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Biomedical subjects

A Banerjee

Publications and source records attributed to A Banerjee.

At least 271 records · Page 15Linked to original sources
PubMed1995

Characterization of a dominant negative mutant of the cell cycle ubiquitin-conjugating enzyme Cdc34.

The yeast Saccharomyces cerevisiae CDC34 gene encodes a ubiquitin-conjugating enzyme that is required for the cell cycle G1/S transition. We show here that a dominant negative Cdc34 protein is generated by simultaneously replacing both Cys95 and Leu99 with Ser residues. Cys95 is an essential catalytic residue that forms a transient thiol ester with ubiquitin during catalysis, and Leu99 is highly conserved among all known ubiquitin-conjugating enzymes. Mutants that encode either an alanine or a serine at one or both of these two positions are inactive. Of these eight mutants, overexpression of CDC34-C95S,L99S in wild type strains was found to block cell growth. Although cells overexpressing Cdc34-C95S,L99S do not exhibit the characteristic multibudded phenotype of cdc34 temperature-sensitive or null mutants, this blockade is relieved by simultaneous overxpression of wild type Cdc34. Purified Cdc34-C95S,L99S protein can be shown to inhibit in vitro ubiquitination of the Cdc34-specific substrate, Cln2 protein. We suggest that Cdc34-C95S,L99S selectively sequesters a subset of Cdc34 substrates or regulators. These findings have implications for the structure/function relationships of ubiquitin-conjugating enzymes, and suggest a general method for identifying components and substrates of specific ubiquitination pathways of eukaryotes.

Amino Acid Sequence↗
PubMed1995

PMNs primed for superoxide release and increased CD11b expression do not sequester in normal lung.

Our previous work has implicated platelet activating factor (PAF)-induced neutrophil (PMN) priming and increased CD11b/CD18 receptor expression in the pathogenesis of lung injury following gut ischemia/reperfusion (I/R). In this model CD11b blockade abrogates lung injury but does not alter PMN priming or pulmonary leukosequestration. We, therefore, hypothesized that PAF-stimulated PMN priming and CD11b expression are insufficient to promote lung PMN sequestration. Normal rat PMNs, labeled with 51Cr, were incubated with PAF (10 ng/ml) to induce priming for superoxide (O2-) generation and enhance CD11b expression. Gut I/R animals underwent superior mesenteric artery occlusion for 45 min. 51Cr-labeled PMNs (2 x 10(7)) were injected iv. Study groups, consisting of (a) normal/control, (b) sham/laparotomy, and (c) gut I/R, were given either normal or PAF-treated PMNs. PAF-primed PMNs had increased 2- release and CD11b expression, but did not sequester in the lungs of normal rats. However, following gut I/R PAF-treated PMNs sequestered in the pulmonary bed. These data suggest that PAF priming for O2- generation and increased CD11b expression are insufficient alone to promote PMN sequestration in the lung. Rather, additional factors generated by gut I/R are necessary for this process.

Animals↗
PubMed1995

Reperfused gut elaborates PAF that chemoattracts and primes neutrophils.

Our in vivo model of mesenteric ischemia/reperfusion (I/R) has shown that the gut serves as a priming bed for neutrophils (PMN). Activation of phospholipase A2 (PLA2) during ischemia temporally precedes PMN sequestration in the gut and the appearance of primed PMN in the portal circulation. Therefore, we hypothesized that reperfused gut secretes platelet activating factor (PAF) via PLA2 activation that is responsible for increased PMN chemotaxis and priming for superoxide (O2-) generation. Sprague-Dawley rats underwent gut ischemia/reperfusion (45 min SMA occlusion/2 hr reperfusion) or sham laparotomy. Distal ileum was harvested, rinsed with bacteriostatic saline/neomycin, and incubated for 1 hr at 37 degrees C in RPMI 1640 and the cell-free supernatant was collected. Normal human PMNs, isolated by plasma-Percoll gradients, were pretreated with or without a PAF receptor antagonist (WEB 2170). Chemotaxis toward gut supernatant was then measured by the agarose method. Additionally, PMNs were preincubated with or without WEB 2170 and their O2- release in response to 1 microM FMLP was measured by the Vmax of SOD-inhibitable cytochrome c reduction. Reperfused gut produced a chemotactic index of 2.1 +/- 0.1 compared to 0.2 +/- 0.9 following sham laparotomy (P < 0.05); this was reduced to 0.4 +/- 0.9 with PAF receptor blockade. Similarly, gut I/R supernatant primed PMNs for O2- (P < 0.05) compared to laparotomy, and this effect was abrogated by a PAF antagonist. These data suggest that reperfused gut can elaborate PAF which chemoattracts and primes PMNs for O2- generation.

