1985 — 1994 |
Swank, Richard T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genetic Control of the Microsomal Glucuronidase Complex @ Roswell Park Cancer Institute
The long term objective of this research is to understand the regulation, at the genetic and molecular level, of the specific subcellular localization of Beta-glucuronidase. Glucuronidase is an unusual acid hydrolase in that it has a dual subcellular location in several mammalian tissues with up to 40% of total cellular glucuronidase present as a complex with the protein egasyn in liver microsomes. The results will contribute to knowledge about the mechanisms of biosynthesis of specific subcellular organelles such as the lysosome; an organelle thought to be involved in disease processes such as tumor cell killing and chronic inflammation, and to knowledge of disease processes involving enzyme mislocalization. The systems to be utilized include inbred mouse strains, congenic mutant and wild mice with alterations in glucuronidase subcellular distribution or in glucuronidase processing. These mutants enable studies difficult to perform by purely biochemical or molecular techniques. Also specific antibodies to glucuronidase and egasyn will be utilized to study turnover, processing and subcellular localization of the glucuronidase-egasyn complex. The specific aims are: (1) to kinetically analyze the turnover of the glucuronidase-egasyn complex; (2) to define the structural features of glucuronidase and egasyn important in the formation and stabilization of the glucuronidase-egasyn complex; (3) to examine a novel regulation by egasyn of the post translational processing of lysosomal glucuronidase; (4) to examine the genetic regulation of the structure and stability of the glucuronidase-egasyn complex in various inbred strains, congenic mutant and wild mice; (5) to examine proteins, other than glucuronidase, complexed with egasyn. Also to determine whether these proteins are altered in the Eg(o/o) mutant both in regard to physical properties and subcellular location; (6) to identify the subcellular and suborganellar location(s) of the glucuronidase-egasyn complex.
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0.906 |
1985 — 1991 |
Swank, Richard T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Megakaryocyte-Platelet Granules in Storage Pool Disease @ Roswell Park Cancer Institute
The long term objective of this research is to identify the basic defects in megakaryocytes and platelets in a series of 7 mouse pigment mutants which have prolonged bleeding time accompanied by platelet storage pool disease (SPD). These mice are strong candidates as animal models for such inherited human bleeding disorders as Chediak-Higashi and Hermansky-Pudlak syndromes. Identification of the altered regulatory steps in these mutants will in turn provide valuable information about steps in normal megakaryocytopoiesis and platelet granule production. The specific aims of the project are: a) To determine if inherited platelet storage pool defects in mouse mutants are due to intrinsic abnormalities in platelet precursor cells or to abnormal systemic poietic factors; b) To study the formation of dense granules, which are greatly decreased in platelets of mutant mice, in megakaryocytes of mutant and normal mice; c) To analyze molecular components of other granules such as alpha granules and lysosomes of platelets and megakaryocytes of normal and mutant mice to determine if the granular defect of mutant mice is restricted to dense granules or if more widespread abnormalities in granule biogenesis and processing occur; d) to define more specifically individual mouse mutants with regard to what is known about particular human SPD; and e) to initiate studies designed to detect the primary defective gene product in mouse models of human platelet storage pool disease. The specific methods proposed include the use of special mouse mutant stocks with SPD which are congenic and coisogenic (genetically identical) to the normal parental mouse except for the chromosomal site of the mutation. These congenic mice enable histocompatible bone marrow engraftment of mice using mutant and normal animals as reciprocal donors and hosts. The genesis of dense granules will be studied morphologically at different developmental stages of bone marrow megakaryocytes after specific incorporation of mepacrine and biochemically after using radiolabelled serotonin. Components of other megakaryocyte granules will be analyzed by immunological methods with specific antibodies to components of alpha granules and lysosomal enzymes.
