Biomedical subjects

R Dhand

Publications and source records attributed to R Dhand.

At least 55 records · Page 3Linked to original sources

PubMed1993

A biosensor approach to probe the structure and function of the p85 alpha subunit of the phosphatidylinositol 3-kinase complex.

Phosphatidylinositol 3-kinase, which generates putative novel second messenger phospholipids, is a heterodimer composed of regulatory adaptor 85-kDa and catalytic 110-kDa subunits. The p85 alpha subunit contains a NH2-terminal src homology (SH) 3 domain, a region with homology to the product of the breakpoint cluster region (bcr) gene, and a COOH-terminal portion of the molecule which contains two SH2 domains, separated by a spacer region. In this study a panel of monoclonal antibodies (mAb) was raised against recombinant bovine p85 alpha to probe its multidomain structure in relation to function. These mAbs were characterized using a BIAcore biosensor instrument. Epitopes for nine mAbs were mapped in relation to the domain structure of p85 alpha using recombinant protein fragments expressed in bacteria. These mAbs were then used to map the sites on p85 alpha which are involved in growth factor receptor binding. Two interesting classes of functional mAbs were identified. First, mAb U14, whose epitope lies within the NH2-terminal SH2 domain of p85 alpha, blocked the interaction of p85 alpha with activated protein-tyrosine kinase receptors. Second, real-time binding experiments using phospholipid-containing vesicles showed that p85 alpha by itself could specifically bind certain phospholipids. Two mAbs (U9 and U15) with epitopes located in the inter-SH2 spacer region blocked the binding of lipids to this site. The relevance of these observations to understanding the relationship of structure to function of p85 and the phosphatidylinositol 3-kinase are discussed.

Amino Acid Sequence↗

PubMed1993

Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.

3T3 Cells↗

PubMed1993

Intracellular levels and extracellular release of lysosomal enzymes from peripheral blood monocytes in pulmonary tuberculosis patients.

The intracellular activity and extracellular release (basal and latex-stimulated) of B-glucuronidase (BG) and N-acetylglucosaminidase (NAG), measured fluorimetrically, were observed to be significantly (P < 0.05) higher in blood monocytes (BM) of untreated pulmonary tuberculosis (TB) patients compared to those of age- and sex-matched controls and Mantoux-positive subjects without any evidence of active disease. After completion of antituberculous therapy, BG and NAG activities declined appreciably (P < 0.05) and their levels became comparable to those in control subjects. The present results suggest the potentiation of the oxygen-independent defense mechanism of BM in pulmonary TB.

Acetylglucosaminidase↗

PubMed1993

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.

Amino Acid Sequence↗

PubMed1993

A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor.

The pleiotropic effects (mitogenesis, motogenesis, and morphogenesis) elicited by hepatocyte growth factor/scatter factor (HGF/SF) are mediated by the activation of the tyrosine kinase receptor encoded by the MET proto-oncogene. Following autophosphorylation, the receptor associates with the p85/110 phosphatidylinositol (PI) 3-kinase complex in vivo and in vitro. By a combination of two complementary approaches, competition with synthetic phosphopeptides and association with Tyr-Phe receptor mutants, we have identified Y-1349 and Y-1356 in the HGF/SF receptor as the binding sites for PI 3-kinase. Y-1349VHV and Y-1356VNV do not conform to the canonical consensus sequence YXXM for PI 3-kinase binding and thus define YVXV as a novel recognition motif. Y-1349 and Y-1356 are located within the C-terminal portion of the HGF/SF receptor and are phosphorylation sites. The affinity of the N- and C-terminal src homology region 2 (SH2) domains of p85 for the phosphopeptides including Y-1349 and Y-1356 is 2 orders of magnitude lower than that measured for Y-751 in the platelet-derived growth factor receptor binding site. However, the closely spaced duplication of the novel recognition motif in the native HGF/SF receptor may allow binding with both SH2 domains of p85, thus generating an efficient docking site for PI 3-kinase. In agreement with this model, we have observed that a phosphopeptide including both Y-1349 and Y-1356 activates PI 3-kinase in vitro.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of a phosphatidylinositol 3-kinase complex from bovine brain by using phosphopeptide affinity columns.

