactives (CNRS/Université de Strasbourg) sought to design particles capable of releasing macromolecules that are only active inside cells. To achieve this, these particles must comply with several, often contradictory, constraints. They must remain stable in the extracellular medium, they must be able to bind to the cells so that they be internalized, but they must be more fragile inside the cells so that they can release their content. Using two polymers designed by the team, the scientists succeeded in creating a “chemical virus” that meets the conditions necessary for the direct delivery of active proteins into cells.

In practice, the first polymer (pGi-Ni2+) serves as a substrate for the proteins that bind to it. The second, recently patented polymer (πPEI), encapsulates this assembly thanks to its positive charges, which bind to the negative charges of pGi-Ni2+. The particles obtained (30-40 nanometers in diameter) are able to recognize the cell membrane and bind to it. This binding activates a cellular response: the nanoparticle is surrounded by a membrane fragment and enters the intracellular compartment, called the endosome. Although they remain stable outside the cell, the assemblies are attacked by the acidity that prevails within this new environment. Furthermore, this drop in pH allows the πPEI to burst the endosome, releasing its content of active compounds.

Thanks to this assembly, the scientists were able to concentrate enough active proteins within the cells to achieve a notable biological effect. Thus by delivering a protein called caspase 3 into cancer cell lines, they succeeded in inducing 80% cell death.

The in vitro results are encouraging, particularly since this “chemical virus” only becomes toxic at a dose ten times higher than that used during the study. Furthermore, preliminary results in the mouse have not revealed any excess mortality. However, elimination by the body of the two polymers remains an open question. The next stage will consist in testing this method in-depth and in vivo, in animals. In the short term, this system will serve as a research tool to vectorize recombinant and/or chemically modified proteins into cells. In the longer term, this work could make it possible to apply pharmaceutical proteins to intracellular targets and contribute to the development of innovative drugs.

To achieve maximum efficacy, the scientists are hoping to combine siRNA (small nucleic acids that specifically target the expression of certain genes) with the proteins, and these could also be delivered using the same particles. Vectorization consists in controlling the distribution of an active substance to a given target by combining it with a vector. This enables delivery of a protein inside a cell via the intermediary of a biocompatible vehicle.

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http://www.sciencedaily.com/releases/2015/08/150828135244.htm  Original web page at Science Daily

Activated neurons produce a protein that protects against nerve cell death. Prof. Dr. Hilmar. When nerve cells die, e.g. as a result of a stroke, Alzheimer’s disease or through age-related processes, the result may be considerable impairments of memory. Earlier studies led by Prof. Bading have shown that brain activity counteracts the death of nerve cells. The NMDA receptor plays a major role at the molecular level. This type of receptor is a molecule set in motion by biochemical neurotransmitters. Due to neuronal activity, the NMDA receptor causes calcium to enter the cell. The calcium signal is spreading within the cell, invades the cell nucleus and switches on a genetic protection programme. Prof. Bading’s group identified this nuclear calcium-induced gene programme a few years ago. “However, it was not clear to us how it leads to a protective shield,” Hilmar Bading explains

The scientists have now discovered the explanation for this — again by studying NMDA receptors. If these receptors are not located at the neuronal junctions, i.e. the synapses, they do not contribute to the protection of cells. On the contrary, they severely damage nerve cells and cause them to die. “Life and death are only a few thousandths of a millimetre away from one another. Outside the synapse the NMDA receptor is no longer Dr. Jekyll, it becomes Mr. Hyde,” Hilmar Bading comments. The current research results show that toxic extrasynaptic NMDA receptors are suppressed through brain activity. The Heidelberg research team has identified activin A as the protein activating this process.

Activin A plays an important role e.g. in the menstruation cycle and in healing wounds. It is produced in the nervous system thanks to neuronal activity. This leads to a reduction in extrasynaptic NMDA receptors and builds up a protective shield, according to Prof. Bading. Activin A also mediates the well-known protective properties of brain-derived neurotrophic factor (BDNF), a signalling molecule that protects existing neurons and synapses, and helps developing new ones. “Activin A can therefore be regarded as a crucial activator of a common neuroprotective mechanism in the brain.”

The discoveries made by the Heidelberg neurobiologists open up new prospects for treating degenerative diseases of the nervous system. In their study they showed that activin A in mice was able to significantly reduce brain damage after a stroke. “Our research results also indicate that activin A may possibly be used to treat Alzheimer’s disease or Huntington’s disease. The characteristic degeneration of nerve cells associated with these two diseases seems to be due to an increased activity of toxic extrasynaptic NMDA receptors,” says Prof. Bading. “In everyday terms the new findings mean: An active brain produces activin A thereby protecting itself from neurodegeneration.”

