Life Sciences Division
1997-98
Progress Report
- Overview
- Mission
- Structure
- Lab Animal Resources
- Operations & Support
- Mouse Genetics Research
- DNA Sequencing Lab
- Gene Expression Chip Lab
- Dosimetry & Biokinetic
- Life Cycle Analysis
- Environmental Assessment
- Biological Sciences
- Functional Genomics & Proteomics
- Structural Biology
- Biomedical Engineering
- Technology Applications
Biochemistry and Biophysics Section
Staff
|
J. P. Alarie |
L. J. Foote |
T. D. Jones |
M. Terzaghi-Howe1 |
Technical Support
| A. R. Ellis M. Y. Mack V. P. Patania* |
Administrative Support
|
S. C. Ayers1 |
J. B. Cooper |
D. Holt |
M. W. Terrell |
Postdoctoral Fellows
|
Z. Chi |
B. Hanson |
Z. Liu |
M. Zhan |
Consultants/Subcontractors
|
J. Fleming |
T. Bunde |
F. Kleinhans |
P. Walsh, Sr. |
Students/Faculty/Visiting Scientists
|
S. Ahamed |
Z. Hu |
A. Nagel |
T.-H. Wang |
*Dual Capacity
1Retired or terminated employment with Life Sciences Division
in 1997 or 1998
2UT/ORNL Collaborating Scientist
3Part-Time
Introduction
Activities in the Biochemistry and Biophysics Section span the fields of biology, chemistry, and physics with particular emphasis on the science at the intersection of these fields; e.g., biochemistry, biophysics, and chemical physics. Basic research is carried out in areas such as protein engineering, establishment of structure-function relationships, x-ray and neutron crystallography of biomolecules, DNA structure, biosensors, disease diagnosis, health effects from chemicals and radiation, plasma physics, microscopy, and optical and mass spectrometry. In addition to the basic research, considerable effort is spent on technology development such as the invention and refinement of novel measurement techniques, instruments, and sensors. Some technologies of recent interest include laser spectroscopy, mass spectrometry, scanning probe microscopies, imaging technology, and biosensors. A recent emphasis has been on potentially high throughput chip technologies and multiplexed sensors. Wireless biomedical devices, developed in collaboration with the I&C Division, represent another important focus for the future.
Biochemistry research focuses on exploration of structure and function relationships of animal and plant proteins. The proteins studies include energy-related enzymes, growth factors important in cancer biology, and DNA repair enzymes. Site-directed mutagenesis, biochemical modification, and genetic techniques are used to produce modified proteins. Extensive studies on the regulation of eukrayotic messenger RNA synthesis and turnover has focused on several exoribonucleases. These enzymes play an important role in regulating RNA levels and function. ORNL has extensive national-resource capabilities for structural biology in neutron scattering and diffraction at the HFIR. The x-ray counterparts of these facilities provide synergistic and complementary structural capabilities. These facilities are being used to determine the structural organization of genomic DNA and associated nuclear proteins through detailed three-dimensional analysis of structures such as the nucleosome core particle. The molecular immunology program focuses on ligands that bind to cell surface molecules to target isotopes to cellular sites. Glycoproteins in the endothelium of tumors are the subject of study of monoclonal antibodies. These target molecules are identified using protein chemistry and molecular biology.
Selected Accomplishments
Flowthrough Genosensor Technology. A high-throughput DNA analysis system based on flowthrough genosensor technology was assembled by researchers in BABS. This system has the ability to monitor thousands of DNA sequences simultaneously, at high speed, due to the unique micro-capillary array design. The flowthrough genosensor chip is comprised of DNA probes attached to the inner walls of micro-capillaries connecting the two faces of a glass or silicon wafer. Robotic technologies for the automated gridding of DNA probes across the genosensor chips have been developed. When a labeled nucleic acid sample is flowed through the chip, the pattern of binding across the chip, analyzed by a CCD imaging system, reveals the presence and relative abundance of specific nucleotide sequences in the sample. Flowthrough chip technology has been patented and licensed. Applications of the technology in genotyping, gene expression analysis, and microbial identification are under development. The technology is expected to help researchers uncover complicated cellular processes such as metabolic pathways and responses to genotoxic or pathogenic exposure and will facilitate diagnosis of genetic and infectious diseases, and accelerate drug discovery.