Specific phosphorylated tyrosine residues in the kinase insert region of the human platelet-derived-growth-factor beta-receptor mediate the formation of multienzyme complexes with this receptor. When phosphorylated, tyrosine residue 751 within the kinase insert region mediates binding of PtdIns 3-kinase to this receptor. A 17-amino-acid peptide containing this tyrosine residue was synthesized, phosphorylated by using epidermal-growth-factor receptor and then coupled to an Actigel matrix. The tyrosine-751 phosphopeptide column is used here as a final affinity step in the purification of the PtdIns 3-kinase from bovine brain to apparent homogeneity. The active resin-bound PtdIns 3-kinase is composed of two polypeptides, p110 and p85, which are elutable with SDS-containing buffers and detectable by silver staining of polyacrylamide gels. The 85 kDa protein is shown to be identical with the recently cloned p85 alpha. Phosphotyrosine is demonstrated to be an essential part of the structure required for binding of both of these proteins and PtdIns 3-kinase activity to this peptide. The active PtdIns 3-kinase complex from bovine brain, but not recombinant p85 subunits, shows specificity for binding to phosphopeptides containing a YXXM consensus sequence. Neither PtdIns 3-kinase activity, nor the complex of p85 and 110 kDa proteins, binds to several other phosphopeptide affinity columns lacking this sequence motif. The selectivity of binding of baculovirus-expressed free p85 alpha subunit of bovine brain PtdIns 3-kinase, the closely related protein p85 beta and purified bovine brain PtdIns 3-kinase to these and other phosphopeptide columns is examined.

Amino Acid Sequence↗

PubMed1992

Expression and characterization of the p85 subunit of the phosphatidylinositol 3-kinase complex and a related p85 beta protein by using the baculovirus expression system.

PtdIns 3-kinase associates with certain activated protein-tyrosine kinase receptors and with the pp60c-src/polyoma middle-T complex, suggesting that the enzyme is involved in growth regulation. The purified PtdIns 3-kinase appears to have two subunits, of 85 kDa and 110 kDa. Structural analysis at protein and cDNA levels revealed two forms of the 85 kDa subunit, one which associates with PtdIns 3-kinase activity termed p85 alpha, and a protein of unknown function, p85 beta. Both 85 kDa proteins contain src-homology regions 2 and 3 (SH2 and SH3), but lack enzymic activity, suggesting that they may be regulatory subunits of PtdIns 3-kinase. To probe their structure and function further, p85 alpha and p85 beta have been expressed and purified in large amounts from insect cells by using baculovirus vectors. Specific antisera detect p85 alpha, but not p85 beta, associated with PtdIns 3-kinase activity in various cell types. Co-expression studies in insect cells have shown that p85 alpha and p85 beta are substrates for the protein-tyrosine kinases of epidermal growth factor, colony-stimulating factor 1 and c-erbB2 receptors and the src family kinase p59c-fyn. Both p85 alpha and p85 beta form tight complexes with these protein-tyrosine kinases as measured by immunoprecipitation and kinase assays in vitro. The specificity of binding of free p85 is less restricted than that of p85 in the active PtdIns 3-kinase complex with the 110 kDa protein. The relevance of these results to growth-factor-induced PtdIns 3-kinase activation is discussed.

Amino Acid Sequence↗

PubMed1992

Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit.

Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits. The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases. Here the characterization of the p110 subunit is presented. cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S. cerevisiae protein involved in the sorting of proteins to the vacuole. p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor. p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.

Amino Acid Sequence↗

PubMed1992

Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed.

Amino Acid Sequence↗

PubMed1992

Oxidative metabolic status of blood monocytes and alveolar macrophages in the spectrum of human pulmonary tuberculosis.

The oxidative metabolic status of blood monocytes (BM) and alveolar macrophages (AM) in patients with active pulmonary tuberculosis (TB) (n = 40) and in successfully treated patients (n = 40) was assessed and compared with that of healthy control subjects (n = 40). Oxygen free radical (OFR) generation, measured by chemiluminescence (CL) and cytochrome c reduction assay and confirmed by using scavengers of different OFR, was suppressed in AM of the pulmonary TB group compared with healthy controls, whereas it was enhanced in BM. Successfully treated patients showed partial recovery of CL and cytochrome c reduction in AM. There was no significant change in BM of patients after having been treated. The overall capacity to generate OFR was markedly suppressed upon in vitro stimulation with latex in both BM and AM of TB patients. The observed suppressed oxidative metabolic activity in BM and AM was further elucidated by studying the molecular mechanism of respiratory burst. The activities of NADPH oxidase and enzymes of the hexose monophosphate (HMP) shunt were significantly (p less than 0.05) decreased in BM and AM of pulmonary TB patients compared with healthy controls. Patients who had been treated showed marked recovery of NADPH oxidase and HMP shunt activity. The present study suggests that tubercle bacilli escape the microbicidal action of macrophages as a result of suppressed OFR generation caused by decreased activity of HMP shunt, leading to decreased levels of NADPH, thereby preventing NADPH oxidase from working at its full capacity.

