Research into chromosome replication reveals details of heredity dynamics

A novel study from Karolinska Institutet has deepened the understanding of how chromosome replication, one of life's most fundamental processes, works. In a long term perspective these results could eventually lead to novel cancer therapies. The study is presented in the prestigious scientific journal Nature.

By studying DNA replication in yeast cells, researchers at Karolinska Institutet have discovered that a protein complex (Smc5/6) helps to release torsional stress created in the DNA molecule when chromosomes are replicated in preparation for a coming cell division.

"Our study also indicates that the stress can propagate more freely along the DNA in a chromosome than was previously thought," says KI professor Camilla Sjögren, head of the team that conducted the study.

The study therefore sheds more light on the mechanisms behind one of life's most fundamental processes. Since topoisomerases, enzymes known to remove replication-related stress in the DNA are common targets for cancer treatments, the finding might eventually lead to new therapies.

When a fertilised egg develops into a complete organism, or when old cells are replaced by new ones, it is done through cell division. If human daughter cells are to survive and develop normally, they must each obtain a full set of 46 chromosomes, which are made of double-stranded DNA helices. Since the original mother cell started as a cell with 46 chromosomes, these must be duplicated before division take place.

During this process, the DNA double helix is separated so that the replication machinery can reach the individual DNA strands. This prising apart of the strands creates stress in the form of over-twisted DNA in the vicinity of the replication zone. If this stress is not removed, replication can be slowed down or even stopped, and this, in turn, can lead to mutagenesis and/or cell death.

"Several modern cancer treatments attack topoisomerases, but there's a problem in that some cancers become resistant to such therapies," says Professor Sjögren. "Now that we've discovered that also the Smc5/6 complex releases the stress which form during the replication process, our results might trigger the development of drugs that target Smc5/6. This could create another tool for inhibiting tumour growth."

More information: Andreas Kegel, Hanna Betts-Lindroos, Takaharu Kanno, Kristian Jeppsson, Lena Ström, Yuki Katou, Takehiko Itoh, Katsuhiko Shirahige & Camilla Sjögren, Chromosome length influences replication-induced topological stress, Nature, AOP 2 March 2011, DOI: 10.1038/nature09791

 

Firefly glow: Scientists develop a safe hydrogen peroxide probe based on firefly luciferin

 A unique new probe based on luciferase, the enzyme that gives fireflies their glow, enables researchers to monitor  hydrogen peroxide levels in mice and thereby track the progression of infectious diseases or cancerous tumors without harming the animals or even having to shave their fur. Developed by researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley, this new bioluminescent probe has already provided the first direct experimental evidence that hydrogen peroxide is continuously made even in a healthy animal.

“We are reporting the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of hydrogen peroxide within living animals,” says Christopher Chang, a chemist who holds appointments with Berkeley Lab’s Chemical Sciences Division and UC Berkeley’s Chemistry Department, as well as the Howard Hughes Medical Institute.

Chang is the corresponding author of a paper in the Proceedings of the National Academy of Science (PNAS) that describes this research. The paper is titled “In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter.” Co-authoring with Chang were Genevieve Van de Bittner, Elena Dubikovskaya and Carolyn Bertozzi.

The PCL-1 probe consists of a light-emitting luciferin molecule enclosed inside a molecular cage of boronic acid. The boronic acid selectively reacts with hydrogen peroxide molecules to release the luciferin, triggering a bioluminescent response in the presence of firefly luciferase. For their studies, Chang and his co-authors worked with transgenic mice that carried the firefly luciferase gene.

“The high sensitivity and selectivity of the PCL-1 probe for hydrogen peroxide, combined with the favorable properties of bioluminescence for in vivo imaging, afford a unique technology for monitoring physiological fluctuations in hydrogen peroxide levels in real-time,” Chang says. “This offers opportunities to dissect the  disparate contributions of hydrogen peroxide to health, aging and disease.”

Hydrogen peroxide is nature’s disinfectant. Cells produce this small but highly reactive molecule to kill invading pathogens. It also plays a critical role in cellular signaling that is essential to the growth, development and physical well-being of humans and other organisms. However, over-production of hydrogen peroxide in cells is the mark of oxidative stress and inflammation, and has been linked to the onset and advancement of cancer and diabetes, and numerous cardiovascular and neurodegenerative diseases.

