Mentor Project Summaries
Joseph Chappell
Project Summary
Chappell laboratory (contact: chappell@uky.edu)
My laboratory (www.uky.edu/Ag/Agronomy/Chappell/welcome.htm) is dedicated to understanding the mechanisms plants use to defend themselves against microbial pathogens. For many years, and like many laboratories, we have focused our attention on how plants regulate the biosynthesis of anti-microbial compounds (antibiotics). Our work has utilized a wide range of experimental strategies including biochemical and chemical characterizations, genetic engineering, structure-function comparisons of genes and proteins, and most recently, very simple physiological experiments to uncover putative signal molecules controlling these processes. Because our focus is on the chemical compounds generated by plants, we also have an interest in the development of plant Natural Products for applications in medicine, agriculture and other industries.
Potential projects within the Chappell laboratory include the characterization of new antibiotic compounds from plants, functional analysis of plant genes coding for the biosynthesis of these anti-microbial compounds, and genetic engineering for novel Natural Products.
Glenn B. Collins
Project Summary
The laboratory, greenhouse and field research and graduate/postgraduate training programs are focused on crop plant improvement through the combination of conventional genetics and plant genetic engineering approaches. The main focus of our research is in two areas: development and use of tissue cell culture systems for introducing useful foreign genes into crop plants; and, the recovery and characterization of the transgenic lines regenerated through seed generations. We currently work with soybean, tobacco, Arabidopsis and tomato.
Randy Dinkins
Project Summary
Research centers around the optimizing gene constructs for plant expression, molecular characterization, and generational advancement of genetically engineered agronomic plant lines. Plants worked with include Arabidopsis, Soybean, Tobacco, and Tomato.
David Hildebrand
Project Summary
Our research program focuses on the general area of plant biochemistry and genetics and the application of biotechnology to crop improvement with particular emphasis on food, lipid and oil quality and new uses of agricultural commodities. This research involves the identification, isolation, cloning and manipulation by plant genetic engineering of agriculturally important genes. The major research thrust is the understanding and manipulation of fatty acid metabolism and triglyceride synthesis.
We are improving triglycerides of oilseeds with emphasis on soybeans for enhanced edible and industrial quality. For improved edible quality we are changing the ratios of the mix of vegetable oil fatty acids reducing both the saturated and polyunsaturated fatty acid percentages with a corresponding increase in monounsaturated fatty acids. This will result in a more healthy and stable product. For industrial uses we are tailoring the triglycerides towards high triunsaturated fatty acid level which would make vegetable oils much more valuable in several industrial products such as "drying oils".
We are also are working toward developing oilseed oils high in epoxy fatty acids which will greatly increase their value for a number of industrial products. Epoxy fatty acids are examples of "oxylipins", or oxygenated products of fatty acids. Another major thrust of this research program is the detailed understanding of oxylipin formation in plant tissues. Most plant tissues form a range of oxylipins. Some oxylipins are very important in the flavor and aroma and therefore general quality of plant derived foods. Some are also important in plant pest defense and defense signaling systems. Others we are working with can be useful new antibiotics and for prevention of food-borne illnesses.
Arthur Hunt
Project Summary
A) Messenger RNA 3' end formation and post-transcriptional events in plants. This research involves the analysis of gene expression in transgenic plants and the development of in vitro approaches for studying mRNA processing and metabolism in plants.
B) Molecular genetics of potyviruses. The goals of this research are to understand how potyvirus genes are expressed, define interactions between different viral gene products and between viral and host gene products, and to develop strategies for engineering potyvirus resistance in plants.
C) Expression of foreign genes in plants. Several collaborative projects involving the expression of foreign genes in plants for particular purposes are in progress. These projects seek to use foreign genes as tools for analyzing biochemical and physiological phenomena in plants.
Todd Pfeiffer
Project Summary
My research focuses on producing improved soybean germplasm and investigating the role of recombination in producing genetic variation. Current germplasm emphasis is on evaluating transgenic and genetic sources of virus resistance, creating high linoleic fatty acid oil sources for potential industrial oil modifications, and introgressing genes from the wild species Glycine soja into soybean. Recombination studies involve mapping method studies utilizing SSR molecular markers in soybean, selection for altered recombination frequencies using visual markers in corn, and analyzing the relationship between recombination and transgressive segregation in soybean using molecular markers.
Guoying Bing
Project Summary
Neuroinflammation Mediated Dopaminergic Cell Death
Parkinson's Disease (PD) is a neurodegenerative disease characterized by loss of the dopamine-containing neurons in the substantia nigra pars compacta (SNpc). Although the cause of neuronal death remains unclear, increasing evidence points to the role of chronic inflammatory processes. Dr. Bing's laboratory has recently found that the dopaminergic neurons in the substantia nigra are selectively susceptible to neuroinflammation induced by lipopolysaccharide (LPS), a bacterial endotoxin that activate microglia. The LPS injection not only caused the activation of microglia, but also resulted in a dose-dependent, selective loss of dopaminergic neurons by apoptosis in the SNpc. We therefore hypothesize that LPS activates microglia in the SN resulting in the release of cytotoxic agents. These agents, in turn, activate signal transduction pathways that cause neuronal degeneration of dopaminergic neurons in the SN. The long-term goal of this study is to validate LPS injection as a new animal model of PD that can be used to elucidate the etiology and molecular mechanisms underlying PD and to develop novel therapeutic treatments for this and other neurodegenerative diseases.
Influence of Xenobiotic Metabolites on the Neuronal Cell Death
Dr. Bing's laboratory has recently found that a high level of microsomal epoxide hydrolase (mEH) expression in the brain of patients with Alzheimer's disease as well as the rat brain following exposure to neurodegenerative agents such as kainic acid and trimethyl-tin. Relatively little is known about the expression and function of mEH in the CNS. However, the dual function of mEH in the activation as well as the inactivation of various reactive compounds from xenobiotic metabolites has important implications regarding its role in brain toxicity. The hypothesize is that mEH plays a crucial role in the biotransformation of endogenous xenobiotics and/or environmental chemicals into more toxic metabolites which may cause neuronal degeneration in specific neuronal populations. The identifying the exact role of mEH as well as the role of xenobiotic metabolites in the brain will be critical in understanding the neurodegeneration induced by exposure to toxic chemicals.
Marilyn Duncan
Project Summary
Neuroendocrine Regulation of Biological Rhythms
This research in my laboratory focuses on the neural regulation of biological rhythms, including annual rhythms, such as breeding cycles and hibernation, and circadian (24 hour) rhythms, such as sleep-wake cycles and circulating hormone rhythms. Our work investigates the roles of two biogenic amines, melatonin and serotonin, in regulating annual and circadian rhythms, respectively.
Our current studies are directed towards elucidating how aging disrupts biological rhythms. Age-related disintegration of circadian rhythms, especially sleep-wake cycles, lowers resistance to disease and impairs memory and cognitive function. This disintegration results from functional losses in the circadian pacemaker located in the hypothalamic suprachiasmatic nuclei (SCN). The expression of circadian rhythms depends upon the pacemaker's ability to integrate endogenous and exogenous time signals. During aging, the circadian pacemaker loses its ability to respond to serotonin, one of the neurotransmitters which normally communicates time signals. In order to elucidate the pre- and post- synaptic mechanisms causing age-related desensitization to serotonin, we are studying synaptic release of serotonin, serontinin receptors, and intracellular second messengers, as well as the interaction of serotinin with neuropeptides. These studies utilize a variety of neurochemical and behavioral techniques, including receptor autoradiography, computerized image analysis, in situ hybridization, immunohistochemistry, animal surgery, and monitoring of locomotor activity rhythms. This research will provide a rational basis for novel treatments of sleep disorders in the elderly population, as well as in shift-workers and jet travelers.
Lothar Jennes
Project Summary
Dr. Jennes' research focuses on the identification of the neuroendocrine mechanisms which control female reproduction. The work centers around gonadotropin releasing hormone (GnRH) which is synthesized by a relatively small number of neurons in the septal - preoptic area and transported to the axon terminals in the mediobasal hypothalamus. Here, GnRH is released into fenestrated capillaries and carried through the blood to the anterior pituitary where GnRH stimulates the release and synthesis of LH and FSH. Our research seeks to identify the neurotransmitters and the receptor subtypes which regulate GnRH expression and release. We study currently monoamine and peptide receptor mRNA expression in GnRH neurons to determine which neurotransmitter activates directly specific membrane receptors on GnRH neurons. In addition to this hormonal action, GnRH has important effects inside the brain where it is involved in the facilitation of certain reproductive behaviors. We recently identified and characterized specific membrane receptors for GnRH in the central nervous system and are currently studying the intracerebral effects of GnRH. We focus on the characterization of second messenger systems that are activated by GnRH binding and on the regulation of GnRH receptor mRNA expression by gonadal steroids. The combination of morphological, physiological and molecular biological provides detailed information on the intracerebral mechanisms regulating the GnRH neuronal system and are important for our basic understanding of how the brain participates in the regulation of hormonal homeostasis.