Adolescent↗

PubMed1992

C-reactive proteins, immunoglobulin profile and mycobacterial antigens in cerebrospinal fluid of patients with pyogenic and tuberculous meningitis.

Cerebrospinal fluid (CSF) samples were collected from 12 patients with pyogenic meningitis (PM), 19 with tuberculous meningitis (TBM), 20 with clinically suspected but not definitely proved cases of tuberculous meningitis (STBM) and 12 normal controls. C-reactive proteins, immunoglobulins G, A, M and mycobacterial antigens were estimated in the CSF samples. Seven out of 51 (13.7%) samples obtained from the patient groups were positive for CRP. Immunoglobulins M and A were significantly raised in the PM group. When the TBM and STBM groups were compared with the controls a highly significant increase was obtained for all immunoglobulins. Mycobacterial antigens/epitopes were identified in 36.8% samples with TBAGB1 and TB68-H monoclonals and in 26.3% with WTB72-A2. In case of patients with suspected TBM, 6.6% were positive with TBAGB1 and WTB72-A2 and 13.3% with TB68-H. However, non-tuberculous patients also reacted with WTB72-A2 (10.5%) and TB68-H (21.0%). This is, to the authors' knowledge, the first report on the presence of CRP in the CSF. Technique for immunoglobulins in CSF is also updated in this paper. We infer that the monoclonal antibody TBAGB1 and immunoglobulins G and A may be safely considered as diagnostic markers of TBM. Estimation of CRP in CSF samples may be made to give a preliminary or additional diagnosis of meningitis regardless of its aetiology.

Antigens, Bacterial↗

PubMed1991

Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase.

Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.

Animals↗

PubMed1990

A randomized trial of fully intermittent vs. daily followed by intermittent short course chemotherapy for childhood tuberculosis.

Fully intermittent short course chemotherapy regimens have been used successfully in adults but not in children. We report the results on 76 children with tuberculosis, excluding central nervous system tuberculosis and primary pulmonary complex. Isoniazid, rifampin and pyrazinamide were used for treatment. They were randomly allocated to Regimen A (52 doses) and Regimen B (94 doses). Overall efficacy of both schedules was greater than 95% in 27 children with lymphatic, 43 with pulmonary and 6 with disseminated tuberculosis. Compliance in 10 children after 2 to 4 months of therapy was poor because rapid improvement was mistaken by parents for cure. Two children died, probably of underlying lung disease. Follow-up for up to 2 years did not reveal any case of relapse or recurrence of the disease. Therapy for 6 months involving administration of only 52 or 94 doses of drugs was found to be economical, effective and safe for treating children with tuberculosis.

Adolescent↗

PubMed1990

Effect of chemotherapy on cell counts in peripheral blood & bronchoalveolar lavage of patients with pulmonary tuberculosis.

Cell counts in peripheral blood and bronchoalveolar lavage fluid were estimated in 38 patients with active tuberculosis; 12 patients with tuberculosis who had successfully completed more than 9 months of chemotherapy with isoniazid, rifampicin, and ethambutol; 10 Mantoux negative bronchitic subjects; and 6 control subjects. There were 50 males and 16 females aged 16-50 yr. Age, haemoglobin, total and differential serum proteins, were comparable in the various groups. Patients with active tuberculosis had higher ESR and significantly raised absolute (2.24 +/- 0.13 x 10(3); P less than 0.05), B (0.56 +/- 0.03 x 10(3); P less than 0.01) and Null lymphocyte counts (0.56 +/- 0.05 x 10(3); P less than 0.01) in blood. After chemotherapy there was no significant change in lymphocyte counts (2.43 +/- 0.21; P greater than 0.05). In bronchoalveolar lavage (BAL) total cell counts were increased five fold in patients with active disease (40.8 +/- 5.79 x 10(4)/ml) and although these showed considerable reduction after therapy they were higher (18.33 +/- 4.73 x 10(4)/ml) than those in controls (8.3 +/- 1.2 x 10(4)/ml; P greater than 0.05). In bronchitic subjects, total cell counts and macrophage counts in BAL fluid were elevated (P less than 0.01; less than 0.05 respectively) but lymphocyte counts were comparable to controls (3.4 +/- 1.35 x 10(4)/ml vs 1.09 +/- 0.19 x 10(4)/ml; P greater than 0.05). Lymphocytosis in the bronchoalveolar lavage fluid of patients with pulmonary tuberculosis persisted even after adequate treatment.

Adolescent↗

PubMed1990

Monocyte/macrophage functions & humoral response in blood & bronchoalveolar lavage fluid of pulmonary tuberculosis patients.

