Joseph Sambrook - US grants

Studies are planned to continue to investigate the structure, expression, and function of tropomyosin in normal and transformed cells. Full-length cDNA clones encoding all five isoforms of tropomyosin from rat embryonic fibroblasts will be isolated and their DNA sequences determined to deduce their primary structure. This information will be used to prepare synthetic peptides to be used as immunogens for making isoform-specific antibodies. Such antibodies will be useful in determining the subcellular distribution of each form of tropomyosin and if this localization is altered upon transformation. Bacteriophage lambda recombinants which carry the genes for rat tropomyosin will be isolated and the general structure and organization of the genes determined. For example, these studies will determine if all five forms of fibroblast tropomyosin arise from separate but related genes or if some arise from a single gene via differential processing of the message. The cloned DNAs will be used to study tropomyosin expression to determine if there are alterations in transcription, processing, and translation of tropomyosin messages in transformed cells. To study the functional significance of the altered pattern of tropomyosins in transformed fibroblasts, tropomyosin will be introduced into living cells by (1) microinjection of purified mRNA or (2) the expression of cloned DNAs encoding tropomyosin, for example, via the metallothionein promoter. These studies will determine if expression of tropomyosin in transformed cells will result in changes in cell shape or organization of microfilaments. (V)

The general aim of our laboratory is to understand the mechanisms which govern the direction and speed with which viral proteins expressed in mammalian cells are transported from their sites of synthesis to their final destinations. We are approaching this problem in two ways (i) by analyzing a series of genetically-engineered mutant and chimeric viral proteins that are defective at different stages of their particular transport pathways (ii) by isolating mutants of mammalian cells that are defective in transporting viral proteins to specific destinations. Recently, two types of amino acid sequences have been shown to be necessary and sufficient to guide proteins to these very different cellular locations. One of these - hydrophobic signal sequences - causes nascent polypeptides to be translocated across the membrane of the rough endoplasmic reticulum, from where they may travel to the Golgi apparatus and then to the cell surface or to cellular vesicular cytoplasmic organelles such as lysosomes. The other, more recently discovered, causes proteins to be transported to and retained in the cell nucleus. These karyophilic signals, like hydrophobic signals, vary greatly in sequence from protein to protein; in their simplest forms they consist of a short tract of largely basic amino acids. The goal of this application is to understand how two viral proteins (SV40 T antigen and influenze virus hemagglutinin) move to their specific destinations: the oncogenic SV40 T antigen is transported both to the nucleus and the plasma membrane of transformed cells. Influenze virus hemagglutinin (HA) is a prototypical integral membrane protein that moves with high efficiency along the conventional exocytotic pathway to the cell surface. In particular we will (i) isolate and characterize cellular mutants that are unable to transport HA to the cell surface along the conventional exocytotic pathway (ii) investigate the properties and intracellular transport of a set of chimeric SV40 T antigens and contain a hydrophobic sequence derived from another protein (influenza virus hemagglutinin) at their N-terminus (iii) test the hypothesis that SV40 T antigen associated with the plasma membrane reaches the cell surface, not by the conventional secretory pathway, but by transport through the cell nucleus (iv) analyze the transport of HA along the exocytotic pathway in lines of mutant CHO cells that display grossly altered patterns of glycosylation.

