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Biomedical subjects

J A Cook

Publications and source records attributed to J A Cook.

At least 199 records · Page 11Linked to original sources
PubMed1991

Cellular glutathione and thiol measurements from surgically resected human lung tumor and normal lung tissue.

Cellular glutathione (GSH) levels were measured from 27 human lung tumor biopsies, enzymatically disaggregated, and compared with cells isolated from normal lung of the same patients. GSH levels from normal lung were similar among patients with a mean value of 11.20 +/- 0.58 (SEM) nmol GSH/mg protein (24 patients) with a range from 6.1 to 17.5 nmol GSH/mg protein. GSH levels varied considerably within and across histological tumor types with the following values: adenocarcinomas, 8.83 +/- 0.96 nmol/mg protein (8 patients); large cell carcinomas, 8.25 +/- 2.51 nmol/mg protein (3 patients); and squamous cell carcinomas, 23.25 +/- 5.99 nmol/mg protein (8 patients). The cyclic GSH reductase assay gave only average GSH values and could not distinguish possible GSH variation among subpopulations of cells isolated. Cell volume measurements and microscopic evaluation of cells isolated from both tumors and normal lung revealed heterogeneity with respect to cell types present. To determine the extent of thiol variation among tumor cell subpopulations, tumor cell suspensions were stained with the thiol-specific stain, monochlorobimane (MCB). The accuracy of MCB staining was tested by flow cytometric analysis of 12 in vitro human tumor cell lines and 3 rodent cell lines. A linear relationship was found between the bimane cellular fluorescence and the cyclic GSH reductase assay for cell lines having less than 80 nmol GSH/mg protein (R2 = 0.82). Above 80 nmol GSH/mg protein the rate of change of the bimane fluorescence intensity with respect to increasing GSH concentrations was much reduced. However, by labeling cells with MCB it was possible to distinguish between cell lines with low versus high GSH content. MCB staining of tumor samples revealed multiple populations of cells with respect to thiol levels. In particular, 2 of 8 squamous cell carcinomas had a proportion of cells with elevated fluorescence intensities (from 10 to 35% of the population) suggesting the presence of cells with greatly elevated thiol levels. These findings underscore the complexity of quantitating intracellular GSH levels from tumor biopsies. The combined use of MCB with flow cytometry and conventional GSH assays may help to delineate subpopulations of cells within tumors with different thiol levels.

Adenocarcinoma↗
PubMed1991

Effect of the LTD4 receptor antagonist LY203647 on endotoxic shock sequelae in the rat.

This study investigated the effect of a novel LTD4 receptor antagonist LY203647 on Salmonella enteritidis endotoxin-induced shock sequelae in anesthetized rats. LY203647 (30 mg/kg i.v.) or vehicle was given 10 min prior to endotoxin (10 mg/kg i.v.) or its vehicle, and the hematocrit, mean arterial pressure and circulating leukocyte counts were determined. LY203647 significantly inhibited endotoxin-induced hemoconcentration up to 90 min post-endotoxin (46.7 +/- 1.3 vs. 51.9 +/- 2.4% at 30 min post-endotoxin, 45.9 +/- 1.1 vs. 53.1 +/- 1.4% at 90 min post-endotoxin, N = 8-9, P less than 0.05). The endotoxin-induced decreases in mean arterial pressure were also attenuated by LY203647, -29 +/- 5 vs. -56 +/- 9 mm Hg at 60 min post-endotoxin and -42 +/- 4 vs. -60 +/- 9 mm Hg at 90 min post-endotoxin (N = 9-10, P less than 0.05). LY203647 also attenuated endotoxin-induced decreases in leukocyte count in arterial blood. A study of differential counts in circulating leukocytes (N = 3) showed that endotoxin induced complete disappearance in circulating neutrophils. The circulating lymphocyte count was decreased by 30 +/- 10 and 41 +/- 1% at 15 and 30 min post-endotoxin, respectively. LY203647 inhibited endotoxin-induced lymphopenia (P less than 0.05) but failed to alter endotoxin-induced neutropenia. These data suggest that LTD4 may play an important role in mediating hemoconcentration, hypotensive and lymphocytopenic sequelae of endotoxin shock.

