I. Center Summary

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. Evolution of the CERH during the last year required several adjustments in structure as detailed below.

Research Cores: The Biostatistics and Epidemiology research core focuses on several interrelated research projects, including development of improved models for risk assessment, sampling and environmental exposures and epidemiology of breast cancer and pregnancy. Investigators of the Chemical Biology core, formerly Cellular and Molecular Biology/Toxicology core, use chemical and biological approaches to address fundamental problems related to the pathogenesis of environmental disease. The Nutrition research core focuses on the study of nutritional and environmental interactions that influence colon cancer, immune-mediated inflammatory disease, and atherosclerotic vascular disease. The Reproductive and Developmental Biology research 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 Biological Mass Spectrometry core was reconfigured as the Protein Technologies core to include more comprehensive approaches for analysis of peptides and proteins. The Biostatistics and Computational core provides extensive network and software support, data management and statistical support for research projects. The DNA Technologies core continues to provide technical assistance for routine DNA synthesis and sequencing, but is being expanded to include DNA Chip technology. The Field Services core assists investigators with development and implementation of sampling strategies and provides analytical services for CERH projects. 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 Transgenic core provides comprehensive support in the generation and characterization of transgenic mice produced by pronuclear injection as well as homologous recombination.

COEP: Outreach activities focus on training and education in human health and the environment in rural Texas. In collaboration with community leaders and lay health educators, efforts are directed at the implementation of educational and community-based research programs in colonias (Spanish for neighborhood) along the Texas-Mexico border. Layman, K-12, and professional education programs focusing on birth defects, cancer, and cardiovascular and renal health are being developed. Services for the local community include the broadcast of a weekly TV show 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. Five projects were funded in 1999 to support collaborative research and enhance the success of grant applications in competing for external funding.

Title: Importance of Mice Lacking Folate Receptor in Birth Defects Research

Significance: The three most common types of human birth defects: cardiac, neural tube defects, such as spina bifida and anencephaly, and oral-facial clefts, including cleft lip and palate, are of multi-factorial origin. These disorders are believed to share both genetic and environmental components to their development. The one single environmental factor that appears to moderate the risk for these birth defects is the vitamin, folic acid. Several large epidemiological studies have demonstrated that women taking a folic acid supplement during pregnancy reduce the risk of birth defects by approximately 50% or more. Unfortunately, not all women benefit from this vitamin intervention, so babies are still born with these birth defects resulting in health care costs that exceed $2 billion dollars annually. At this time it is not known how folic acid works to reduce the prevalence rates for these birth defects. If the mechanism of folic acid action was understood there might be a solution for women who fail to benefit from vitamin supplementation. Folate binding protein 1 and 2 (Folbp1 and Folbp2) knockout mice developed by CERH investigators are unique animals that have lost the ability to transport folic acid into their cells. Without supplemental folic acid, their progeny have lethal neural tube and craniofacial birth defects. The mothers provided with large amounts of folic acid during pregnancy have progeny to develop normally. Given that folic acid can interact with environmental agents to influence birth defect outcomes, these studies demonstrate the importance of these genes in regulating environmentally induced birth defects.  

References:

Finnell RH, Shaw GM, Greer KA, Barber RA, Piedrahita JA, and Lammer EJ. (1998). Neural tube and craniofacial defects with special emphasis on folate pathway genes. Critical Reviews Oral Biological Medicine 9:38-53.

Piedrahita JA, 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.

Title: DNA Damage, Repair, and Removal and its Relationship to Colon Cancer

Significance: Colon cancer is the second leading cause of death from cancer in the United States today. Diet is thought to play an important role in its prevention and its occurrence. CERH investigators have shown that fish oil (high in n-3 fatty acids) is more protective of colon cancer development than corn oil (high in n-6 fatty acids). In addition, the combination of fish oil and pectin (a highly fermentable fiber) has a synergistic protective effect against colon cancer. The mechanism behind this protective effect during the stage of tumor promotion is an enhancement of cell death (apoptosis) rather than a decrease in cell proliferation. More recent investigations have focused on the very earliest stage of cancer development (0 to 12 hours after the injection of a cancer-causing chemical) to see if the fish oil/pectin diet was also protective at this time point. A number of important observations resulted from the series of experiments. Fish oil/pectin protected against DNA damage in the distal colon (during the 12 h time period) more than corn oil/pectin. There appears to be an interaction between DNA repair and apoptosis in that as apoptosis decreases, the expression of DNA repair enzyme increases. In addition, a different diet response to DNA damage was observed in the proximal versus the distal colon that may be important to the interpretation of site-specific colon cancers. 

References:

Fan, Y.Y., Zhang, J., Barhoumi, R., Burghardt, R.C., Turner, N.D., Lupton J.R., and Chapkin, R.S. (1999). Antagonism of CD95 (APO-1/Fas) signaling blocks butyrate induction of apoptosis in young adult mouse colonic (YAMC) cells. American Journal of Physiology 277 (Cell Physiology 46): C310 C319.

Hong, M.Y., Chapkin, R.S., Wild, C.P., Morris, J.S., Wang, N., Carroll, R.J., Turner, N.D., and Lupton, J.R. (1999). Relationship between DNA adduct levels, repair enzyme and apoptosis as a function of DNA methylation by azoxymethane. Cell Growth & Differentiation 10:749-758.

Lupton, J.R., Chang, W. C-L., Hong, M.Y., and Chapkin, R.S. (1999). Fat/fiber interactions on colonic cytokinetics: relationship to colon cancer. Asia Pacific Journal of Clinical Nutrition: 8:S37-S40.

Description:

Thhe 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 the health concerns of citizens in rural communities of the State of Texas and the nation. The routine activities coordinated by the Administrative Core include: scheduling of Executive Committee and Research 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.

• Members:

• 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, Department of Veterinary Physiology & Pharmacology and Department of Environmental and Occupational Health

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

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

• Marcy Whited, B.B.A.
  Administrative Assistant, Department of Veterinary Physiology and Pharmacology

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

 

Internal and External Advisory Committees:

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 provides advice and guidance on scientific and administrative concerns and also serves as an advocate for the CERH within the Texas A&M University System. This committee meets biannually and includes:

• Robert Kennedy, Ph.D.
  Vice-President for Research and Associate Provost for Graduate Studies
  Office of Graduate Studies & Research
  Texas A&M University
  College Station, Texas 77843-1112

• Edward Hiler, Ph.D.
  Vice Chancellor and Dean
  College of Agricultural & Life Sciences
  Texas A&M University
  College Station, Texas 77843-2142

• H. Richard Adams, Ph.D.
  Dean, College of Veterinary Medicine
  Texas A&M University
  College Station, Texas 77843-4461

• Richard Ewing, Ph.D.
  Dean, College of Science
  Texas A&M University
  College Station, Texas 77843-3257

• Roderick McCallum, Ph.D.
  Interim Vice President for Health Affairs and Dean of Medicine
  College of Medicine
  Texas A&M University System Health Science Center
  College Station, Texas 77843-1114

• Ciro V. Sumaya, M.D.
  Dean, School of Rural Public Health
  Texas A&M University System Health Science Center
  College Station, Texas 77843-1256

 

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. Members of the External Advisory Committee include:

• Daniel Acosta, Ph.D.
  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

 

Biostatistics and Epidemiology Research Core Report

Goals and Objectives:

The goal of the Biostatistics and Epidemiology Research Core is to develop new biostatistical methods related to environmental and rural health, and to perform epidemiologic studies to examine the relationship between risk factors as disease. The specific objectives of the members of the research core in the past year have been as follows.

1. In conjunction with the Nutrition Research Core, to develop biostatistical methods to help understand the origin of colon cancer, and factors, such as apoptosis, affected by diet.

2. To estimate the risk and origin of breast cancer associated with molecular interactions of various pregnancy-related proteins.

3. To develop biostatistical methods to analyze correlated longitudinal and spatial data using subsampling methods.

4. 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.

5. To develop biostatistical methods for apportionment of the sources of environmental air pollutants and toxicants.

Members:

Raymond J. Carroll, Ph.D., Director
Distinguished Professor, Department of Statistics

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

Barbara Richardson, Ph.D.
Assistant Professor, Department of Biostatistics and Epidemiology and Veterinary Anatomy and Public Health

Michael Sherman, Ph.D.
Assistant Professor, Department of Statistics

Clifford Spiegelman, Ph.D.
Professor, Department of Statistics

Naisyin Wang, Ph.D.,
Associate Professor, Department of Statistics

Soujin Wang, Ph.D.
Professor, Department of Statistics

• Key Words:

Measurement Error

Non-linear Models

Missing Data

Longitudinal Data Analysis

Spatial Modeling

Mixed Linear Models

Receptor Modeling

Chemometrics

Breast Cancer Research

Genetic Epidemiology

• Progress Report:

In addition to the collaborations among members internal to the Biostatistics and Epidemiology Research Core, and especially among the members of the Department of Statistics, the CERH has led to the development of several major new collaborations during the past two years.

Dr. James A. Calvin is currently working with Dr. K.C. Donnelly, of the Chemical Biology Research Core, on a study of pesticide exposure in young children in South Texas. He also collaborates with Dr. K. Ramos on the development of new statistical tools to evaluate DNA transfection and ectopic gene expression data.

Dr. Naisyin Wang is currently on sabbatical leave at the University of Washington. She and Dr. Raymond Carroll have been working on a research project that involves faculty in the Nutrition (Chapkin, Lupton, and Turner) and Biostatistics and Epidemiology Research Cores. They have cast the problem as involving multivariate mixed models and have developed new statistical methods to investigate relationships between high DNA adduct levels in proximal parts of the colon and its association with distal regions. One of the more exciting findings is the robustness of the results obtained. These investigators hypothesized that a variety of methods from parametric to nonparametric analysis ought to give the same answers to this problem, and that indeed is what was found. The collaboration has lead to development of statistical methods not previously considered by other investigators. The current focus is on the relationship of DNA adduct levels and apoptosis. This involves a mixture of classical random effects modeling and binary random affects modeling, as well as nonparametric versions of both. One paper has been published from this collaboration to date, and three papers have been submitted for publication. In addition, a grant proposal based on this work was submitted to the National Institutes of Health in February, with Drs. Carroll, Suojin Wang, Mallick and Lupton as co-investigators, and received a fundable score (5.6 percentile).

Dr. Suojin Wang has collaborated with Center investigators (R. J. Carroll, Naisyin Wang and Rosemary Walzem). His Ph.D. student C. Huang also has had a great learning experience of working with different investigators as a statistical consultant at the Statistical Help Desk.

Because of the existence of the CERH, Dr. Michael Sherman has been able to continue his research in subsampling and analysis of spatial data. In particular, he has greatly benefited from his collaboration with Dr. Sherry Bame (Departments of Urban Planning and Health Policy and Management) and Dr. K. Ramos of the Chemical Biology Research Core in an ongoing study of links between end stage renal failure and nephrotoxins in drinking water. The CERH has provided startup funding for the group to sample sites for their metal level in counties of both high and low risk of renal failure.

Dr. Clifford Spiegelman worked with G. Cote’s lab in the bioengineering department to increase detection levels for glucose sensors and cancer detection. Besides a number of papers, 2 patents were filed with the A&M licensing office. One provides a new approach to calibrations for glucose sensors that decrease detection limits by half. The other identifies important wave numbers for glucose sensoring. The ability to find important wave numbers means that smaller and cheaper and more accurate glucose sensors (for diabetics) can be built.

• Publications:

Cameron, B.D., Coté, G.L., McShane, M.J., Motamedi, M. and Spiegelman, C.H. (1999). A novel peak-hopping stepwise feature selection method with raman spectroscopy. Analytica Chemica Acta, 388, 251-264.

Carroll, R.J. (1999). Measurement error: a review. In Uncertainties in Radiation Dosimetry and Their Impact on Dose Response Analysis, E. Ron, editor. National Cancer Institute Press.

Carroll, R. J. (1999). Risk assessment with subjectively derived doses. In Uncertainties in Radiation Dosimetry and Their Impact on Dose Response Analysis, E. Ron, editor. National Cancer Institute Press.

Carroll, R. J., Roeder, K. and Wasserman, L. (1999). Flexible parametric measurement error models. Biometrics, 55, 44-54.

Edwards, J.C., Maldonado, F.G. and Calvin, J.A. (1999). The effects of differently weighting interview scores on the admission of underrepresented minority medical students. Academic Medicine, 74, 59-61.

Henry, R., Park, E. (1999). Comparing a new algorithm with the classic methods for estimating the number of factors. Chemometrics and Intelligent Laboratory Systems, 48, 91-97.

Hong, M. Y., Chapkin, R. S., Wild, C. P., Morris, J. S., Wang, N., Carroll, R. J., Turner, N. D. and Lupton, J. R. (1999). Relationship between DNA adduct levels, repair enzyme and apoptosis as a function of DNA methylation by azoxymethane. Cell Growth and Differentiation, 10,749-758.

Iturria, S., Carroll, R. J. and Firth, D. (1999). Multiplicative measurement error estimation: estimating equations. Journal of the Royal Statistical Society, Series B, 61, 547-562.

Lemke, S.L., Mayura, K., Ottinger, S.E., McKenzie, K. S., Wang, N., Fickey, C., Kubena, L.F. and Phillips, T.D. (1999). Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay. Journal of Toxicology and Environmental Health, 56, 283-295.

Ottinger, S.E., Mayura, K., Lemke, S.L., McKenzie, K. S., Wang, N., Kubena, L.F. and Phillips, T.D. (1999). Utilization of electrochemically generated ozone in the degradation and detoxification of benzo[a]pyrene. Journal of Toxicology and Environmental Health, 56, 565-583.

Schafer, D. W., Stefanski, L. A. and Carroll, R. J. (1999). Consideration of measurement errors in the international radiation study of cervical cancer. In Uncertainties in Radiation Dosimetry and Their Impact on Dose Response Analysis, E. Ron, editor. National Cancer Institute Press.

Sherman, M. (1999). Batch Variance Estimators for the Median of Simulation Output, Operations Research Letters, 23, 129-134.

Sherman, M. (1999). Inferences for spatial statistics and asymptotic normality, Far East Journal of Statistics, 3, 275-293.

Wang, N., Lin, X. and Gutierrez, R.G. (1999). A bias correction regression calibration approach in generalized linear mixed measurement error models. Communications in Statistics, Theory and Methods, 28, 217-233.

Wang, S.J. (1999). Importance sampling, Updated Encyclopedia of Statistical Sciences. Vol. 3, pp. 350--355.

 

 

Goals and Objectives:

The overall goal of the Chemical Biology Research Core is to develop and promote research activities that focus on the application of chemical and biological approaches to study fundamental processes related to the pathogenesis of environmental disease. In November, Dr. Hagan Bayley assumed leadership of the core and begun to work closely with other core investigators to promote intra- and inter-core collaborative research activities spanning the area of chemical biology. Drs. Sacchettini and Wilson were recruited as full members, and Dr. Dangott as an associate member, to strengthen the protein biology and gene regulation capabilities of the core.

The research efforts of core members focus on three areas of activity, namely, molecular signaling, molecular detection systems and neuro-endocrinology. The specific objectives of the members of the research core in the past year have been as follows.

1. To identify mechanisms of gene deregulation by environmental chemicals (Busbee, Castiglioni, Ramos, Safe, Wilson).

2. To define molecular mechanisms of chemical toxicity (Bayley, Busbee, Castiglioni, Derr, Dees, Donnelly, Johnson, Kier, Phillips, Ramos, Russell, Sacchettini, Safe, Wild, Wilson).

3. To investigate the influence of environmental factors on aging (Busbee, Johnson, Kier, Wilson).

4. To develop biologically based molecular detection systems that help define the genetic basis of human disease (Bayley, Busbee, Castiglioni, Derr, Donnelly, Kier, Phillips, Ramos, Russell, Sacchettini, Safe, and Wild).

5. To develop molecular technologies for environmental decontamination and protection (Donnelly, Phillips, Safe, Wild).

6. To delineate molecular mechanisms of action of environmental estrogens (Busbee, Kier, Safe).

These efforts are of direct relevance to rural communities where the incidence of exposures to agricultural chemicals, food-borne toxicants, and ambient contaminants is often elevated relative to the general population.

