ne of the hallmarks of cancer cells is their uncontrolled growth. So, hoping to find a way to rein in this behavior, researchers are looking at modifying proteins called growth factors--molecules that trigger the process of cellular reproduction--to slow or stop cancer's progress.
One of these molecules is epidermal growth factor, or EGF. As its name suggests, EGF was first found in skin cells, but it works in a variety of tissues throughout the body. Because it promotes cell growth, EGF has been used to speed the healing of severe burns, stomach ulcers, and the lens of the eye after cataract surgery.
In 1986 ORNL researcher Salil Niyogi and his colleagues in the Biology Division cloned the gene for human EGF. Their aim was, and still is, to better understand the structure and function of EGF by studying the effects of minor variations in the sequence of amino acids that make up the EGF protein. Some of these altered, or mutant, forms of EGF could be useful in preventing normal EGF from fulfilling its role as the "on" switch for cellular reproduction--serving instead as "off" switches for cancer growth.
To determine which amino acids to change, Niyogi and his team look at several different aspects. Because EGF is shared, with some variations, among many species, researchers studying the similarities and differences in the structure of these molecules can begin to identify points along human EGF's amino acid sequence at which changes could make a critical difference.
Another way of locating promising sites for changes is to examine the structure of the molecule as revealed by nuclear magnetic resonance (NMR) studies--the same type of technique used for medical imaging in many hospitals. In fact, a diagram of the structure of EGF based on collaborative studies between ORNL and Rutgers University recently appeared on the cover of the British journal Protein Engineering.
Drawing on this information and experience with other proteins, researchers can determine the steps the cell goes through in building an EGF molecule and many of the critical junctures in that process. "Look at the antiparallel beta sheet structure," Niyogi says, tracing one of the crooked horseshoe shapes on a diagram of the EGF molecule. "These structures at either end of the linear molecule are quite close together in the EGF-like molecules of many species, suggesting that both the shape of the molecule and the composition of these structures are important enough to be conserved."
Once they have altered the composition of the EGF gene, Niyogi and his team measure the performance of the protein it produces. "We measure function by first measuring how well EGF binds to its protein receptor on the cell membrane," Niyogi says. "Once the molecule binds to the receptor, it transmits a biochemical signal to the inside of the cell. This signal, known as the protein-tyrosine kinase reaction, begins a series of biochemical reactions--a cascade turns on--leading to the synthesis of DNA, RNA, and protein and, finally, to cell division." Niyogi stresses that this entire process is started by the binding of EGF to the receptor on the cell membrane, telling the cell it's time to grow and divide. If the EGF molecule fails to bind to the receptor, or if it fails to send the necessary signal to the cell, the cell will not reproduce.
Six major sites are involved in binding EGF to its receptor. When Niyogi's team makes a change in the protein, they look at the effects of the change on both EGF's structure and its ability to bind to its receptor. The structural work has been done in collaboration with Guy Montelione of Rutgers University. "He's a protein structure person and one of the first to determine the structure of EGF," says Niyogi. Using NMR, this group has determined that, when substitutions are made for each of the amino acids at these six sites, the structure of the molecule is mostly unchanged. "Therefore," Niyogi says, "we believe that these sites are mostly functional, rather than structural."
To determine how these sites bind EGF to its receptors, Niyogi and his team created a series of double-site mutations by simultaneously replacing both amino acids at any two of the sites. They found that each site binds to the receptor independently. "Previous investigators thought that a particular site was the key," Niyogi says, "but the protein binds like a hand in a glove--all the fingers are important.
"Most of the time," he continues, "when you change an amino acid in a protein, nothing improves. You make it worse because you are tampering with Mother Nature." Despite this admonition, Niyogi and his colleagues have discovered a double-site mutation that results in an altered EGF protein that binds to its receptor twice as strongly as normal EGF.
Other intriguing EGF mutants produced at ORNL include molecules that, even when they occupy all of the EGF receptors, still do a poor job of stimulating the tyrosine kinase reaction that spurs cell growth. "It's like the car is running," says Niyogi, "but it can't get into high gear." Mutations like these competetively inhibit normal EGFs stimulation of the receptor's tyrosive kinase reaction and, therefore, could act as antigrowth substances by preventing normal EGF molecules from initiating cell growth. "These agents may be effective as growth inhibitors with a little more engineering to make them bind more strongly to cell receptors," Niyogi adds. "But we still have some basic research to do with normal and cancer cells before we can tell whether an EGF mutant might be able to control cancer."
