Tuesday, April 12, 2011

Protein adaptation shows that life on early earth lived in a hot, acidic environment

A new study reveals that a group of ancient enzymes adapted to substantial changes in ocean temperature and acidity during the last four billion years, providing evidence that life on Early Earth evolved from a much hotter, more acidic environment to the cooler, less acidic global environment that exists today.

The study found that a group of ancient enzymes known as thioredoxin were chemically stable at temperatures up to 32 degrees Celsius (58 degrees Fahrenheit) higher than their modern counterparts. The enzymes, which were several billion years old, also showed increased activity at lower -- which correspond to greater acidity.

"This study shows that a group of ubiquitous proteins operated in a hot, acidic environment during early life, which supports the view that the environment progressively cooled and became more alkaline between four billion and 500 million years ago," said Eric Gaucher, an associate professor in the School of Biology at the Georgia Institute of Technology.

The study, which was published April 3 in the advance online edition of the journal Nature Structural & Molecular Biology, was conducted by an international team of researchers from Georgia Tech, Columbia University and the Universidad de Granada in Spain.

Major funding for this study was provided by two grants from the National Aeronautics and Space Administration to Georgia Tech, a grant from the National Institutes of Health to Columbia University, and a grant from the Spanish Ministry of Science and Innovation to the Universidad de Granada.

Using a technique called ancestral sequence reconstruction, Gaucher and Georgia Tech biology graduate student Zi-Ming Zhao reconstructed seven ancient thioredoxin enzymes from the three domains of life -- archaea, bacteria and eukaryote -- that date back between one and four billion years old.

To resurrect these enzymes, which are found in nearly all known modern organisms and are essential for life in mammals, the researchers first constructed a family tree of the more than 200 thioredoxin sequences available from the three domains of life. Then they reconstructed the sequences of the ancestral thioredoxin enzymes using statistical methods based on maximum likelihood. Finally, they synthesized the genes that encoded these sequences, expressed the ancient proteins in the cells of modern Escherichia coli bacteria and then purified the proteins.

"By resurrecting proteins, we are able to gather valuable information about the adaptation of extinct forms of life to climatic, ecological and physiological alterations that cannot be uncovered through fossil record examinations," said Gaucher.

The reconstructed enzymes from the Precambrian period -- which ended about 542 million years ago -- were used to examine how environmental conditions, including pH and temperature, affected the evolution of the enzymes and their chemical mechanisms.

"Given the ancient origin of the reconstructed thioredoxin enzymes, with some of them predating the buildup of atmospheric oxygen, we thought their catalytic chemistry would be simple, but we found that thioredoxin enzymes use a complex mixture of chemical mechanisms that increases their efficiency over the simpler compounds that were available in early geochemistry," said Julio Fernández, a professor in the Department of Biological Sciences professor at Columbia University.

Fernández led a team that included Columbia University postdoctoral researchers Raul Perez-Jimenez, Jorge Alegre-Cebollada and Sergi Garcia-Manyes, and graduate student Pallav Kosuri in using an assay based on single molecule force spectroscopy to measure the activity level of the thioredoxin enzymes under different pH levels.

For their experiments, the researchers used an atomic force microscope to pick up and stretch an engineered in a solution containing thioredoxin. They first applied a constant force to the protein, causing it to rapidly unfold and expose its disulfide bonds to the thioredoxin enzymes. The rate at which a thioredoxin snipped the disulfide bonds determined the enzyme's level of efficiency.

The study results showed that the three oldest thioredoxin enzymes -- those thought to have inhabited Earth 4.2 to 3.5 billion years ago -- were able to operate in lower pH environments than the modern thioredoxin enzymes.

"Our analysis indicates that ancient thioredoxin enzymes were well adapted to function under acidic conditions and that they maintained their high level of activity as they evolved in more alkaline environments," said Fernández.

