MIT engineers have developed a new, highly efficient way to pair up cells so they can be fused together into a hybrid cell.

The new technique should make it much easier for scientists to study what happens when two cells are combined.  For example, fusing an adult cell and an embryonic stem cell allows researchers to study the genetic reprogramming that occurs in such hybrids.

The researchers, led by a collaboration between Joel Voldman, associate professor of electrical engineering and computer science, and Rudolf Jaenisch, professor of biology and a member of the Whitehead Institute, report the new technique in the Jan. 4 online edition of Nature Methods.

The work was spearheaded by two postdoctoral associates, Alison Skelley, who worked in Voldman’s lab, and Oktay Kirak, who works with Jaenisch.  Skelley and Kirak are lead authors of the Nature Methods paper.  Heikyung Suh, a technical associate in the Whitehead Institute, is also an author of the paper.

The team’s simple but ingenious sorting method increases the rate of successful cell fusion from around 10 percent to about 50 percent, and allows thousands of cell pairings at once.

Though cell fusion techniques have been around for a long time, there are many technical limitations, said Voldman.

Getting the right cells to pair up before fusing them is one major obstacle.  If scientists are working with a mixture of two cell types, for example A and B, they end up with many AA and BB pairings, as well as the desired AB match.

Researchers had previously trapped cells in tiny cups as they flow across a chip.  Each cup can hold only two cells, but there is no way to control whether the cups capture an A and a B, two As or two Bs.

In contrast, the cell-trapping cups on Voldman and Jaenisch’s new sorting device are arranged strategically to capture and pair up cells of different types.

First, type A cells are flowed across the chip in one direction and caught in traps that are large enough to hold only one cell.  Once the cells are trapped, liquid is flowed across the chip in the opposite direction, pushing the cells out of the small cups and into larger cups across from the small ones.

Once one A cell is in each large cup, type B cells are flowed into the large cups.  Each cup can only hold two cells, so each ends up with one A and one B. After the cells are paired in the traps, they can be joined together by an electric pulse that fuses the cell membranes.

In addition to helping with studies of stem cell reprogramming, this technique could be used to study interactions between any types of cells.  “It’s a very general type of device,” said Voldman.

Shredded extracellular matrix (ECM) is toxic to neurons. Chen et al. Reveal a new mechanism for how ECM demolition causes brain damage. The study will appear in the December 29, 2008 issue of The Journal of Cell Biology (www.jcb.org).

A stroke or head injury kills large numbers of neurons through a process called excitotoxicity. A surge of the neurotransmitter glutamate jolts receptors such as the kainate receptor and stimulates cell death. Enzymes add to the death toll by chopping up ECM near the injury site. How ECM breakdown takes out neurons was mysterious. The standard view was that neurons perished because they got separated from the ECM as it dissolved.

Chen et al. Found otherwise when they engineered mice to lack the ECM component laminin in the hippocampus, a brain region often damaged by stroke or injury. If cells languished after parting from the ECM, the researchers reasoned that mice missing laminin would suffer more damage from excitotoxicity. But when excitotoxicity was spurred with an injection of kainate—a molecule that, like glutamate, activates the kainate receptor—the laminin-lacking mice showed less brain damage. After a dose of diced laminin, however, the mutant mice were vulnerable to kainate, indicating that the fragments are the culprit in cell death.

The researchers discovered that chopped-up ECM kills cells by ramping up production of one subunit of the kainate receptor, known as KA1. They speculate that hiking the amount of KA1 subunits might make the receptor more sensitive and thus more likely to trigger an overreaction by the cell.

Although drugs that obstruct the glutamate receptor slow brain cell death, they can lead to serious cognitive impairment and even coma. The study suggests that drugs that block KA1 might provide an alternative way to save brain cells after stroke or head trauma.

Molecular targets identified by a Spanish research team may hold the key to freedom for some sufferers of kidney disease. A new study published in Disease Models & Mechanisms (DMM), reveals the cellular signals which cause one treatment for kidney failure to lose its usefulness over time.

One of the most devastating aspects of kidney failure is the strict, time-consuming treatment regimen. Normally, healthy kidneys take on the role of filtering and cleaning the blood. Therefore patients with diseased kidneys traditionally need to attend a dialysis clinic to have their blood cleaned through a special filter. This treatment requires three regular clinic visits per week, with each session lasting three to five hours.

An alternative to this treatment involves creation of an “artificial kidney” in a process known as peritoneal dialysis (PD). Fluid is inserted into the abdominal cavity, and the blood vessel-rich cavity lining, the peritoneum, acts as a filter for the blood. Exchanges of dialysis fluid can take place at home, thus freeing patients of a rigid schedule of clinic visits.