Members:

Hagan Bayley, Ph.D., Director
Professor and Head, Department of Medical Biochemistry and Genetics

David Busbee, Ph.D.Professor,
Department of Veterinary Anatomy and Public Health

Evelyn Castiglioni, Ph.D.Professor and Head,
Department of Veterinary Anatomy and Public Health

Larry Dangott, Ph.D., Associate Member
Research Scientist, Department of Chemistry

Les Dees, Ph.D.Professor,
Department of Veterinary Anatomy and Public Health

James Derr, Ph.D.Assistant Professor,
Department of Veterinary Pathobiology

Kirby Donnelly, Ph.D.Associate Professor,
Departments of Veterinary Anatomy and Public Health, Soil and Crop Sciences, and Environmental and Occupational Health

Larry Johnson, Ph.D.Professor,
Department of Veterinary Anatomy and Public Health

Ann Kier, D.V.M.Professor and Head,
Department of Veterinary Pathobiology

Timothy Phillips, Ph.D.Professor,
Department of Veterinary Anatomy and Public Health

Steve Safe, D.Phil.
Distinguished Professor, Departments of Veterinary Physiology and Pharmacology and Environmental and Occupational Health

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

David Russell, Ph.D.Professor,
Department of Chemistry

Jim Sacchettini, Ph.D.Professor,
Department of Biochemistry and Biophysics

Jim Wild, Ph.D.Professor and Head,
Department of Biochemistry and Biophysics

Emily Wilson, Ph.D.Assistant Professor,
Department of Medical Physiology

Key Words:

Aging

Aryl Hydrocarbon Receptor

Biosensors

Dioxin

Endocrine Disruption

Gene Regulation

Molecular Detoxification

Nitroaromatics

Polycyclic Aromatic Hydrocarbons

Pesticides

Progress Report:

Regular meetings served to promote scientific interactions and formal collaborations within the core, as well as with other CERH members. The major benefit realized by core members continues to be the increased access to state-of–the-art technologies that facilitate studies to elucidate fundamental biological events involved in cell determination, growth, differentiation, aging, as well as the onset and progression of diseases that share an environmental etiology. Tangible measures of success include breakthroughs in the identification of novel transcription factors and co-activators involved in the regulation of xenobiotic-responsive genes, understanding of gene/protein structure and function relationships, neuro-endocrine/toxicant interactions, and genotoxicity of complex polycyclic aromatic hydrocarbon mixtures. These research activities often relied upon the use of core facilities. In collaboration with members of the Reproductive and Developmental Biology Research Core and the Biostatistics and Epidemiology Research Core, core members continue to promote a major initiative to investigate the link between environmental exposures and birth defects at the genetic and epidemiologic levels. These efforts lead to submission of a research grant application to develop community-based research programs in the Lower Rio Grande Valley. In addition, two new grant applications were submitted to expand CERH outreach efforts in rural communities in collaboration with members of the Chemical Biology research core. One of the applications is to develop a partnership between public schools and the CERH in order to disseminate information and research findings on environmental heath. The project is under the direction of Dr. Larry Johnson and was funded by NIEHS.

Research in the Bayley laboratory is aimed at devising biosensors for the identification and quantification of toxic substances in physiological fluids, foodstuffs and the environment. The Bayley group has focused on "stochastic sensing", in which the current through a single pore is monitored, allowing the determination of not only the concentration of an analyte, but also its identity from its distinctive current signature. A novel form of genetic engineering, non-covalent targeted chemical modification, has been used to modify the bacterial pore-forming protein staphylococcal a-hemolysin and place a binding site for organics within the conductive pathway. His expertise will benefit many members of the core interested in the study of structure/function relationships at the protein level and these opportunities will be explored aggressively in the next year.

Work in Dr. Donnelly’s laboratory has established that risk assessment of genotoxic potential for complex mixtures of polycyclic aromatic hydrocarbons should not be based solely on benzo(a)pyrene since metabolism or repair mechanisms can markedly influence toxicological outcomes. This work supports collaborations between Drs. Castiglioni, Donnelly, Ramos, and Safe, which have been funded by Agency Toxic Substances and Disease Registry (ATSDR) to study neurological, renal, and immunologic effects of complex mixtures. In separate studies, Dr. Castiglioni’s group has shown that lead (Pb) exposures induces the glucose-regulated protein (GRP78) in C6 rat glioma cells, an astrocyte-like cell line that accumulates Pb in culture. Given that Pb strongly binds to GRP78, a molecular chaperone in the endoplasmic reticulum, these investigators have suggested that Pb triggers increased GRP78 synthesis to maintain protein trafficking and overcome inhibition of protein function. The novelty of the Pb induction response resulted in a formal collaboration between Drs. Castiglioni and Ramos to define molecular mechanisms of stress protein induction by heavy metals. A joint Superfund Program Project grant application (in which Drs. Burghardt, Donnelly, Finnell, Phillips, and Safe are involved) was submitted seeking support for this collaboration.

Investigators in the Dees laboratory continued to work to define the mechanisms involved in the ability of insulin like growth factor-1 (IGF-1) to initiate the onset of puberty in rats and primates by inducing secretion of luteinizing hormone releasing hormone luteinizing hormone. In these studies, alcohol was shown to cause significant suppression of IGF-1 in female Rhesus monkeys, an event associated with blockade of increased pulsatile secretion of luteinizing hormone. In other studies using the rat, this group showed changes in the synthesis of specific isoforms of nitric oxide synthase within the ovary during pubertal development. A new grant application was funded (ES 9627-01) to support efforts directed at the elucidation of low level Pb effects on the neuro-endocrine axis. These studies will determine if insult occurs during pre- or post-natal time periods and what is/are the specific mechanisms of action.

In the Ramos laboratory, efforts during the past year continue to focus on the study of functional interactions between the aryl hydrocarbon receptor (AhR) and transactivation of electrophile response-cis acting elements (EpRE) within the regulatory region of toxicant inducible genes. Supershift experiments showed that CCAAT enhancer binding protein-beta (C/EBP-beta) binds to both EpRE and AhRE, a finding consistent with the presence of C/EBP sites within those consensus sequences. Surprisingly, the AhR itself was detected in the protein complex binding to the EpRE in the absence of an AhR binding site. These data suggest that C/EBP-beta and the AhR participate in the regulation of rat GST-Ya gene in mammalian cells. Drs. Dangott, Ramos, and Russell have entered into a collaboration using Matrix Assisted Laser Desorption Ionization- Mass Spectrometry (MALDI-MS) and Edman’s sequencing for definitive identification of critical transacting factors involved in the response triggered by aromatic hydrocarbons. Strong research collaboration between the Wilson and Ramos laboratories has focused on the study of NF-kB regulated expression of integrins and matrix proteins. This collaboration was funded by an institutional collaborative grant and will convert to RO1 funding from the NIH in April 2000.

Research efforts in the Safe laboratory focus on mechanisms of breast cancer cell growth and development of new AhR based drugs for treatment of this devastating disease. Initial interest in breast cancer research evolved from the study of toxic chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), agents that bind the AhR and exhibit antiestrogenic activity. TCDD also inhibited the induction of several genes by estradiol and studies were designed to determine the molecular mechanism of cross talk between the AhR and estrogen receptor signaling pathways. An important new finding was that GC-rich Spl protein binding sites are critical cis-genomic sites required for estrogen (ER) actions. Using a series of constructs containing wild type and mutant 5’-flanking sequences from the c-fos promoter, it was shown that a GC-rich motif containing an imperfect Spl-binding site was required for hormone-induced activity. This sequence also bound Spl protein, and co-incubation with the ER enhanced Spl-DNA binding. Ongoing studies in this laboratory have identified several other E2-responsive genes regulated via ER/Spl interactions and the differential cell- and gene promoter-specific effects of ER /Spl are also being investigated. Several studies on the molecular biology of estrogen action are being carried out in collaboration with Drs. Burghardt and Piedrahita, and their respective facility cores. The Safe laboratory also collaborates with the Castiglioni, Ramos, and Turner laboratories, among others.

This has been a very productive, collaborative year in the Wild laboratory relative to the development of enzyme-based biosensors that can be used to identify and discriminate between various classes of neurotoxic pesticides and chemical threat agents being used by terrorist groups and nations around the world. This collaboration involves Drs. Castiglioni and Harris, members of the Chemical Biology and Nutrition research cores of the CERH. In addition, it has been possible to rationally design enzymes with enhanced structural and catalytic capabilities by new combinatorial technologies that can be used as covalently bound catalysts in fire-fighting foams for the detoxification of pesticides and chemical threat agents. These efforts have lead to a broad appreciation of the applied opportunities to use enzymes to remove highly toxic neurologic inhibitors from surface and air-borne sources through bioremediation applications of hydrolytic enzymes from a variety of biological sources.

Publications:

Alejandro, N.F. and Ramos, K.S. (1999). Functional, morphological and molecular alterations induced by benzo(a)pyrene in the adult rat kidney. Toxicological Sciences 48, 29.

Alexander, D., Donnelly, K.C., and Tiffany-Castiglioni. E. (1999). Development of in vitro screening assays for potentially neurotoxic polycyclic aromatic hydrocarbons in C6 rat glioma cells. The Toxicologist 48:287.

Balendiran, G.K., Molina, J.A., Xu, Y., Torres-Martinez, J. Stevens, R. Focia, P.J., Eakin, A.E., Sacchettini, J.C. and Craig III, S.P. (1999). Ternary complex structure of human HGPRTase, PRPP, and Mg²⁺ and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding. Protein Science 8, 1023-1031.

Barhoumi, R., Ramos, K.S., Safe, S.H., Phillips, T.D. and Burghardt, R.C. (1999). Analysis of the subcellular distribution and cytotoxicity of benzo(a)pyrene in rat liver cells. Toxicological Sciences 48, 15.

Bayley, H. (1999). Designed membrane channels and pores. Current Opinions in Biotechnology 10, 94-103.

Bayley, H. (1999). Protein therapy: delivery guaranteed. Nature Biotechnology 17, 1066-1067.

Boronin, A.M., Ermakova, I.T., Sakharovsky, V.G., Grechkina, G.M., Starovoitov, I.I., Autenreith, R.L. and Wild, J.R. (2000). Ecologically Safe Destruction of Mustard-Lewisite Mixtures from the Russian Chemical Stockpile. Journal of Chemical Techonology and Biotechnology 75:1-7.

Castro-Rivera, E. and Safe, S. (1999). Comparative mechanisms of activation of estrogen receptor a by estrogen and 4’-hydroxytamoxifen. Gulf Coast Society of Toxicology, Houston, TX.

Castro-Rivera, E. and Safe, S. (1999). Estrogen- and antiestrogen- (through AhR) responsiveness in ECC-1 endometrial cancer cell Lines. 5^th Annual Meeting of Texas Forum on Female Reproduction, Houston, TX, May 13-14.

Castro-Rivera, E., Wormke, M. and Safe, S. (1999). Estrogen and aryl hydrocarbon responsiveness of ECC-1 endometrial cancer cells. Molecular Cellular Endocrinology 150:11-21.

Cheley, S., Braha, O., Lu, X., Conlan,S. and Bayley, H. (1999). A functional protein pore with a "retro" transmembrane domain. Protein Science 8, 1257-1267.

Chen, I., Hsieh, T., Thomas, T. and Safe, S. (1999). Identification of estrogen-induced genes downregulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin by coupling suppression subtractive hybridization and cDNA microarrays. Gulf Coast Society of Toxicology, Houston, TX.

Chen, I-C. and Safe, S. (1999). Identification of estrogen-induced genes downregulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 human breast cancer cells by suppression subtractive hybridization. 5^th Annual Meeting of Texas Forum on Female Reproduction, Houston, TX, May 13-14.

Chen, Y-H. and Ramos, K.S. (1999). Negative regulation of the rat GST-Ya gene in vascular smooth muscle cells. Toxicological Sciences 48, 273.

Chen, Y-H. and Ramos, K. S. (1999). Negative regulation of rat GST-Ya via antioxidant/electrophile response element is directed by a C/EBP site. Biochemical and Biophysical Research Communications 265, 18-23.

Cho, T., Wild, J.R., Tiffany-Castiglioni, E., and Donnelly, K.C. (1999). The use of OPH enzyme for the detoxification of methyl parathion. The Toxicologist 48:185.

Crow, R.T., Rosenbaum, B., Smith R., Ramos, K.S. and Sulikowsky, G.A. (1999). Landomycin A inhibits G₁/S cell cycle progression and induces apoptosis. Bioorganic and Medicinal Chemistry Letters, 9,1663-1666.

Cunin, R., Rani, C.S., VanVliet, F., Wild, J.R.. and Wales, M.E. (1999). Intramolecular signal transmission in enterobacterial aspartate transcarbamylases. (II): engineering cooperativity and allosteric regulation in the aspartate transcarbamylase of erwinia herbicola. Journal of Molecular Biology, 294:1401-1411.

Dees, W.L., Hiney, J.K. and Srivastava, V. (1999). Alcohol's effects on female puberty: The role of lGF-1. Alcohol Health Research World 22:165-169.

diSioudi, B.D., Grimsley, J.K., Lai, K. and Wild, J.R. (1999). Modification of near-active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity. Biochemistry 38:2866-2872.

diSioudi, B.D., Miller, C.L., Lai, K., Grimsley, J.K. and Wild, J.R. (1999). Rational design of organophosphorus hydrolase for altered substrate specificities. Chemico-Biological Interactions. 119:211-223.

Duan, R. and Safe, S. (1999). Estrogen-mediated activation of c-fos protooncogene through protein binding the serum response element. Gulf Coast Society of Toxicology, Houston, TX.

Duan, R. and Safe, S. (1999). Transcription activation of c-fos protooncogene by 17b -estradiol: mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated inhibition. 5^th Annual Meeting of Texas Forum on Female Reproduction, Houston, TX, May 13-14.

Duan, R., Porter, W., Samudio, I., Vyhlidal, C., Kladde, M. and Safe, S. (1999) Transcriptional activation of c-fos protooncogene by 17b -estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition. Molecular Endocrinology 13:1511-1521.

Eicken, C., Krebs, B. and Sacchettini, J. S. (1999). Catechol oxidase – structure and activity. Current Opinion in Structural Biology. 9, 677-683.

Falahatpisheh, M.H. and Ramos, K.S. (1999). Nephrotoxicity assessment of selected polycylic aromatic hydrocarbons. Toxicological Sciences 42, 27.

Fan, Y.Y., Ramos, K.S. and Chapkin, R.S. (1999). Modulation of atherogenesis by dietary gamma-linolenic acid. In: Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation and Related Diseases, 4. (Honn, K.U., ed.), Plenum Press, New York. pp. 485-492.

Figueroa, I. and Russell, D.H. (1999). Matrix-assisted laser desorption ionization hydrogen/deuterium exchange studies to probe peptide conformational changes. Journal of the American Society for Mass Spectrometry 713-719.

Flounders, A.W., Singh, A.K., Volponi, J.V., Carichner, S.C., Wally, K., Schoeniger, J.S., Simonian, A.L. and Wild, J.R. (1999). Development of sensors for direct Ddetection of organophosphates: Sol-gel modified field effect transistor with immobilized organophosphate hydrolase. Biosensors & Bioelectronics 14:713-720.

Garcia, S. and Donnelly, K.C. (1999). Genotoxicity analysis of contaminated environmental media. In: G.I. Sunahara, A.Y. Renoux, C.L. Gaudet, C. Thellen and A. Pilon (Eds.) Environmental Analysis of Contaminated Sites: Toxicological Methods and Approaches. John Wiley & Sons, Publ.

Grimsley, J.K., diSoudi, B.D. and Wild, J.R. (1999). Enzyme engineering for the improved degradation of organophosphorus neurotoxins. Biotechnology International 2:235-242.

Gu, L., Braha, O., Conlan, S. Cheley, S. and Bayley, H. (1999). Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686-690.

Hanneman, W.H., Legare, M.E., Hong, S.H., Barhoumi, R., Burghardt, R.C., Safe, S.H., and Tiffany-Castiglioni, E. (1999). Sustained increase in intracellular calcium but not CYP1A1 gene activity in rat astroglial cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Seventeenth International Neurotoxicology Conference, Little Rock, Arkansas, October 17-20.

Hiney, J.K., Dearth, R., Lara, F., Wood, S., Srivastava, V. and Dees, W.L. (1999). Effects of ethanol on leptin secretion and the leptin-induced release from juvenile female rats. Alcohol: Clinical and Experimental Research 23:1785-1792.

Holderman, M.T., Miller, K.P. and Ramos, K.S. (1999). Protein interactions with the electrophile response element induced by benzo(a)pyrene and related oxidants. Toxicological Sciences 48, 273.

Kasianowicz, J.J., Burden, D.L., Han, L.C., Cheley, S., and Bayley, H. (1999). Genetically engineered metal ion binding sites on the outside of a channel's transmembrane beta barrel, Biophysical Journal 76, 837-845.

Kerzee, J.K. and Ramos, K.S. (1999). Role of the aryl hydrocarbon receptor and P450 metabolism in the redox regulation of c-Ha-ras by benzo(a)pyrene in vascular smooth muscle cells. Toxicological Sciences 48, 272.

Kerzee, J.K. and Ramos, K.S. (1999). Role of the aryl hydrocarbon receptor in the regulation of c-Ha-ras in vascular smooth muscle cells by benzo(a)pyrene, a tobacco smoke constituent. The FASEB Journal 13, A197.

Kim, J-W., Rainina, E.I., Engler, C.R. and Wild, J.R. (1999). Processing efficiency of immobilized non-growing bacteria: biocatalytic modeling and experimental analysis. Canadian Journal of Chemical Engineering 77:883-892.

Kim, K.H. and Safe, S. (1999). Enhanced DNA-independent transcriptional activity of zinc finger domain - deleted mouse estrogen receptor through the estrogen receptor/Sp1 protein interaction. Gulf Coast Society of Toxicology, Houston, TX.

Kurokawa, H., Dewan, J. C., Mikami, B., Sacchettini, J. C. and Hirose, M. (1999). Crystal structure of hen apo-ovotransferrin. Journal of Biological Chemistry 274, 28445-28452.

Lee, J-E., Sethi-Gupta, M. and Safe, S. (1999). 3’,4’-Dimethoxyflavone as an aryl hydrocarbon receptor antagonist in breast cancer cells. Gulf Coast Society of Toxicology, Houston, TX.

Letcher, R.J., van Holsteijn, I., Drenth, H.J., Norstrom, R.J., Bergman, A., Safe, S., Pieters, R. and van den Berg, M. (1999). Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells. Toxicology and Applied Pharmacology 160:10-20.

Li, X., Porter, W., Castro-Rivera, E., Chin, Q., Duan, R., Saville, B., Sun, G., Xie, W. and Safe, S. (1999). Effects of ligand-structure on estrogen/antiestrogen induction via estrogen receptor/Sp1 interactions with GC-rich promoter elements. Gulf Coast Society of Toxicology, Houston, TX.