To measure the temperature range in which the enzymes operated, professor Jose Sanchez-Ruiz and graduate student Alvaro Inglés-Prieto from the Departamento de Química-Física at the Universidad de Granada in Spain used a technique called differential scanning calorimetry. This method measures the stability of enzymes by heating the enzymes at a constant rate and measuring the heat change associated with their unfolding.

The researchers found that the ancient proteins were stable at temperatures up to 32 degrees Celsius higher than the modern thioredoxins. The experiments showed that the enzymes exhibited higher temperature stability the older they were. The results provide evidence that ancestral thioredoxins adapted to the cooling trend of ancient oceans, as inferred from geological records.

"Our results confirm that life has the remarkable ability to adapt to a wide range of historical environmental conditions; and by extension, life will undoubtedly adapt to future environmental changes, albeit at some cost to many species," said Gaucher.

This study also showed that the experimental resurrection of ancient proteins together with the sensitivity of single-molecule techniques can be a powerful tool for understanding the origin and evolution of life on Earth.

The researchers are currently using this strategy to assess other enzymes to get a clearer picture of what life was like on Early Earth. They are also applying these tools to the field of biotechnology, where enzymes play important roles in many industrial processes.

"The functions and characteristics we observed in the ancestral enzymes show that our techniques can be implemented to generate improved enzymes for a wide range of applications," added Perez-Jimenez.

Provided by Georgia Institute of Technology (news : web)

Research identifies on-off switch for key 'factor' in heart disease and cancer

Scientists at the University of Hull have identified a cellular 'on-off' switch that may have implications for treating cardiovascular disease and cancer.

The team has found the mechanism which controls the inclusion of a protein called tissue factor into endothelial microparticles, tiny vesicles which are released from in the lining of blood vessels.

"Although tissue factor is part of the body's natural healing process, helping create clots to stop bleeding and repair injuries, high levels circulating in the blood stream can be harmful," says lead researcher Dr Camille Ettelaie. "Excessive tissue factor is linked to , including the formation of irregular and higher risk of thrombosis, leading to and stoke."

Dr Ettelaie and co-researcher Dr Mary Collier found that two tandem within tissue factor work like an 'on-off switch' within the cells, controlling how and when it is incorporated into the microparticles and released. When a phosphate molecule is added to the first one of these two amino acids, the process starts and when added to the other, it stops.

By blocking the addition of the phosphate to the first amino acid, the researchers were able to stop the process – opening up the possibility of controlling when and how much tissue factor is released in microparticles.

"The aim of the research was to see if there might be a way to control the output of tissue factor from endothelial cells into microparticles," says Dr Ettelaie "This project focused on the vascular system and is helpful in controlling , but tissue factor is also released in microparticles from cancer cells and linked to cell proliferation – so our findings could have implications for treating cancer as well.

"Tissue factor is exploited by cancer cells – they use it to speed up their growth directly, and also increase the growth of blood capillaries which supply the tumour with nutrients – but if levels of tissue factor are too high within a cell, then the cell will die. If we could use this switch to stop cancer cells getting rid of excess tissue factor, it might be possible to kill them without causing detrimental effect to the body's normal cells."

The findings from the research – which was partly funded by Yorkshire Cancer Research and the Castle Hill Hospital Cancer Trust Fund – are published in the latest issue of Journal of Biological Chemistry (April 8).

Provided by University of Hull

Formaldehyde: Poison could have set the stage for the origins of life

Formaldehyde, a poison and a common molecule throughout the universe, is likely the source of the solar system's organic carbon solids—abundant in both comets and asteroids. Scientists have long speculated about the how organic, or carbon-containing, material became a part of the solar system's fabric. New research from Carnegie's George Cody, along with Conel Alexander and Larry Nittler, shows that these complex organic solids were likely made from formaldehyde in the primitive solar system. Their work is published online April 4 by the Proceedings of the National Academy of Sciences.

