104 results on '"Novak, E K"'
Search Results
2. Molecular markers near the mouse brachymorphic (bm) gene, which affects connective tissues and bleeding time
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Rusiniak, M. E., O’Brien, E. P., Novak, E. K., Barone, S. M., McGarry, M. P., Reddington, M., and Swank, R. T.
- Published
- 1996
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3. Molecular markers near two mouse Chromosome 13 genes, muted and pearl, which cause platelet storage pool deficiency (SPD)
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O'Brien, E. P., Novak, E. K., Zhen, L., Manly, K. F., Stephenson, D., and Swank, R. T.
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- 1995
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4. Molecular map of Chromosome 19 including three genes affecting bleeding time: ep, ru, and bm
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O'Brien, E. P., Novak, E. K., Keller, S. A., Poirier, C., Guénet, J.-L., and Swank, R. T.
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- 1994
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5. Porphyromonas gingivalis platelet aggregation activity: outer membrane vesicles are potent activators of murine platelets
- Author
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Sharma, A., Novak, E. K., Sojar, H. T., Swank, R. T., Kuramitsu, H. K., and Genco, R. J.
- Published
- 2000
6. High-resolution genetic mapping of the gunmetal gene which regulates platelet production
- Author
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O’Brien, E. P., Zhen, L., Jiang, S. Y., Novak, E. K., and Swank, R. T.
- Published
- 1996
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7. The Murine Chediak-Higashi Mutation and Other Murine Pigmentation Mutations
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Brandt, E. J., Swank, R. T., Novak, E. K., Gershwin, M. Eric, editor, and Merchant, Bruce, editor
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- 1981
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8. Long-range elastic interactions and equilibrium shapes of curved ferroelastic domain walls in crystals
- Author
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N. A. Pertsev, J. Novak, E. K. H. Salj
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Physics and Astronomy (miscellaneous) ,Metals and Alloys ,General Materials Science ,Condensed Matter Physics ,Electronic, Optical and Magnetic Materials - Published
- 2000
9. The effect of various fungicides on the active transport of D-arabinose in Procandida albicans
- Author
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Teren, J, primary and Novak, E . K ., additional
- Published
- 1970
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10. Long-range elastic interactions and equilibrium shapes of curved ferroelastic domain walls in crystals
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A. Pertsev, J. Novak, E. K. H. Salj, N., primary
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- 2000
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11. Survival and Lung Pathology of Mouse Models of Hermansky- Pudlak Syndrome and Chediak-Higashi Syndrome
- Author
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McGarry, M. P., primary, Reddington, M., additional, Novak, E. K., additional, and Swank, R. T., additional
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- 1999
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12. An integrated genetic map of the pearl locus of mouse chromosome 13.
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Seymour, A B, primary, Yanak, B L, additional, O'Brien, E P, additional, Rusiniak, M E, additional, Novak, E K, additional, Pinto, L H, additional, Swank, R T, additional, and Gorin, M B, additional
- Published
- 1996
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13. Expression of egasyn-esterase in mammalian cells. Sequestration in the endoplasmic reticulum and complexation with beta-glucuronidase.
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Novak, E K, primary, Baumann, H, additional, Ovnic, M, additional, and Swank, R T, additional
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- 1991
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14. Analysis of atherosclerosis susceptibility in mice with genetic defects in platelet function.
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Paigen, B, primary, Holmes, P A, additional, Novak, E K, additional, and Swank, R T, additional
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- 1990
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15. Linkage of loci affecting a murine liver protein and arylsulfatase B to chromosome 13.
- Author
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Elliott, R. W., Daniel, W. L., Taylor, B. A., and Novak, E. K.
- Published
- 1985
16. Effects of ammonia on processing and secretion of precursor and mature lysosomal enzyme from macrophages of normal and pale ear mice: evidence for two distinct pathways.
- Author
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Brown, J A, Novak, E K, and Swank, R T
- Abstract
Lysosomal enzymes have been shown to be synthesized as microsomal precursors, which are processed to mature enzymes located in lysosomes. We examined the effect of ammonium chloride on the intracellular processing and secretion of two lysosomal enzymes, beta-glucuronidase and beta-galactosidase, in mouse macrophages. This lysosomotropic drug caused extensive secretion of both precursor and mature enzyme forms within a few hours, as documented by pulse radiolabeling and molecular weight analysis. The normal intracellular route for processing and secretion of precursor enzyme was altered in treated cells. A small percentage of each precursor was delivered to the lysosomal organelle slowly. Most precursor forms traversed the Golgi apparatus, underwent further processing of carbohydrate moieties, and were then secreted in a manner similar to secretory proteins. The lag time for secretion of newly synthesized beta-galactosidase precursor was notably longer than that for the beta-glucuronidase precursor. The source of the secreted mature enzyme was the lysosomal organelle. Macrophages from the pale ear mutant were markedly deficient in secretion of mature lysosomal enzyme but secreted precursor forms normally. These results suggest that ammonia-treated macrophages contain two distinct intracellular pathways for secretion of lysosomal enzymes and that a specific block in the release of lysosomal contents occurs in the pale ear mutant.
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- 1985
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17. Lumenal location of the microsomal beta-glucuronidase-egasyn complex.
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Brown, J, Novak, E K, Takeuchi, K, Moore, K, Medda, S, and Swank, R T
- Abstract
Mouse liver beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the beta-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within the vesicle lumen or only weakly bound to the inside of the vesicle membrane. This conclusion was derived from release studies using low concentrations of Triton X-100 or controlled sonication. Both the intact complex and free egasyn were released in parallel with lumenal proteins, not with intrinsic membrane components. Also, beta-glucuronidase was protected from digestion by proteinase K by the membrane of microsomal vesicles. The hydrophilic nature of both the complex and free egasyn was confirmed by phase separation experiments with the detergent Triton X-114. Egasyn is one of an unusual group of esterases that, despite being located within the lumen or only weakly bound to the lumenal surface of the endoplasmic reticulum, do not enter the secretory pathway.
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- 1987
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18. The gene mutated in cocoa mice, carrying a defect of organelle biogenesis, is a homologue of the human Hermansky-Pudlak syndrome-3 gene.
- Author
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Suzuki T, Li W, Zhang Q, Novak EK, Sviderskaya EV, Wilson A, Bennett DC, Roe BA, Swank RT, and Spritz RA
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- Alleles, Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Cells, Cultured, Chromosome Mapping, Cloning, Molecular, DNA chemistry, DNA genetics, Female, Gene Expression, Genes genetics, Hair Color genetics, Heterozygote, Homozygote, Humans, Intracellular Signaling Peptides and Proteins, Male, Melanocytes cytology, Melanocytes metabolism, Melanocytes ultrastructure, Melanosomes metabolism, Mice, Mice, Inbred C3H, Mice, Inbred C57BL, Mice, Mutant Strains, Microscopy, Electron, Molecular Sequence Data, Mutation, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Tissue Distribution, Membrane Proteins genetics, Organelles metabolism
- Abstract
Hermansky-Pudlak syndrome (HPS) is a group of human disorders of organelle biogenesis characterized by defective synthesis of melanosomes, lysosomes, and platelet dense granules. In the mouse, at least 15 loci are associated with mutant phenotypes similar to human HPS. We have identified the gene mutated in cocoa (coa) mice, which is associated with an HPS-like mutant phenotype and thus represents a strong candidate for human HPS. Analysis of coa-mutant mice and cultured coa-mutant mouse melanocytes indicates that the normal coa gene product is involved in early stages of melanosome biogenesis and maturation.
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- 2001
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19. 5'-UTR structural organization, transcript expression, and mutational analysis of the human Rab geranylgeranyl transferase alpha-subunit (RABGGTA) gene.