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0.906 |
1986 |
Swank, Richard T |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
System For Analysis and Synthesis of Dna and Peptides @ Roswell Park Cancer Institute
high performance liquid chromatography; peptide chemical synthesis; nucleic acid chemical synthesis; biomedical facility;
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0.906 |
1992 — 2004 |
Swank, Richard T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Animal Models in Inherited Hemorrhagic Diseases @ Roswell Park Cancer Institute |
0.91 |
1995 — 1999 |
Swank, Richard T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
2 Genes Affecting Platelet Function @ Roswell Park Cancer Institute
DESCRIPTION: Normal platelet production and platelet function are critical to the maintenance of vascular integrity. Abnormalities in these processes contribute to several important inherited and acquired platelet disorders such as thrombocytopenia and platelet storage pool disease (SPD). Recent studies indicate that (a) inherited thrombocytopenia due to lowered rates of platelet production is common in humans and (b) that SPD is a very common cause of inherited prolonged bleeding in humans. Current treatment of these disorders by platelet transfusions contains a risk of transmission of blood borne infectious diseases and of alloimmunization of patients. Nevertheless, knowledge of genes regulating platelet production and platelet of SPD is very limited. The specific Aims are to: 1) identify and partially characterize the gm gene by a positional cloning approach; 2) simultaneously approach the identification and partial characterization of the gm gene by a candidate gene approach; and 3)initiate studies to identify the sdy gene by a positional candidate approach. Molecular markers located within 0.3 centiMorgans (3 X 105 base and pairs) of the gm and sdy genes will be identified by comparing the segregation of each gene and microsatellite and/or CDNA markers in interspecific mouse backcrosses.
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0.906 |
1998 — 2000 |
Swank, Richard T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Analysis of a Hermansky-Pudlak Syndrome Gene @ Roswell Park Cancer Institute Corp
DESCRIPTION (Adapted from applicant's abstract): The inherited human syndromes of oculocutaneous albinism and in particular the subform termed Hermansky Pudlak Syndrome (HPS) cause considerable morbidity and mortality, yet knowledge of the genes which cause the syndrome and efficacious treatments are minimal. In contrast, studies in the mouse have identified at least 14 different genes which directly cause HPS. The broad long-term objective of this research is to utilize the genetic advantages of the mouse to clone and characterize the mouse HPS and corresponding human homologs to better understand the causes and ultimately devise therapies for severe forms of human HPS. In particular, it is proposed to clone and partially characterize one of the more defined mouse HPS genes, ruby eye. The ruby eye gene has intrinsic interest, not only because it causes HPS, but also because it regulates the biogenesis/processing/secretion of three subcellular organelles; melanosomes, lysosomes and platelet dense granules. The specific aims of the proposal are to: (1) identify the mouse ruby eye (ru) gene by positional/candidate gene approaches; (2) isolate the human homolog corresponding to the ruby eye cDNA and test for alteration in this gene in HPS kindreds; and (3) partially characterize the expression and regulation of the mouse ruby eye gene.
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0.91 |
2000 — 2004 |
Swank, Richard T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Inherited Diseases Affecting Platelet Function @ Roswell Park Cancer Institute Corp
Knowledge of genes responsible for inherited diseases which affect platelet function and synthesisis is minimal. Consequently, there is a lack of precise molecular diagnostic tests as well as efficacious treatments. The Hermansky-Pudlak Syndromes (HPS) are a group of recessively inherited diseases of humans that cause prolonged bleeding, platelet storage pool deficiency and lung fibrosis, leading to considerable morbidity and premature death. Recent studies have demonstrated that the disease is multigenic in humans and mice. One of the mouse genes, gunmetal (gm), is of additional interest in that it reduces rates of platelet synthesis due to a mutation in the Rab geranylgeranyl transferase (RabGGTase) gene. The long term goals of this proposal are: a) to utilize the inherent advantages of inbred mice to identify additional HPS genes at the molecular level, b) to apply this knowledge to identify mutations in human HPS patients not yet molecularly defined and c) ultimately to devise effective diagnostic approaches and therapies for the disease. A secondary goal is to contribute to