Chang and his group have shown that hydrogen peroxide can serve as a highly effective signaling agent for in vivo imaging. To this end, they’ve developed a series of hydrogen peroxide fluorescent tags for tracking small-molecule oxygen metabolites in living cells, tissue and organisms. With their new PCL-1 probe, they were able to study mice with prostate cancers and monitor fluctuations in the hydrogen peroxide generated by cancerous cells based on the amount of light emitted by the probe.

“The PCL-1 probe enables us to study the chemistry in living animals as cancers and other diseases progress,” Chang says. “We can use the probe to look at the same mouse over time to see how see how therapeutics and other treatments affect its physiology, without having to do biopsies or sacrifice the animal. This is a significant advance over previous hydrogen peroxide probes.”

In addition to doing no harm to the animal, Chang and his group wanted a probe that could simultaneously detect hydrogen peroxide signals from multiple regions or the entire organism. They also wanted a probe that could detect intracellular signals, and preferred not having to remove fur or skin to detect a signal from a specific tissue of interest. They elected to pursue bioluminescence because of its favorable properties for in vivo imaging.

“The fact that in nature fireflies use the luciferin enzyme to communicate by light inspired us to adapt this same strategy for pre-clinical diagnostics,” Chang says. “Bioluminescence from the catalytic transformation of firefly luciferin by the firefly luciferase enzyme exhibits a high efficiency for photon production and a 612 nanometer emission frequency that provides a detectable bioluminescent signal in all organs of a mouse. The PCL-1 probe is small enough to travel through a mouse’s body andits red-shifted luminescent reaction with luciferase allows for deep tissue signal penetration with an optical readout.”

Chang and his colleagues are now working to improve the sensitivity of the PCL-1 probe. They would also like to refine their methodology to be able to simultaneously examine multiple biomarkers.

Provided by Lawrence Berkeley National Laboratory (news : web)

 

Cancer-causing virus exploits key cell-survival proteins

A cancer-causing retrovirus exploits key proteins in its host cells to extend the life of those cells, thereby prolonging its own survival and ability to spread, according to a new study by researchers at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James) and Ohio State's College of Veterinary Medicine.

The human T-lymphotropic virus type-1 (HTLV-1), which causes adult T-cell leukemia and lymphoma, produces a protein called p30 that is essential for the retrovirus to establish an infection. This study found that this viral protein targets two important cell proteins: ATM, a key player in a cell's response to DNA damage, and REG-gamma, which marks proteins within the cell for destruction.

"Our findings suggest that the p30 viral protein prolongs the survival of host cells through this interaction with ATM and REG-gamma, and the longer a virus-infected cell survives, the better chance the virus has to spread, " says principal investigator Michael Lairmore, DVM, PhD, professor of veterinary biosciences and associate director for shared resources at the OSUCCC – James.

The findings were published recently in the Journal of Biological Chemistry.

An estimated 20 million people worldwide are infected by HTLV-1, and about five percent of them will develop adult T-cell leukemia or lymphoma, or one of a variety of inflammatory disorders.

Lairmore and his colleagues used cell lines and a variety of biochemical assays to identify cellular binding partners of p30. They discovered the following:
p30 specifically binds to cellular ATM (ataxia-telangiectasia mutated), a key regulator of DNA damage responses and cell cycle control, and to REG-gamma, a nuclear proteasome activator.
Under stressful conditions, p30 levels are associated with lower ATM levels and increased cell survival.
The expression of p30 changes in concert with expression of REG-gamma, suggesting that overexpression of REG-gamma enhances levels of p30.
p30 forms a complex with ATM and REG-gamma.

 

Turning bacteria into butanol biofuel factories

Enlarge

The enzyme pathway by which glucose is turned into n-butanol is set against the silhouette of an E. coli bacterium. The pathway, taken from Clostridium bacteria and inserted into E. coli, consists of five enzymes that convert acetyl-CoA, a product of glucose metabolism, into n-butanol (C4H9OH).