However, the filtration ability of the peritoneum can lose efficiency over time, requiring patients to discontinue PD. In order to understand this change in the peritoneum, scientists Raffaele Strippoli, Miguel del Pozo and colleagues examined the dialysis fluid from PD patients, and identified molecular signals that cause abnormal changes in the peritoneum. They also found that pharmacologically disrupting these signals causes these abnormal cells to revert back to their original state, as they normally existed in the abdominal cavity lining.

These findings support further research on maintaining the effectiveness of PD, and indicate that perhaps even former PD patients could once again have an option to use PD rather than traditional hemodialysis. Additionally, the cellular changes studied in the peritoneum are similar to cell transformations in tumor formation and inflammation. Their findings may aid in greater understanding of cell change in these situations, as well.

Measuring an electrical current in an organism is pretty straightforward. All you need is an electrode. Measuring the flow of chemicals in cells or live tissue, however, is much more difficult because the molecules diffuse, mix with one another, and interact with their surroundings.

So to help understand biological processes, university researchers have invented a new device, the “chemistrode,” that makes it possible to stimulate, record, and analyze molecular signals at high resolution—in terms of precisely when, where, and in what sequence the signals occurred.

The chemistrode will help researchers study any surface that responds to chemical stimulation, including cells, tissue, biofilms and catalytic surfaces. It may also help neurologists, cardiologists, and endocrinologists study and diagnose diseases, according to those who developed the device in the Ismagilov Lab in the Department of Chemistry at the University of Chicago. Researchers in the Lab have already used it to measure how a single murine islet responds to glucose.

The developers have begun to apply for a patent on the new device, and their research describing it will be published online Oct. 27, 2008, by the Proceedings of the National Academy of Science. (The paper will appear in the print version of the prestigious journal on Nov. 4, 2008.)

“An analogue of the electrode, the chemistrode is a droplet-base microfluidic device that will provide exciting opportunities to study stimulus-response dynamics in chemistry and biology,” said Rustem Ismagilov, Associate Professor in Chemistry, who conceived the device, coined its name, and heads up the team that developed it.

Previous techniques for stimulating and measuring chemical reactions in organisms relied on laminar flow, which allows the chemicals in question to intermingle and disperse, making them hard to control and measure. The new V-shaped device, on the other hand, traps the chemicals in water droplets and suspends the droplets in a fluorocarbon carrier fluid. This keeps the chemical-laden droplets intact, allowing a controlled stream of stimulating chemicals to enter on one end of the device and a steady stream of distinct resultant chemicals to be captured on the other end. The chemical-laden droplets can be analyzed immediately or stored for future analysis. Furthermore, the droplets can be split up for parallel study by different techniques.

“The inspiration for this work was the microelectrode, but the key to its success was encapsulating the chemicals in aqueus droplets so that the chemicals could be delivered to and picked up from the reactive site in a controllable, measurable fashion,” said Delai Chen, a graduate student in the Department of Chemistry and Institute for Biophysical Dynamics at the University of Chicago. Chen was one of the four lead authors of the PNAS research paper, along with University post-doctoral researchers Wenbin Du and Ying Liu, and graduate student Weishan Liu.

A year and one-half in the making, the chemistrode is compatible with traditional methods of culturing cells and tissues because—like the electrode—it can be used on any surface. The device is used by being brought into contact with the surface of a cell or tissue under investigation. An array of tiny droplets containing chemical stimuli is then delivered to the sample; chemical reactions occur or molecules are released from the sample, as in the case of a hormone; and the resultant chemical-laden droplets are carried away. All the while, the fluorocarbon carrier fluid remains in contact with the droplets and shields them from the wall of the device.

“The chemistrode offers a time-resolved, high-fidelity record of molecular stimulation and response dynamics,” Ismagilov said. “Our PNAS paper describes the physical principles that guide the operation of the chemistrode. It also implements the chemistrode to test the feasibility of each step and the compatibility of this platform with living cells.”

For now, the device “allows you to look very hard and precisely at living cells in a dish, but it has the potential to be used in whole organisms, as well,” said Louis Philipson, a professor in the Department of Medicine and co-author on the paper. “The chemistrode offers real-time input-output analysis captured in excellent resolution. As such, it will facilitate research in a lot of areas and holds the potential for widespread applications in medicine.

“The development of this device is a wonderful example of the lack of walls at the University of Chicago,” Philipson added. “Here, physicians can interact with other scientists in unconventional ways and bring together different kinds of technology. The result is new ways of looking at things and new answers to old problems.”