Niyogi and his team think they have a good understanding of how EGF interacts with its receptor and how to make EGF mutants that inhibit the chemical cascade that leads to cell growth. Their next goal is to produce cells with mutant receptors to learn how receptors work. "This would help us understand both how normal cell growth takes place,"says Niyogi, "and how things get out of control, resulting in a cancer."
Niyogi is also quick to point out the invaluable contributions of his co-workers: the cloning of human EGF and its mutagenesis by Dave Engler and Rise Matsunami; elegant mutagenesis studies by Steve Campion, Doug Tadaki, and Krishnadas Nandagopal; fruitful collaborations with researchers in John Cook's laboratory, including cell culture studies by Melinda Hauser; and the scientific contribution of senior staff scientist Audrey Stevens, who mentored Matsunami, suggested EGF as the protein for study, and provided advice and inspiration for the project's success.--Jim Pearce
Terzaghi-Howe's research in this area focuses on determining the effects of alpha particle radiation on both intact rat tracheas and cultures of cells removed from rat tracheas. "The reason we are working with rat tracheal cells," she says, "is that their diameter and cell structure are very similar to those of cells in the area of the human lung that is most affected by radon."
Radon and its close relatives emit primarily alpha radiation and are blamed for an estimated 7000 to 30,000 lung cancer deaths in the United States each year. Studies like Terzaghi-Howe's improve researchers' ability to gauge the environmental and occupational risks posed by radon and other radionuclides.
Her latest work involves exposing tracheal cells and tissue to three different radiation sources: plutonium-238, polonium-210, and americium-241. Although all of these radionuclides emit alpha radiation, their differences are probably more important than their similarities.
As they decay, americium and plutonium isotopes produce not only alpha particles but also low-energy gamma particles; these radionuclides are also relatively long-lived, with half-lives measured in years. On the other hand, polonium, one of the products of radon decay, is a nearly pure alpha emitter, and its half-life is measured in days.
These contrasting qualities come into play in Terzaghi-Howe's research for several reasons. First, it is not entirely clear that alpha particles alone can induce cancer. "It is very difficult to transform cells with alpha radiation," says Terzaghi-Howe. "My suspicion is that other things interacting with alpha particles, like the chemicals in cigarette smoke or gamma radiation, might make the difference."
One of the problems Terzaghi-Howe faced in setting up this experiment was that polonium emits alpha particles at a slower rate than the other two sources, making it likely that cell and tissue samples would dry out before being completely irradiated. Terzaghi and her colleagues chose to irradiate samples on the surface of the polonium source, rather than at a distance, as is usually done. The greater alpha particle fluence--the number of particles passing through a given area--closer to the source ensured that the polonium-exposed samples would encounter the same number of alpha particles as the other samples.
To test the hypothesis that some attribute of intact tissue or the environment of a live animal is responsible for inhibiting tumor development, the intact tracheas were irradiated and then implanted under the skin of mice to approximate an in vivo environment. The cultured tracheal cells were suspended in a liquid and then irradiated.
Inspection of the irradiated samples revealed precancerous changes in cell structure in some of the samples but not in others. "We did the same experiments with plutonium-238, polonium-210, and americium-241 at the same alpha particle fluences," says Terzaghi-Howe, "and we got no neoplastic transformation from the polonium isotope, but we did get transformations in both the cell suspensions and the tracheal implants that had been exposed to the plutonium and americium.
"We don't know what the explanation for this is," says Terzaghi-Howe. "All of these experiments are aimed at determining whether we can detect an alpha-gamma interaction effect that causes cellular changes to take place--or an effect based on the position of the sample relative to the source."
The distance a cell is from the radiation source can make a big difference. When an alpha particle is emitted, the farther along its track it is, the more energy it dumps. So it's conceivable that the sources were located at distances from the samples where, by chance, they either did or did not cause cell transformations to take place."