Lindahl, L.S., Bird, L., Legare, M.E., Mikeska, G., Bratton, G.R., and Tiffany-Castiglioni, E. (1999). Differential ability of astroglia and neuronal cells to accumulate lead: Dependence on cell type and on degree of differentiation. Toxicological Sciences, 50:236-243.

Lindahl, L.S., Bird, L., Legare, M.E., Mikeska, G., Bratton, G.R., and Tiffany-Castiglioni, E. (1999). Differential ability of astroglia and neuronal cells to accumulate lead: Dependence on cell type and on degree of differentiation. Seventeenth International Neurotoxicology Conference, Little Rock, Arkansas, October 17-20.

Lindahl, L.S., Contreras, Y., Mikeska, G., Bratton, G.R., and Tiffany-Castiglioni, E. (1999). The ability of glutathione and glutathione-altering agents to modulate brain lead accumulation in the developing rat pup. Seventeenth International Neurotoxicology Conference, Little Rock, Arkansas, October 17-20.

Lu, K.P. and Ramos, K.S. (1999). Identification of genes differentially expressed in benzo(a)pyrene treated vascular smooth muscle cells. Toxicological Sciences 48, 272.

Lücke, C., Fushman, D., Ludwig, C., Hamilton, J.A., Sacchettini, J.C. and Rüterjans, H. (1999). A comparative study of the backbone dynamics of two closely related lipid binding proteins: bovine heart fatty acid binding protein and porcine ileal lipid binding protein. Molecular Cellular Biochemistry 192, 109-121.

Mayura, K., Dwyer, M.R., McKenzie, K.S., Washburn, K.S., Huebner, H.J., Donnelly, K.C.. Kubena, L.F., and Phillips T.D. (1999). Evaluation of the toxicity of polycyclic aromatic hydrocarbons derived from coal tar utilizing the Chick embryotoxicity screening test (CHEST).

McDougal, A.J. and Safe, S. (1999). Antiestrogenic and antitumorigenic activities of diindolylmethane analogs. 5^th Annual Meeting of Texas Forum on Female Reproduction, Houston, TX, May 13-14.

McDougal, A.J. and Safe, S. (1999). Aryl hydrocarbon receptor-mediated inhibition of mammary tumor growth in an athymic nude mouse model bearing MCF-7 cell xenografts. Gulf Coast Society of Toxicology, Houston, TX.

Miller, K.P., Holderman, M.T. and Ramos, K.S. (1999). Further characterization of EpRE binding proteins and their function in vascular smooth muscle cells. Toxicological Sciences 48, 134.

Morawietz, H., Ma, Y.-H., Vives, F., Wilson, E., Sukhatme, V.P., Holtz, J., and Ives, H.E., (1999). Mechanical strain induces early growth response gene-1 expression in vascular smooth muscle cells. Circulation Research 84 678-687.

Morrow, M.D., McDougal, A. and Safe, S. (1999). Methylene-substituted 1,1’-dimethyldiindolylmethane analogs as inhibitors of carcinogen-induced mammary tumor growth in rodents. Gulf Coast Society of Toxicology, Houston, TX.

Murphy, E.J., Edmondson, R.D., Russell, D.H., Colles, S., and Schroeder, F. (1999). Isolation and characterization of two distinct forms of liver fatty acid binding protein from the rat. Biochimica et Biophysica Acta, 413-425.

Nguyen, T.A. and Safe, S. (1999). Cell and promoter-specific interactions of steroid receptor coactivators with estrogen receptor a (ERa ) and ERa /Sp1. Gulf Coast Society of Toxicology, Houston, TX.

Nguyen, T.A. and Safe, S. (1999). Physical interactions: estrogen receptor (ER)/Sp1 protein and ER coactivators. 5^th Annual Meeting of Texas Forum on Female Reproduction, Houston, TX, May 13-14.

Nguyen, T.A., Hoivik, D., Lee, J-E. and Safe, S. (1999). Interactions of nuclear receptor coactivator/corepressor proteins with the aryl hydrocarbon receptor complex. Archives of Biochemistry and Biophysics 367:250-257.

Pallaroni, L., Saville, B., Lee, J-E., Stoner, M., Gaido, K. and Safe, S. (1999). Cell context-dependent estrogen receptor a (ERa ) agonist and ERb antagonist activities of methoxychlor metabolites. Gulf Coast Society of Toxicology, Houston, TX.

Qian, Y, Harris, E.D., Zheng, Y., and Tiffany-Castiglioni, E. (1999). Lead (Pb) targets the molecular chaperone GRP78 in C6 rat glioma cells. Seventeenth International Neurotoxicology Conference, Little Rock, Arkansas, October 17-20.

Qian, Y., Falahatpisheh, M.H., Ramos, K., and Tiffany-Castiglioni, E. 1999). Expression of 78 kD glucose-regulate protein (GRP78) in Pb and Hg-exposed rat C6 and SSC-1 cells. Gulf Coast Chapter Society of Toxicology Annual Meeting, Houston, TX, November 19-20.

Qian, Y., Harris, E.D., Zheng, Y., and Tiffany-Castiglioni, E. (1999) Lead (Pb) targets A 78 kD glucose-regulated protein (GRP78) in C5 rat glioma cells. Gulf Coast Chapter Society of Toxicology Annual Meeting, Houston, TX, November 19-20.

Qian, Y., Mikeska, G., Harris, E.D., Bratton, G.R., and Tiffany-Castiglioni, E. (1999). Effect of lead exposure and accumulation on copper homeostasis in cultured C6 rat glioma cells. Toxicology and Applied Pharmacology. 138:41-49.

Qian, Y., Zheng, Y., and Tiffany-Castiglioni, E. (1999). Pb tolerance in C6 rat glioma cells. The Toxicologist 48:244.

Qin, C. and Safe, S. (1999). Transcriptional activation of ornithine decarboxylase gene expression by estrogens in MCF-7 breast cancer cells. Gulf Coast Society of Toxicology, Houston, TX.

Qin, C., Singh, P. and Safe, S. (1999). Transcriptional activation of insulin-like growth factor binding protein 4 by 17 b-estradiol in MCF-7 cells: role of estrogen receptor-Sp1 complexes. Endocrinology 140:2501-2508.

Ramamoorthy, K., Gupta, M.S., Sun, G., McDougal, A. and Safe, S.H. (1999). 3,3’,4,4’-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary and in human breast cancer cells. Carcinogenesis 20:115-123.

Ramos, K.S. (1999). Health effects in Vietnam veterans of exposure to herbicides. In Veterans and Agent Orange III. Health Effects of Herbicides Used in Vietnam. National Academy Press.

Ramos, K. S. (1999). Redox regulation of c-Ha-ras and osteopontin signaling in vascular smooth muscle cells: implications in chemical atherogenesis. Annual Reviews of Pharmacology and Toxicology. 29, 243-265.

Ramos, K.S. (1999). Regulation of gene expression via the electrophile response element. Toxicological Sciences, 48, 167.

Ramos, K.S. (1999). Regulation of mammalian gene expression via the antioxidant response element. XVII Congresso Nacional de Investigacion Biomedica, Universidad Autonoma de Nuevo Leon.

Ramos, K.S. (1999). Science of Dangerous Materials: Integrated Emergency Weapons of Mass Destruction Response. Bush School of Government and Public Services, Texas A&M University.

Ramos, K.S., Chen, Y.-H., Holderman, M.T. and Miller, K.P. (1999). Cell and promoter specific patterns of gene regulation via the electrophile response element. Toxicological Sciences 48, 168.

Randerath, K., Randerath, E., Zhou, G.D., Supunpong, N., He, L.Y., McDonald, T.J. and Donnelly, K.C. (1999). Genotoxicity of complex PAH mixtures recovered from contaminated lake sediments as assessed by three different methods. Environmental Molecular Mutagenesis 33:303-312.

Rooney, A.P., Honeycutt, R.L., Davis, S.K., and Derr, J.N. (1999). Evaluating a putative bottleneck in a population of bowhead whales from patterns of microsatellite diversity and genomic disequilibria. Journal of Molecular Evolution. 49:682-690.

Rooney, A.P., Merrit, D.B., and Derr, J.N. (1999). Microsatellite diversity in captive bottlenose dolphins (Tursiops truncatus). Journal of Heredity 90:228-331.

Rozwarski, D.A., Vilcheze, C., Sugantino, M., Bittman, R. and Sacchettini, J.C. (1999). Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) in complex with NAD⁺ and a C16 fatty acyl substrate. Journal of Biological Chemistry 274, 15582-15589.

Russell, R.J., Pishko, M.V., Simonian, A.L. and Wild, J.R. (1999). Poly(ethylene glycol) hydrogel-encapsulated fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins. Analytical Chemistry. 71:4909-4912.

Safe, S. (1999). Ah receptor: activation and toxicity. British Toxicology Society Annual Meeting, Oxford, England.

Safe, S. (1999). Controversies and research in endocrine disruptors. The Annual Meeting of the American College of Occupational and Environmental Medicine, San Antonio, TX.

Safe, S. (1999). Crosstalk between aryl hydrocarbon receptor and estrogen receptor signaling pathways and antiestrogenic action of AhR agonists. Keystone Conference on Endocrine Disruptors, Tahoe City, CA.

Safe, S. (1999). Endocrine disruptors and human health - an update. The 19^th International Symposium on Halogenated Environmental Organic Pollutants and POPs, Venice, Italy.

Safe, S. (1999). Endocrine disruptors: the significance for human and mammals — the current view. Endocrine Disruption, Brussels, Belgium.

Safe, S. (1999). Environmental estrogens and human health. The 37^th Hanford Symposium on Health and the Environment, Richland, WA.

Safe, S. (1999). Environmental estrogens. In: Encyclopedia of Reproduction (E. Knobil and J.D. Neill, eds.), Academic Press, NY. Vol. 1, pp. 1100-1103.

Safe, S. (1999). Organochlorine contaminants and breast cancer - transcriptional activation of estrogen-induced gene expression. International Symposium on Breast and Prostate Cancer, German Society of Endocrinology, Elmau Castle, Klais, Germany.

Safe, S. (1999). Risk assessment of POPs - problems. Workshop on Persistent Manufactured Chemicals, the United Nations Environment Program, Geneva, Switzerland.

Safe, S., Wargovich, M.J., Lamartinierre, C.A. and Mukhtar, H. (1999). Symposium on mechanisms of action of naturally-occurring anticarcinogens. Toxicological Sciences 52:1-8.

Samudio, I., Vyhlidal, C. and Safe, S. (1999). Transcriptional activation of DNA polymerase a by estrogen in MCF-7 cells requires interaction of estrogen receptor a /Sp1 with a GC-rich element. Gulf Coast Society of Toxicology, Houston, TX.

Saville, B., Wormke, M. and Safe, S. (1999). Ligand-activated estrogen receptor a (ERa )/Sp1 action in breast cancer cells is dependent on the activation function 1 domain of ERa . Gulf Coast Society of Toxicology, Houston, TX.

Simonian, A.L., diSioudi, B.D. and Wild, J.R. (1999). An enzyme-based biosensor for the direct determination of diisopropylfluorophosphate Analytic Chimica Acta. 389:189-196.

Springman, K., Mayura, K. McDonald, T., Donnelly, K.C., Kubena, L., and Phillips, T. (1999). Organoclay adsorption of wood-preserving waste from groundwater: Analytical and Toxicological Evaluations. Toxicology and Environmental Chemistry. 100:1-13.

Srivastava, V., Hiney, J.K. and Dees, W.L. (1999). Effects of ethanol on the intraovarian insulin-like growth factor- 1 system in the prepubertal female rat. Alcohol: Clinical and Experimental Research 23:293-300.

Srivastava, V., Hiney, J.K., Rettori, V. and Dees, W.L. (1999). Effects of ethanol on intraovarian nitric oxide production in the prepubertal female rat. Journal of Endocrinology 161:69-75.

Stoner, M. and Safe, S. (1999). 17b -Estradiol (E2) negatively regulates vascular endothelial growth factor (VEGF) gene expression in HEC1-A human endometrial carcinoma cell line. 5^th Annual Meeting of Texas Forum on Female Reproduction, Houston, TX, May 13-14.

Stoner, M., Wang, F., Samudio, I., Vyhlidal, C., Kladde,M., Nguyen, T. and Safe, S. (1999). Downregulation of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor a and Sp3 proteins. Gulf Coast Society of Toxicology, Houston, TX.

Sun, G. and Safe, S. (1999). Mechanism of Ah receptor agonist in inhibition of estrogen-induced retinoic acid receptor a 1 gene expression in MCF-7 human breast cancer cells. 5^th Annual Meeting of Texas Forum on Female Reproduction, Houston, TX, May 13-14.

Sun, G., Samudio, I. and Safe, S. (1999). Inhibition of estrogen-induced retinoic acid receptor a 1 gene expression by TCDD - mechanisms of action. Gulf Coast Society of Toxicology, Houston, TX.

Tiffany-Castiglioni, E. and Lindahl, L. (1999). Complementarity and usefulness of in vitro approaches in lead toxicology: Commentary on a Forum position paper. Neurotoxicology 20:713-718.

Tiffany-Castiglioni, E., Ehrich, M., Dees, L., Costa, L.G., Kodavanti, P.R.S., Lasley, S.M., Oortgiesen, M., and Durham, H.D. (1999). Bridging the gap between in vitro and in vivo models for neurotoxicology. Toxicological Sciences, 51:178-183.

Vyhlidal, C. and Safe, S. (1999). Transcriptional regulation of transforming growth factor by a 17b -estradiol alone and in combination with 2,3,7,8-tetrachlorodibenzo-p-dioxin in human breast cancer cells. 5^th Annual Meeting of Texas Forum on Female Reproduction, Houston, TX, May 13-14.

Vyhlidal, C., Mach, C. and Safe, S. (1999). Regulation of transferrin gene expression by 17b -estradiol in human breast cancer cells. Gulf Coast Society of Toxicology, Houston, TX.

Wales, M.E. and Wild, J.R. (1999). Aspartate Transcarbamoylase. In Encyclopedia of Molecular Biology. Ed. Tom Creighton. Publisher Academic Press, NY. p196-201.

Wales, M.E., Madison, L.L., Glaser, S.S. and Wild, J.R. (1999). Divergent allosteric patterns establish a new regulatory paradigm for aspartate transcarbamoylase. Journal Molecular Biology. 294:1387-1400.

Wang, F., Duan, R. and Safe, S. (1999). Transcriptional activation of cathepsin D gene expression by growth factors. Gulf Coast Society of Toxicology, Houston, TX.

Wang, F., Wang, W. and Safe, S. (1999). Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex. Biochemistry 38:11490-11500.

Wang, W., Dong, L., Saville, B. and Safe, S. (1999). Transcriptional activation of E2F1 gene expression by 17b -estradiol in MCF-7 cells is regulated by NF-Y - Sp1/estrogen receptor interactions. Molecular Endocrinology 13:1373-1387.

Ward, T.J., Bielawski, J.P., Davis, S.K., Templeton, J.W., and Derr, J.N. (1999). Identification of domestic cattle hybrids in wild cattle and bison species: A general approach using mtDNA markers and the parametric bootstrap. Animal Conservation 2:51-57.

Wilson, E. (1999). Integrin signaling by mechanical forces in vascular smooth muscle cells. Young Vascular Biology Meeting, San Antonio TX.

Wilson, E. (1999). Regulation of Smooth Muscle Phenotype by Mechanical Forces. Inserm Unit 144 Paris, France.

Wilson, E. (1999). 2^nd Workshop on Vascular Biology and Mechanical Factors – Modulation of vascular smooth muscle response to mechanical strain by extracellular matrix-integrin interaction: Paris, France.

Wilson, E. (1999). Regulation of Smooth Muscle Phenotype by Mechanical Forces. Halle Germany.

Wormke, M., Castro-Rivera, C., Chen, I. and Safe, S. (1999). Estrogen and aryl hydrocarbon receptor expression and crosstalk in human Ishikawa endometrial cancer cells. Gulf Coast Society of Toxicology, Houston, TX.

Xie, W., Duan, R. and Safe, S. (1999). Estrogen induces adenosine deaminase gene expression in MCF-7 human breast cancer cells: role of estrogen receptor-Sp1 interactions. Endocrinology 140:219-227.

Xie, W., Duan, R. and Safe, S. (1999). Insulin-like growth factor-1 induces adenosine deaminase in MCF-7 human breast cancer cells through estrogen receptor-Sp1 interactions. Gulf Coast Society of Toxicology, Houston, TX.

Yoon, K., Pellaroni, L., Ramamoorthy, K., Gaido, K. and Safe, S. (1999). Ligand structure-dependent differences in activation of estrogen receptor a in human HepG2 liver and U2 osteogenic cancer cell lines. Gulf Coast Society of Toxicology, Houston, TX.

Zhang, Y., Autenrieth, R.L., Bonner, J.S., Harvey, S.P. and Wild, J.R. (1999). Biodegradation of neutralized sarin. Biotechnology and Bioengineering 64:221-231.

 

Goals and Objectives:

There have been no changes to the objectives of the Nutrition research core. The primary goal of the Nutrition research core is to understand the role of nutritional factors in outcomes associated with environmental exposures to toxic chemicals or anticarcinogens, especially as it relates to colonic cancer. Members of the core serve as an interface between the nutritional problems observed in rural communities and the molecular mechanisms of disease development. Core activities have supported research initiatives of core members and associate members, and through their body of expertise, core faculty have assisted in the performance of other Center projects that together addressed the goal of the Center — improving environmental and rural health. The specific objectives of the members of the research core in the past year have been as follows.