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http://www.sciencedaily.com/releases/2015/08/150824065021.htm  Original web page at Science Daily

A molecular chaperone has been found to inhibit a key stage in the development of Alzheimer’s disease and break the toxic chain reaction that leads to the death of brain cells, a new study shows. The research provides an effective basis for searching for candidate molecules that could be used to treat the condition. It may not actually be too difficult to find other molecules that do this, it’s just that it hasn’t been clear what to look for until recently. A good tactic now is to search for other molecules that have this same highly targeted effect and to see if these can be used as the starting point for developing a future therapy.

A molecule that can block the progress of Alzheimer’s disease at a crucial stage in its development has been identified by researchers in a new study, raising the prospect that more such molecules may now be found. The report shows that a molecular chaperone, a type of molecule that occurs naturally in humans, can play the role of an “inhibitor” part-way through the molecular process that is thought to cause Alzheimer’s, breaking the cycle of events that scientists believe leads to the disease. Specifically, the molecule, called Brichos, sticks to threads made up of malfunctioning proteins, called amyloid fibrils, which are the hallmark of the disease. By doing so, it stops these threads from coming into contact with other proteins, thereby helping to avoid the formation of highly toxic clusters that enable the condition to proliferate in the brain. This step — where fibrils made up of malfunctioning proteins assist in the formation of toxic clusters — is considered to be one of the most critical stages in the development of Alzheimer’s in sufferers. By finding a molecule that prevents it from occurring, scientists have moved closer to identifying a substance that could eventually be used to treat the disease. The discovery was made possible by an overall strategy that could now be applied to find other molecules with similar capabilities, extending the range of options for future drug development.

The research was carried out by an international team comprising academics from the Department of Chemistry at the University of Cambridge, the Karolinska Institute in Stockholm, Lund University, the Swedish University of Agricultural Sciences, and Tallinn University. Their findings are reported in the journal Nature Structural & Molecular Biology. Dr Samuel Cohen, a Research Fellow at St John’s College, Cambridge, and lead author of the report, said: “A great deal of work in this field has gone into understanding which microscopic processes are important in the development of Alzheimer’s disease; now we are starting to reap the rewards of this hard work. Our study shows, for the first time, one of these critical processes being specifically inhibited, and reveals that by doing so we can prevent the toxic effects of protein aggregation that are associated with this terrible condition.”

Alzheimer’s disease is one of a number of conditions caused by naturally occurring protein molecules folding into the wrong shape and then sticking together — or nucleating — with other proteins to create thin filamentous structures called amyloid fibrils. Proteins perform important functions in the body by folding into a particular shape, but sometimes they can misfold, potentially kick-starting this deadly process. Recent research, much of it by the academics behind the latest study, has however suggested a second critical step in the disease’s development. After amyloid fibrils first form from misfolded proteins, they help other proteins which come into contact with them to misfold and form small clusters, called oligomers. These oligomers are highly toxic to nerve cells and are now thought to be responsible for the devastating effects of Alzheimer’s disease. This second stage, known as secondary nucleation, sets off a chain reaction which creates many more toxic oligomers, and ultimately amyloid fibrils, generating the toxic effects that eventually manifest themselves as Alzheimer’s. Without the secondary nucleation process, single molecules would have to misfold and form toxic clusters unaided, which is a much slower and far less devastating process. By studying the molecular processes by which each of these steps takes effect, the research team assembled a wealth of data that enabled them to model not only what happens during the progression of Alzheimer’s disease, but also what might happen if one stage in the process was somehow switched off. “We had reached a stage where we knew what the data should look like if we inhibited any given step in the process, including secondary nucleation,” Cohen said. “Working closely with our collaborators in Sweden — who had developed groundbreaking experimental methods to monitor the process — we were able to identify a molecule that produced exactly the results that we were hoping to see in experiments.”

