Biostatistics and Community Health Research Core (formerly Biostatistics and Epidemiology)

Chemical Biology Research Core

Nutrition Research Core

Reproductive and Developmental Biology Research Core

Analytical Services Facility Core (formerly Field Services)

Biostatistics and Computational Services Facility Core

Genomics and Bioinformatics Facility Core (formerly DNA Technologies)

Image Analysis Facility Core

Protein Technologies Facility Core

Transgenics Facility Core

Introduction: The Center for Environmental and Rural Health (CERH) provides a forum for promotion of outstanding basic and applied science programs focusing on the impact of environmental factors on human health and disease in rural communities. Research efforts of CERH investigators are supported by centralized core facilities that advance the scientific discovery process, enhance the quality of research programs, and attract young faculty into the field of environmental health sciences. The names of three cores were changed in 1999 to reflect the activities of CERH investigators.

Research Cores: The Biostatistics and Community Health (formerly Biostatistics and Epidemiology) core focuses on interrelated research projects to develop mathematical models to analyze complex data sets arising from environmental health studies, improve models for risk assessment, and investigate causes and methods of prevention of the most prevalent diseases in rural Texas. Investigators of the Chemical Biology core use chemical and biological approaches to address fundamental problems related to the pathogenesis of environmental diseases such as atherosclerosis, cancer, infections, neurologic disorders, and renal failure. The Nutrition core focuses on the study of nutritional and environmental interactions that influence atherosclerosis, cancer, and immune-mediated inflammatory diseases. The Reproductive and Developmental Biology core focuses on the adverse impacts of environmental agents on all aspects of reproduction and development including gametogenesis, conception, pregnancy and embryonic morphogenesis.

Facilities Cores: Six facility cores support the research programs of members, their students, postdoctoral fellows and staff. The Analytical Services core (formerly Field Services) assists investigators with the development and implementation of environmental sampling strategies and detection of DNA adducts and drug metabolites. The Biostatistics and Computational Services core provides extensive network and software support, data management and statistical support for research projects. The Genomics and Bioinformatics core (formerly DNA Technologies) provides support for analysis of global gene expression using DNA chip technology. The Image Analysis core provides a host of non-invasive imaging tools to probe the role of environmental factors on cellular homeostasis along with traditional digital microscopic imaging and camera-ready digital printing. The Protein Technologies core offers comprehensive approaches for analysis of peptides and proteins. The Transgenic core provides support in the generation and characterization of transgenic mice produced by pronuclear injection or homologous recombination.

Community Outreach and Education: Outreach activities focus on training and education in human health and the environment in rural Texas. In collaboration with community leaders and lay educators, efforts are directed at the implementation of community-based educational programs in "colonias" along the Texas-Mexico border. Grades K-12 and professional education programs are also being developed in collaboration with multiple units. Services for the local community include broadcast of a weekly TV segment in the Bryan/College Station viewing area, production of articles focusing on environmental health issues for bilingual education, and collaborations with established local community outreach programs.

Pilot Projects: The CERH provided financial support for several research projects consistent with the major scientific themes of the Center. Four projects were funded in 2000 to support collaborative research and enhance the success rate of grant applications submitted to secure external funding.

Title: A New Mechanism of Estrogen Action

Significance: Endogenous estrogenic hormones such as 17-beta-estradiol (E2) are required for multiple functions including development of the male and female reproductive tract, vascular development, bone growth and aspects of brain function. In addition, estrogens play an important role in development and growth of breast and endometrial cancer in women and research in the Safe laboratory focuses on understanding the mechanism of estrogen action in human breast and endometrial cancer cell lines. The classical mechanism of E2-induced gene expression involves initial binding to the estrogen receptor (ER), interaction of bound ER dimers to genomic estrogen response elements (EREs) in target gene promoters followed by upregulated gene expression. Results of recent studies in the Safe laboratory have shown that E2 can induce (or repress) multiple genes in breast and endometrial cancer cells through a new mechanism in which ER does not directly bind DNA but interacts with the transcription factor Sp1 (or other Sp-like proteins) to modulate gene expression. ER/Sp1-mediated transactivation has been identified as a major pathway regulating genes in breast cancer cells, and subsequent studies in other laboratories have demonstrated that many other receptors also act through binding the Sp1 protein. Drugs and gene therapy can now target this activation pathway as novel mechanism-based therapies for treatment of breast and endometrial cancer in women.

• Saville, B., Wormke, M., Wang, F., Nguyen, T., Enmark, E., Kuiper, G., Gustafsson, J.-A., and Safe, S. Ligand-, cell- and estrogen receptor subtype (alpha/beta)-dependent activation at GC-rich (Sp1) promoter elements. Journal of Biological Chemistry 275, 5379-5387, 2000.
• Stoner, M., Wang, F., Wormke, M., Nguyen, T., Samudio, I., Vyhlidal, C., Marme, D., Finkenzeller, G., and Safe, S. Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3 proteins. Journal of Biological Chemistry 275, 22769-22779, 2000.
• Xie, W., Duan, R., Chen, I., Samudio, I., and Safe, S. Transcriptional activation of thymidylate synthase by 17-beta-estradiol in MCF-7 human breast cancer cells. Endocrinology 141, 2439-2449, 2000.

Title: Novel Sensors of Oxidative Stress

Significance: Exposure to environmental chemicals is often associated with oxidative stress and activation/repression of signaling pathways involved in cellular homeostasis. Research in the Ramos laboratory on the molecular biology of oxidant signaling pathways recently showed that low levels of polycyclic aromatic hydrocarbon interfere with mammalian expression of c-Ha-ras, GSTA1 and L₁Md retrotransposon by disruption of functional interactions between aryl hydrocarbon receptor and proteins that bind antioxidant/electrophile response like-elements (ARE/EpRE) in the regulatory region of these genes. CCAAT/enhancer-binding protein (C/EBP)-beta, CREB binding protein, a serum albumin like-redox sensitive protein and members of the Zn-finger superfamily have been identified as novel ARE/EpRE interacting proteins. These proteins sense alterations in redox balance within the intracellular compartment and convey signals to the nucleus that disrupt normal gene transcription. In the case of L₁Md retrotransposon, recently published work is among the first to establish a molecular link between genetic and epigenetic events following induction of mutator phenotypes in response to oxidative injury. Altered expression of these genes may play a significant role in human diseases such as atherosclerosis, end-stage renal disease, and renal cancer.

• Chen, Y.H., and Ramos, K.S. A CCAAT/enhancer binding protein (C/EBP) site within antioxidant electrophile response element (ARE/EpRE) along with CREB binding protein (CBP) participate in the negative regulation of rat GST-Ya gene in vascular smooth muscle cells. Journal of Biological Chemistry 275, 27366-27376, 2000.

• Kerzee, J.K., and Ramos, K.S. Activation of c-Ha-ras by benzo(a)pyrene in vascular smooth muscle cells involves redox stress and aryl hydrocarbon receptor. Molecular Pharmacology 58, 152-158, 2000.

• Lu, K.P., Hallberg, L.M., Tomlinson, J., and Ramos, K.S. Benzo(a)pyrene activates L₁Md retrotransposon and inhibits DNA repair in vascular smooth muscle cells. Mutation Research 454, 35-44, 2000.

Description: The Administrative Core functions to facilitate research, service and outreach activities for CERH investigators and to ensure fiscal integrity of the Center. The core provides leadership in environmental health to the Texas A&M community and promotes expansion of outstanding environmental health research programs that address health concerns of citizens in rural communities of the State of Texas and beyond. The routine activities coordinated by the Administrative Core include: scheduling of Scientific Advisory Group and Facility Core meetings; coordination of biannual and annual meetings of the Internal and External Advisory Boards, respectively; coordination of the Visiting Speakers Program; development of the annual CERH thematic scientific conference; development of contacts and interactions with other EHS Centers and NIEHS staff; administrative and scientific support of collaborative CERH-sponsored grant proposals; coordination of pilot project program call for proposals, review of facility core operations, preparation of CERH annual report; and development of contacts with State and Federal elected officials and their staff to ensure that they are aware of the CERH and its potential services.

• Kenneth S. Ramos, Ph.D., Center Director, Professor, Departments of Veterinary Physiology and Pharmacology, Medical Physiology and Environmental and Occupational Health

• Stephen H. Safe, Ph.D., Deputy Director, Distinguished Professor, Departments of Veterinary Physiology & Pharmacology, Biochemistry and Biophysics and Environmental and Occupational Health

• Robert C. Burghardt, Ph.D., Associate Director, Professor, Department of Veterinary Anatomy and Public Health

• Jeannie Bowman, A.S., Administrative Assistant, Department of Veterinary Physiology and Pharmacology

• Kim Daniel, B.S., Staff Assistant, Department of Veterinary Physiology and Pharmacology

• Yvonne Kovar, Business Administrator, Department of Veterinary Physiology and Pharmacology

• Lorna Safe, B.S., Administrative Assistant, Department of Veterinary Physiology and Pharmacology

• Ruth Yeager, Staff Assistant, Department of Veterinary Physiology and Pharmacology

Internal Advisory Committee: The Internal Advisory committee consists of Deans from Colleges whose faculty are CERH investigators and the Vice-President for Research and Associate Provost for Graduate Studies. This Committee meets biannually to provide advice and guidance on scientific and administrative concerns and also serves as an advocate for the CERH within the Texas A&M University System.

Richard Ewing, Ph.D., Chair

Vice-President for Research and Associate Provost for Graduate Studies

Office of Graduate Studies & Research

Texas A&M University

College Station, Texas 77843-1112

H. Richard Adams, Ph.D.

Dean, College of Veterinary Medicine

Texas A&M University

College Station, Texas 77843-4461

Nancy Dickey, M.D.

Interim Dean of Medicine

College of Medicine

Texas A&M University System Health Science Center

College Station, Texas 77843-1114

Edward Hiler, Ph.D.

Vice Chancellor and Dean

College of Agricultural & Life Sciences

Texas A&M University

College Station, Texas 77843-2142

Joseph Newton, Ph.D.

Interim Dean, College of Science

Texas A&M University

College Station, Texas 77843-3257

Ciro V. Sumaya, M.D.

Dean, School of Rural Public Health

Texas A&M University System Health Science Center

College Station, Texas 77843-1256

 

External Advisory Committee: The External Advisory Committee provides critical input regarding CERH operations, thematic development, and evolution. The committee meets once a year usually during early spring and when possible, participates in the activities of the annual CERH meeting.

Daniel Acosta, Ph.D., Chair

Dean, College of Pharmacy

University of Cincinnati

3223 Eden Ave

Cincinnati, OH 45267-004

 

Steven D. Clarke, Ph.D.

M.M. Love Chair of Nutritional, Cellular, and Molecular Sciences

Department of Human Ecology – GEA-117/A2700

The University of Texas

Austin, TX 78712

 

Dennis M. Bier, Ph.D.

Director, Children’s Nutrition Research Center

Department of Pediatrics

Baylor College of Medicine

1100 Bates Street

Houston, TX 77030

 

Roger O. McClellan, Ph.D.

1111 Cuatro Cerros S.E.

Albuquerque, NM 87123

Edward R. McCabe, Ph.D.

Department of Pediatrics

UCLA - School of Medicine

10833 Le Conte Avenue, Room 22-412 MDCC

Los Angeles, CA 90095

 

The goal of the Biostatistics and Community Health Research Core is to develop new biostatistical methods related to environmental and rural health, and to perform community-based research studies to examine the relationship between risk factors and disease. The specific objectives of the members of the research core are:

• To develop biostatistical methods to help understand the origin of colon cancer, and how factors, such as apoptosis, are affected by chemicals and diet.
• To develop biostatistical methods to analyze correlated longitudinal and spatial data using subsampling methods.
• To link spatial and measurement error techniques to improve assessment of environmental exposures. Also, more generally, to develop new biostatistical methods for problems having missing and incorrectly measured data, including population-based pharmacokinetic modeling.
• To develop biostatistical methods for apportionment of the sources of environmental air pollutants and toxicants.
• To investigate the causes, mechanisms and methods of prevention of the most prevalent diseases in rural Texas and other rural areas.
• To study the delivery of health services to people with chronic diseases and chronic physical disabilities in rural communities, particularly as it relates to conditions involving a significant environemental component.
• To analyze data derived from complex multi-stage samples to evaluate health outcomes and health services used in rural communities.