Jim Turner, a physicist in ORNL's Health Sciences Research Division, is developing a computer model that takes the energy transfer characteristics of alpha particles into consideration to optimize the differences among the three sources. His model also predicts the chances that a cell will be hit by both an alpha and a gamma particle. "The chances of that happening are ridiculously low," says Terzaghi-Howe. "So, if gamma radiation is important in causing changes in cells, then it's not because a cell needs to be hit by both alpha and gamma particles. It could be that something else happens to the unhit cells."
Another dilemma that Terzaghi-Howe points out is that, if an alpha particle passes through a cell nucleus, the cell is killed before it can become a tumor cell. "If that's true," she asks, "what's transforming cells? Either the near-miss cells are affected, or it's another effect altogether."
Despite the lack of an obvious explanation for why good cells go bad, Terzaghi-Howe maintains her enthusiasm for the research. "These experiments are intriguing," she says. "There's clearly something happening here that we don't quite understand, but there are more puzzles than answers at this point."--Jim Pearce
The enzyme, known as Rubisco, is a key component of the process of photosynthesis, used by all plants and some bacteria to convert carbon dioxide in the air into food. Rubisco provides the critical chemical link that enables plants to combine carbon dioxide molecules absorbed from the air, each containing a single carbon atom, with five-carbon sugar molecules. This reaction produces two three-carbon sugars--a net gain of a carbon atom. Then, using energy drawn from sunlight, these simple sugars are transformed into complex carbohydrates, like starch. These carbohydrates nourish the plant, and plants, directly or indirectly, nourish us.
Rhenium-188 Video Clip (QuickTime, 1.4 minutes, 2 MB)
"So," you might ask, "if something has worked for three billion years, why mess with it?"
"It's a question of efficiency," says Hartman. "Photosynthesis is a very inefficient process, and improving the efficiency of this enzyme could increase plant growth, crop yields, and biomass energy production."
Rubisco's main problem is that it doesn't do a very good job of distinguishing between oxygen and carbon dioxide. As a result, it's about as likely to react with one as the other, even though the oxygen reaction apparently serves no biological purpose. This laissez faire approach to chemical reactions on the part of the enzyme cuts some plants' capacity for processing carbon dioxide in half.
It also has several chemical inefficiencies that render it only about 1% as effective as other enzymes at promoting its assigned reaction--the transformation of carbon dioxide to sugar. Hartman points to the fact that the human body contains 50,000 enzymes, most of which are much more efficient than Rubisco. "It seems reasonable," he says, "that we should be able to improve the efficiency of this enzyme."
He is realistic, however, about the difficulty of the task facing him. "When a scientist projects improvement on Mother Nature, you should be skeptical," he says. Part of the reason Hartman counsels caution is that Rubisco has a much bigger job than most other enzymes.
A typical enzyme's job is to make or break a single chemical bond. For example, pepsin, an enzyme that works primarily in the stomach, is devoted to breaking proteins apart into amino acids so they can be absorbed by the body. "On the other hand," says Hartman, "Rubisco is responsible for catalyzing six distinct partial reactions in a single stage of photosynthesis. Its inefficiency may be a reflection of the difficulties of designing a single enzyme to execute all of this chemistry."
To unravel the mysteries surrounding how Rubisco works, Hartman and his research team use recombinant DNA technology to alter the enzyme's 475-amino-acid-long structure (see sidebar). By changing one acid at a time they can monitor the effect of each change on the enzyme's performance. Because there are 20 amino acids, there are about 9000 different ways to replace a single amino acid along the length of the Rubisco molecule. Rather than creating 9000 variations of the enzyme, Hartman's group has relied heavily on information about Rubisco's three-dimensional structure provided by X-ray crystallography and functional information drawn from earlier chemical studies. This information, particularly a knowledge of which areas of the enzyme are binding sites for substrates, has enabled Hartman and his group to make educated guesses about which amino acids to substitute to effect the desired changes in Rubisco.
Of the dozens of altered versions of Rubisco produced by the group, several can distinguish between carbon dioxide and oxygen somewhat better than the normal form of the enzyme--which is half the battle. But, so far, none of the group's creations can match the rate at which unaltered Rubisco promotes the carbon dioxide reaction.
