The
training faculty provides wide-ranging expertise in most of the
areas that are central to contemporary protein chemistry in both
its basic and applied aspects. These include:
A short consideration of the opportunities available in each of the training laboratories follows:
M. E. Black. Being at the crossroad of DNA synthesis, nucleotide metabolizing enzymes are key players in the cell life cycle. Because of their crucial role in replication and cell viability, many nucleotide metabolizing enzymes are targets for chemotherapeutic drugs and anti-infective compounds. Perhaps most intriguing to me is their application in gene therapy protocols to sensitize cells to nucleoside analogs for tumor ablation. A more recent interest of mine is the molecular basis of drug resistance within such drug targets that are often associated with treatment failure. Despite the central role these enzymes play in cell viability, very few are actively studied at the molecular level. I seek to explore the molecular basis of enzyme function using a combination of molecular evolution (random mutagenesis) to generate novel functional enzymes and molecular modeling based on structures derived from x-ray diffraction studies. A practical extension of these studies is to exploit the random mutagenesis approach to create mutants with improved substrate or prodrug activities. Such mutants not only reveal important functional motifs but are also highly desirable for enhancing gene directed pro-drug therapies in the treatment of cancer. Additional approaches used include pathway engineering, site-directed mutagenesis and chimeric gene construction. For a list of current publications, please see: http://www.pharmacy.wsu.edu/PharmSci/black.html
K. A. Brayton. My research uses genomics to elucidate mechanisms of pathogen-host interactions. In collaboration with Don Knowles (USDA) and Guy Palmer (WSU) we are sequencing the genome of the cattle pathogen Anaplasma marginale using a Bacterial Artificial Chromosome (BAC) based clone by clone sequencing strategy. Annotation begins as the sequence of each clone is completed. To date, we have sequenced and annotated 83% of the 1.2 Mb genome. The preliminary data from this project has elucidated the split operon structure of the ribosomal RNA genes of Anaplasma, and the pseudogene structure for the msp2 and msp3 gene families, which resulted in identification of the gene conversion mechanism for the generation of antigenic diversity of these proteins. In addition, a superfamily of outer membrane proteins that are thought to be important for pathogen persistence in the immunocompetent host has been identified.
W. C. Brown. Dr. Wendy Brown's research emphasis is on understanding the bovine helper T cell response to vector-borne hemoparasites and defining protective immune mechanisms and targeted protein antigens. Because Babesia bovis and Anaplasma marginale reside exclusively within erythrocytes, the focus is on MHC class II-restricted, CD4 + T cell responses, IgG responses, and macrophage activation. Specific studies involve understanding immunological control mechanisms of Babesia and Anaplasma infections, defining helper T cell responses to conserved and variable regions of immunogenic proteins, using helper T lymphocytes from immune cattle to identify novel protective antigens that can be used in a subunit or nucleic acid based vaccine, defining the role of antigenic variation in helper T cell epitopes in persistent infection by these organisms, and determining ways to modulate immune responses. A proteomics approach towards identifying novel A. marginale outer membrane proteins that stimulate memory CD4 + T cell responses is being pursued, facilitated by the nearly completed sequencing of the genome of this organism. These major areas of research emphasis provide opportunities for graduate students to study immune mechanisms against different types of parasitic pathogens in an outbred species, and provide a unique training environment that combines molecular and cellular approaches towards understanding the host-parasite interaction that results in protective immunity or successful parasitism.
J. Browse.One of the research programs in my laboratory encompasses a diverse set of projects that have at their base our investigation of the biosynthesis and function of membrane and storage lipids in plants using Arabidopsis as a model. We have several research projects that focus on the roles of membrane lipids in the cell biology and physiology of plants using a large number of mutants with alterations in the lipid composition of their membranes. A second program uses mutational analysis and molecular genetics to investigate lipid structure and membrane function in the model nematode Caenorhabditis elegans.