James Matthews
Project Summary
Nutrient Transport Studies
The capacity of nutrient absorption across cell membranes ultimately dictates the supply of nutrients available for metabolic processes. This laboratory focuses on the identification of specific proteins that function to transport amino acids and small peptides across cell membranes of cattle and sheep, and the metabolic enzymes that utilize the transported substrates. After generation of molecular reagents required to identify specific transport proteins and enzymes, how the expression of mRNA protein, and activity is regulated by diet (substrate) and (or) age (ontogeny) is evaluated. Excellent opportunities often arise from these research activities for undergraduate students to learn a variety of molecular and cellular biological research techniques, including RT-PCR, cloning, Northern and immunoblot analyses, and whole-cell transport.
Young-In Chi
Project Summary
Molecular basis of mutations found in inherited human diseases
The complete human genome sequence and modern genetic tools allow us to facilitate the identification of all genes that contribute to human diseases (www.ncbi.nlm.nih.gov/Omim). Random nucleotide mismatch, insertion or deletion during DNA replications can lead to many different types of mutations such as frame shift, nonsense (truncation) or missense (point) mutations. Among these, missense mutations and the encoded single amino acid substitutions are more instructive as site-specific measures of protein function.
We are utilizing the method of X-ray crystallography to understand the molecular basis of these point mutations found in the culprit gene products, especially transciption factors and mediators, from their three-dimensional structures. Transcription factors are central players in all developmental processes and also in adult homeostasis. Because transcription factors orchestrate the manufacture of proteins that make up living tissues and regulate all of the body™s functions, any genetic defects in them can lead to serious illnesses such as developmental defects, metabolic disorders and malignant tumors. Because the structure dictates protein™s functions, these structures of the proteins and complexes will shed lights on how these mutations cause the loss of protein functions at the molecular level, and aid us to design agonists or antagonists once we identified key residues for their functions.
The projects in progress include a transcription factor HNF1a (Hepatocyte Nuclear Factor1a) whose mutations are the most common Mendelian cause of diabetes mellitus, and another transcription factor LMX1B (LIM-homeodomain factor1B) whose mutations are responsible for Nail-patella syndrome, and a putative transcription factor AIRE (Auto Immune Regulator) whose mutations cause onset of various multi-organ autoimmune diseases. More projects will be considered and the accumulated findings and knowledge will be added to the resources available in the public domain.
Structural studies on the mechanisms of cAMP-dependent gene regulation
Many physiological events in a living cell require signal transduction pathways and inducible gene expressions as final readouts. These orchestrated cellular communications involve various protein- protein, protein-DNA, and protein-ligand interactions.
The cyclic AMP-responsive gene regulations represent a good model for ligand-induced signal transduction and gene activation. Upon ligand binding to a Gprotein-coupled receptor, cAMP is produced and initiates a cascade of signal transduction results in the activations of a set of transcription factors known as the cyclic AMP- responsive element binding (CREB) protein family. These transcription factors control the expression of a large number of genes in response to various signaling pathways leading to physiological functions such as memory, circadian rhythm and metabolic pathways. Activation by CREB can occur classically upon phosphorylation at an essential regulatory site (Ser133 in CREB) and the subsequent interaction with the ubiquitous coactivator CREB- binding protein (CBP), or through interaction with a recently identified tissue-specific coactivator ACT (activator of CREM in testis) in a phosphorylation-independent manner. These interactions would further trigger the formation of the transcription initiation complex by recruiting the basal components of the transcriptional machinery.
Our laboratory utilizes a tool of X-ray crystallography to visualize the molecular details of the complexes with CREB, DNA, coregulators and other mediators regulating the cAMP-responsive gene expressions. These efforts will aid us to understand the mechanisms that relay the signal to the main players of the transcriptional machinery, and the mechanisms by which selectivity is achieved in the identification of target genes, as well as the routes adopted to ensure tissue-specific activation upon physiological changes and needs.
Trevor Creamer
Project Summary
The main interests of this laboratory are protein structure and function. We are studying both aspects protein structure and interactions between proteins. We accomplish this using a variety of computational and experimental methods. The specific questions we are currently tackling include: 1) what are the physical determinants of secondary structure in peptides and proteins and how do these relate to interactions between proteins, and 2) what are structures and functions are associated with low-complexity protein sequences ? We are also continuing our long-standing studies of aspects of the protein-folding problem. All of this research can be characterized as ...
Structural Bioinformatics and Experimental Studies of Protein Structure and Function.
Proteins are the cellular machinery. The vast majority of biological processes in cells are controlled and/or performed by proteins. Often this involves proteins interacting with one another. These interactions are usually very specific: a given protein might interact only with one or two other specific proteins. How these proteins recognize each other in the crowded cell environment and how they physically interact with one another are of great interest. We are particularly interested in proline-rich regions of sequence (PRR's) that act as protein-protein interaction domains. Such sequences, which are generally >30% proline in content, are known to interact with small, modular protein domains such as SH3, WW and WVH1 domains. They are involved in numerous vital processes such as cell motility, signal transduction, transcription and the immune system.
PRR's are often assumed to adopt the left-handed polyproline II (PPII) helical conformation. This is the same conformation as adopted by a single strand of collagen. We are elucidating the physical determinants of PPII helix formation using both computational and experimental methods. Computational methods include computer simulations, conformational searches and surveys of known structures. Our main experimental tools are circular dichroism (CD) and NMR spectroscopy.
Low-complexity sequences are protein sequences that are highly enriched in one or a handful of residues types. PRR's are low-complexity sequences enriched in proline. These are surprisingly common - to the point that we feel they must, at least sometimes, be playing important functional roles. Very little is known about the functional roles, or structural tendencies of these sequences. We are examining the occurrence of low-complexity sequences in fully-sequenced genomes, and are embarking on studies of their conformational properties (our work on PRR's is an initial step).
Finally, we are studying the energetics behind protein folding. Our particular emphasis is on the contributions of conformational entropy to the determinants of protein structure.
Robert Dickson
Project Summary
Signal transduction pathways that regulate stress resistance and cellular aging.
My laboratory studies signal transduction pathways and processes that regulate cellular aging and resistance to stresses including heat and starvation. We are particularly interested in the AGC family of protein kinases that include the phosphoinositide-dependent protein kinases (Pkh1 and Pkh2). Pkh1 and Pkh2 regulate downstream protein kinases including Pkc1, Ypk1 and Sch9, which in turn regulate stress responses and cellular aging. How these protein kinases are regulated and the cellular processes they control are unknown for the most part. Ongoing studies aim to: (1) understand how Pkh1 and Pkh2 are activated and deactivated; (2) understand how Pkh1 and Pkh2 regulate Pkc1, Ypk1 and Sch9 activity; and (3) uncover cellular processes regulated by Pkc1, Ypk1 and Sch9 and identify the protein substrates of these kinases. Insight from these studies should help us to understand the cellular basis of aging and stress resistance and may uncover new drug targets for treatment of aging, cancer, cardiovascular disease and neurological disorders.
Rebecca Dutch
Project Summary
Viral entry into cells
Many major human pathogenic viruses (including HIV, herpes simplex virus, easles virus and Ebola virus) are packaged in a membrane. In order for these viruses to infect cells, specific viral proteins promote fusion of the viral membrane with the membrane of the host cell. This process of protein-mediated membrane fusion is the major focus of our work. We study fusion proteins from several different viral systems. First, we are examining the fusion protein from Hendra virus, a newly emerged disease in the paramyxovirus family that is highly pathogenic to both horses and humans. Also, we are studying the F protein from the paramxyovirus SV5, for which multiple mutants have been made and for which a partial atomic structure is known. Finally, we are initiating study of the more complicated membrane fusion event promoted by glycoproteins from herpes simplex type 1. Our long-term goal is to understand the specific molecular events in these important membrane fusion processes.
Richard McCann
Project Summary
The actin cytosleleton and cellular architecture in health and disease.
The actin cytoskeleton is involved in cell motility, cytoplasmic organization, and cell adhesion through the coordinated interaction of many different cytoskeleton-associated proteins. We concentrate on the structure, function, regulation, and cellular roles of a particular class of modular cytoskeletal proteins containing an actin-binding domain known as the I/LWEQ module.
The I/LWEQ module protein talin exists as two isoforms (Talin-1 and Talin-2) in mammalian cells. These proteins have different physiological roles during cellular differentiation, which we are elucidating using several model systems, including skeletal muscle cell lines, primary chicken cardiomyocytes, cortical neurons, and lung cancer cell lines. Dysregulation of Talin-2 may be responsible for several heart and skeletal myopathies and an inherited form of Lou Gehrig's disease (amyotrophic lateral sclerosis, ALS5). Dysregulation of Talin-1 is associated with the lethal progression of lung cancer. The other principal branch of the I/LWEQ module superfamily includes Hip1 (huntingtin interacting protein-1). As a binding partner of huntingtin, Hip1 is implicated in the etiology of Huntington disease. We address these subjects at the molecular, genetic, and cellular levels.
Michael Mendenhall
Project Summary
Cell division is a highly complex process requiring the coordinated synthesis and assembly of all the components needed to form a copy of the original cell. Genetic defects that result in the misregulation of any of these events, when not lethal, often lead to the production of damaged daughter cells. In metazoans, individuals carrying these defects have increased risks of having birth defects or cancer.