• Raymond J. Carroll, Ph.D., Director, Distinguished Professor, Department of Statistics
• Robert J. Buchanan, Ph.D., Professor, Department of Health Policy and Management
• James A. Calvin, Ph.D., Professor and Head, Department of Statistics
• Kirby C. Donnelly, Ph.D., Professor, Departments of Environmental and Occupational Health and Veterinary Anatomy and Public Health
• Catherine Hawes, Ph.D., Professor, Department of Health Policy and Management
• Bani Mallick, Ph.D., Assistant Professor, Department of Statistics
• Marlynn L. May, Ph.D., Distinguished Lecturer, Center for Housing and Urban Development
• Charles D. Phillips, Ph.D., Professor, Department of Health Policy and Management
• Michael Sherman, Ph.D., Assistant Professor, Department of Statistics
• Naisyin Wang, Ph.D., Associate Professor, Department of Statistics
• Soujin Wang, Ph.D., Professor, Department of Statistics

• Chemometrics
• Genetic Epidemiology
• Longitudinal Data Analysis
• Measurement Error
• Missing Data
• Mixed Linear Models
• Non-linear Models
• Receptor Modeling
• Rural Health Research
• Spatial Modeling

Progress Report: Drs. Carroll, Chapkin, Lupton, Mallick, Turner and N. Wang (Biostatistics and Nutrition) have cooperated extensively on problems of nutrition and cancer. This relationship has expanded to six faculty, along with their graduate students working, as a team. Three Ph.D.'s, Christian Galindo (Statistics), Jeffrey Morris (Statistics) and Mee Young Hong (Nutrition), and multiple papers have resulted from this collaboration. Two biostatistics students are supported to help in the analysis of data arising from on-going experiments. Three NCI grants have become multidisciplinary and were awarded continued funding after competitive review as a result of Core-sponsored activities.

Drs. Carroll (P.I.), Lupton, Mallick and S. Wang are funded for development of new statistical methodology focusing on the hierarchical structure of the Chapkin-Lupton-Turner experiments. Drs. Carroll, Chapkin, Lupton (P.I.s), Turner and Wu are also funded for comparison of small and large intestinal epithelial cells to determine causes for the development of colon cancer, and Carroll, Chapkin, Lupton and Turner for understanding why n-3 fatty acids establish permissive conditions for butyrate-induced apoptosis.

More recently, Drs. Carroll, Chapkin, Lupton, Turner and N. Wang submitted a training grant proposal with the goal of training statistically oriented individuals to function as independent researchers in a multidisciplinary environment. To achieve this goal, a team specializing in Biostatistics, Statistics, Bioinformatics/Biomedical Imaging and the biology of nutrition and cancer was assembled. Through a combination of didactic coursework, seminars and research experiences, trainees will make important contributions in the development of statistical methods targeted to experiments in nutrition and cancer, functioning as true collaborators in teams of biologists, instead of merely as a specialist in setting sample sizes and performing data analysis of simple experiments. This grant received an outstanding priority score and official notification of funding has been received.

Drs. Mallick and Calvin of the Department of Statistics are members of the NIEHS-funded Superfund Basic Research Program (SBRP) grant "Procedures to Assess the Hazards of a Superfund Site." There are more than 31,000 uncontrolled hazardous waste sites in the United States, including 1,236 sites on the National Priorities List. After extensive clean up efforts, only 422 sites have been environmentally improved and less than 70 sites have been remediated. A major focus of the Texas A&M SBRP research has been on the integrated development of bioremediation techniques. From the basic science to the field setting, research projects have been designed to study these issues. Field experiments led by Drs. Autenrieth and Donnelly, both in Texas and in Azerbaijan, are to be used to develop models to describe environmental exposure to a variety of toxicants. Data from the Texas sites are being used to create models that will be further tested with data from Azerbaijan. These models will recognize both the spatial and measurement errors incumbent in such problems, and will also be used to aid the design of site selection in Azerbaijan. In another project, remediation techniques using thin film clays are being studied to improve adsorption properties and to better target specific toxic substances. The SBRP is a highly interdisciplinary venture involving multiple CERH members.

The Center for Housing and Urban Development (CHUD) is a recognized leader in community development and outreach, particularly in borderland settlements along the Texas-Mexico border called "colonias". Among the awards received are the Nationwide finalist in the "Innovations in American Government" award program sponsored by the Ford Foundation and administered by Harvard University’s John F. Kennedy School of Government in partnership with the Council for Excellence in Government (award winner pending), the President’s Council on Sustainable Development Award for being a "Texas Most Sustainable Community Development Project", the prestigious Hammer Award from the National Partnership for Reinventing Government, and the Secretary’s Honor Award for outstanding performance as part of the Partnership for Change Team in improving health, nutrition and housing conditions for residents of impoverished Texas colonias. The Corporation for National Service has placed fifty VISTA members working with CHUD’s Community Resource Centers in the El Paso, Laredo, and Lower Rio Grande Valley areas. Likewise, thirty full-time AmeriCorps members are on the CHUD staff in these three areas of the border. The strong interactions between CHUD, COEP, and CERH basic scientists have greatly enhanced the breadth of environmental health research and education efforts on the border.

Bordelon, N.R., Donnelly, K.C., King, L.C., Wolf, D.C., Reeves, W.R., and George, S.E. Bioavailability of the genotoxic compounds in coal tar contaminated soils in Fisher 344 rats. Toxicological Sciences 56, 37-48, 2000.

Cho, T.H., Wild, J.R., and Donnelly, K.C. Utility of organophosphorus hydrolase enzyme for the remediation of mutagenicity of methylparathion. Environmental Toxicology and Chemistry 19, 2022-2028, 2000.

Gail, M.H., Pfeifer, R., Van Houwelingen, H.C., and Carroll, R.J. On meta-analytic assessment of surrogate outcomes. Biostatistics 1, 231-246, 2000.

Garcia, S.S., and Donnelly, K.C. Genotoxicy analysis of contaminated environmental media. In: Environmental Analysis of Contaminated Sites: Toxicological Methods and Approaches, (Sunahara, G.I., Renoux, A.Y., Guadet, C.L., Thellen, C., and Pilon, A., eds), John Wiley & Sons, ltd., Sussex, England, 2000.

Lin, X., and Carroll, R.J. Nonparametric function estimation for clustered data when the predictor is measured without/with error. Journal of the American Statistical Association 95, 520-534, 2000.

Ruckstuhl, A., Welsh, A.H., and Carroll, R.J. Nonparametric function estimation of the relationship between two repeatedly measured variables. Statistica Sinica 10, 51-72, 2000.

Spiegelman, C.H., Bennett, J.F., Vannucci, M.J., and Cote, G.L. A transparent tool for seemingly difficult calibrations: the parallel calibration method. Analytical Chemistry 72, 135-140, 2000.

Wang, C.Y., Wang, N., and Wang, S. Regression analysis when covariates are regression parameters of a random effect model. Biometrika 56, 487-496, 2000.

The goal of the Chemical Biology Research Core is to understand signaling pathways important in cellular homeostasis and disease, and to determine the modulatory effects of environmental toxicants and dietary factors on these processes. The core also focuses on molecular aspects of toxicology and environmental biology, as it relates to the development of novel molecular detection systems, remediation and detoxification, protein characterization, and protein engineering. Members of this Core promote scientific collaborations within this group, with investigators in other CERH Research Cores, and with other collaborators on-campus and in other university/research laboratories. Drs. Lori Bernstein, Arthur Johnson and Terry Thomas are new members of the Core and the CERH.

The specific objectives of the research core are:

• To determine the mechanisms associated with tissue-specific oxidative stress injury and altered gene expression associated with these processes.
• To investigate modulation of kinase dependent pathways and downstream effects including their role in tumor development.
• To determine molecular mechanisms of ligand activated receptors and their differential interactions with endogenous compounds and environmental contaminants.
• To investigate tissue specific modulation of gene expression by environmental contaminants and dietary factors.
• To define molecular mechanisms of action of several environmental chemicals including aromatic hydrocarbons, pesticides, environmental estrogens/antiestrogens, lead, and mercury.
• To develop biologically based molecular detection systems for analytes of interest in environmental biology.
• To develop molecular technologies for environmental decontamination and protection.

• Stephen H. Safe, D. Sc., Director, Distinguished Professor, Departments of Veterinary Physiology and Pharmacology, Biochemistry and Biophysics and Environmental and Occupational Health
• Hagan Bayley, Ph.D., Professor and Head, Department of Medical Biochemistry and Genetics
• Lori Bernstein, Ph.D., Assistant Professor, Department of Medical Pathology
• David Busbee, Ph.D., Professor, Department of Veterinary Anatomy and Public Health
• Arthur Johnson, Ph.D., Professor, Department of Medical Biochemistry and Genetics
• Ann Kier, D.V.M., Professor and Head, Department of Veterinary Pathobiology
• Timothy Phillips, Ph.D., Professor, Department of Veterinary Anatomy and Public Health
• Kenneth S. Ramos, Ph.D., Professor, Departments of Veterinary Physiology and Pharmacology, Medical Physiology and Environmental and Occupational Health
• James Sacchettini, Ph.D., Professor, Department of Biochemistry and Biophysics
• Terry Thomas, Ph.D., Professor and Head, Department of Biology
• Evelyn Tiffany-Castiglioni, Ph.D., Professor and Head, Department of Veterinary Anatomy and Public Health
• James Wild, Ph.D., Professor and Head, Department of Biochemistry and Biophysics
• Emily Wilson, Ph.D., Assistant Professor, Department of Medical Physiology

• Aging
• Aryl Hydrocarbon Receptor
• Biosensors
• Dioxin
• Endocrine Disruption
• Gene Regulation
• Molecular Detoxification
• Nitroaromatics
• Polycyclic Aromatic Hydrocarbons
• Pesticides

Progress Report: The Texas A&M University Superfund Basic Research Program (SBRP) has been funded since 1989. Research over this period focused on development and validation of bioassays for hazard and risk assessment of contaminants associated with oil and wood-preserving waste sites including polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and dibenzofurans, chlorinated phenolics and other endocrine-disrupting environmental contaminants. Planning for the SBRP grant renewal application was lead by CERH investigators and greatly benefited from collaborative interactions directly linking various CERH cores and facilities. The renewal grant application submitted in 1999 focused, in part, on developing methods for more effective hazard and risk assessment of complex mixtures. However, individual research projects and facility cores were completely revised to include basic molecular toxicology research, exposure assessment and modeling, site remediation, population studies and outreach; most of these initiatives involved CERH investigators.

Dr. Steve Safe continues to be the PI for the SBRP and also serves as PI on a project focused on endocrine-active environmental contaminants and other dietary phytoestrogens. It is hypothesized that different structural classes of xenoestrogens and naturally occurring estrogenic compounds exhibit "unique" estrogenic activities and unique biology that cannot be determined using traditional bioassays. The major aims of this project are development of bioassays to characterize estrogenic compounds and their action on ER alpha and ER beta, and investigation of ER-subtype specific gene expression. Results of initial studies show that four metabolites of methoxychlor uniquely interact with three receptors and exhibit ER alpha agonist, ER beta antagonist and androgen receptor antagonist activities. These studies will give new mechanism-based data on xenoestrogen-mediated ER action and provide a more rational basis for risk/hazard assessment of these compounds.

Two members of the Chemical Biology Research Core (Drs. Ramos and Tiffany-Castiglioni) collaborated in development of a new project to study regulation of glucose-regulated protein 78 (grp78) in renal and brain cells. The hypothesis being tested is that grp78 is regulated transcriptionally following lead and mercury induced alterations in signal transduction, and that this response affords cytoprotection against subsequent chemical injury. Treatment regimens used in their studies model toxicologically relevant exposures in humans by use of repeated dosing of cells with lead acetate and HgCl₂ at sub-cytotoxic concentrations over a period of days.