Researchers have new insight into the mechanisms that underlie a pathological increase in the size of the heart.  The research, published by Cell Press in the October 24th issue of the journal Molecular Cell, may lead to the development of new strategies for managing this extremely common cardiac ailment that often leads to heart failure.

High blood pressure, heart valve disease and heart attacks can lead to a abnormal thickening of the heart muscle, called myocardial hypertrophy.  At the molecular level, signals driving myocardial hypertrophy, such as elevated levels of catecholamine hormones (i.e. adrenaline), activate the Myocyte Enhancer Factor (MEF) proteins.  This alters gene expression in heart muscle cells and induces an adverse developmental paradigm known to cardiologists as the “fetal gene response”.

“Previous research has shown that the signaling pathways leading to MEF2 are altered during pathological cardiac hypertrophy,” says senior study author Dr. John D. Scott, a Howard Hughes Medical Institute Investigator from the Department of Pharmacology at the University of Washington.  “Although we know that enzymes called histone deacetylases (HDACs) control MEF2 activity, it was not clear that HDACs and MEF2 were integrated into a larger signaling unit.”

To further identify the molecular mechanisms associated with cardiac hypertrophy, Dr. Scott and colleagues studied cardiac A-Kinase Anchoring Proteins (AKAPs), which are known to play a critical role in organizing signaling complexes in response to catecholamine hormones and transmitted signals within cells.

The researchers found that AKAP-Lbc functions as a scaffolding protein that selectively directs catecholamine signals to the transcriptional machinery to potentiate the hypertrophic response.  “Our study supports a model where AKAP-Lbc facilitates activation of protein kinase D, which in turn phosphorylates the histone deacetylase HDAC5 to promote its export from the nucleus.  The reduction in nuclear HDAC5 favored MEF2 transcription and the onset of cardiac hypertrophy.”

These studies reveal a role for AKAP-Lbc in which increased expression of the anchoring protein selectively amplifies a signaling pathway that drives cardiac muscle cells to a pathophysiological outcome.  “It will be important to explore the role of the AKAP-Lbc/PKD/HDAC5 signaling pathway in whole animal models to establish whether AKAP-Lbc is a valid biomarker for hypertrophic cardiomyopathy and to determine which genes are initiated upon up-regulation of the anchoring protein,” offers Dr. Scott.

Scientists at Dana-Farber Cancer Institute have identified a previously undetected trigger point on a naturally occurring “death protein” that helps the body get rid of unwanted or diseased cells. They say it may be possible to exploit the newly found trigger as a target for designer drugs that would treat cancer by forcing malignant cells to commit suicide.Loren Walensky, MD, PhD, pediatric oncologist and chemical biologist at Dana-Farber and Children’s Hospital Boston, and colleagues report in the Oct. 23 issue of the journal Nature that they directly activated this trigger on the “executioner” protein BAX, killing laboratory cells by setting in motion their self-destruct mechanism.

The researchers fashioned a peptide (a protein subunit) that precisely matched the shape of the newly found trigger site on the killer protein, which lies dormant in the cell’s interior until activated by cellular stress. When the peptide docked into the binding site, BAX was spurred into assassin mode. The activated BAX proteins flocked to the cell’s power plants, the mitochondria, where they poked holes in the mitochondria’s membranes, killing the cells. This process is called apoptosis, or programmed cell death.

“We identified a switch that turns BAX on, and we believe this discovery can be used to develop drugs that turn on or turn off cell death in human disease by targeting BAX,” said Walensky, who is also an assistant professor of pediatrics at Harvard Medical School.

BAX is one of about two dozen proteins known collectively as the BCL-2 family. The proteins interact in various combinations leading to either the survival of a cell or its programmed self-destruction. Cancer cells have an imbalance of BCL-2 family signals that drives them to survive instead of dying on command.

The late Stanley Korsmeyer, MD, an apoptosis research pioneer and Walensky’s Dana-Farber mentor, had suggested that killer proteins like BAX could be activated directly by “death domains,” termed BH3, contained within a subset of BCL-2 family proteins. He hypothesized that this activating interaction was a fleeting “hit-and-run” event, making it especially challenging for scientists to study the phenomenon.

As suspected, the proposed BAX-activating interactions could not be captured by traditional methods. “When you tried to measure binding of the BH3 subunits to BAX, you couldn’t detect the interaction,” explained Walensky. He recognized, however, that the BH3 peptides being used in the laboratory didn’t retain the coiled shape of the natural BH3 domains that participate in BCL-2 family protein interactions. Walensky and his colleagues pioneered the design of “stapled” BH3 peptides, which contain a chemical crosslink that locks the peptides into their natural coiled shape. With biologically active shape restored, the stapled BH3 peptides bound directly to BAX and triggered its killer activity.