• To identify diet components that help reduce or delay onset of colon carcinogenesis (Chapkin, Lupton, Turner, Wu).

• To evaluate non-invasive biomarkers of colon cancer as a means of monitoring dietary anticarcinogens (Chapkin, Lupton, Turner).

• To determine how dietary minerals exacerbate the response to environmental toxins (Harris).

• To evaluate mechanisms of intracellular lipid transport and metabolism (Schroeder).

• To assess the mediation of androgen-stimulated human prostate epithelial growth by fibroblast growth factor (McKeehan).

• To elucidate the cellular and molecular mechanisms by which diet modulates immune-mediated inflammatory disease, and alters host resistance to infectious pathogens (Chapkin, McMurray).

Members:

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

Wallace L. McKeehan, Ph.D., Associate Member
Professor, Institute of Bioscience and Technology

David N. McMurray, Ph.D.
Professor, Department of Medical Microbiology and Immunology

Friedhelm Schroeder, Ph.D.
Professor, Department of Veterinary Physiology and Pharmacology

Nancy D. Turner, Ph.D., Associate Member
Research Assistant Professor, Department of Animal Science

Guoyao Wu
Associate Professor, Department of Animal Science

Environmental carcinogens or mutagens

Phytochemicals

Colon cancer

Prostate cancer

Minerals

Immune responsiveness

Biomarkers

 

Members of the Nutrition research core have come to better understand the research activities of all the other members of the core, as well as to have an appreciation of the research of members from other cores through jointly held CERH meetings. During the meetings we also have learned of the opportunities available through the various facility cores. The most recent core meeting concentrated on mechanisms to increase the transfer of our research efforts to the activities of the COEP. The results of our increased collaborations and access to facility cores are described below.

Interaction between Drs. Lupton, Chapkin, Turner, Wu of the Nutrition Research Core, Drs. Carroll and Wang of the Biostatistics Epidemiology Research Core, and Dr. Burghardt of the Reproductive and Developmental Biology Core has led to the submission of four new proposals to the National Cancer Institute of NIH. One proposal was designed to compare the differences between small intestine and large intestine cancer development. It is currently undergoing an Accelerated Executive Review (Submitted September 26, 1999). In addition, a proposal was submitted to the Charlotte Geyer Foundation on October 1, 1999, to support acquisition of additional preliminary data in support of this NIH proposal. A full proposal was resubmitted to NIH on November 1. Another proposal submitted to NIH (October 1, 1998, resubmission July 1, 1999) seeks to determine if diet can decrease the expression of cyclooxygenase II, and thereby increase apoptosis leading to a reduction in colon cancer. In an attempt to develop a less invasive technique for detection of colon cancer, Drs. Chapkin, Lupton and Carroll submitted a proposal to NIH to study the effectiveness (in humans) of a patent-pending technique that they have developed for cancer evaluation in animals. Dr. Carroll initiated a proposal to develop statistical techniques to analyze data from nutrition and cancer studies, which involves Dr. Lupton as a co-investigator. Besides the NIH proposals, Drs. Turner, Chapkin, Lupton, Carroll and Wang submitted a proposal to the Texas Higher Education Coordinating Board to use isolated mRNA from rectal eluate of patients undergoing colonoscopy for analysis of expression of key proteins involved in colon cancer development.

In addition to studying colon cancer, Dr. Chapkin, in collaboration with Dr. McMurray, studies the influence of select polyunsaturated fatty acids on T-cell intracellular signal transduction. Their NIH-funded research program is in the first year of work and is progressing such that publications should be produced within the year. Dr. Chapkin also collaborates with Dr. Ramos in studies to evaluate the influence of nutritional gamma linolenic acid on atherogenesis.

Dr. McKeehan is striving to determine how fibroblast growth factor mediates androgen-induced growth in stromal epithelial cells of the prostate. Mechanistic studies to determine the signaling pathways in this process resulted in 10 papers and abstracts from this laboratory. He continues to collaborate with Dr. Bazer of the Reproductive and Developmental Biology Research Core.

Dr. Schroeder works in collaboration with Drs. Kier from the Chemical Biology research core in determining the dynamics of lipid transport across membranes, within cells, and their metabolism. Specific emphases include binding to sterol carrier proteins, fatty acid binding proteins in various tissue beds, and metabolism in the liver. This area of research is supported by their joint NIH project. Their studies also include aspects of both the structure and function of intracellular lipid binding proteins, for which the expertise of Dr. Russell of the Chemical Biology Research Core has been invaluable. In addition, Dr. Schroeder has collaborated with Dr. Piedrahita of the Reproductive and Developmental Biology research core in the development of a gene targeted mouse in which the SCP-2 gene is ablated or overexpressed.

Drs. Turner and Lupton are collaborating with Dr. Nancy Ing of the Reproductive and Developmental Biology Core to evaluate the effect of dietary phytoestrogens on colon cancer development. The work was funded by a Pilot project grant to Dr. Turner, as well as additional outside funding acquired from the Houston Live Stock Show and Rodeo. Dr. Turner and Dr. Safe (Chemical Biology Research Core) are determining the role of a phytochemical prevalent in cruciferous vegetables in regulation of colon cell cytokinetics and induction of apoptosis. External funding will be pursued to further support the development of this research program in the coming year. Both of these research areas have resulted in abstracts, one presented at the annual meeting of the American Institute of Cancer Research (September, 1999) and another to be presented at the Experimental Biology meetings in 2000.

Dr. Wu's research emphases are the study of arginine metabolism in pig enterocytes and the regulation of nitric oxide synthesis in endothelial cells. Research found that proline is a major substrate for arginine and polyamine synthesis in enterocytes of suckling piglets. In addition, it was found that glucocorticoids play an important role in regulating intestinal expression of type II arginase. Another key finding was that dietary arginine deficiency impairs constitutive and inducible nitric oxide synthesis in young rats. It was also found that impaired nitric oxide synthesis in endothelial cells of diabetic BB rats is due to a deficiency of tetrahydrobiopterin. The final finding was that intestinal arginine synthesis is underdeveloped in preterm piglets. His collaborations with Drs. Bazer and Ing from the Reproductive and Developmental Biology Core generated a great deal of these findings.

 

Atshaves, B.P., Petrescu, A.D., Starodub, O., Roths, J.B., Kier, A.B., and Schroeder, F. (1999). Expression and intracellular processing of the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase in transfected mouse L-cell fibroblasts. Journal of Lipid Research 40:610-622.

Avdulov, N.A., Chochina, S.V., Igbavboa, U., Warden C.S., Schroeder, F., and Wood, W.G. (1999). Lipid binding to sterol carrier protein-2 is inhibited by ethanol. Biochimica et Biophysica Acta 1437:37-45.

Battershill, J.M., Greig, J.B. and Lupton, J.R. (1999). Miscellaneous substance: salatrim (short- and long-chain acyltriglyceride molecules). In: Evaluation of Certain Food Additives and Contaminants. WHO Technical Report Series 884, World Health Organization, Geneva, pp. 23-25.

Carney, A.D., Turner, N.D., Chapkin, R.S. and Lupton, J.R. (1999). Dietary inositol hexaphosphate reduces aberrant crypt formation in the colon of azoxymethane-injected rats. Presented at the 9^th Annual AICR Research Conference, Washington, DC., September 2-3.

Carney, A.D., Turner, N.D., Chapkin, R.S., and Lupton, J.R. (1999). Phytate helps prevent colon cancer. Proceedings of the TAMU Student Research Week, p. 25, Spring.

Chao, H., Billheimer, J.T., Kier, A.B., and Schroeder, F. (1999). Microsomal long chain fatty acyl CoA transacylation: Differential effect of sterol carrier protein-2. Biochimica et Biophysica Acta 1439:371-383.

Chapkin, R.S., Fan, Y.Y., Zhang, J., Barhoumi, R., Burghardt, R.C. and Lupton, J.R. (1999). Cell proliferation, apoptosis and signaling pathways as biomarkers for colon carcinogenesis. Toxicology Letters, Suppl. 1:15, W2-4.

Chapkin, R.S., Fan, Y.Y., Zhang, J., Barhoumi, R., Burghardt, R.C., Davidson, L.A., Turner, N.D., and Lupton, J.R. (1999). Antagonism of CD95 (APO-1/Fas) signaling blocks butyrate induction of apoptosis in young adult mouse colonic (YAMC) cells. Presented at Gastrointestinal Tract VIII: Signaling, Transport & Integration Conference, Copper Mountain, CO, July 25-30.

Chapkin, R.S., McMurray, D.N. and Jolly, C.A. (1999). Dietary n-3 polyunsaturated fatty acids modulate T-lymphocyte activation: Clinical relevance in treating diseases of chronic inflammation. In: Nutrition and Immunology: Principles and Practice. (Gershwin, M.E., German, B. and Keen, C. eds.), Plenum Publishing, New York, pp. 121-134.

Davidson, L.A., Lupton, J.R., Jiang, Y.H. and Chapkin, R.S. (1999). Carcinogen and dietary lipid regulate ras expression and localization in rat colon without affecting farnesylation kinetics. Carcinogenesis 20:785-791.

Dick, E.S., Davidson, L.A., Lupton, J.R. and Chapkin, R.S. (1999). Effect of docosahexaenoic acid on ras post-translational processing and localization in a transgenic mouse colonic cell line. FASEB J. 13:A584.

Dillon, E.L., Knabe, D.A., and Wu, G. (1999). Lactate inhibits citrulline and arginine synthesis from proline in pig enterocytes. American. Journal of Physiology 276: G1079-G1086.

Fan, Y.Y., Ramos, K.S. and Chapkin, R.S. (1999). Modulation of atherogenesis by dietary gamma-linolenic acid. In: Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation and Related Diseases, 4. (Honn, K.U., ed.), Plenum Press, New York. pp. 485-492.

Fan, Y.-Y., Zhang, J., Barhoumi, R., Burghardt, R.C., Turner, N.D., Lupton, J.R. and Chapkin, R.S. (1999). Antagonism of CD95 signaling blocks butyrate induction of apoptosis in young adult mouse colonic cells. American Journal of Physiology 277:C310-C319.

Flynn, N.E., Meininger, C.J., Kelly, K., Ing, N.H., Morris, S.M., Jr. and Wu, G. (1999). Glucocorticoids mediate the enhanced expression of intestinal type II arginase and argininosuccinate synthase in postweaning pigs. Journal of Nutrition 129: 799-803.

Frolov, A., So, P.T.C., Petrescu, A., Gratton, E. and Schroeder, F. (1999). Multiphoton excitation imaging of naturally occurring fluorescent sterol (dehydroergosterol) in intact cells. 43^rd Annual Biophysical Society Meeting, Baltimore, MD, Feb. 13, Biophysical Journal 76:A99.

Hong, M.Y., Chapkin, R.S., Carroll, R.J., Wang, N., Turner, N.D., Morris, J.S., Davidson, L.A. and Lupton, J.R. (1999). Fish oil is protective against colon tumorigenesis by two distinct mechanisms in a site specific manner. Proceedings of the TAMU Student Research Week, p. 52.

Hong, M.Y., Chapkin, R.S., Carroll, R.J., Wang, N., Turner, N.D., Morris, J.S., Davidson, L.A. and Lupton, J.R. (1999). Fish oil is protective against colon tumorigenesis by two distinct mechanisms in a site specific manner. FASEB J. 13:A540.

Huang, H., Ball, J., Billheimer, J.T., and Schroeder, F. (1999). Interaction of the sterol carrier protein-2 amino terminus: role of membrane curvature. Biochemical Journal 344:593-603.

Huang, H., Ball, J., Billheimer, J.T., and Schroeder, F. (1999). Structure and function of the sterol carrier protein-2 amino-terminus. Biochemistry 38:13231-13243.

Kan, M., X. Wu, F. Wang and W.L. McKeehan. (1999). Specificity for fibroblast growth factor (FGF) determined by the heparan sulfate subunit of a heparan sulfate-receptor kinase binary complex. Journal of Biological Chemistry 274: 15947-15952.

Lu, W., Luo, Y., Kan, M. and McKeehan, W.L. (1999). Fibroblast growth factor-10: a second candidate stromal to epithelial cell andromedin in prostate. Journal of Biological Chemistry 274: 12827-1283.

Lupton, J.R. and Turner, N.D. (1999). Carbohydrates - Dietary fiber. In: Biochemical & Physiological Bases of Human Nutrition. (Stipanuk, M., ed.), Saunders Co., Philadelphia, PA, pp. 143-154.

Lupton, J.R. and Turner, N.D. (1999). Potential protective mechanisms of wheat bran fiber. American Journal Medicine 106 (1A) 24S-27S.

Lupton, J.R., Chang, W.C.L., Hong, M.Y. and Chapkin, R.S. (1999). Fat/fiber interactions on colonic cytokinetics: Relationship to colon cancer. Asia Pacific Journal of Clinical Nutrition (Suppl.) S37-S40.

Lupton, J.R., Chapkin, R.S., Turner, N.D., Chang, W.C.L., Hong, M.Y., Carroll, R.J., Wang, N. and Morris, J.S. (1999). Fat/fiber interactions and their effect on colon cancer. Invited presentation to the Center for Environmental and Rural Health Annual Meeting, May 25.

McArthur, M.J., Atshaves, B.P., Frolov, A., Foxworth, W.D., Kier, A.B., and Schroeder, F. (1999). Cellular uptake and intracellular trafficking of long chain fatty acids. Journal of Lipid Research 40:1371-1383.

McKeehan, W.L., Wu, X. and Kan, M. (1999). Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex. Journal of Biological Chemistry 274: 21511-21514.

McMurray, D.N., Jolly, C.A. and Chapkin, R.S. (1999). Effect of dietary fatty acids on T-cell activation an d T-cell receptor (TcR) mediated signaling in a murine model. Presented at the NIH, Macronutrients and Infectious Diseases: Cellular and Molecular Immunomodulatory Mechanisms Meeting, Bethesda, MD, September 16-17.

Murphy, E.J., Edmondson, R.D., Russell, D.H., Colles, S., and Schroeder, F. (1999). Isolation and characterization of two distinct forms of liver fatty acid binding protein from the rat. Biochimica et Biophysica Acta 1436:413-425.

Murray, N.R., Davidson, L.A., Chapkin, R.S., Gustafson, W.C., Schattenberg, D.G. and Fields, A.P. (1999). Overexpression of protein kinase C beta II in the colonic epithelium causes hyperproliferation and increased sensitivity to colon carcinogenesis. Journal of Cell Biology 145:699-711.

Nakano, K., Fukabori, Y., Itoh, N., Kan, M., McKeehan, W.L. and Tamanaka, H. (1999). Androgen-stimulated human prostate epithelial growth mediated by stromal-derived fibroblast growth factor-10. Endocrine Journal. 46:405-413.

Nakano, K., Taniguchi, A., Kan, M. and McKeehan, W.L. (1999). Improved recovery of active radiolabeled TGFb 1 by TGFb receptor type III affinity chromatography. In Vitro Cellular and Developmental Biology-Animal 35: 241-243.

Pu, L., Annan, R.S., Carr, S.A., Frolov, A., Wood, W.G., Spener, F., and Schroeder, F. (1999). Isolation and identification of a native fatty acid binding protein from mouse brain. Lipids 34:363-373.

Pu, L., Igbavboa, U., Wood, W.G., Roths, J.B., Kier, A.B., Spener, F., and Schroeder, F. (1999). Expression of fatty acid binding proteins is altered in aged mouse brain. Molecular Cellular Biochemistry 198:69-78.

Sumner, L.W., Wolf, B.P., Russell, D.H., Dick, E.S., Davidson, L.A., Lupton, J.R. and Chapkin, R.S. (1999). Characterization of human ras protein by MALDI-TOF-MS. Presented at The American Society for Mass Spectrometry and Allied Topics Meeting, Dallas, TX. June.

Turner, N.D., Zhang, J., Davidson, L.A., Chapkin, R.S., Safe, S. and Lupton, J.R. (1999). Diindolylmethane reduced HT-29 colon cancer cell number by decreasing proliferation and increasing apoptosis. Presented at the 9^th Annual AICR Research Conference, Washington, DC., September 2-3.

Wang, F., Lu, W., McKeehan, K., Mohamedali, K., Gabriel, J.L., Kan, M. and McKeehan, W.L. (1999). Common and specific determinants for fibroblast growth factors (FGF) in the ectodomain of the receptor kinase complex. Biochemistry 38:160-171.

Wood, W.G., Schroeder, F., Avdulov, N.A., Chochina, S.V., and Igbavboa, U. (1999). Recent advances in brain cholesterol dynamics: transport, domains, and alzheimers disease. Lipids 34:225-234.

Wu, G., Flynn, N.E., Flynn, S.P., Jolly, C.A. and Davis, P.K. (1999). Dietary protein or arginine deficiency impairs constitutive and inducible nitric oxide synthesis by young rats. Journal of Nutrition 129: 1347-1354.

Wu, G., Ott, T.L., Knabe, D.A. and Bazer, F.W. (1999). Amino acid composition of the fetal pig. Journal of Nutrition 129: 1031-1038.

Zoran, D.L., Turner, N.D., Burkholder, W.J. and Lupton, J.R. (1999). Effects of insoluble dietary fiber on fecal short chain fatty acids and nitrogen in normal cats. Journal of Veterinary Internal Medicine 13:263.