Animals↗
PubMed1995

Pulmonary hypertension in acute lung injury is due to impaired vasodilation with intact vascular contractility.

The major hemodynamic feature of acute lung injury (ALI) is pulmonary hypertension. Both endothelial-dependent and -independent pulmonary vasorelaxation is impaired in ALI due to endotoxemia. We hypothesized that endotoxemia selectively impairs relaxation of the pulmonary artery but does not impair contractility of pulmonary vascular smooth muscle (VSM). Our purpose was to determine the effect of endotoxemia (ETX) on the contractile response of pulmonary VSM to (1) tubular depolarization (KCl), (2) alpha 1-adrenoreceptor stimulation (phenylephrine, PE), (3) 5HT2 receptor stimulation (serotonin, 5HT), and (4) prostaglandin F2 alpha receptor stimulation. Pulmonary artery rings were isolated from rats 6 hr after injection of ETX, 20 mg/kg ip (n > or = 6), or saline (n > or = 6) and suspended on tensiometers in individual organ baths. Endothelial-dependent cGMP-mediated relaxation was determined using the receptor agonist acetylcholine (ACh) in rings preconstricted with PE. Dose-response curves were generated to each contractile agonist. Statistical comparison was performed using one-way ANOVA with post hoc Bonferonni-Dunn, P < 0.05 accepted as significant. Relaxation to ACh was 96.4 +/- 1.3% in controls vs 21.4 +/- 3.1% (P < 0.05) in endotoxin-treated rats. Endotoxin did not affect the maximal tension in response to the contractile agonists nor did it change the concentration required to produce 50% contraction (EC50). From these data we conclude that endotoxemia causes a decrease in vasorelaxation to the endothelial-dependent receptor agonist acetylcholine but does not impair agonist-induced contractility of pulmonary VSM. This suggests that pulmonary hypertension in ALI is mediated by impairment of pulmonary vasodilation with preservation of VSM contractility.

Acute Disease↗
PubMed1995

Cardiac preconditioning protects against irreversible injury rather than attenuating stunning.

The purpose of this experiment was to determine if cardiac preconditioning (PC) mediates protection by attenuating stunning or preventing irreversible injury. Inherent in the definition of myocardial stunning is the ability to respond to catechol stimulation after ischemia/reperfusion (I/R). Irreversibly injured myocardium cannot respond to catechols. We hypothesized that alpha 1-adrenergic-stimulated PC is mediated through a functional protection against reversible injury. We investigated this hypothesis in the isolated, buffer-perfused rat heart subjected to global ischemia (20 min, 37.5 degrees C) and reperfusion (40 min). The PC group received an alpha 1-adrenergic stimulus (norepinephrine, 0.5-1.0 microM, 2 min) 10 min prior to ischemia. Control hearts were perfused normoxically for 80 min. Developed pressure (DP) and heart rate were recorded continuously. To determine maximal myocellular function, all hearts received a beta-adrenergic pathway stimulus (forskolin (FSK), 100 microM bolus) at end reperfusion. The ability to improve DP in response to FSK was indicative of reversible dysfunction (stunning). Failure to attain the maximal DP established in normoxic controls was utilized as a measure of irreversible dysfunction. Recovery was assessed as a percentage of initial DP. The results suggest that (1) PC protects against an I/R injury (recovery: I/R, 50.1%; PC + I/R, 76.0%; P < 0.05); (2) all groups exhibit reversible dysfunction (all increased DP in response to FSK); (3) when maximally stimulated, I/R hearts are unable to develop pressures similar to those of normoxic controls, suggesting irreversible injury; and (4) PC hearts, however, attained similar maximal pressures compared to controls. We conclude that alpha 1-adrenergic PC improves postischemic cardiac function by preventing irreversible injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Selective inhibition of cyclic adenosine monophosphate-mediated pulmonary vasodilation by acute hypoxia.