Subsegmental bronchoalveolar lavage (BAL) was performed in 33 patients with active pulmonary tuberculosis and five control subjects. Phagocytosis by monocytes and alveolar macrophages was studied, and in addition serum and BAL immunoglobulin and complement levels were also determined. The phagocytic activity of blood monocytes was depressed in pulmonary tuberculosis patients as compared to controls, 37.8 +/- 2.3 per cent; 50.7 +/- 4.2 per cent and 32.9 +/- 3.6 per cent for sheep RBC's, latex and Staphylococcus aureus respectively compared to 66.7 +/- 6, 54.8 +/- 2.2 and 68.3 +/- 3.5 per cent respectively in controls; the differences being significant for sheep RBC's (P less than 0.05) and Staph. aureus (P less than 0.001). However, phagocytosis was not impaired in BAL macrophages (P greater than 0.05). In patients no significant alteration in serum immunoglobulin and complement levels was observed except that levels of C4 component of complement were increased in patients with far advanced lesions (98.5 +/- 33.7 mg/dl compared to 78.7 +/- 7.9 mg/dl; P less than 0.05). While IgM and C4 component of complement could not be detected in BAL fluid the levels of IgA were significantly increased in pulmonary tuberculosis patients (65.5 +/- 50.5 mg/dl compared to 39.9 +/- 13.3 mg/dl in control; P less than 0.05). Since IgA secreted in the BAL fluid is mostly synthesised locally, increased levels of this immunoglobulin could be of value in determining activity of the disease.

Adult↗

PubMed1989

Enzyme levels in bronchoalveolar lavage fluid and serum of active pulmonary tuberculosis patients.

Bronchoalveolar lavage (BALF) was found to be a useful index of cell damage. It has been observed that the enzymes in BALF could give an idea of cell damage in a pulmonary disease. Acid phosphatase, alkaline phosphatase, leucine aminopeptidase and gamma-glutamyl-transpeptidase were assessed in mild, moderate, and severe pulmonary tuberculosis patients and Mantoux-negative normal controls. Activities of these enzymes were found to be higher in patients and were increasing with the severity of the disease. Increase in these enzymes in pulmonary tuberculosis patients could be attributed to lung tissue damage.

Acid Phosphatase↗

PubMed1988

Factors influencing the cellular response in bronchoalveolar lavage and peripheral blood of patients with pulmonary tuberculosis.

Subsegmental bronchoalveolar lavage was performed in 30 patients with active pulmonary tuberculosis and six control subjects. Total leucocyte count, absolute lymphocyte count, count of polymorphonuclear leucocytes, T and B lymphocytes were determined in peripheral venous blood. These parameters and macrophage counts were also determined in bronchoalveolar lavage fluid. Variations in the cellular responses were correlated with patients' age, sex, nutritional status and duration of symptoms as well as radiological severity of disease. Patients of both sexes (seven female) showed similar responses. Decreased cell counts in peripheral blood were observed in patients aged 31 to 40 years. Well-nourished patients (n = 19) had higher counts of lymphocytes in peripheral blood and polymorphonuclear leucocytes in bronchoalveolar lavage fluid. The duration of symptoms had a significant influence on cellular responses. In blood, lymphocyte counts were increased in those with symptoms of shorter duration but reduced in those symptomatic for more than 6 months. In bronchoalveolar lavage fluid also all cellular elements were increased in those symptomatic for less than 6 months but a decline followed in those with symptoms for longer duration. Patients with minimal disease radiologically showed higher total leucocyte counts in blood, whereas those with moderately advanced lesions had elevated absolute lymphocyte counts. T cell lymphopenia was observed in blood of patients with far advanced disease. The inflammatory response in bronchoalveolar lavage fluid, however, increased in parallel with the severity of disease. Patients with far advanced lesions showed marked inflammation irrespective of duration of symptoms. Thus, the pattern of inflammation in bronchoalveolar lavage fluid was not similar to that in peripheral blood, particularly in patients with far advanced lesions.

Adolescent↗

PubMed1988

False-positive reactions with enzyme-linked immunosorbent assay of Mycobacterium tuberculosis antigens in pleural fluid.

The value of enzyme-linked immunosorbent assay (ELISA) for the diagnosis of tuberculous pleural effusion has not been defined. We performed ELISA by a double antibody sandwich technique with anti-BCG antibody in the solid phase to detect Mycobacterium tuberculosis antigen in pleural fluid from 36 patients with pleural effusion (tuberculosis 15, lung cancer 12, miscellaneous 9). Pleural fluids from 12 of the tuberculosis patients, 12 of the cancer patients and one patient in the miscellaneous group had optical densities above the cut-off point. False-positive reactions in patients with lung cancer limit the usefulness of ELISA with conventional anti-BCG antibody for detection of M. tuberculosis antigen.

Adult↗