May 2001
Stella Tsirka, Ph.D.
Downstream Targets of the TPA Plasmin Cascade in Excitotoxin Induced Neuronal Death in the Mouse Hippocampus Efficacy of MMP Inhibitors
Pfizer, Inc. 02/01 – 12/01
The tPA/plasmin extracellular proteolytic cascade can promote
excitotoxin-induced neurona ldeath in the mouse hippocampus. We have shown that this death can be prevented if inhibitors of tPA or plasmin are infused into the brain parenchyma prior to the excitotoxic injury. Since plasmin is known to activate protolytically extracellular matrix metalloproteases (MMPs), we are interested in investigating whether delivery of inhibitors of MMPs into the brain can also protect from the excitotoxic insult, thus involving MMPs in the events that lead to this type of cell death.
January 2001
Charles Iden
Acquisition of a MALDI/TOF Mass Spectrometer System
National Institutes of Health
This proposal requests funds for the purchase of a PE Biosystems
Voyager-DE STR MALDI/TOF Mass Spectrometer and Data System. This high resolution instrument is equipped with a reflectron analyzer and is capable of accurate mass analysis of molecular ions and fragment ions forms through post source decay. It will be located in the SUNY- Stony Brook Mass Spectrometer Facility and will significantly promote the research objectives and laboratory productivity of fourteen distinguished scientists from SUNY-Stony Brook and Brookhaven National Laboratory who comprise the major users group. The mass spectrometer system will make directly available to these investigators important new techniques for the analysis of biomolecules which are presently unavailable locally and inaccessible or difficult to obtain from other organizations. MALDI/TOF is unsurpassed for the structural analysis of
molecules with critical biomedical consequences, including biopolymers such as proteins, oligonucleotides, and oligosachharides and a diverse group of compounds that are non-volatile, labile, or thermally unstable. Accurate mass analysis of protein molecular ions and determination of proteolytic fragments from proteins will be employed to identify proteins by automated computer searching internet-based data banks. Negative ion detection for oligodeoxynucleotides, modified oligomers, and DNA-protein complexes will be essential for several investigators. Automated sample analysis from large multi-well plates will ensure high throughput and optimal utilization of the instrument.
The instrument will be operated and maintained by trained personnel at the USB Mass Spectrometer Facility, and the Director will schedule daily operation and provide technical expertise for the users. An advisory committee will ensure access to the major users and other PHS-supported investigators and will be responsible for the long term operation of the instrument. The Stony Brook administration has made a significant contribution toward the purchase of the instrument and to the installation and operation over its expected lifetime.
May 2000
Miguel Berrios
The Cell Biology of Oxidative DNA Damage and Repair
National Cancer Institute, 4/00 – 3/05
Oxidative DNA damage induced by reactive oxygen species has been associated with aging and age-related diseases, as well as several forms of human cancer. 8-Oxoguanine is a lesion that has been used as a marker for oxidative DNA damage. 8-Oxoguanine has been shown to be mutagenic in vivo and in vitro. Recently, mogg1, a murine 8-oxoguanine-DNA repair enzyme was cloned and over expressed in transgenic animals. Although extensive information has been accumulated on the substrate specificity and the repair mechanism of mogg1 and its isoforms, there is little information regarding their biochemical properties, regulation during the cell cycle and subcellular distribution in cells growing under normal and oxidative stress conditions. Similarly, there is little information regarding the intranuclear distribution of ogg1 and its molecular relationships with chromatin and structural components of the nucleus. We propose to address these questions by raising monospecific antibodies directed against purified bacterially-expressed wild-type recombinant mogg1. We will use these antibodies to determine the subcellular localization of ogg1 and biochemically characterize nuclear and cytoplasmic pools of the enzyme derived from mammalian tissue culture cells as well as the liver of wild-type and transgenic mice over expressing mogg1. We will also characterize the induction of oxidative DNA damage in nutrient deprived cells by determining whether levels of 8-oxoguanine correlate with the synthesis of ogg1 and heat shock proteins and evaluating the cell cycle-dependent regulation of ogg1. Finally, we will colocalize 8-oxoguanine nuclear "hot spots" with ogg1, chromatin and structural protein components of the nucleus in tissue culture cells and tissues from wild-type and transgenic mice over expressing mogg1.