Defining how the activating peptides docked on BAX remained a formidable catch-22. In order to solve the structure of an interaction complex, it needed to be stable enough for analysis. In this case, the BH3 binding event itself triggers BAX to change its shape and self-associate to perform its killer function, rendering the activating interaction unstable by definition.

What if, Walensky proposed, you could set up the interaction of BH3 and BAX under laboratory conditions that caused it to be more stable or proceed in slow motion? The plan was to adjust the potency of the stapled BH3 peptide so that, according to Walensky, “it was good enough to bind BAX, yet activate it just a bit more slowly so that we could actually study the interaction.” The researchers would then look for any detectable shift in the three-dimensional structure of the BAX protein to help point them to the docking site.

The researchers used nuclear magnetic resonance (NMR) spectroscopy to monitor the arrangement of atoms in the protein. First authors of the Nature paper Evripidis Gavathiotis, PhD, of Walensky’s laboratory and Motoshi Suzuki, PhD, of Nico Tjandra, PhD,’s laboratory at the National Institutes of Health, succeeded in generating pure BAX protein that could be put into solution with the stapled BH3 peptide — the latter in increasing concentrations until it initiated a BH3-BAX interaction. Gavathiotis and Suzuki used the NMR technique to spot a group of BAX amino acids, the building blocks of proteins, which were affected by the addition of the stapled BH3 peptide.

“The discrete subset of amino acids that shifted upon exposure to the stapled BH3 peptide mapped to a completely unanticipated location on BAX,” said Walensky. The long-elusive binding site on BAX that initiates its killer activity was revealed. “Because BAX lies at the crossroads of the cell’s decision to live or die, drugs that directly activate BAX could kill diseased cells like in cancer and BAX-blocking drugs could potentially prevent unwanted cell death, such as in heart attack, stroke, and neurodegeneration,” said Walensky.

In a paper published today in Nature, the team led by Professor Nigel Robinson have revealed a mechanism that ensures the right metal goes to the right protein. Proteins are essential and involved in just about every process in living cells.

Life, microbe, plant or human, is a painstaking assembly of trillions of atoms. The atoms include metals such as copper and manganese which act as catalysts in proteins. The proteins wrap around the metal atoms.

The research team has shown that to ensure a copper and a manganese protein wrap around the correct metal atoms they do this in different parts of the cell, in zones which contain different metals. Therefore, which protein attaches to which metal is determined by where the folding action takes place in the cell.

Previously, a common view was that the right metals were simply those which were most attracted to the protein, but in this work that is not the case.

Professor Nigel Robinson at Newcastle University who led the research says: “This has taken us one step closer to understanding why metals and proteins assemble in the ways they do.”

“One motive behind the work is pure curiosity, but as so many proteins need metals this type of work has many potential uses - for example, in synthetic biology which is striving to produce green power from bacteria by using energy from sunlight to produce hydrogen gas, a process which needs nickel and iron.

“It may also help in diseases such as Alzheimers where there are unexplained links to proteins binding metals such as copper. There’s also application in controlling infections by Staphylococcus aureus; a bacterium which our bodies defences succeed - or sometimes fail - in killing by removing manganese and zinc from abscesses.”

The researchers have shown that the way the metals attach is identical for a protein that binds manganese to one that binds copper. In both cases the metals bind inside protein barrels with the same type of metal-attractions.

Carrying out the work in a blue-green algae, a cyanobacterium, the team has been able to show that a protein requiring copper transports to the periplasm, the outer area of the cell, where it then folds around the available metal, which is copper.

Conversely, manganese but not copper atoms are found in the cytosol, in the middle of the cell. The team has demonstrated that a protein requiring manganese folds in the cytosol. The manganese protein is then transported to the periplasm having first trapped its manganese.

The cyanobacterium organism was chosen because it has a high demand for these two metals which are required for proteins involved in photosynthesis. These metals were chosen because they lie towards opposite ends of a chemical series called the Irving-Williams series, such that selecting these metals for proteins should be especially demanding.

In the work funded by the BBSRC, the Newcastle University team first developed a new approach to discover metal-binding proteins. This is now being swiftly applied to lots of other types of living cells and other essential metals (zinc, nickel, cobalt, iron). Unexpectedly, x-ray crystal structures showed that the identified proteins, MncA for manganese and CucA for copper, were both cupins (Latin for barrels) with identical sets of atoms for binding to the metals. Consistent with the chemical series, a ten-thousand times excess of manganese over copper was needed to fill the MncA barrel with manganese when folding is done in the laboratory.