J. E. Bruce. Our laboratory is developing and applying new analytical capabilities and strategies to exploit high performance mass spectrometry to overcome today’s barriers to systems-level comprehension of biological organisms. We are interested in using these advanced capabilities to provide information on posttranslational modifications and noncovalent interactions on a proteome-wide scale. This information will constitute a critical component of systems biology research that current technology and approaches can not provide. Our research and development efforts will provide major impact on biological, biomedical, and health-related research fields, by identifying relevant posttranslational and interaction networks critical for disease progression, adverse drug reaction and toxicology, as well as normal system function
R. B. Croteau. Croteau, a member of the National Academy of Sciences, directs a very productive research program in chemistry and enzymology of terpenoid biosynthesis in plants. Terpenoids are of practical and commercial value to man as pharmaceuticals, flavors, fragrances and industrial raw materials and function in plant defense against pathogen infection and herbivore attack. Thus training in this laboratory would be particularly relevant to biotechnology. Graduate students obtain rigorous training, firmly grounded in organic chemistry and focused on fundamental problems of metabolic regulation, reaction mechanisms and enzymology.
J. L. Dahl.The main focus of my research is understanding how the pathogenic bacterium Mycobacterium tuberculosis is capable of persisting in the human host for decades before initiating active infections. Over a third of the planet is infected with M. tuberculosis with most cases of tuberculosis arising from dormant bacilli initiating growth once the immune system is compromised. One gene that has been implicated in the survival of M. tuberculosis in the host is relA which codes for a global regulatory protein. Deletion of relA from M. tuberculosis results in an unprecedented decline in survival in infected mice. RelA has been studied in Escherichia coli and shown to regulate upwards to 80 different genes in both positive and negative fashion. My laboratory is interested in defining the RelA regulon in M. tuberculosis with the ultimate goal that genes regulated by RelA may be potential targets of rational drug or vaccine design against tuberculosis. We are employing the cutting edge techniques of microarray and proteomic analyses to identify what genes are regulated by RelA. The proteomic approach we are using involves DIGE (differential gel electrophoresis) technology which has not previously been applied to M. tuberculosis but is yielding exciting results in our laboratory. Understanding the RelA regulon of M. tuberculosis will involve integrating the results of microarray, proteomic, and reporter-gene (GFP) analyses.
W. B. Davis.Over the past several years, we have investigated the kinetics and thermodynamics of charge injection into DNA, reactions which mimic the introduction of oxidative damage into duplex DNA. These studies were performed using DNA oligomers covalently modified by an acridine chromophore. We believe these DNA oligomers have the potential to be useful in several different classes of experiments. 1) The influence of protein-DNA interactions on the dynamics of charge transfer in DNA. 2) FRET studies of the structure and dynamics of DNA-protein complexes. 3) Modulation of DNA-protein binding by covalent modifications of DNA-binding proteins, eg. acetylation and phosphoylation. In addition, we are currently looking at telomeric DNA sequences tagged with acridines in order to study the dynamics of these unique structures under different environmental conditions (i.e. mono vs. divalent cations, temperature, etc.)
L. M. Gloss. My lab is interested in the folding and assembly of proteins and enzymes. We work on two types of model systems: 1) oligomeric, DNA-binding proteins with all helical, segment-swapped dimerization interfaces, including eukaryotic and archae histones, E. coli FIS (Factor of Inversion Stimulation), and the cyanobacterial Zn-dependent repressor, SmtB; and 2) enzymes from the archae extreme halophiles, particularly dihydrofolate reductase. Our research uses spectral techniques such as circular dichroism and fluorescence, as well as enzymmological studies.
M. D. Griswold. Griswold's laboratory is prominent in the area of reproductive biology for its emphasis on protein biochemistry and the effective use of techniques of DNA manipulation to investigate the mechanisms by which hormones alter gene expression and thus produce cell differentiation. Trainees in this research program would gain experience in the use of protein chemistry to study hormones, their receptors as well as the modification and targeting of secreted proteins.