Dr. Phillips has long been interested in the development of innovative chemical strategies for the detection and detoxification of environmental chemicals. In particular, the laboratory has employed molecular modeling techniques to design various phyllosilicate clays for detoxification of aflatoxins and halogenated aromatic hydrocarbons. The findings are of direct relevance to rural communities where incidence of this type of exposure is often elevated. One aim of the laboratory is to understand surface chemistry and mechanisms involved in the interactions of these poisons with phyllosilicate clay minerals. For example, it has been shown that a processed phyllosilicate clay can prevent the adverse effects of aflatoxins in young animals when included in their diets at a level as low as 0.5% (w/w). Further work has indicated that this clay mineral acts to selectively and tightly adsorb ligands containing a ketolactone, or bislactone functionality (e.g., aflatoxins B and G series). A proposed mechanism of aflatoxin binding by phyllosilicate clay is the formation of symmetrical, resonance-stabilized chelate by the dicarbonyl system of the aflatoxins with certain metal ions in the clay. Moreover, this reaction occurs in the gastrointestinal tract of animals, resulting in a notable reduction in the bioavailability of aflatoxin to the blood and target organs. Similar approaches are being used to develop and investigate: (1) novel porous solids for the sorption of diverse toxins and microbes; (2) multicomponent adsorption, separation and water filtration systems; and (3) phyllosilicate clay-based bioremediation and diagnostic strategies. Sophisticated chemical techniques for the spectral elucidation of molecular structure (including capillary GC/ quadrupole mass spectrometry, diffuse reflectance infrared FT spectroscopy and UV-visible and fluorescence spectrophotometry) are routinely used to probe and delineate the location of various ligand binding sites on phyllosilicate clay particles, the type of binding, and chemical reaction products. A wide range of tools is used for analytical separations, and isothermal and surface analysis. Prototypic models of relevant processes are optimized using molecular visualization and simulation. Computational analysis is used to investigate surface-ligand interactions in these models.

Dr. Kier’s laboratory successfully transfected transformed fibroblast cells with the cDNA encoding the various lipid transfer proteins to obtain overexpression of fatty acid, fatty acyl CoA, and sterol carrier proteins. Immuno-colocalization and confocal microscopy identified intracellular organelles where these proteins are targeted and in collaboration with the Schroeder laboratory (Nutrition Research Core) she overexpressed sterol carrier proteins in transgenic mice. By applying confocal and multiphoton imaging technologies, the first images of a naturally occurring fluorescent sterol in living cells were generated. Additional imaging studies established that sterol carrier protein expression dramatically inhibits HDL mediated reverse cholesterol transport in living cells. Transfected cells are now being utilized to examine the effects of environmental toxicants that might interact with these proteins to alter normal lipid metabolism and/or expression resulting in cellular toxicity.

A toxicogenomics initiative has been launched at Texas A&M involving interactions between CERH and several system-wide components. Drs. Terry Thomas and Kenneth S. Ramos provide leadership for this initiative. High throughput gene expression analysis, or transcriptional profiling, has emerged as a critical tool for the utilization of genome sequence information. This is done using DNA microarrays, or gene chips. In the case of DNA microarrays, robots are used to place DNA representing thousands of individual genes in a precise order or array on glass microscope slides, hence the term DNA microarray. Each gene has a specific address on the microarray, and its expression as messenger RNA (mRNA) can be queried using fluorescently labeled hybridization probes. This allows comparisons of mRNA populations from two different developmental or growth states, e.g. normal and disease state. Using powerful computer programs, transcription profiles of all genes on the microarray can be developed. This profile is a signature of a developmental or growth state. This information can then be used to design more comprehensive experiments to determine gene function or define previously unknown gene regulatory networks. DNA microarray experiments generate large amounts of data that must be stored, processed and analyzed, and displayed. This requires diverse computational capabilities from image processing and validation to cluster analysis to database construction. As part of institutional initiatives and the NIEHS Toxicogenomics Consortium grant application currently in preparation by several members of this core (Dougherty, Ramos, Safe and Thomas), the CERH is developing the computational infrastructure necessary for DNA microarray analysis. Dr. Dougherty, Director of the Computer Assisted Medical Diagnostic Imaging Laboratory (CAMDI) and one of the developers of ArraySuite- microarray analysis software developed at the National Human Genome Research Institute (NHGRI), has facilitated application of the multiple data analysis strategies developed at NHGRI to microarray data generated at Texas A&M University.

A potentially revolutionary technique for analyte detection, known as stochastic sensing, is under development in Dr. Bayley’s laboratory. Sensor elements are made using engineering transmembrane protein pores. Analyte molecules modulate the ionic current driven through the engineered pores by a transmembrane potential. Stochastic sensing, which uses currents from single pores, is an especially attractive prospect. This approach yields both the concentration and identity of an analyte, the latter from its distinctive current signature. Further, several analytes can be detected simultaneously with a single sensor element. In one example of stochastic sensing, the bacterial pore-forming protein staphylococcal alpha-hemolysin has been altered to permit the detection of divalent metal cations by using mutagenesis to place a cation-binding site within the conductive pathway. In a second example, the hemolysin pore has been modified with cyclodextrins, which act as non-covalent molecular adapters, to allow the detection of a variety of small organic molecules. Finally, functionalized polymers have been anchored within the hemolysin pores to construct sensor elements for the detection of biological macromolecules. This work, published in Nature in 1999, has generated considerable interest among the chemical analysis community. Dr. Bayley’s research is also focused on the design and synthesis of biomolecular materials, a rapidly growing interdisciplinary area in which the properties of molecules found in nature are mimicked or extended to produce materials with unusual properties. An additional goal is to manufacture and dispose of materials by environmentally benign methods of low energy cost. The heterologous expression of protein-based materials is an attractive option being explored, as well as related approaches for making materials that form porous sheets, fibers, adhesives and elastomers. For example, this laboratory has initiated engineering studies on S layers, the robust porous proteinaceous envelopes that surround many bacterial cells. One goal is to obtain porous monolayers containing molecular switches for use in biosensors. In another example, cDNAs encoding abductin, a protein found in the highly elastomeric inner hinge ligaments of bivalve mollusks have been sequenced. Long-term goals include the engineering of elastomers with novel properties, based on these sequences and on chimeras with other proteins, for films, coatings and energy storage devices.

In the Johnson laboratory, it was recently discovered that the protein-conducting channel in the endoplasmic reticulum (ER) membrane (the aqueous translocon pore) is the largest (40-60 Å in diameter) discovered to date in a membrane that must maintain a permeability barrier. The pore is sealed by the ribosome during translocation to maintain the permeability barrier of the ER membrane. The Johnson laboratory discovered that when the ribosome is released at the end of translation, the pore contracts to a diameter of only 9-15 Å, and the ribosome-free pore is then sealed, directly or indirectly, at its lumenal end by BiP, a soluble lumenal protein previously thought to function solely as a chaperone. In mammals, both secretory proteins and membrane proteins are synthesized by ribosomes bound to translocons. The molecular machinery must therefore distinguish between a nascent secretory protein that passes through the membrane and a nascent transmembrane protein that is inserted into the bilayer. The shift in translocon function from translocation to integration is triggered by the appearance of a transmembrane (TM) sequence in a nascent membrane protein, and it was discovered that this functional transition occurs while the TM sequence is still inside the ribosome. Thus, the ribosome, not the translocon, first recognizes the TM sequence. The ribosome initiates a highly coordinated sequence of structural changes at the translocon that elicit the conversion from translocation to integration without compromising the membrane’s permeability barrier. These changes include the breaking of the ribosomal seal at the translocon to allow the cytoplasmic domain of a nascent membrane protein to enter the cytosol, but this occurs only after the other (lumenal) end of the pore is closed. It was also shown that when a TM sequence of a nascent membrane protein is inserted into the translocon, it is not immediately released into the hydrophobic interior of the phospholipid bilayer as is generally believed. Instead, a TM sequence binds to a translocon protein(s) after entering the translocon, and remains bound until translation terminates or another TM sequence enters the translocon. The technologies developed to perform this work are now being applied in several collaborations with members of the CERH.

Dr. Sacchettini’s laboratory studies interactions between proteins and their ligands or substrates. Several techniques are used for examination of the molecular details of these types of interactions including x-ray crystallography, microcalorimetry and molecular biology. With funding from the pilot project program, the Sacchettini laboratory has also been developing human transthyretin (TTR) amyloid disease inhibitors. Human amyloid disorders, including familial amyloid polyneuropathy, familial amyloid cardiomyopathy and senile systemic amyloidosis are linked to deposition of insoluble TTR in peripheral nerves and heart tissues. Hydroxy-PCBs bind TTR with high affinity. In collaboration with Dr. Safe, new TTR ligands based on a hydroxybipheny backbone are currently being investigated and x-ray crystallographic analysis and binding assays show that some congeners exhibit high affinity for TTR.

Dr. Wild's research group focuses on the molecular characterization and manipulation of specific enzymes involved in the detoxification of pesticides and organophosphate nerve gases. Studies on the mechanism of detoxification of organophosphate neurotoxins primarily involve the genetic and biochemical analysis of the plasmid-borne organophosphorus-degrading gene of Pseudomonas diminuta and its unique broad-spectrum organophosphate hydrolase. The bacterial gene encodes a single organophosphorus hydrolase (OP anhydrase, EC 3.8.1), which is capable of stereospecific hydrolysis of organophosphorus neurotoxins ranging from insecticides such as parathion and diazinon to mammalian neurotoxins such as diisopropylfluorophosphate (DFP) and sarin (a chemical warfare neurotoxin). The organophosphorus degrading genes (opd) from two different plasmids in the soil bacteria P. diminuta and Flavobacterium have been sequenced and their structural organizations are being characterized. The cloned genes have been expressed in a number of biological systems from bacteria to insect tissue culture to native soil fungi, and the enzyme has been purified and characterized from several different sources. Site-directed mutagenesis has been utilized to define the nature of amino-terminal signal sequence and the bimetallic binding site(s). The derived understanding of the function of the various histidine and cysteine residues of the enzyme have been used to design regional mutagenic strategies with randomized oligonucleotides to further define the active site and manipulate the substrate specificities of the enzyme. Applications of this system are currently being developed and already have led to the development of an enzyme-based biosensor that is capable of the direct and discriminatory detection of various OP neurotoxins. In addition, the gene has been productively inserted into plants and soil fungi for the purpose of developing an in situ decontamination system for environmental bioremediation. Detoxification with the hydrolase received special attention in 1998 through the publication of a paper in Nature describing the incorporation of the enzyme into foams that are readily applied using firefighting equipment.

Alejandro, N. F., Parrish, A. R., Bowes, R.C. III, Burghardt, R.C., and Ramos, K.S. Phenotypic profiles of cultured glomerular cells following repeated cycles of hydrocarbon injury. Kidney International 57, 1571-1580, 2000.

Bielec, P., Gallagher, D., Yang, Y.P., Womack, J., Davis, S., Taylor, J., and Busbee, D. Assignment of crystalline beta-polypeptide 1 (CRYBA1) to Atlantic bottlenose dolphin chromosome band 16p11 by in situ hybridization. Cytogenetics and Cell Genetics 89, 96-97, 2000.

Boronin, A.M., Ermakova, I.T., Sakharovsky, V.G., Grechkina, G.M., Starovoitov, I.I., Autenreith, R.L., and Wild, J.R. Ecologically safe destruction of mustard-lewisite mixtures from the Russian chemical stockpile. Journal of Chemical Technology and Biotechnology 75, 1-7, 2000.

Chak, R.K.F., Thomas, T.L., Quatrano, R.S., and Rock, C.D. The genes AB11 and AB12 are involved in abscisic acid- and drought-inducible expression of the Daucus carota L. Dc3 promoter in guard cells of transgenic Arabidopsis thaliana (L.) Heynh. Planta 210, 875-883 2000.

Chen, Y.H., and Ramos, K.S. A CCAAT/enhancer binding protein (C/EBP) site within antioxidant electrophile response element (ARE/EpRE) along with CREB binding protein (CBP) participate in the negative regulation of rat GST-Ya gene in vascular smooth muscle cells. Journal of Biological Chemistry 275, 27366-27376, 2000.

Davis, A.J., Sepuri, N.B., Holder, J., Johnson, A.E., and Jensen, R.E. Two intermembrane space TIM complexes interface with different domains of Tim 23p during its import into mitochondria. Journal of Cell Biology 150, 1271-1282, 2000.

Gadda, G., Banerjee, A., Dangott, L., and Fitzpatrick, P.F. Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide. Journal of Biological Chemistry 275, 31891-31895, 2000.

Gaido, K.W., Maness, S.C., McDonnell, D.P., Dehal, S.S., Kupfer, D., and Safe.S. Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta, and androgen receptor: Structure-activity studies. Molecular Pharmacology 58, 852-858, 2000.