Once folded, the manganese site is buried, the metal trapped inside the protein, and so the manganese protein can subsequently co-exist with the copper protein because its’ metal becomes impervious to replacement by metals further up the Irving-Williams series.

The work exemplifies a cell overcoming the metal binding preferences of proteins.

The new discipline of synthetic biology aims to engineer cells to carry out useful tasks, for example to generate valuable compounds. Because metals are the catalysts for so much of biology, knowing how to engineer a supply of the right metals to the right proteins will be important to the success of these ventures.

In the tiny realm of nanotechnology, scientists have used a wide variety of materials to build atomic scale structures.  But just as in the construction business, nanotechnology researchers can often be limited by the amount of raw materials.  Now, Biodesign Institute at Arizona State University researcher Hao Yan has avoided these pitfalls by using cells as factories to make DNA based nanostructures inside a living cell.

The results were published in the early online edition of the Proceedings of the National Academy of Sciences.

Yan specializes in a fast-growing field within nanotechnology -commonly known as structural DNA nanotechnology -that uses the basic chemical units of DNA, abbreviated as C, T, A, or G, to self-fold into a number of different building blocks that can further self-assemble into patterned structures.

“This is a good example of artificial nanostructures that can be replicated using the machineries in live cells” said Yan.  “Cells are really good at making copies of double stranded DNA and we have used the cell like a copier machine to produce many, many copies of complex DNA nanostructures.”

DNA nanotechnologists have made some very exciting achievements during the past five to 10 years.  But DNA nanotechnology has been limited by the need to chemically synthesize all of the material from scratch.  To date, it has strictly been a test tube science, where researchers have developed many toolboxes for making different DNA nanostructures to attach and organize other molecules including nanoparticles and other biomolecules.

“If you need to make a single gram of a DNA nanostructure, you need to order one gram of the starting DNA materials.  Scientists have previously used chemical methods to copy branched DNA structures, and there has also been significant work in using long-stranded DNA sequences replicated from cells or phage viruses to scaffold short helper DNA sequences to form 2-D or 3-D objects,” said Yan, who is also a professor in the Department of Chemistry and Biochemistry at ASU.

“We have always dreamed of scaling up DNA nanotechnology.  One way to scale that it up is to use the cellular system because simple DNA can be replicated inside the cell.  We wanted to know if the cell’s copy machine could tolerate single stranded DNA nanostructures that contain complicated secondary structures.”

To test the nanoscale manufacturing capabilities of cells, Yan and his fellow researchers, Chenxiang Lin, Sherri Rinker and Yan Liu at ASU and their collaborators Ned Seeman and Xing Wang at New York University went back to reproducing the very first branched nanostructure made up of DNAa cross-shaped, four-arm DNA junction and another DNA junction structure containing a different crossover topology.

To copy these branched DNA nanostructures inside a living cell, the ASU and NYU research team first shipped the cargo inside a bacteria cell.  They cut and pasted the DNA necessary to make these structures into a phagemid, a virus-like particle that infects a bacteria cell.  Once inside the cell, the phagemid used the cell just like a photocopier machine to reproduce millions of copies of the DNA.  By theoretically starting with just a single phagemid infection, and a single milliliter of cultured cells, Yan found that the cells could churn out trillions of the DNA junction nanostructures.

The DNA nanostructures produced in the cells were also found to fold correctly, just like the previously built test tube structures.  According to Yan, the results also proved the key existence of the DNA nanostructures during the cell’s routine DNA replication and division cycles.  “When a DNA nanostructure gets replicated, it does exist and can survive the complicated cellular machinery.  And it looks like the cell can tolerate this kind of structure and still do its job.  It’s amazing,” said Yan.

Yan acknowledges that this is just the first step, but foresees there are many interesting DNA variations to consider next.  “The fact that the natural cellular machinery can tolerate artificial DNA objects is quite intriguing, and we don’t know what the limit is yet.”

Yan’s group may be able to change and evolve DNA nanostructures and devices using the cellular system and the technology may also open up some possibilities for synthetic biology applications.

“I’m very excited about the future of DNA nanotechnology, but there is a lot of work to be done.  An interesting research topic to pursue is the interface of DNA nanostructures with live cells; it is full of opportunities,” said Yan.

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.