Retinoids are potent anticarcinogenic agents which inhibit the formation of carcinogen induced preneoplastic lesions (hyperplasia, squamous metaplasia) and cause regression of preformed lesions in epithelial tissues. 13-cis-Retinoic acid has demonstrated potent chemopreventive activity coupled with diminished host toxicity in animal studies. 13-cis-Retinoic acid is currently undergoing clinical trials in individuals who are at identifiable high risk for development of epithelial cancers. The prospect for long-term administration of this retinoid necessitates a detailed investigation of its in vivo metabolism. Furthermore, the consequences of chronic 13-cis-retinoic acid therapy on the physiological metabolism of endogenous retinoids must be evaluated. We propose to establish a rat model of chronic 13-cis-retinoic therapy and to use this model to study the in vivo metabolism of this retinoid. The objectives of the proposed research are: 1) the investigation of the metabolites of 13-cis-retinoic acid in selected retinoid target-tissues of chronically treated rats; 2) the characterization of the chemical structure and anticarcinogenic activity of these metabolites and 3) the evaluation of the effects of chronic 13-cis-retinoic acid treatment on certain aspects of phy siological retinol metabolism. Metabolism studies will be conducted with high-performance liquid chromatography (HPLC) methods developed by the authors for the separation of retinoids. The individual in vivo metabolites of 13-cis-retinoic acid will be purified by HPLC and identified by absorbance spectroscopy, mass spectroscopy, nuclear magnetic resonance spectroscopy, an chemical synthesis. The anticarcinogenic activity of the metabolites will be tested in two retinoid-sensitive assays: the epidermal ornithine decarboxylase assay and the embryonal carcinoma differentiation assay. Information gained from the correlation of metabolite structure with anticancer activity would not only help to delineate the in vivo metabolism of 13-cis-RA, but could provide insight useful for the design of synthetic retinoids with enhanced chemopreventative activity, increased biological half-lives, and perhaps selective localization. Three important aspects of vitamin A homeostasis will also be investigated in 13-cis-retinoic acid treated rats; liver vitamin A stores, serum retinol levels, and the steady-state concentrations of retinyl esters, retinol, and tetinoic acid in several vitamin A-target organs. These experiments will be used to pobe the effects of chronic 13-cis-retinoic acid therapy on the metabolism of endogenous retinoids.

fibrinolytic therapy; enzyme biosynthesis; fibrinolysis; myocardial infarction; zymogens; mutant; enzyme mechanism; anticoagulants; antifibrinolytic agents; plasminogen activator; radiotracer; site directed mutagenesis; disease /disorder model; model design /development;

The Ischemic Heart SCOR at The University of Texas Southwestern Medical Center at Dallas will continue research directed at elucidating pathophysiological mechanisms operative during myocardial ischemia and evolving myocardial infarction. Research work in this SCOR is built upon the principle that from the elucidation of fundamental mechanisms responsible for myocardial ischemia and its consequences will come improved patient care and rehabilitation, and the ability to prevent myocardial infarction in some individuals at risk. The experimental studies that we have proposed represent continuations of productive research efforts that have been ongoing within this Program during the previous 14-1/2 years. However, 4 new research sections have been added to the Clinical Section (Project 2) of our Ischemic SCOR Program, including new studies that will elucidate mechanisms responsible for fish oil diets reducing the risk of restenosis after coronary angioplasty; studies that will demonstrate whether serotonin and thromboxane accumulation at sites of coronary artery injury and stenosis are important physiologically in altering coronary vascular resistance and flow; and studies that will help develop nuclear magnetic resonance (NMR) imaging and spectroscopy for the noninvasive detection of myocardial ischemia and to estimate perfusion, evaluate segmental and global ventricular function, and detect atherosclerotic plaques. In addition, new research studies have been added that will utilize molecular biology methods to: (a) evaluate gap junction proteins and their role in the regulation of cell-to-cell communication and calcium binding and determine how their function is altered with ischemic cell injury (Project 8) and (b) study the interaction between tissue plasminogen activator and its endogenous inhibitor and develop mutant tissue plasminogen activators that may be evaluated in relevant animal models (and later patients) for their thrombolytic potential (Project 9). A new research section that will evaluate basic mechanisms contributing to the development of congestive heart failure has also been added (Project 10). We have also added new research studies to evaluate the role of proteases in myocyte and vascular injury (Project 7) and study the effects of myocardial ischemia on the cardiac microcirculation (Project 4). Coronary heart disease remains a major health hazard in contemporary western society. This Ischemic SCOR Program has been productive and made important contributions toward: (a) the elucidation of basic pathophysiologic mechanisms involved in the evolution of cellular and vascular damage during myocardial ischemia and infarction; (b) the development of sensitive methods for the identification of acute myocardial ischemia and infarction and estimating their extent; and (c) providing improved prognostic insight so that selected patients at risk for future ischemic heart disease complications may be identified prior to their development.