Much of the coordination of cell cycle events is achieved through the control of the cyclin-dependent protein kinases (CDKs), of which p34cdc2 is the best known. Two principal means of controlling this kinase have been described: (1) activating and inhibiting phosphorylations and dephosphorylations of the protein kinase itself and (2) regulated synthesis and degradation of the cyclin components. We have identified a third mechanism: inhibition by complex formation with a 32 kD protein, encoded by the yeast SIC1 gene, that blocks access to the CDK substrates. Genetic disruption of SIC1 results in viable cells that generate chromosomal abnormalities that include breakage and loss at a high rate. These events occur in a "stem cell" type pattern in which the chromosomal abnormalities appear to occur in only one of the daughter cells at each division leaving one cell that is always normal. Overproduction of the SIC1 protein in vivo arrests cell division with a pattern that suggests that a specific subset of the cyclin-CDK complexes are being inhibited, a pattern that agrees with our biochemical analysis. We postulate that SIC1 part of a system of inhibitory constraints that when lost leads to premature cell division that in turn produces the chromosomal aberrations. Recent studies in animal cells indicate that tumor suppressors such as retinoblastoma and p53 may be involved in similar types of constraints on cell division. These proteins are also known to form physcial associations with various cyclins in normal cells.
We are continuing a multifaceted analysis of this gene and its gene product in yeast cells to learn how and when it interacts with the kinase and how it is in turn regulated. In addition we have obtained evidence that SIC1 is highly conserved and are extending our studies to vertebrate systems.
Carole Moncman
Project Summary
Striated muscles are responsible for a majority of the motile events in large multi-cellular organisms. Cardiac muscle is responsible for pumping blood throughout an entire organism and is capable of sustaining continuous contractile activity for over 100 years. Skeletal muscles are responsible for all voluntary movements. While these two muscle types differ in their functions, both muscle types achieve their goals via the same functional unit: the striated myofibrils. The striated myofibrils are a highly ordered array of numerous filamentous networks that contain 100's of proteins.
Several decades of muscle research have led to the identification of major myofibrillar proteins and have even elucidated the mechanisms responsible for the production of force and contractile activity in these tissues.. The genetic basis for muscular disorders, such as familial hypertrophic cardiomyopathy, muscular dystrophy, and nemaline myopathy , have also been identified. Minor changes in the primary structure or targeting signals of a single protein component of the striated myofibrils can have dramatic effects on the organization and function of the entire tissue.
Yet with all this research, little is known about the course of events responsible for the formation of this near crystalline lattice found in the striated myofibrils. A major goal of our research is to elucidate the mechanisms involved in myofibril assembly. To this end, we have a identified a novel member of the nebulin family of actin binding proteins that is found exclusively in cardiac muscle, nebulette. We have determined the temporal pattern of expression of this protein and have characterized its complete cDNA. Using the jellyfish green fluorescent protein as a tag, we have expressed truncated forms of nebulette in cardiomyocytes and have assessed the role of the different domains of this protein in the assembly and function of the cardiac myofibrils. Our current research efforts are geared toward fully characterizing the molecular interactions of nebulette by the identifying binding partners using the yeast two hybrid system and complete analysis of known interactions. This work will involve the use of molecular genetic techniques for expression of proteins in both prokayrotic and eukaryotic systems, as well as, biochemical and biophysical techniques to assess the molecular interactions.
Kevin Sarge
Project Summary
Heat shock transcription factor 1, or HSF1, is a critically important DNA-binding protein whose ability to bind is activated when the cell is exposed to any of a multitude of different types of stress conditions. It functions to turn on the expression of a family of heat shock protein genes which the cell needs to survive the stress. However, despite the critical importance of this HSF1 function little is known about how stress actually converts the HSF1 protein from its inactive state to the active DNA-binding form.
Recently we made the exciting discovery that stress causes HSF1 to become modified at a particular lysine residue by a 97 amino acid protein called SUMO-1. As shown in the figure below, our data suggests that SUMO-1 modification is the long-sought mechanism by which stress activates HSF1 DNA-binding ability, whch we think occurs because SUMO-1 attachment causes a conformational change in the HSF1 polypeptide. Interestingly, this SUMO modification also causes HSF1 to localize in punctate bodies in the nucleus, the function of which is currently unknown but which we are investigating. Our laboratory is actively probing the function and regulation of SUMO modification of HSF1 to further understand this exciting new development.
Another important area of research in our laboratory is the investigation of how a related HSF called HSF2 interacts with and regulates the function of protein phosphatase 2A, or PP2A. PP2A plays a critical role in regulating many signal transduction pathways in the cell including regulating the MAP kinase pathway and controlling cell division. This HSF2-PP2A interaction is very exciting to us because it could explain previous findings that HSF proteins, in addition to regulating heat shock protein expression, are also required for cell division. We are currently extending this work to probe the role of PP2A in regulating these critical cellular processes.
Haining Zhu
Project Summary
Our research falls in an exciting new field called ???proteomics???, the study of ???proteome??? that is the complete pool of protein molecules in an organism, tissue or cell. It is known that proteins interact with each other as complexes to function properly (e.g. polymerase holoenzyme, transcription complexes, etc.) and that proteins communicate with each other in signal transduction pathways to respond to stimuli (e.g. MAP kinase cascade, insulin receptor
cascade etc.). Therefore, it is important to understand how proteins interact and function together. We are using mass spectrometry coupled with protein/peptide separation techniques to achieve a
comprehensive understanding of the proteome. We combine the proteomic approach with various molecular biology techniques to study metalloenzymes and metal ions in human diseases such as amyotrophic lateral sclerosis (ALS, Lou Gehrig???s disease, a fatal neurodegenerative diseases) and prostate cancer. We use
proteomic approach to study how mutations in a metallo-protein called Cu,Zn-superoxide dismutase cause ALS. We also study the relationship between zinc ion and apoptosis and the role of zinc ion metabolism in the pathogenesis of prostate cancer.Our group is new and has the state-of-art mass spectrometry instrument and liquid chromatography and 2-dimensional gel electrophoresis equipments. We also use various molecular biology and biochemistry techniques (e.g. PCR, cloning, protein assays, cell
culture etc.). We provide a fun and challenging learning environment for undergraduate researchers. More information can be found at this website:
http://www.mc.uky.edu/biochemistry/Department/faculty/zhu.html
Philip Bonner
Project Summary
Development and regeneration of the vertebrate nervous system includes differentiation of neurons and their subsequent elaboration of axons and dendrites. These neuronal processes elongate and form branches to contact various kinds of pre- and post-synaptic cells. There are numerous influences that modulate axonal and dendritic elaboration based on relatively accessible signaling systems. Neurons commonly use protein tyrosine kinases to regulate axon outgrowth, pathfinding, and synapse location and formation. Similar signaling systems are used by cells during regeneration, especially of peripheral nervous system neurons following traumatic damage. We use cultured chick embryo motor and sensory neurons to examine ways in which axon growth and branching may be controlled during development and regeneration. Interference with normal signaling pathways has revealed alterations in axonal and dendritic growth and arborization patterns. We are especially interested in the use of botulinum toxin to better understand mechanisms that regulate axon branching. Botox is a neurotoxin that blocks synaptic vesicle release by interference with calcium mediated tyrosine kinase(s) and causes pronounced axon branching in vivo and in vitro. Human patients suffering various spasmodic muscle disorders such as blepharospasm can be treated with toxin for relief of symptoms but motor axon branching and growth of sprouts to muscle fibers results in formation of new synapses and the eventual return of symptoms. Axon morphogenesis is also affected by estrogen receptor activity. Our experimental systems are cultured chick embryo neurons and whole embryos treated in ovo and wholemount-stained to reveal alterations in innervation patterns. Pilot experiments to test the usefulness of Drosophila larvae are underway.
Rebecca Kellum
Project Summary
The research in my lab is focused on chromosome structure and function. Much of the work on this subject has been aimed at understanding how the local chromatin environment of a gene promoter modulates expression of the gene. The emphasis in my lab is on more global aspects of nuclear architecture that impact multiple levels of chromosome structure and function in addition to gene transcription.
One research project in the lab focuses on the highly compact chromatin (heterochromatin) found at the centric and telomeric regions of the chromosomes. We are using a combination of biochemistry, genetics, cell biology, and molecular biology research tools to study heterochromatin structure and assembly. These studies have pointed to a role for the DNA replication origin-binding Origin Recognition Complex (ORC) and a heterochromatin sequence-binding protein (HP1/ORC-Associated Protein, HOAP) in the assembly of heterochromatin.
A second research project in the lab makes use of the same combination of research tools to study the protein composition of specialized nucleoprotein structures that can block the action of a transcriptional regulatory element on a promoter. These elements are thought to define the limits of independent domains of gene regulation throughout the genome.
Judith Lesnaw
Project Summary
My research program is focused on understanding the mechanisms of negative-stand RNA virus transcription and replication. Our model system is the transcription complex of vesicular stomatitis virus (VSV) which contains a large multifunctional protein. We are integrating genetic, biochemical and recombinant DNA technologies to the identification of functional domains within the VSV transcription complex. Specific approaches include: identification of functional and structural mutational lesions in spontaneous mutants; generation of site-directed mutations targeted to conserve regions which we recently identified by direct RNA sequencing; and enzyme active site-directed photoaffinity labeling. Knowledge that emerges from these studies will direct the design of new therapeutic agents for a wide array of human pathogens, and will contribute to our understanding of proteins that contain useful constellations of enzyme activities.
