Biomedical subjects

V Rajapurohitam

Publications and source records attributed to V Rajapurohitam.

3 recordsLinked to original sources

PubMed2001

The mouse osteopetrotic grey-lethal mutation induces a defect in osteoclast maturation/function.

The osteopetrotic grey-lethal (gl) mouse mutant displays many similarities to the human malignant autosomal-recessive form of osteopetrosis. In this study, we show that the gl osteopetrotic bone phenotype is characterized by the presence of numerous differentiated multinucleated osteoclasts. A significant increase in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was detected in vivo, suggesting induction of differentiation in the osteoclast lineage as a compensatory mechanism. These gl osteoclast cells demonstrated a defective cytoskeletal reorganization and an underdeveloped ruffled border, a membrane structure essential for active bone resorption. Accordingly, resorption activity of these cells is markedly impaired by four- to tenfold as evaluated with the pit formation assay. This low bone resorption in gl osteoclasts is highly reminiscent of the loss in key enzymes, V-ATPase or cathepsin-K, and in signaling factors, Src or TRAF-6, which were shown not to be significantly altered in gl osteoclasts. Thus, independently of a deficiency in V-ATPase, Src, cathepsin-K, and TRAF-6, the gl mutation results in increased number of osteoclasts, characterized by a disrupted cytoskeleton and an underdeveloped ruffled border.

Acid Phosphatase↗

PubMed2001

Role of c-Src in the regulation of vascular contraction and Ca2+ signaling by angiotensin II in human vascular smooth muscle cells.

OBJECTIVE: Tyrosine kinases, typically associated with growth-signaling pathways, also play a role in Ang II-stimulated vascular contraction. However the specific kinases involved are unclear. We hypothesize here that c-Src, a non-receptor tyrosine kinase, is an important upstream regulator of vascular smooth muscle cell (VSMC) Ca2+ signaling and associated vascular contraction induced by Ang II. METHODS: Cultured VSMCs from resistance arteries of healthy subjects were studied. Human VSMCs electroporated with anti-c-Src antibody and c-Src-deficient VSMCs from small arteries of c-Src knockout mice (Src-/-mVSMCs) were also investigated. Intracellular free Ca2+ concentration ([Ca2+]i), c-Src activity and IP3 production were measured by fura 2, immunoblot and radioimmunoassay respectively. Contraction was examined in intact rat small arteries. RESULTS: Ang II rapidly increased VSMC c-Src activity, with peak responses obtained at 1 min. Ang II induced a biphasic [Ca2+]i response (Emax = 636 +/- 123 nmol/l). The initial [Ca2+]i transient, mediated primarily by Ca2+mobilization, was dose-dependently attenuated by the selective Src inhibitor, PP2, but not by PP3 (inactive analogue). Ang II-elicited [Ca2+]i responses were blunted in cells electroporated with anti-c-Src antibodies and in c-Src-/-mVSMCs. Src inhibition decreased Ang II-induced generation of IP3 in human VSMCs. Ang II dose-dependently increased vascular contraction (Emax = 40 +/- 6.5%). These responses were attenuated by PP2 (Emax = 7.8 +/- 0.08%) but not by PP3 (Emax = 35 +/- 4.5%). CONCLUSIONS: Our findings identify c-Src as an important regulator of VSMC [Ca2+]i signaling and implicate a novel contractile role for this non-receptor tyrosine kinase in Ang II-stimulated vascular smooth muscle.

Angiotensin II↗

PubMed1999

Activation of a UBC4-dependent pathway of ubiquitin conjugation during postnatal development of the rat testis.

During spermatogenesis, germ cells undergo mitotic and meiotic divisions to form haploid round spermatids which mature to functional elongated spermatozoa. During this process there occurs remodeling of cell structure and loss of most of the cytoplasm and a large fraction of cellular proteins. To evaluate the role of the ubiquitin proteolytic system in this protein loss, we measured levels of ubiquitinated proteins and rates of ubiquitin conjugation in extracts of testes from rats of different ages. Endogenous ubiquitin-protein conjugates increased till day 30 and then reached a plateau. In parallel, there was a progressive increase in the rate of conjugation of ubiquitin to proteins in testis extracts from these animals. To test the importance of two major ubiquitin conjugating enzyme families in the conjugation, immunoprecipitation of UBC2 or UBC4 from 10- and 30-day-old testis extracts was carried out and the remaining conjugation activity in supernatants was assayed. Depletion of either enzyme family resulted in decreased conjugation. However, most of the conjugation activity and, more importantly, the increased conjugation during development were UBC4-dependent. Immunocytochemistry demonstrated a marked increase in expression of UBC4 in spermatids, consistent with the UBC4-dependent activation of conjugation seen in vitro. In situ hybridization studies evaluated the contribution of various UBC4 isoforms to this induction. UBC4-1 mRNA was expressed in most cells. UBC4-2 mRNA was restricted to germ cells with high levels of expression in round and elongated spermatids. UBC4-testis had previously been shown to be expressed only in spermatids. Our data suggest that induction of various UBC4 isoforms activates overall conjugation and plays an important role in the cellular remodeling and protein loss occurring during spermatogenesis.

Age Factors↗