In the long run, determining how to increase the efficiency of Rubisco could have profound implications for agriculture and biomass energy production. It has also been suggested that increasing Rubisco's use of carbon dioxide could decrease levels of the gas in the atmosphere, reducing the likelihood of global warming while promoting plant growth if the improved version of the enzyme were incorporated into crops and other plants on a large scale.
"That would be nice," Hartman adds, "but nothing like that is going to happen today, this week, or even this year," In the short run, his team is focusing on understanding how enzymes tell the difference between potential reactants, such as carbon dioxide and oxygen, and what factors control how quickly or slowly they work--a reasonable goal, particularly since the enzymes have a three-billion-year head start.--Jim Pearce
Town gas was the vaporous result of the slow heating and cooling of coal or oil--a process that left a lot of incompletely burned material in the form of tar. Because tar wasn't good for much, utility companies usually dumped it somewhere--often on the outskirts of town. Over the years, these dumps were often buried, forgotten, or overrun by expanding cities and their suburbs.
Recently, however, interest in coal tar has been renewed as researchers have found that it is rich in polycyclic aromatic hydrocarbons (PAHs), many of which are mutagens or carcinogens. These findings have made tar dumps a major liability for some utility companies, spurring studies of the effects of these sites on the environment. A major question has been whether PAHs absorbed by plants could come into contact with humans or even enter the food chain.
PAH molecules come in a variety of sizes. Initial studies of larger, heavier PAHs, particularly benzo[a] pyrene, showed that they were not absorbed from the soil by vegetation. However, few of these studies clearly addressed the behavior of lighter-weight PAH molecules. To help determine whether lightweight PAHs could be absorbed by plants, the Electric Power Research Institute funded a study by Environmental Sciences Division researchers Barbara Walton and Anne Hoylman on the subject. "Coal tars break down over time when oxygen is present," says Walton. "However, lighter PAHs are most likely to remain at older sites, particularly if they are buried and don't have any contact with oxygen. This isolation prevents them from being broken down further through oxidation."
Hoylman, then a University of Tennessee doctoral student working at ORNL, designed an experiment to measure plant uptake, or absorption through roots, of PAHs. The key to this study was an experimental design that isolated the aboveground foliage from the roots and soil, thereby providing a clear route for the uptake of PAHs tagged with radioactive carbon-14.
"We wanted to determine where the PAHs were going in the plant and soil systems," Hoylman says. "If we established that they were being absorbed by the plants, then there would be a reason to be concerned, and further research might be required."
To create a worst-case scenario for the uptake of PAHs by the plants in their study, Hoylman and Walton tailored their experimental setup to favor the absorption of PAHs. First, because PAHs are likely to attach themselves to organic material, the researchers reduced the amount of organic carbon in the soil, increasing the likelihood that the plants would take up the radiolabeled PAHs. Also, they chose to grow white sweetclover plants, a plant found at the site, which has a large root mass and, as a result, a high likelihood of absorbing PAHs.
Under these optimized conditions, the plants were grown in airtight flasks that isolated the stems and leaves from the soil and any PAHs released from the soil into the air. This approach ensured that the presence of PAHs in the stems and leaves of the plant would be a result of absorption through the plant's roots. The specially designed flasks also ensured that any carbon-14 released into the air was trapped and measured.
At the end of the study, the soil and the plants were analyzed. Hoylman and Walton found that the bulk (86%) of the PAHs remained in the soil. Of the PAHs that were absorbed by the plants, the lightest and most water-soluble, naphthalene, had the highest concentration (0.5%), remaining primarily in the roots, with progressively lower concentrations in the stems and leaves. Three times more naphthalene was absorbed, on the average, than any of the other three hydrocarbons: pyrene, phenanthene, and fluoranthene.
So although a small quantity of lightweight PAHs was absorbed by the plants' root systems, only a fraction was transported to the plant's stem and leaves. "We established conditions to enhance the uptake of PAHs," says Hoylman, "and we saw that the root tissue of plants could be a potential 'sink' for PAHs. But, overall, it was a very limited mechanism, given that we established conditions to enhance uptake." This finding has led to the hypothesis that roots, in conjunction with microbes that inhabit the area around them, act as a defense mechanism for plants against absorption of toxic chemicals, such as PAHs.