C. Her. My laboratory is interested in the application of the state-of-the-art genomics and proteomics approaches to address fundamental questions related to mammalian DNA mismatch repair pathways. In particular, we are interested in the understanding of the molecular mechanisms underlying mismatch repair genes in human cancer, as well as to study the roles of mismatch repair proteins in homologous recombination and other biological pathways facilitated by the identification and characterization of protein-protein and protein-DNA interactions. All current on-going projects in the laboratory utilize a combination of molecular biology/genomics, protein engineering/proteomics, and gene targeting approaches.
C. F. Ivory. My primary research interests are in developing instrumentation and protocols for high-performance biological separations, at scales ranging from grams down to nanograms, especially using electric fields. Current projects use various techniques, including counteracting gradient electrofocusing as a replacement for isoelectric focusing, in multi-dimensional separations cascades capable of isolating low-abundance proteins from complex mixtures, e.g., tissue and cell homogenates, blood sera and fermentation broths.
J. P. Jones.The Jones laboratory investigates benign biosynthetic pathways for the oxidation of hydrocarbons. His laboratory uses experimental, computational and predictive models for the cytochrome P450 enzymes that can be used as tools in risk management and the prediction of metabolic disposition of drugs and environmental contaminants in humans. In particular, his research focuses on the prediction of human drug toxicity caused by P450-mediated bioactivation. Students in his laboratory receive excellent and extensive training in protein chemistry, enzymology and toxicoproteomics.
M. L. Kahn.We are interested in the biochemistry, genetics and physiology of intermediary metabolism in the nitrogen-fixing symbiosis between Sinorhizobium meliloti and alfalfa. Current funded projects in the laboratory seek to clone and genetically manipulate the 6200 predicted proteins in S. meliloti, investigate the basis of dicarboxylic acid transport in the bacteria and the mechanisms of energy transduction that lead to nitrogenase. We are also investigating the genetics of the plant contribution to a productive symbiosis.
C. H. Kang. We are trying to identify the alteration in the structure of DNA induced by variety of DNA damage. We are also studying the means of recognition of altered DNA by a repair enzyme and the interaction of DNA repair enzymes with a damage containing DNA. In the last several years, the linkage between mutations in DNA repair genes to some cancers has become increasingly evident. A better understanding of the details of DNA damage and repair will help pave the way to risk assessment based on the mechanics of carcinogenesis. Currently we are carrying out X-ray studies of several enzymes involved in a process that maintains the integrity of the genome including nucleotide excision repair of damaged DNA.
K. H. Kim.I am interested in understanding why vitamin A is essential for testis function during embryonic and postnatal development. As vitamin A signals through the retinoid receptors, we are interested in understanding the regulation and function of these receptors during testis development. The research plan includes the study of protein phosphorylation of the retinoid receptors and identification of target genes and proteins regulated by the retinoid receptors. In addition, we found two inhibitory mechanisms for the retinoid receptor signaling pathway, one involving phthalate plasticizers, the class of chemicals considered to be one of the most abundant environmental contaminants and also endocrine disruptors, and the other involving alcohol, a well-established risk factor for teratogenicity in the fetus. The study of these inhibitory mechanisms revealed even more valuable information about the role of retinoid receptors during embryonic and postnatal testis development. Elucidation of the regulation and function of the retinoid receptors in the testis could lead to development of diagnostic tools and therapeutic drugs in the treatment of human male infertility or development of male contraceptives.
D. L. Kramer.My laboratory seeks to understand how plants convert light energy into forms usable for life, how these processes function at both molecular and physiological levels, how they are regulated and controlled, how they define the energy budget of plants and the ecosystem and how they have adapted through evolution to support life in extreme environments.
J. M. Lee.Our lab is working to optimize the production and stabilization of mammalian proteins secreted from transgenic plant suspension cultures. This includes the characterization of productivity levels under various environmental conditions and the influence of stabilizers in attaining higher productivity levels. This also includes the design of reactor systems to enhance the production and recovery of these secreted proteins. .