The results indicated that the molecule, Brichos, effectively inhibits secondary nucleation. Typically, Brichos functions as a “molecular chaperone” in humans; a term given to “housekeeping” molecules that help proteins to avoid misfolding and aggregation. Lab tests, however, revealed that when this molecular chaperone encounters an amyloid fibril, it binds itself to catalytic sites on its surface. This essentially forms a coating that prevents the fibrils from assisting other proteins in misfolding and nucleating into toxic oligomers. The research team then carried out further tests in which living mouse brain tissue was exposed to amyloid-beta, the specific protein that forms the amyloid fibrils in Alzheimer’s disease. Allowing the amyloid-beta to misfold and form amyloids increased toxicity in the tissue significantly. When this happened in the presence of the molecular chaperone, however, amyloid fibrils still formed but the toxicity did not develop in the brain tissue, confirming that the molecule had suppressed the chain reaction from secondary nucleation that feeds the catastrophic production of oligomers leading to Alzheimer’s disease. By modelling what might happen if secondary nucleation is switched off and then finding a molecule that performs that function, the research team suggests that they have discovered a strategy that may lead to the identification of other molecules that could have a similar effect. “It may not actually be too difficult to find other molecules that do this, it’s just that it hasn’t been clear what to look for until recently,” Cohen said. “It’s striking that nature — through molecular chaperones — has evolved a similar approach to our own by focusing on very specifically inhibiting the key steps leading to Alzheimer’s. A good tactic now is to search for other molecules that have this same highly targeted effect and to see if these can be used as the starting point for developing a future therapy.

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http://www.sciencedaily.com/releases/2015/02/150217114547.htm Original web page at Science Daily

A group of researchers from the University of Helsinki and the Universitat Autònoma de Barcelona have been able to experimentally reproduce morphological changes in mice which took millions of years to occur. in nature Through small and gradual modifications in the embryonic development of mice teeth, induced in the laboratory, scientists have obtained teeth which morphologically are very similar to those observed in the fossil registry of rodent species which separated from mice millions of years ago. To modify the development of their teeth, the team from the Institute of Biotechnology of the University of Helsinki worked with embryonic teeth cultures from mice not coded by the ectodysplasin A (EDA) protein, which regulates the formation of structures and differentiation of organs in the embryo throughout its development. The teeth obtained with these cultures which present this mutation develop into very basic forms, with very uniform crowns. Scientists gradually added different amounts of the EDA protein to the embryonic cells and let them develop. The researchers observed that the teeth formed with different degrees of complexity in their crown. The more primitive changes observed coincide with those which took place in animals of the Triassic period, some two hundred million years ago. The development of more posterior patterns coincides with the different stages of evolution found in rodents which became extinct already in the Palaeocene Epoch, some 60 million years ago. Researchers have thus achieved experimentally to reproduce the transitions observed in the fossil registry of mammal teeth. The team of scientists were able to contrast the shape of these teeth with a computer-generated prediction model created by Isaac Salazar-Ciudad, researcher at the UAB and at the University of Helsinki, which reproduces how the tooth changes from a group of equal cells to a complex three-dimensional structure, with the full shape of a molar tooth, calculating the position of space of each cell. The model is capable of predicting the changes in the morphology of the tooth when a gene is modified, and therefore offers an explanation of the mechanisms that cause these specific changes to occur in the shape of teeth throughout evolution. The research appears in Nature.

http://www.sciencedaily.com/  Science Daily

http://www.sciencedaily.com/releases/2014/07/140730133255.htm  Original web page at Science Daily

In two separate papers given early online release today by the journal Science — which is publishing the papers this coming Friday, Professors Amy Wagers and Lee Rubin, of Harvard’s Department of Stem Cell and Regenerative Biology (HSCRB), report that injections of a protein known as GDF11, which is found in humans as well as mice, improved the exercise capability of mice equivalent in age to that of about a 70-year-old human, and also improved the function of the olfactory region of the brains of the older mice — they could detect smell as younger mice do. Rubin and Wagers each said that, baring unexpected developments, they expect to have GDF11 in initial human clinical trials within three to five years. Postdoctoral fellow Lida Katsimpardi is the lead author on the Rubin group’s paper, and postdocs Manisha Sinha and Young Jang are the lead authors on the paper from the Wagers group. Both studies examined the effect of GDF11 in two ways. First, by using what is called a parabiotic system, in which two mice are surgically joined and the blood of the younger mouse circulates through the older mouse. And second, by injecting the older mice with GDF11, which in an earlier study by Wagers and Richard Lee, of Brigham and Women’s Hospital who is also an author on the two papers released today, was shown to be sufficient to reverse characteristics of aging in the heart. Doug Melton, co-chair of HSCRB and co-director of HSCI, reacted to the two papers by saying that he couldn’t “recall a more exciting finding to come from stem cell science and clever experiments. This should give us all hope for a healthier future. We all wonder why we were stronger and mentally more agile when young, and these two unusually exciting papers actually point to a possible answer: the higher levels of the protein GDF11 we have when young. There seems to be little question that, at least in animals, GDF11 has an amazing capacity to restore aging muscle and brain function,” he said. Melton, Harvard’s Xander University Professor continued, saying that the ongoing collaboration between Wagers, a stem cell biologist whose focus has been on muscle, Rubin, whose focus is on neurodegenerative diseases and using patient generated stem cells as targets for drug discover, and Lee, a practicing cardiologist and researcher, “is a perfect example of the power of the Harvard Stem Cell Institute as an engine of truly collaborative efforts and discovery, bringing together people with big, unique ideas and expertise in different biological areas.”