Reproductive and Developmental Biology Research Core Report

Goals and Objectives:

Human populations are being exposed to an increasing number of potentially harmful agents through the environment; therefore, there is a clear need to understand the mechanisms by which exogenous compounds interact with critical aspects of reproduction and early embryonic development in humans, domestic animals and wildlife populations. The rate at which potentially hazardous reproductive toxicants enter the environment is rapidly outpacing our ability to effectively evaluate their safety. In addition to compounds introduced into the environment by human intervention, naturally occurring toxicants like phytoestrogens in plants and mold can impact reproduction and development. There are also reproductive effects of estrogen agonists in the environment that may positively or negatively impact the reproductive success. Both possibilities must be better understood

The Reproduction and Developmental Biology Core seeks to:

• Understand the impact of environmental toxicants on all aspects of reproduction and development.

• Identify and characterize genes conferring susceptibility to environmentally induced congenital malformations.

• Develop methods for the genetic modification of small and large mammals.

• Define molecular regulation of steroid receptors by environmental effectors.

• Determine the action of environmental toxicants on various aspects of cellular signal transduction.

• Determine how environmental factors affect brain development.

• Identify genes which are transcribed and translated during normal preimplantation development.

Members:

Fuller W. Bazer, Ph.D., Director
Professor, Department of Animal Science, Veterinary Anatomy and Public Health and Institute of Biosciences and Technology

James West, Ph.D., Associate Director
Professor, Department of Medical and Neurobiology

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

Nancy Ing, D.V.M., Ph.D.
Assistant Professor, Department of Animal Science

Laurie Jaeger, D.V.M., Ph.D.
Assistant Professor, Department of Veterinary Anatomy and Public Health

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

Mark Westhusin, Ph.D.
Associate Professor, Department of Veterinary Physiology and Pharmacology

1. Key Words:

Transgenics

Teratogens

Cloning

Pre-implantation

Genetic Sensitivity

Steroid Receptors

Uterine Environment

Signal Transduction

Neuronal Differentiation

Nuclear Transplantation

Progress Report:

The CERH has provided the basis for members of the Center to interact and create synergies in research efforts. Highlights of newly created or expanded collaborations are described below.

Drs. Abbott and Miranda collaborate to investigate the roles of intrinsic versus extrinsic factors responsible for Purkinje cell and granule cell death in the leaner cerebellum through the use of long-term cerebellar slice cultures. Dr. Richard Finnell also benefited from this collaboration to develop a collaborative relationship between Dr. Rajesh Miranda using an embryonic model of neural tube closure defects for which they received local funding-Risk Factors for Neural Tube Defects: Developing Strategies for Prevention-Texas A&M University Interdisciplinary Research Initiative Grant. This new collaboration was written into Dr. Finnell’s renewal (submitted November, 1998) application for his grant entitled: Fetal antiepileptic drug syndrome: a molecular analysis. National Institutes of Dental Research 2R01 DE 11303-05. Dr. R.H. Finnell, P.I.; Dr. J.A. Calvin, Dr. R. Miranda, Co-Investigators.

Collaborative interactions between Drs. Finnell and Piedrahita on folic acid and the development of neural tube defects resulted in production of founder mice lacking folate binding protein genes (FBP-1 and FBP-2). They have been determining the phenotype of these mice and exploring the impact of environmental arsenic as a model toxicant to learn more how a genetically sensitive population, those with genetically engineered folate transport defects, might respond to the developmental challenge. The focus is on how additional downstream genes might be abnormally regulated under this environmental perturbation. This work is supported by NIEHS grant. (Folate receptor knockouts, arsenate and birth defects, ES/HD35396: Dr. R.H. Finnell, Principal Investigator ; Dr. J.A. Piedrahita, Co-Principal Investigator) Initial observations in this research program concerning the development of craniofacial defects have led to the submission of a new grant application involving members of this research core. (Folate Receptors and Craniofacial Malformations, R01 ES/DE 09846: Dr. R.H. Finnell, Principal Investigator; Dr. J.A. Piedrahita, Co-Principal Investigator; Dr. R.C. Burghardt, collaborating investigator) Dr. Burghardt has contributed state-of-the-art imaging technologies to identify cellular changes occurring in response to the genetic manipulations. This collaborative research program would not have occurred in the absence of support from the Center

Drs. Safe and Finnell (former Director of the Reproductive and Developmental Biology Research Core) are working on Arnt protein as a prognostic indicator of survival in breast cancer patients. Using this genetic marker they are testing the hypothesis that AhR mediated signaling pathways may be useful as prognostic factors for mammary and potentially other forms of cancer in study populations, both in Texas and Nebraska, as well as in an environmentally challenged foreign population (Baku, Azerbaijan). Funding for this new collaborative effort is being sought through the NIH Superfund Research granting mechanism, as well as through local cancer research funding venues.

Biochemical signaling between the uterus and conceptus (embryo and its associated membranes) is essential for maintenance of ovarian corpora lutea (CL) and their continuous secretion of progesterone, which is essential for establishment and maintenance of pregnancy. Dr. Fuller Bazer, in collaboration with Drs. Laurie Jaeger, Nancy Ing, Tom Spencer and Robert Burghardt have been working together in characterizing the uterine environment with respect to steroid receptor regulation and integrin-mediated attachment, adhesion and signal transduction. Drs. Spencer, Miranda and Bazer are also working on effects of exposure of lambs to progestins during the neonatal period to block epigenetic events essential for development of uterine glands and normal uterine morphology. Drs. Westhusin, Piedrahita and Burghardt are working to evaluate and develop non-invasive imaging approaches to evaluate oocyte quality in several animal species. These efforts have resulted in submission of both USDA and NIH grant applications. In addition, Dr. Bazer’s collaborations with Dr. Piedrahita’s laboratory center on a project involving the isolation of porcine embryonic stem cells. The extent of the collaboration includes shared reagents, expertise, and a steady flow of graduate students moving between the laboratories on a regular basis. The success of these projects has led to support from biotechnology firms to build a new farrowing facility for the transgenic sows. The ability to attract grants and contracts to support these collaborative projects is due, in no small measure, to the support provided by the CERH.

Drs. Jaeger and Bazer are Co-Principal Investigators on Dr. Guoyao Wu’s (member of Nutrition Research Core) funded CERH pilot project (Arginine synthesis in the Fetal Pig Small Intestine). This project is to understand how endogenous and nutritional factors alter development of the small intestine and subsequent fetal somatic growth. A grant has been submitted to NIH for additional funding (Protein Nutrition and Fetal Intestinal Growth, RO1 DK56241: Dr. L.A Jaeger, PI; Dr. G. Wu, Co-PI; Dr. F.W. Bazer and Dr. N.H. Ing, collaborating investigators). Dr. Jaeger is also using the Image Analysis Laboratory’s laser confocal imaging capabilities to study conceptus adhesion molecules and this resulted in pilot data for a grant application to the National Science Foundation and to the United States Department of Agriculture. Vital imaging services of the Core are used to examine effects of estrogenic compounds of cultured porcine trophoblast cells in a project supported by the Texas Higher Education Coordinating Board.

Drs. Abbott and West continue to collaborate on a project to further delineate effects of alcohol exposure on the early postnatal development of the brain. The study of morphologic cellular changes at the ultrastructural level provides a clearer understanding of functional changes in individual cells when exposed to toxins such as alcohol, especially effects on cerebellar Purkinje cells within 24-48 hours after acute alcohol exposure. This type of exposure ultimately leads to Purkinje cell death and is supported by a NIH-NIAAA grant (Fetal Alcohol Syndrome - Third Trimester Model: Dr. West, (PI): Dr. Abbott, collaborating investigator). This collaboration between Dr. West and Abbott has been strengthened by the infrastructure afforded by the CERH. Additionally, Dr. Abbott is studying the process of cell death using an animal model, the leaner mouse, that exhibits excessive apoptosis of specific cells in the postnatal cerebellum, including Purkinje cells, granule cells and Golgi cells. The direct overlap of Purkinje cell death in this model with the alcohol model being studied by Dr. West provides exciting possibilities for comparison between these two models for similarities and differences by which Purkinje cells undergo cell death. The CERH core facilities have provided excellent resources for the members of Dr. Abbott's lab to design and construct DNA probes to look for mRNA expression for cell death associated proteins as well as calcium binding proteins. This work is supported by a NIH-NINDS K08 award (K08 NS1681-05) to Dr. Abbott and forms the basis for a recent R01 proposal submission to NIH-NINDS (November 1, 1998: Calcium channel mutations and Purkinje cell function; R01 NS38187). Dr. Abbott has also recently initiated collaborative efforts with Dr. Ramos to study the impact of environmental factors in nephrogenesis and the role of the AhR in this process.

In addition to primary collaborative activities with member of the Reproductive and Developmental Biology Research Core, Dr. Burghardt interacts with members of the Nutrition (Drs. Lupton, Chapkin, Harris and Turner) and Chemical Biology Research Cores (Ramos, Safe, Phillips, Tiffany-Castiglioni, Donnelly and Busbee). These collaborative interactions involve the development and adaptation of laser cytometric approaches including fluorescence deconvolution, confocal and multiphoton microscopy to mechanistic analysis of cellular physiology and pathophysiology.

4. Publications:

Andrews, D.L., Williams, G.S., Mahoney, J.C., and West, J.R. (1999). DNA fragmentation during exposure of rat cerebella to ethanol in vitro. Journal of Neurobiology, 32:82-92.

Burghardt RC, Barhoumi, R., Sanborn, B.M. and Andersen, J. (1999) Oxytocin-induced Ca2+ responses in human myometrial cells. Biology of Reproduction 60:78-82.

Cheema, Z. F., Wade, S., Sata, M., Walsh, K., Sohrabji, F. and Miranda, R.C. (1999). Fas/Apo [apoptosis]-1 and associated proteins in the differentiating cerebral cortex: induction of caspase-dependent cell death and activation of NF-kB. Journal of Neuroscience 19, 1754-1770.

Chen, W-J.A., Parnell, S.E., and West, J.R. (1999). The effects of alcohol and nicotine on the developing olfactory bulb: loss of mitral cells and changes in neurotransmitter levels. Alcoholism: Clinical and Experimental Research, 23:18-25, 1999.

Dekaney, C.M., Wu, G. and Jaeger, L.A. (1999). Ornithine aminotransferase activity and mRNA expression in porcine fetal small intestine. FASEB J. 13:734.4.

DeSousa P.A., Winger Q.A., Hill J., Jones K., Watson A.J. and Westhusin M.E. (1999). Reprogramming of fibroblast mRNA expression following nuclear transfer in bovine embryos. Cloning 1: 63-69.

Fan YY, Zhang, J., Barhoumi, R., Burghardt, R.C., Turner, N.D., Lupton, J.R. and Chapkin, R.S. (1999). Antagonism of CD95 (APO-1/Fas) signaling blocks butyrate induction of apoptosis is colonic cells. American Journal of Physiology. 277 (Cell Physiol.46): C310-C319.

Flynn, N.E., Meininger, C.J., Kelly, K., Ing, N.H., Morris, S.M. and Wu, G. (1999). Glucocorticoids play an important role in mediating the enhanced expression of intestinal type II arginase and arginosuccinate lyase in postweaning pigs. Journal of Nutrition 129: 799-803.

Hsiao, S-H., West, J.R., Mahoney, J.C., and Frye, G.D. (1999). Postnatal ethanol exposure blunts upregulation of GABA_A receptor currents in Purkinje neurons. Brain Research, 832:124-135.

Ing, N. H. and Ott , T.L. (1999). Estradiol up-regulates estrogen receptor messenger RNA by increasing its stability. Biology of Reproduction 60: 134-139.

Johnson GA, Burghardt, R.C., Flemming, J-AGW., Taylor, K.M., Newton, G.R., Bazer, F.W. and Spencer, T.E. (1999). Development and characterization of immortalized ovine endometrial cell lines. Biology of Reproduction 61: 1324-1330.

Johnson GA, Burghardt, R.C., Spencer, T.E., Newton, G.R., Ott, T.L. and Bazer, F.W. (1999). Ovine osteopontin II. Osteopontin and a_vb₃ integrin expression in the ovine uterus and conceptus during the peri-implantation period. Biology of Reproduction 61: 892-899.

Johnson GA, Spencer, T.E., Burghardt, R.C. and Bazer, F.W. (1999) Ovine osteopontin I. Cloning and expression of ovine osteopontin mRNA in the uterus during the peri-implantation period. Biology of Reproduction 61: 884-891.

Johnson GA, Spencer, T.E., Hansen, T.R., Austin, K.J., Burghardt, R.C., and Bazer, F.W. (1999). Expression of the interferon tau inducible ubiquitin cross-reactive protein in the ovine uterus. Biology of Reproduction 61: 312-318.

Johnson, G.A., Burghardt, R.C., Taylor, K.M., Fleming, J-AGW., Bazer, F.W. and Spencer, T.E. (1999). Development and characterization of immortalized ovine endometrial cell lines. Biology of Reproduction 61:1324-1330.

Luo, J., West, J.R., Cook, R.T., and Pantazis, N.J. (1999). Ethanol induces cell death and cell cycle delay in cultures of pheochromocytoma PC12 cells. Alcoholism: Clinical and Experimental Research, 23: 644-656.

Maier, S.E., Miller, J.A. and West, J.R. (1999). Prenatal binge-like alcohol exposure in the rat: region-specific deficits in brain growth. Neurotoxicology and Teratology, 21:285-291.

McAlhany, Jr., R.E, Miranda, R.C., Finnell, R.H. and West, J.R. (1999). Alcohol decreases glial-derived neurotrophic factor (GDNF) release and alters GDNF-stimulated SHC phosphorylation without altering GDNF mRNA levels in the developing cerebellum. Alcoholism: Clinical and Experimental Research 23(10), 1691-1697.

Piedrahita, J.A., Oetama, B., Bennett, G., Waes, J.V., Lacey, S.W., 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.

Rhyu, I.J., Abbott, L.C., Walker, D.B. and Sotelo, C. (1999). An ultrastructural study of granule cell/Purkinje cell synapses in tottering (tg/tg), leaner (tgla/tgla) and compound heterozygous, tottering/leaner (tg/tgla) mice. Neuroscience 90(3):717-728.

Rhyu, I.J., Oda, S-I., Uhm, C-S., Kim, H., Suh, Y-S. and Abbott, L.C. (1999). Morphologic investigation of rolling mouse Nagoya (tg rol/ tg rol) cerebellar Purkinje cells: an ataxic mutant revisited. Neurosci. Lett. 266:49-52.

Robertson, J.A., Bhattacharyya, S. and Ing, N.H. (1999). Tamoxifen acts as an oestrogen agonist in endometrium of the ewe. J. Steroid Biochemistry and Molecular Biology 67: 285-292.

Spencer, T.E., Bartol, F.F., Bazer, F.W., Johnson, G.A. and Joyce, M.M. (1999). Identification and characterization of glycosylation dependent cell adhesion molecule 1 (GlyCAM-1) expression in the ovine uterus. Biology of Reproduction 60: 241-250.

Spencer, T.E., Gray, C.A., Joyce, M.M., Jenster, G., Wood, C.G., Bazer, F.W., Wiley, A.A. and Bartol, F.F. (1999). Discovery and characterization of genes expressed in the endometrial epithelium using the ovine uterine gland knockout model. Endocrinology 140:4070-4080.

Spencer, T.E., Gray, C.A., Ott, T.L., Johnson, G.A., Ramsey, W.S. and Bazer, F.W. (1999). Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on endometrial gene expression of cyclic ewes. Biology of Reproduction 61:464-470.

Spencer, T.E., Stagg, A.G., Taylor, K.M., Johnson, G.A., Gertler, A., Gootwine, E., Bazer, F.W. (1999). Effects of recombinant ovine interferon tau, placental lactogen and growth hormone on ovine endometrial function. Biology of Reproduction 61:1409-1418.

Wade, S.B, Oommen, P., Conner, W.C., Earnest, D. and Miranda, R.C. (1999). Overlapping and divergent actions of estrogen and the neurotrophins on cell fate and p53-dependent signal transduction in conditionally immortalized cerebral cortical neuroblasts. Journal of Neuroscience 19, 6994-7006.

Westhusin M.E. (1999). Technology of Cloning: Experience with Cattle. American Association of Tissue Banks, Clearwater Beach, Florida, March 22.

Westhusin M.E. (1999). Applications of Cloning to Human Medicine: The Good, The Bad, The Ugly. FDA, Bethesda, Maryland, January 14.

Westhusin M.E. (1999). Cloning Cattle: The Current State of the Art. University of Calgary, Calgary, Alberta, Canada, March 20.

Facility Cores

Biostatistics & Computational Services Facility Core Report

The goal of this core is to support four different activities of the CERH.

• Help Desk: The first line of support comes from the Help Desk maintained in the CERH main offices. Support is provided for short-term statistical design and analysis questions. Services are provided at no charge. At the current time we have one graduate student staffing the desk on a no appointment basis for 10 hour per week. The core also supports long-term collaborative research by covering 25% of a graduate research assistant for those research teams whose work involves more extensive statistical modeling and analysis.

• Statistical Consulting: Support is provided for long-term statistical design and analysis.

• Computational Services: A web site has been developed that is used to support Center’s outreach activities. A three-minute streaming video has been added to the site to inform individuals as to the purpose and goals of the CERH. 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 is currently being developed. This system also benefits investigators who can request services from facility core. Facility Cores along with the Administrative Core will be able to track services rendered and schedule services in a more efficient manner.

