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Biomedical subjects

A W Murray

Publications and source records attributed to A W Murray.

At least 109 records · Page 6Linked to original sources
PubMed1992

Examination of the role of the proteolytically-activated form of protein kinase C in the differentiation of human haemopoietic cells.

In neutrophils, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the translocation of the Ca(++)- and phospholipid-dependent protein kinase, protein kinase C (PK-C) from the soluble to the particulate fraction. At the same time there was a corresponding increase in the amount of Ca(++)- and phospholipid-independent protein kinase activity recovered in the soluble fraction. This soluble Ca(++)- and phospholipid-independent protein kinase presumably reflects proteolytic activation of the particulate associated PK-C. Bone marrow and undifferentiated HL-60 cells also translocated PK-C to the particulate fraction in response to TPA but did not accumulate the soluble Ca(++)- and phospholipid-independent form of the enzyme. Similar results were obtained using HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO), recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) or 1 alpha,25-dihydroxyvitamin D3. There was also no significant change in either the number or time of expression of differentiation-specific cell surface antigens observed on HL-60 cells induced to differentiate with either DMSO, 1 alpha,25-dihydroxyvitamin D3 or TPA in the presence of cyclosporin A, an agent reported to inhibit the proteolytic breakdown of PK-C to the Ca(++)- and phospholipid-independent form. Likewise, cyclosporin A did not affect the rate of extent of differentiation of primary bone marrow cell cultures. These results suggest that the proteolytically activated and phospholipid-independent form of PK-C is probably not involved in haemopoietic cell differentiation.

Biological Transport↗
PubMed1991

Phorbol ester-stimulated hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase D in HeLa cells. Evidence that the basal turnover of phosphoglycerides does not involve phospholipase D.

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [3H]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.

Choline↗
PubMed1991

Phosphorylation and chromatin mechanics: the central importance of substrate conformation in determining the patterns of HL-60 nuclear phosphorylation.

HL-60 nuclear autophosphorylation was studied in vitro under circumstances in which the conformation of chromatin was manipulated with both polyamines and DNAse-I. A general re-arrangement of the phosphorylation patterns occurred as polyamines were removed and nuclei were observed to expand. DNAse-I treatment reduced these phosphorylation patterns to a much simpler configuration indicating that the responding substrates were DNA-associated. It was concluded that substrate conformation was the main determining factor in the control of nuclear protein phosphorylation. These results suggest a method of general utility for the identification of truly nuclear proteins by the characteristics of their phosphate acceptor activity.

Cell Line↗
PubMed1991

Phorbol ester induces phosphorylation and down-regulation of connexin 43 in WB cells.

Western blot analysis indicated that WB cells contained multiple proteins of molecular mass 42-47 K which reacted with anti-connexin 43 antibody. Incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 5 min caused the inhibition of dye transfer and the appearance of reactive material of 50 K which was abolished by acid phosphatase. Longer term incubation with TPA caused loss of connexin 43 protein.

Animals↗
PubMed1991

Feedback control of mitosis in budding yeast.

We have investigated the feedback control that prevents cells with incompletely assembled spindles from leaving mitosis. We isolated budding yeast mutants sensitive to the anti-microtubule drug benomyl. Mitotic arrest-deficient (mad) mutants are the subclass of benomyl-sensitive mutants in which the completion of mitosis is not delayed in the presence of benomyl and that die as a consequence of their premature exit from mitosis. A number of properties of the mad mutants indicate that they are defective in the feedback control over the exit from mitosis: their killing by benomyl requires passage through mitosis; their benomyl sensitivity can be suppressed by an independent method for delaying the exit from mitosis; they have normal microtubules; and they have increased frequencies of chromosome loss. We cloned MAD2, which encodes a putative calcium-binding protein whose disruption is lethal. We discuss the role of feedback controls in coordinating events in the cell cycle.

Amino Acid Sequence↗
PubMed1991

The neutrophil respiratory burst. Responses to fatty acids, N-formylmethionylleucylphenylalanine and phorbol ester suggest divergent signalling mechanisms.

The oxygen-dependent respiratory burst is a key neutrophil function required for the killing of bacteria. However, despite intensive investigation, the molecular events which initiate the respiratory burst remain unclear. Recent reports have suggested the agonist-induced hydrolysis of cellular phosphatidylcholine (PtdCho) by phospholipase D may be an essential requirement for initiating or mediating the respiratory burst. We have investigated the effects of the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLF), the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and the polyunsaturated fatty acids arachidonic [20:4 (n-6)] and docosahexaenoic [22:6 (n-3)] acids in light of this hypothesis. Ethanol-inhibited superoxide production in response to 20:4, 22:6 and fMLF, in a dose-dependent fashion, suggesting an involvement of phospholipase D. The phosphatidic-acid phosphohydrolase inhibitor DL-propranolol completely inhibited superoxide production induced by both 20:4 and 22:6, and partially inhibited the response to TPA. In contrast, superoxide production in response to fMLF was increased by propranolol. fMLF and TPA, but not the fatty acids, stimulated phospholipase D as indicated by the accumulation of phosphatidic acid and, in the presence of ethanol, phosphatidylethanol derived from PtdCho. Extracellular Ca2+ was found to be an essential requirement for fMLF-induced superoxide production. However, responses to the fatty acids were dramatically enhanced under Ca(2+)-free conditions. Responses to TPA were independent of the extracellular Ca2+ concentration. Both fatty acids and fMLF, but not TPA, mobilised Ca2+ from intracellular stores, a response insensitive to the effects of both ethanol and propranolol. These results show that, unlike fMLF and TPA, the fatty acids do not cause hydrolysis of PtdCho by phospholipase D. However, the data indirectly suggests that the fatty acids may initiate the phospholipase-D-catalysed hydrolysis of phospholipids other than PtdCho.