Acetophenones↗
PubMed1991

Differential specificity of monochlorobimane for isozymes of human and rodent glutathione S-transferases.

Monochlorobimane (MCB) has been used as a glutathione (GSH) specific fluorescent probe capable of delineating GSH heterogeneity in cellular systems. Generally, low concentrations of MCB (less than 50 microM) have been used to quantitatively label GSH in rodent cell lines. Incubation of the hamster cell lines, CHO AB1 and V79, with 10 microM MCB labeled 75 and 39% of the reduced GSH pool, respectively. In contrast, incubation of 7 different human cell lines with 10 microM MCB labeled less than 4% of the total reduced GSH pool. The human cell lines required 1000 microM MCB to label an average of 73% of the GSH pool (range, 60-88%). When using 1000 microM MCB to label GSH, flow cytometry results from 7 different cell lines (human and rodent) were in good agreement with high performance liquid chromatography and standard spectrophotometric analysis with regards to a rank ordering of the GSH content determined for each cell line. The human glutathione S-transferases B2B2, B1B2, psi, pi, and the rat transferases 1-2, 3-3, and 3-4 were isolated and purified for steady state kinetic analysis with MCB and GSH as the primary substrates. The human basic transferases, B1B2 and B2B2, had Km values for MCB of 354 and 283 microM and Vmax values of 33.3 and 34.6 mumol bimane-GSH/min/mg protein, respectively. The rat basic transferase 1-2 showed similar kinetic results with a Km of 199 microM and a Vmax of 35.5 mumol bimane-GSH/min/mg protein. The human neutral transferase (psi) had a Km for MCB of 204 microM with a Vmax of 6.5 mumol bimane-GSH/min/mg protein. In contrast, MCB has a high affinity for the rat neutral transferase with a Km of 2.6 microM and a Vmax of 35.1 mumol bimane-GSH/min/mg protein. The human acidic transferase (pi), the predominate transferase found in most human tumor cell lines, has a Km of 264 microM for MCB and a Vmax of 1.99 mumol bimane-GSH/min/mg protein. The kcat/Km values indicated that MCB is an excellent substrate for the rat neutral transferases while the human pi glutathione S-transferase showed the least reactivity. Collectively the data indicate that MCB fails to label GSH at lower concentrations (less than 50 microM) in human cell lines because of the reduced affinity of MCB for the human transferases and possibly also due to differences in glutathione S-transferase isozyme expression between rodent and human cell lines.

Animals↗
PubMed1991

Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli.

Expression plasmids encoding random sequence mutant proteins of insulin-like growth factor II (IGFII) were constructed by cassette mutagenesis, to improve the efficiency of IGFII synthesis in Escherichia coli. A pool of oligodeoxyribonucleotide linkers containing random trinucleotide sequences were used to introduce second-codon substitutions into the gene encoding Met-Xaa-Trp-IGFII in expression vectors. E. coli RV308 cells transformed with these vectors synthesized IGFII at levels varying from 0-22% of total cell protein. This variable synthesis is a function of the random second-codon sequence and its corresponding amino acid, Xaa. Our data showed that mRNA stability, protein stability and translational efficiency all contributed to variable expression levels of Met-Xaa-Trp-IGFII in E. coli. Furthermore, an efficiently synthesized IGFII mutant protein, Met-His-Trp-IGFII, was converted to natural sequence IGFII by a simple oxidative cleavage reaction.

Amino Acid Sequence↗
PubMed1991

Kinetics of superoxide production by stimulated neutrophils.

Oxygen-derived active species and superoxide radical in particular are generated and excreted upon granulocyte activation and are instrumental in host defense against bacterial and fungal infections. Associated with the activation of neutrophils is an apparent transitory oxy-radical production. Evidence from independent methods has previously suggested that radical production peaks shortly following neutrophil stimulation and decays within minutes. However, since neutrophil function in the body might reasonably be expected to last beyond the few minutes following stimulation, cessation of the production of oxy-radicals is unexpected. In an attempt to reconcile this discrepancy, the formation kinetics of superoxide by stimulated human neutrophils was reinvestigated by three independent methods: electron spin resonance, chemiluminescence, and ferricytochrome c reduction. The present results demonstrate that under appropriate experimental conditions stimulated neutrophils have the capacity to produce superoxide for several hours. The reasons for the previously reported "apparent" ephemeral nature of oxy-radical formation are discussed.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Right ventricular pump dysfunction with acute experimental septic shock.