As Melton noted, GDF11 is naturally found in much higher concentration in young mice than in older mice, and raising its levels in the older mice has improved the function of every organ system thus far studied. Wagers first began using the parabiotic system in mice 14 years ago as a post doctoral fellow at Stanford University, when she and colleagues Thomas Rando, of Stanford, Irina Conboy, of UC Berkley, and Irving Weissman, of Stanford, observed that the blood of young mice circulating in old mice seemed to have some rejuvenating effects on muscle repair after injury. Last year she and Richard Lee published a paper in which they reported that when exposed to the blood of young mice, the enlarged, weakened hearts of older mice returned to a more youthful size, and their function improved. And then working with a Colorado firm, the pair reported that GDF11 was the factor in the blood apparently responsible for the rejuvenating effect. That finding has raised hopes that GDF11 may prove, in some form, to be a possible treatment for diastolic heart failure, a fatal condition in the elderly that now is irreversible, and fatal. “From the previous work it could have seemed that GD11 was heart specific,” said Wagers, “but this shows that it is active in multiple organs and cell types… Prior studies of skeletal muscle and the parabiotic effect really focused on regenerative biology. Muscle was damaged and assayed on how well it could recover,” Wagers explained. She continued: “The additional piece is that while prior studies of young blood factors have shown that we achieve restoration of muscle stem cell function and they repair the muscle better, in this study, we also saw repair of DNA damage associated with aging, and we got it in association with recovery of function, and we saw improvements in unmanipulated muscle. Based on other studies, we think that the accumulation DNA damage in muscle stem cells might be reflect an inability of the cells to properly differentiate to make mature muscle cells, which is needed for adequate muscle repair.

Read more: http://www.sciencedaily.com/releases/2014/05/140504133205.htm

http://www.sciencedaily.com/  Science Daily

May 27, 2014

http://www.sciencedaily.com/releases/2014/05/140504133205.htm Original web page at Science Daily

When cannibals ate brains of people who died from prion disease, many of them fell ill with the fatal neurodegenerative disease as well. Likewise, when cows were fed protein contaminated with bovine prions, many of them developed mad cow disease. On the other hand, transmission of prions between species, for example from cows, sheep, or deer to humans, is — fortunately — inefficient, and only a small proportion of exposed recipients become sick within their lifetimes. A study published on April 3rd in PLOS Pathogens takes a close look at one exception to this rule: bank voles appear to lack a species barrier for prion transmission, and their universal susceptibility turns out to be both informative and useful for the development of strategies to prevent prion transmission. Prions are misfolded, toxic versions of a protein called PrP, which in its normal form is present in all mammalian species that have been examined. Toxic prions are “infectious”; they can induce existing, properly folded PrP proteins to convert into the disease-associated prion form. Prion diseases are rare, but they share features with more common neurodegenerative diseases like Alzheimer’s disease.

Trying to understand the unusual susceptibility of bank voles to prions from other species, Stanley Prusiner, Joel Watts, Kurt Giles and colleagues, from the University of California in San Francisco, USA, first tested whether the susceptibility is an intrinsic property of the voles’ PrP, or whether other factors present in these rodents make them vulnerable. The scientists introduced into mice the gene that codes for the normal bank vole prion protein, thereby generating mice that express bank vole PrP, but not mouse PrP. When these mice get older, some of them spontaneously develop neurologic illness, but in the younger ones the bank vole PrP is in its normal, benign folded state. The scientists then exposed young mice to toxic misfolded prions from 8 different species, including human, cattle, elk, sheep, and hamster. They found that all of these foreign-species prions can cause prion disease in the transgenic mice, and that the disease develops often more rapidly than it does in bank voles. The latter is likely because the transgenic mice express higher levels of bank vole PrP than are naturally present in the voles. The results show that the universal susceptibility of bank voles to cross-species prion transmission is an intrinsic property of bank vole PrP. Because the transgenic mice develop prion disease rapidly, the scientists propose that the mice will be useful tools in studying the processes by which toxic prions “convert” healthy PrP and thereby destroy the brain. And because that process is similar across many neurodegenerative diseases, better understanding prion disease development might have broader implications.

