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Biomedical subjects

R Dhand

Publications and source records attributed to R Dhand.

At least 37 records · Page 2Linked to original sources
PubMed1997

Surfactant convertase action is not essential for surfactant film formation.

A serine-active enzyme, "surfactant convertase", is required for the conversion of surfactant from the tubular myelin (TM) form to the small vesicular (SV) form. This transformation involves at least two steps, the conversion of TM to a surface-active film at the air-fluid interface and the reorientation of the film into the surface-inactive SV form; we asked if convertase was required for the first of these steps. Rat and mouse TMs were pretreated with diisopropyl fluorophosphate (DFP) to inactivate endogenous convertase activity or with vehicle and then were analyzed for their ability to lower surface tension in vitro as an index of the conversion of TM to a surface film. DFP pretreatment did not alter the ability of TM preparations to lower surface tension, as assessed by pulsating bubble, and it did not affect the behavior of TM in a surface balance. In an experiment designed to test the ability of TM to feed a surface film to exhaustion, TMs that had been pretreated with DFP or vehicle performed similarly. These experiments show that convertase activity is not required for the conversion of TM to a monolayer and suggest, instead, that convertase acts at a post surface film stage.

Animals↗
PubMed1996

Binding to the platelet-derived growth factor receptor transiently activates the p85alpha-p110alpha phosphoinositide 3-kinase complex in vivo.

Ligand stimulation of the platelet-derived growth factor (PDGF) receptor results in its association with phosphoinositide 3-kinase activity and a corresponding synthesis of 3'-phosphorylated lipids. Early studies that examined this interaction in vivo employed anti-phosphotyrosine antiserum or antiserum against the PDGF receptor. The recent identification of multiple isoforms of both the regulatory and the catalytic subunit of the enzyme have led us to utilize antisera against p85alpha and p110alpha to characterize the association of this particular phosphoinositide 3-kinase complex with the PDGF receptor following ligand stimulation of murine fibroblasts. Both the p85alpha and p110alpha subunits rapidly associated with the ligand-activated receptor resulting in a transient, 2-fold increase in the total pool of p110alpha lipid kinase activity. This association was stable for 15 min after initial stimulation. Subsequently, both subunits began to dissociate from the receptor with similar kinetics. By 60 min this process was complete, demonstrating that p85alpha and p110alpha both associate with the receptor and dissociate from the receptor as a dimeric complex. At this time, marked PDGF receptor down-regulation was observed. Immunoprecipitation from metabolically labeled cells revealed that p85alpha is constitutively phosphorylated on serine residues in quiescent cultures. Upon PDGF stimulation, this phosphorylation upon serine residues was maintained in addition to tyrosine phosphorylation of this subunit. No phosphorylation of the p110alpha subunit was detected in either quiescent or PDGF-stimulated cells. Quantitation of Western blot analysis demonstrated that only 5% of the total pool of p85alpha associated with the PDGF receptor upon ligand stimulation. The 2-fold increase in the lipid kinase activity measured in immunoprecipitates using either anti-p85alpha or anti-p110alpha antiserum therefore reflects a far greater increase in the specific activity of the enzyme upon its association with the PDGF receptor.

3T3 Cells↗
PubMed1996

Aerosol delivery from a metered-dose inhaler during mechanical ventilation. An in vitro model.

Successful bronchodilator therapy with a metered-dose inhaler (MDI) in intubated, mechanically ventilated patients requires adequate delivery of aerosol to the lower respiratory tract. We determined the effect of ventilator mode, inspiratory flow pattern, humidity, and spontaneous respiratory effort on albuterol delivery in a model of the trachea and bronchi. The model was ventilated through an endotracheal tube during controlled mechanical ventilation (CMV), assist control (AC), pressure support (PS), and continuous positive airway pressure (CPAP), separately with a dry and humidified ventilator circuit. Delivery of albuterol administered by a MDI and spacer on filter placed at the ends of the bronchi was measured by spectrophotometry (246 nm). Under dry conditions and with a frequency of 10 breaths/min, albuterol delivery with CMV (VT, 800 ml; 30.3 +/- 3.4%), AC (VT, 800 ml; 31.9 +/- 1.3%), PS 10 cm H2O (VT, 700 ml; 28.8 +/- 4.5%), or PS 20 cm H2O (VT, 800 ml; 30.9 +/- 1.8%) was lower than that observed with simulated spontaneous breaths with CPAP (VT, 800 ml; 39.2 +/- 1.4%) (p < 0.01 for all modes). Delivery was greater under dry (28.8 to 39%) than under humidified conditions (15.9 to 20.2%) (p < 0.005 in all modes). Albuterol delivery showed a linear correlation with both inspiratory time and duty cycle (r > 0.91). Lower respiratory tract delivery of aerosol from a MDI varied from 4.9 to 39.2%. We conclude that in addition to other known factors such as dose, type of spacer, and its position the technique of administering MDIs in mechanically ventilated patients markedly influences lower respiratory tract aerosol delivery.