"We may owe our existence on this planet to interstellar formaldehyde," Cody said. "And what's ironic about it is that formaldehyde is poisonous to on Earth."

During the early period of the inner solar system's formation, much of the organic carbon that wasn't trapped in primitive bodies was lost to space, along with much of the water. Prior to this study numerous competing ideas emerged to explain the existence of primitive organic solids. Cody, of the Geophysical Laboratory, along with Alexander and Nittler, of the Department of Terrestrial Magnetism, and the team decided to study primitive solar system objects using advanced methods. What they discovered clearly pointed to a polymer formed from formaldehyde.

They tested their conclusion with experiments to reproduce the type of organic matter found in carbonaceous chondrites, a type of organic-rich meteorite, starting with formaldehyde. They found that their formaldehyde-synthesized organic material was not only similar to that found in carbonaceous chondrites, but also similar to organic material found in a comet named 81P/Wild 2, pieces of which were collected in space by NASA's Stardust mission, as well as in interplanetary dust particles, or particles from space that likely originated from comets and asteroids.

Their results make sense, because is relatively abundant throughout the galaxy and the polymerization process would have been possible under conditions of the primitive solar system.

"Establishing the likely origin of the principal source of in primitive bodies is extremely satisfying," Cody said.

Provided by Carnegie Institution

New process turns waste chicken feathers into biodegradable plastic

Nearly 3 billion pounds of chicken feathers are plucked each year in the United States -- and most end up in the trash. Now, a new method of processing those feathers could create better types of environmentally-friendly plastics.

" are one of those materials that is still basically waste," said Yiqi Yang, a researcher at the University of Nebraska-Lincoln and one of the authors of the new research. Feathers are mostly made of keratin, the that's responsible for the strength of wool, hair, fingernails, and hooves, he added. So they "should be useful as a material."

Past efforts to create plastic from feathers resulted in products that didn’t hold up mechanically or weren't completely water-resistant, said Yang’s University of Nebraska colleague Narenda Reddy, who also worked on the project.

To make the new plastic, the researchers started with chicken and turkey feathers that had been cleaned and pulverized into a fine dust. They then added chemicals that made the keratin molecules join together to form long chains -- a process called polymerization. The team presented their work March 24 at a meeting of the American Chemical Society in Anaheim, California.

The plastic they made was stronger than similar materials made from starch or soy proteins, and it stood up to water. Moreover, high temperature treatment of the feathers at the start of the process would blast out any possible contamination, such as from bird flu, according to Reddy.

The new material is a thermoplastic. "We can use heat and melt it to make different products," said Reddy. Heating it to a modest -- for industrial manufacturing -- 170 degrees Celsius allows the plastic to be molded into some desired shape, and it can be melted and remolded many times. Unlike most thermoplastics, which are petroleum-based, chicken-feather plastic uses no fossil fuels, the researchers said.

The feather-based plastic could be used for all kinds of products, from plastic cups and plates to furniture. In addition to making use of feathers that would otherwise end up in landfills, it is highly biodegradable.

This and other new sources of plastic may signal a shift in the way people think about packaging, said Walter Schmidt, a scientist with the Department of Agriculture’s Agricultural Research Service in Beltsville, Md., who works on making a different kind of plastic from feathers. "With foods, almost everyone understands half-life and shelf-life. No one expects milk in the fridge to be good three months after purchase."

Yet we rarely think of packaging in the same way, said Schmidt. "Stuff floats around in the ocean [or] is mixed in landfills that stay there for generations. A far better solution is to make less mess in the first place and to have that material naturally recycle in a reasonable amount of time." Although feathers are known to be tough, he added, there are no archeological sites containing reservoirs of feathers, showing that they break down over time.

The usefulness of any biopolymer, like the feather plastic, depends on the cost and versatility of the end product, said Schmidt, adding that when the price of oil increases, bio-alternatives become more attractive.