http://www.sciencedaily.com/  Science Daily

April 29, 2014

http://www.sciencedaily.com/releases/2014/04/140403212519.htm  Original web page at Science Daily

Get ready: The “new genetics” promises to change faulty genes of future generations by introducing new, functioning genes using “designer sperm.” A new research report appearing online in The FASEB Journal, shows that introducing new genetic material via a viral vector into the sperm of mice leads to the presence and activity of those genes in the resulting embryos. This new genetic material is actually inherited, present and functioning through three generations of the mice tested. This discovery — if successful in humans — could lead to a new frontier in genetic medicine in which diseases and disorders are effectively cured, and new human attributes, such as organ regeneration, may be possible. “Transgenic technology is a most important tool for researching all kinds of disease in humans and animals, and for understanding crucial problems in biology,” said Anil Chandrashekran, Ph.D., study author from the Department of Veterinary Clinical Sciences at The Royal Veterinary College in North Mimms, United Kingdom. To achieve these results, Chandrashekran and colleagues used lentiviruses to generate transgenic animals via the male germ line. When pseudotyped lentiviral vectors encoding green fluorescent protein (GFP) were incubated with mouse spermatozoa, these sperm were highly successful in producing transgenics. Lentivirally-transduced mouse spermatozoa were used in vitro fertilization studies and when followed by embryo transfer, at least 42 percent of founders were transgenic for GFP. GFP expression was detected in a wide range of murine tissues, including testis and the transgene was stably transmitted to a third generation of transgenic animals.

“Using modified sperm to insert genetic material has the potential to be a major breakthrough not only in future research, but also in human medicine,” said Gerald Weissmann, M.D., Editor-in-Chief of The FASEB Journal. “It facilitates the development of transgenic animal models, and may lead to therapeutic benefits for people as well. For years we have chased effective gene therapies and have hit numerous speed bumps and dead ends. If we are able to alter sperm to improve the health of future generations, it would completely change our notions of ‘preventative medicine.'”

Science Daily
January 7, 2014

Original we page at Science Daily

A peptide similar to the one pictured was effective at treating type-2 diabetes in a small clinical trial. Joe Chabenne, Faming Zhang, Richard DiMarchi/Indiana University An experimental diabetes treatment that packs the action of two natural hormones into a single injectable agent has been shown to successfully lower blood sugar in humans, monkeys and rodents. Marking a new approach in the treatment of the disease, the currently unnamed molecule also seems likely to cause fewer gastrointestinal side effects in humans than did other diabetes medicines. “We aimed for achieving the best glycaemic control with as little effect on the gut as possible,” says Richard DiMarchi, a biomolecular scientist at Indiana University in Bloomington, and a member of the international team that publishes the results today in Science Translational Medicine. The molecule, which targets receptors for the two hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), was developed in DiMarchi’s lab. Swiss pharmaceutical company Roche, based in Basel, supported the research and has licensed the agent.

As part of the study, 44 patients with type 2 diabetes received once-weekly injection of the dual-action molecule at various doses for six weeks while nine others received placebo injections. Blood tests showed a dose-dependent response; at the highest doses, a standard marker of blood glucose levels dropped an average of 1.1 percentage points from the baseline (which ranged from 7.4% to 7.9%; normal levels are below 5.7% in non-diabetic patients). In the placebo group, the marker dropped by just 0.16 points. There were no significant changes in body weight in the human trial, but the animal studies suggest that a long-term treatment at higher doses could also treat obesity. Obese mice receiving the highest doses of the molecule lost nearly 19% of their body weight in just one week, compared with about 9% for mice treated with equivalent amounts of a commonly prescribed diabetes drug called liraglutide. Both GLP-1 and GIP naturally respond to spikes in blood sugar by stimulating insulin production. GLP-1 also decreases appetite and suppresses glucagon, a hormone that raises blood sugar. Several current diabetes drugs, including exenatide and liraglutide, work by mimicking GLP-1. But as DiMarchi notes, about 10–30% of people taking such drugs develop gastrointestinal distress, including nausea, flatulence and sometimes vomiting. In the latest trial, just two people in the treatment group complained of mild nausea.