Aerosols↗
PubMed1996

Dose-response to bronchodilator delivered by metered-dose inhaler in ventilator-supported patients.

In nonintubated patients, metered-dose inhalers (MDIs) are accepted as the most convenient, efficient, and cost effective method of administering inhaled bronchodilators. Recent studies have demonstrated the efficacy of MDIs in ventilator-supported patients; however, the optimal dose of a bronchodilator from a MDI is unknown. We determined the response to increasing doses of albuterol administered by a MDI and cylindrical spacer to 12 mechanically ventilated patients with chronic obstructive pulmonary disease (COPD). Four, eight, and 16 puffs of albuterol were given at 15-min intervals. Rapid airway occlusion were performed before and at 5-min intervals after albuterol for 80 min. Respiratory mechanics were also measured for 60 min in another group of seven patients with COPD who received four puffs of albuterol. Significant decrease in airways resistance occurred after administration of albuterol (p < 0.001). The decrease in airway resistance with four puffs of albuterol was comparable to that observed with cumulative doses of 12 puffs (p = 0.12) and 28 puffs (p = 0.25). Heart rate increased significantly (p < 0.01) after a cumulative dose of 28 puffs. The decrease in airway resistance was sustained for 60 min in the group that received only four puffs of albuterol (p < 0.003). In conclusion, four puffs of albuterol given by a MDI and spacer provided the best combination of bronchodilator effect and safety in stable mechanically ventilated patients with COPD.

Administration, Inhalation↗
PubMed1996

Serum albuterol levels in mechanically ventilated patients and healthy subjects after metered-dose inhaler administration.

In mechanically ventilated patients, systemic blood levels of inhaled drugs reflect absorption from the lower respiratory tract alone since, unlike nonintubated patients, oropharyngeal and gastrointestinal absorption cannot occur. To determine the efficiency of aerosol administration by a metered-dose inhaler (MDI), we measured serum albuterol levels after administration by a MDI and spacer to nine mechanically ventilated patients (10 puffs) and to 10 healthy subjects (six puffs). Serum albuterol levels (+/- SEM) quantitated by high-performance liquid chromatography and electrochemical detection were: 0.09 +/- 0.04 mg/ml/puff at baseline, 0.66 +/- 0.10 at 5 min, 0.98 +/- 0.10 at 10 min, 0.56 +/- 0.08 at 15 min, and 0.37 +/- 0.03 at 30 min in mechanically ventilated patients versus zero at baseline, 0.89 +/- 0.12 at 5 min, 1.27 +/- 0.13 at 10 min, 0.84 +/- 0.09 at 15 min, and 0.53 +/- 0.07 at 30 min in control subjects (p > or = 0.07 at 5, 10, and 30 min; p < or = 0.05 at baseline and at 15 min). Area under the curve (AUC0-30) in the mechanically ventilated patients was 16.8 +/- 1.4 versus 23.4 +/- 1.9 ng/ml/puff x min in control subjects (p = 0.014). In summary, administration of albuterol with a MDI achieved a profile of serum levels in mechanically ventilated patients similar to that in healthy control subjects, but the peak serum level and systemic bioavailability (AUC0-30) were lower in the patients. In conclusion, serum levels reliably assess lower respiratory tract deposition of albuterol, and show that MDIs are more efficient for aerosol delivery in mechanically ventilated patients than was previously reported in studies using radiolabeled aerosols.

Aged↗
PubMed1996

Bronchodilator delivery with metered-dose inhalers in mechanically-ventilated patients.