(PhysOrg.com) -- While ethanol is today's major biofuel, researchers aim to produce fuels more like gasoline. Butanol is the primary candidate, now produced primarily by Clostridium bacteria. UC Berkeley chemist Michelle Chang has transplanted the enzyme pathway from Clostridium into E. coli and gotten the bacteria to churn out 10 times more n-butanol than competing microbes, close to the level needed for industrial scale production.

University of California, Berkeley, chemists have engineered bacteria to churn out a gasoline-like biofuel at about 10 times the rate of competing microbes, a breakthrough that could soon provide an affordable and “green” transportation fuel.

The advance is reported in this week’s issue of the journal Nature Chemical Biology by Michelle C. Y. Chang, assistant professor of chemistry at UC Berkeley, graduate student Brooks B. Bond-Watts and recent UC Berkeley graduate Robert J. Bellerose.

Various species of the Clostridium bacteria naturally produce a chemical called n-butanol (normal butanol) that has been proposed as a substitute for diesel oil and gasoline. While most researchers, including a few biofuel companies, have genetically altered Clostridium to boost its ability to produce n-butanol, others have plucked enzymes from the bacteria and inserted them into other microbes, such as yeast, to turn them into n-butanol factories. Yeast and E. coli, one of the main bacteria in the human gut, are considered to be easier to grow on an industrial scale.

While these techniques have produced promising genetically altered E. coli bacteria and yeast, n-butanol production has been limited to little more than half a gram per liter, far below the amounts needed for affordable production.

Chang and her colleagues stuck the same enzyme pathway into E. coli, but replaced two of the five enzymes with look-alikes from other organisms that avoided one of the problems other researchers have had: n-butanol being converted back into its chemical precursors by the same enzymes that produce it.

The new genetically altered E. coli produced nearly five grams of n-buranol per liter, about the same as the native Clostridium and one-third the production of the best genetically altered Clostridium, but about 10 times better than current industrial microbe systems.

“We are in a host that is easier to work with, and we have a chance to make it even better,” Chang said. “We are reaching yields where, if we could make two to three times more, we could probably start to think about designing an industrial process around it.”

“We were excited to break through the multi-gram barrier, which was challenging,” she added.

Graduate student Brooks Bond-Watts and post-doctoral fellow Jeff Hanson examine cultured E. coli used to produce the biofuel n-butanol. (Photo by Michael Barnes)

Among the reasons for engineering microbes to make fuels is to avoid the toxic byproducts of conventional fossil fuel refining, and, ultimately, to replace fossil fuels with more environmentally friendly biofuels produced from plants. If microbes can be engineered to turn nearly every carbon atom they eat into recoverable fuel, they could help the world achieve a more carbon-neutral transportation fuel that would reduce the pollution now contributing to global climate change. Chang is a member of UC Berkeley’s year-old Center for Green Chemistry.

The basic steps evolved by Clostridium to make butanol involve five enzymes that convert a common molecule, acetyl-CoA, into n-butanol. Other researchers who have engineered yeast or E. coli to produce n-butanol have taken the entire enzyme pathway and transplanted it into these microbes. However, n-butanol is not produced rapidly in these systems because the native enzymes can work in reverse to convert butanol back into its starting precursors.

Chang avoided this problem by searching for organisms that have similar enzymes, but that work so slowly in reverse that little n-butanol is lost through a backward reaction.

“Depending on the specific way an enzyme catalyzes a reaction, you can force it in the forward direction by reducing the speed at which the back reaction occurs,” she said. “If the back reaction is slow enough, then the transformation becomes effectively irreversible, allowing us to accumulate more of the final product.”

Chang found two new enzyme versions in published sequences of microbial genomes, and based on her understanding of the enzyme pathway, substituted the new versions at critical points that would not interfere with the hundreds of other chemical reactions going on in a living E. coli cell. In all, she installed genes from three separate organisms – Clostridium acetobutylicum, Treponema denticola and Ralstonia eutrophus — into the E. coli.