Green, L.D., Derr, J.N., and Knight, A. Mitochondrial DNA affinities of the peoples of North-Central Mexico. The American Journal of Human Genetics 66, 989-98, 2000.

Hackeng, T.M., Yegneswaran, S., Johnson, A.E., and Griffin, J.H. Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S. Biochemistry 349, 757-764, 2000.

Herrera, P., Burghardt, R.C., and Phillips, T.D. Adsorption of Salmonella enteritidis by cetyl pyridinium exchanged montmorillonite clays. Veterinary Microbiology 74, 259-272, 2000.

Heuck, A.P., Hotze, E.M., Tweten, R.K., and Johnson, A.E. Mechanism of membrane insertion of a multimeric beta-barrel protein: perfringolysin O creates a pore using ordered and coupled conformational changes. Molecular Cell Biology 6, 1233-1242, 2000.

Holderman, M.T., Miller, K.P., and Ramos, K.S. Activation of nuclear protein binding to the antioxidant/electrophile response element in vascular smooth muscle cells by benzo(a)pyrene. Biochemical and Biophysical Research Communications 267, 12-16 2000.

Huebner, H.J., Mayura, K., Pallaroni, L., Ake, C.L., Lemke, L., Herrera, P., and Phillips, T.D. Development and characterization of a carbon-based composite material for reducing patulin levels in apple juice. Journal of Food Protection 63, 106-110, 2000.

Johnson, A.E., and Haigh, N.G. The ER translocon and retrotranslocation: is the shift into reverse manual or automatic? Cell 102, 709-712, 2000.

Kerzee, J.K., and Ramos, K.S. Activation of c-Ha-ras by benzo(a)pyrene in vascular smooth muscle cells involves redox stress and aryl hydrocarbon receptor. Molecular Pharmacology 58, 152-158, 2000.

Koomen, J.M., and Russell, D.H. UV-MALDI MS Characterization of 2,5-dihydroxybenzoic acid induced changes in the isotope ratios of oligonucleotides. Journal of the American Society of Mass Spectrometry 35, 1025-34, 2000.

Koomen, J.M., Russell, W.K., Hettick, J.M., and Russell, D.H. Improvement of resolution, mass accuracy, and reproducibility in reflected mode DE-MALDI-TOF analysis of DNA using fast evaporation-overlayer sample preparations. Analytical Chemistry 72, 3860-6, 2000.

Kumar, N.V., and Bernstein, L.R. Rapid detection of zymomonas mobilis redox activity using 5-cyano-2,3-tolyl-tetra-zolium chloride (CTC). BioTechniques 29, 418-424, 2000.

Lee, J-E., and Safe, S. 3΄4΄-dimethoxyflavone as an aryl hydrocarbon receptor antagonist in human breast cancer cells. Toxicological Sciences 58, 235-242, 2000.

Lu, K.P., Hallberg, L.M., Tomlinson, J., and Ramos, K.S. Benzo(a)pyrene activates L1Md retrotransposon and inhibits DNA repair in vascular smooth muscle cells. Mutation Research 454, 35-44, 2000.

McCallum, C.D., Do, H., Johnson, A.E., and Frydman, J. The interaction of the chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TriC) with ribosome-bound nascent chains examined using photo-cross-linking. Journal of Cell Biology 149, 591-601, 2000.

McDougal, A., Gupta, M.S., Ramamoorthy, K., Sun, G., and Safe, S.H. Inhibition of carcinogen-induced rat mammary tumor growth and other estrogen-dependent responses by symmetrical dihalo-substituted analogs of diindolylmethane. Cancer Letters 151, 169-179, 2000.

Miller, K.P., Chen, Y-H, Hastings, V., Bral, C.M., and Ramos, K.S. Profiles of antioxidant/electrophile response element (ARE/EpRE) nuclear protein binding and c-Ha-ras transactivation in vascular smooth muscle cells treated with oxidative metabolites of benzo(a)pyrene. Biochemical Pharmacology 60, 1285-1296, 2000.

Nuccio, M.L., Hsieh, T-F., and Thomas, T.L. The use of RT-PCR differential display in single-celled organisms and plant tissues. In: Differential Display: A Practical Approach (Leslie, R. and Robertson, H., eds), Oxford University Press, Oxford, 83-99, 2000.

Peluso, P., Herschlag, D., Nock, S., Freymann, D.M., Johnson, A.E., and Walter, P. Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor. Science 288, 1640-1643, 2000.

Nathan, I., Dizdaroglu, M., Bernstein, L., Junker, U., Lee, C-K., Muegge, K., and Durum, S. Induction of oxidative DNA damage in U937 cells by TNF or Anti-FAS stimulation. Cytokine 12, 881-997, 2000.

Qian, Y., Harris, E.D., Zheng, Y., and Tiffany-Castiglioni, E. Lead targets GRP78, a molecular chaperone, in C6 rat glioma cells. Toxicology and Applied Pharmacology 163, 260-266, 2000.

Safe, S. Endocrine disruptors. New toxic menace? In: Earth Report: Revisiting the True State of the Planet (Bailey, R., ed), McGraw Hill, New York, 189-202, 2000.

Safe, S. Endocrine disruptors and human health – Is there a problem? An update. Environmental Health Perspectives 108, 487-493, 2000.

Safe, S. Reproductive effects: endocrine disruption. In: Risk Assessment in the Food Chain of Children (Aggett, P.J. and Kuiper, H.A., eds) Nestec Ltd., Vevey/Lippincott Williams & Wilkins, Philadelphia, PA, 35-46, 2000.

Safe, S. Xenoestrogens and human health. Is there a problem? The argument against. Progress in Environmental Science 2, 21-30, 2000.

Safe, S., Wormke, M., and Samudio, I. Mechanisms of inhibitory aryl hydrocarbon receptor-estrogen receptor crosstalk in human breast cancer cells. Journal of Mammary Gland Biology and Neoplasia 5, 295-306, 2000.

Saville, B., Wormke, M., Wang, F., Nguyen, T., Enmark, E., Kuiper, G., Gustafsson, J-Å., and Safe, S. Ligand-, cell-, and estrogen receptor subtype (alpha/beta)-dependent activation at GC-rich (Sp1) promoter elements. Journal of Biological Chemistry 275, 5379-5387, 2000.

Shepard, L.A., Shatursky, O., Johnson, A.E., and Tweten, R.K. The mechanism of pore assembly for a cholesterol-dependent cytolysin: Formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins. Biochemistry 39, 10284-10293, 2000.

Stoner, M., Wang, F., Wormke, M., Nguyen, T., Samudio, I., Vyhlidal, C., Marme, D., Finkenzeller, G., and Safe, S. Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3 proteins. Journal of Biological Chemistry 275, 22769-22779, 2000.

Vyhlidal, C., Samudio, I., Kladde, M.P., and Safe, S. Transcriptional activation of transforming growth factor alpha by estradiol: Requirement for both a GC-rich site and an estrogen response element half-site. Journal of Molecular Endocrinology 24, 329-338, 2000.

Wang, F., Duan, R., Chirgwin, J., and Safe, S.H. Transcriptional activation of cathepsin D gene expression by growth factors. Journal of Molecular Endocrinology 24, 193-202, 2000.

Wormke, M., Castro-Rivera, E., Chen, I., and Safe, S. Estrogen and aryl hydrocarbon receptor expression and crosstalk in human Ishikawa endometrial cancer cells. Journal of Steroid Biochemistry and Molecular Biology 72, 197-207, 2000.

Xie, W., Duan, R., Chen, I., Samudio, I., and Safe, S. Transcriptional activation of thymidylate synthase by 17-beta estradiol in MCF-7 human breast cancer cells. Endocrinology 141, 2439-2449, 2000.

Yoon, K., Pellaroni, L., Ramamoorthy, K., Gaido, K., and Safe, S. Ligand structure-dependent differences in activation of estrogen receptor alpha in human HepG2 liver and U2 osteogenic cancer cell lines. Molecular and Cellular Endocrinology 162, 211-220, 2000.

The primary goal of the Nutrition Research Core is to promote active collaboration among members of the Nutrition core and the other Center cores, primarily focusing on gene-environment interactions. The individual scientists in the Nutrition Research Core serve as the interface between nutritional problems observed in the field and the molecular mechanisms behind those specific problems. Of particular interest is the role of dietary factors in outcomes associated with environmental exposures to toxic chemicals, especially as it relates to colon cancer and cardiovascular disease.

The specific research aims of the core include:

• Identify diet components that reduce or delay onset of chemically induced and "spontaneous" colon carcinogenesis, and thus determine potential diet interventions that ameliorate the impacts of environmentally derived insults (Chapkin, Lupton, Turner, Wu).
• Evaluate non-invasive biomarkers of colon cancer as a means of monitoring food born anticarcinogens (Chapkin, Lupton).
• Determine how over- and under-consumption of dietary minerals (e.g. copper and iron) may exacerbate the response to environmental toxins (Harris).
• Evaluate how previous nutritional status affects the response to environmental challenges, and if the response can be modulated by changes in diet (Chapkin, Harris, McMurray).
• Elucidate the cellular and molecular mechanisms by which diet modulates immune-mediated inflammatory disease, and alters host resistance to infectious pathogens (Chapkin, McMurray).
• Identify diet components that reduce atherosclerotic cardiovascular disease (Chapkin, Schroeder, Walzem).
• Determine dietary influences on fetal small intestinal development and enzyme expression (Wu).

• Joanne R. Lupton, Ph.D., Director, Regent's Professor, Department of Animal Science
• Robert S. Chapkin, Ph.D., Associate Professor, Department of Animal Science
• Edward D. Harris, Ph.D., Professor, Department of Biochemistry and Biophysics
• David N. McMurray, Ph.D., Professor, Department of Medical Microbiology and Immunology
• Friedhelm Schroeder, Ph.D., Professor, Department of Veterinary Physiology and Pharmacology
• Guoyao Wu, Associate Professor, Department of Animal Science

• Atherosclerosis
• Biomarkers
• Colon cancer
• Environmental carcinogens or mutagens
• Immune responsiveness
• Minerals
• Phytochemicals

Progress Report: The collaboration between members of the Nutrition Core (Drs. Lupton, Chapkin, Turner, Walzem and Wu) and members of the Biostatistics and Community Health Research Core (Drs. Carroll, Mallick, and Wang) has resulted in several key publications in the area of colon cancer and submission of papers in statistical theory and applications, one new NIH grant, one new National Space Biomedical Research Institute grant, and the continuation of two NIH grants. A training grant proposal was submitted by members of the Nutrition Core (Lupton, Chapkin, Turner, Walzem, and Wu) in collaboration with Dr. Carroll (Biostatistics and Community Health Research Core) to train biostatisticians who are knowledgeable about carcinogenesis and the relevance of diet in disease occurrence/prevention. This grant was recently funded by the NIH as one of the first of its kind in the nation. Collaborations between Drs. Chapkin and McMurray also resulted in a new NIH grant.

Dr. Harris has been engaged in research aimed at delineating the molecular defect in Menkes disease. He has cloned and sequenced the protein ATP7A, a P-type ATPase that is specifically involved in transmembrane copper movement and is known to be defective in human infants expressing Menkes disease. With this gene, he hopes to correct the molecular defect in the human cell model and the murine model of the disease. He also hopes to determine if a controlled deletion can duplicate the symptoms of Menkes disease. A fourth objective is to determine if overexpression of Menkes gene is deleterious to copper homeostasis in cells. Finally, success with the Menkes gene will encourage an attempt at a similar manipulation with the gene for Wilson’s disease. This gene is also a key factor controlling intracellular copper homeostasis.

Dr. McMurray’s work with a guinea pig model of pulmonary tuberculosis has revealed that chronic, moderate protein deficiency is associated with profound impairment of T lymphocyte functions that render the malnourished animals less responsive to vaccination and less able to control pulmonary infection with virulent tubercle bacilli. Protein-deprived guinea pigs fail to expand antigen-reactive T lymphocyte clones due, in part, to blunted IL-2 production, and fail to activate their macrophages appropriately. This latter effect is due to the apparent failure of malnourished animals to make macrophage-activating cytokines (e.g., IFN-gamma, TNF-alpha) and/or to the inappropriate production of macrophage deactivating cytokines, such as TGF-beta. These observations have great significance for the role of dietary protein in enhancing vaccine efficacy and reducing the incidence of tuberculosis in malnourished human populations, such as those along the Texas-Mexico border.