The results of this study also provide some encouragement for power utilities and others who ask, "Is it okay for people to use these areas?" The answer may be "yes."--Jim Pearce
"For every ten dollars normally spent running the air conditioner, two dollars will be saved," says Fang Chen, leader of ORNL's Thermal and Environmental Technology Group in the Laboratory's Energy Division.
The patented new design, created by Chen and division colleague Vince Mei, is compatible with coolants that do not contain chlorofluorocarbons (CFCs), which are believed to contribute to ozone depletion in the upper atmosphere and which are being phased out.
If the design is fully implemented in the U.S. auto industry, Chen says, the increased energy efficiency could save about 1.29 billion liters (340 million gallons) of fuel a year nationwide and could cut annual carbon dioxide emissions by more than 2.9 billion kilograms (6.5 billion pounds). Annual fuel cost savings to the consumer would exceed $400 million a year at a fuel price of $1.20 per gallon. Chen said he and his ORNL group are now talking with two U.S. automakers and with air-conditioner manufacturers about licensing the technology.
The improvement involves adding a simple, inexpensive heat-exchange device to existing air-conditioner designs. The device allows the coils in an air conditioner's evaporator to be "overfed" with liquid coolant, increasing cooling capacity. Any excess liquid coolant exiting the evaporator is rerouted back into the same heat exchanger for vaporization, protecting the unit's compressor and decreasing its work load, thus lowering energy consumption and CO2 emissions.
The 20% efficiency boost and the associated savings may be applicable to home air-conditioning units. If the ORNL device were added to each home air conditioner, American consumers would save $5 billion and the carbon dioxide output would be cut by 12.2 billion kilograms (27 billion pounds). Further studies to apply the technology to stationary air conditioners, home heat pumps, and refrigerators are pending. Development of the new heat-exchange device has been funded by DOE's Office of Transportation Technologies.--Wayne Scarbrough
Team members working in conjunction with ORNL's Cooperative Forest Studies Program have erected 18 open-top chambers made of clear plastic and aluminum near TVA's Norris Dam Reservation. The purpose of these chambers is to help scientists learn how tropospheric ozone--caused by emissions from motor vehicles, petroleum refineries, coal-burning power plants, and many other industries--interacts with mature northern red oak trees and seedlings.
"The chambers allow us to monitor and regulate the exposure of trees and seedlings to ground-level ozone, while comparing responses," says Paul Hanson of ORNL's Environmental Sciences Division. "Although we have learned that the leaves of mature trees are somewhat more sensitive to ozone, the key goal of our study is to determine whether seedling studies can be used to extrapolate data for mature tree responses."
Currently, it is not known how accurately seedling responses will reflect responses of mature trees and forest ecosystems to air pollution. The information derived from the ORNL-TVA study, however, will be developed into plant response models that will help researchers better understand tree and forest ecosystem responses to air pollution.
When exposing trees to elevated ozone levels, researchers use computer software to compare the average ozone concentration of ambient air with that in the chamber to calculate amounts required for maintaining the desired elevated ozone concentration in the chamber.
"After harvesting the seedlings," says Jim Kelly, TVA project manager for the study, "we'll then use another nearby chamber site to also study red oak, yellow poplar, and shortleaf pine trees that were planted 3 years ago to simulate a forest."
Some 24 model forest ecosystems then will be exposed to various levels of ozone, as well as soil temperature and moisture treatments. The chambers, which are 9 meters (28 feet) high by 4 meters (12 feet) in diameter, will accommodate measurements over several growing seasons to show researchers how tree responses vary over the years.
"We believe this project will eventually help utility industries and federal agencies because the electric power industry and the biological science community have long had the goal of being able to predict the responses of ecosystems to a changing atmosphere," Kelly says.
The ORNL-TVA project is sponsored by TVA and the Electric Power Research Institute.--Brian Daly
Currently, the fiber-optic sensors are being used in the construction of a new science and engineering research facility at the University of Tennessee at Knoxville.
ORNL researchers have embedded optical fibers in the concrete support beams of the UT building to measure the strain on the concrete before and after the post-tensioning process and throughout the life of the building. If a crack forms in a concrete slab, the silicone rubber fiber deforms as the crack shifts. The changes in the amount of light going through the fiber then can be monitored to gather information about the characteristics of the crack and the building's superstructure. The flexible fiber-optic sensors also have many other uses, including measuring the strain and pressure on bridges and in airplanes.