Chang is optimistic that by improving enzyme activity at a few other bottlenecks in the n-butanol synthesis pathway, and by optimizing the host microbe for production of n-butanol, she can boost production two to three times, enough to justify considering scaling up to an industrial process. She also is at work adapting the new synthetic pathway to work in yeast, a workhorse for industrial production of many chemicals and pharmaceuticals.

Provided by University of California - Berkeley (news : web)

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Biomarker could make diagnosing knee injury easier, less costly, othopaedists say

 A recently discovered biomarker could help doctors diagnose a common type of knee injury, according to a new study.

A team of researchers led by Gaetano Scuderi, MD, clinical assistant professor of orthopaedic surgery at Stanford University School of Medicine and an orthopaedic surgeon at Stanford Hospital & Clinics, has confirmed that a particular protein complex appears in patients with painful meniscal tears. The finding, to be published Feb. 16 in The Journal of Bone and Joint Surgery, could be used to prevent needless surgery and to save billions of dollars in medical-imaging costs.

The menisci are two crescent-shaped pieces of cartilage in each knee joint. Contact sports, such as football, as well as sports that involve lot of pivoting, such as basketball and tennis, increase the risk of the cartilage tearing. It is also prone to tear as a result of natural degeneration, meaning older people are at increased risk. Meniscal tears are painful and usually accompanied by swelling and stiffness. Sometimes the knee joint feels as though it is locked in place.

Patients are generally advised to elevate and apply ice to the knee, as well as to take a break from physical activity that could aggravate the injury. These measures might not be enough, however, so patients can undergo a minimally invasive procedure, arthroscopic surgery, to trim away or repair the meniscus.

But identifying whether a patient's knee pain stems from a meniscal tear, as opposed to joint arthritis or another type of leg injury, is difficult. For example, in an older patient, magnetic-resonance imaging might reveal an abnormal-looking meniscus that doctors mistake as evidence of a painful tear, even though it is just due to natural degeneration from lots of wear over the years. For such a patient, who is perhaps really suffering from joint arthritis, meniscal surgery would offer no relief.

Knee pain also can stem from other parts of the body. For example, a young athlete who complains of symptoms similar to those of a torn meniscus may undergo a costly MRI that reveals no cartilage abnormalities. In reality, an injured hip ligament could be causing the knee to hurt. "It's like someone with heart disease feeling pain in his left shoulder," Scuderi said.

In the study, Scuderi and his co-authors found that the biomarker appeared in the knee fluid of 30 patients who had suffered a painful meniscal tear. It was not present in the knees of 10 asymptomatic patients. The biomarker, a fibronectin-aggrecan complex, holds out the promise of allowing orthopaedists to quickly and accurately diagnose whether the source of a patient's discomfort is a meniscal tear, as opposed to another type of injury or abnormality, simply by taking a sample of knee fluid. It could thus obviate the need for expensive medical scans and help to prevent surgery that does not address the true cause of a patient's pain.

"The challenge is not identifying molecular markers of cartilage degeneration, dozens of which are now known," said co-author Raymond Golish, MD, PhD, who recently completed a fellowship in spine surgery at Stanford. "The difficulty is in finding markers that correlate with painful injuries, as opposed to age-related degeneration that is painless. This study is a big step in that direction."

Scuderi and his colleagues undertook the prospective study to validate their findings from an earlier study in which they first noted the presence of the protein complex in patients with torn menisci and knee pain. (Those results were published in the July 2010 issue of Clinical Biochemistry.)

The researchers are now running experiments to confirm that the biomarker does not show up in other types of knee injuries, such as ACL tears unaccompanied by meniscal tears. They also are studying whether the protein complex, which is implicated in knee inflammation, could serve as a therapeutic target. "We could envision several things, such as blocking the fibronectin and aggrecan protein fragments from coming together to form a complex, or interfering with the activation of white blood cells at the site," Scuderi said.

Provided by Stanford University Medical Center (news : web)

Scientists create cell assembly line

 

Borrowing a page from modern manufacturing, scientists from the Florida campus of The Scripps Research Institute have built a microscopic assembly line that mass produces synthetic cell-like compartments.

The new computer-controlled system represents a technological leap forward in the race to create the complex membrane structures of biological cells from simple chemical starting materials.