BACKGROUND: Adult respiratory distress syndrome is characterized by hypoxia and acute pulmonary hypertension. Therefore we examined the effect of acute hypoxia on the mechanisms of pulmonary vasodilation. METHODS: Isolated rat pulmonary artery rings were suspended on tensiometers in a balanced salt solution. A normoxic gas mixture was bubbled through the solution (21% O2, 5% CO2, 74% N2). Rings were preconstricted with phenylephrine, and the following mechanisms of pulmonary vascular smooth muscle relaxation were studied in a random order: (1) endothelial-dependent cyclic guanosine monophosphate-mediated (acetylcholine, 10(-9) to 10(-6) mol/L), (2) endothelial-independent cyclic guanosine monophosphate-mediated (nitroprusside, 10(-9) to 10(-6) mol/L), and (3) beta-adrenergic receptor cyclic adenine monophosphate-mediated (isoproterenol, 10(-9) to 10(-6) mol/L). Separate rings were preconstricted with phenylephrine, and the gas was switched to a hypoxic mixture (0% O2, 5% CO2, 95% N2). After vasoconstriction to hypoxia reached a plateau, the response to the maximal effective dose of the above vasodilators (10(-6) mol/L) was determined in a random order. Statistical analysis was done with one-way analysis of variance with post hoc Bonferroni-Dunn correction. A p value of less than 0.05 was accepted as significant. RESULTS: Endothelial-dependent and -independent cyclic guanosine monophosphate-mediated relaxation was the same in normoxia and hypoxia. On the other hand, hypoxia inhibited beta-adrenergic receptor cyclic adenine monophosphate-mediated pulmonary vasorelaxation (97.5% +/- 2.5% versus 71.5% +/- 2.3% in hypoxia; p < 0.01). CONCLUSIONS: These data suggest that hypoxia selectively inhibits beta-adrenergic cyclic adenine monophosphate-mediated pulmonary vasorelaxation. This dysfunction of the normal mechanism of pulmonary vasodilation may contribute to the pulmonary hypertension seen in adult respiratory distress syndrome.

Acetylcholine↗
PubMed1995

Postinjury neutrophil priming and activation: an early vulnerable window.

BACKGROUND: Generation of extracellular, cytotoxic superoxide anion (O2-) by polymorphonuclear neutrophils (PMNs) contributes to an unbridled inflammatory response that can precipitate multiple organ failure (MOF). Release of O2- is markedly enhanced when activated PMNs have been previously "primed" by inflammatory mediators, such as those expressed after trauma. We therefore hypothesized that PMN priming occurs as an integral part of the early inflammatory response to trauma. METHODS: PMNs were obtained from 17 high-risk patients with torso trauma at 3, 6, 12, 24, 48, and 72 hours after injury, as well as from 10 healthy donors, and the in vitro release of O2- was quantitated with a kinetic, superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay. PMN O2- release was measured in the presence and absence of 1 mumol/L N-formyl-methionyl-leucyl-phenylalanine (fMLP) and after priming and activation with 20 nmol/L platelet-activating factor (PAF) and 1 mumol/L fMLP, respectively. RESULTS: In vitro PMN O2- release was used to determine whether postinjury PMNs were (1) activated in vivo, (2) primed in vivo, or (3) primable in vitro. Unstimulated PMNs from trauma patients spontaneously expressed modest amounts of O2- in vitro from 6 to 48 hours after injury, suggesting endogenous activation. Also, fMLP-activated PMNs collected between 3 and 24 hours after injury expressed more O2- than controls (p < or = 0.02), indicating in vivo, trauma-related priming. Furthermore, postinjury PMNs were maximally primed in vivo (i.e., in vitro exposure to PAF before fMLP activation failed to significantly enhance O2- release) as compared to PMNs treated with fMLP. CONCLUSIONS: These data indicate that major torso trauma (first hit) primes and activates PMNs within 3 to 6 hours after injury. Consequently, we postulate that postinjury priming of PMNs may create an early vulnerable window during which a second hit (e.g., a secondary operation or delayed hemorrhage) activates exuberant PMN O2- release, rendering the injured patient at high risk for MOF.