The post-tensioning of a beam involves putting more than 50,000 pounds of tension on the concrete to reinforce the post-tensioning strands (braided wire within a plastic jacket). The goals of the project are to better understand the dynamics of the post-tensioning process and to monitor the integrity of a building's superstructure over time.
By observing changes inside the beams as tension increases and as the building ages, civil engineers will gain new information that will enable them to design better and safer buildings, highways, bridges, dams, and other concrete structures. Embedded optical fibers also are sensitive to vibration and may be used to improve the accuracy of early warning systems for earthquakes.--Angela C. Swatzell
ORNL scientists are lending their expertise to a consortium of 27 companies that have formed a task force to scrutinize the phenomenon of "spray drift."
The ORNL team has developed a unique means to measure critical qualities of droplets during the milliseconds of their formation. Their discoveries will be used to help determine proper droplet size for specific mixtures as they are blasted from a spray nozzle. The knowledge gained from the measurements will help ensure that what is sprayed from above will land where it is intended.
"This is good for the health of the people in the area, good for protecting the groundwater, and good from the standpoint of saving money through less waste," said Osman Basaran, a chemical engineer and group leader in ORNL's Chemical Technology Division.
The nature of how droplets of a sprayed solution will drift through the air obviously has something to do with the prevailing winds. But there are other factors, including evaporation, droplet composition, viscosity, flow rate through the spray nozzle, and a property called dynamic surface tension, which is the quality that relates to the ability of a droplet to maintain a uniform shape instead of breaking up.
Through years of research focused on the chemical and petroleum industries, ORNL has become a leader in measuring these subtle surface forces when chemicals are manufactured by separation processes, such as when kerosene or gasoline are derived from crude oil. "This expertise made ORNL a natural choice for the spray-drift studies," Basaran said.
"Just as the petroleum industry wants to ensure the best separation processes, agricultural companies and regulatory agencies want a sound scientific basis for predicting the drift of chemical sprays," he said. "Each of the 27 companies in these studies has its area of expertise in providing this basis."
But existing means of measuring dynamic surface tension, for example, of gas bubbles during their relatively slow formation and ascent through a more dense surrounding liquid, proved useless for studying droplets sprayed at high pressure.
To make their assessments, the ORNL team uses a system of precision pumps and flow regulators, and electronics to digitize information from the growing droplets. A powerful video camera, capable of recording 12,000 partial images per second, is focused on a tiny, needle-like capillary from which the droplets emerge.
On a video monitor in the laboratory, the droplets appear as large, shimmering globes of fluid. They are actually not much bigger than the period that ends this sentence.
As droplets form at the end of the capillary, the scientists determine surface tension by measuring simultaneously the curvature of the droplet and the pressure difference between the inside and outside of the droplet. Capillaries having small enough diameters cause the droplets to maintain the shape of a partial sphere throughout their formation, which aids in measurements. (A vibration table is used throughout the experiment to minimize or eliminate unwanted movement of the droplets.)
Dynamic surface tension of droplets depends in large part on the surfactant, or additive such as a pesticide, in the liquid. As droplets form, the movement of surfactants within the droplet may lag behind the expansion of the droplet's surface. This effect changes the surface tension and, thus, will affect spray drift.
Basaran and his colleagues Michael Harris and Xiaoguang Zhang, also of the Chemical Technology Division, will use the new measurement techniques on several solutions to determine dynamic surface tension and other parameters.
"It's important to get baseline measurements for various solutions that are representative of what the agricultural industry might be spraying," Basaran said, explaining that in the future, agricultural companies will be required to tell regulatory agencies before spraying begins what droplet size will result for a particular spray.
"With the data being produced at ORNL and by others in this project," Basaran said, "sprayers will be able to predict spray drift with more confidence because they will be able to better control the factors that determine drift. That's good not only for the companies involved but for everyone who lives near the area being sprayed."
Basaran said that the ORNL drift data may also be applicable to other work involving sprayed solutions, such as spray-painting aircraft and buildings.--Wayne Scarbrough
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