Nicholas McLetchie
Project summary
The main objectives of this project are; 1) to detect individual variation in measurable plant traits, 2) to test if this variation can be explain by the sex of the individual plant and, 3) determine if these differences can explain why some population contain only one sex or mainly one sex. Methodologies include, growing and maintaining plants in a greenhouse or growth chamber, measuring plant traits under the microscope or with a rule, and sometimes DNA fingerprinting.Students will be assisting others with on going projects. The exact tasks can include: inputting data into Microsoft Excel, image analysis, caring for plants, DNA extractions and general laboratory duties. I try to get student to be involved in all of the steps of the scientific process.
Stephen L. Dobson
Project Summary
Possible research projects for undergraduates:
1) the use/development of a molecular technique for discriminating
between important mosquito disease vectors2) differential genome skimming of obligate, endosymbiotic bacteria
(requires use of PCR and cloning techniques)3) experiments related to in vitro cultivation of Wolbachia bacteria
(will provide experience with tissue culture, immunofluorescent
staining, and microscopy).
Reddy Palli
Project Summary
Hormonal regulation of insect development
Insect pests compete with us for food, and additionally can transmit human and livestock diseases. Therefore, there has been a continuous demand for the development of insect control methods that are target-specific. Juvenile hormone (JH) and ecdysteroids are the major hormones that regulate various developmental events including molting, metamorphosis and reproduction during insect life cycle. Since these hormones are not present in vertebrates, they represent attractive targets for the development of environmentally friendly insect control methods. During the last two decades there has been good progress in understanding the molecular basis of 20-hydroxyecdysone (20E) action. Non-steroidal ecdysone agonists have been discovered, and four of them are being used for the control of pests. Although, the biological action of JH in regulating metamorphosis and in controlling reproduction in adults is well studied, the molecular basis of JH action is poorly understood. The main objectives of our project are to identify receptor proteins that play critical roles in JH signal transduction and develop applications for their use in agriculture and medicine. Receptors can used to develop screening assays for identification of more potent and target-specific insecticides and they can also be used for development of gene switches for regulation of transgene expression in plants, animals and humans.
Examples of undergraduate projects within our study are:
1. Functional analysis of ecdysone receptor-based gene switches in human cells (supported by RheoGene Inc.).
2. Identification of protein kinases involved in phosphorylation of nuclear proteins that bind to juvenile hormone response elements in Drosophila cells.
3. Identification of phosphatases involved in dephosphorylation of nuclear proteins that bind to juvenile hormone response elements in Drosophila cells.
David Wagner
Project Summary
Population and evolutionary biology in forest trees.
Current work includes:
(i) the molecular basis and population genetic structure of chloroplast and mitochondrial DNA polymorphisms;
(ii) tests of cytonuclear disequilibrium (as well as the geographical structure of this disequilibrium) among the three major eucaryotic genomes (chloroplast, mitochondrial, and nuclear);
(iii) spatial patterns of organellar and nuclear genetic diversity within populations; and
(iv) molecular phylogeny. Taxa of interest include North American, European, and Asian species of pine (Pinus), hemlock (Tsuga), and oak (Quercus) genera.
Glenn Telling
Project Summary
Unlike conventional infectious agents, prions lack genetic material and are composed largely, if not exclusively of an abnormally folded version of the prion protein (PrP). The prion diseases are transmissible neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD) in humans, scrapie of sheep and bovine spongiform encephalopathy (BSE) or 'mad cow disease'. It is now clear that a new variant of CJD (vCJD), which is affecting increasing numbers of young adults and teenagers in the United Kingdom, is the human manifestation of BSE. The major focus of our group is to decipher the molecular and cellular mechanisms of prion pathogenesis using transgenic and in vitro approaches. Expression of PrP transgenes in mice has been an extremely effective means of studying prion diseases. Indeed much of what we understand about the mechanism of prion propagation and the molecular basis of prion strains and species barriers derives from transgenic approaches (Campbell et al., 2000; Telling, 2000). A significant portion of our research effort is currently geared towards producing a transgenic mouse model for studying chronic wasting disease, a highly contagious prion disease of deer and elk of the US,. Although studies in transgenic mice have been extremely informative, there is an urgent need for additional in vitro models of prion diseases. In particular, the ability to study the molecular and cellular mechanisms of human prion disease in cell culture would be extremely valuable. More generally, these studies are expected to cast considerable light on the fundamental mechanisms of neurodegeneration that are relevant to other degenerative brain diseases.
Lynnette Dirk
Project Summary
Peptide deformylase, an enzyme which hydrolyzes the formyl group from the translation-initiating methionine of nascent proteins in bacteria and plant organelles, is being investigated as a target for development of novel antibiotics and herbicides. Research of the plant enzymes ongoing in the lab involves enzymatic and structural characterization of the active site including the ligated metal.
Molecular techniques are utilized to generate mutant forms of the enzyme for further characterization. Functional consequences of the removal of the enzyme from plants continue to be investigated.
A. Bruce Downie
Project Summary
Seeds comprise 70% of the human diet world-wide and make up the bulk of the feed used to produce livestock that constitute a considerable proportion of the other 30% of our diet. The two areas of seed biology I chose to study are seed longevity (how the seed survives dehydration) and seed germination (what processes are required to progress to the point where the nascent root exits the seed). The orchestration of the switch from seed development to seed maturation and then to germination all involve a radical alteration of gene transcriptional activity. These alterations result in profound physiological changes that permit most seeds to survive dehydration to 5% moisture content, extend longevity in this dry state for considerable periods, and finally undergo a fascinating alteration upon imbibition commencing with an intense metabolic activity and culminating in the completion of seed germination and the establishment of the next generation of plants. I examine, at the physiological and molecular level, how the seed thus fulfills its function as a propagule.
Robert Houtz
Project Summary
Our research is focused on providing a detailed enzymological, molecular, and functional analysis of the chloroplast-localized post-translational processing enzyme responsible for the site-specific methylation of Lys-14 in the LS of Rubisco. The formation of enzymatically catalyzed site-specific trimethyllysyl residues has significant effects on functional attributes of several other target protein substrates. Two well described examples are the site-specific methylation of Lys-77 in cytochrome c by a cytochrome c specific N-methyltransferase, which results in a 4-fold increase in import by isolated mitochondria, and methylation of Lys-115 in calmodulin by a calmodulin specific N-methyltransferase, which results in a 3-fold reduction in NAD kinase activating activity. The formation of trimethyllysyl residues in both cytochrome c and calmodulin has been reported to decrease suceptibility to proteolysis, by blocking a potential ubiquitination site in calmodulin, and by increased resistance to non-specific proteases in cytochrome c. However, there has been no clear unifying hypothesis with regards to the in vivo functional significance of covalent modification of the epsilon-amine group of specific lysyl residues in proteins by these highly specific protein N-methyltransferases. This may be in part do to the functional diversity of the target protein substrates, which encompass proteins with structural, regulatory, and enzymatic properties. Indeed it has even been proposed that protein methylase III enzymes may have co-evolved along with the respective protein substrates.
Richard Greenberg
Project Summary
The research projects are divided into 3 areas: (1) basic research on the natriuretic and diuretic properties of uroguanylin (with Dr. Steven Carrithers), (2)in vitro and in vivo atherogenic effects of HIV proteases inhibitors (with Dr Eric Smart), and (3)clincal research trials involving HIV treatments, antibiotic treatments of in-patient infections (fungal, pneumonia, enterococcal infection, and skin infections), treatment of Clostridium difficile diarrhea, and vaccine trials to prevent Shingles, HIV, hepatitis B, pneumococcal infection, and Variola. The Infectious disease clinical research team includes full time laboratory technicians working in a CLIA certified microbiology laboratory, 2 RN's, and dedicated CRAs and regulatory staff. The Uroguanlin research collaborates with Drs. Ott and Jackson in the Department of Physiology and is funded by a VA merit grant. The Shingles vaccine project is funded by a VA COOP study agreement.
J. Scott Bryson
Project Summary
My laboratory has been interested in studying the mechanisms responsible for the induction of murine cyclosporine A-induced syngeneic graft-versus-host disease (SGVHD). Ongoing work in this model has ranged from describing the regulation and pathogenesis associated with the induction and adoptive transfer of this disease, to analysis of T cell clones isolated from diseased animals. It has been shown clinically that a beneficial consequence of GVHD following bone marrow transplantation (BMT) is the generation of anti-tumor or graft-versus-leukemia (GVL) responses. With this in mind, we are beginning to examine the GVL immune potential of SGVHD against transplantable murine tumors. A second area of interest is to study the effects of aging on the development of GVHD following BMT. Clinically, older patients have increased morbidity and mortality due to GVHD. Using a murine GVHD system, preliminary data has suggested that aged recipients of young adult donor cells have an increased incidence of GVHD when compared with young recipients animals. The mechanisms for this finding are unknown at the present time and will require further characterization.