Members:

James A. Calvin, Ph.D.,
DirectorProfessor and Head, Department of Statistics

James Snell, Ph.D., Co-Director
Department of Veterinary Anatomy and Public Health

Kendra Brown, B.S.
Graduate Assistant Research, Department of Statistics

Jeff Morris, B.S.
Graduate Assistant Research, Department of Statistics

 

1. 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 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 these activities research teams are able to perform more complete analyses and design more cost effective experiments. Texas A&M University does not provide a statistical consulting service and, thus, without the CERH, Center investigators would not have any access to statistical support without creating personal contacts and paying for any and all contacts, regardless of the request. As the
f the facility.

The Computational Services component of this facility core is being developed as a platform for archiving and retrieving data that result from the individual and collaborative activities of Center investigators. The archival storage/retrieval system has been designed to store any electronic file, be it a flat data file, an image file, a word processing document or a spreadsheet. The system functions such that each investigator will have his/her own secure storage hierarchy which will allow oversight over all members of the lab without requiring that the investigator be the only member of the lab allowed to store data. Stored files are given generation numbers so that new versions of the same file can be stored without destroying older versions. There are also keywords and an abstract 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 entire CERH system looking for other researchers working in the same area. If another researcher has used the same keyword(s) then contact information will be supplied so that 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 also been established. This allows for easier communication for and to teams spread throughout the Texas A&M campus and sites in greater Houston.

 

 

The goal of the oligonucleotide synthesis, sequencing and analysis component of the Facility Core is to provide the equipment, expertise and software to support Center investigators in a multitude of data collection technologies involving nucleic acids. These technologies include: oligonucleotide primer design and synthesis, PCR amplification of template DNAs, construction of genomic and cDNA libraries and automated DNA sequence analysis. During the last year, the Facility Core was expanded to include microarray technology as part of the services offered by the core. Dr. Terry Thomas, Head of the Department of Biology and Director of the Laboratory for Functional Genomics, has been recruited to guide these efforts. The genomic resources available to investigators include cDNA library construction, arraying and curation; high throughput (HT) EST analysis and transcriptional profiling. This is a developing area in which increased levels of interest and activity are anticipated for the next year.

The specific objectives of the core include the following:

• Provide assistance in the overall strategies, design, and synthesis of oligonucleotides.

• Provide the necessary expertise for sample preparation and sequence determination of template DNAs.

• Provide a low cost and highly efficient service determining the nucleotide sequence of DNA molecules.

• Provide an automated system using capillary electrophoresis and microarrays for genotyping, expression analysis and mutation detection.

• Provide computer assisted database searching, analysis, and interpretation of DNA sequence and gene expression data.

Members:

James Derr, Ph.D., Co-Director
Assistant Professor, Department of Veterinary Pathobiology

Terry Thomas, Ph.D.,
Co-DirectorProfessor and Head, Department of Biology

C.E. Kolenda, B.S.
Laboratory Manager, Department of Veterinary Pathobiology

Jamie Schroeder,B.S.
Laboratory Technician, Department of Veterinary Pathobiology

Facilities and Equipment:

• Applied Biosystems 377 automated DNA/RNA sequencer

• Applied Biosystems model 310 capillary electrophoresis system

• PerSeptive Biosystems Expedite oligosynthesizer with a MOSS attachment

• GeneAmp 5700 sequence detection system and associated benchtop and hand-held laboratory equipment.

• Qbot (Genetix, Inc.) – robotic array with 50,000-100,000 in 384 well microtiter plates.

• BioMek (Beckman Coulter) – high-density nylon filters are made using the Qbot or BioMek robots for screening by virtual subtraction.

• Template preparation (BioMek 2000), reaction assembly (Robbins Hydra 96), HT PCR and HT sequencing.

• Two ABI 377 (Perkin Elmer) sequencers available.

• An ABI3700 96 channel capillary electrophoresis sequencer.

• Gridding robot from Genemachines (trademark) gridding robot is one of only a few commercially available robots capable of fabricating DNA microarrays. It uses a 16 "pin" configuration to deposit sub-nanoliter volumes of liquid in 100 um spots on as many as 110 glass slides at 200 um spacing.

• A confocal laser scanning device (ScanArray 3000; General Scanning, Inc.) is used to detect and quantify hybridization. The ScanArray 3000 is specifically designed to scan DNA microarrays fabricated on glass slides. (1" x 3") using the Cy3 and Cy5 fluorescent labels. Scan rates of 4 min per 20 x 20 mm arrays also rapid data acquisition at 10 um resolution.

• Genomic Solutions Visage HDG software mounted on a Sun Microsystem Ultra 10 and the Array Suite that runs on top of IPLab Spectrum Image Analysis software. The latter software was obtained from the DHHS/NIH Human Genome Project and is running on a Macintosh G3 and is now marketed and supported by Scananlytics, the source of IPLab.

Usage and Benefits:

This service core facility is targeted at both the small laboratories that are interested in taking maximum advantage of automated sequencing, genotyping and oligonucleotide synthesis, as well as research groups pursuing large-scale cDNA and genome sequencing projects. This facility has helped established more interaction among individual research laboratories by combining oligonucleotide synthesis, DNA sequencing and genotyping in one convenient and centrally located area. In the current funding year, CERH investigators from the Nutrition Research, Reproductive and Developmental Biology, and Chemical Biology research cores have taken advantage of the expertise and capabilities of this core facility. To date, the DNA Technologies Laboratory has provided various services to 14 separate research projects for Drs. Abbott, Bazer, Chapkin, Tiffany-Castiglioni, Finnell, Harris, Ing, Kier, McMurray, Piedrahita, Ramos, Safe, Schroeder, and Westhusin. Functional genomic efforts represent a more recent development, but so far projects are being developed to support the programs of Drs. Chapkin, Lupton, Turner, Safe, and Ramos. Increased use of these technologies, particularly via the Pilot Project Program, is anticipated in the next year of funding.

Field Services Facility Core Report

Description:

The conduct of investigations into the impact of environmental contaminants on rural health requires collaborations between a broad range of basic and applied disciplines. An important component of epidemiologic studies is the availability of information to define contaminant concentrations in the environment, while mechanistic studies often require analytical measurements defining the kinetics of metabolite production. In addition, the organized production and evaluation of data from all of these studies requires the establishment of a detailed Quality Assurance/Quality Control (QA/QC) program and the implementation of Standard Operating Procedures for routine laboratory techniques. The primary purpose of the QA/QC program is to improve the precision, accuracy and reproducibility of data generated by Center investigators.

The specific objectives of the Field Service Core Facility are to:

• Provide routine analytical support including sample preparation, extraction and standard analytical measurements to all Center investigators.

• Provide assistance to Center investigators in the development and implementation of a sampling strategy for field investigations conducted in support of epidemiologic studies.

• Assist in the development and implementation of Quality Assurance Project Plans for all Center research.

The Field Services facility core has provided support to investigators in the Chemical Biology, Reproductive & Developmental Biology, and Biostatistics and Epidemiology research cores of the CERH. Since inception of the CERH, the core has been involved in 20 different projects to support several investigations. 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 of Excellence at Rutgers University to support several jointly funded EPA initiatives.

Members:

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

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

         Bokelman, M.S.
         QA/QC Officer, Department of Veterinary Anatomy and Public Health

Thomas McDonald, Ph.D.
Associate Research Scientist, Department of Civil Engineering

 

Facilities and Equipment:

Field Sampling and safety equipment – includes full face respirators, boots, coveralls and various disposable equipment. Also, trowels for surface soil sampling, an extension for surface water sampling, and an HEPA vacuum cleaner for sampling indoor house dust.

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

2 – GCs with EC, FID and NP detectors – these Gas Chromatographs 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 eventually be combined with an Accelerated Solvent Extractor for efficient sample preparation.

Tecator Soxhlet Extractor – used for extraction of soils, sediments and solid waste. Reduced the time and solvent required for the standard Soxhlet extraction.

Greenhouse space – approximately 600 ft² of greenhouse space are available for sample preparation or 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 (in collaboration with Dr. T. McDonald) – this equipment is available through a collaboration with the Environmental Engineering analytical laboratory. The GC/MS is primarily used 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.

Usage and Benefits:

Two projects were initiated prior to the start of the CERH and 20 projects have been directly supported by CERH activities. Three additional projects are still in the proposal stage. The 20 projects supported by the CERH include 8 projects providing field services (Drs. Calvin, Shalat, Donnelly, Ramos and Bame), 9 projects providing analytical support (Drs. Ramos, Tiffany-Castiglioni, Safe, Phillips, Busbee, Donnelly and Burghardt) and 3 projects providing QA/QC support (Drs. Shalat, Tiffany-Castiglioni, Ramos, Finnell).

Analytical support has focused on standards preparation (for a variety of projects), fractionation of complex mixtures (for ATSDR and NIEHS funded projects), and analysis of benzo(a)pyrene metabolites (for NIEHS funded projects). In order to understand both the mechanism of benzo(a)pyrene toxicity, and the importance of various metabolic polymorphisms, information describing the production of metabolites and their corresponding toxicity is of great value. The initial data suggests that the two major metabolites in renal glomerular mesangial cells include a 3-hydroxy benzo(a)pyrene and 1, 6-benzo(a)pyrene-dione. Additional studies are planned to investigate the impact of binary mixtures (benzo(a)pyrene and chrysene) on metabolite formation, and to study the characteristics and concentrations of metabolites produced by several different cell lines.

In the spring of 1999, the core completed support of a pilot study funded by the CERH on End Stage Renal Disease. Support included collection of samples from multiple households in four counties, including Jackson, Titus, Victoria and Abilene. Drinking water samples were collected from households of individuals with end stage renal disease and analyzed for heavy metals. The sampling was conducted in three of the four counties with the assistance of science teachers from local schools. This collaboration facilitated the collection of multiple samples from each household; and allowed high school science teachers to be involved in the research project. In addition, core staff members in collaboration with COEP presented a seminar on "Careers in Environmental Toxicology" at each of the high schools. Similar types of collaborative programs are presently being organized in Laredo and McAllen.

The most recent project support by the Field Services Facility Core is a collaborative study to monitor childhood exposure to pesticides. The project is supported by the USEPA through Dr. S. Shalat at Rutgers University. Sampling conducted by the core will take place in several Hispanic neighborhoods in the Laredo area. A total of 60 households will be selected for the study. A group from Rutgers will film children during daily activities to monitor hand-to-mouth activities as an indication of potential exposures to soil and household dust. The core will collect household dust, surface soils (from play areas outside) and hand-wipe samples to be analyzed for pesticides. In addition, urine samples will be collected and shipped to the ATSDR in Atlanta for analysis of pesticide metabolites. The goal of the study is to provide a more accurate model for estimating intake and absorption of pesticides by young children.

Space and equipment has been acquired to begin P-postlabeling studies. It is anticipated that the initial testing of tissues prepared in Dr. Randerath’s laboratory at Baylor College of Medicine will begin in January. The postlabeling technique should be available to CERH investigators in early March. The core has also recruited Dr. Thomas McDonald to provide assistance with routine chemical analysis, particularly in the area of mass spectrometry of origin analytes. Through Dr. McDonald, the core has access to several GC-MSs, as well as additional equipment for metals analysis; and, a GC-MS-MS for analysis and identification.

Image Analysis Facility Core Report

The Image Analysis Service Core provides CERH investigators with access to state-of-the-art microscopy and image analysis services. The specific objectives are to provide instrumentation and service for:

• All aspects of specimen preparation for ultrastructural analysis and immunocytochemistry.

• Digital imaging, image processing and analysis.

• Quantitative single and multiparameter steady-state analysis of vital fluorescence endpoints within living and/or stabilized cells and tissues.

• Quantitative single and multiparameter kinetic analysis of endpoints of cellular homeostasis mechanisms.

The mission of the Image Analysis Core is fully integrated with the activities of the CERH. Statistical models are being explored to study toxicant effects on cellular signal transduction with particular emphasis on analysis intracellular calcium oscillations. Image Analysis staff are working with CERH investigators to continue development and application of new methods to enhance mechanistic assessment of cellular physiology and pathophysiology. Improvements in analytical microscopy resources remain a priority and the efforts during the past year reflect this activity. The purchase of a multiple photon microscope is planned for the current fiscal year. This technology will facilitate the adaptation of mechanistic analysis of cytotoxicity mechanisms developed for use at the individual cell level to the tissue level using precision cut tissue slices.

Robert C. Burghardt, Ph.D.,
DirectorProfessor, Departments of Veterinary Anatomy & Public Health and Medical Physiology

Rola Barhoumi, Ph.D.,
Associate DirectorResearch Scientist, Department of Veterinary Anatomy & Public Health

Youssef Mouneimne, Ph.D.
Research Scientist, Department of Veterinary Anatomy & Public Health

Miles Frey, B.S.Technician II,
Department of Veterinary Pathobiology

Stephen Lundback, B.S.Technician II,
Department of Veterinary Anatomy & Public Health

Facilities and Equipment:

Zeiss 10C high resolution Transmission Electron Microscope with top entry stage. Accessories include goniometer stage, cyro- specimen stage, and micro-dose focusing control.

Fluorescence microscopy and spectroscopy:

Meridian Ultima Interactive Laser Cytometer/Scanning Laser Confocal Microscope. Multi-line, UV/Visible Coherent Enterprise^TM argon ion laser capable of simultaneous excitation with two wavelengths, 3 high quantum efficiency photomultiplier tubes for detection, and an array of detection filter sets to provide fluorochrome versatility

Meridian InSIGHT Point Laser Scanning Confocal Microscope. A Zeiss Axioplan microscope interfaced with 100 mW argon ion and 75 mW Krypton ion lasers, capable of direct ocular viewing in real time and real color (i.e., at video rates).

Scanalytics CELLscan^TM Fluorescence Deconvolution Workstation supported by a Zeiss Axoiplan inverted fluorescence microscope with 100 W mercury source, a cooled CCD camera, widefield image capture, and image deblurring software. This system is being upgraded with a Photometrix Quantix CCD camera and Pentium III computer to upgrade fluorescence deconvolution imaging.

Digital Imaging and Image Analysis Workstation consisting of a Zeiss Axioplan 2 Research Microscope interfaced with a Hamamatsu 3 chip color camera supported by a Power Macintosh G3 Computer, 850 Monitor, a Kodak XLS 8650 PS Digital Printer and Epson Stylus Color Printer, and a Epson, Expression 636 scanner.

Zeiss PMIII Light Microscope equipped for bright field, phase contrast, fluorescence and Nomarski differential interference contrast microscopy and video imaging.

Bio-Tek FL600FA Fluorescence/Absorbance Reader, supporting flexible kinetic assays with top and bottom probes, a fluorescence excitation and emission range of 300-635 nm and 350-700+ nm respectively and probe diameters of 5, 3, 1.5 and 1.0 mm.

PixCell II Laser Capture Microdissection System utilizes a microscope fitted with an infrared laser to attach cells of interest onto a film that can be transferred to a tube for extraction of DNA, RNA or protein and subsequent molecular analysis.

Usage and Benefits:

The CERH investigators who have utilized services of the Image Analysis Service Core are listed below by Research Core.

Chemical Biology Research Core: Drs. Ken Ramos, David Busbee, Evelyn, Tiffany-Castiglioni, K.C. Donnelly, Larry Johnson, Timothy Phillips, and Stephen Safe.

Reproductive and Developmental Biology Research Core: Drs. Louise Abbott, Fuller Bazer, Robert Burghardt, Nancy Ing, Laurie Jaeger, Jorge Piedrahita, Thomas Spencer, and Mark Westhusin.

Nutrition Research Core: Drs. Joanne Lupton, Robert Chapkin, Nancy Turner, and Edward Harris.

Biostatistics and Epidemiology Research Core: Drs. Jim Calvin and Barbara Richardson

This service core represents a major investment in state-of-the-art cellular imaging technology. A new instrument, the PixCell II Laser Capture Microdissection System, was obtained through an invitation directed by Dr. Abbott of the Reproductive and Developmental Biology Research Core. Image Analysis Core staff with the help of other CERH investigators is currently seeking new imaging instrumentation. The availability of this shared equipment to individual investigators is a cost-efficient way to provide advanced imaging technologies to all laboratories. Highly trained members of the Image Analysis Core staff are working closely with CERH investigators to develop and apply new experimental methods to support mechanistic assessment of cellular physiology and pathophysiology. Complications caused by the complexity of instrumentation, the large data sets that result from experimental analyses involving digital images acquired over various intervals of time, and statistical analysis of the large data sets are minimized through the core laboratory approach. The relatedness of the work of CERH investigators continues to drive applications development and the development of a corporate memory of experimental approaches that add to the cost effectiveness. Center investigators have either led new instrumentation initiatives or have a major input into directing the development of new resources. Opportunities for the Image Analysis Core Director and Associate Director to meet with investigators from individual Research Cores and discuss instrumentation capabilities, technical expertise and current and developing applications has enhanced the exchange of information making the facility more faculty-driven.

During the past year, the monthly allocation of instrument time has been consumed in the generation of data for projects covered by the mission of the CERH including:

• Analysis of natural and chemically modified sorbent materials used for remediation of toxicant-contaminated water and soil (electron microscopy).

• Analysis of ozonated metabolites of benzo[a]pyrene (confocal microscopy and multiparameter kinetic analysis of vital endpoints).

• Analysis of the subcellular partitioning, metabolism, and toxicity of benzo[a]pyrene and TCDD (confocal microscopy; multiphoton microscopy; multiparameter kinetic analysis of intracellular pH, plasma membrane and mitochondrial function; calcium homeostasis and oscillations).

• Analysis of estrogen and progesterone receptor expression in conceptus tissues as targets for endocrine disruptors and evaluation of environmental estrogens on conceptus trophectoderm cells (digital imaging and image analysis, brightfield and darkfield in situ hybridization; immunocytochemistry, multiparameter kinetic analysis of vital fluorescence endpoints).