Michael Frohman
Role of Drosophila Phospholipase D in Cellularization
National Institutes of Health, 01/00 – 12/03
During the past several years, a superfamily of phosphatidylcholine-hydrolyzing Phospholipase D (PLD) genes conserved from prokaryotes to mammals has been described. Mammalian PLDs are activated by G-protein-coupled receptor and tyrosine kinase receptor signal transduction pathways. The biochemical step mediated by PLD and the second messenger generated by it are fairly well characterized, but functional roles for PLDs although thought to be important are not well defined except in yeast, where intriguing results have been generated. These results, together with less definitive mammalian studies, suggest that PLD promotes specialized types of membrane biogenesis as it relates to vesicular trafficking from the Golgi and plasma membrane. This proposal addresses potential roles of PLD in Drosophila melanogaster embryogenesis in cellularization, which is a specialized form of membrane biogenesis originating from the Golgi. Roles in germ cell migration will also be examined for other reasons. We have mapped and cloned a PLD gene from Drosophila (denoted dPLD). Similar to mammalian PLD, dPLD appears to be regulated by Protein Kinase C-stimulated pathways. Unlike mammalian PLD but similar to yeast PLD, dPLD is not stimulated by the small G-protein ARF. dPLD-specific antisera reveal that it is expressed in germ cells and in the periphery of the embryo during cellularization. We propose 1) to define the mechanisms that regulate dPLD; 2) to characterize dPLD spatial expression and subcellular localization at high resolution in wild type embryos and embryos with defects in cellularization; 3) to generate and characterize loss-of-function dPLD alleles; 4) to examine the consequence of overexpression of wild-type and mutant PLD during cellularization and germ cell migration. Ultimately, we seek to understand what cell biological pathways dPLD mediates, to model the more complex cell biological and physiological roles undertaken by the mammalian PLDs, and to bridge the growing knowledge concerning PLD mechanism of action in yeast and mammals using technological approaches unique to Drosophila.
Michael Frohman and Andrew Morris
Mammalian Phospholipase D Genes
Gift from Parke Davis, 2000 –
Abstract unavailable
Michael Frohman
Physiological and Cellular Roles of Signal transducing Phospholipase D
National Science Foundation – INT Japan US Cooperative Research, 04/00 – 03/02
Phospholipase D (PLD) is a recently identified mammalian signal transducer that is believed to play important roles in agonist-induced cellular responses including membrane vesicular trafficking (secretion and endocytosis), proliferation,and differentiation. Recent studies have demonstrated to some extent the mechanism through which PLD is regulated. However, the physiological and cellular functions of PLD remain to be clarified. The aims of this project are to determine the signaling pathway(s) in which PLD is involved and the consequence(s) of PLD activation, using biochemical, molecular biological, cell biological, and transgenic approaches. To address these aims, we will identify effectors downstream of PLD activation and determine how their activity changes in the absence of PLD, or when wild type, dominant negative, and constitutively active PLD mutants are over-expressed. Dr. Frohman's group is developing mice and flies (Drosophila) lacking PLD genes, and Dr. Kanaho's group has established mammalian cellular assays for several PLD downstream effectors. Exchange visits by the principle investigators and group members will provide training opportunities for each group in new technologies and hopefully will lead to new insights into physiological and cellular roles for PLD through the cross-fertilization.