Nancy Webb
Project Summary
My laboratory focuses on the molecular events that lead to cardiovascular disease (atherosclerosis). It has been recognized for many decades that one of the major risk factors for developing atherosclerosis is high concentration of low density lipoprotein (LDL) in the blood. However, LDL by itself does not readily cause atherosclerosis. This suggests that LDL must be modified in order to produce a harmful effect. We study secretary phospholipase A2 (sPLA2) enzymes that are produced by cells within the vessel wall in response to inflammatory stimuli. According to our model, these enzymes hydrolyze phospholipids on the surface of the LDL particle. This destabilizes the particle and causes it to aggregate. These aggregated particles become trapped in the vessel wall and are engulfed by macrophages, which leads to a massive accumulation of lipid and fatty lesions in the heart. We test our model uses two approaches. For one approach, the effect of sPLA2 modifications of LDL is studied in vitro. The interaction of sPLA2-modified LDL and cultured macrophage cells are used for these analyses. For the second approach, genetically altered strains of mice are used to determine whether increased or decreased phospholipase activity in vivo leads to changes in the extent of atherosclerosis.
Indu B. Maiti
Project Summary
My research interest is to improve the technology for manipulating plant genes and genetic materials on the molecular level. The objective is to have better ways to identify and manipulate plant genetic materials in order to modify and improve plants and to produce new products in plants. In this category it meets the following criteria:
" Vectors systems potentially useful in plant genetic engineering.
" Enhancement of expression of single and multiple foreign genes in plants.
" Engineering plants for the production of new materials.
I have coordinated the molecular virology and plant genetic engineering projects; my laboratory has been involved in designing and testing plant genetic engineering tools useful for introducing economically important traits in plants. These projects have been highly productive and very useful in KTRDC program ('new uses', alternative use of tobacco). From these projects we have generated a number of patented intellectual properties.
At present we are engaged primarily in application of these plant genetic engineering tools developed at the KTRDC to introduce valuable traits in plants through the following collaborative projects:
(1) Expression of novel modified TSP14 insecticidal genes in transgenic tobacco. (Collaboration with Dr. Bruce A. Webb, Department of Entomology, University of Kentucky)
(2) Expression of antifungal victoriocin genes in tobacco. (Collaboration with Dr. Said A. Ghabrial, Dept. Plant pathology, University of Kentucky)
(3) Expression of cis-motif viral genes that inhibit insect growth in tobacco.
(Collaboration with Dr. Bruce A. Webb, Department of Entomology, University of Kentucky)
(4) Expression of eukaryotic peptide deformylase genes AtDEF1 and AtDEF2 in tobacco to develop novel herbicide strategy. (Collaboration with Dr. Mark A. Williams, Department of Horticulture, University of Kentucky, Lexington, KY 40546)
(5) Developing novel technology for purification of transgene products in tobacco
(Collaboration with Dr. Czarena Crofcheck, Department of Biosystems & Agricultural Engineering, University of Kentucky, Lexington, KY 40546)
Jeff Davidson
Project Summary
Pyrimidines are essential to normal cellular metabolism, proliferation and viability and to development. The work proposed here is focused on the first three steps in the de novo synthesis of pyrimidines (i.e. the enzymes carbamyl phosphate synthetase [CPSase], aspartate transcarbamylase [ATCase], and dihydroorotase). In mammals, these three enzymes are found in a single translation product of ~240,000 daltons called CAD. CAD is exciting because of its multienzymatic nature, its requirement to form homomultimers to have ATCase activity, its allosteric regulation of the rate of pyrimidine biosynthesis and because having the first three steps on a single polypeptide raise the possibility of channeling. These aspects of hamster CAD are the focus of the three specific aims described in this proposal. The first aim is to establish evidence that the CPSase and ATCase domains interact not only in defining the kinetic characteristics of the CPSase but also in channeling the product of the first enzyme to the substrate binding site of the second enzyme. Emphasis will be placed on using a CAD protein with an ATCase defective in its ability to form CAD multimers. In the second aim, site-directed mutagenesis and informatics will be used to identify the subdomains of the allosteric region involved in binding the positive effector PRPP and the negative effector, UTP. In addition, the studies may help identify residues involved in transmitting information about effector binding back to the active sites of the CPSase. Ultimately, being able to develop a crystal structure of CAD may provide information on how this protein, its multiple enzymatic domains and allosteric regulation function. Therefore, the third aim is to purify milligram quantities of hamster Cad protein from tissue culture cells and to define the optimum conditions for growing high quality crystals. The experiments described in this proposal should provide new date on the structure and function of a key multienzymatic protein, on this ability to channel pathway intermediates and on the structure and function of a key multienzymatic protein, on its ability to channel pathway intermediates and on the structure of the domain involved in regulating the first step in pyrimidine biosynthesis. These studies may provide important new information that may be applicable to other multienzymatic proteins and to the many other enzymes whose activities have recently been shown to require a multimeric structure.
Robert Geraghty
Project Summary
Our overall goal is to understand how alphaherpesviruses, such as herpes simplex virus 1 and 2 (HSV), enter susceptible cells. HSV enters specific cells within the host by first binding to cell-surface receptors and then fusing with the cell membrane. The binding and fusion events are performed by a subset of the viral envelope glycoproteins. An aspect of virus entry that is of particular interest in my laboratory is understanding how the viral envelope glycoproteins interact with their respective receptors, such as the viral glycoprotein D (gD)/Nectin-1 interaction. We use cell culture and in vitro models to study gD/Nectin-1 interactions and mutational analysis is employed to determine regions of both proteins important for binding and virus entry. A second aspect of entry in which we are interested is the molecular mechanism of the fusion event. We conduct mutational analysis on the glycoproteins likely to be involved in virus-to-cell fusion (glycoproteins B, H, and L) and these mutants are tested in virus entry and direct fusion assays in cell culture to shed light on the role each glycoprotein plays in the fusion process. A thorough understanding of the intimate details of virus entry will facilitate the design of anti-viral therapies to block virus entry thereby preventing the virus from ever gaining access to the cell.
Brett Spear
Project Summary
Our research interest is in the area of mammalian gene regulation; in particular, we are interested in transcriptional regulation in the liver during development and disease. Two experimental systems are being used for these studies. First, liver-specific regulation of the mouse alpha-fetoprotein (AFP) and H19 genes is being investigated using biochemical and molecular genetic strategies in tissue culture cells and transgenic mice. In vitro biochemical studies allow us to characterize the interplay between transcription factors and AFP/H19 regulatory elements such as promoters and enhancers. Reporter constructs are analyzed in liver cell lines to further explore the consequences of these interactions. Finally, to fully understand aspects of developmental regulation, we introduce DNA constructs into the mouse germline to produce transgenic animals. In addition, using the tools and resources of the human genome project, we used a positional cloning approach to locate and clone Afr1, a regulator of AFP/H19 expression. Now that we have identified the Afr1 gene, much of our work will focus on the mechanisms by which Afr1 regulates its target genes. Our long-term objective is to understand the complex processes that control gene expression during mammalian development and will ultimately elucidate how specific organs such as the liver arise from a precursor cell population.
In collaboration with Dr. Howard Glauert, we are also studying how transcription factors may contribute to liver cancer. In particular, we are interested in the link between chemical carcinogens, oxidative stress, and the transcription factor NF-kB. Using transgenic and gene knock-out mice, we are using methods to block NF-kB activity specifically in the liver. These mice provide an in vivo model system to study the role of NF-kB in hepatocarcinogenesis.
Brian Stevenson
Project Summary
Lyme disease is caused by the spirochetal bacterium Borrelia burgdorferi. Spread by the bites of certain tick species, it is the most common arthropod-borne disease in the United States. B. burgdorferi has evolved mechanisms by which it can infect both mammalian and arthropod hosts, and be efficiently transmitted between these two very different types of animals. To do so, B. burgdorferi senses its environment and responds accordingly by producing proteins appropriate for each step in the infectious cycle. We are investigating regulatory mechanisms by which B. burgdorferi controls synthesis of infection-associated proteins. Interactions between bacterial and host proteins that confer resistance to host innate immune responses are also being studied. Current and future research projects include characterization of DNA-binding proteins that regulate gene expression, analyses of gene promoter and terminator structures, determination of specific protein expression patterns throughout the bacterial host-vector infectious cycle, and examination of the B. burgdorferi quorum sensing system.
Susan Straley
Project Summary
Dr. Straley's lab studies environmentally requlated virulence gene expression and bacterium-cell signaling by the human plague pathogen Yersinia pestis. A set of virulence operons is coordinately regulated by inputs of temperature, calcium, and signaling through contact with eucaryotic cells such as phagocytes. Dr. Straley's lab is studying both the regulation of these operons and the immunomodulatory mechanisms of the virulence protein products of these operons. Novel aspects of the regulation are the central role of a secretion mechanism for virulence proteins and its regulation by calcium or contact with a phagocyte. A second layer of regulation is the sorting of virulence proteins into ones that are vectorially secreted into phagocytes vs. ones that are targeted to the extracellular media.
Stephen Zimmer
Project Summary
Cell division is a highly complex process requiring the coordinated synthesis and assembly of all the components needed to form a copy of the original cell. Genetic defects that result in the misregulation of any of these events, when not lethal, often lead to the production of damaged daughter cells. In metazoans, individuals carrying these defects have increased risks of having birth defects or cancer.