Metered-dose inhalers (MDIs) provide several advantages over nebulizers, including ease of administration, decreased cost, reliability of dosing, and freedom from contamination. However, this method of aerosol delivery has been considered ineffective in mechanically-ventilated patients because most of the aerosol deposits in the endotracheal tube and ventilator circuit. A smaller amount of aerosol from a MDI is deposited in the lower respiratory tract of mechanically-ventilated patients than in ambulatory patients, although recent studies show that a significant bronchodilator effect can still be achieved. When employed optimally, significant bronchodilation occurs with as little as 4 puffs of a sympathomimetic aerosol. Multiple factors influence the efficacy of MDIs in mechanically-ventilated patients. The method of connecting the MDI canister to the ventilator circuit has a marked effect on aerosol delivery, and other factors include the timing of MDI actuation, ventilator mode, tidal volume, circuit humidity, and duty cycle. With a proper technique of administration, a MDI serves as an effective, convenient, and safe method for delivering bronchodilator aerosols in mechanically-ventilated patients.

Administration, Inhalation↗
PubMed1995

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Amino Acid Sequence↗
PubMed1995

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Amino Acid Sequence↗
PubMed1995

Bronchodilator delivery by metered-dose inhaler in ventilator-supported patients.

The optimal dose and technique for administration of bronchodilators with a metered-dose inhaler (MDI) in mechanically ventilated patients have not been established. We studied the efficacy and safety of 10 puffs (90 micrograms/puff) of albuterol administered by an MDI in seven mechanically ventilated patients with chronic obstructive pulmonary disease (COPD). Rapid airway occlusions at constant flow inflation were performed before and at 5-min intervals after administration of albuterol for 60 min. Significant decreases in maximum (Rrsmax; p < 0.01) and minimum inspiratory resistance (Rrsmin; p < 0.01) were present at 5 min and persisted for 60 min after administration of albuterol (p < 0.01 for both parameters). Rrsmax indicates maximal inspiratory resistance while Rrsmin represents the ohmic flow resistance. Intrinsic positive end-expiratory pressure decreased significantly (p < 0.05) 15 min after albuterol administration. Heart rate, blood pressure, and arterial oxygenation did not show significant change after albuterol. In summary, 10 puffs of albuterol given by an MDI and spacer produced significant bronchodilation in ventilator-supported patients with COPD, without producing side effects. In conclusion, higher doses of albuterol given by an MDI and spacer could be used routinely in mechanically ventilated patients with COPD.

Administration, Inhalation↗
PubMed1995

Using physiologic end points to assess innovations in mechanical ventilation.

It is our view that new ventilatory methods should be withheld from clinical practice until there has been adequate evaluation of their effect on physiologic variables and the link to long-term outcomes has been established. In the past, premature and over-enthusiastic acceptance of ventilatory strategies may have resulted in patient discomfort and even harm, and this can be minimized by a more careful evaluation of the physiologic effects of such innovations before their acceptance into clinical practice.

Diffusion of Innovation↗
PubMed1994

Biochemical characterization of the free catalytic p110 alpha and the complexed heterodimeric p110 alpha.p85 alpha forms of the mammalian phosphatidylinositol 3-kinase.

The regulatory (p85 alpha) and catalytic (p110 alpha) subunits of the mammalian phosphatidylinositol 3-kinase have been expressed in insect cells using the baculovirus sytem. The free catalytic subunit p110 alpha and the coexpressed heterodimeric complex of p85 alpha and p110 alpha were purified and their enzymological properties compared. While many kinetic parameters were similar, the coexpressed complex was found to have a 20-fold higher Km for ATP in comparison with the free catalytic subunit p110 alpha using phosphatidylinositol 4,5-bisphosphate as a substrate; no significant difference was detectable when phosphatidylinositol was used. Reconstitution of the p110 alpha.p85 alpha complex in vitro showed that it had the properties of the free p110 alpha and not the p110 alpha.p85 alpha in vivo complex. Therefore, a post-translational modification dependent upon the presence of the regulatory subunit p85 alpha rather than the physical subunit interaction itself is responsible for the observed properties of the lipid kinase activity of the p110 alpha.p85 alpha complex. Phosphatase treatment of the purified lipid kinase complex reduced the high Km for ATP, suggesting that a phosphorylation of the heterodimeric complex (p85 alpha.p110 alpha) caused this effect. This mode of regulation is discussed in the context of lipid kinase activation in vivo.

Animals↗
PubMed1994

Phosphatidylinositol-3-OH kinase as a direct target of Ras.