Right ventricular (RV) function has been poorly characterized at the onset of acute bacterial septic shock. Using a volumetric thermodilution catheter, previously validated in our laboratory, we serially measured RV function in a porcine model of acute septic shock. Six pigs (21.3 +/- 0.9 kg) were instrumented and Pseudomonas aeruginosa (3.4 x 10(8) cfu/ml, 0.3 ml/20 kg/min) was infused. RV ejection fraction, RV volumes, cardiac output, arterial pressure, central venous pressure, and pulmonary arterial pressure were measured at baseline and at 30, 60, 120, and 240 min after starting the bacterial infusion. RV ejection fraction and stroke volume were decreased at 30 min compared to baseline (21 +/- 5 vs 43 +/- 5% and 14 +/- 3 vs 23 +/- 3 ml, respectively; P less than 0.05) and remained depressed throughout the experiment. Mean arterial pressure was significantly reduced at 60, 120, and 240 min compared to baseline (P less than 0.05). There was a significant increase in pulmonary vascular resistance (1771 +/- 493 vs 301 +/- 99 dyn-sec-cm-5 at 30 min; P less than 0.05) and RV stroke work (5.7 +/- 1.1 vs 2.3 +/- 0.3 gm-m/beat at 30 min; P less than 0.05) while no significant change in RV end-diastolic volume or central venous pressure was observed. Thus, a decrease in RV pump performance was associated with an increase in afterload and no change in preload. These results suggest that severe RV pump dysfunction occurs early in acute septic shock. This was manifested by significant reductions in RV ejection fraction and increased in stroke work.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Endotoxin tolerance differentially alters hemodynamic responses to a thromboxane A2 mimetic and phenylephrine.

Repeated sublethal doses of endotoxin render rats tolerant to lethal doses of endotoxin and reduce thromboxane (Tx) A2 synthesis. Endotoxin-tolerant rats are also more resistant to lethal doses of U46619, a stable TxA2 mimetic. These observations raised the possibility that tolerance may alter hemodynamic responses to TxA2 via one or more mechanisms. Mean arterial pressure (MAP) responses to i.v. injections of the stable TxA2 mimetic U46619 at doses ranging from 0.17 to 8.4 micrograms/kg were determined. Despite an initial lower systemic vascular resistance in tolerant rats compared to control rats (2.4 +/- 0.3 vs 3.1 +/- 0.2 mm Hg/ml/min/100 g of body weight, respectively, p less than 0.05), the maximum pressor response to U46619 was significantly greater (p less than 0.05) at the higher doses of U46619 in tolerant rats compared to control rats. Tolerant and control rats also exhibited qualitatively different changes in MAP in response to U46619. Control rats manifested an initial hypotensive response (15 s) not observed in tolerant rats. In contrast, tolerant rats exhibited no depressor response, but a higher peak pressor response to U46619 than that seen in controls. Since prostaglandins may modulate vascular responses to U46619, subsequent studies were conducted in indomethacin-pretreated or essential fatty acid (EFA) deficient rats that were depleted of arachidonic acid substrate. Either indomethacin or EFA deficiency significantly prevented the initial hypotensive response in control rats, suggesting that prostaglandins may mediate this response to U46619. In additional studies, the MAP response in tolerant and control rats to the alpha 1-adrenergic agonist phenylephrine was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1991

Superoxide production by stimulated neutrophils: temperature effect.