DESCRIPTION (Adapted from the applicant's abstract): The endoplasmic reticulum, the first organelle in the secretory pathway, carries out a multiplicity of functions including folding and glycosylation of newly-synthesized polypeptides, assembly of multimeric proteins, storage of Ca++ ions and packaging of secretory proteins into vesicles that are targeted to the next organelle in the secretory chain. The long-term goal of the investigators work is to understand how each of these individual functions is accomplished and how they are coordinated in a way that allows them to be carried coherently. The proposed work couples the advantages of two experimental eukaryotic systems: mammalian cells (or in vitro systems derived from them) for biochemical analysis, and the yeast Saccharomyces cerevisae for genetic analysis. The investigators specific aims are: to analyze the relationship between the structure and function of BiP, the chief chaperone protein of the ER. Using the recently-elucidated three-dimensional structure of N-terminal domain of a closely-related protein (hsc70) as a guide, the investigators will analyze the biochemical and physiological properties of a series of site-directed mutants that (i) alter the amino acids involved in ATP binding and hydrolysis (ii) prevent movement of the hinge region of the molecule (iii) eliminate Ca++ binding sites (iv) modify the sequences within the substrate recognition domain. To analyze in detail the physiological and biochemical properties of a novel form of peptidyl prolyl isomerase (sig-PPI) that appears to be located in the ER of yeast. To explore the role of Ca++ in the maintenance of ER integrity and function. In particular, Dr. Sambrook will investigate the biochemical and physiological behavior of yeast cells carrying mutations in genes coding for three proteins (Ca++ ATPase, calreticulin, and the receptor for Ins (1,4,5)P3 that are thought to be involved in the storage of Ca++ in the ER and in the flux of Ca++ across the ER membrane.

DESCRIPTION (Adapted from the applicant's abstract): The endoplasmic reticulum, the first organelle in the secretory pathway, carries out a multiplicity of functions including folding and glycosylation of newly-synthesized polypeptides, assembly of multimeric proteins, storage of Ca++ ions and packaging of secretory proteins into vesicles that are targeted to the next organelle in the secretory chain. The long-term goal of the investigators work is to understand how each of these individual functions is accomplished and how they are coordinated in a way that allows them to be carried coherently. The proposed work couples the advantages of two experimental eukaryotic systems: mammalian cells (or in vitro systems derived from them) for biochemical analysis, and the yeast Saccharomyces cerevisae for genetic analysis. The investigators specific aims are: to analyze the relationship between the structure and function of BiP, the chief chaperone protein of the ER. Using the recently-elucidated three-dimensional structure of N-terminal domain of a closely-related protein (hsc70) as a guide, the investigators will analyze the biochemical and physiological properties of a series of site-directed mutants that (i) alter the amino acids involved in ATP binding and hydrolysis (ii) prevent movement of the hinge region of the molecule (iii) eliminate Ca++ binding sites (iv) modify the sequences within the substrate recognition domain. To analyze in detail the physiological and biochemical properties of a novel form of peptidyl prolyl isomerase (sig-PPI) that appears to be located in the ER of yeast. To explore the role of Ca++ in the maintenance of ER integrity and function. In particular, Dr. Sambrook will investigate the biochemical and physiological behavior of yeast cells carrying mutations in genes coding for three proteins (Ca++ ATPase, calreticulin, and the receptor for Ins (1,4,5)P3 that are thought to be involved in the storage of Ca++ in the ER and in the flux of Ca++ across the ER membrane.