The differential distribution of specific proteins in axons or dendrites underlies the specialized functions of these neurites.  At least two mechanisms can create a polarized distribution of centrally produced transmembrane proteins: (1) segregation of proteins into distinct vesicles that are specifically targeted to the appropriate domain, and (2) unsorted transport followed by specific endocytosis of inappropriately expressed proteins.  This week, Bushlin et al.  Suggest that the latter mechanism can be regulated by proteins that associate with endocytic vesicles and determine their cargoes.  Knockdown of proteins involved in endocytic vesicle formation, AP180 or clathrin assembly lymphoid myeloid protein (CALM), inhibited axon or dendrite formation, respectively.  Knockdown of either protein caused VAMP2—an axonal synaptic vesicle protein that is normally endocytosed from dendrites—to be expressed in all processes, supporting a role in the establishment of polarity.  CALM knockdown also reduced surface expression of a secreted protein, suggesting that it may be involved in secretory as well as endocytic pathways.

Last week we learned that blocking expression of the AMPA receptor GluR1 subunit in motor neurons reduces dendrite growth, leading to impaired motor function.  This week, Zhou et al.  Begin to unravel the molecular mechanisms tying GluR1 to activity-dependent dendritic growth.  Intracellularly, glutamate receptors interact with membrane-associated guanylate kinases (MAGUKs)—scaffolding proteins that form the postsynaptic density and anchor signaling and other effector molecules near receptors.  GluR1 interacts with the MAGUK synapse-associated protein 97 (SAP97).  Overexpression of SAP97 increased dendritic branching, whereas SAP97 knockdown decreased branching in motor neurons.  This effect was blocked by an AMPA receptor antagonist.  Interaction between GluR1 and SAP97 was required for either to enhance dendritic branching, but only because the interaction localizes SAP97 to the plasma membrane.  If SAP97 was targeted to the membrane by the addition of a palmitoylation sequence, its effects on dendritic branching were restored in the absence of direct interaction with GluR1.

An international team of researchers led by Professors Mark Caulfield and Patricia Munroe, from the William Harvey Research Institute at Barts and The London School of Medicine and Dentistry with Chris Cheeseman at the University of Alberta in Canada and Kelle Moley at the University of Washington in USA, have shown that the SLC2A9 gene, which encodes a glucose transporter, is also a high-capacity urate transporter, and thus possibly a new drug target for gout.  Their findings are published in this week’s PloS Medicine (7 October 2008).

Several urate transporters have already been identified but recently, using an approach called genome-wide association scanning, Caulfield and others found that some genetic variants of a human gene called SLC2A9 are more common in people with high serum urate levels than in people with normal levels.  SLC2A9 encodes a glucose transporter (a protein that helps to move the sugar glucose through cell membranes) and is highly expressed in the kidney’s main urate handling site.  Professor Caulfield and his team investigated the possibility that the protein made by the SLC2A9 gene might be a urate transporter and tested whether genetic variations in SLC2A9 might be responsible for the association between serum urate levels and high blood pressure.

The team first expressed SLC2A9 in frog eggs, a type of cell that does not have its own urate transporter.  They found that SLC2A9 transported urate about 50 times faster than glucose, and that glucose facilitated SLC2A9-mediated urate transport.  Similarly, over expression of SLC2A9 in human embryonic kidney cells more than doubled their urate uptake.  Conversely, when the researchers used a technique called RNA interference to reduce the expression of mouse SLC2A9 in mouse cells that normally makes this protein, urate transport was reduced.  Researchers then looked at two genetic variations within SLC2A9 that vary between individuals (so-called single polynucleotide polymorphisms) in nearly 900 men who had had their serum urate levels and urinary urate excretion rates measured.  They found that certain genetic variations at these two sites were associated with increased serum urate levels and decreased urinary urate excretion.  Finally, the researchers used a statistical technique called meta-analysis to look for an association between one of the SLC2A9 gene variants and blood pressure.  In two separate meta-analyses that together involved more than 20,000 participants in several studies, there was no association between this gene variant and blood pressure.

Overall, these findings indicate that SLCA9 is a high capacity urate transporter, and suggest that this protein plays an important part in controlling serum urate levels.  They provide confirmation that common genetic variants in SLC2A9 affect serum urate levels to a marked degree, although they do not show exactly which genetic variant is responsible for increasing serum urate levels.  They also provide important new insights into how the kidneys normally handle urate and suggest ways in which this essential process may sometimes go wrong.  The findings could eventually lead to new treatments for gout and possibly for other diseases that are associated with increased serum urate levels.