Activation of neutrophils results in a one-electron reduction of oxygen to produce the superoxide anion and other oxygen-derived, microbicidal species. Evidence from many kinetic studies of oxygen-derived radicals generated by stimulated neutrophils in vitro shows that radical production is optimal at 37 degrees C but only lasts several minutes and then rapidly subsides. These findings support the widely held perception that the neutrophil's "oxidative burst" is a transitory event that peaks within minutes of stimulation and ends shortly thereafter. However, while some studies have shown that under controlled conditions stimulated neutrophils can generate superoxide continuously for several hours, others have observed that the superoxide formation by neutrophils stimulated in buffer at 37 degrees C does not persist. To reconcile the conflicting findings and to better understand neutrophil function, we have reinvestigated the effect of temperature on the kinetics of radical generation by PMA-stimulated cells. Electron paramagnetic resonance spectroscopy coupled with spin-trapping and SOD-inhibitable ferricytochrome c reduction were used to monitor superoxide production by neutrophils stimulated at either 25 degrees C or 37 degrees C in RPMI 1640 medium or in Hank's balanced salt solution. When oxygen was supplied continuously, neutrophils stimulated at 25 degrees C in buffer or in medium generated superoxide for several hours but at 37 degrees C, particularly in HBSS, O2- formation strikingly and rapidly decreased. This cessation of superoxide generation was reversible by lowering the temperature back to 25 degrees C. These data imply that in vivo neutrophils may be capable of generating oxy-radicals for prolonged periods. In part, our results may also explain the often observed termination of neutrophil-derived radical formation in vitro and help to dispel the perception that neutrophil-derived oxy-radical production is an ephemeral phenomenon.

Cyclic N-Oxides↗
PubMed1991

Effect of a LTD4 receptor antagonist in porcine septic shock.

The effect of a selective LTD4 receptor antagonist SK & F104353 was studied in septic pigs anesthetized with isoflurane. Yorkshire pigs (25.2 +/- 2.3 kg) were instrumented and monitored for cardiac output (CO), mean arterial pressure (MAP), systemic vascular resistance (SVR), mean pulmonary arterial pressure (MPAP), pulmonary vascular resistance (PVR), renal artery blood flow (RABF), renal vascular resistance (RVR), arterial PO2, and extravascular lung water (EVLW). Blood samples were also collected for platelet, white blood cell and hematocrit determinations and plasma was assayed for thromboxane (TX) B2. Sepsis was induced by infusion of Pseudomonas aeruginosa (3 x 10(8) CFU%kg/h) for 2 h. Cardiovascular and hematologic data were determined at 30 min intervals for 4 h. Groups were infused with either SK & F104353 (3 mg/kg/h; n = 5) or drug vehicle (n = 6) beginning 15 min prior to infusion with the P. aeruginosa. In the vehicle group beginning at -90 min after sepsis induction, there was a 30 +/- 7% decrease of CO, a 27 +/- 5.0% decrease of MAP, and a 44 +/- 7% decrease of RABF, whereas, MPAP increased to 147 +/- 37% and plasma TXB2 increased from less than 200 pg/ml to 3,049 +/- 367 pg/ml (P less than 0.05). The EVLW and hematocrit increased (P less than 0.05), and the arterial PO2, white blood cell count, and platelet count decreased with the severity of the sepsis. In pigs pretreated with SK & F104353 the MAP and RABF were transiently improved (P less than 0.05), and the decrease in arterial PO2 was delayed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Effect of LTB4 receptor antagonists in endotoxic shock in the rat.

This study examined 1) the effects of infusion of LTB4 and 2) the potential role of LTB4 in the sequelae to endotoxic shock in the rat. Control rats were anesthetized with Ketamine/xylazine and given LTB4 (2 micrograms/kg) bolus i.v. followed by a 1 microgram/kg/min infusion for 10 min. LTB4 induced systemic hypotension and a three-fold increase in circulating band neutrophils which contributed to a 70% increase (P less than 0.05) in the total peripheral neutrophil count. LTB4 did not cause changes in circulating mature (segmented) neutrophils, lymphocytes, platelets, or hematocrits. Pretreatment (1 min) with LY233978, an LTB4 antagonist (10 mg/kg bolus i.v.), inhibited LTB4-induced systemic hypotension (-16.1 +/- 6.1 mmHg [n = 3] vs. -38.8 +/- 5.9 mmHg [n = 4], P less than 0.05). Salmonella enteritidis endotoxin (10 mg/kg bolus i.v.) induced systemic hypotension, hemoconcentration, leukopenia, and thrombocytopenia, which was greatest at 5 and 15 min postendotoxin. The leukopenia was characterized by lymphopenia, band neutropenia, and segmented neutropenia. LY233978 (10 mg/kg bolus i.v. immediately before endotoxin administration and followed by an infusion at 0.67 mg/kg/min for 90 min) attenuated endotoxin-induced hemoconcentration at 60 and 90 min postendotoxin (P less than 0.05), and systemic hypotension at 15 min postendotoxin (P less than 0.05). The LTB4-receptor antagonist LY255283 (10 mg/kg bolus i.v., 10 min before endotoxin followed by a 5 mg/kg bolus i.v. 30 min postendotoxin) completely inhibited endotoxin-induced systemic hypotension and partially attenuated endotoxin-induced hemoconcentration from 15 min to 90 min postendotoxin (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Effects of a linseed oil enriched diet on endotoxin-induced sequelae: differential in vitro and in vivo effects.