Ras (p21ras) interacts directly with the catalytic subunit of phosphatidylinositol-3-OH kinase in a GTP-dependent manner through the Ras effector site. In vivo, dominant negative Ras mutant N17 inhibits growth factor induced production of 3' phosphorylated phosphoinositides in PC12 cells, and transfection of Ras, but not Raf, into COS cells results in a large elevation in the level of these lipids. Therefore Ras can probably regulate phosphatidylinositol-3-OH kinase, providing a point of divergence in signalling pathways downstream of Ras.

Animals↗
PubMed1994

The catalytic subunit of phosphatidylinositol 3-kinase is a substrate for the activated platelet-derived growth factor receptor, but not for middle-T antigen-pp60c-src complexes.

The interaction of phosphatidylinositol 3-kinase (PI 3-K) with polyoma-virus middle-T antigen-pp60c-src (mT:cSrc) complexes and with the platelet-derived growth factor (PDGF) receptor has been investigated. Firstly, we undertook reconstitution studies, using proteins derived from a baculovirus expression system. The p110 catalytic subunit of the PI 3-K associated with tyrosine kinases only when complexed with the p85 alpha regulatory subunit. Both p85 alpha and p110 were substrates of the PDGF receptor. In contrast, only the p85 alpha subunit was detectably phosphorylated when PI 3-K was associated with mT:cSrc. Secondly, we studied PI 3-K in mammalian cells. In mT-antigen-transformed NIH-3T3 cells neither p85 alpha nor p110 was phosphorylated on tyrosine residues in vivo, even though p85 alpha was a substrate in kinase assays in vitro. In quiescent NIH-3T3 cells, PI 3-K showed detectable activity in vitro; PDGF stimulation resulted in a rapid and transient association of PI 3-K with the receptor, which was correlated with a transient increase in intrinsic P13-K activity (approx. 2-fold). The activated PDGF receptor phosphorylated p110 in vitro, at one major site. In vivo, PDGF stimulation induced tyrosine phosphorylation of p110 that persisted for at least 1 h after stimulation. Immunodepletion of the PDGF receptor from stimulated cell lysates showed that p110 was released from the receptor in a tyrosine-phosphorylated form. From these results we conclude that (i) the mT:cSrc complex and the PDGF receptor differ in their association with PI 3-K activity, (ii) PDGF receptor appears to activate PI 3-K in vivo both by relocation of the enzyme and by stimulation of its intrinsic activity, and (iii) tyrosine phosphorylation of the p110 subunit by the PDGF receptor may play a role in PI 3-K regulation in some circumstances.

3T3 Cells↗
PubMed1994

PI 3-kinase: structural and functional analysis of intersubunit interactions.

Phosphatidylinositol (PI) 3-kinase has an 85 kDa subunit (p85 alpha) which mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. Here p85 alpha and a related protein p85 beta are shown to form stable complexes with recombinant p110 in vivo and in vitro. Using a panel of glutathione S-transferase (GST) fusion proteins of the inter-SH2 region of p85, 104 amino acids were found to bind directly the p110 protein, while deletion mutants within this region further defined the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85 alpha protein in mouse L cells showed it was unable to bind PI 3-kinase activity in vivo. Mapping of the complementary site of interaction on the p110 protein defined 88 amino acids in the N-terminal region of p110 which mediate the binding of this subunit to either the p85 alpha or the p85 beta proteins. The inter-SH2 region of p85 is predicted to be an independently folded module of a coiled-coil of two long anti-parallel alpha-helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3-kinase complex is discussed.

Amino Acid Sequence↗
PubMed1994

PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.

Phosphatidylinositol 3-kinase (PI 3-kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein-serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N-terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3-kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter-subunit serine phosphorylation in the regulation of the PI 3-kinase in vivo.

Animals↗
PubMed1993

The GTPase dynamin binds to and is activated by a subset of SH3 domains.

Src homology 3 (SH3) domains have been implicated in mediating protein-protein interactions in receptor signaling processes; however, the precise role of this domain remains unclear. In this report, affinity purification techniques were used to identify the GTPase dynamin as an SH3 domain-binding protein. Selective binding to a subset of 15 different recombinant SH3 domains occurs through proline-rich sequence motifs similar to those that mediate the interaction of the SH3 domains of Grb2 and Abl proteins to the guanine nucleotide exchange protein, Sos, and to the 3BP1 protein, respectively. Dynamin GTPase activity is stimulated by several of the bound SH3 domains, suggesting that the function of the SH3 module is not restricted to protein-protein interactions but may also include the interactive regulation of GTP-binding proteins.

Amino Acid Sequence↗
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↗