Dr. Schroeder’s laboratory has successfully transfected transformed fibroblast cells with the cDNA encoding the various lipid transfer proteins to obtain overexpression of fatty acid, fatty acyl CoA, and sterol carrier proteins. Immunocolocalization and confocal microscopy identified the intracellular organelles where these proteins are targeted. In collaboration with Dr. A. Kier (Chemical Biology Core), he overexpressed sterol carrier proteins in transgenic mice. By applying confocal and multiphoton imaging technologies in their laboratories, the first images of a naturally occurring fluorescent sterol in living cells have been obtained. Furthermore, sterol carrier protein expression dramatically inhibited HDL mediated reverse cholesterol transport in living cells. The transfected cells can now be utilized to examine the effects of environmental toxicants that might interact with these proteins and thereby alter the normal lipid metabolism and/or expression of toxicity.

Atshaves, B.P., Starodub, O., Roths, J.B., McIntosh, A., Petrescu, A., Kier, A.B., and Schroeder, F. Sterol carrier protein-2 alters high density lipoprotein mediated cholesterol efflux. Journal of Biological Chemistry 275, 36852-36861, 2000.

Bell, J., Donovan, J.L., Wong, R., Waterhouse, A.L., German, J.B., Walzem, R.L., and Kasim-Karakas, S.E. (+)-Cathechin in human plasma after ingestion of a single serving of reconstituted red wine. American Journal of Clinical Nutrition 71, 103-8, 2000.

Chapkin, R.S., Fan, Y.Y., and Lupton, J.R. Effect of diet on colonic programmed cell death: molecular mechanism of action. Toxicology Letters 112-113, 411-414, 2000.

Chapkin, R.S., McMurray, D.N., and Jolly, C.A. Dietary n-3 polyunsaturated fatty acids modulate T-lymphocyte activation: Clinical relevance in treating diseases of chronic inflammation. Nutrition and Immunology: Principles and Practice 121-134, 2000.

Collett, E.D., Davidson, L.A., Lupton, J.R., and Chapkin, R.S. Dietary fish oil reduces colon cancer risk. Current Organic Chemistry 4, 1157-1168, 2000.

Davidson, L.A., Brown, R.E., Chang, W-C.L., Lupton, J.R., Morris, J.S., Wang, N., Carroll, R.J., Turner, N.D., and Chapkin, R.S. Morphodensitometric analysis of protein kinase C beta II expression in the rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis. Carcinogenesis 21, 1513-1519, 2000.

Flynn, N.E, Knabe, D.A, Mallick, B.K., and Wu, G. Postnatal changes of plasma amino acids in suckling pigs. Journal of Animal Science 78, 2369-2375, 2000.

Frolov, A., Petrescu, A., Atshaves, B.P., So, P.T.C., Gratton, E., Serrero, G., and Schroeder, F. High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. Journal of Biological Chemistry 275, 12769-12780, 2000.

Gallegos, A.M., Schoer, J.K., Starodub, O., Kier, A.B., Billheimer, J.T., and Schroeder, F. A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria. Chemistry and Physics of Lipids 105, 9-29, 2000.

German, J.B., and Walzem, R.L. The Health Benefits of Wine. Wine and Health 20, 461-593, 2000.

Harris, E.D. Cellular copper transport and metabolism. Annual Review of Nutrition 20, 291-310, 2000.

Hong, M.Y., Lupton, J.R., Morris, J.S., Wang, N., Carroll, R.J., Davidson, L.A., Elder, R.H., and Chapkin, R.S. Dietary fish oil reduces O⁶-methylguanine DNA adduct levels in the rat colon in part by increasing apoptosis during tumor initiation. Cancer Epidemiology, Biomarkers and Prevention 9, 819-826, 2000.

Jolly, C.A., Chao, H., Kier, A.B., Billheimer, J.T., and Schroeder, F. Sterol carrier protein-2 suppresses microsomal acyl-CoA hydrolysis. Molecular and Cellular Biochemistry 205, 83-90, 2000.

Jolly, C.A., Wilton, D.A., and Schroeder, F. Microsomal fatty acyl CoA transacylation and hydrolysis: fatty acyl CoA species dependent modulation by liver fatty acyl CoA binding proteins. Biochimica et Biophyica Acta 1483, 185-197, 2000.

Lupton, J.R., and Turner, N.D. Carbohydrates - Dietary fiber. M. Stipanuk (ed.). In: Biochemical & Physiological Bases of Human Nutrition. Saunders Co., Philadelphia, PA. Chapter 8, pp. 143-154, 2000.

Lupton, J.R. Is fiber protective against colon cancer? Where the research is leading us. Nutrition 16, 558-561, 2000.

Maier, S.M., Turner, N.D., and Lupton, J.R. Serum lipids in hypercholesterolemic men and women consuming oat bran and amaranth products. Cereal Chemistry 77, 297-302, 2000.

Jang, J.H., Shin, K.H., Park, Y.J., Lee, R.J., McKeehan, W.L., and Park, J.-G. Novel transcripts of fibroblast growth factor receptor 3 reveal aberrant splicing and activation of cryptic splice sequences in colorectal cancer. Cancer Research 60, 4049-4052, 2000.

Kepka-Lenhart, D., Mistry, S., Wu, G., and Morris, S.M. Arginase I: a limiting factor for nitric oxide and polyamine synthesis by activated macrophages? American Journal of Physiology 279, R2237-R2242, 2000.

McMurray, D.N. A non-human primate model for preclinical testing of new tuberculosis vaccines. Clinical Infectious Diseases 30 Supplement 3:S210-2. Review, 2000.

McMurray, D.N., Jolly, C.A., and Chapkin, R.S. Effect of dietary n-3 fatty acids on T cell activation and T cell receptor mediated signaling in a murine model. Journal of Infectious Disease 182, S103-7, 2000.

Meininger, C.J., Kelly, K.T., Li, H., Haynes, T.E., and Wu, G. Glucosamine inhibits inducible nitric oxide synthesis. Biochemical and Biophysical Research Communications 279, 234-239, 2000.

Meininger, C.J., Marinos, R.S., Hatakeyama, K., Martinez-Zaguilan, R., Rojas, J.D., Kelly, K.A., and Wu, G. Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency. Biochemical Journal 349, 353-356, 2000.

Li, H., Meininger, C.J., and Wu, G. Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection. Journal of Chromatography B746, 199-207, 2000.

Murakami, S., Kan, M., McKeehan, W.L., and de Crombrugghe, B. Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proceedings National Academy of Sciences 97, 1113-1118, 2000.

Murphy, E.J., Prows, D.R., Stiles, T., and Schroeder, F. Liver and intestinal fatty acid-binding protein expression increases phospholipid content and alters phospholipid fatty acid composition in L-cell fibroblasts. Lipids 35, 729-738, 2000.

Murphy, E.J., Stiles, T., and Schroeder, F. Sterol carrier protein-2 expression alters phospholipid content and fatty acyl composition in L-cell fibroblasts. Journal of Lipid Research 41, 788-796, 2000.

Reddy, M.C.M., Majumdar, S., and Harris, E.D. Evidence for a Menkes-like protein with a nuclear targeting sequence. Biochemical Journal 350, 855-863, 2000.

Schoer, J.K., Gallegos, A.M., McIntosh, A.L., Starodub, O., Kier, A.B., Billheimer, J.T., and Schroeder, F. Lysosomal membrane cholesterol dynamics. Biochemistry 39, 7662-7677, 2000.

Schroeder, F., Frolov, A., Starodub, O., Atshaves, B.P., Russell, W., Petrescu, A., Huang, H., Gallegos, A, McIntosh, A., Tahotna, D., Russell, D.H., Billheimer, J., Baum, C., and Kier, A.B: Pro-sterol carrier protein-2: Role of the N-terminal presequence in structure, function, and peroxisomal targeting. Journal of Biological Chemistry 275, 25547-25555, 2000.

Shearer, G.C., Joles, J.A., Jones, Hardin, Jr., Walzem, R.L., and Kaysen, G.A. Estrogen effects on triglyceride metabolism in analbuminemic rats. Kidney International 57, 2268-2274, 2000.

Starodub, O., Atshaves, B.P., Schoer, J., Roths, J.B., Kier, A.B., and Schroeder, F. Sterol carrier protein-2 immunocolocalization in endoplasmic reticulum and stimulation of phospholipid formation. American Journal of Physiology 279, C1259-C1269, 2000.

Thorngate, F.E., Rudel, L.L., Walzem, R.L., and Williams, D.L. Low levels of extrahepatic nonmacrophage ApoE inhibit atherosclerosis without correcting hypercholesterolemia in ApoE-deficient mice. Arteriosclerosis, Thrombosis, and Vascular Biology 20, 1939-1945, 2000.

Wu, G., Meininger, C.J., Kelly, K., Watford, M., and Morris, S. A cortisol surge mediates the enhanced expression of pig intestinal pyrroline-5-carboxylate synthase during weaning. Journal of Nutrition 130, 1914-1919, 2000.

Wu, G., Flynn, N.E., Knabe, D.A., and Jaeger, L.A. A cortisol surge mediates the enhanced polyamine synthesis in porcine enterocytes during weaning. American Journal of Physiology 279, R554-559, 2000.

Wu, G., and Meininger, C.J. Arginine nutrition and cardiovascular function. Journal of Nutrition 130, 2626-2629, 2000.

Wu, G, Meininger, C.J., Knabe, D.A., Bazer, F.W., and Rhoades, J.M. Arginine nutrition in development, health and disease. Current Opinions in Nutrition and Metabolic Care 3, 59-66, 2000.

Wu, G., Flynn, N.E., and Knabe, D.A. Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets. American Journal of Physiology, Endocrinology and Metabolism 279, E395-402, 2000.

Wu, G., Haynes, T.E., Li, H., and Meininger, C.J. Glutamine metabolism in endothelial cells: Ornithine synthesis from glutamine via pyrroline-5-carboxylate synthase. Comparative Biochemistry and Physiology, Part A, 126, 115-123, 2000.

Reproductive and Developmental Biology Research Core                         Return to top

The specific research aims of the Reproductive and Developmental Biology Research Core are to:

• Understand the impact of environmental toxicants on reproduction and development (Abbott, Burghardt, Johnson, Martin, McKeehan, Miranda, Piedrahita, Westhusin, Sohrabji, Spencer).
• Identify and characterize genes conferring susceptibility to environmentally induced congenital malformations (Abbott, Ing, Jaeger, Johnson, Martin, McKeehan, Miranda, Piedrahita, Sohrabji, Spencer, Westhusin).
• Develop methods for the genetic modification of small and large animals (Martin, Piedrahita, Westhusin).
• Define molecular regulation of steroid hormone receptors by environmental effectors (Bazer, Ing, Jaeger, Miranda, Sohrabji, Spencer).
• Determine the action of environmental toxicants on various aspects of cellular signal transduction (Bazer, McKeehan, Miranda, Sohrabji, Spencer).
• Determine how environmental factors affect brain development (Abbott, Miranda, Sohrabji, Spencer, West).
• Identify genes that are transcribed and translated during normal pre-implantation and peri-implantation development (Bazer, Burghardt, Ing, Jaeger, Spencer, Westhusin).