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Li W, Detter JC, Weiss HJ, Cramer EM, Zhang Q, Novak EK, Favier R, Kingsmore SF, and Swank RT
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- 5' Untranslated Regions analysis, Alkyl and Aryl Transferases chemistry, Alkyl and Aryl Transferases metabolism, Animals, Base Sequence, Cloning, Molecular, DNA Mutational Analysis, Disease Models, Animal, Exons genetics, Genetic Testing, Humans, Introns genetics, Lysosomes metabolism, Lysosomes pathology, Melanosomes metabolism, Melanosomes pathology, Mice, Molecular Sequence Data, Organelles metabolism, Organelles pathology, Platelet Storage Pool Deficiency pathology, Polymorphism, Single Nucleotide genetics, Protein Subunits, RNA Splice Sites genetics, Reverse Transcriptase Polymerase Chain Reaction, Syndrome, 5' Untranslated Regions genetics, Alkyl and Aryl Transferases genetics, Mutation genetics, Platelet Storage Pool Deficiency enzymology, Platelet Storage Pool Deficiency genetics, Transcription, Genetic
- Abstract
Hermansky-Pudlak syndrome (HPS) is a recessively inherited disease with dysfunction of several related subcellular organelles including platelet-dense granules, melanosomes, and lysosomes. Our recent identification of the mutation in murine Rab geranylgeranyl transferase alpha-subunit gene (Rabggta) in one mouse model of HPS, the gunmetal mouse, suggested that human patients with similar phenotypes might have mutations in the human orthologous RABGGTA gene. This prompted reanalysis of the 5'-untranslated structure of the human RABGGTA gene in normal individuals and in patients with deficiencies of platelet-dense granules (alphadelta-SPD), alpha granules (alpha-SPD or gray platelet syndrome, GPS) or alpha plus dense granules (alphadelta-SPD). We report the complete sequence of intron alpha of RABGGTA and demonstrate that exon alpha is immediately upstream of intron alpha. The exon/intron structural organization of the 5'-untranslated region (UTR) of human RABGGTA was found to be similar to that of the mouse Rabggta gene. However, exons alpha and introns alpha are not homologous between mouse and human. Features of the 5'-UTR of RABGGTA suggest it is a housekeeping gene. While obvious disease-causing mutations of human RABGGTA were not found in our existing SPD patients by sequencing its entire coding region, several polymorphisms of RABGGTA including a putative cryptic splicing mutation in intron 4 were identified. Knowledge of the 5'-UTR structure of RABGGTA and its common polymorphisms will be useful for mutation screening or linkage analysis in patients with albinism, thrombocytopenia, or platelet SPD., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
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20. Genomic structure of the mouse Ap3b1 gene in normal and pearl mice.
- Author
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Feng L, Rigatti BW, Novak EK, Gorin MB, and Swank RT
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- Adaptor Protein Complex alpha Subunits, Adaptor Proteins, Vesicular Transport, Alleles, Animals, Base Sequence, DNA, Complementary, Exons, Gene Deletion, Gene Duplication, Introns, Mice, Mice, Inbred C3H, Mice, Inbred C57BL, Mice, Mutant Strains, Molecular Sequence Data, Mutation, RNA, Tandem Repeat Sequences, Hermanski-Pudlak Syndrome genetics, Hypopigmentation genetics, Membrane Proteins genetics, Monomeric Clathrin Assembly Proteins
- Abstract
The mouse hypopigmentation mutant pearl is an established model for Hermansky-Pudlak syndrome (HPS), a genetically heterogenous disease with misregulation of the biogenesis/function of melanosomes, lysosomes, and platelet dense granules. The pearl (Ap3b1) gene encodes the beta3A subunit of the AP-3 adaptor complex, which regulates vesicular trafficking. The genomic structure of the normal Ap3b1 gene includes 25 introns and a putative promoter sequence. The original pearl (pe) mutation, which has an unusually high reversion rate on certain strain backgrounds, has been postulated to be caused by insertion of a transposable element. Indeed, the mutation contains a 215-bp partial mouse transposon at the junction point of a large tandem genomic duplication of 6 exons and associated introns. At the cDNA level, three pearl mutations (pearl, pearl-8J, and pearl-9J) are caused by deletions or duplications of a complete exon(s)., (Copyright 2000 Academic Press.)
- Published
- 2000
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21. A mutation in Rab27a causes the vesicle transport defects observed in ashen mice.
- Author
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Wilson SM, Yip R, Swing DA, O'Sullivan TN, Zhang Y, Novak EK, Swank RT, Russell LB, Copeland NG, and Jenkins NA
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- Albinism, Oculocutaneous, Animals, Biological Transport genetics, Blood Platelets pathology, Chromosome Mapping, Cytoplasmic Granules parasitology, Disease Models, Animal, Gene Library, Genetic Complementation Test, Intermediate Filament Proteins metabolism, Melanocytes ultrastructure, Mice, Mice, Inbred C3H, Mice, Mutant Strains, Muridae, Protein Binding, RNA Splicing, Skin cytology, Syndrome, rab27 GTP-Binding Proteins, Hair Color genetics, Intracellular Membranes metabolism, Melanocytes metabolism, Myosin Heavy Chains, Myosin Type V, rab GTP-Binding Proteins genetics
- Abstract
The dilute (d), leaden (ln), and ashen (ash) mutations provide a unique model system for studying vesicle transport in mammals. All three mutations produce a lightened coat color because of defects in pigment granule transport. In addition, all three mutations are suppressed by the semidominant dilute-suppressor (dsu), providing genetic evidence that these mutations function in the same or overlapping transport pathways. Previous studies showed that d encodes a major vesicle transport motor, myosin-VA, which is mutated in Griscelli syndrome patients. Here, using positional cloning and bacterial artificial chromosome rescue, we show that ash encodes Rab27a. Rab GTPases represent the largest branch of the p21 Ras superfamily and are recognized as key players in vesicular transport and organelle dynamics in eukaryotic cells. We also show that ash mice have platelet defects resulting in increased bleeding times and a reduction in the number of platelet dense granules. These defects have not been reported for d and ln mice. Collectively, our studies identify Rab27a as a critical gene for organelle-specific protein trafficking in melanocytes and platelets and suggest that Rab27a functions in both MyoVa dependent and independent pathways.
- Published
- 2000
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22. Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis.
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Detter JC, Zhang Q, Mules EH, Novak EK, Mishra VS, Li W, McMurtrie EB, Tchernev VT, Wallace MR, Seabra MC, Swank RT, and Kingsmore SF
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- Animals, Chromosomes, Artificial, Yeast, Genotype, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Molecular Sequence Data, Phenotype, Protein Prenylation, Alkyl and Aryl Transferases genetics, Blood Platelets cytology, Cell Division genetics, Mutation, rab GTP-Binding Proteins metabolism
- Abstract
Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders.
- Published
- 2000
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23. Abnormal vesicular trafficking in mouse models of Hermansky-Pudlak syndrome.