basic knowledge of genes which regulate the synthesis and trafficking of subcellular organelles such as platelet dense granules, lysosomes and melanosomes. The Specific Aims of the proposal are to: 1) complete the identification and partial characterization of the mouse sandy (sdy) HPS gene; 2) identify and partially characterize the mouse light ear (le) HPS gene; 3) identify and partially characterize the mouse muted (mu) HPS gene; 4) isolate the human homologues of cloned mouse HPS genes and test for alterations of these genes in human HPS kindreds: 5) clarify the mechanism(s) by which decreased RabGGTase activity causes abnormalities in platelet synthesis and organelle function. Large interspecific mouse backcrosses are used to construct high resolution genetic maps of each mouse HPS gene. Physical maps of the critical genetic regions will be constructed by selection of BACs (bacterial artificial chromosomes) which span the critical genetic intervals. Genes within BACs will be identified by exon trapping, cDNA selection and whole BAC sequencing. Candidate genes will be sequenced to detect mutations. The same genes will be analyzed in human HPS patients, not yet molecularly diagnosed, by cDNA sequencing and related techniques to determine if mutations in these genes are the cause of HPS in these patients. The consequences of a reduction in levels of RabGGTase in gunmetal mice will be analyzed by comparing levels of geranylgeranylation of multiple Rab GTPases and by comparing the subcellular distribution of Rabs in tissues of gunmetal and normal mice.
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0.91 |
2001 — 2005 |
Swank, Richard T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Analyses of Hermansky-Pudlak Syndrome Genes @ Roswell Park Cancer Institute Corp
DESCRIPTION (provided by applicant): The proposed research is part of a comprehensive research effort to molecularly define genes of the mouse, which cause oculocutaneous albinism (OCA), and in particular, the multigenic subform of OCA termed Hermansky-Pudlak Syndrome (HPS). HPS is a genetically heterogeneous, recessively inherited disease, which causes visual defects, hemorrhaging and significantly shortened lifespan due to fibrotic lung disease. A long-term goal is to devise diagnostic and therapeutic strategies for HPS, which presently has no efficacious treatment. The second long-term goal is to understand the mechanism of action of genes responsible for the normal biosynthesis of specialized mammalian subcellular organelles such as melanosomes, platelet dense granules and lysosomes. Two mouse HPS mutants, ruby eye and ruby eye-2, are of special interest, not only because they accurately model HPS, but also because they are phenotypic mimic mutants. Their molecular characterizations are therefore expected to lead to identification of a common pathway and perhaps a common protein complex involved in the disease. The specific aims of this proposal are to: 1) complete the identification and partial characterization of the mouse ruby eye (ru) HPS gene; 2) identify and partially characterize the closely related ruby eye-2 (ru2) HPS gene; 3) isolate human homologues of the cloned ru and ru2 HPS genes and test for alterations of these genes in human kindreds; and 4) identify interactions between the products of the ru and ru2 genes. A multidisciplinary positional/candidate cloning approach will be used to accomplish these aims including construction of high-resolution genetic maps through the use of large interspecific mouse backcrosses and construction of high-resolution physical maps through identification of overlapping contigs of RPCI-23 bacterial artificial chromosomes (BACs). Critical BACs containing the gene of interest will be identified through insertion into recipient transgenic mutant mice. Transcripts within critical BACs will be identified by a combination of exon trapping, cDNA selection and complete BAC sequencing. Transcripts containing the ruby eye and ruby eye-2 mutations will be identified by qualitative and quantitative approaches including complete cDNA sequencing and ribonuclease protection assays, respectively. To identify patients with mutations in the corresponding human genes, the cDNA sequence of the human homologue of each gene and its expression level will be determined in normal individuals and in HPS patients with no mutations in known HPS genes. Each gene will be partially characterized by assays of transcript and protein tissue distribution together with subcellular localization measurements by immunofluorescence techniques. Interaction of the ruby eye and ruby eye-2 genes will be measured by a variety of phenotypic assays in mice bred to be doubly mutant for each gene together with direct tests for interaction of their protein products by co-immunoprecipitation techniques and yeast two-hybrid approaches.
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0.91 |