New Generation of Intelligent Sensors. The next revolution in microminiature electronic devices will be in the area of intelligent sensors. Exploiting the sensitivity of micromachined springboards used in the atomic force microscope, researchers have shown that a variety of physical, chemical, and biological components in the environment can be perceived at extremely small levels. Arrays of such sensors now combined on a single silicon chip provide a powerful and yet very inexpensive means for simultaneous measurement. Several companies have licensed or are in the process of licensing the cantilever technology, which is expected to impact areas of environmental monitoring, industrial control, and consumer products.
ORNL Technology Puts Knoxville Company in Remediation Fast Lane. Slow and expensive commercial laboratory tests of water or soil may become obsolete because of a new instrument developed by the Advanced Monitoring Development Group and licensed to Environmental Systems Corporation. The Knoxville company has purchased the commercial rights to the technology to be used in the Luminoscope, a field-portable instrument designed to detect, measure, and monitor levels of gasoline, oil, polychlorinated biphenyls (PCBs) and pesticides down to parts per million levels in soil or in water. The instrument fits inside a suitcase-size carrying case and has a battery pack. A laptop computer provides instrument control, data analysis, spectral display and data storage.
Surface-Enhanced Raman Gene Probe for HIV Detection. The Advanced Monitoring Development Group has reported for the first time the use of surface-enhanced Raman (SERS) active labels for primers used in polymerase chain reaction (PCR) of specific target DNA sequences. This method has the potential for combining the spectral selectivity and high sensivitity of the SERS technique with the inherent molecular specificity offered by DNA sequence hybridization. The effectiveness of the detection scheme is demonstrated using the gag gene sequence of the human immunodeficiency virus (HIV). The SERS gene probe technology, which can make use of multiple probes for simultaneous detection of multiple biological targets, is currently developed for improved DNA mapping using BAC close methods.
Antibody-Based Nanosensor for Single-Cell Measurement. Optical sensors with nanoscale dimensions are powerful tools that are capable of providing selective identification of biochemical compounds at ultra-trace levels in biological systems. Research staff in the Advanced Monitoring Development Group have recently developed an antibody-based nanosensor for measurement of benzo(a)pyrene tetrol (BPT), a metabolite of the carcinogen benzo(a)pyrene inside a single cell. The antibody has recognition/binding sites for specific molecular structures of the antigen and "fit" the unique antigen such that hollows, protrusions, planes, and ridges of the antigen and antibody are complementary. One can then develop antibodies to recognize molecular structures of chemicals, biochemicals, and microorganism components. The inherent selectivity of these antibodies can be utilized as specific "detectors" to identify many analytes of interest that are present at ultra-trace levels in single cells for studying gene expression or for medical diagnostics. Combining the exquisite specificity of biological recognition probes (antibodies) and the excellent sensitivity of laser-based optical detection, optical biosensors are capable of detecting and differentiating bio-chemical constituents of complex systems in order to provide unambiguous identification of a variety of diseases.
Monoclonal Antibodies Developed in Life Sciences Laboratory. The integrins are a family of cell-surface proteins involved in cell attachment. Researchers in the Molecular Immunology Group developed monoclonal antibodies (MAb) which recognize two members of the family and can be used to identify these proteins and their role in cellular functions. Although not patented, a bailment transfer mechanism was developed to "sell" the antibodies to companies so that they can be made available commercially. Three companies have acquired a total of four of these MAbs. In the last several years previous to these agreements, the group's laboratory has supplied over 300 samples of these reagents to other scientists.
Protein Crystal Annealing. Cryocrystallography has become the method of choice for macromolecular crystallography because of the many advantages conferred by cryogenic data collection. The only significant disadvantage, in some cases, is increased mosaic spread as a result of flash cooling. In crystals containing large unit cells, increased mosaicity can make data reduction difficult to impossible due to reflection overlap. Scientists in the Physical Biosciences Group have developed a process that, when applied to a flash-cooled crystal, will often lower the mosaic spread. The process has been applied to a number of different macromolecular crystals. Refined values of mosaicity have been observed to improve by greater than a factor of two, and resolution may also improve. Experiments demonstrate that the molecular structure is unaffected by the annealing process. The process has been successfully applied to crystals grown using a number of precipitants. Crystals have been flash cooled using a variety of cryoprotectants and also by using Paratone N to remove surface solution from the crystal. The process is simple, reproducible, and promises to routinely improve data quality in flash-cooled crystals of biological macromolecules. It should also extend the application of cryogenic data collection to a wider range of challenging crystals and simplify the handling of flash-cooled crystals.