Much of the coordination of cell cycle events is achieved through the control of the cyclin-dependent protein kinases (CDKs), of which p34cdc2 is the best known. Two principal means of controlling this kinase have been described: (1) activating and inhibiting phosphorylations and dephosphorylations of the protein kinase itself and (2) regulated synthesis and degradation of the cyclin components. We have identified a third mechanism: inhibition by complex formation with a 32 kD protein, encoded by the yeast SIC1 gene, that blocks access to the CDK substrates. Genetic disruption of SIC1 results in viable cells that generate chromosomal abnormalities that include breakage and loss at a high rate. These events occur in a "stem cell" type pattern in which the chromosomal abnormalities appear to occur in only one of the daughter cells at each division leaving one cell that is always normal. Overproduction of the SIC1 protein in vivo arrests cell division with a pattern that suggests that a specific subset of the cyclin-CDK complexes are being inhibited, a pattern that agrees with our biochemical analysis. We postulate that SIC1 part of a system of inhibitory constraints that when lost leads to premature cell division that in turn produces the chromosomal aberrations. Recent studies in animal cells indicate that tumor suppressors such as retinoblastoma and p53 may be involved in similar types of constraints on cell division. These proteins are also known to form physical associations with various cyclins in normal cells.
We are continuing a multifaceted analysis of this gene and its gene product in yeast cells to learn how and when it interacts with the kinase and how it is in turn regulated. In addition we have obtained evidence that SIC1 is highly conserved and are extending our studies to vertebrate systems.
John Littleton
Project Summary
High throughput screening for biologically active compounds from plants.
Plants are potentially a very valuable resource for natural products that have value as pharmaceuticals or nutraceuticals. However, most pharmaceutical companies have given up the use of plants in drug discovery because the methods that are commonly used to test and analyze compounds in plants are outdated and slow. Pharmaceutical drug discovery is now mostly based on "combinatorial chemistry" with "high throughput pharmacological screening" of the resulting hundreds of thousands of novel chemicals that results. However, there is very little evidence that this approach is going to be successful in generating new drugs, and many scientists are advising the industry to return to natural product research. This research group is dedicated to innovations that will make plant drug discovery able to compete with synthetic chemistry. For example, genetic engineering of plants is capable of generating compound diversity that might rival combinatorial chemistry, but the problem of how to evaluate the thousands of compounds that are generated remains. One way in which this can be achieved is to develop high throughput pharmacological screening for natural products in plant extracts, and this approach defines the ABT 395 projects available in this laboratory. The primary screening program is run by Dr Trent Rogers under the direction of Dr Littleton.
The focus of the high throughput screens currently in use is on interactions with receptor proteins that regulate nerve cell excitability and/or viability. Compound libraries or plant extracts can currently be very rapidly evaluated for interactions with membrane receptors (e.g. nicotinic receptors for acetylcholine (nicAChRs) or glutamate/NMDA receptors) or for interactions with nuclear receptors (e.g. glucocorticoid or estrogen receptors). In all cases the technology is capable of distinguishing between receptor subtypes, and these primary screens are performed using robotic handling techniques with 96 well plate reading or fluorescence capacity. All screens are performed "blind" using quality control measures typical of the pharmaceutical industry. Screens for several other types of receptor activity have been used in the past or are planned for the future. A major recent development is the "differential screen" in which mixtures of active compounds (for example in plant extracts) can be distinguished on the basis of activity at two or more closely related receptors. This is of particular value in establishing novelty in extracts for genetically engineered plants which is one focus of this laboratory. Secondary screens for excitation or inhibition, and for effects on neuronal viability, use both simple and complex (organotypic) primary cultures of rat brain together with fluorescent imaging techniques. These high throughput pharmacological screening methods have been developed to support a very strong drug discovery program at the University of Kentucky. For example, much of the research at the Kentucky Tobacco Research and Development Center (which houses Dr Littleton's laboratories) now focuses on novel natural products from genetically modified plants, and most of the screens were developed in response to this initiative. However, the screens are quite capable of adaptation to synthetic compounds and are increasingly being used for this purpose in collaboration with other researchers. The experience gained over the last 5 years forms a very valuable resource for the development and performance of high throughput screens directed at many other molecular targets. ABT 395 students may be exposed to any of the above screening projects or may take part in development of novel screens or in GC/MS analysis of active compounds detected in plant extracts by existing screens.
Craig Miller
Project Summary
Dr. Miller investigates oral disease caused by chronic and recurrent infections of DNA viruses. The lab focuses on the oncogenic role of human papillomavirus in oral squamous cell carcinoma, and the molecular mechanisms of reactivation of herpes simplex virus from latency. Ongoing studies use neurally differentiated PC12 cells in culture as hosts of a HSV-1 latent-like state. Genes critical to the reactivation process are evaluated by exposing cells to stimulatory agents that cause reactivation and release of virus. Genes activated and repressed are identified using reverse transcriptase polymerase chain reaction (RT-PCR), viral deletion mutants, the chloramphenicol acetyl transferase assay (CAT) and luciferase assay. Other technologies used to perform these studies include use of cell culture, direct plaque assay, mutant construction, immunocytochemistry, in situ hybridization, Northern blot analyses, electrophoretic mobility shift assay and the polymerase chain reaction.
Herpes virus latency and reactivation. http://www.mc.uky.edu/microbiology/miller.asp
(see web site for additional information)
Possible research projects for undergraduates:
Lab bench experience in virology
Students enrolled in BIO395 who are interested in working with one of the College of Dentistry faculty mentors listed below are asked to contact Dr. Karen Novak (323-8705, knova2@uky.edu) prior to selecting their mentor.
Jeffrey L. Ebersole
Project Summary
(1) To characterize the use of a murine model for studies delineating the bacterial and host components that contribute to both soft and hard tissue destruction caused by these pathogens. We have used this model to provide comparative characteristics of soft tissue destruction and host responses by various oral pathogens. Additionally, the model has been utilized to evaluate alterations in virulence capacity induced by in vitro and in vivo environmental conditions. This model system is also be used to discriminate the characteristics of the host response that may provide innate and acquired immune protection from the virulence of these microorganisms.
(2) To utilize the nonhuman primate model of periodontitis to evaluate host-bacterial interactions in the chronic inflammatory disease. We have used this model system of infection to examine the local and systemic host responses, which may control destructive periodontal infections. The nonhuman primate model has been used to manipulate the components and kinetics of the host inflammatory/immune response. Additionally, this model is now being used to explore the oral-systemic infection linkage.
(3) To delineate the macromolecules of these bacteria that elicit the production/secretion of various host inflammatory mediators (ie. arachidonate metabolites) and cytokines (ie. IL-1ß, IL-6, IL-8, GM-CSF) from non-immune cells (ie. gingival fibroblasts, epithelial cells). This has included the isolation and characterization of various proteins (eg. leukotoxin, momps, heme binding proteins, cystalysin), LPS molecules (eg. P. gingivalis, A. actinomycetemcomitans, C. rectus, T. pectinovorum), and structures (eg. outer membrane vesicles, fimbria, capsular polysaccharide) from periodontal pathogens. These have been used to evaluate the capacity of individual bacteria to stimulate pro- and anti-inflammatory host responses.
(1) Examination of host responses to oral treponemes associated with HIV periodontitis
(2) Determination of host responses linking oral disease to systemic health problems
L. Kesavalu
Project Summary
Research interests to study the role of oral periodontal bacteria (Porphyromonas gingivalis, Tannerella forsythensis, and Treponema denticola) in the progression of periodontitis and host-response induction. We use murine models (rat periodontitis, mice abscess model, and calvarial bone resorption model) to examine specific bacterial pathogenesis such as host transcriptome response, oral polybacterial synergism, and environmental factors altered virulence expression. In addition, studying the potential role of dietary n-3 polyunsaturated fatty acid (PUFA) regulation on molecular host responses to oral infection with monobacterial and polybacterial pathogenic consortium in rat periodontitis model and nonhuman primate model of periodontitis.
Possible research projects for undergraduates:
1. To determine the effect of synergistic pathogenic consortium P. gingivalis, T. forsythensis, and T. denticola infection in an in vivo rat model of alveolar bone resorption.
2. To examine the effect of dietary n-3 fatty acid on gingival tissue expression of proinflammatory (TNF¥â, IL-1©, IL-6), anti-inflammatory (IL-10, TGF©1, antioxidants) biomolecules, and T cell phenotypes (Th1, Th2) induced by P. gingivalis, T. denticola and T. forsythensis infection.
3. To examine the effect of dietary n-3 FATTY ACID modulation of synergistic pathogenic consortium P. gingivalis, T. forsythensis and T. denticola infection in an in vivo diabetic rat model of alveolar bone resorption.
4. To examine the effect of dietary n-3 FATTY ACID and ANTI-OXIDANT modulation of synergistic pathogenic consortium P. gingivalis, T. forsythensis and T. denticola infection in an in vivo rat model of alveolar bone resorption.
5. To examine the effect of dietary n-3 FATTY ACID and ANTI-OXIDANT modulation of synergistic pathogenic consortium P. gingivalis, T. forsythensis and T. denticola infection in DIABETIC rat model of alveolar bone resorption.
6. To determine interbacterial synergistic virulence effects of P. gingivalis, T. forsythensis, and T. denticola in an in vivo calvarial bone resorption model.
7. To determine the host genomics or transcriptome responses to interbacterial synergistic virulence effects of P. gingivalis, T. forsythensis, and T. denticola in an in vivo calvarial bone resorption model.