N. G. Lewis. The main interests of the Lewis laboratory lies within gaining a detailed understanding of the biochemical basis of various phenylpropanoid radical-radical coupling modes, of those involved in lignan formation, lignin initiation and biopolymer assembly, and of how gene function and gene evolution has occurred, particularly with respect to land plant evolution. A second goal is in gaining knowledge as to how the various genes function in a particular woody plant species to provide the different reinforced tissues and organs unique to the land plants, e.g., sapwood, heartwood, the vascular apparatus, branching tissues, etc. A third goal is in defining how the various medicinally important lignan skeleta are formed in various plant species, with many having antibacterial, antifungal, antiviral and anticancer properties.
N. S. Magnuson.My primary research interest is determining the normal function the proto-oncogene, Pim-1 kinase, determining how it causes cancer and determining how its expression is regulated under normal physiological conditions and during tumorigenesis.
J. H. Nilson.Currently, we are deciphering the critical elements and factors responsible for correct temporal, spatial, and hormonal regulation of the a subunit and LHb genes. We have established that the proximal promoter-regulatory region of either gene is regulated appropriately by GnRH, estrogen, and androgen when studied in transgenic mice. We have also established that two different combinatorial arrays of regulatory elements target expression of the a subunit gene to either gonadotropes or trophoblasts. A similar but distinct combinatorial array of regulatory elements directs expression of the LHb gene to gonadotropes. Having validated the physiological significance of these regulatory elements, we are now cloning their cognate DNA-binding proteins and determining the basis of their interaction with components of the core transcriptional complex. We are also interested in using transgenic technology to develop mouse models that mimic human diseases specific to the reproductive endocrine axis.
T. W. Okita. One project is centered on elucidating the structure-function of the starch regulatory enzyme ADPglucose pyrophosphorylase at both the biochemical level and physiological levels. Past and ongoing metabolic engineering efforts have resulted in new approaches to increase plant productivity and yields. A second project deals with understanding how RNAs are targeted to specific subdomains of the cortical endoplasmic reticulum.
G. H. Palmer. Dr. Palmer and colleagues are using a combined genomic and proteomic approach to identify new vaccine targets and are developing novel vaccine delivery systems to optimize the immune response. The key to the approach is identifying the immune cells that kill the microbe and then using these cells in functional assays in a comprehensive search of all microbial proteins. Using a proteomics approach combined with the complete microbial genome sequences allows identification of the vaccine candidate. This approach differs markedly from those previously used to identify candidate proteins in that it directly couples the immune function to protein identification—without bias as to location or function of the protein itself. The goal is to develop new vaccines against microbial pathogens and use immunization to protect animal and public health. Microbes currently being targeted include tick-transmitted pathogens of animals and humans and bacterial agents of risk for use in bioterrorism.
R. Reeves. The primary focus of our laboratory is the study of protein-DNA interactions involved with regulation of gene transcription in human cells. This interest has led us to investigate the role of the HMGA (formerly called HMG-I/Y) nonhistone proteins in modulating chromatin structure and gene function. The HMGA proteins have been referred to as “hubs of nuclear function” because of their involvement in such diverse biological processes as the control of chromosome structure and gene transcription, regulation of DNA replication and repair and serving as a host-supplied co-factor for retroviral integration into the genome, among others. The genes coding for HMGA proteins are bona fide oncogenes whose over-expression is a diagnostic feature of many different types of human cancers. Our laboratory employs a range of biochemical, biophysical and biological techniques to investigate the molecular mechanisms by which HMGA proteins exert their biological effects in both normal and malignant cells.
J.C. Rogers.Storage vacuoles are distinct organelles separate from lytic vacuoles (lysosomes). We work to define how plant cells make and maintain separate storage and lytic vacuoles, and how membrane and proteins are targeted specifically to each.