Enhanced Electron Attachment to Highly-Excited Molecules Using a Plasma Mixing Scheme. Researchers have developed a novel plasma mixing technique to achieve enhanced electron attachment to highly-excited states of a variety of molecules. In this scheme, long-lived metastable states of inert gases are produced in a glow discharge and are extracted into an adjoining discharge-free region (target region); a suitable molecular gas is fed into the target region where they undergo excitation transfer from the inert gas metatstable states. The highly excited molecules thus produced attach slow electrons that are also extracted to the target region from the discharge region. Researchers observed negative ion formation in a variety of gases including methane, nitric oxide, and some fluorocarbons. In addition to the production of negative ions, this technique automatically leads to the formation of radicals (molecular fragments).
This technique may have a variety of applications including (1) an inexpensive negative ion (neutral beam) source, (2) the means to efficiently produce radicals for plasma processing of materials, and (3) the means to dissociate molecules in plasma remediation of volatile toxic compounds. Two patent disclosures have been filed regarding the last two applications.
A Novel Energy-Efficient Plasma Chemical Process for the Destruction of Volatile Toxic Compounds. Researchers have reported several achievements in developing a novel energy-efficient plasma chemical process for the destruction of volatile toxic compounds (VTC). The basic physics/chemistry involved in the dissociative electron attachment to highly excited molecules was unraveled in a plasma mixing apparatus. The researchers have developed a methodology for the evaluation of VTC destruction in a plasma mixing apparatus and completed a baseline study on the destruction of VTCs using a DC glow discharge apparatus.
Tumor Blood Vessels Targeted with Radioisotopes in Cancer Therapy. Therapy of metasteses from solid tumors remains as a major problem in curing carcinoma--the major cause of death from cancer in humans. Much current work has focused on tumor blood vessels as a target for directed therapy. Inhibition of the growth of new blood vessels has shown to keep tumors at bay, but does not actually cure the cancer. Working in a mouse model system, researchers from BABS and ATS have used radioisotopes targeted to tumor blood vessels to kill not only the cells lining the vessels, but also the tumor cells they serve. In the most recent experiments, the effects of an alpha-particle emitter, 213Bi, and a beta-particle emitter 90Y were compared. Alpha particles can travel only about 10 cell thicknesses in tissues but are extremely destructive in their short path length, whereas beta particles can travel 100 times farther, but are less destructive per unit path length.
The data show that mice bearing artificial metasteses in their lungs can be cured of the tumors with vascular targeting of either radioisotope; however, the beta-particle emitter causes more damage to the adjacent normal lung as well as nearby organs. These results indicate that for radioimmunotherapy by vascular targeting to small tumors, up to 2000 cells or so, the "surgical strike" of a vessel targeted alpha-particle emitter is a more focused and specific agent for therapy.
Cloning and Characterization of an Enzyme Integral Tophotosynthetic Assimilation of Atmospheric Carbon Dioxide. The seemingly trite bumper sticker, "have you hugged a plant today?", actually reflects the profound truth that the entire animal kingdom requires plants for survival. We depend on plants for oxygen that we breathe, food that we eat, shelter that protects us from the elements, and medicinals that ward off disease. Fossilized plants fuel our automobiles, generate electricity, and provide innumerable consumer products and industrial materials. All of these benefits are consequences of the photosynthetic machinery by which plants capture and utilize the energy from sunlight to produce carbohydrates from atmospheric carbon dioxide (CO2). Photosynthetic conversion of atmospheric CO2 to carbohydrates, which is the only biospheric avenue for sequestration of this predominant greenhouse gas, is dependent on the concerted action of multiple enzymes that comprise the Calvin cycle. As inefficiency of this biosynthetic pathway severely curtails potential plant growth and yield, application of genetic engineering toward improved efficiency offers the prospect of enhanced biomass for energy, food, and global carbon management. Reaching such an ambitious, long-term goal is predicated on comprehensive understanding of mechanism and interplay of the requisite enzymes. Although some of the Calvin cycle enzymes are well-characterized, others have been glaringly neglected. For example, ribulose-5-phosphate epimerase, which is essential for the regeneration of the substrate for CO2 fixation and also provides for critical linkage between distinct metabolic pathways, has never even been isolated from plants due to its extreme instability and low natural abundance. Scientists in the division have overcome these impediments by cloning the gene that encodes the spinach epimerase, expressing the gene in Escherichia coli, identifying conditions for stabilizing the epimerase, and isolating the overproduced enzyme. Structural, catalytic, and stability parameters of the purified, biologically active epimerase have been characterized, thereby paving the way for future mechanistic studies.