• Fuller W. Bazer, Ph.D., Director, Professor, Department of Animal Science, Veterinary Anatomy and Public Health and Institute of Biosciences and Technology
• Louise Abbott, D.V.M., Ph.D., Associate Professor, Department of Veterinary Anatomy and Public Health
• Robert Burghardt, Ph.D., Professor, Department of Veterinary Anatomy and Public Health
• Les Dees, Ph.D., Professor, Department of Veterinary Anatomy and Public Health
• Larry Johnson, Ph.D., Professor, Department of Veterinary Anatomy and Public Health
• James Martin, M.D., Assistant Professor, Institute of Biosciences and Technology
• Wallace L. McKeehan, Professor, Institute of Biosciences and Technology
• Rajesh Miranda, Ph.D., Assistant Professor, Department of Medical Anatomy and Neurobiology
• Jorge Piedrahita, Ph.D., Associate Professor, Department of Veterinary Anatomy and Public Health
• Thomas E. Spencer, Ph.D., Assistant Professor, Institute of Biosciences and Technology
• James West, Ph.D., Professor, Department of Medical and Neurobiology
• Mark Westhusin, Ph.D., Associate Professor, Department of Veterinary Physiology and Pharmacology

• Cloning
• Genetic Sensitivity
• Neuronal Differentiation
• Nuclear Transplantation
• Pre-implantation
• Signal Transduction
• Steroid Receptors
• Teratogens
• Transgenics
• Uterine Environment

Progress Report: Dr. Louise Abbott obtained the Laser Capture Microdissection System from Arcturus. This system is maintained by the Image Analysis core facility and is used by various CERH members to initiate various research projects. Dr. Fuller W. Bazer determined that: 1) endometrial gland epithelial cell morphogenesis and secretory activity is sequential and requires down-regulation of progesterone receptor, undefined permissive effects of interferon tau, and effects on placental lactogen and placental growth hormone; 2) interferon tau activates multiple members of the signal transducers and activators of transcription (STAT) and suppresses transcription of genes for estrogen receptor, and perhaps oxytocin receptor, through a novel and unreported mechanism involving STAT proteins and gamma activated sequence elements in the promoter regions of these target genes to allow pregnancy recognition; 3) osteopontin is a major secretory product of sheep uterine gland epithelium that binds integrins on conceptus trophectoderm to allow for implantation and perhaps placentation; 4) only stromal cells of the uterus express receptors for progesterone and that, in sheep, uterine stromal cells express fibroblast growth factor (FGF)-10 and hepatocyte growth factor to affect proliferation and/or function of uterine epithelia, while FGF-7 is expressed only by tunica intima of blood vessels; and 5) in the pig uterus FGF-7 is expressed by uterine lumenal and glandular epithelia in response to estrogens produced by trophectoderm, and that FGF-7 stimulates both proliferation and differentiated cell function by trophectoderm of the conceptus. A new USDA Grant to study FGF-7 in pigs, and a competitive renewal of the NIH grant to study effects of interferon tau on hormone receptor expression were obtained.

Dr. Robert C. Burghardt identified a complex set of integrin receptor subunits and extracellular matrix (ECM) proteins present at the maternal/conceptus interface in pigs and sheep. Two integrin receptors highly expressed at the peri-implantation period, alpha V beta 3 and alpha 4 beta 1, are expressed in human endometrium during the window of implantation. Dysregulation of these receptors may lead to infertility in the female, especially affecting women with luteal phase deficiency, endometriosis, hydrosalpinges, recurrent pregnancy loss and unexplained infertility. Unique immortalized cell lines derived from uterine luminal epithelium, glandular epithelium, stroma and myometrium from pigs and sheep, in collaboration with Drs. Bazer and Spencer, along with porcine and ovine trophectoderm cell lines, developed in Dr. Jaeger’s laboratory, express the same integrin receptor subunit profiles in vitro that are present during uterine receptivity in domestic animals and humans. Thus a novel vitro model system was developed to monitor functional interactions of integrin receptors on conceptus trophectoderm and uterine epithelial cells with several ECM ligands identified in vivo.

Strategies to analyze frequency encoded calcium signaling were used to characterize uterotonin-induced calcium oscillations that result in muscle contraction, and to identify intracellular and extracellular pools of calcium responsible. Analytical hardware and software developed for these studies are the subject of a patent application and an NSF Instrumentation Development proposal. This technology is being applied to the analysis of altered intracellular calcium homeostasis by environmental toxicants including studies of uptake, subcellular partitioning, and metabolism of aryl hydrocarbon receptor-binding ligands (using benzo[a]pyrene as a model compound), and their effects on cellular homeostasis.

Dr. Les Dees determined that: 1) alcohol ingestion inhibits secretion of puberty related hormones and alters the pattern of menstrual development in developing rhesus monkeys; 2) leptin acts centrally to induce prepubertal luteinizing hormone secretion and alcohol blocks this effect; 3) follicular stimulating hormone-releasing-hormone stimulates gonadotropin secretion in cattle; 4) alcohol modulates both nitric oxide synthase and insulin like growth factor-1 (IGF-1) intraovarian systems; and 5) IGF-1 of peripheral origin stimulates luteinizing hormone secretion to accelerate onset of female puberty, and alcohol can block this effect of IGF-1.

Dr. Laurie A. Jaeger determined that: 1) exposure of pre-implantation trophoblast to estradiol depletes cellular glutathione and decreases mitochondrial membrane potential; 2) estrogen and progesterone receptors are present in pre-implantation porcine conceptus; and 3) estrogen down-regulates expression of estrogen receptor alpha in porcine trophectoderm cells. Porcine and ovine trophectoderm cell lines were developed to permit mechanistic studies of normal and perturbed conceptus development and implantation (e.g., toxicities) during early pregnancy, and assays were developed to assess adhesion mediated by integrins and ECM between trophoblast and uterine cells.

Dr. Larry Johnson determined that the level of spermatogenesis in North American men has declined in recent years, and established outreach education programs for enhancing environmental and health science education in grades 6-8 in rural public schools.

Dr. James Martin’s research is on molecular mechanisms controlling cell growth and differentiation in the context of vertebrate embryogenesis and causes of birth defects. He studies the roles of homeobox genes in cell growth and differentiation within the craniofacial skeleton using targeted gene mutations through "knockout" technology and related transgenic techniques to express genes of interest in mice. Another interest is to understand how environmental factors, such as teratogens, interact with the genome to generate congenital defects. Two immediate goals are to understand cellular and molecular functions of the paired -related homeobox gene, Mhox, during craniofacial development and to define the DNA sequences which control expression of the Mhox gene in craniofacial primordia. Mice homozygous for a loss-of-function mutation in the Mhox gene do not have specific skull bones such as the temporal and occipital bones. Genetic interactions between Mhox and other paired-related homeobox genes, such as S8 and goosecoid, and different combinations of prx genes appear to regulate the formation of different craniofacial skeletal elements. One region of the Mhox gene, called an enhancer, regulates gene expression in a subset of Mhox-expressing cells in craniofacial primordia of mouse embryos. This enhancer provides an important tool to dissect molecular mechanisms controlling patterning of the developing facial structures.

Dr. Jorge A. Piedrahita: 1) developed transgenic pig chimeras using primordial germ cell-derived cell lines; 2) elucidated the apoptotic pathway affecting in vitro survival of porcine primordial germ cells in culture; 3) elucidated the importance of follicular size on the efficiency of nuclear transfer in cattle; and 4) produced four cattle clones that were born. Drs. Piedrahita and Finnell also had a significant 1999 paper published in Nature Genetics [Piedrahita J.A., Oetama B, Bennett G, Waes JV, Lacey SW, Kamen B, Richardson J, Lark R, and Finnell R. 1999. Inactivation of the folate binding protein genes disrupts neural tube closure. Nature Genetics, 23:228-232]. A manuscript describing newly identified folate receptors as potential candidate genes for neural tube defects is currently in preparation. These manuscripts describe the development of FOLBP1 and FOLBP2 knockout mice.

Dr. Thomas E. Spencer discovered: 1) that endometrial glands are required for the ovine uterus to support the estrous cycle and establishment of pregnancy by using the novel Uterine Gland Knock Out (UGKO) ewe model; 2) that endometrial glands in adult uterus express novel and known mRNAs that provide a basis for a genomics approach to enhancing uterine capacity and embryotrophic potential, as well as potential tools for gene targeting; 3) prolactin, estradiol-17 beta, prolactin receptors and estrogen receptor alpha are potential regulators of endometrial gland morphogenesis in neonatal ewes; and 4) that endometrial gland morphogenesis and function during pregnancy requires down-regulation of the progesterone receptor by progesterone itself and sequential actions of conceptus hormones including interferon tau, placental lactogen and placental growth hormone. Dr. Spencer was funded by grants from USDA and a CERH Pilot Project Grant. He received a score that placed him in the 5^th percentile for funding from NIH beginning in March 2001 to study "Mechanisms Regulating Uterine Morphogenesis". He is also Co-PI with Dr. Bazer on a grant that received a 6^th percentile score for funding beginning December 1, 2000 from NIH to study "Interferon Tau Regulates Uterine Hormone Receptors."

Abbott, L.C., and Sotelo, C. Ultrastructural analysis of catecholaminergic innervation in weaver and normal mouse cerebellar cortices. Journal of Comparative Neurology 526, 316-329, 2000.

Barhoumi, R., Mouneimne, Y., Ramos, K.S., Safe, S.H., Phillips, T.D., Centonze, V.E., Ainley, C., Gupta, M.S., and Burghardt, R.C. Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line. Toxicological Sciences 53, 264-270, 2000.

Barthel, R., Piedrahita, J.A., McMurray, D.N., Payeur, J., Baca, D., Guemes, F.S., Perumaalla, V.S., Ficht, T.A., Templeton, J.W., and Adams, G. Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 in cattle from herds with naturally occurring tuberculosis. American Journal of Veterinary Research 61, 1140-1144, 2000.

Bennett, G.D., Wlodarczyk, B., Calvin, J.A., Craig, J.C., and Finnell, R.H. Valproic acid-induced alterations in growth and neurotrophic factor. Reproductive Toxicology 14, 1-11, 2000.

Bergwerff, M., Gittenberger-de Groot, A.C., Wisse, L.J., DeRuiter, M.C., Wessels, A., Martin, J.F., Olson, E.N., and Kern, M.J. Loss of function of the Prx1 and Prx2 homeobox genes alters architecture of the great elastic arteries and ductus arteriosus. Vichows Arch 436, 12-19, 2000.

Blanchard, T.L., Johnson, L., and Roser, A.J. Increased germ cell loss rates and poor semen quality in stallions with idiopathic testicular degeneration. Journal of Equine Veterinary Science 20, 263-265, 2000.

Bowen, J.A., and Burghardt, R.C. Cellular mechanisms of implantation in domestic farm animals. Cell and Developmental Biology 11, 93-104, 2000.

Cheema, Z.F., West, J.R., and Miranda, R.C. Ethanol induces Fas/Apo [apoptosis]-1 and cell suicide in the developing cerebral cortex. Alcoholism: Clinical Experimental Research 24, 535-543, 2000.

Chen, C., Spencer, T.E., and Bazer, F.W. Expression of hepatocyte growth factor and its receptor c-met in the ovine uterus. Biology of Reproduction 62, 1844-1850, 2000.

Chen, C., Spencer, T.E., and Bazer, F.W. Fibroblast growth factor-10: A stromal mediator of epithelial function in the ovine uterus. Biology of Reproduction 63, 959-966, 2000.

Cudd, T.A., Wasser, J.S., Chen, W.J.A., and West, J.R. Brain energy phosphate responses to alcohol exposure in neonatal rats: an in vivo ³¹P-NMR study. Alcoholism: Clinical and Experimental Research 24, 865-872, 2000.

Dearth, R.K., Hiney, J.K., and Dees, W.L. Leptin acts centrally to induce the prepubertal secretion of luteinizing hormone in the female rat. Peptides 21, 387-392, 2000.

Dees, W.L., Dissen, J.K., Hiney, F.L., and Ojeda, S.R. Alcohol ingestion inhibits the increased secretion of puberty-related hormones in the developing female rhesus monkey. Endocrinology 141,1325-1331, 2000.

Dove, L.E., Nahm, S.S., Murchison, D., Abbott, L.C., and Griffith, W.H. Altered calcium homeostasis in cerebellar purkinje cells of Leaner mutant mice. Journal of Neurophysiology 84, 513-524, 2000.

Finnell, R.H., Gelineau-van Waes, J., Bennett, G.D., Barber, R.C., Wlodarczyk, B., Shaw, G.M., Lammer, E.J., Piedrahita, J.A., and Eberwine, J.H. Genetic basis of susceptibility to environmentally-induced birth defects. Toxicology for the Next Millennium, R.J. Isfort and J. Lederberg, Eds. New York Academy of Sciences, 919, 261-277, 2000.

Gray, C.A., Bartol, F.F., Taylor, K.M., Wiley, A.A., Ramsey, W.S., Ott, T.L., Bazer, F.W., and Spencer, T.E. The ovine uterine gland knockout model: effects of gland ablation on the estrous cycle. Biology of Reproduction 62, 448-456, 2000.

Gray, C.A., Taylor, K.M., Bazer, F.W., and Spencer, T.E. Mechanism regulating Norgestomet inhibition of endometrial gland morphogenesis in the neonatal ovine uterus. Molecular Reproduction and Development 57, 67-78, 2000.