• Study of uterine biology during the periimplantation period as targets for endocrine disruptors. Endpoints include uterine secretion of osteopontin, interferon-inducible ubiquitin cross-reactive protein, receptors including, integrin, prolactin, EGF, FGF-10, and HGF, glycoconjugate markers of uterine receptivity (brightfield/darkfield in situ hybridization, immunocytochemistry, digital imaging and image analysis).

• Development of functional assays for integrin-mediated attachment and signal transduction between trophectoderm and uterine epithelium (immunocytochemistry and fluorescence deconvolution).

• Dietary factor modulation of colonocyte proliferation and apoptosis and action of dietary lipids on Ha-ras expression (fluorescence detection of cell cycle and apoptosis markers, detection of reactive oxygen species and mitochondrial damage leading to apoptosis, measurement of intracellular GSH and pH).

• Phenotypic profiles of cultured adult and embryonic renal glomerular cells following repeated cycles of hydrocarbon injury (electron microscopy; brightfield imaging and image analysis).

• Analysis of apoptosis in primordial germ cells (electron microscopy).

• Analysis of daily sperm production and abnormal spermatozoa (electron microscopy).

• Analysis of GFP constructs (confocal and fluorescence deconvolution).

• Analysis of the GSH levels in mouse spleen cells challenged with virus and glyconutritionals (confocal microscopy and multiparameter kinetic analysis).

• Nuclear translocation of transcription factors, e.g. STAT proteins (immunocytochemistry fluorescence deconvolution and confocal microscopy.

• Digital imaging and preparation of camera ready plates for manuscripts and slides for oral presentations.

Protein Technologies Facility Core Report (Formerly Biological Mass Spectrometry Facility Core)

Description:

Services offered by the core were expanded to include a more comprehensive analysis of protein structure and function. Dr. Larry Dangott, an experienced protein biochemist and director of the Protein Chemistry Laboratory, was recruited as Co-Director. The core works to expedite basic and applied research by providing state-of-the-art analytical and preparative protein chemical and consulting services, including automated N-terminal sequencing, amino acid analysis and electrophoretic and chromatographic protein separations of proteins and peptides. The Protein Chemistry Laboratory also offers a wide range of ancillary techniques for protein/peptide identification and micro-characterization including protein fragmentation and reversed phase HPLC fingerprinting. The core is also actively involved in developing new approaches and protocols for advanced technology in protein characterization to further the Center's research goals.

The Laboratory for Biological Mass Spectrometry complements the Protein Chemistry Laboratory offered to CERH members. The laboratory provides modern mass spectrometry capabilities to CERH researchers, including several high resolution time-of-flight (TOF) mass spectrometry instruments. Although methods are still under development to enhance the sensitivity of detection of biomolecules, they are currently being used in a number of projects, e.g., to identify individual proteins from partially purified calcium transport channel proteins found in mouse brains in collaboration with Dr. Louise Abbott. We are also using a derivative of this experimental method in combination with H/D exchange chemistry to study protein folding and solvent dependent protein folding.

The objectives of the Protein Technologies Core are:

• Provide modern technologies focused on characterization of proteins and other biological molecules.

• Assist in the design, execution and date interpretation of experiments.

Members:

David H. Russell, Ph.D.,
Director,Professor, Department of Chemistry

Larry Dangott, Ph.D.,
Co-Director,Research Scientist, Protein Chemistry Laboratory

William K. Russell, Ph.D.,
Collaborations Coordinator,Research Scientist, Department of Chemistry

Kent J. Gillig, Ph.D.
Research Scientist, Department of Chemistry

Jianhong Wang, Ph.D.
Research Scientist, Department of Chemistry

Virginia Johnson, M.S.
Research Associate, Protein Chemistry Laboratory

Julius L. Apuy, B.S.
Laboratory Technician, Department of Biochemistry

John Koomen, B.S.
Laboratory Technician, Department of Chemistry

Zee-Yong Park, B.S.
Laboratory Technician, Department of Chemistry

Facilities and Equipment:

Time-of-flight mass spectrometers: PerSeptive Biosystems, Inc. Voyager Elite XL high resolution matrix-assisted laser desorption ionization (MALDI). Mass resolution of greater than 10,000 can be achieved up to m/z ratios of 10,000 with mass measurement accuracy of less than 10 ppm.

Electrospray ionization (ESI) TOF mass spectrometer: This is a unique instrument for direct analysis of solutions containing biological samples. The instrument is especially well suited for studies of protein-protein and protein-small molecule interactions.

High-resolution tandem TOF instrument equipped with photodissociation: This instrument is used for developing peptide sequencing using mass spectrometry. Ionization is achieved by using either ESI or MALDI and the fragment ion mass spectrum (used to extract the amino acid sequence) can be obtained using metastable ion, collision-induced dissociation, and/or photodissociation.

Ion mobility-TOF instruments: There are two prototype instruments designed and built in our laboratory that can be used for studies of the size or conformation of proteins and peptides. These instruments are well suited for analyzing complex mixtures of proteins and protein digests.

Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry: Two FTICR instruments are used for collaborations and applications research. FTICR provide unique capabilities for analysis of large biomolecules. One FTICR is equipped with capabilities for ion mobility measurements, determination of volumes of gas-phase ions similar to gel-electrophoresis.

The Protein Chemistry Laboratory has the following state-of-the-art equipment for use by researchers in the CERH.

Hewlett Packard G1005A Protein Sequencer

Hewlett Packard AminoQuant Amino Acid Analyzer

Hewlett Packard 1100 Liquid Chromatography system

Model G1315A Diode Array Detector

Model G1312A Binary Pump

Model G1313A Autosampler

Model G1316A Thermostated Column Heater

Hewlett Packard Model 1046A Fluorescence Detector

Hewlett Packard Protein Chemistry Workstation

PerSpective Biosystems Voyager MALDI-TOF

Pharmacia Explorer 10 Liquid Chromatograph

Pharmacia IPGPhor Isoelectric focusing unit

Waters Model 626 Liquid Chromatography System

Model 626 Pump

Model 486 Tunable Absorbance Detector

Model 600S Controller

Waters Model 600 Liquid Chromatography System (delivered, awaiting installation)

LC Packings Probot

Savant Model AES1010Speed Vac Concentrator

Gilson Model FC204Fraction Collector

 

Usage and Benefits:

Several CERH members have benefited from the services provided by the core. Dr. Louise Abbott used the facilities to identify proteins from complex mixtures. Dr. R. Chapkin and Dr. F Schroeder used the facilities to study fatty acid binding proteins and binding sites. Dr. K. Ramos used the facilities to identify proteins that activate electrophile response elements in the 5’- regulatory region of xenobiotic regulated genes. Each of these projects involved the development of specific methods for a specific protein or class of proteins, and eventually these methods will be modified for more general applications. CERH efforts catalyzed extensive working relationships between the Protein Chemistry Laboratory and the Laboratory for Biological Mass Spectrometry components of the core.

Transgenics Facility Core Report

The goal of the transgenic core facility is to support Center investigators in the generation and characterization of transgenic mice produced by pronuclear injection as well as homologous recombination in a more cohesive and cost-efficient manner.

Specific objectives of the Transgenics Facility Core are:

• Assists with the design and fabrication of DNA constructs for pronuclear injection and homologous recombination.

• Provides services for the inactivation of specific genes by homologous recombination in embryonic stem cells.

• Provides the ability to generate transgenic mice by pronuclear injection.

• Generates germ line chimeras from selected transgenic ES cell lines via blastocyst injection and breeding of chimeric mice.

• Provides assistance with screening and maintenance of transgenic mouse lines and create segregating or non-segregating congenic inbred strains.

• Assists with the morphological and pathological analysis of transgenic lines.

• Provides computer-assisted image acquisition, and image editing, data collection and analysis.

To facilitate interactions with the research cores, presentations were made to each of the research facilities indicating the available services and procedures for requesting core services. Additional contact information such as phone numbers and e-mails were provided for the directors as well as key staff personnel. A handout with more detailed information was also provided.

 

Members:

Jorge A. Piedrahita, Ph.D., Co-Director
Associate Professor, Department of Veterinary Anatomy and Public Health

Ann Kier, Ph.D., Co-Director
Professor and Head, Department of Veterinary Pathobiology.

Andrew Beitman, B.S.
Technician I, Department of Veterinary Anatomy and Public Health.

Karen Carpenter, B.S.
Research Assistant, Department of Veterinary Pathobiology

William Foxworth, Ph.D.
Research Scientist, Department of Veterinary Pathobiology

Mark McArthur, D.V.M.
Lecturer, Department of Veterinary Pathobiology

Regina Weaks, B.S.
Research Scientist, Department of Veterinary Anatomy and Public Health

John Roths, M.S.
Associate Research Scientist, Department of Veterinary Pathobiology

 

Molecular Biology Division: Tissue culture hoods and incubators, Recombinant DNA-associated equipment for electrophoresis, PCR, sequencing, and Southern analysis

Developmental Biology Division: State-of-the-art microinjection equipment.

Morphology/pathology Division. State-of-the-art morphometric analysis with light, fluorescence, laser scanning confocal microscopy using NIH Image and Metamorph software, video digital capture and electronically linked black and white and color glossy printers.

3. Usage and Benefits:

Although only in its second year of full operation, the facility is already being used by several CERH investigators. The person in charge of day-to-day molecular biology and construction services is Ms. Regina Weaks, Assistant Research Scientist. Additionally, Dr. Piedrahita spends approximately 10% of his time directing Ms. Weaks, as well as meeting and advising other faculty interested in using the transgenic core facility. Other services of the core involving either blastocyst or pronuclear injections are carried out on alternate days throughout the week in the Injection Facility. These services, as well as morphology/pathology support have been offered at no charge to CERH investigators, including microinjections, mouse purchases, mouse per diem, morphometric analyses, and pathology consultation. The resources and services of the core (personnel, supplies, and mouse costs) are leveraged with funds from Dr. Kier’s grants and the Department of Veterinary Pathobiology.

Interactions with Dr. Richard Finnell have resulted in the generation of three targeted inactivations in folate pathway genes. Two of these inactivations have progressed all the way to the germ line with a publication in Nature Genetics. The third mutation, the reduced folate pathway, has been completed, and several chimeras have now been produced and are being tested for germ line transmission. Further blastocyst injections are ongoing weekly. CERH funds were used for personnel and supplies and mouse per diem, such that there was no cost to the CERH investigators (Finnell and Piedrahita).

Interactions with Dr. Steve Safe have identified a potential gene to target the mouse estrogen receptor. The gene has now been cloned and partial sequence indicated that the clone isolated by Dr. Piedrahita and staff members of the core was the correct clone. Additionally a polymorphism in the cloned gene when compared to the GeneBank sequence has been identified. Determination of the prevalence of this polymorphism in several strains of mice is ongoing. A construct will be submitted for blastocycst injection shortly (Safe, Piedrahita).

Interactions with Fred Schroeder have resulted in several papers and manuscripts concerning the role of Sterol Carrier Protein-2 (SCP-2) in cholesterol and fatty acid trafficking within the cell. Co-localization experiments have for the first time shown that SCP-2 is located not only in peroxisomes, but in Golgi, plasma membranes and endoplasmic reticulum, where in vitro experiments have shown high affinity binding in pathways which would facilitate fatty acid and /or cholesterol metabolism. A targeted construct to inactivate the function of the gene coding for SCP-2, made in Dr. Kier’s lab with the advice of Dr. Piedrahita, is currently being injected into mouse blastocysts. Several chimeric litters have been produced, but cannibalized. A second construct is currently being injected by Drs. Maeda/Smithies’ group at the University of North Carolina Medical Center in a joint collaboration to further train our technician, Karen Carpenter, in state of the art injection and husbandry techniques. Ms. Carpenter recently returned from a very productive training session in Drs. Maeda/Smithies’ core transgenic facility.

A cDNA coding for the full length protein, as well as specific ligand binding components of SCP-2 is also being injected into pronuclei for overexpression of SCP-2 in mice. Overexpressed SCP-2 in ES and LM cells have shown up and down regulation of several key lipid carrier proteins (see publications and manuscripts). One overexpression mouse line has been produced, and several pregnancies are ongoing. Pronuclear injections will continue until 3 lines are obtained. Other sterol carrier protein constructs are being designed currently for pronuclear injection this year. One NIH RO1 competitive renewal was awarded for this work this year (Schroeder and Kier), a KO8 application was submitted (McArthur, Schroeder, Kier) and a new RO1 application (Kier and Schroeder) are planned for 2000.

Interactions with Joanne Lupton resulted in an Interdisciplinary grant from Texas A&M being funded to generate rats with specific overexpressed kinase isoforms (Lupton, Kier, and Foxworth). Few facilities have the capability of generating rats, and Dr. Foxworth, a former staff member of the core, submitted a technique manuscript based upon his preliminary work in generating eggs/embryos in sufficient number from rats. Soon, cDNAs coding for these particular kinases will be ready to be injected into the rats by these new methods. It is important to note that the recent departure of Dr. Foxworth has not significantly slowed progress of the core with Ms. Carpenter continuing to hone her skills. The recent addition of a small percent effort of Dr. McArthur, a comparative pathologist, will allow us to add a small component of pathology analysis for investigators as well.

Consultations by John Roths with Dr. Ramos’ lab for morphometric analysis of Ras and cadherin expression in various cell lines have involved both quantitative densitometry using NIH image and qualitative imaging for image acquisition for fluorescence photomicrographs. Other consultations by John Roths for morphometric analysis have included personnel from the laboratories of Drs. Safe, Schroeder, Kier, Piedrahita, Chapkin, and Lupton.

Consultations by Dr. Mark McArthur, a laboratory animal comparative pathologist, with Dr. Safe’s lab (Derek Morrow) for morphometric analysis of tumor types in a model of mammary carcinogenesis (DMBA in 55 day old virgin Sprague Dawley females) that they are using to test a therapeutic agent. Mr. Morrow has been meeting with John Roths for approximately 3 times per week for several months to be trained in recognizing the different tumor types that arise in the model, characteristics of malignancy, stratification of selection of tissue area for histologic slides, and normal histology of rat mammary glands.

The Department of Veterinary Pathobiology has committed a tenure-track position to replace the non-tenure tract position previously occupied by Dr. Foxworth. A Search Committee has composed a job advertisement recently published in Science, and we are pleased to have three excellent applicants thus far in this area of high demand for experienced personnel. In addition, the Department of Veterinary Pathobiology has contributed almost half a million dollars to the facility equipment and mouse per diem for the core facility. This facility serves other investigators at Texas A&M, but NIEHS investigators have first priority. All NIEHS investigators requesting service have been accommodated, with first priority, and free of charge. Clearly, the NIEHS Center has benefited from the commitment of the Department of Veterinary Pathobiology to this Core, the DNA Core, and a Peptide Synthesis Core, which is being integrated into the Protein Technologies Facility Core. The total investment by the Department of Veterinary Pathobiology for all these facilities, equipment, and personnel is over 2.5 million dollars in the past 5 years.

Increased communication between staff members have resulted in an increased awareness of the potential of Transgenic technologies in CERH research programs. As communications continue to be facilitated by the existence of the Center, it is expected that the efficiency in time and money provided by the core will become even more obvious each year. This type of methodology requires highly trained individuals and is not easily transferred to individual investigators’ laboratories. As such, this type of core is not a high volume core facility. It is anticipated, however, that as the capability and volume continue to grow the services made available by this Facility Core will increasingly provide a new avenue of experimental approach to many areas in environmental and rural health research.

COEP

Overall Goal:

The main goal of the Community Outreach and Education Program (COEP) is to help translate major research findings in the environmental health sciences to concepts that can be adopted by rural communities in Texas, mainly in the colonias along the Texas-Mexico border. A Brazos Valley Community Outreach Counterpart Initiative has also been established to address health concerns in our immediate vicinity. Ultimately, the goal of our COEP is to educate communities to reduce potential environmental exposures associated with human illness and to provide health care providers and the community with scientifically sound information to deal with environmental issues. The major objectives of the COEP are:

To disseminate culturally appropriate education and communication materials related to general principles of toxicology and environmental health at Community Resources Centers (CRC’s).

• To coordinate town meetings with residents in the target communities to discuss issues related to toxicology, nutrition, and environmental health and to obtain information about their concerns.

• To develop and coordinate a monthly TV program to address environmental health issues in both English and Spanish.

• To publish a monthly health column on local and regional newspapers in both English and Spanish dealing with environmental health issues.

• To organize and co-sponsor an Annual Conference on environmental health related issues in which local health practitioners as well as leaders of the community, and the general public comes together with Center investigators.

Irma N. Ramos, M.D., COEP Director
Research Scientist, Department of Veterinary Physiology and Pharmacology

         K.C. Donnelly, Ph.D.
         Associate Professor, Departments of Veterinary Anatomy and Public Health,
         Soil and Crop Sciences, and Environmental and Occupational Health

Larry Johnson, Ph.D.
Professor, Department of Veterinary Anatomy and Public Health

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

Mark Sicilio, M.D., Associate Member
Department of Pediatrics, Scott and White Clinic and Department of Family Medicine, Texas A&M University System Health Science Center

Mary Wolf, M.B.A., R.N.,
Associate MemberDepartment of Social and Behavioral Sciences, School of Rural and Public Health

Marlynn May, Ph.D.
Department of Sociology and Center for Housing and Urban Development

Current Collaborations:

• Texas A&M University School of Rural Public Health

• Texas Agricultural Extension Service

• Texas Engineering Extension Service

• Community Partnership Board

• Texas Department of Health

• Texas A&M University College of Veterinary Medicine

• Texas A&M University Baylor College of Dentistry

• Texas A&M University College of Medicine

• Center for Housing and Urban Development

• Bush School of Government and Public Service

• KBTX TV-3, Bryan/College Station

Highlights 1999:

The COEP continues working toward the implementation of the COEP major objectives. During this year significant progress has been made in advancing the goals of the Center.