DiMarchi and his colleagues have previously shown that combining GLP-1 and oestrogen could reverse risk factors for diabetes in mice. But there have been few attempts to create drugs that harness the power of GIP. The hormone clearly has a natural role in controlling blood sugar, says DiMarchi, so it should not be ignored in drug development. “The best pharmacology replicates physiology,” he says. “This a key that works on both locks.” By combining human trials with thorough rodent and monkey studies in a single paper, the researchers have “delivered a complete package”, says Philipp Scherer, chair of diabetes research at the University of Texas Southwestern Medical Center in Dallas. “Usually, if you just have complete rodent data, you can make quite a splash.” However, the suggestion that the treatment causes relatively few side effects needs to be confirmed in a larger clinical trial, he says. DiMarchi says that the next step will be a long-term study that compares the latest approach to an established GLP-1 mimic. “This is still years away from being an approved drug,” he adds.

Nature
November 12, 2013

Original web page at Nature

The body’s immune system is set up much like a home security system; it has sensors on the outside of cells that act like motion detectors — floodlights — that click on when there’s an intruder rustling in the bushes, bacteria that seem suspect. For over a decade researchers have known about one group of external sensors called Toll-like receptors that detect when bacteria are nearby. Now, researchers at the University of North Carolina School of Medicine have identified a sensor pathway inside cells. These internal sensors are like motion detectors inside a house; they trigger an alarm that signals for help — a response from the immune system. This research, published in the Sept. 13, 2013 issue of the journal Science, indicates that both exterior and interior sensors work together to detect the same component of bacterial cell membranes, a molecule called lipopolysaccharide or LPS. By showing how the immune system distinguishes between suspicious activity and real threats, the study could lead to new therapies for septic shock — when the immune system overreacts to a bacterial infection to such an extent that it causes more harm than good.

“During the defense against an infection you want to be able to differentiate between the bacteria that stay on the outside of the cell and the ones that get inside,” said senior study author Edward A. Miao, MD, PhD, assistant professor of microbiology and immunology. “You can think of the exterior sensors as a yellow alert; they tell us that bacteria are present. But these bacteria could either be simple ones in the wrong place, or very dangerous ones that could cause a serious infection. The interior sensors act as a red alert; they warn us that there are bacteria with ill intent that have the genetic capacity to invade and manipulate our cells.” The body responds to a bacterial infection by increasing blood vessel permeability near the area under attack, which allows immune system cells to leave the bloodstream and seek and destroy the bacteria. Fluid also leaks into the area surrounding the infection, causing characteristic swelling. This is beneficial in fighting infection, but when the infection gets out of hand and these immune response occur throughout the body, blood pressure plummets, overtaxing the heart and leading to organ failure and often death. This increasingly prevalent syndrome, known as septic shock, afflicts over 750,000 people each year in the United States at a cost of nearly $17 billion. About half of the cases of septic shock are caused by bacteria that produce LPS, also known as endotoxin. In fact, much of what is known about endotoxic shock comes from studying animals injected with high doses of LPS. For example, previous studies pinpointed the role of the Toll-like receptor 4 gene (TLR4) as a sensor on the outside of cells; mice without that gene resisted endotoxic shock.

In a study published in January 2013, also in the journal Science, Miao and his colleagues showed that a sensor called caspase-11 sounds an alert when bacteria enter a cell. However, it wasn’t clear which of the thousands of molecules that make up a bacterial cell triggers that new sensor. In the current study, Miao and his colleagues investigated which bits of foreign material were being detected. They took apart and delivered different chunks of bacteria into the cytoplasmic compartment inside the cell. To their surprise, they found that the caspase-11 sensor inside the cell was detecting the same molecule, LPS, as the TLR4 sensor outside the cell. The researchers wondered whether there was a link between these two sensors. Through a number of experiments in animal models of sepsis, Miao’s team showed that the exterior and interior alarms work together through a two-step defense mechanism: LPS is first seen on the outside of the cell by TLR4, which sets the interior caspase-11 alarm into a watchful state. At very high doses, the LPS crosses into the cell, tripping the caspase-11 alarm. The end result is the generation of the red alert signal, which causes the cell to explode, a form of cell death called pyroptosis. During an infection, the immune system essentially burns the house down around the invading bacteria, depriving it of a place to replicate, and exposing it to more potent immune defenses. During sepsis, however, too much fire leads to the onset of shock. Miao says that figuring out how these two sensors get activated in response to a bacterial infection could help researchers develop new ways of preventing or treating septic shock, a condition that kills about half its victims. “The septic shock we see in patients is probably a lot more complicated than what we see in this experimental system,” said Miao. “The next question we need to ask is whether these same sensors are going off in people with septic shock, and if so, is there a way to block them so we can keep patients from dying.”