- Author
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Swank RT, Novak EK, McGarry MP, Zhang Y, Li W, Zhang Q, and Feng L
- Subjects
- Animals, Cloning, Organism, Disease Models, Animal, Hermanski-Pudlak Syndrome genetics, Humans, Mice, Mice, Mutant Strains, Hermanski-Pudlak Syndrome metabolism
- Abstract
Hermansky-Pudlak Syndrome (HPS) is a group of related multigenic recessively inherited disorders which causes abnormalities in the biosynthesis and/or function of three related organelles; melanosomes, platelet-dense granules and lysosomes. These lead, in turn, to hypopigmentation, prolonged bleeding and ceroid deposition. Positional cloning strategies have identified five mouse HPS genes. Two orthologous human diseases (HPS1 and HPS2) have likewise been identified. At least four of the five mouse genes encode proteins involved in the regulation of intracellular vesicle trafficking. The pearl (HPS2) and mocha genes encode the beta3A and delta subunits, respectively, of the AP-3 adaptor complex, which captures organelle membrane proteins at the trans-Golgi apparatus. The protein products of the pallid and gunmetal genes are also important components of the vesicular trafficking machinery. The former interacts with a t-SNARE, syntaxin13, and the latter is the alpha subunit of Rab geranylgeranyltransferase, which renders Rab proteins sufficiently lipophilic to function at their target membranes. The pale ear (HPS1) gene encodes a ubiquitously expressed protein of unknown function. Recent physiological studies have shown that mouse HPS mutants, like their human HPS counterparts, have variably reduced lifespans and may have lung abnormalities.
- Published
- 2000
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24. Analyses of proteins involved in vesicular trafficking in platelets of mouse models of Hermansky Pudlak syndrome.
- Author
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Richards-Smith B, Novak EK, Jang EK, He P, Haslam RJ, Castle D, Whiteheart SW, and Swank RT
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- Albinism, Oculocutaneous genetics, Animals, Antigens, Surface metabolism, Biological Transport, Carrier Proteins genetics, Carrier Proteins metabolism, Chromosome Mapping, Disease Models, Animal, GTP-Binding Proteins metabolism, Golgi Matrix Proteins, Humans, Intracellular Signaling Peptides and Proteins, Membrane Proteins metabolism, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Mutant Strains, Munc18 Proteins, Muridae, N-Ethylmaleimide-Sensitive Proteins, Nerve Tissue Proteins metabolism, Protein Isoforms metabolism, Proteins analysis, Proteins genetics, Qa-SNARE Proteins, Qb-SNARE Proteins, Qc-SNARE Proteins, Syntaxin 1, Albinism, Oculocutaneous metabolism, Blood Platelets metabolism, Cytoplasmic Granules metabolism, Proteins metabolism, Vesicular Transport Proteins, rab GTP-Binding Proteins
- Abstract
Hermansky Pudlak syndrome (HPS) is an autosomal recessive inherited disorder characterized by defects in synthesis and/or secretion of three related subcellular organelles: melanosomes, platelet-dense granules, and lysosomes. In the mouse, mutant forms of any of 14 separate genes result in an HPS-like phenotype. The mouse pearl and mocha genes encode subunits of the AP3 adaptor protein complex, confirming that HPS mutations involve proteins regulating intracellular vesicular trafficking. Therefore, expression of several additional proteins involved in vesicular transport was examined by immunoblotting of platelet extracts from HPS mutant and control mice. Platelet levels of SCAMPS (secretory carrier membrane proteins), Rab11, Rab31, NSF (N-ethylmaleimide-sensitive fusion protein), syntaxin 2, syntaxin 4, munc18c, and p115/TAP (p115/transcytosis-associated protein) were not significantly altered in several different HPS mutants. However, gunmetal (gm/gm) platelets contained decreased amounts of SNAP-23. The Snap23 gene was mapped to mouse chromosome 5, demonstrating it cannot encode the gm gene, which maps to chromosome 14. It is likely therefore that the gm gene functions upstream of SNAP-23 in vesicular trafficking., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
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25. Increased incidence and analysis of emperipolesis in megakaryocytes of the mouse mutant gunmetal.
- Author
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McGarry MP, Reddington M, Jackson CW, Zhen L, Novak EK, and Swank RT
- Subjects
- Animals, Bone Marrow Cells cytology, Bone Marrow Cells physiology, Cell Count, Female, Femur, Flow Cytometry, Male, Megakaryocytes cytology, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Mutation, Ploidies, Spleen cytology, Spleen physiology, Megakaryocytes physiology, Phagocytosis
- Abstract
Mutant gunmetal (gm/gm) mice exhibit prolonged bleeding, platelet granule defects, abnormal megakaryocyte demarcation membranes, and thrombocytopenia. The number of megakaryocytes in gm/gm mice is increased substantially. Also, the percentage of gm/gm megakaryocytes exhibiting emperipolesis is increased. However, the number of emperipolesed cells per megakaryocyte is not. EC are of several hematopoietic lineages, with a slight skew to granulocytes, and include mature, primitive, and degenerating cells. No significant differences in the types of emperipolesed cells were observed between mutant mice and their normal gm/+ or +/+ counterparts. The increased incidence of emperipolesis in gm/gm megakaryocytes is controlled by the megakaryocyte genotype, not systemic factors. A significant practical finding of these studies was the demonstration that increased emperipolesis results in a significant "right shift" in megakaryocyte ploidy determined by flow cytometry., (Copyright 1999 Academic Press.)
- Published
- 1999
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26. Abnormal expression and subcellular distribution of subunit proteins of the AP-3 adaptor complex lead to platelet storage pool deficiency in the pearl mouse.
- Author
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Zhen L, Jiang S, Feng L, Bright NA, Peden AA, Seymour AB, Novak EK, Elliott R, Gorin MB, Robinson MS, and Swank RT
- Subjects
- Adaptor Protein Complex alpha Subunits, Adaptor Proteins, Vesicular Transport, Albinism, Oculocutaneous blood, Albinism, Oculocutaneous genetics, Albinism, Oculocutaneous pathology, Animals, Biological Transport, Blood Platelets physiology, Blood Platelets ultrastructure, Gene Expression, Mice, Mice, Inbred C3H, Mutation, Platelet Storage Pool Deficiency blood, Platelet Storage Pool Deficiency pathology, Membrane Proteins biosynthesis, Membrane Proteins genetics, Monomeric Clathrin Assembly Proteins, Platelet Storage Pool Deficiency genetics
- Abstract
The pearl mouse is a model for Hermansky Pudlak Syndrome (HPS), whose symptoms include hypopigmentation, lysosomal abnormalities, and prolonged bleeding due to platelet storage pool deficiency (SPD). The gene for pearl has recently been identified as the beta3A subunit of the AP-3 adaptor complex. The objective of these experiments was to determine if the expression and subcellular distribution of the AP-3 complex were altered in pearl platelets and other tissues. The beta3A subunit was undetectable in all pearl cells and tissues. Also, expression of other subunit proteins of the AP-3 complex was decreased. The subcellular distribution of the remaining AP-3 subunits in platelets, macrophages, and a melanocyte-derived cell line of pearl mice was changed from the normal punctate, probably endosomal, pattern to a diffuse cytoplasmic pattern. Ultrastructural abnormalities in mutant lysosomes were likewise apparent in mutant kidney and a cultured mutant cell line. Genetically distinct mouse HPS models had normal expression of AP-3 subunits. These and related experiments strongly suggest that the AP-3 complex regulates the biogenesis/function of organelles of platelets and other cells and that abrogation of expression of the AP-3 complex leads to platelet SPD.
- Published
- 1999
27. cDNA sequence and mapping of the mouse Copb gene encoding the beta subunit of the COPI coatomer complex.