• Environmental Health Science Curriculum for Lay Public

Outreach efforts in the Lower Rio Grande Valley focus on the development of culturally appropriate training/education program for Promotoras (community educators) working in the colonias. A bilingual curriculum suitable for Promotora education has been developed. This program focuses on the human health effects of environmental hazards, particularly emphasizing reproductive and cardiovascular outcomes, occupational safety, and genetic susceptibility to birth defects and cancer. The COEP will also provide training/education programs for health practitioners (clinicians), public health professionals and service providers on diagnosis and management of environmentally-related diseases. Emphasis will be given to health effects of commonly encountered pollutants in this region. The program will be implemented this Spring in selected rural communities in the Lower Rio Grande Valley, specifically in Cameron and Hidalgo counties.

• Development of Educational Written Materials

The COEP continues with the production of written materials (brochures, articles) for community distribution, TV dissemination. Web-Site publications include: Preventing Carbon Monoxide Poisoning (Como Prevenir Intoxicación con Monoxido de Carbono), Environmental Tobacco Smoke and the Health of Children, Adolescents and Adults (Contaminación Ambiental Secundaria al Humo del Cigarrillo y la Salud de Niños, Adolescentes y Adultos), Sun Safety-Tips (El Sol y la Salud de su Piel). Holiday Health Highlights. The web site address is cerh@tamu.edu. TV-Show Presentations for which written articles are available include: Preventing Carbon Monoxide Poisoning, Preventing Food Poisoning; Allergic Rhinitis; Ticks Bites: Preventing Lyme Disease; Swimmer’s Ears; Child Adolescent Nutrition; Children and Cholesterol; How to Prevent Autumn and Winter Allergy; Environmental Tobacco Smoke: Children/Adolescents/Adults, Sun Safety Tips, Preventing Poison Ivy/Oak/Sumac, Asthma and the Environment.

• Environmental Health Topics in Public Middle School Science Curriculum

In collaboration with Dr. Larry Johnson the COEP is working to incorporate rural environmental health issues into the middle school science curriculum in rural communities in Texas. This is a NIH funded initiative in collaboration with the Colleges of Education and Veterinary Medicine provide rural communities in Texas education related with the environment health and also information on how to prevent human illness that have an environmental component associated. A web site has been developed, for more information on this project the address is: http://PEER.tamu.edu/.

Brochures:

Preventing Food Poisoning

Como Prevenir Intoxicación Alimenticia

Holiday Health Highlights

Consejos de Salud Durante la Navidad

Environmental Tobacco Smoke and Children

Contaminación Ambiental Secundaria al Humo de Cigarrillo y la Salud de su Niño

Birth Defects: Environmental Factors and Folic Acid

Defectos Congénitos: Factores Ambientales y Ácido Folico

Preventing Carbon Monoxide Poisoning

Como Prevenir Intoxicación con Monóxido de Carbono

 

Web site Publications:

Preventing Carbon Monoxide Poisoning

Como Prevenir Intoxicación con Monóxido de Carbono

Environmental Tobacco Smoke (ETS) and the Health of Children, Adolescents, and Adults

Contaminación Ambiental Secundaria al Humo del Cigarrillo (ETS)

Y la Salud de Niños, Adolescentes y Adultos

Sun-Safety Tips

El Sol y la Salud de su Piel

Holiday Health Highlights

Consejos de Salud para Temporada de la Navidad

 

Articles Written for Distribution:

Allergic Rhinitis

Ticks Bites: Preventing Lyme Disease

Swimmer’s Ears

Child Adolescent Nutrition

Children and Cholesterol

How to Prevent Autumn and Winter Allergy

Preventing Poison Ivy/Oak/Sumac

Asthma and the Environment

 

Irma N. Ramos, M.D., COEP Director
Center for Environmental and Rural Health
Texas A&M University
College Station, TX 77843-4455

Phone: 409-458-1291
Fax: 409-862-8942
Email: iramos @cvm.tamu.edu

Pilot Projects Funded in 1999:

Title: Detection of environmental estrogens by stochastic sensing

Principal Investigator: Orit Braha, Ph.D., Research Scientist

Co-Investigator: Hagan Bayley, Professor

Department of Medical Biochemistry and Genetics

Description: This is a new pilot project.

This project will determine whether stochastic sensing with biosensors based on engineered ion channels can be used to detect presumed and accepted environmental estrogens.

Stochastic sensing is based on the detection of individual binding events between analyte molecules and a single receptor, which acts as a biosensor element. In the present study, the receptor is the channel protein hemolysin (HL). The read-out is fluctuations in electrical current through the channel, which report binding events. The frequency of the events gives the concentration of the analyte. The nature of the binding events (e.g. magnitude and duration) provides a signature for identification of the analyte. The recent discovery that cyclodextrins can act as molecular adapters in the HL channel and mediate the detection of a variety of organic molecules has been employed. New results indicate that cyclodextrin allows for detection of endogenous estrogens (e.g. estriol and estrone).

Title: Birth defects arising in early development: alcohol/nutritional interactions.

Principal Investigator: Timothy A. Cudd, D.V.M., Ph.D., Assistant Professor,

Department of Veterinary Physiology and Pharmacology

Description: This is a new pilot project.

Alcohol has been established as an important teratogen throughout development, especially during organogenesis and later in the third trimester. However, almost no attention has been given to the effects of alcohol during the first few days of life. It is not uncommon for women to consume alcohol during the period of conception and early development, and then abstain when pregnancy is detected. Yet, this early period of development may be a time when alcohol exposure mediates important negative actions on the conceptus. Information about the effects of alcohol on the conceptus during the period of early development is important for the proper advisement of women of child bearing age who consume alcohol and who may potentially become pregnant. The purpose of this proposal is to establish a murine model system to investigate the effects of alcohol on the conceptus during the period of fertilization and pre-implantation.

Title: PCB – protein interactions.

Principal Investigator: James C. Sacchettini, Professor

Department of Biochemistry and Biophysics

Description: This is a new project.

The goal of this new pilot project on human transthyretin (TTR) is to use structural biology and biophysical techniques to determine the three dimensional conformation of the protein-hydroxy-polychlorinated biphenyl (PCB) complex. Structural studies will provide a wealth of information on the conformational changes that take place when a hydroxy PCB binds to TTR, as well as the key molecular interactions/contacts that take place in the active site of the protein. The structural information of the hydroxy PCB-TTR complexes can then be taken into account for the development of novel therapeutics for TTR associated amyloidosis. To date, the recombinant expression and crystallization conditions of TTR have been optimized, yielding gram quantities of pure tetrameric protein which forms large crystals suitable for x-ray diffraction. Of all compounds studied so far, 3,5,3',5'-tetrachloro-4, 4'-dihydroxy biphenyl has the highest affinity for TTR and amyloid repression. The structural information obtained has lead to the discovery of new, and potentially less toxic, PCB analogues. One publication is already in press and one has been submitted to Nature Structural Biology.

Title: Organophosphate Effects on Neuronal Differentiation

Principal Investigator: Evelyn Tiffany-Castiglioni, Professor and Head, Department of Veterinary Anatomy and Public Health

Description: This is a new pilot project.

A human cell culture model will be characterized to distinguish between two classes of organophosphate neurotoxicants (OPs): those that induce acute neurotoxicity (ending in recovery or death) vs. those that induce delayed, long-term polyneuropathy. These classes are often distinguishable by their inhibitory effects on neuronal esterases: acute neurotoxicity is associated with the inhibition of acetylcholinesterase (AChE), and OP-induced delayed neurotoxicity (OPIDN) with the inhibition of both AChE and neuropathy target esterase (NTE). The SY5Y human neuroblastoma cell line selected is the best non-animal candidate model available for cellular investigations of OP neurotoxicity, based on known effects of OPS on its relevant esterase activities. Novel assays will address differential responses of SY5Y cells to mipafox, which induces OPIDN, and paraoxon, which induces acute toxicity but does not induce OPIDN. The amount and time course of expression of specific proteins (immunoblots) and their mRNAs (Northern blots) known to be associated with neurite extension will be measured. This cell culture system should prove a valuable screening device for neurotoxic activity in environmental samples containing OPs or other neurotoxicants that affect the same targets.

Title: Oxidative Stress Disrupts Cadherin/Catenin Complexes

Principal Investigator: Alan R. Parrish, Assistant Professor

Department of Medical Pharmacology and Toxicology

Description: This is a new pilot project.

Cell-cell adhesion is predominately mediated by the cadherin superfamily responsible for the regulation of calcium-dependent cell-cell adhesion in association with catenins. Despite the importance of the cadherin/catenin proteins, little attention has focused on the effect of environmental stress on this complex. Preliminary data suggests that oxidative stress disrupts normal protein interactions of the E-cadherin catenin complex in liver slices. Interestingly, this effect was specific for certain populations of hepatocytes, with no effect on some parenchymal cells or on bile duct epithelium. As cadherin-dependent cell adhesion in the liver is also mediated by N-cadherin and catenin, it is hypothesized that oxidative stress selectively disrupts cadherin/catenin complexes. In this pilot project, precison-cut mouse liver slices will be challenged with diamide or tert-butylhydroperoxide. The impact of chemically-induced oxidative stress on protein interactions of each cadherin/catenin complex in the liver will be assessed by biochemical and histological techniques.

Previous Pilot Projects:

Title: ESRD Risk Correlated With Metal Nephrotoxins In Drinking Water

Principal Investigator: Sherry I. Bame, Associate Professor, Department of Urban Planning and Social and Behavioral Sciences

Co-Principal Investigator: K. C. Donnelly, Associate Professor, Department of Veterinary Anatomy and Public Health

Funding Year: 9/98 - 9/99

Description:

The aim of this study was to correlate exposure to nephrotoxins (lead, mercury, cadmium & arsenic) in drinking water and end stage renal disease (ESRD) risk identified in two "hot spot" counties (highest ESRD incidence ratios and highest environmental water toxin levels), and as a control, in two "cold spot" counties (lowest ESRD risk counties with the lowest toxin levels). ESRD patients were measured in actual exposure to metal toxins in their household drinking water. In this pilot study of 12 households in 4 counties, it was found that metal toxin levels were not above the minimum threshold level. Despite the negative findings, a variety of factors to improve study design and proposing hypotheses of chronic low-level exposure to nephrotoxins and ESRD risk were learned for future research. This preliminary study led to a subsequent study to correlate ESRD risk with public drinking water data across Texas.

Positive Outcomes:

Presentations:

NIH-NIEHS / CERH Conference poster presentation (2nd place award)

Working Papers:

Bame, S., Donnelly, K.C., Autenrieth, R., Harriss, R., Morgan, R., Ramos, K., Richardson, B., Rogers, G. and Sherman, M. (2000). Distribution of ESRD risk and metal nephrotoxins in Texas.

Bame, S., Richardson, B., and Sherman, M. (2000). Distribution of ESRD risk and metal nephrotoxins in Texas. Geographic distribution of a chronic disease: Measuring ESRD risk .

Bame, S., Donnelly, K.C., Autenrieth, R., Harriss, R., Morgan, R., Ramos, K., Richardson, B., Rogers, G. and Sherman, M. (2000). Policy implications of measuring disease risk: The case of end-stage rental disease in Texas.

Other Funding:

Texas A&M University Research Enhancement Grant (9/98-5/99: $2000)

Southern Arizona Foundation (9/99-8/00: $24,953).

Title: Environmental Detection of Water-borne Parasites"

Principal Investigator: Karen Snowden, Assistant Professor, Department of Pathobiology

Funding year: 9/98 – 8/99

Description:

The Environmental Protection Agency protocol for parasite detection in environmental samples (EPA 821-R-97-023) was evaluated for its ability to concentrate and detect microsporidian parasites. Only 3 to 8% of spores were recovered from parasite-spiked water samples. Elution protocols to recover parasites from filter cartridges were inadequate, while modified protocols improved parasite recovery (maximum 15%). Numerous spores were not retained by 1.0 µm-pore filters but were retained using 0.2/0.45 µm-pore filters. Separation of parasites from filter-concentrated eluates using immunomagnetic beads labeled with polyclonal parasite-specific antisera was not successful. Antigen-specific affinity-purified antibodies are being prepared to increase sensitivity of parasite recovery. Alternatively, preliminary experiments with flow cytometric detection of parasites in suspension are proving promising. To evaluate viability of parasites retrieved from environmental samples, an in vitro infectivity assay using slide culture chambers with monolayers of RK-13 rabbit kidney cells was successfully developed.

Positive Outcomes:

Presentation:

Snowden, K. (1999). Environmental detection of water-borne parasites, poster presentation at the 2^nd annual meeting of the Gulf Coast Tropical Medicine Association. UTMB, Galveston, TX. June 25-27.

Proposal submitted:

Underdiagnosed Causes of Infectious Diarrheas in Brazilian Children: submitted January, 1999, to the Wellcome Trust-Burroughs Wellcome Fund Infectious Diseases Initiative.

 

 

Title: Dietary Fibers/Phytoestrogens and Colon Carcinogenesis

Principal Investigator: Nancy D. Turner

Research Assistant Professor

Department of Animal Science

Funding Year: 1/99-12/99

Description:

Recent data suggest estrogen inhibits colon cancer and that estrogen receptor (ER) expression is reduced in colon tumors. This project determined if phytoestrogens maintain ER expression in colon samples (normal and tumor) from rats consuming wheat bran or oat bran and injected with saline or carcinogen (AOM). There were no differences in saline rats. Oat bran reduced ER levels by 19% in normal tissues from AOM-injected rats. Tumors had little ER protein, except in crypt remnants. Wheat bran tumors had more crypt remnants, and ER expression was maintained in those cells. Further work will determine the mechanism whereby phytoestrogens affect ER expression. Another research focus was the effect of diindolylmethane (DIM) on proliferation and apoptosis in the HT-29 colon cancer cell line. DIM and 4-Cl-DIM decreased proliferation and increased apoptosis in this system. A proposal is being produced to make the same measurements in vivo using the rat AOM-model of colon cancer.

Positive Outcomes:

Presentations:

Data from these experiments were presented at the 9^th Annual AICR meeting (DIM) and was used to prepare an abstract for the 2000 Experimental Biology meeting (ER).

Turner, N.D., Zhang, J., Davidson, L.A., Chapkin, R.S., Safe, S and Lupton, J.R. (1999). Diindolylmethane reduced HT-29 colon cancer cell number by decreasing proliferation and increasing apoptosis. Presented at the 9^th Annual AICR Research Conference, September 2-3, Washington, DC.

Turner, N.D., Ing, N.H., Carroll, R.J. and Lupton, J.R. (2000). Estrogen receptor levels in rats consuming wheat bran and injected with azoxymethane. FASEB J. (Submitted).

Additional Support:

Houston Live Stock Show and Rodeo (1/99-12/99: $19,000).

 

 

Title: Arginine Synthesis in the Fetal Pig Small Intestine

Principal Investigator: Guoyao Wu, Associate Professor

Department of Animal Science

Funding Year: 9/98 - 8/99

Description:

Arginine is deficient in preterm infants, and is associated with cardiovascular, pulmonary and intestinal dysfunction. Hypoargininemia in preterm infants may result from underdeveloped intestinal synthesis of citrulline and arginine. Results demonstrated that enterocytes of fetal pigs synthesize citrulline from glutamine and proline as early as day 60 of gestation, but could not convert citrulline into arginine until day 113 of gestation. The activities of arginine-synthetic enzymes were predominantly located in villus enterocytes and were also found in crypt enterocytes. Pyrroline-5-carboxylate synthase, carbamoyl-phosphate synthase I, and ornithine carbamoyltransferase (mitochondrial enzymes) were co-localized in mucosal epithelium. Findings indicate that intestinal arginine synthesis was negligible before the perinatal period provides a new biochemical basis for explaining hypoargininemia in preterm infants. The results have important implications for intrafetal (e.g., intra-amniotic) feeding of the fetus with impaired placental function, and for improving arginine nutrition in preterm infants and neonates.

Positive Outcomes:

Publications:

Wu, G., Pond, W.G., Flynn, S.P., Ott, T. & Bazer, F.W. (1998). Maternal dietary protein deficiency decreases nitric oxide synthase and ornithine decarboxylase activities in placenta and endometrium of pigs during early gestation. Journal of Nutrition 128: 2395-2402.

Wu, G., Ott, T.L., Knabe, D.A. & Bazer, F.W. (1999). Amino acid composition of the fetal pig. Journal of Nutrition 129: 1031-1038.

Dekaney, C.M., Wu, G., Jaeger L.A. (1999). Ornithine aminotransferase activity and mRNA expression in porcine fetal small intestine. FASEB Journal 13: A1010 (Abstract)

Wu, G., Meininger, C.J., Knabe, D.A., Bazer, F.W. & Rhoads, J.M. (2000). Arginine nutrition in development, health and disease. Current Opionions in Clinical Nutrition and Metabolic Care 4: 1-8.

Grant Applications to NIH:

Wu, G. (1999). Corticosteroids and fetal arginine synthesis.

Jaeger, L.A. and Wu, G. (1998). Maternal protein nutrition and fetal intestinal growth.

Additional Support:

Meininger, C.J. and Wu, G. ($252,000) Juvenile Diabetes Foundation to study: Arginine Metabolism in Endothelial Cells.