Endotoxin stimulates macrophages to synthesize membrane-associated inflammatory mediators including eicosanoids and procoagulant activity (PCA). Alpha linolenic acid, a component of linseed oil, is metabolized to eicosapentaenoic acid, which may replace arachidonic acid in membrane phospholipids. Thus, ingestion of linseed oil may alter the generation of eicosanoids, PCA and other membrane-dependent responses. This study compared the in vitro endotoxin-induced synthesis of eicosanoids and expression of PCA by peritoneal macrophages obtained from rats fed a control diet and rats fed a diet enriched with linseed oil. The effect of endotoxin on in vivo plasma eicosanoid concentrations, leukogram, and microvascular permeability were determined. Endotoxin (5 ug/ml) stimulated synthesis of iTxB2, i6-keto PGF1 alpha and expression of PCA by macrophages in vitro. These in vitro responses of macrophages from linseed oil fed rats were significantly less than those of macrophages from control rats. In contrast, there were no significant differences in in vivo responses to endotoxin. The fatty acid composition of total lipids and phospholipids in liver and plasma from linseed oil fed rats and control rats were not different. These composite data suggest several possibilities: (1) linseed oil may have effects independent of alpha-linolenic acid on macrophage function, (2) linseed oil may alter the fatty acid composition of macrophage phospholipids prior to changing that of other tissues, and (3) the reduced in vitro responses of peritoneal macrophages may not reflect the systemic responses to endotoxin.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Sensitization of low-dose-rate irradiation by nonlethal hyperthermia.

To assess whether hyperthermia could radiosensitize cells irradiated at a low dose rate, Chinese hamster V79 cells were simultaneously heated and irradiated at 0.86 Gy/h. The data showed that heat treatments at 39 and 40 degrees C, which did not induce heat killing alone or high-dose-rate radiosensitization, resulted in enhanced cell killing with low-dose-rate irradiation. The dose-modification factor (ratio of the slopes of the curves for low dose rate and high dose rate) was reduced to 1.8 at 39 degrees C and 1.4 at 40 degrees C, compared to a value of 2.1 at 37 degrees C. These data indicate that nonlethal heat treatments can cause enhanced radiosensitization under low-dose-rate conditions. The implications of these results for interstitial thermoradiotherapy are discussed.

Animals↗
PubMed1990

Determination of amifloxacin and two of its principal metabolites in plasma and urine by high-performance liquid chromatography using automated column switching.

The automated determination of amifloxacin and two of its principal metabolites in human plasma and urine by column-switching high-performance liquid chromatography is described. Plasma or urine samples, diluted 1:1 with 0.5 M sodium citrate buffer pH 2.5, were directly injected onto a cation-exchange pre-column. Following a 2.0-min wash of the pre-column with water at a flow-rate of 1.1 ml/min, the effluent from the pre-column was directed to the analytical column by a column-switching device. The precision of the plasma and urine methods ranged from a +/- 1.9 to +/- 3.6% for all compounds. The accuracies of the methods were within a range of -3.3% to 6.4% of the nominal values for all compounds. Linear responses were observed for all the standards in the range 0.10-5.0 micrograms/ml for plasma and 0.50-100 micrograms/ml for urine for all three compounds. The minimum quantifiable levels were 0.10 and 0.50 micrograms/ml for plasma and urine, respectively. The analytical methods may be used to quantify amifloxacin and the piperazinyl-N-desmethyl and piperazinyl-N-oxide metabolites in plasma and urine samples obtained from humans, monkeys, dogs and rats.