- Author
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LI W, Elliott RW, Novak EK, and Swank RT
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chromosome Walking, Contig Mapping, DNA Primers, Mice, Molecular Sequence Data, Radiation Hybrid Mapping, Sequence Analysis, DNA, Coat Protein Complex I genetics, Coatomer Protein genetics
- Abstract
COPI-coated vesicles are involved in retrograde-directed selective transport of proteins from the Golgi complex to the endoplasmic reticulum (ER) as well as mediate anterograde transport of cargo proteins within the Golgi or in endosomal trafficking. The COPI protein complex contains an ADP-ribosylation factor (ARF1) and seven coatamer subunits (alpha, beta, beta', gamma, delta, epsilon, zeta-COP). The localization and function of human beta subunit of coatamer (COPB) suggests it is likely a candidate gene of ruby-eye-2 (ru2), which is a mouse model of human Hermansky-Pudlak syndrome characterized by the dysfunction of several subcellular organelles. In this study, we determined the entire coding sequence of mouse (Copb) cDNA by combining an overlapping mouse EST contig with EST walking. beta-COP was found highly conserved in mouse, rat, and human, and it is ubiquitously expressed in mouse. The Copb gene was mapped to mouse Chr 7 at a position of 53.3 cM by radiation hybrid mapping. Our RH mapping data, sequencing of RT-PCR products, and Western blotting exclude the Copb gene as a candidate for ru2.
- Published
- 1999
- Full Text
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28. Survival and lung pathology of mouse models of Hermansky-Pudlak syndrome and Chediak-Higashi syndrome.
- Author
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McGarry MP, Reddington M, Novak EK, and Swank RT
- Subjects
- Animals, Disease Models, Animal, Female, Genotype, Homozygote, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Mutation, Survival Rate, Albinism, Oculocutaneous genetics, Albinism, Oculocutaneous pathology, Chediak-Higashi Syndrome genetics, Chediak-Higashi Syndrome pathology, Lung pathology
- Abstract
Hermansky-Pudlak Syndrome (HPS), a recessively inherited disease in humans, affects the biosynthesis/processing of the related intracellular organelles: lysosomes, melanosomes, and platelet dense granules. The disease is multigenic in both humans and mice where 14 separate genes have been demonstrated to be causative. Patients often die prematurely with severe lung abnormalities. Patients with the related Chediak-Higashi Syndrome (CHS) likewise have significantly reduced life spans. Long-term survival and lung histomorphology were analyzed in a pilot experiment involving several genetically defined singly and doubly mutant mouse HPS mutants and the beige CHS mutant to determine whether these parameters are altered in the mouse models. The mutants differed widely in both longevity and lung architecture. Mice doubly homozygous for the pale ear and ruby eye or for the muted and pearl genes had the shortest life spans with none surviving the two-year experimental duration. Life spans were similarly severely reduced in the beige and gunmetal mutants. Intermediate life spans were apparent in the pearl, pallid, and cocoa mutants whereas minimal effects were noted in ruby eye, muted, light ear, and cocoa mutants. Enlarged air spaces were noted in histologic sections of lungs of several of the mutants. For the most part, the severity of lung abnormalities was inversely proportional to the long-term survival of these various mutants, suggesting that lung pathology may contribute to mortality, as has been suggested for human HPS patients.
- Published
- 1999
- Full Text
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29. The beta3A subunit gene (Ap3b1) of the AP-3 adaptor complex is altered in the mouse hypopigmentation mutant pearl, a model for Hermansky-Pudlak syndrome and night blindness.
- Author
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Feng L, Seymour AB, Jiang S, To A, Peden AA, Novak EK, Zhen L, Rusiniak ME, Eicher EM, Robinson MS, Gorin MB, and Swank RT
- Subjects
- Adaptor Protein Complex beta Subunits, Adaptor Proteins, Vesicular Transport, Alleles, Amino Acid Sequence, Animals, Base Sequence, COS Cells, Cloning, Molecular methods, Contig Mapping, DNA, Complementary chemistry, DNA, Complementary genetics, Female, Gene Expression, Male, Mice, Mice, Inbred C3H, Mice, Inbred C57BL, Mice, Mutant Strains, Molecular Sequence Data, Mutation, RNA, Messenger genetics, RNA, Messenger metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid, Tissue Distribution, Transcription, Genetic, Albinism, Oculocutaneous genetics, Genes genetics, Hypopigmentation genetics, Membrane Proteins genetics, Monomeric Clathrin Assembly Proteins, Nerve Tissue Proteins genetics, Night Blindness genetics, Phosphoproteins genetics
- Abstract
Lysosomes, melanosomes and platelet-dense granules are abnormal in the mouse hypopigmentation mutant pearl. The beta3A subunit of the AP-3 adaptor complex, which likely regulates protein trafficking in the trans - Golgi network/endosomal compartments, was identified as a candidate for the pearl gene by a positional/candidate cloning approach. Mutations, including a large internal tandem duplication and a deletion, were identified in two respective pearl alleles and are predicted to abrogate function of the beta3A protein. Significantly lowered expression of altered beta3A transcripts occurred in kidney of both mutant alleles. The several distinct pearl phenotypes suggest novel functions for the AP-3 complex in mammals. These experiments also suggest mutations in AP-3 subunits as a basis for unique forms of human Hermansky-Pudlak syndrome and congenital night blindness, for which the pearl mouse is an appropriate animal model.
- Published
- 1999
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30. Analysis of the Kit and Pdgfra genes in the patch-extended (Phe) mutation.
- Author
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Stephenson DA, Novak EK, and Chapman VM
- Subjects
- Animals, Brain metabolism, Heterozygote, Mice, Mice, Inbred Strains, Phenotype, Phosphorylation, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, RNA, Messenger analysis, Receptor, Platelet-Derived Growth Factor alpha, Sequence Deletion genetics, Transcription, Genetic genetics, Mutation genetics, Pigmentation genetics, Proto-Oncogene Proteins c-kit genetics, Receptors, Platelet-Derived Growth Factor genetics
- Abstract
The patch (Ph) locus allele, patch-extended (Phe), has significantly less pigmentation than the original mutation and homozygotes have been known to survive to term. Analysing intersubspecific F1 hybrids, we were able to demonstrate that Phe is a deletional mutation encompassing the platelet-derived growth factor receptor alpha subunit (Pdgfra). The deletion does not appear to extend into the coding sequence of the Kit gene (a related tyrosine kinase receptor). However, we were able to demonstrate that, while the Kit gene is transcribed, it does not encode a functionally active receptor.
- Published
- 1998
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31. Mouse models of Hermansky Pudlak syndrome: a review.
- Author
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Swank RT, Novak EK, McGarry MP, Rusiniak ME, and Feng L
- Subjects
- Animals, Bleeding Time, Disease Models, Animal, Humans, Mice, Organelles, Phenotype, Pigmentation, Albinism, Oculocutaneous genetics, Albinism, Oculocutaneous physiopathology
- Abstract
Hermansky Pudlak Syndrome (HPS) is a recessively inherited disease affecting the contents and/or the secretion of several related subcellular organelles including melanosomes, lysosomes, and platelet dense granules. It presents with disorders of pigmentation, prolonged bleeding, and ceroid deposition, often accompanied by severe fibrotic lung disease and colitis. In the mouse, the disorder is clearly multigenic, caused by at least 14 distinct mutations. Studies on the mouse mutants have defined the granule abnormalities of HPS and have shown that the disease is associated with a surprising variety of phenotypes affecting many tissues. This is an exciting time in HPS research because of the recent molecular identification of the gene causing a major form of human HPS and the expected identifications of several mouse HPS genes. Identifications of mouse HPS genes are expected to increase our understanding of intracellular vesicle trafficking, lead to discovery of new human HPS genes, and suggest diagnostic and therapeutic approaches toward the more severe clinical consequences of the disease.