Karen Novak
Project Summary
Research area and interests:
Periodontal (gum) disease affects a large percentage of the US population. Work in our laboratory focuses on pathogenic properties of microorganisms associated with periodontal disease. Specific projects are outlined below:
1) Characterization of the mechanisms by which Actinobacillus actinomycetemcomitans (a pathogen associated with certain forms of periodontal disease) transports potential virulence factors from the intracellular to the extracellular environment.
2) Evaluation of bacterial typing systems in the genetic characterization of oral pathogens. Bacterial typing systems are fundamental for epidemiologic study of colonizing clones or for tracing the transmission of microorganisms from one individual to another. There is evidence that using multiple typing systems is more reliable and provides a higher discriminatory power than any one typing system alone in genetic analysis of bacteria. We hypothesized that a select panel of typing systems previously not used with oral pathogens would allow discrimination of different bacterial species as well as different strains within a given species. We have been investigating four polymerase chain reaction (PCR)-based methods in examining genetic variability of eight pathogens associated with periodontitis (Porphyromonas gingivalis; Prevotella intermedia; Actinobacillus actinomycetemcomitans; Tannerella forsynthesis; Treponema denticola; Peptostreptococcus micros; Fusobacterium nucleatum; Selenomonas noxia). The methods were: 1) arbitrarily-primed (AP)-PCR; 2) repetitive extragenic palindromic (REP)-PCR; 3) enterobacterial repetitive intergenic consensus (ERIC)-PCR; and 4) BOX-PCR. 3) Evaluation of the efficacy of antimicrobial peptides against oral biofilms. With the increasing development of antimicrobial resistance to traditional antibiotics, new technologies are needed to combat infections that cause human disease. Oral infections are rarely life threatening but have a significant impact on quality of life, economics, and oral and systemic health. The use of systemic antibiotics for the management of oral infections, especially in cases of the biofilm-associated diseases dental caries and periodontal disease, has not received considerable support because of the implications for the emergence of antibiotic resistance in medically important pathogens. However, the use of topical chemotherapeutic agents is gaining considerable support but the development of such agents is lagging behind the potential applications. We are evaluating the efficacy of natural and synthetic antimicrobial peptides against oral pathogens, grown both planktonically and in biofilms, in an attempt to develop new agents to fight oral infections. Possible research projects for undergraduates: Characterization of protein profiles from wild-type and mutant strains of Actinobacillus actinomycetemcomitans. Molecular characterization of periodontal microorganisms Evaluation of efficacy of antimicrobial peptides against oral biofilms.
Isabel Mellon
Project Summary
Our broad objective is to understand the molecular mechanisms cells use for removing DNA damage. Nucleotide excision repair (NER) is one process that helps cells tolerate exposure to various DNA-damaging agents present in the environment by removing helix-distorting lesions from cellular genomes. Its role in ameliorating the carcinogenic consequences of DNA damage is supported by the observation that patients with the genetic disease xeroderma pigmentosum are defective in NER and are also predisposed to skin cancer. One interesting feature of NER is that it is enhanced in expressed genes. In bacteria, yeast and mammalian cells, ultraviolet light-induced pyrimidine dimers are selectively removed from the transcribed strands of active genes. This feature of NER appears to be ubiquitous and is generally termed transcription-coupled repair (TCR). Our research efforts are focused on characterizing the mechanism, generality and biological consequences of deficiencies in TCR. Studies in E. coli have been emphasized because of the availability of mutants that we have used to identify genes required for the process. To this end we discovered a novel overlap between NER and DNA mismatch repair and found that mutations in mismatch repair genes abolish TCR in E. coli. These results have implications for the mechanism of TCR and its potential role in carcinogenesis. Deficiencies in mismatch repair have been linked to a common cancer predisposition syndrome in humans, hereditary nonpolyposis colorectal cancer (HNPCC). More recently we extended this observation to human cells and demonstrated that several mismatch repair deficient tumor cell lines and HNPCC-derived lymphoblastoid cell are also deficient in TCR. These results imply that deficiencies in TCR and exposure to carcinogens in the environment may contribute to the etiology of tumors associated with genetic defects in mismatch repair. We are currently employing genetic and biochemical approaches to elucidate the role of mismatch repair in TCR.
Scott Diamond
Project Summary
Cancers of the reproductive and endocrine systems result from the failure of the normal systems for establishing cell-identity, growth-control, which depend upon the precise regulation of tissue-specific gene expression. Thus, these cancers are at root a phenomenon of mis-regulation of gene expression. Tissue-specific gene expression is accomplished by a combination of cell-type-specific and ubiquitous gene regulators, many of which are produced as arrays of closely related isoforms. In order to understand precisely how gene expression is controlled, we must understand both the functional effects of small structural differences between isoforms and the mechanisms(s) by which tissue-specific transcription factors confer specificity of target gene expression to ubiquitous factors. Our laboratory takes a number of biochemical, molecular genetic and functional proteomic approaches to these problems, including protein interaction and structure-function analyses. Current projects in the laboratory utilize the anterior pituitary somatolactotroph and the regulation of prolactin hormone gene expression as a model system in which to identify the structural determinants of transcription factor isoform-specific function and to characterize the specific molecular complex that includes both the ubiquitous estrogen receptor alpha and the tissue-specific transcription factor Pit-1.
Ming Cui Gong
Project Summary
Research in our laboratory focuses on elucidating the molecular mechanisms regulating vascular smooth muscle function under normal and diabetic conditions.
A variety of neurotransmitters, hormones and local reagents regulate vascular smooth muscle contraction and relaxation by binding to their respective specific receptors located at the plasma membrane. The force output is produced by the sliding of the myofilaments located in the cytoplasm of smooth muscle cells. One of our goals is to understand, at the molecular and cellular level, how the binding of various neurotransmitters and hormones to their receptors located at the plasma membrane activates and relays the contractile or relaxing signals to the myofilaments located in the cytoplasm. Current projects focus on the rhoA, rho-kinase and phosphatase pathway, and its interaction with the phospholipase A2 and arachidonic acid pathway.
Another major goal of our laboratory is to elucidate the molecular mechanisms responsible for vascular smooth muscle hyperreactivity under diabetic condition. Sixteen million Americans have type II diabetes, and vascular complications are the most common causes of morbidity and mortality in diabetic patients. Disturbances in vascular functionality are detected in early diabetic patients. We and others found that vascular smooth muscle tissue isolated from type II diabetic animals exhibit a significantly increased contractile response to stimuli. The molecular mechanisms that underlie such vascular smooth muscle hyperreactivity are unclear. Currently we are testing the potential causative role of up-regulated prostaglandin H synthase (PGHS, also termed COX) in the vascular smooth muscle hyperreactivity.
State-of-the-art techniques including microarray, real-time PCR, small interference RNA and adenoviral mediated gene transfer are combined with classical physiological and biochemical methods to pursue our goals.
Marianan Nikolova-Karakashian
Project Summary
Our Laboratory studies the function and regulation of metabolism of sphingolipids, a class of second messengers that participate in cellular response to stress and inflammation. We use multifaceted approach that includes the following specific projects:
Project 1: Role of ceramide in up-regulation of acute phase proteins during inflammation and aging.Acute phase proteins like C-reactive protein, a1 acid glycoprotein and serum Amyloid A are markers and independent indicators for inflammation, aging and aging-associated disease like atherosclerosis. Ceramide, which is generated during numerous stress conditions including aging and acute phase response of liver to inflammation is part of the signaling cascade leading to induction of mRNA levels of these proteins. The major focus of this project is to understand the mechanisms that regulate ceramide generation during inflammation and aging and how the excess ceramide affects the cellular responses. Our in vitro approach includes the use of (i) adenovirus-mediate gene transfer to overexpress candidate sphingomyelinases in primary hepatocytes and hepatocellular cell lines, (ii) different cell biology approaches to evaluate their subcellular localization, and (iii) state-of-the art analytical techniques to quantify the mass of sphingolipids. The end-point analyses in this project are different methods to assess the level of mRNA including RT-PCR, the activity of transcription factors from the C/EBP, NFkB and Ap-1 families as well as the activation state of other down-stream components of the signaling cascade, including MAP kinases. In parallel, we apply an in vivo approach involving studies on the acute phase response in acid sphingomyelinase knockout mice.
Project 2: Role of ceramide in circulating lipoproteins for endothelial dysfunction and apoptosis.In addition to being intracellular second messengers, ceramide is present in serum lipoproteins and its levels are up-regulated during systemic inflammation and other stress conditions. The goal of this project is to identify the functional significance of these increases. The experimental approach that we use includes (i) experiments on isolation and characterization of serum lipoproteins from humans and rodents, (ii) induction of specific modification of the levels of ceramide in Low-density lipoproteins using biophysical tools, (iii) investigation on up-take and metabolism of ceramide enriched LDL by human microvascular cells, and (iv) the cellular consequences of ceramide-enriched lipoproteins on cell survival and apoptosis. Knockout mice are also used to confirm the conlusions from cell culture studies in physiological context.
http://www.mc.uky.edu/physiology/people/faculty/karakashian%20research.asp
Melinda Wilson
Project Summary
Estrogen action in the nervous and cardiovascular systems
The gonadal steroid hormone estrogen is essential for reproduction. It plays a critical role in the brain and the pituitary in regulating the sequence of hormonal events that result in female reproduction. In recent years, however, we have begun to appreciate that estrogen also exerts many critical effects on non-reproductive systems as well. Estrogen appears to play a protective role against a variety of neurodegenerative conditions as well as provide protection against cardiovascular disease. As our lifespan continues to increase, women are spending a greater proportion of their lives in a hypoestrogenic state. Thus, it will be critical to understand the tissue-specific mechanisms of estrogen action to help design strategies of replacement that will provide protection in the brain without increasing potentially deleterious effects of long-term estrogen therapy in other tissues.