Calcium↗
PubMed1991

Cyclin is degraded by the ubiquitin pathway.

Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle. When a region in the N terminus of cyclin is fused to a foreign protein, it produces a hybrid protein susceptible to proteolysis at mitosis. During the course of degradation, both cyclin and the hybrid form conjugates with ubiquitin. The kinetic properties of the conjugates indicate that cyclin is degraded by ubiquitin-dependent proteolysis. Thus anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.

Amino Acid Sequence↗
PubMed1991

Examination of cardiorespiratory changes during upper gastrointestinal endoscopy. Comparison of monitoring of arterial oxygen saturation, arterial pressure and the electrocardiogram.

Critical events including hypoxaemia, arrhythmias and myocardial ischaemia may occur more frequently during endoscopic procedures than during anaesthesia. A study was undertaken to assess the cardiovascular changes and to evaluate suitable monitoring techniques to detect critical events during sedation and endoscopy. Twenty patients scheduled to undergo a prolonged endoscopic procedure which required deep sedation were studied. Continuous recordings of electrocardiogram, heart rate and arterial oxygen saturation were made and arterial pressure was recorded at one-minute intervals. The study commenced immediately before administration of sedatives, continued for the duration of the examination and for one hour following the examination. Oxygen saturation decreased in all patients during the examination to a mean of 82.9% (SD 11.9), and remained below baseline for the duration of the examination and into the recovery period. Statistically significant increases and reductions of systolic arterial pressure and rate-pressure product were found during the procedures compared with baseline values recorded before administration of sedatives. Sixteen of the 20 patients developed tachycardia during the examination. Ten patients developed ectopic foci which were supraventricular, ventricular or both in origin. Electrocardiogram changes resolved during the recovery period. Myocardial ischaemia was assessed by S-T segment depression and a significant correlation was found between S-T segment depression and hypoxaemia, although the magnitude of the S-T depression was small and may not have been detected clinically. No correlation was found between S-T segment depression and arterial pressure, heart rate or rate-pressure product.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Effect of 22-32 carbon n-3 polyunsaturated fatty acids on superoxide production in human neutrophils: synergism of docosahexaenoic acid with f-met-leu-phe and phorbol ester.

Docosahexaenoic (22:6 n-3) and eicosapentaenoic acid (20:5 n-3) stimulated the oxygen-dependent respiratory burst in intact neutrophils in a dose-dependent manner as measured by either superoxide dismutase (SOD)-inhibitable cytochrome c reduction and lucigenin-dependent chemiluminescence. A number of longer chain hexaenoic acids isolated from ram testis (22 to 32 carbon fatty acids) showed a diminishing response with increasing carbon chain length. 22:6 acted synergistically to enhance the responses to two other neutrophil agonists, f-met-leu-phe (FMLP) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Whereas 22:6-induced chemiluminescence was markedly inhibited by pre-treatment with cytochalasin B, the response to FMLP was augmented, while TPA-induced activation was largely unaffected by cytochalasin B. 22:6-induced activation of neutrophils is independent of protein kinase C, as 22:6, unlike TPA, did not cause the membrane translocation of this enzyme. Furthermore, the putative protein kinase C inhibitor H-7 [1-(5-isoquinolinylsulphonyl)-2-methylpiperazine] had little effect on 22:6-induced chemiluminescence. In contrast, 22:6-induced activation was extremely sensitive to the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], indicating that calmodulin-dependent enzymes may be involved in the responses to 22:6. These results suggest that different mechanisms are involved in the 22:6-, FMLP- and TPA-induced activation of neutrophil NADPH-oxidase.

Calmodulin↗
PubMed1990

Phorbol esters modulate the turnover of both ether- and ester-linked phospholipids in cultured mammalian cells.

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the metabolism of ester- and ether derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in HeLa and HEL-37 cells. TPA stimulated the incorporation of [3H]choline into diacyl-, alkylacyl- and alkenylacy/PC in HeLa cells, but inhibited the incorporation of [3H]ethanolamine into the corresponding derivatives of PE. TPA also stimulated the incorporation of [3H]ethanolamine into lysoPE and the release of labelled ethanolamine and phosphoethanolamine from HeLa cells prelabelled with [3H]ethanolamine. All responses to TPA were abolished in HeLa cells preincubated with the phorbol ester and which were deficient in protein kinase C. In HEL-37 cells TPA stimulated label incorporation into both ester- and ether-forms of PE. The marked effects of TPA on ether-lipid metabolism raises the possibility that hydrolysis products of this class of lipid are important in transmembrane signalling pathways.

Animals↗
PubMed1990

Arterial oxygen saturation during upper gastrointestinal endoscopy: the effects of a midazolam/pethidine combination.

This study was performed to assess the severity and duration of hypoxia during and after prolonged upper gastrointestinal endoscopy. Twenty patients were studied using a non-invasive pulse oximeter. Significant reductions in oxygen saturation were noted during endoscopy and remained below the preexamination level during the one hour period after the examination. This study indicates that pulse oximetry is a useful monitor for detection of hypoxia during and after endoscopy.

Adult↗