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R Dhand

Publications and source records attributed to R Dhand.

At least 19 recordsLinked to original sources
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↗
PubMed1988

High speed photographic analysis of aerosols produced by metered dose inhalers.

The design of pressurized metered dose inhalers (MDI) used to assess asthma is variable. We have examined the aerosol spray flumes generated by four commercially available MDI products using high speed video photography. For this purpose a moulded jacket was designed which could hold the inhaler in an immovable position during actuation. Fresh inhalers were fitted in the jacket after thorough shaking and three successive actuations 30 s apart were filmed with a high speed video camera (200 frames s-1). The aerosol, ejected at high velocity into calm room air, was seen to have a 'jet' phase followed by a 'cloud' phase as a result of particle dispersion. Filming was continued till the flume could no longer be visualized on the TV monitor. High speed photography was used to record flumes seen on the video monitor, to enable characterization of flume appearance, dimensions and mean velocity.

Aerosols↗
PubMed1988

Use of visual analogue scales for assessment of the severity of asthma.

Use of visual analogue scales (VAS) can provide reliable assessment of dyspnea. In order to determine if VAS can be used for clinical assessment of asthma we administered the scale to 40 stable asthmatics and found a good correlation (r = -0.72) with the peak expiratory flow rate (PEFR) readings expressed as per cent of predicted normal. The scales were also used to assess severity in 12 acutely ill asthmatics. Values of VAS and PEFR were recorded at the time of admission and twice daily thereafter till discharge from the hospital. The correlation of the two parameters was less than that obtained in stable asthmatics, but graphic parallelism was remarkable.

Adolescent↗
PubMed1988

Single dose kinetics of rifampicin and isoniazid in well-nourished and malnourished patients of tuberculosis.

A single dose kinetics of isoniazid and rifampicin alone, and combination was studied in well-nourished and malnourished patients of tuberculosis. The elimination half-life of isoniazid was significantly increased when administered in combination with rifampicin in well-nourished and malnourished patients, while no significant difference was observed in any of the pharmacokinetic parameters of rifampicin in combination with isoniazid or alone in both groups of patients. Results are discussed on the basis of pharmacokinetic drug interaction.

Acetylation↗