The actions of steroid hormones are mediated at the cellular level via intracellular receptors that act as transcription factors or indirectly modulate transcription by interacting with other signal transduction pathways. Thus, the study of steroid hormone action provides an exciting opportunity to study physiology at the molecular level. My research focuses on the mechanisms by which estrogen functions in non-reproductive systems. Specifically, we are examining the ability of estrogen to act as a protective factor in the brain as well as in the cardiovascular system.
We are currently investigating how estrogen may protect the brain from HIV proteins. These proteins are secreted from infected non-neuronal cells causing neurotoxic injury and ultimately HIV associated dementia. We have previously shown that estrogen can prevent neuronal apoptosis by both suppressing pro-apoptotic signaling pathways and enhancing anti-apoptotic signaling pathways following injury. Also we have shown that estrogen can suppress the expression of HIV transcription and thus the release of the toxic proteins. Taken together, estrogen has multiple mechanisms of neuroprotection.
Estrogen action in the cardiovascular system is quite complex. Premenopausal women have a lower risk of cardiovascular disease as compared to men. However, recent studies have implicated hormone replacement therapy in adverse cardiovascular events. We have recently begun studies to understand the role of estrogen in atherosclerotic lesion formation. We are using a model of HIV protease inhibitor treatment to induce atherosclerosis and to study estrogen's effects. An understanding of the similarities and differences of estrogen action in cells of the central nervous system and the cardiovascular system will eventually allow us to develop better strategies of hormone replacement.
Mark Farman
Project Summary
In the Farman lab, we study rice blast - the most serious disease of rice worldwide. One aspect of our research is focused on understanding how the fungus Magnaporthe grisea is able to grow inside a living host plant. We are addressing this question by looking for genes that are required for growth in plants yet are dispensable for growth on simple media. Undergraduate projects associated with these studies will involve creation and analysis of fungal mutants, as well as some DNA isolation and characterization. A specific goal will be to use PCR to clone and sequence genes that are disrupted by the mutagenic process.
A second project seeks to identify genes that occur at the ends of fungal chromosomes, as we hypothesize that these regions are involved in fungal adaptation to different ecological niches. We have isolated several chromosome ends from two fungi, Magnaporthe grisea and Neurospora crassa, and are determining their nucleotide sequences. Analysis of the these sequences enables us to identify genes that are close to the chromosome ends, which, in turn, yields insight into possible roles in adaptation. Undergraduate projects linked with this research effort will entail sequence analysis of a large DNA clone. This will be accomplished by shearing the clone into small piece, sub-cloning the fragments, robotic preparation of sequencing templates, robotic sequence reaction set-up and sequence analysis and interpretation. Please visit the Plant Pathology website for information about Graduate Study opportunities.
Peter Nagy
Project Summary
Plant virus replication
We use Tombusviruses, small model RNA viruses of plants, to identify the viral and host factors in replication and to unravel the mechanism of virus replication. RNA replication (multiplication) is the central process in virus infections, which in case of Tombusviruses is a robust process, and it leads to the production of millions of progeny viruses in a day per infected cells. Better understanding of virus replication is expected to lead to improved antiviral strategies and enhanced resistance against virus diseases in plants. The basic discoveries made with Tombusviruses are expected to influence studies on replication of important human and animal pathogens.
Plant virus recombination
The second area of major emphasis in Nagy's research program is to understand the mechanism of virus evolution. This area is also important since viruses can change dramatically via recombination, which, in turn, can lead to emergence of new viruses and strains. The new recombinant viruses may elude host defenses initially due to their unique features. RNA recombination in viruses can also hinder the use of viruses as gene delivery systems or gene expression vectors. We are working on the dissection of the mechanism of RNA recombination, which may lead to development of recombination predicting software, safer virus vectors and lead to improved vaccination programs. Please visit the Plant Pathology website for information about Graduate Study opportunities.
Christopher Schardl
Project Summary
" Symbiosis of grasses and bioprotective fungi
" Evolution of symbiotic mutualism and pathogenesis
" Taxonomy links
" Molecular biology of plant-fungus interactions
" Chemistry and Biosynthesis of protective alkaloids by mutualistic symbionts
" Publications; a representative list of recent pubs.
Acknowledgment of Government Support
Please visit the Plant Pathology website for information about Graduate Study opportunities.
Lisa Vaillancourt
Project Summary
In our lab we work with the fungus Colletotrichum graminicola, which causes anthracnose disease of corn. Symptoms of anthracnose include lesions on the leaves and rotting of the corn pith tissue. We want to understand how the plant and the fungus "communicate" with each other during the first stages of the disease interaction. It is during the earliest stages of a disease interaction that the plant and the pathogen recognize one another, and their respective responses to this event determine whether the outcome of the interaction will be a diseased or a healthy plant. We are specifically working to clone and characterize genes from the fungus that are involved in its ability to sense and respond to its environment, and/or that are required for pathogenic growth. Our long term goal is to manipulate the genes or gene products that we identify in order to control this disease in the field.
Fungal genetics; molecular regulation of fungal development and of fungus-plant disease interactions; anthracnose leaf blight and stalk rot in corn; biological control of corn diseases; development of infection structures in the corn anthracnose fungus, Colletotrichum graminicola; genetic regulation of sexual reproduction in fungi.
Please visit the Plant Pathology website for information about Graduate Study opportunities.
Ernest Bailey
Project Summary
Immunology and genetics of domestic animals, especially the horse. Current areas of research include molecular studies on the major histocompatibility complex, gene mapping, genetic influences on fertility and development, immune mediated infertility in stallions and genetic variation in immune responses of horses.
Thomas Chambers
Project Summary
My lab is an international reference laboratory for equine influenza, one of the most common infectious respiratory diseases of horses. The disease is similar to human influenza except that it is not seasonal, and the biology of the equine influenza viruses is also similar to that of human influenza viruses, particularly in that both are characterized by mutation leading to emergence of new virus variants. Ongoing research in my laboratory is directed at: (1) deciphering the genetic and antigenic variation among different strains of equine influenza virus; (2) developing improved ways to stimulate effective host immunity; (3) studies of the basic biology of influenza virus including the cellular and molecular mechanisms of viral pathogenesis. Much of our recent work has been on vaccine development and evaluation, and on maternal antibody interference with vaccination of young animals. We are now also evaluating maternal antibody interference in connection with West Nile virus vaccination of horse foals.
Kathryn Trembicki Graves
Project Summary
My research program is associated with the Equine Parentage and Genetic Research Lab in the Department of Veterinary Science. We have a number of projects centered on identifying mutations/markers for heritable disease in horses and other animals. Some of these projects are performing a linkage disequilibrium study in Peruvian Pasos for Degenerative Suspensory Ligament Desmitis and sequencing the laminin alpha-3 gene in American Saddlebreds affected with epitheliogenesis imperfecta. We also are sequencing several genes associated with coat color in horses.
David W. Horohov
Project Summary
Dr. Horohov's research program focuses on the identification and characterization of equine cytokines and their role in protective and pathologic immune responses. Cytokines are small, hormone-like proteins that regulate various aspects of the immune system. His group has cloned, sequenced and expressed a number of cytokine genes from the horse and developed assays to detect their presence in various biological samples. These assays are being used to characterize the protective immune responses of horses to bacterial, viral and parasitic infections, as well as the pathologic response in autoimmune and allergic diseases. He also has an interest in the effect of age on the immune system. Age-associated variations in immune responsiveness have been identified in a number of species, including the horse. Both neonatal and aged horses exhibit increased susceptibility to a variety of infectious agents due, in part, to the immature status of the foal's immune system and an age-related decline in immune function in older animals. Studies are underway to characterize the nature of these defects in the immune systems of foals and geriatric horses in hopes of finding better ways to protect these animals from infectious diseases.
Daniel Howe
Project Summary
Equine Protozoal Myeloencephalitis (EPM) is a neurologic disease of horses that is caused by the obligate intracellular parasite Sarcocystis neurona. To gain a better understanding of how S. neurona is able to infect host cells, evade the immune response, and cause disease, my research is focused on determining the molecular and genetic composition of this pathogen. To accomplish this goal, we are conducting an expressed sequence tag (EST) sequencing project on S. neurona, which has thus far generated approximately 8500 gene sequences. This EST database is now being explored to identify parasite molecules that serve as virulence factors and/or elicit significant immune responses in infected animals.
Charles Issel
Project Summary
Research interests: Epidemiology, diagnosis and control of infectious diseases. Our program currently is centered on the equine lentivirus, especially to divine methods to stimulate broad protective immunity through understanding viral genetics and host responses to viral antigens in this persistent lentivirus model for HIV.