- Published
- 1998
- Full Text
- View/download PDF
32. The murine misty mutation: phenotypic effects on melanocytes, platelets and brown fat.
- Author
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Sviderskaya EV, Novak EK, Swank RT, and Bennett DC
- Subjects
- Adenosine Triphosphate metabolism, Animals, Bleeding Time, Blood Platelets metabolism, Cell Division drug effects, Cell Division genetics, Melanocytes drug effects, Mice, Mice, Inbred C3H, Mice, Inbred C57BL, Mutation, Phenotype, Phenylthiourea pharmacology, Pigmentation genetics, Adipose Tissue, Brown abnormalities, Genes genetics, Hair Color genetics, Melanocytes cytology
- Abstract
Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, suggesting a developmental mutation. A report in abstract has also suggested effects on white fat and body weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet serotonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and immortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced numbers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, overproduced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimulating hormone, which normally promote melanin production. The misty gene product may be involved in adenine nucleotide metabolism or signaling.
- Published
- 1998
- Full Text
- View/download PDF
33. The mouse pale ear (ep) mutation is the homologue of human Hermansky-Pudlak syndrome.
- Author
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Gardner JM, Wildenberg SC, Keiper NM, Novak EK, Rusiniak ME, Swank RT, Puri N, Finger JN, Hagiwara N, Lehman AL, Gales TL, Bayer ME, King RA, and Brilliant MH
- Subjects
- Amino Acid Sequence, Animals, Chromosome Mapping, Disease Models, Animal, Humans, Mice, Mice, Mutant Strains, Molecular Sequence Data, Sequence Alignment, Sequence Homology, Amino Acid, Albinism, Oculocutaneous genetics, Mutation
- Abstract
The recessive mutation at the pale ear (ep) locus on mouse chromosome 19 was found to be the homologue of human Hermansky-Pudlak syndrome (HPS). A positional cloning strategy using yeast artificial chromosomes spanning the HPS locus was used to identify the HPS gene and its murine counterpart. These genes and their predicted proteins are highly conserved at the nucleotide and amino acid levels. Sequence analysis of the mutant ep gene revealed the insertion of an intracisternal A particle element in a protein-coding 3' exon. Here we demonstrate that mice with the ep mutation exhibit abnormalities similar to human HPS patients in melanosomes and platelet-dense granules. These results establish an animal model of HPS and will facilitate biochemical and molecular analyses of the functions of this protein in the membranes of specialized intracellular organelles.
- Published
- 1997
- Full Text
- View/download PDF
34. Biosynthesis of renin in mouse kidney tumor As4.1 cells.
- Author
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Jones CA, Petrovic N, Novak EK, Swank RT, Sigmund CD, and Gross KW
- Subjects
- Animals, Cell Compartmentation, Cytoplasmic Granules metabolism, Enzyme Precursors metabolism, Fluorescent Antibody Technique, Indirect, Glycoproteins biosynthesis, Mice, Molecular Weight, Renin metabolism, Tumor Cells, Cultured, Kidney enzymology, Renin biosynthesis
- Abstract
As4.1, a renin-expressing cell line isolated from a mouse renal tumor, was characterized for synthesis, processing, storage and secretion of renin polypeptides. Metabolic labeling, immunoprecipitation and SDS/PAGE analysis revealed that renin was secreted into the culture supernatant predominantly in the form of prorenin which migrated as products of 42-47 kDa. The predominant intracellular renin was processed into two chains, of 33-34 and 5 kDa. N-glycanase treatment removed N-linked oligosaccharides and yielded products of 41 kDa for prorenin and 31-32 kDa for the heavier chain of two-chain renin. The N-terminus of the constitutively secreted prorenin was determined by automated Edman degradation to be Leu22 while the N-terminus of the heavy chain was Ser72. Renin polypeptides constituted 3.1 +/- 1.4% (mean percentage of total precipitable radioactivity +/- SD) of de-novo-synthesized protein secreted into the medium and 0.2 +/- 0.17% retained intracellularly. Extrapolation of renin activity assays suggest that a single cell stores approximately 680 fg of active renin. A slow incremental release into the medium of processed renin heavy chain was detected by immunoprecipitation and SDS/PAGE. Renin activity assays confirmed the release of approximately 4 fg prorenin and 0.32 fg active renin cell(-1) h(-1). Indirect immunofluorescence demonstrated intracellular renin to be distributed in a punctate pattern. Renin was found to be colocalized with the lysosomal marker, beta-glucuronidase, by double-fluorescent labeling. These cells have enabled characterization of glycosylated mouse renin-1 and may prove a valuable tool for studying intracellular trafficing of renin and associated processing enzymes.
- Published
- 1997
- Full Text
- View/download PDF
35. A novel modifier gene for plasma von Willebrand factor level maps to distal mouse chromosome 11.
- Author
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Mohlke KL, Nichols WC, Westrick RJ, Novak EK, Cooney KA, Swank RT, and Ginsburg D
- Subjects
- Animals, Base Sequence, Crosses, Genetic, DNA Primers, Enzyme-Linked Immunosorbent Assay, Genetic Carrier Screening, Genotype, Homozygote, Humans, Mice, Molecular Sequence Data, Polymerase Chain Reaction, von Willebrand Diseases blood, von Willebrand Factor metabolism, Chromosome Mapping, Mice, Inbred Strains genetics, Polymorphism, Restriction Fragment Length, von Willebrand Diseases genetics, von Willebrand Factor biosynthesis
- Abstract
Type 1 von Willebrand disease (VWD), characterized by reduced levels of plasma von Willebrand factor (VWF), is the most common inherited bleeding disorder in humans. Penetrance of VWD is incomplete, and expression of the bleeding phenotype is highly variable. In addition, plasma VWF levels vary widely among normal individuals. To identify genes that influence VWF level, we analyzed a genetic cross between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold difference in plasma VWF level. DNA samples from F2 progeny demonstrating either extremely high or extremely low plasma VWF levels were pooled and genotyped for 41 markers spanning the autosomal genome. A novel locus accounting for 63% of the total variance in VWF level was mapped to distal mouse chromosome 11, which is distinct from the murine Vwf locus on chromosome 6. We designated this locus Mvwf for "modifier of VWF." Additional genotyping of as many as 2407 meioses established a high resolution genetic map with gene order Cola1-Itg3a-Ngfr-Mvwf/Gip-Hoxb9-Hoxb1++ +-Cbx'rs2-Cox5a-Gfap. The Mvwf candidate interval between Ngfr and Hoxb9 is approximately 0.5 centimorgan (cM). These results demonstrate that a single dominant gene accounts for the low VWF phenotype of RIIIS/J mice in crosses with several other strains. The pattern of inheritance suggests a gain-of-function mutation in a unique component of VWF biosynthesis or processing. Characterization of the human homologue for Mvwf may have relevance for a subset of type 1 VWD cases and may define an important genetic factor modifying penetrance and expression of mutations at the VWF locus.
