Biomedical subjects
Publications and source records attributed to H Nandurkar.
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Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized. These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking. The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases. PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores. A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases. Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity. We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity. Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.
Mice lacking thrombopoietin (TPO) or its receptor c-Mpl are severely thrombocytopenic, consistent with a dominant physiological role for this cytokine in megakaryocytopoiesis. However, these mice remain healthy and show no signs of spontaneous hemorrhage, implying that TPO-independent mechanisms for platelet production exist and are sufficient for hemostasis. To investigate the roles of cytokines that act through the gp130 signaling chain in the residual platelet production of mpl (-/-) mice, mpl (-/-)IL-6(-/-), mpl(-/-)LIF(-/-), and mpl(-/-)IL-11Ralpha(-/-) double-mutant mice were generated. In each of these compound mutants, the number of circulating platelets was no lower than that observed in mice lacking only the c-mpl gene. Moreover, the deficits in the numbers of megakaryocytes and megakaryocyte progenitor cells in the bone marrow and spleen were no further exacerbated in mpl(-/-)IL-6(-/-), mpl(-/-)LIF(-/-), or mpl(-/-)IL-11Ralpha(-/-) double-mutant mice compared with those in Mpl-deficient animals. In single IL-6(-/-), LIF(-/-), and IL-11Ralpha(-/-) mutant mice, platelet production was normal. These data establish that, as single regulators, IL-6, IL-11, and LIF have no essential role in normal steady-state megakaryocytopoiesis, and are not required for the residual megakaryocyte and platelet production seen in the c-mpl(-/-) mouse. (Blood. 2000;95:528-534)
We have investigated the compartmentalization of aminopeptidase-N-like activity in various blood fractions obtained from patients with acute lymphoid (ALL) or myeloid (AML) leukemia. The primary difference appears not to be the absolute level of overall activity, but rather the relative proportions of the different forms of activity detected. Thus, despite similar levels of total aminopeptidase-N-like activity detected in cells from different leukemic groups, true aminopeptidase-N/CD13 activity was only detected in cells derived from AML patients. Even in these patients, however, most of the detected aminopeptidase-N-like activity ( > 80%) could not be attributed to aminopeptidase-N/CD13. In marked contrast, plasma from leukemic patients also contained substantial total aminopeptidase-N-like activity, of which (irrespective of leukemic group) most could be attributed to aminopeptidase-N/CD13. Whilst slightly higher levels of total activity were obtained in plasma from AML patients compared to ALL patients, there was no difference in the relative proportion attributable to aminopeptidase-N/CD13 (approximately 80% of total aminopeptidase-N-like activity). Evaluation of total aminopeptidase-N-like activity present in whole blood gave differential patterns, and whilst only a proportion (20-40% of total aminopeptidase-N-like activity) could be attributed to true aminopeptidase-N/CD13, blood from patients with CD13+ AML showed the greatest activity so attributable. In total, our results outline the complexities of peptidase activities present within blood of leukemic individuals, and may, in part, explain the variability of previous studies attempting to associate prognostic features with phenotypic expression of CD13.
In clinical chemistry, determination of serum alanine aminopeptidase (AAP) levels has been found to be useful for detecting or confirming biliary obstructions from either intra- or extra-hepatic disorders. In haematology, 'gp150' is a surface-expressed protein molecule, recognised by monoclonal antibodies belonging to the so-called 'Cluster of Differentiation' (CD-) 13, which has independently been found to be a useful marker of myeloid leukaemia in addition to providing potential prognostic value. The current report links these two independent research streams and provides evidence that the hepatobiliary disease marker, serum AAP, predominantly comprises a circulating isoform of 'gp150'. Thus, a (CD-13) monoclonal antibody raised to, and specifically reactive with, cell surface myeloid 'gp150' is able to specifically and almost completely block serum (or plasma) AAP activity otherwise observed in its absence. This holds true for serum (or plasma) derived both from normal individuals or from patients suffering hepatic dysfunction, with or without associated biliary obstruction. In the case of patients with obstructive jaundice, raised levels of AAP are observed, which fall to near normal levels following preincubation with this monoclonal antibody. In addition, data are presented to support the view of varying isoforms of AAP within flowing blood. Finally, preliminary data is provided on AAP activity in cases of leukaemia. These studies should thus prove of use to clinical laboratories investigating the involvement of AAP activity in various pathological processes.
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