As concerns over the environment and shortages of raw materials grow, creative thinking about waste products takes on greater importance. "Think of a Styrofoam coffee cup," said Schmidt. "It is used for maybe 10-15 minutes and discarded; one can dig up a Styrofoam cup from a landfill 200 years from now, wash it out, and reuse it. This is an example of a lousy design." A better design is an ice cream cone: "The container lasts a little longer than the ice cream in it."

Although more work is needed to bring the new plastic into large-scale production, chicken feathers could soon be moving from the coop to the cup.

Provided by Inside Science News Service (news : web)

Understanding methods of assessing botulinum neurotoxin exposure

Popular in cosmetic medicine for making tiny frown wrinkles go away, botulinum neurotoxin (BoNT) is, perhaps, more notorious for historic outbreaks of respiratory paralysis and death. Traditionally, medical responders have relied on the highly sensitive, but slow, mouse assay to confirm the presence of BoNT and determine its serotype. But today, research emphasis has turned to developing in vitro immunoassay techniques with high sensitivity and rapid sample results -- both important for potential exposure.

"Each immunoassay approach has its own advantages and disadvantages," said Dr. Jay Grate, a Laboratory Fellow at the Pacific Northwest National Laboratory. "Some techniques are slow and some are fast; some are extremely sensitive and some are less so, but they are all useful."

To gain a better understanding of the breadth and depth of immunoassay techniques for BoNT, Grate and his research team reviewed current scientific literature to focus on the most promising approaches. The team's review is described as an invited cover article in the journal Trends in . (TrAC, November 2010.)

Natural cases of BoNT intoxication can occur through consumption of , infection of an open wound, and intestinal infection in infants. Intoxication can also occur through inhalation, raising concerns about use of this potent neurotoxin as a tool of warfare or terrorism. In fact, the U.S. Centers for Disease Control has listed BoNT as one of the six highest-risk threat agents for bioterrorism. According to Grate, a better understanding of immunoassay techniques for BoNT is important for public safety and national security.

"In the event of an outbreak of BoNT intoxication—whether natural, accidental, or through intentional release—it will be critical to have rapid, sensitive assays to assess a large number of samples," said Grate.

The team was invited to author an article for the TrAC article after publishing four primary research papers about BoNT detection from 2004 to 2009. In review, the team documented the progress that scientists around the world have made to develop more sensitive and rapid assays. This, says Grate, involves tremendous effort in capturing the toxin from the sample and concentrating it to a small volume to generate a strong signal.

The team's review concludes that longer assay times continue to yield the lowest achievable limits of detection. However, as research continues to replace the sluggish mouse assay, rapid new techniques are much more sensitive than in the past. For example, luminescence-detection methods have largely replaced colorimetric enzyme-amplified methods, and microfluidic formats have emerged for very sensitive assays. Plate-based formats continue to be important, as they are suitable for robotic automation. And one of the key trends that emerged from the review was a growing focus on solid phases, such as beads, for heterogeneous immunoassays.

"This technique, which helps separate the components of the assay, is not a trend that's unique to this field, but people have applied it in this area of research to achieve very low detection limits," explained Grate. "The use of solid phases has been an integral part of our approach for as long as we've been developing biodetection systems. So this review confirmed that this is a strong approach."

Grate and his research team will continue to research immunoassay techniques to rapidly screen for BoNT and other toxins.

"This is a dynamic and evolving field where researchers are all working toward better public health and safety," said Grate. "I don't think anybody—yet—has the final answer."

More information: Grate JW, et al. 2010. "Advances in Assays and Analytical Approaches for Botulinum Toxin Detection." Trends in Analytical Chemistry. TrAC 29(10):1137-1156. DOI:10.1016/j.trac.2010.07.005

Provided by Pacific Northwest National Laboratory (news : web)

New device promises safer way to deliver powerful drugs

A new drug delivery device designed and constructed by Jie Chen, Thomas Cesario and Peter Rentzepis promises to unlock the potential of photosensitive chemicals to kill drug-resistant infections and perhaps cancer tumors as well.