- Published
- 1996
- Full Text
- View/download PDF
36. High-resolution genetic mapping of the gunmetal gene which regulates platelet production.
- Author
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O'Brien EP, Zhen L, Jiang SY, Novak EK, and Swank RT
- Subjects
- Animals, Genetic Markers, Mice, Molecular Sequence Data, Polymorphism, Restriction Fragment Length, Blood Platelets, Chromosome Mapping, Genes, Recessive genetics, Mice, Inbred C57BL genetics, Mutation genetics
- Published
- 1996
- Full Text
- View/download PDF
37. Inherited prolonged bleeding time and platelet storage pool deficiency in the subtle gray (sut) mouse.
- Author
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Swank RT, Reddington M, and Novak EK
- Subjects
- Adenosine Triphosphate metabolism, Animals, Bleeding Time veterinary, Blood Coagulation Disorders physiopathology, Blood Platelets metabolism, Blood Platelets ultrastructure, Collagen pharmacology, Cytoplasmic Granules physiology, Cytoplasmic Granules ultrastructure, Kidney metabolism, Lysosomes enzymology, Lysosomes metabolism, Mice, Platelet Aggregation drug effects, Platelet Storage Pool Deficiency physiopathology, Serotonin analysis, Blood Coagulation Disorders veterinary, Blood Platelets physiology, Mice, Mutant Strains, Platelet Storage Pool Deficiency veterinary, Rodent Diseases physiopathology
- Abstract
A high proportion of mouse mutants with diluted pigmentation have severely prolonged bleeding times due to platelet storage pool deficiency. The deficiency is associated with concomitant abnormalities in platelet dense granules and coat pigment granules. The coat color of the subtle gray (sut) mouse is diluted to a relatively minor degree. Analysis of platelet serotonin concentration established that this dense granule component similarly is reduced a relatively small amount in this mutant. The subtle gray mouse thus allowed a test of the hypothesis that relatively small changes in platelet dense granule contents may cause discernible increases in bleeding times. Bleeding times of mutant mice were significantly prolonged (3.4-fold) in comparison with those in normal sut/+ controls. These bleeding times were significantly reduced in comparison with other mouse pigment dilution mutants with more severe storage pool deficiency. These results establish the subtle gray mouse as an appropriate animal model for mild storage pool deficiency and human Hermansky-Pudlak syndrome. They indicate, together with related experiments, that bleeding times are highly sensitive to concentrations of platelet dense granule components such as serotonin.
- Published
- 1996
38. von Willebrand disease in the RIIIS/J mouse is caused by a defect outside of the von Willebrand factor gene.
- Author
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Nichols WC, Cooney KA, Mohlke KL, Ballew JD, Yang A, Bruck ME, Reddington M, Novak EK, Swank RT, and Ginsburg D
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Crosses, Genetic, Genes, Mice, Mice, Inbred Strains, Molecular Sequence Data, Mice, Mutant Strains genetics, von Willebrand Diseases genetics, von Willebrand Factor genetics
- Published
- 1995
39. Inherited thrombocytopenia caused by reduced platelet production in mice with the gunmetal pigment gene mutation.
- Author
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Novak EK, Reddington M, Zhen L, Stenberg PE, Jackson CW, McGarry MP, and Swank RT
- Subjects
- Animals, Base Sequence, Blood Platelets metabolism, Bone Marrow Transplantation, Cell Size, Cell Survival, Cytoplasmic Granules ultrastructure, Female, GTP-Binding Proteins biosynthesis, Hair Color genetics, Hematopoiesis, Intracellular Membranes ultrastructure, Male, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Molecular Sequence Data, Ploidies, Polymerase Chain Reaction, Thrombocytopenia pathology, Thrombocytopenia therapy, Blood Platelets pathology, Disease Models, Animal, Megakaryocytes pathology, Thrombocytopenia genetics
- Abstract
Hereditary macrothrombocytopenia and prolonged bleeding times are associated with the recessive mouse pigment dilution gene gunmetal (gm). Other platelet abnormalities include a mild storage pool deficiency and abnormal expression of two low-molecular-weight guanosine triphosphate binding proteins. These studies were designed to further elucidate the cause of the macrothrombocytopenia. The life span of gunmetal mouse platelets was not significantly different from normal. However, rates of platelet synthesis, measured by sulfate incorporation, were decreased to 25% of normal values. Bone marrow transplantation of normal marrow cells corrected the thrombocytopenia. Furthermore, direct morphologic analysis of mature mutant marrow megakaryocytes by transmission electron microscopy showed reductions in the normal cytoplasmic demarcation membrane system, areas of abnormal membrane complexes, and an increased incidence of emperipolesis. Mutant platelets were relatively more heterogeneous in size and contained unusual elongated and striated inclusions. Mutant megakaryocyte numbers were increased threefold to fivefold over normal numbers in marrow and spleen. Thus, the efficiency of platelet production from gunmetal megakaryocytes is reduced by an order of magnitude. Mutant marrow had a greater proportion of 32N and a smaller proportion of 8N megakaryocytes. Collectively, the results indicate that the gunmetal gene acts intrinsically in megakaryocytes and that an abnormality in this gene causes significant qualitative and quantitative effects on platelet production.
- Published
- 1995
40. von Willebrand disease in the RIIIS/J mouse is caused by a defect outside of the von Willebrand factor gene.
- Author
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Nichols WC, Cooney KA, Mohlke KL, Ballew JD, Yang A, Bruck ME, Reddington M, Novak EK, Swank RT, and Ginsburg D
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Disease Models, Animal, Enzyme-Linked Immunosorbent Assay, Mice, Mice, Inbred BALB C, Molecular Sequence Data, von Willebrand Diseases etiology, von Willebrand Factor analysis, von Willebrand Diseases genetics, von Willebrand Factor genetics
- Abstract
An animal model for human type I von Willebrand disease (vWD) has been previously described in the inbred mouse strain RIIIS/J. Murine vWD is characterized by a prolonged bleeding time, normal von Willebrand factor (vWF) multimer distribution, autosomal dominant inheritance, and proportionately decreased plasma vWF antigen, ristocetin cofactor, and factor VIII (FVIII) activities. To study the molecular genetics of murine vWD, a portion of the vWF gene surrounding exon 28 was cloned, sequenced, and used to develop two informative DNA sequence polymorphisms for rapid genotyping by DNA polymerase chain reaction. RIIIS/J mice were crossed with PWK/Ph mice, an inbred line of Mus musculus musculus, and the F1 progeny backcrossed to the parental PWK/Ph strain. vWF antigen levels in F1 mice were not significantly different from the parental RIIIS/J strain but were markedly decreased compared with the parental PWK/Ph mice. Genetic linkage analysis of 104 backcross progeny showed no correlation between vWF antigen level and vWF genotype. These data indicate that murine vWD is caused by a defect at a novel genetic locus, distinct from the murine vWF gene. The distribution of vWF antigen levels among backcross progeny suggests the presence of one major dominant vWD gene in the RIIIS/J mouse with possible modifying contributions from one or more additional minor loci. These observations may provide new insights into the molecular basis and variable expressivity of human vWD.
- Published
- 1994
41. The signal for retention of the egasyn-glucuronidase complex within the endoplasmic reticulum.
- Author
-
Zhen L, Baumann H, Novak EK, and Swank RT
- Subjects
- Amino Acid Sequence, Animals, Biological Transport, Carboxylic Ester Hydrolases isolation & purification, Cell Compartmentation, Cells, Cultured, Esterases genetics, Glucuronidase isolation & purification, Immunohistochemistry, Membrane Glycoproteins isolation & purification, Molecular Sequence Data, Orosomucoid metabolism, Sequence Deletion, Sequence Homology, Amino Acid, Carboxylic Ester Hydrolases genetics, Carboxylic Ester Hydrolases metabolism, Endoplasmic Reticulum metabolism, Glucuronidase metabolism, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Protein Sorting Signals
- Abstract
Egasyn is localized within the lumen of the endoplasmic reticulum (ER) where it complexes with and thus causes sequestration of a considerable portion of beta-glucuronidase. Egasyn has an HTEL sequence at its carboxyl terminus rather than the KDEL sequence that serves as a retention signal for many ER lumenal proteins. To determine whether the HTEL sequence acts as an ER retention signal and/or functions in complex formation, HTEL-deleted egasyn was expressed in mammalian cell lines. The majority of HTEL-deleted egasyn was secreted, while wild type egasyn was retained in the ER. Furthermore, the egasyn HTEL sequence, when added to the carboxyl termini of two secretory proteins, mouse esterase, Es-N, and rat alpha 1-acid glycoprotein (AGP), caused retention of both proteins within the ER, demonstrating that the HTEL sequence is both necessary and sufficient for retention of egasyn and, by extension, the egasyn-glucuronidase complex within the ER. Other carboxyl terminal tetrapeptides including HIEL and HVEL, naturally occurring in other ER lumenal proteins, were also sufficient for ER retention of AGP, while HTEHT and HTEHK were inefficient in ER retention. The HTEL sequence, in contrast, is not required for egasyn-glucuronidase complex formation. Further, the complex is apparently unstable outside the ER since it was not visible in the medium of cells transfected with egasyn lacking the HTEL sequence despite abundant secretion of this egasyn. These results show that it is possible to localize proteins within the lumen of the ER if they form complexes with ER lumenal proteins containing an intrinsic ER retention sequence.