Adolescent↗
PubMed1995

Closed mitral valvotomy and elective ventilation in the postoperative period: effect of mild hypercarbia on right ventricular function.

OBJECTIVES: It is customary to extubate patients immediately after closed mitral valvotomy. These patients often have deranged respiratory function caused by chronic lung congestion. The left ventricular function may also be subnormal after valvotomy in some patients. Therefore, elective ventilation for some duration in the postoperative period can be beneficial to these patients. This work is an attempt to find whether elective ventilation should be preferred over immediate extubation in these patients. DESIGN: A prospective randomized study. SETTING: The study was performed in a tertiary care hospital, and the patients are referred from the northern states of India. PARTICIPANTS: One hundred patients undergoing elective closed mitral valvotomy were included in the initial part of the study. Ten more patients were studied to evaluate the effect of mild hypercarbia on right ventricular function after closed mitral valvotomy. INTERVENTIONS: One hundred patients were divided into two groups of 50 each. Group 1 consisted of patients in whom the neuromuscular blockade was reversed at the end of surgery with neostigmine and atropine and the trachea was extubated. In group 2, the residual neuromuscular paralysis was not reversed and the patients were electively ventilated in the postoperative period for an average duration of 5 hours and 29 minutes +/- 1 hour and 58 minutes. In all the patients in both the groups, electrocardiogram, direct arterial blood pressure, and oxygen saturation were continuously monitored, and arterial blood gases were measured intermittently throughout the study period. Because the results showed that there was mild hypercarbia, 30 minutes after extubation in group 1, 10 more patients were studied to evaluate the effect of mild hypercarbia on right ventricular function after surgery. Patients were ventilated after surgery (F1O2 = 1) to maintain normocarbia (PaCO238.6 +/- 3.4 mmHg). Mild hypercarbia PaCO251.5 +/- 3.7 mmHg) followed by normocarbia (PaCO2 40 +/- 2.5 mmHg) was induced by adjusting the ventilator rate with a constant tidal volume. Standard hemodynamic measurements were performed at each stage. MEASUREMENTS AND MAIN RESULTS: Although all the patients maintained satisfactory and stable hemodynamics in the postoperative period, the PaCO2 at the end of 30 minutes of extubation was significantly higher in group 1 (48.1 +/- 5.3 mmHg) as compared with group 2 (40.2 +/- 4.3 mmHg, p < 0.001). Mild hypercarbia significantly increased pulmonary vascular resistance (p < 0.01), mean pulmonary arterial pressure (p < 0.001), right ventricular stroke work (p < 0.01), right ventricular systolic pressure (p < 0.01), and right ventricular end-diastolic pressure (p < 0.001). The effect was not totally reversible with CO2 washout as all parameters except right ventricular end-diastolic pressure and pulmonary vascular resistance continued to remain significantly higher when normocarbia was restored. The significant changes in systemic hemodynamics produced by hypercarbia were increases in cardiac index, mean arterial pressure, and pulmonary capillary wedge pressure. CONCLUSIONS: Avoidance of even mild hypercarbia, therefore, appears advisable in the early postoperative period because of potential impedence to right ventricular ejection. Continuous monitoring of end-tidal CO2 and frequent blood gas analyses should be practiced, and elective ventilation should be considered in patients with long-standing disease and pulmonary hypertension.

Adolescent↗