Animals↗
PubMed1990

Altered responses to modulators of guanine nucleotide binding protein activity in endotoxin tolerance.

The effects of cholera toxin or pertussis toxin and nonhydrolyzable GTP analogs on Salmonella enteritidis endotoxin stimulation of iTxB2 and i6-keto-PGF1 alpha synthesis in control and endotoxin tolerant rat peritoneal macrophages were determined. Pretreatment with pertussis toxin alone had no effect on basal macrophage iTxB2 or i6-keto-PGF1 alpha production, but pertussis toxin (0.1, 1.0 and 10 ng/ml) significantly inhibited endotoxin-stimulated iTxB2 and i6-keto-PGF1 alpha synthesis. Pretreatment with cholera toxin, which did not affect basal iTxB2 or i6-keto-PGF1 alpha synthesis, significantly enhanced endotoxin-induced synthesis of iTxB2 and i6-keto-PGF1 alpha. The effects of pertussis and cholera toxin with or without endotoxin were significantly (P less than 0.05) less in macrophages from endotoxin tolerant rats compared to control macrophages. GTP[gamma-S] (100 microM) significantly increased iTxB2 synthesis and significantly augmented endotoxin-stimulated iTxB2 synthesis in control macrophages (P less than 0.05). However, in macrophages from endotoxin tolerant rats the effect of GTP[gamma-S] on iTxB2 synthesis was significantly less (P less than 0.05) compared to control macrophages. Collectively, these data suggest that: (1) guanine nucleotide binding regulatory proteins mediate endotoxin-stimulated arachidonic acid metabolism in rat peritoneal macrophages; and (2) endotoxin tolerance induces alterations in guanine nucleotide binding protein activity.

Animals↗
PubMed1990

Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency.

Infection of cells by HIV can result in a period of quiescence or latency which may be obviated by treatment with inducing agents such as 5-azacytidine. Evidence from these experiments demonstrate the existence of two CpG sites in the HIV LTR which can silence transcription of both reporter genes (CAT) and infectious proviral DNA when enzymatically methylated. This transcriptional block was consistently overcome by the presence of the trans-activator tat without significant demethylation of the HIV LTR. These results suggest that DNA hypermethylation of the HIV LTR may change the binding characteristics between LTR sequences and cellular proteins, thereby suppressing HIV LTR transcription and modulating viral expression.

Animals↗
PubMed1990

Role of tympanic neurectomy in otalgia.

Tympanic neurectomy was first described 44 years ago. Although its indications have subsequently increased, it has not become a popular procedure. This paper concentrates on its use in treating otalgia. The history of tympanic neurectomy, the anatomy of the tympanic plexus and the aetiology of otalgia with specific reference to the tympanic plexus are discussed and a case of bilateral otalgia due to glossopharyngeal neuralgia successfully treated by staged, bilateral tympanic neurectomy reported. It is suggested tympanotomy performed under local anaesthesia with selective stimulation of the intra-tympanic nerves may lead to accurate diagnosis and treatment of the various forms of neuralgic otalgia.

Adolescent↗
PubMed1990

Acute ENT admission in Kawasaki disease.

Kawasaki disease (mucocutaneous lymph node syndrome) is an acute vasculitis of childhood carrying a 1-2 per cent mortality from cardiovascular complications. Despite the extensive literature on Kawasaki disease in paediatric journals, there has been a paucity of documentation in the otolaryngology literature. This is despite the fact that Kawasaki disease may present as an otolaryngological emergency before the diagnosis is established. We describe three cases of Kawasaki disease, all of which presented to the ENT department of this hospital within a period of two months. These cases illustrate the slow evolution characteristic of the disease and highlight the difficulties of diagnosis in the initial febrile stage. We emphasize the importance of considering the diagnosis when treating a young child with a pyrexia resistant to antibiotics, as prompt introduction of therapy may decrease the risk of fatal coronary artery or cardiac involvement.

Acute Disease↗