Hill, J.R., Burghardt, R.C., Jones, K., Long, C.R., Looney, C.R., Prior, J., Shin, T-Y, Spencer, T.E. Thompson, J.A., Winger, Q.A., and Westhusin, M.E . Evidence for placental abnormality as the major cause of mortality in first trimester somatic cell cloned bovine fetuses. Biology of Reproduction 63, 1787-1794, 2000.

Johnson, G.A., Spencer, T.E., Burghardt, R.C., Joyce, M.M., and Bazer, F.W. Interferon-tau and progesterone regulate expression of ubiquitin cross-reactive protein in the ovine uterus. Biology of Reproduction 62, 622-627, 2000.

Johnson, G.A., Spencer, T.E., Burghardt, R.C., Taylor, K.M., Gray, C.A., and Bazer, F.W. Progesterone modulation of osteopontin gene expression in the ovine uterus. Biology of Reproduction 62, 1315-1321, 2000.

Johnson, L., Varner, D.D., Roberts, M.E., Smith, T.L., Keillor, G.E., and Scrutchfield, W.L. Efficiency of spermatogenesis: a comparative approach. Animal Reproduction Science 60, 471-480, 2000.

Ka, H., Spencer, T.E., Johnson, G.A., and Bazer, F.W. Keratinocyte growth factor: Expression by endometrial epithelia of the porcine uterus. Biology of Reproduction 62, 1772-1778, 2000.

Lee, C-K., Weaks, R.L., Johnson, G.A., Bazer, F.W., and Piedrahita, J.A. Effects of protease inhibitors and antioxidants on in vitro survival of porcine primordial germ cells. Biology of Reproduction 63, 887-897, 2000.

Lee, C-K., Weaks, R.L., Scales, N., Newton, G., Bazer, F.W., Westhusin, M., and Piedrahita, J.A. Isolation and genetic transformations of primordial germ cells (PGC) – derived cells from cattle, goats, rabbits, and rats. Asian-Australian Journal of Animal Science 13, 587-594, 2000.

Mallo, M., Schrewe, H., Martin, J.F., Olson, E.N., and Ohnemus, S. Assembling a functional tympanic membrane: signals from the external acoustic meatus coordinate development of the malleal manubrium. Development 127, 4127-4136, 2000.

Martin, J.F., and Olson, Eric N. Identification of a prx1 limb enhancer. Genesis 26, 225-229, 2000.

McAlhany, Jr. R.E., West, J.R., and Miranda, R.C. Glial-derived neurotrophic factor (GDNF) prevents ethanol-induced apoptosis and Jun kinase phosphorylation. Developmental Brain Research 119, 209-216, 2000.

Piedrahita, J.A. Targeted modification of the domestic animal genome. Theriogenology 53, 105-116, 2000.

Miller, S., Kennedy, D., Thomson, J., Han, F., Smith, R., Ing, N., Piedrahita, J., and Busbee, D. A rapid and sensitive reporter gene system using green fluorescent protein expression to detect chemicals with estrogenic activity. Toxicological Sciences 55, 69-77, 2000.

Reardon, W., Smith, A., Honour, J.W., Hindmarsh, P., Das, D., Rumsby, G., Nelson, I., Malcolm, S., Adès, L., Sillence, D., Kumar, D., DeLozier-Blanchet, C., McKee, S., Kelly, T., McKeehan, W.L., Baraitser, M., and Winter, R.M. Evidence for digenic inheritance in some cases of Antley-Bixler syndrome? Journal of Medical Genetics 37, 26-32, 2000.

Silber, S.J., Johnson, L., Verheyen, G., and Van Steirteghem, A. Round spermatid injection. Fertility and Sterility 73, 897-900, 2000.

Snowden K., Logan, K., and Phalen, D. Isolation and characterization of an avian isolate of Encephalitozoon hellem. Parasitology 121, 9-14, 2000.

Stewart, M.D., Johnson, G.A., Burghardt, R.C., Schuler, L.A., Bazer, F.W., and Spencer, T.E. Prolactin receptor and uterine milk protein expression in the ovine uterus. Biology of Reproduction 62, 1779-89, 2000.

Taylor, K.M., Gray, C.A., Stewart, M.D., Joyce, M.M., Bazer, F.W., and Spencer, T.E. Neonatal ovine uterine gland development involves alterations in expression of receptors for progesterone, prolactin and estrogen. Biology of Reproduction 63, 1192-1204, 2000.

Uematsu, F., Kan, M., Wang, F., Jang, J-H., Luo, Y., and McKeehan, W.L. Ligand binding properties of binary complexes of heparin and immunoglobulin-like modules of FGF receptor 2. Biochemical and Biophysical Research Communications 272, 830-836, 2000.

Scott, C.J., Pereira, A.M., Rawson, J.A., Simmons, D.M., Rossmanith, W.G., Ing, N.H., and Clarke, I.J. The distribution of progesterone receptor immunoreactivity and mRNA in the preoptic area and hypothalamus of the ewe: Upregulation of progesterone receptor mRNA in the mediobasal hypothalamus by oestrogen. Journal of Neuroendocrinology 12, 565-575, 2000.

Yu, C., Wang, F., Kan, M., Jin, C., Jones, R.B., Weinstein, M., Deng, C-X., and McKeehan, W.L. Elevated cholesterol metabolism and bile acid synthesis in mice lacking membrane tyrosine kinase receptor FGFR4. Journal of Biological Chemistry 275, 15482-15489, 2000.

Facility Cores                                                                         Return to top      

Analytical Services Facility Core Objectives: Research investigating the impact of environmental factors on rural public health requires collaborations between multiple disciplines. For example, interpretation of community health studies is facilitated by collaborative efforts between community-based researchers, epidemiologists and toxicologists to evaluate disease frequency and estimates of exposures. The interpretation of in vitro and in vivo studies to investigate mechanisms through which chemicals produce their toxic effect is enhanced by the availability of data to identify toxic metabolites. Finally, organized production and evaluation of data from these studies require establishment of a detailed Quality Assurance/Quality Control (QA/QC) program and the implementation of Standard Operating Procedures (SOPs) for routine laboratory techniques to improve the precision, accuracy and reproducibility of data generated by Center investigators.

The specific aims of the Analytical Services Facility Core are to:

• Provide analytical support including sample preparation, extraction and standard analytical measurements to Center investigators.
• Provide assistance to Center investigators in the development and implementation of a sampling strategy for field investigations to estimate exposures in support of community-based research studies.
• Assist in the development and implementation of SOPs and/or Quality Assurance Project Plans for Center researchers.

The Facility Core supports investigators in the Chemical Biology, Reproductive and Developmental Biology, and Biostatistics and Community Health Research Cores of the CERH. The majority of these projects have involved analytical services or assistance in the collection of field samples. The core also collaborates with the NIEHS Center at Rutgers University to support several jointly funded EPA initiatives.

· K. C. Donnelly, Ph.D., Director, Associate Professor, Departments of Veterinary Anatomy and Public Health and Environmental and Occupational Health

· A. Bokelman, QA/QC Officer, Department of Veterinary Anatomy and Public Health

· L. Y. He, Ph.D., Research Chemist, Department of Veterinary Anatomy and Public Health

· T. J. McDonald, Ph.D., Associate Research Scientist, Department of Civil Engineering

· G. D. Zhou, M.D., Associate Research Scientist, Department of Veterinary Anatomy and Public Health

· Field Sampling and Safety Equipment – includes full face respirators, boots, coveralls, and various disposable equipment. Trowels for surface soil sampling, an extension for surface water sampling, and a HEPA vacuum cleaner for sampling indoor house dust.

· HPLC with PhotoDiode Array Detector – High Pressure Liquid Chromatography equipped with a PhotoDiode Array detector for trace analysis of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. The Core maintains several HPLC columns for both trace chemical analysis and preparative scale separation of complex mixtures.

· Two Gas Chromatographs (GCs) with EC, FID and NP detectors – these GCs are used for routine chemical analysis. The detectors are analyte specific, with the NP detector primarily used for detection of organophosphate insecticides, the FID for analysis of simple PAH mixtures, and the EC detector for chlorophenols and other halogenated hydrocarbons.

· Zymark Turbovap Concentrator – used for reducing solvent volume of sample extracts. This unit will be combined with an Accelerated Solvent Extractor for efficient sample preparation.

· Tecator Soxhlet Extractor – used for extraction of soils, sediments and solid waste. This equipment reduces time and solvent required for standard Soxhlet extraction.

· Greenhouse Space – approximately 600 square feet of space is available for sample preparation in bioremediation studies. The greenhouse is temperature controlled and includes a rainfall simulator for collection of runoff water from contaminated soils.

· GC/MS and LC/MS-MS – The GC-MS is available for quantitative analysis of environmental samples, while the LC/MS-MS is used for trace analysis and analysis of unknown samples such as PAH degradation products produced from treatment with ozone.

· Tracor 540 GC – with a flame ionization detector and an electron capture detector, another Tracor 540 GC with a nitrogen-phosphorous detector and a Hewlett-Packard 5890 GC with a flame ionization detector.

Usage and Benefits: The Analytical Services Core collaborates with Dr. J. Calvin of the Biostatistics and Community Health Research Core to provide support for the Rio Bravo Health Child Project. This study is designed to provide a more accurate model for estimating intake and absorption of pesticides by small children (aged 6 months to 4 years). Although most exposure models treat children as small adults, numerous studies have demonstrated that the rates of absorption, distribution and metabolism in children are very different from adults. The Rio Bravo (Rio Bravo is a small colonia south of Laredo, Texas) study is designed to measure not only intake and excretion of pesticides in children, but also behavioral activities that may influence intake. The Core developed procedures, trained field staff (Promotoras), and assisted in sample collection and processing. The initial stages of the project, a series of SOPs were developed (in English and Spanish) for the collection of house dust, hand wipe and urine samples, as well as procedures for conducting the interview and videotaping of the children. Core personnel also conducted training on sampling in the field. The Analytical Services Facility staff also receives and processes samples for analysis. Dr. T. McDonald is analyzing hand wipe and house dust samples, and the urine samples are being processed at the Centers for Disease Control by Dr. L. Needham.

The Rio Bravo study involves a total of four sampling periods (spring and fall for two years). Samples collected during the spring of 2000 were analyzed for more than 20 organochlorine and organophosphate insecticides. The data indicate that DDT was the most common chemical detected. DDT and DDT metabolites were detected in 20 of 20 house dust samples and 35 of 37 dermal rinse samples. Although the organophosphate (OPs) pesticides were detected less frequently, concentrations of OPs were generally 10 to 100-fold higher than were observed for the organochlorines. Results from a second round of sampling conducted in the fall of 2000 are being processed, and additional sampling is scheduled for spring and fall of 2001. When combined with data from the analysis of urine and videotapes (to enumerate hand-to-mouth activities), these studies will provide valuable information to characterize the rate and extent of pesticide absorption and excretion in small children.

The Analytical Services Facility Core has developed an HPLC method to quantify metabolites of PAHs in renal cells. Dr. K. Ramos (Chemical Biology) is the PI of a study investigating the toxicity of BaP, and mixtures of BaP with chrysene and anthracene, in renal cells. Although humans are frequently exposed to complex mixtures in the environment, the majority of the existing toxicity database has been developed for individual chemicals. The initial objective of this study was to evaluate the renal glomerular and epithelial cell toxicity of individual chemicals alone and in binary mixture. BaP or anthracene increased mitochondrial fragility and cellular death of glomerular cells, while epithelial cells were preferentially affected by chrysene. Binary and ternary mixtures of these chemicals yielded antagonistic interactions. Facility Core staff completed HPLC analysis to investigate the mechanisms responsible for this interaction. The results indicated that 3-hydroxy benzo(a)pyrene and benzo(a)pyrene 1,6-dione are the major BaP metabolites detected in glomerular cells exposed to a single chemical, but that the concentration of metabolites is below detection limits when cells are exposed to a binary or ternary mixture of hydrocarbons. Understanding the mechanism of inhibition is important for developing accurate measures of the risk associated with exposure to complex mixtures. Additional studies are planned with Clone 9 hepatic cells and SY5Y neural cells.

A new technology available through the Analytical Services Facility Core is DNA postlabeling. Studies are in progress to investigate the affect of pentachlorophenol (PCP) on the formation of BaP adducts in the liver and lung of infant and adult mice. Adduct levels were consistently lower in infant mice exposed to a binary mixture. At the highest dose of PCP, BaP adduct levels in infant liver 6 hours after administration decreased seven-fold. However, in adults an increased level of DNA adducts were observed in hepatic tissues 24 hours after administration of the mixture. The data indicate that PCP potentiated DNA adduct formation in adult, but not infant mice. Similar results were observed with lung tissue.