- Published
- 1993
- Full Text
- View/download PDF
42. Inherited abnormalities in platelet organelles and platelet formation and associated altered expression of low molecular weight guanosine triphosphate-binding proteins in the mouse pigment mutant gunmetal.
- Author
-
Swank RT, Jiang SY, Reddington M, Conway J, Stephenson D, McGarry MP, and Novak EK
- Subjects
- Animals, Bleeding Time, Blood Platelets drug effects, Blood Platelets ultrastructure, Cytoplasmic Granules drug effects, Cytoplasmic Granules metabolism, GTP-Binding Proteins genetics, GTP-Binding Proteins isolation & purification, Glucuronidase blood, Lysosomes drug effects, Lysosomes enzymology, Mice, Mice, Mutant Strains, Molecular Weight, Organelles drug effects, Organelles metabolism, Serotonin blood, Species Specificity, Thrombin pharmacology, beta-Galactosidase blood, Blood Platelet Disorders blood, Blood Platelet Disorders genetics, Blood Platelets metabolism, GTP-Binding Proteins metabolism, Organelles ultrastructure, Pigmentation Disorders genetics
- Abstract
Gunmetal (gm/gm) is a recessively inherited mouse pigment dilution mutant that has high mortality and poor reproductive rates. In these studies, several hematologic defects were found associated with the mutation, including prolonged bleeding times, together with thrombocytopenia and increased platelet size. A unique feature is the presence of simultaneous abnormalities in two platelet organelles, dense granules and alpha-granules. The dense granule component serotonin is present at about half the normal concentration, as are visible dense granules. Three alpha-granule components (fibrinogen, platelet factor 4, and von Willebrand factor) are also significantly reduced. Thus, in several respects the gunmetal mutant resembles the human gray platelet syndrome. A novel abnormality in expression of low molecular weight guanosine triphosphate (GTP)-binding proteins occurs in platelets of gunmetal. In Western blot assays, two additional GTP-binding proteins of 28.5 and 25 Kd were detected. The abnormal expression of GTP-binding proteins is, like the hematologic defects, genetically recessive and is tissue specific. Liver, kidney, brain, spleen, macrophages, and neutrophils have normal GTP-binding protein expression. The additional GTP-binding proteins are soluble. The data indicate that platelet formation and platelet organelle biogenesis are under common genetic control and that abnormal regulation of GTP-binding proteins may affect one or both processes.
- Published
- 1993
43. Characterization and functional expression of a cDNA encoding egasyn (esterase-22): the endoplasmic reticulum-targeting protein of beta-glucuronidase.
- Author
-
Ovnic M, Swank RT, Fletcher C, Zhen L, Novak EK, Baumann H, Heintz N, and Ganschow RE
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Carboxylic Ester Hydrolases chemistry, Carboxylic Ester Hydrolases isolation & purification, Carboxylic Ester Hydrolases metabolism, Chromosomes, Human, Pair 8, Cloning, Molecular, DNA isolation & purification, Female, Gene Expression, Humans, Liver enzymology, Male, Membrane Glycoproteins chemistry, Membrane Glycoproteins isolation & purification, Membrane Glycoproteins metabolism, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Organ Specificity genetics, Polymerase Chain Reaction, Restriction Mapping, Sequence Alignment, Carboxylic Ester Hydrolases genetics, Endoplasmic Reticulum enzymology, Glucuronidase metabolism, Membrane Glycoproteins genetics
- Abstract
Egasyn (esterase-22), a member of the nonspecific carboxylesterase multigene family (E.C. 3.1.1.1), is the endoplasmic reticulum (ER)-targeting protein of beta-glucuronidase. We utilized the polymerase chain reaction (PCR) in the eventual isolation of murine egasyn cDNAs. PCR primers were based upon: (1) partial amino acid sequences derived from egasyn peptides and (2) a conserved active site region shared by carboxylesterases. The amino acid sequence deduced from the PCR product matched that obtained from egasyn protein. This product was utilized as a probe to screen a cDNA library. Two cDNAs whose composite sequence encoded an open reading frame of 562 amino acids were isolated. A message size of 1700-2000 bp was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses detected mRNA in liver, kidney, lung, and submandibular gland, but not in spleen, brain, and testes. Genetic mapping confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Transfection of COS cells with the 2022-bp cDNA resulted in the expression of esterase activity, which comigrated on native gels with liver esterase-22. The features of the deduced amino acid sequence of the egasyn cDNA are compared with previously characterized carboxylesterases and with other lumenal ER proteins.
- Published
- 1991
- Full Text
- View/download PDF
44. Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants muted and mocha.
- Author
-
Swank RT, Reddington M, Howlett O, and Novak EK
- Subjects
- Animals, Ear, Inner abnormalities, Mice, Platelet Storage Pool Deficiency complications, Mice, Mutant Strains physiology, Platelet Storage Pool Deficiency genetics
- Abstract
Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormalities. Both mutants had prolonged bleeding times accompanied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by labeling platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adenosine triphosphate and aggregation was reduced. Lysosomal enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simultaneously affect melanosomes, lysosomes, and platelet dense granules.
- Published
- 1991
45. Sandy: a new mouse model for platelet storage pool deficiency.
- Author
-
Swank RT, Sweet HO, Davisson MT, Reddington M, and Novak EK
- Subjects
- Animals, Bleeding Time, Blood Platelets ultrastructure, Chromosome Mapping, Crosses, Genetic, Female, Male, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Phenotype, Disease Models, Animal, Mutation, Pigmentation genetics, Platelet Storage Pool Deficiency genetics
- Abstract
Sandy (sdy) is a mouse mutant with diluted pigmentation which recently arose in the DBA/2J strain. Genetic tests indicate it is caused by an autosomal recessive mutation on mouse Chromosome 13 near the cr and Xt genetic loci. This mutation is different genetically and hematologically from previously described mouse pigment mutations with storage pool deficiency (SPD). The sandy mutant has diluted pigmentation in both eyes and fur, is fully viable and has prolonged bleeding times. Platelet serotonin levels are extremely low although ATP dependent acidification activity of platelet organelles appears normal. Also, platelet dense granules are extremely reduced in number when analysed by electron microscopy of unfixed platelets. Platelets have abnormal uptake and flashing of the fluorescent dye mepacrine. Secretion of lysosomal enzymes from kidney and from thrombin-stimulated platelets is depressed 2- and 3-fold, and ceroid pigment is present in kidney. Sandy platelets have a reduced rate of aggregation induced by collagen. The sandy mutant has an unusually severe dense granule defect and thus may be an appropriate model for cases of human Hermansky-Pudlak syndrome with similarly extreme types of SPD. It represents the tenth example of a mouse mutant with simultaneous defects in melanosomes, lysosomes and/or platelet dense granules.
- Published
- 1991
- Full Text
- View/download PDF
46. Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants.