Professor Mark Caulfield said: “This MRC funded study shows how a team of international researchers can find a completely unsuspected mechanism for urate handling in the kidney.  Such discoveries could pave the way for new medicines.”

New research suggests that the identification and examination of key cell signaling events required for initiation and progression of cancer might be best accomplished at the single cell level.  The research, published by Cell Press in the October issue of the journal Cancer Cell, provides new insight that may lead to better diagnosis and treatment of some complex cancers.

Recent advances in flow cytometry, a technique that allows detailed examination of individual cells, have enabled simultaneous measurement of cell type and signaling pathways.  Lead study authors Dr. Garry P. Nolan from the Stanford University School of Medicine and Dr. Mignon L. Loh from the UCSF Children’s Hospital and the Helen Diller Family Comprehensive Cancer Center were interested in determining whether examination of cellular signaling abnormalities caused by genetic mutations associated with cancer could provide a precise correlation between aberrant signaling events and disease physiology.

“We had a strong hunch that we could use ‘deranged’ cellular signaling to track how cancer cell populations behave at diagnosis through therapy, as well as during remission or return of the cancer,” explains Dr. Nolan.  “By measuring how signaling proteins respond to certain stimuli at diagnosis and which are modified by resistant cancers, we are essentially monitoring key highways that cancers use to drive their own growth.  The advantage of diagnosing a patient’s cancer at the single cell level provides us an approach for early detection of cancer and yield insights into how cancer cells are responding or adapting to therapy.  A byproduct of the single cell technique, when appropriately extended, is that we should eventually be able to predict those pathways cancer cells might be using to circumvent current therapies and more intelligently direct the patient towards alternative treatments.”

The researchers focused on juvenile myelomonocytic leukemia (JMML), an aggressive myeloproliferative disorder of young children.  JMML is difficult to diagnose and has a complex molecular profile.  Although genetic lesions impacting Ras signaling and alterations downstream of the activated GM-CSF receptor (both linked with inappropriate cell growth and survival) have been linked with JMML, there are very few methods for identifying therapeutic agents and assessing efficacy in JMML patients.

The researchers used flow cytometry to profile signaling at the single cell level, including molecules associated with GM-CSF and Ras signaling, for the presence of primary JMML cells with altered signaling behavior that correlated with disease physiology.  Cells samples came from JMML patients, healthy individuals and patients with other myeloproliferative disorders, some who had initially been diagnosed with JMML.  An unexpected STAT5 signaling signature was seen in most of the JMML patients, suggesting a critical role for JAK-STAT signaling in the biological mechanism of this cancer and suggesting potential targets for future therapies.

“This work successfully used single-cell profiling to follow patients over time and show that disease status in JMML – at diagnosis, remission, relapse and transformation – was indicated by a subset of cells with an abnormal signaling profile,” says Dr. Loh.  “Revealing cell subpopulations, even rare cells, that are associated with disease opens additional avenues for measuring minimal residual disease, assessing biochemical effects of targeted therapies at the single cell level and understanding drug actions and mechanisms of diseases of heterogeneous origins and manifestations in diverse patient populations.”

To understand where fat comes from, you have to start with a skinny mouse.  By using such a creature, and observing the growth of fat after injections of different kinds of immature cells, scientists at the Howard Hughes Medical Institute and Rockefeller University have discovered an important fat precursor cell that may in time explain how changes in the numbers of fat cells might increase and lead to obesity.  The finding, published online in this week’s issue of the journal Cell, could also have implications for understanding how fat cells affect conditions such as diabetes and cardiovascular disease.

“The identification of white adipocyte progenitor cells provides a means for identifying factors that regulate the proliferation and differentiation of fat cells,” says senior author Jeffrey Friedman, who is the Marilyn M. Simpson Professor at Rockefeller and a Howard Hughes Medical Institute investigator.

Obesity, a major public health problem in the United States and increasingly in much of the Western world, results, in part, from an increase in the mass and number of white fat cells.  Because white fat cells are post-mitotic, meaning that they cannot divide, scientists have hypothesized that a population of fat precursor cells must exist in the fat depot in order to produce new fat cells.  But identifying these fat precursor cells has been difficult.

With the assistance of researchers in Rockefeller’s Flow Cytometry Resource Center, first author Matt Rodeheffer, a postdoctoral associate in Friedman’s Laboratory of Molecular Genetics, used a cell sorting technique called fluorescence-activated cell sorting, or FACS, to search for cell populations that could produce fat in cell cultures and identified two such populations.

To determine if these cells could develop into fat cells in living animals, Rodeheffer injected these cell populations into the fat depots of a genetically engineered mouse, developed at NIH, called fatless, which lacks white fat and mimics a condition in humans called lipodystrophy that also results in diabetes.