Additional studies were conducted to investigate the rate of DNA adduct formation in trp53 wild type and knockout mice. In knockout mice, relative adduct levels were comparable following administration of BaP or a mixture of BaP and PCP. However, in liver from the wild type mice the relative adduct level following administration of the binary mixture was approximately 25% the level observed following administration of BaP alone. Studies of this nature provide information that can be used to understand the mechanisms of complex mixture interactions. These data are ultimately useful for improving the methods for risk assessment of environmental mixtures.

Biostatistics and Computational Services Facility Core Objectives: The goal of this core is to support five different activities of CERH investigators in all Research Cores.

· Help Desk – The first line of support for short-term statistical design and analysis questions comes from the Help Desk maintained in the CERH main offices. Services are provided at no charge on a no-appointment basis. One research assistant staffs the desk ten hours per week. The core also supports long-term collaborative research by funding 25% effort of a research assistant for those research teams whose work involves more extensive statistical modeling and analysis.

· Statistical Consulting – Services are provided for long-term statistical design and analysis and to instruct researchers on new statistical methods applicable to their research activities.

· Computational Services – A web site (www.cerh@tamu.edu) highlighting the Center’s outreach and education efforts has been developed. A three-minute streaming video features the focus and goals of the CERH and a 12-month interactive calendar advises members of upcoming seminars, meetings and conferences. An archival storage/retrieval system is being developed that will give researchers access to a secure data storage facility.

· Request for Services System – An on-line service request system for all facility cores has been developed. Facility Cores, as well as the Administrative Core are able to track services rendered and schedule efficiently.

· Data Management – A data management framework has been developed to facilitate data analysis as well as provide secure storage and backup for enhanced data integrity. Programming services to aid in statistical analysis are also available.

Members:

• James A. Calvin, Ph.D., Director, Professor and Head, Department of Statistics

• Michael T. Longnecker, Ph.D., Associate Director, Professor, Department of Statistics

• James Snell, D.V.M., Ph.D., System Administrator, Department of Veterinary Anatomy and Public Health

• Kyeong Eun Lee, Research Assistant, Department of Statistics

• Cristy Spinka, Research Assistant, Department of Statistics

• Veerabhadran Baladandayuthapani, Research Assistant, Department of Statistics

Facilities and Equipment:

· Compaq PC Server that supports the CERH mail system, web site and data archival system

· PC in Help Desk office to support statistical consulting activities

· PC used for web site development

· HP LaserJet printer in Help Desk office to support statistical consulting activities

Usage and Benefits: Statistical support of the Center investigators is the primary mission of this facility. Through Core activities, research teams are able to perform complete analyses and design cost effective experiments. Texas A&M University does not provide a statistical consulting service and, thus, without the CERH, Center investigators would not have access to statistical support without creating personal contacts and paying for any and all contacts, regardless of the request. As the Center continues to grow this service is becoming a dominant activity of the facility.

The Computational Services component of the facility core provides a platform for archiving and retrieving data resulting from the individual and collaborative activities of Center investigators. The archival storage/retrieval system has been designed to store all types of electronic files, including flat data, image, word processing, or spreadsheet files. The system functions such that each investigator has his/her individual secure storage hierarchy and allows oversight by the laboratories personnel. Stored files are given generation numbers so that new versions of the same file can be stored without destroying older versions. There are keywords and abstracts associated with each file.

The key word mechanism is searchable on two levels. The investigator can search his/her own data system for files or search the complete CERH system for related research being done by other CERH investigators. If another project uses the same keyword(s) contact information is provided so researchers can communicate.

This facility core has developed a CERH electronic mail system that is integrated with each member’s own regular mail system. Group aliases for research and facility cores, as well as any other recognized group have been established. This facilitates communication spread throughout the Texas A&M campus and sites in greater Houston.

Collaborations between Drs. J. Lupton and R. Chapkin (Nutrition), with support from the Biostatistics and Computational Facility Core staff, have led to productive interactions. The basic goal of this work is to understand the responses of regions of the small and large intestines and the mediating effects of diet in experimentally induced tumorigenesis.

The research team of Drs. Sherman and C. Phillips (Biostatistics and Community Health) recently initiated a review of assisted living facilities and conditions. Their main interest is assessing which factors of the assisted living facility best determine successful outcomes for the elderly. Following the model set by Carroll et al., the Biostatistics and Computational Facility Core staff support their effort through consultant services.

Dr. Calvin is working with Dr. Donnelly (Biostatistics and Community Health) on experiments studying the effectiveness of microorganisms in bioremediation, and with Dr. Safe (Chemical Biology) on the synergistic properties of certain drug combinations. Dr. Longnecker is working with Dr. K. Ramos in developing approaches to evaluate the biological relevance of in vitro gene reporter experiments.

Dr. Calvin continues to collaborate with Drs. Safe and A. McDougal on studies of the combined antitumorigenic activity of tamoxifen and 6-methyl-1, 3, 8-trichlorodibenzofuran (MCDF) in a rat mammary tumor model. Analysis of the data suggests that combinations of 6-MCDF and tamoxifen synergistically inhibit mammary tumor and this type of combined therapy has now been patented. A paper describing this work has been submitted to Cancer Research.

Genomics and Bioinformatics Facility Core Objectives: The goal of the Genomics and Bioinformatics Facility Core is to support the global gene expression research efforts of CERH investigators. During the second year, the Facility Core was expanded to include microarray technology as part of the services offered by the core. Due to the need for functional genomics capabilities, Dr. Terry Thomas, Head, Department of Biology and Director, Laboratory for Functional Genomics, was recruited to guide these efforts. Subsequently, in consultation with CERH members and the Internal/External Advisory Boards, it was decided that services previously provided by the DNA Facility Core could be obtained at competitive prices elsewhere, and that the primary needs of CERH investigators were in global gene expression analysis. Hence, the core was renamed the Genomics and Bioinformatics Facility Core and Dr. Thomas was named director. The current goals of the Genomics and Bioinformatics Facility Core are to provide all CERH investigators access to state-of-the-art functional genomics and bioinformatics resources.

The specific aims of the core are to:

· Facilitate CERH investigator efforts to obtain or develop unigene sets for construction of relevant DNA microarrays.

· Construct DNA microarrays that will facilitate CERH Research Core objectives.

· Coordinate gene expression experiments of CERH Core users.

· Compile and display expression data.

· Acquire or develop new technologies to advance global gene expression analysis capabilities. This includes developing and comparing alternative and new transcriptional profiling platforms.

· Terry Thomas, Ph.D., Director, Professor and Head, Department of Biology

· Edward Dougherty, Ph.D., Director of Computer Assisted Medical Diagnostics Imaging Laboratory (CAMDI), Professor, Department of Electrical Engineering

· Phillip Beremand, Ph.D., Associate Research Scientist, Department of Biology

· Laurie Davidson, Ph.D., Research Associate, Department of Animal Science

· Jamie Schroeder, B.S., Research Scientist, Department of Biology

The following resources are available to CERH investigators:

· cDNA library construction, arraying and curation – Directionally cloned representative cDNA libraries are constructed and robotically arrayed at a density of 50,000 – 100,000 clones in 384 well microtiter plates using a Qbot (Genetix, Inc.). High-density nylon filters are made using the Qbot or the BioMek (Beckman Coulter) robots for screening by virtual subtraction. The Genomics and Bioinformatics Facility Core serves as a technical resource for library construction and screens using limiting amounts of material (a few hundred cells). Using the Qbot, libraries can also be rearrayed following virtual subtraction screens. These services are provided on a fee-for-service basis.

· High throughput (HT) EST analysis – HT methods are available for template preparation (BioMek 2000), reaction assembly (Robbins Hydra 96), HT PCR and HT sequencing. Two ABI 377 (Perkins Elmer) sequencers are available; thus >70,000 ESTs can be generated per year. Additional sequencing capacity (>20,000 reactions) is available on a ABI 3700 96 channel capillary electrophoresis sequencer housed in the Borlaug Southern Crop Improvement Center. One-pass sequences are determined from each cDNA; the estimated sequence length per EST is 400-500 nucleotides.

· Transcriptional profiling – The facility has assembled a toolbox with enabling technologies for the global analysis of gene expression programs. The two-color fluorescent hybridization microarray system developed by Brown and colleagues at Stanford is used because it is one of the most flexible and robust transcriptional profiling technologies available in an academic setting. A gridding robot from Genemachines is in operation. The Genemachines OmniGrid gridding robot was recently upgraded (9/16/00). The robot uses up to 48 "pins" to develop sub-nanoliter volumes of liquid in approximately 100 micrometer diameter features on as many as 100 glass slides (1"x 3") at 100 micrometer spacing. This allows fabrication of DNA microarrays with up to 40,000 gene sequences per slide. Hybridization is detected using cDNA probes synthesized with one of two color fluorescent deoxyribonucleotide precursors (Cy3 and Cy5). A confocal laser scanning device (ScanArray® 3000; General Scanning Lunonics, Inc.) is used to detect and quantify hybridization signals. The ScanArray® 3000 is specifically designed to scan DNA microarrays fabricated on glass slides using the Cy3 and Cy5 fluorescent labels. Scan rates of 4 min per 20 x 20 µm arrays allow rapid data acquisition at 10µm resolution.

· Real Time PCR – Once cDNAs of interest are identified, experiments to confirm that a particular clone shows the expression pattern indicated by the array are required. Quantitative PCR using an internal standard to monitor each reaction and allow comparisons between different reactions will thus be completed.

· Informatics – The Facility has its own informatics capability for EST analysis and transcriptional profiling. Databases are searchable with standard search algorithms, including several versions of BLAST. The internal databases available use two software packages for analysis of the microarray data. One is Visage HDG Analyzer from Genomic Solutions that runs on a Sun Microsystems ULTRA 10 workstation. The second is ArraySuite, an application developed at the National Human Genome Research Institute (NHGRI) for the IPLab software that runs on Rower MacG4s. Dr. Dougherty recently joined the CERH to provide leadership in bioinformatics and to apply the multiple data analysis strategies developed and used at NHGRI. Several other commercial software packages will be acquired with grant funds over the next five year period to enhance and expand downstream analysis capabilities, including GeneSpring (Silicon Genetics, Inc.) and Spotfire (Spotfire, Inc.) Under the leadership of Dr. Dougherty, a robust bioinformatics cluster is being developed for use by CERH investigators for microarray analysis. His efforts are closely aligned with the Biostatistics and Computational Services Facility Core to synergize the overall effort.

· Genemachines Omnigrid robot (upgraded 9/16/00) for preparing DNA microarrays

· ScanArray 3000 fluorescence scanner (GSI Lumonics) for scanning DNA microarrays

· Three (3) ABI 377 (Perkin Elmer) automated sequencers (96 well format)

· BioMek 2000 (Beckman Coulter) robot for plate replication, filter preparation and repetitive pipetting

· Qbot (Genetic, Inc.) for picking and arraying (rearraying) bacterial colonies and HD filter preparation (This is a shared instrument located elsewhere on campus)

· Multiple 96 well PCR machines

· Robbins Hydra 96 liquid handling robot for reaction assembly, etc.

· One (1) Sun ULTRA 10, three (3) Pentium II PCs, one (1) Pentium III dual processor PC and one (1) Mac G4

CAMDI

· Six (6) networked Dell workstations

· Two (2) Unix workstations

· Three (3) Mac Power PCs

Usage and Benefits: Four research projects are ongoing and briefly described below.

As part of this initiative, studies are planned to evaluate gene expression profiling over time to predict experimentally verifiable phenotypic characteristics (i.e. DNA damage, aberrant crypt foci and tumors) that are relevant to colon tumor initiation, promotion, and progression. Rats are being exposed to an environmental carcinogen and dietary modifiers of tumorigenesis and examined over time (0 hour, 12 hour, 10 week, and 36 week post carcinogen exposure). RNA from the diet and carcinogen treatment groups will be compared with a single standard to find and quantify subtle regulatory relationships among genes.

The endometrial glands of the pregnant ovine uterus express genes, such as growth factors and cytokines, that encode secretory proteins. These gland-derived proteins directly regulate conceptus survival and growth. A uterine gland knockout (UGKO) sheep