Photosensitive chemicals are molecules that release single oxygen atoms and chemical radicals when illuminated. These radicals are very active chemically, and can rip apart and destroy bacteria, said Peter Rentzepis, a professor of chemistry at University of California, Irvine.

Yet photosensitive chemicals are not approved for use in the United States, and used relatively rarely in Europe. This is because they are highly toxic and difficult to activate beneath the skin, since light only penetrates a few millimeters into the body.

Photosensitive chemicals also cause severe reactions, including headaches, nausea, and light sensitivity for 30 days. They kill healthy cells as well as bacteria. Although several have therapeutic potential, they are too toxic for human use by injection.

The researchers solved this problem with an optical fiber-based device that can deliver very small amounts of photosensitive chemicals to internal organs with pinpoint accuracy.

The device consists of three components. The first is an imaging component similar to the charge coupled devices (CCDs) in digital cameras. It enables a physician to guide the device to the infection.

A 1-millimeter-diameter flexible optical fiber attached micro sized high-power LED or provides the light for the CCD. Once the physician positions the device, the same shines with greater intensity to activate the medicine.

The third component is a hollow tube connected to a syringe of medicine to deliver the medicine to the infection. Rentzepis adds , a thickening agent used in surgical soaps, to keep the medicine from spreading to healthy cells.

Pulling the syringe backwards creates a vacuum that sucks up any remaining chemical after the procedure.

"We can insert the instrument through the nose, bowels, mouth, or almost any opening and direct it where we want," Rentzepis said. "It lets us deliver very small amounts of these chemicals right to an infection or tumor, then remove them before they damage healthy cells."

The researchers plan to test the device on animals with infections and cancer.

Provided by American Institute of Physics

Nature helps to solve a sticky problem

The arrays of fine adhesive hairs or 'setae' on the foot pads of many insects, lizards and spiders give them the ability to climb almost any natural surface. Research by James Bullock and Walter Federle from the University of Cambridge in England found that the different forces required to peel away these adhesive hairs from surfaces are what allows beetles to adhere to diverse surfaces, thereby reducing the risk of detachment. Their study, published online in the Springer journal Naturwissenschaften – The Nature of Science, reports the first adhesive force measurements from single microscopic setae in a live animal.

The adhesive hairs on the feet of leaf beetles are known to take three distinct shapes; pointed, flat (spatula-tipped) and disk-like. They are arranged in specific patterns across the beetle's feet, indicating different biological functions for each hair type. Given their small size (only 1/200th of a millimeter across), there existed no way to determine their individual properties. Bullock and Federle therefore devised a method for measuring the in vivo stickiness of each hair using a fine glass cantilever. By observing the deflection of the cantilever through a microscope, the force needed to detach each hair was calculated.

Results in male beetles showed that the disk-like hairs adhered with the highest force, followed by spatula-tipped and then pointed hairs. Disk-like hairs were also stiffer than either flat or pointed hairs, likely providing stability to the pad. Bullock and Federle suggest that it is these disk-like hairs in particular which allow male to achieve strong adhesion on smooth surfaces. This ability is also important for the males during copulation to hold on firmly to the back of the females. The other hair types, being easier to 'un-stick', may help the beetle to rapidly detach its feet when running upside down.

Before these natural structures can be replicated as synthetic adhesives, a better understanding of their detailed function is needed. The authors conclude, "The question of how forces in natural adhesive systems run from the single-hair to the whole-animal level is a central, unresolved problem. Its understanding will be a prerequisite for the design of bio-inspired synthetic adhesives."

More information: Bullock JMR and Federle W (2011). Beetle adhesive hairs differ in stiffness and stickiness: In vivo adhesion measurements on individual setae. Naturwissenschaften – The Science of Nature. DOI:10.1007/s00114-011-0781-4

Provided by Springer