Science Daily
October 1, 2013

Original web page at Science Daily

Memory improved in mice injected with a small, drug-like molecule discovered by UCSF San Francisco researchers studying how cells respond to biological stress. The same biochemical pathway the molecule acts on might one day be targeted in humans to improve memory, according to the senior author of the study, Peter Walter, PhD, UCSF professor of biochemistry and biophysics and a Howard Hughes Investigator. The discovery of the molecule and the results of the subsequent memory tests in mice were published in eLife, an online scientific open-access journal, on May 28, 2013. In one memory test included in the study, normal mice were able to relocate a submerged platform about three times faster after receiving injections of the potent chemical than mice that received sham injections. The mice that received the chemical also better remembered cues associated with unpleasant stimuli — the sort of fear conditioning that could help a mouse avoid being preyed upon. Notably, the findings suggest that despite what would seem to be the importance of having the best biochemical mechanisms to maximize the power of memory, evolution does not seem to have provided them, Walter said. “It appears that the process of evolution has not optimized memory consolidation; otherwise I don’t think we could have improved upon it the way we did in our study with normal, healthy mice,” Walter said.

The memory-boosting chemical was singled out from among 100,000 chemicals screened at the Small Molecule Discovery Center at UCSF for their potential to perturb a protective biochemical pathway within cells that is activated when cells are unable to keep up with the need to fold proteins into their working forms. However, UCSF postdoctoral fellow Carmela Sidrauski, PhD, discovered that the chemical acts within the cell beyond the biochemical pathway that activates this unfolded protein response, to more broadly impact what’s known as the integrated stress response. In this response, several biochemical pathways converge on a single molecular lynchpin, a protein called eIF2 alpha. Scientists have known that in organisms ranging in complexity from yeast to humans different kinds of cellular stress — a backlog of unfolded proteins, DNA-damaging UV light, a shortage of the amino acid building blocks needed to make protein, viral infection, iron deficiency — trigger different enzymes to act downstream to switch off eIF2 alpha. “Among other things, the inactivation of eIF2 alpha is a brake on memory consolidation,” Walter said, perhaps an evolutionary consequence of a cell or organism becoming better able to adapt in other ways.

Turning off eIF2 alpha dials down production of most proteins, some of which may be needed for memory formation, Walter said. But eIF2 alpha inactivation also ramps up production of a few key proteins that help cells cope with stress. Study co-author Nahum Sonenberg, PhD, of McGill University previously linked memory and eIF2 alpha in genetic studies of mice, and his lab group also conducted the memory tests for the current study. The chemical identified by the UCSF researchers is called ISRIB, which stands for integrated stress response inhibitor. ISRIB counters the effects of eIF2 alpha inactivation inside cells, the researchers found. “ISRIB shows good pharmacokinetic properties how a drug is absorbed, distributed and eliminated, readily crosses the blood-brain barrier, and exhibits no overt toxicity in mice, which makes it very useful for studies in mice,” Walter said. These properties also indicate that ISRIB might serve as a good starting point for human drug development, according to Walter. Walter said he is looking for scientists to collaborate with in new studies of cognition and memory in mouse models of neurodegenerative diseases and aging, using ISRIB or related molecules. In addition, chemicals such as ISRIB could play a role in fighting cancers, which take advantage of stress responses to fuel their own growth, Walter said. Walter already is exploring ways to manipulate the unfolded protein response to inhibit tumor growth, based on his earlier discoveries. At a more basic level, Walter said, he and other scientists can now use ISRIB to learn more about the role of the unfolded protein response and the integrated stress response in disease and normal physiology.