E. A. Sheldon. My laboratory is investigating the regulation and function of hsp27 in epithelial cells using molecular and biochemical methods, as well as fluorescence microscopy. We recently reported that hsp27 can associate with basolateral cell junctions in epithelial cell monolayers and co-localize with actin, at the level of the light microscope, at this site. We have now expressing serine and cysteine hsp27 mutants in cultured epithelial cells and are testing these mutants for their ability to interact with cytoskeletal complexes and promote epithelial cell survival and protect epithelial function from disruptions in cells exposed to heat shock, reactive oxygen species and toxins. These studies employ cell fractionation followed by immunoblotting and assays of apoptosis and cell survival using LDH release and fluorimetric quantification of nucleic acid. In addition, we conduct immuolocalization studies and are getting fluorescent proteins f wild-type and mutant hsp27. We plan to test interaction of these proteins with fluorescent fusion proteins of cytoskeletal proteins using fluorescence resonance energy transfer (FRET) methods.
M. Skinner. Dr. Skinner is the Director of both the Center for Reproductive Biology and the newly created Center for Integrated Biotechnology. His research is focused on the investigation of how different cell types in a tissue interact and communicate to regulate cellular growth and differentiation, with emphasis in the area of reproductive biology. The cell of interest and specific interactions investigated have an integral role in controlling the development of the spermatozoa and oocyte.
M. J. Smerdon. Dr. Smerdon has a strong background in physical biochemistry and molecular biology that he has used to great advantage in the study of repair of DNA damage and the role of protein-DNA interactions in DNA repair. This work has substantial relevance to carcinogenesis and the action of environmental mutagens and carcinogens. Trainees with an interest in physical science, biochemistry or molecular biology would find this lab an excellent choice.
B. J. Van Wie. There are currently two major focuses in this laboratory. The first is that of chemical and biochemical sensors for diagnostics and environmental monitoring. As a result of a recent 2000/2001 sabbatical with Dr. David Kidwell of the Naval Research Laboratory collaboration continues on miniature hand-held device technology with telemetry circuitry for water quality monitoring. Ion-selective electrode and enzyme amperometric electrode approaches are used. The second focus consists of a cross-disciplinary effort with William C. Davis of the WSU Veterinary Microbiology & Pathology Department and with the Southern Research Institute that is focused on prophylactic human antibody production and study of the immune response to biological pathogens. The latter work will employ a novel centrifugal bioreactor to study productivity and mixed cell cultures. The device was patented by Washington State University.
J. J. Wyrick.Cells respond to environmental or developmental signals by reprogramming the expression of specific genes throughout their genome. We wish to understand the mechanisms by which this occurs. To do so, we have used genomics-based approaches to decipher how gene expression is regulated in the model eukaryote, S. cerevisiae, with the hope that the lessons we learn in yeast will be applicable in mammalian systems. Ongoing projects include the (1) use of DNA microarrays to profile genome-wide mRNA levels in various transcription factor and histone mutant strains; (2) analysis of global protein-DNA interactions using DNA microarrays; (3) the development of various bioinformatics tools to analyze the microarray data, including an online microarray database; (4) the development of new functional genomic tools to investigate changes in genomic chromatin structure. These projects involve the development and use of cutting-edge biotechnology to investigate how genome expression is regulated in eukaryotic cells.
L. Xun. Xun’s lab focuses on two research areas. One is on the biochemistry of microbial degradation and biotransformation of xenobiotics, and the other is on how Escherichia coli senses and responds to redox changes. Students in this laboratory receive excellent training in areas of microbiology and biochemistry that are relevant to environmental biotechnology.
S. Zhou. The research in our lab is in organic chemistry, biochemistry and protein engineering, centering on two areas pertaining to biotechnology. The first area focuses on mechanistic study and inhibitor design for biologically important enzymes. We are currently studying the bacterial enzyme LuxS, which plays a pivotal role in bacterial quorum sensing, biofilm formation, virulence regulation and metabolism. Absent in humans, the LuxS enzyme is an attractive target for anti-infective agent development. The second area involves characterization of protein post-translational modifications and selective derivatization of proteins. More specifically, we are interested in carboxylate esterification of proteins. S-Adenosylmethionine-dependent methylation of protein carboxylic acids play diverse roles in damaged protein repair, signaling and regulation. We are developing methods to selectively derivatize protein carboxylate esters. These methods will not only allow us to detect and quantify protein carboxylate esters, but also introduce desired functional groups into proteins at selected sites.