- Author
-
McGarry MP, Novak EK, Reddington M, and Swank RT
- Subjects
- Animals, Bleeding Time, Bone Marrow pathology, Genetic Therapy, Glucose-6-Phosphate Isomerase blood, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Platelet Count, Platelet Storage Pool Deficiency blood, Platelet Storage Pool Deficiency genetics, Bone Marrow Transplantation, Chimera, Platelet Storage Pool Deficiency surgery
- Abstract
We have previously shown mouse platelet storage pool deficiency (SPD) to be associated with lesions at eight different genetic loci, each of which is sufficient to produce murine SPD. We have also shown that normal bleeding times and normal platelet functions are restored when mice with SPD are transplanted with marrow from normal mice. Conversely, when normal mice are transplanted with mutant marrow, they present symptoms of SPD. In order to determine the amount of normal platelets needed to prevent the prolonged bleeding times associated with SPD, we established stable mixed chimeric mice by transplanting various ratios of normal and mutant marrow into lethally irradiated host animals. The proportion of normal input marrow correlated well with the proportion of normal peripheral red blood cells and platelets determined in chimerae 100 days after transplantation using direct morphology and electrophoretic variants of glucose phosphate isomerase to identify normal and mutant cell populations. The proportions of normal input marrow were also reflected in the proportions of platelets with normal and mutant platelet morphology in the chimerae. This confirms that the platelet abnormality in SPD is intrinsic to the stem cell population from which the platelets are derived. When bleeding times were determined in the mixed chimeric mice, a surprisingly high percentage of normal platelets (greater than 50% and sometimes greater than 75%) were needed to stop bleeding. These results suggest that the mutant platelets in the mixed chimeric mice may interfere with normal platelet aggregation patterns. They also raise some important considerations in devising treatment for SPD. Bleeding episodes in human SPD are normally treated by platelet transfusion. The results suggest that, at least in some cases, transfusions may not be effective. Also, in future gene therapy of this disease, it is like that a functional gene will have to be present in greater than 50% of stem cells for therapy to be effective.
- Published
- 1990
47. The RIIIS/J inbred mouse strain as a model for von Willebrand disease.
- Author
-
Sweeney JD, Novak EK, Reddington M, Takeuchi KH, and Swank RT
- Subjects
- Animals, Antigens deficiency, Bleeding Time, Blood Platelets physiology, Blood Platelets ultrastructure, Cytoplasmic Granules ultrastructure, Deamino Arginine Vasopressin pharmacology, Deamino Arginine Vasopressin therapeutic use, Factor VIII metabolism, Factor XI metabolism, Mice, Mice, Inbred C57BL, Mice, Inbred Strains, Microscopy, Electron, Partial Thromboplastin Time, Platelet Aggregation, Platelet Count, Serotonin blood, von Willebrand Diseases drug therapy, von Willebrand Factor immunology, Disease Models, Animal, von Willebrand Diseases blood
- Abstract
Mice of the RIIIS/J inbred strain have prolonged bleeding times (greater than 15 minutes) after experimental injury when compared with normal C57BL/6J mice (1.8 minutes) and other strains of mice. The prolonged bleeding time was accompanied by normal platelet counts. Platelet aggregation with collagen and agglutination with ristocetin were not significantly altered in RIIIS/J mice. Also, platelets from RIIIS/J mice had normal serotonin content and normal numbers of dense granules by electron microscopy. Thus, the bleeding abnormality is not due to platelet storage pool deficiency as has been found in several other mouse mutants. The activated partial thromboplastin time (APTT) which plasma from RIIIS/J mice was prolonged compared with normal mice, and factor VIII:C activity and von Willebrand antigen levels were one half to one third that of normal mouse plasma. Factor XI activity was also significantly deficient (levels at 42% to 64% of normal). Plasma of RIIIS/J mice contained the full complement of multimers of von Willebrand factor, although each multimer was lower in concentration compared with that in normal mice. Platelet alpha-granule von Willebrand antigen levels were similar to those of normal mice. The prolonged bleeding time of RIIIS/J mice was corrected by treatment with desmopressin. Heterozygous C57BL/6J x RIIIS/J F1 animals had low plasma von Willebrand antigen levels like the RIIIS/J parent and had variable bleeding times. Inheritance of the bleeding tendency was as an incomplete dominant, autosomal trait. These data indicate the RIIIS/J strain is a suitable animal model for type IA von Willebrand disease.
- Published
- 1990
48. Analysis of atherosclerosis susceptibility in mice with genetic defects in platelet function.
- Author
-
Paigen B, Holmes PA, Novak EK, and Swank RT
- Subjects
- Animals, Arteriosclerosis blood, Blood Platelets analysis, Disease Models, Animal, Disease Susceptibility, Female, Mice, Mice, Inbred C57BL blood, Mice, Mutant Strains blood, Mutation, Serotonin analysis, Arteriosclerosis genetics, Blood Platelets physiology, Mice, Inbred C57BL genetics, Mice, Mutant Strains genetics
- Abstract
To determine whether platelets contribute to the development of atherosclerosis, we compared the severity of atherosclerosis in susceptible C57BL/6 mice carrying either a normal or a variant phenotype for platelet function. Five genetically distinct mutants with increased bleeding times and abnormal dense granules were used: maroon (ru-2mr), light ear (le), ruby eye (ru), beige (bg1), and pale ear (ep). After a 14-week consumption of an atherogenic diet, three mutants had significantly less disease involvement than the control: light ear, maroon, and ruby eye. In contrast, pale ear ahd lesions similar to control animals. After 48 weeks, the two mutants with the least degree of atherosclerosis at 14 weeks, light ear and ruby eye, showed greater than 50% survival. In contrast, no animals from the beige, pale ear, or the normal C57BL/6 strains survived. To determine whether a specific biochemical component of platelet function is related to atherosclerosis, we measured serotonin found in dense granules. Serotonin showed no correlation with each mutant's atherosclerosis susceptibility. These results indicate that some particular component of platelet function affects atherosclerosis. That component is intact in pale ear, moderately affected in beige and maroon, and severely affected in light ear and ruby eye. The identity of that component remains an interesting question whose answer may provide further insight into the atherosclerotic disease process.
- Published
- 1990
- Full Text
- View/download PDF
49. Pigmentation and lysosome function in mice homozygous for both pale ear and beige-J pigment genes.
- Author
-
Novak EK, Swank RT, and Meisler MH
- Subjects
- Animals, Crosses, Genetic, Female, Lysosomes enzymology, Mice, Mice, Inbred C57BL genetics, Mutation, Lysosomes physiology, Mice, Mutant Strains genetics, Skin Pigmentation
- Published
- 1980
- Full Text
- View/download PDF
50. The mouse pale ear pigment mutant as a possible animal model for human platelet storage pool deficiency.
- Author
-
Novak EK, Hui SW, and Swank RT
- Subjects
- Adenosine Diphosphate blood, Adenosine Triphosphate blood, Animals, Cytoplasmic Granules, Female, Lysosomes enzymology, Male, Mice, Platelet Count, Serotonin deficiency, Blood Platelet Disorders physiopathology, Disease Models, Animal, Ear, Mice, Inbred C57BL genetics, Mutation
- Abstract
The mouse pigment mutant pale ear, ep/ep, which has a defect in kidney lysosomal enzyme secretion, had prolonged bleeding on experimental injury. Platelet counts and platelet protein did not differ from normal. There was, however, a deficiency in the platelet dense granule contents, serotonin, ATP, and ADP. Furthermore, a marked reduction of platelet dense granules was observed by electron microscopy. The results suggest that pale ear is a useful animal model in the study of platelet storage pool disease. Studies on this mutant and other pigment mutants have established that one gene can regulate at least three subcellular organelles, including the melanosome, the lysosome, and the platelet dense granule.
- Published
- 1981
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