Laser Desorption Mass Spectrometry for Dynamic Mutation Analysis with Clinical Samples. Working with researchers at UT Medical Center, the Photophysics Group recently developed laser desorption mass spectrometry (LDMS) for dynamic mutation analysis with clinical samples, and gave the first demonstration for Huntington Disease (HD) and denatorubral-pallidoluysian atrophy (DRPLA), which are two major genetic neurodegenerative diseases. With LDMS, the number of trinucleotide repeats can be rapidly and reliably measured. The analysis time by LDMS per sample can be a few seconds per sample versus minutes to hours for conventional gel electrophoresis. With LDMS, no radioactive or dye tagging are required. Thus, the cost for analysis can be significantly lower. It has been found that trinucleotide expansion is associated with many other serious genetic diseases. Among those diseases are Spinobulbar muscular atrophy (Kennedy disease), spinocerebellar ataxias, Fragile X, myotonic dystrophy, and FRAZE. LDMS is expected to be able to detect any of the above diseases. Since the analysis time is shorter and the cost can be lower, LDMS has the potential for population screening for these diseases. In addition to the detection of dynamic mutation, the ORNL researchers also cooperated with staff from the FBI Laboratory the Academia Sinica in Taiwan to apply this technology for DNA fingerprinting for forensic applications. LDMS was successfully used for DNA typing of short tandem repeat (STR) for different loci from several human samples for person identification. These results indicate LDMS can become an important tool for DNA fingerprinting for forensic applications in the future.
Licensing Agreement with Graviton, Inc., for Research in Micromachined Sensors. Culminating progress in developing novel micromachined sensors, an agreement for a multiyear research effort has been reached between ORNL and Graviton, Inc. of San Diego, CA. Graviton's license of intellectual property will allow commercial development in a number of fields. The licensing agreement represents the largest commitment of funds by an outside firm for technology transfer. As a part of the agreement, funds in from Graviton will be used for research supporting advanced sensing techniques. The novel concepts for sensing by microcantilevers originated several years ago in a DOE-funded basic research program. A recent internally funded program involving Life Sciences, I&C, and CASD successfully demonstrated the world's first palm-sized, wireless, multiple-input sensor.
Life Sciences Division Part of Initiative for Superconducting Transformer. A Superconductivity Partnership Initiative was signed on September 1, 1998, by the DOE and Waukesha Electric Systems, Waukesha, Wisconson, that is a mega boost toward next-generation transformers that are vastly more efficient, reliable, and compact.
The goal of the three-year $6-million cooperative agreement is to design, build and test a prototype transformer rated at 5 mega-volt-ampere with a 10-mega-volt-ampere overload--or emergency-- capability. One megawatt will light 10,000 100-watt light bulbs. The initiative pools the resources of ORNL, Intermagnetics General, and Rochester Gas and Electric with those of transformer manufacturer Waukesha.
The 5/10-mega-volt-ampere superconducting transformer will be a scaled-down version of the final product, a 30-mega-volt-ampere commercial unit that will weigh half that of a conventional transformer. Furthermore, the superconducting transformer will not contain the thousands of gallons of cooling and insulating oil, a potential fire and environmental hazard. Superconducting transformers could be in wide use in about 20 years. The goal of the project is to fund cutting-edge research on difficult but important engineering problems.
Tasks include helping to develop high-voltage bushings that operate at cryogenic temperatures and conducting studies on electrical insulation materials, geometries, and sub-scale testing to verify the transformer design.