David L. Williams
Atherosclerosis and Peripheral Apoprotein E Synthesis
National Institutes of Health, 03/00 - 02/04
The long-term goals of this research are to understand the functional basis for the expression of apolipoprotein (apo) E in peripheral tissues. ApoE plays a major role in systemic cholesterol metabolism by serving as a ligand for the removal of cholesterol-laden plasma lipoproteins by hepatic receptors. ApoE is atypical of most plasma apolipoproteins in being synthesized at high levels in many tissues. ApoE has a variety of biological activities that are distinct from its action to promote the hepatic clearance of remnant lipoproteins. In cultured steroidogenic cells apoE alters cholesterol utilization for hormone synthesis and promotes cholesteryl ester accumulation. ApoE also has a variety of actions on cells that are typical components of atherosclerotic lesions, namely macrophage, endothelial cells, T-lymphocytes, and smooth muscle cells. Many of these effects may be anti-atherogenic within the vascular wall. This proposal consists of 4 aims that are focused on activities of apoE that influence cellular cholesterol metabolism in adrenal cells and activities that are anti-atherogenic within the vascular wall. Experiments in Aim 1 will test the hypothesis that low levels of systemic apoE act within the vascular wall to block an early step in atherosclerotic lesion development. These experiments employ transgenic mice that express apoE selectively in the adrenal gland of apoE-deficient mice. These mice have plasma apoE levels too low to correct the hypercholesterolemia of the apoE-deficient mouse but nevertheless protect against atherosclerosis development. Aim 2 will explicitly test the hypothesis that low levels of apoE provide protection against atherogenesis initiated by apoB48 lipoproteins but do not protect against atherogenesis due to apoB100 lipoproteins. The experiments in Aim 3 test the hypothesis that adrenal gland apoE expression increases adrenal cholesteryl ester storage and diminishes steroid production. Parameters of adrenal cholesterol metabolism will be determined in mice that express apoE selectively in the adrenal gland in comparison to their non-transgenic littermates. Studies in Aim 4 will test the physiological importance of a newly discovered apoE-stimulated pathway for the selective uptake of low density lipoprotein cholesteryl ester. In cultured cells this pathway requires cell surface proteoglycans but is independent of scavenger receptor BI. Mice expressing apoE selectively in the adrenal gland will be used to evaluate this pathway in vivo. Information gained in these studies will be important for understanding the mechanisms by which apoE alters cellular cholesterol metabolism and inhibits the development of atherosclerosis.
David L. Williams
Role of Scavenger Receptor BI in Cellular Cholesterol Metabolism
National Institutes of Health, 02/00 -01/04
The mechanisms of cholesterol transfer between cells and lipoproteins are central to our understanding of cholesterol accumulation in vascular wall cells during atherosclerosis development. The formation of lipid-laden foam cells within the arterial intima is a hallmark of atherosclerotic disease. Previous studies showed that free cholesterol (FC) is transferred between cells and lipoproteins by an aqueous diffusion pathway and by an apolipoprotein-dependent microsolubilization of membrane FC and phospholipid (PL). Recent studies have identified a novel FC transfer pathway that involves the cell surface receptor, scavenger receptor BI (SR-BI). The SR-BI-dependent pathway greatly accelerates the flux of FC between cells and lipoproteins and may play key roles in cholesteryl ester (CE) accumulation and FC efflux from vascular cells and in the transfer of CE and FC from lipoproteins to the liver. This proposal is focused on the mechanisms by which SR-BI alters cellular cholesterol metabolism. Aim 1 will employ domain swapping and mutagenesis approaches to identify SR-BI domains responsible for the effects of SR-BI on FC efflux, changes in plasma membrane lipid composition and organization, and HDL binding. Aim 2 will develop cell culture models in which SR-BI expression is under control of a tetracycline-regulated promoter to permit tightly regulated expression of SR-BI in COS7 cells and in the J774 macrophage model foam cell. Aim 3 will investigate the relationships between SR-BI expression and the intracellular metabolism of cholesterol in cells with inducible SR-BI expression and in macrophage derived from wild type and SR-BI-deficient mice. These experiments will test the effects of SR-BI on FC and CE accumulation from various lipoproteins and distinguish between SR-BI-mediated effects on cholesterol uptake from those on CE hydrolysis and FC efflux from the cell. PL-enriched HDL will be used to test the hypothesis that large PL-enriched HDL are more efficient acceptors for SR-BI-mediated FC efflux as compared to PL-depleted HDL. The effects of SR-BI on lipid domains of the plasma membrane will be evaluated by determinations of the PL, FC, and sphingomyelin composition of plasma membrane and membrane subfractions prepared by detergent based and non-detergent based protocols. These experiments will provide new information that is fundamental to our understanding of SR-BI function, its role in the regulation of cellular cholesterol metabolism, and its impact on the formation of lipid-laden foam cells in the vascular wall.