Science Daily
July 9, 2013

Original web page at Science Daily

A potential new treatment strategy for patients with Charcot-Marie-Tooth disease is on the horizon, thanks to research by neuroscientists now at the University at Buffalo’s Hunter James Kelly Research Institute and their colleagues in Italy and England. The institute is the research arm of the Hunter’s Hope Foundation, established in 1997 by Jim Kelly, Buffalo Bills Hall of Fame quarterback, and his wife, Jill, after their infant son Hunter was diagnosed with Krabbe leukodystrophy, an inherited fatal disorder of the nervous system. Hunter died in 2005 at the age of eight. The institute conducts research on myelin and its related diseases with the goal of developing new ways of understanding and treating conditions such as Krabbe disease and other leukodystrophies. Charcot-Marie-Tooth or CMT disease, which affects the peripheral nerves, is among the most common of hereditary neurological disorders; it is a disease of myelin and it results from misfolded proteins in cells that produce myelin. The new findings sere published online earlier this month in The Journal of Experimental Medicine.

They may have relevance for other diseases that result from misfolded proteins, including Alzheimer’s disease, Parkinson’s, multiple sclerosis, Type 1 diabetes, cancer and mad cow disease. The paper shows that missteps in translational homeostasis, the process of regulating new protein production so that cells maintain a precise balance between lipids and proteins, may be how some genetic mutations in CMT cause neuropathy. CMT neuropathies are common, hereditary and progressive; in severe cases, patients end up in wheelchairs. These diseases significantly affect quality of life but not longevity, taking a major toll on patients, families and society, the researchers note. “It’s possible that our finding could lead to the development of an effective treatment not just for CMT neuropathies but also for other diseases related to misfolded proteins,” says Lawrence Wrabetz, MD, director of the institute and professor of neurology and biochemistry in UB’s School of Medicine and Biomedical Sciences and senior author on the paper. Maurizio D’Antonio, of the Division of Genetics and Cell Biology of the San Raffaele Scientific Institute in Milan is first author; Wrabetz did most of this research while he was at San Raffaele, prior to coming to UB.

The research finding centers around the synthesis of misfolded proteins in Schwann cells, which make myelin in nerves. Myelin is the crucial fatty material that wraps the axons of neurons and allows them to signal effectively. Many CMT neuropathies are associated with mutations in a gene known as P0, which glues the wraps of myelin together. Wrabetz has previously shown in experiments with transgenic mice that those mutations cause the myelin to break down, which in turn, causes degeneration of peripheral nerves and wasting of muscles. When cells recognize that the misfolded proteins are being synthesized, cells respond by severely reducing protein production in an effort to correct the problem, Wrabetz explains. The cells commence protein synthesis again when a protein called Gadd34 gets involved. “After cells have reacted to, and corrected, misfolding of proteins, the job of Gadd34 is to turn protein synthesis back on,” says Wrabetz. “What we have shown is that once Gadd34 is turned back on, it activates synthesis of proteins at a level that’s too high — that’s what causes more problems in myelination. “We have provided proof of principle that Gadd34 causes a problem with translational homeostasis and that’s what causes some neuropathies,” says Wrabetz. “We’ve shown that if we just reduce Gadd34, we actually get better myelination. So, leaving protein synthesis turned partially off is better than turning it back on, completely.”

In both cultures and a transgenic mouse model of CMT neuropathies, the researchers improved myelin by reducing Gadd34 with salubrinal, a small molecule research drug. While salubrinal is not appropriate for human use, Wrabetz and colleagues at UB and elsewhere are working to develop derivatives that are appropriate. “If we can demonstrate that a new version of this molecule is safe and effective, then it could be part of a new therapeutic strategy for CMT and possibly other misfolded protein diseases as well,” says Wrabetz. And while CMT is the focus of this particular research, the work is helping scientists at the Hunter James Kelly Research Institute enrich their understanding of myelin disorders in general. “What we learn in one disease, such as CMT, may inform how we think about toxins for others, such as Krabbe’s,” Wrabetz says. “We’d like to build a foundation and answer basic questions about where and when toxicity in diseases begin.” The misfolded protein diseases are an interesting and challenging group of diseases to study, he continues. “CMT, for example, is caused by mutations in more than 40 different genes,” he says. “When there are so many different genes involved and so many different mechanisms, you have to find a unifying mechanism: this problem of Gadd34 turning protein synthesis on at too high a level could be one unifying mechanism. The hope is that this proof of principle applies to more than just CMT and may lead to improved treatments for Alzheimer’s, Parkinson’s, Type 1 diabetes and the other diseases caused by misfolded proteins.”

Science Daily
May 14, 2013

Original web page at Science Daily