"Biology is full of synthetic targets that have inspired chemists for more than a century," said Brian Paegel, Scripps Research assistant professor and lead author of a new study published in the Journal of the American Chemical Society. "The lipid membrane assemblies of cells and their organelles pose a daunting challenge to the chemist who wants to synthesize these structures with the same rational approaches used in the preparation of small molecules."

While most cellular components such as genes or proteins are easily prepared in the laboratory, little has been done to develop a method of synthesizing cell membranes in a uniform, automated way. Current approaches are capricious in nature, yielding complex mixtures of products and inefficient cargo loading into the resultant cell-like structures.

The new technology transforms the previously difficult synthesis of cell membranes into a controlled process, customizable over a range of cell sizes, and highly efficient in terms of cargo encapsulation.

The membrane that surrounds all cells, organelles and vesicles – small subcellular compartments – consists of a phospholipid bilayer that serves as a barrier, separating an internal space from the external medium.

The new process creates a laboratory version of this bilayer that is formed into small, cell-sized compartments.

How It Works

"The assembly-line process is simple and, from a chemistry standpoint, mechanistically clear," said Sandro Matosevic, research associate and co-author of the study.

A microfluidic circuit generates water droplets in lipid-containing oil. The lipid-coated droplets travel down one branch of a Y-shaped circuit and merge with a second water stream at the Y-junction. The combined flows of droplets in oil and water travel in parallel streams toward a triangular guidepost.

Then, the triangular guide diverts the lipid-coated droplets into the parallel water stream as a wing dam might divert a line of small boats into another part of a river. As the droplets cross the oil-water interface, a second layer of lipids deposits on the droplet, forming a bilayer.

The end result is a continuous stream of uniformly shaped cell-like compartments.

The newly created vesicles range from 20 to 70 micrometers in diameter—from about the size of a skin cell to that of a human hair. The entire circuit fits on a glass chip roughly the size of a poker chip.

The researchers also tested the synthetic bilayers for their ability to house a prototypical membrane protein. The proteins correctly inserted into the synthetic membrane, proving that they resemble membranes found in biological cells.

"Membranes and compartmentalization are ubiquitous themes in biology," noted Paegel. "We are constructing these synthetic systems to understand why compartmentalized chemistry is a hallmark of life, and how it might be leveraged in therapeutic delivery."

More information: "Stepwise Synthesis of Giant Unilamellar Vesicles on a Microfluidic Assembly Line," was published February 10, 2011. For more information, see http://pubs.acs.or … 21/ja109137s

Green chemistry offers route towards zero-waste production

 Novel green chemical technologies will play a key role helping society move towards the elimination of waste while offering a wider range of products from biorefineries, according to a University of York scientist.

Professor James Clark, Director of the University's Green Chemistry Centre of Excellence, will tell a symposium at the Annual meeting of the American Association for the Advancement of Science (AAAS) that the use of low environmental impact green chemical technologies will help ensure that products are genuinely and verifiably green and sustainable.

He says the extraction of valuable chemicals from biomass could form the initial processing step of many future biorefineries.

"We have shown that wax products with numerous applications, can be extracted from crop and other by-products including wheat and barley straws, timber residues and grasses, using supercritical carbon dioxide – a green chemical technology that allows the production of products with no solvent residues," he says.

"The extracted residues can be used in applications including construction as well as in bioprocessing."

Low-temperature microwaves can also be used to pyrolyse biomass, allowing greater control over the heating process. The process results in significant energy savings and produces high quality oils, and oils and solids with useful chemical properties.

Professor Clark says that combining continuous extraction with microwave irradiation, it is possible separate an aqueous phase leaving the oils cleaner, less acidic and with lower quantities of other contaminants such as alkali metals. The oils have significant potential as feedstocks for making chemical products as well as for blending into transport fuels.

"Our microwave technology can also be tuned to produce bio-chars with calorific values and physical properties that make them suitable for co-firing with coal in power-stations," he adds.

More information: Professor Clark will be among the speakers the session 'Biorefinery: Toward an Industrial Metabolism' at the Annual Meeting of the AAAS, Washington, D.C. on Friday, 18 February, 2011.

Provided by University of York