Rodeheffer found that only one of the isolated cell populations, which express the CD24 cell-surface marker protein, produced fat tissue in the fatless mouse.  This population normally represents only .08 percent of the non-adipocyte population in adipose tissue.

An imaging assay recently developed by co-author Kivanç Birsoy, a graduate student in Friedman’s laboratory, enabled Rodeheffer to observe the CD24-expressing cells form fat in a living animal.  Birsoy’s technique uses another animal strain called the leptin-luciferase mouse, in which the visibly detectable marker luciferase is expressed under the control of the promoter of the gene that produces the hormone leptin.  In this mouse strain the luciferase marker gene only switches on in mature fat cells, and provides a non-invasive way of watching immature fat cell precursors develop into mature fat cells in a living animal over time.

“I injected the CD24+ cells which represent a very small population of cells in normal adipose tissue into a site where the fat would normally develop in the fatless mouse, and I found that a normal sized fat depot forms at the site of injection,” says Rodeheffer.

Rodeheffer also found that the injection of the fat-producing cells corrects the fatless mouse’s diabetes, and the fat cells secrete adipocyte-specific signaling proteins called cytokines.  Both of these results confirm that the cells produced in the fatless mouse are functional fat cells.

“This finding gives us a better understanding of the basic biology of adipose tissue and opens the door for us and for other researchers to be able to study these cells in living animals and determine the molecular factors that regulate formation of adipose tissue,” says Rodeheffer.  “We then can potentially study how the growth and differentiation of these cells are regulated in obesity and determine whether or not the molecular events that are involved in the regulation of adipose tissue are contributing factors to other pathologies, such as diabetes and cardiovascular disease, that are associated with obesity and metabolic syndrome.”

A bacteria cell’s ‘crisis command centre’ has been observed for the first time swinging into action to protect the cell from external stress and danger, according to new research out today (3 October) in Science.

The research team behind today’s study says that finding out exactly how bacteria respond and adapt to stresses and dangers is important because it will further their understanding of the basic survival mechanisms of some of the most resilient, hardy organisms on Earth.

The crisis command centre in certain bacteria cells is a large molecule, dubbed a ’stressosome’ by the scientists behind today’s research.  These cells have around 20 stressosomes floating around inside them, and although scientists knew they played an important role in the cell’s response to stressful situations, the complexities of this process had not been fully understood until now.

If a bacteria cell finds itself in a dangerous situation for example, if the temperature or saltiness of the bacteria’s environment reach dangerous levels which threaten the survival of the bacteria -a warning signal from the cell’s surface is transmitted into the cell.

Using cutting edge electron microscopy imaging techniques the authors of the new research observed that the stressosomes receive this warning signal, and in response several proteins called RSBT break away from the large stressosome.  This breakaway triggers a cascade of signals within the cell which results in over 150 proteins being produced proteins which enable the cell to adapt, react and survive in its new environment.

Professor Marin van Heel from Imperial College London’s Department of Life Sciences, one of the corresponding authors of the study, explains: “The cascade of events inside bacteria cells that occurs as a result of stressosomes receiving warning signals leads to particular genes inside the cell being transcribed more.  This means that some genes already active inside the cell are ‘turned up’ so that levels of particular proteins in the cell increase.  These changes to the protein make-up of the cell enable it to survive in a hostile or challenging environment.”

Dr Jon Marles-Wright from Newcastle University says: “Our work shows that cells respond to signals much like a dimmer on a light switch.  Now we’ll be building on this to work out how nature controls that dimmer switch.  We wouldn’t have been able to carry out this work without access to the Diamond synchrotron Light Source which has enabled us to examine the structures of individual stressosome proteins at atomic resolution.”

Dr Tim Grant, one of Imperial’s post doctoral researchers, adds that the key to bacteria cells’ success at surviving in rapidly changing environments is their speedy response: “The cell’s stressosomes are very good at their job as crisis command centres because they provide a very fast effective response to danger.  The chain reaction they kickstart produces results really quickly which enables bacteria to adapt to changes in their surroundings almost instantaneously.”

The team is now planning to collect very high resolution data of the stressosome complex on the world’s newest high-resolution cryo electron microscope, the FEI “KRIOS” that has just been installed in the Max Planck Institute in Martinsried, Germany.  Improving the resolution of the stressosome structure by a factor of two will lead to a resolution range normally only attainable by X-ray crystallography and will allow the researchers to directly see the amino-acid components of this fascinating complex.