PubMed Health⌕ Search

Biomedical subjects

A W Murray

Publications and source records attributed to A W Murray.

At least 19 recordsLinked to original sources
PubMed1992

Basal and phorbol-ester-stimulated turnover of phosphatidylcholine in HeLa cells involve different pathways.

HeLa cells prelabelled with [3H]lysoplatelet-activating factor (lyso-PAF) accumulated phosphatidic acid (PtdOH) when incubated in the presence of either propranolol (an inhibitor of PtdOH phosphohydrolase) or phorbol ester. In the presence of ethanol, phorbol ester but not propranolol stimulated the accumulation of phosphatidylethanol, an index of phospholipase D activity. Incubation of cells with [3H]lyso-PAF led to a rapid accumulation of label in diacylglycerol (DG) followed by a delayed accumulation in PtdOH. It is concluded that propranolol-induced PtdOH accumulation is derived from DG by DG kinase and does not involve phospholipase D.

Cell Membrane↗
PubMed1992

Creative blocks: cell-cycle checkpoints and feedback controls.

Before division, cells must ensure that they finish DNA replication, DNA repair and chromosome segregation. They do so by using feedback controls which can detect the failure to complete replication, repair or spindle assembly to arrest the progress of the cell cycle at one of three checkpoints. Failures in feedback controls can contribute to the generation of cancer.

Animals↗
PubMed1992

S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28.

In somatic cells, entry into mitosis depends on the completion of DNA synthesis. This dependency is established by S-phase feedback controls that arrest cell division when damaged or unreplicated DNA is present. In the fission yeast Schizosaccharomyces pombe, mutations that interfere with the phosphorylation of tyrosine 15 (Y15) of p34cdc2, the protein kinase subunit of maturation promoting factor, accelerate the entry into mitosis and abolish the ability of unreplicated DNA to arrest cells in G2. Because the tyrosine phosphorylation of p34cdc2 is conserved in S. pombe, Xenopus, chicken and human cells, the regulation of p34cdc2-Y15 phosphorylation could be a universal mechanism mediating the S-phase feedback control and regulating the initiation of mitosis. We have investigated these phenomena in the budding yeast Saccharomyces cerevisiae. We report here that the CDC28 gene product (the S. cerevisiae homologue of cdc2) is phosphorylated on the equivalent tyrosine (Y19) during S phase but that mutations that prevent tyrosine phosphorylation do not lead to premature mitosis and do not abolish feedback controls. We have therefore demonstrated a mechanism that does not involve tyrosine phosphorylation of p34 by which cells arrest their division in response to the presence of unreplicated or damaged DNA. We speculate that this mechanism may not involve the inactivation of p34 catalytic activity.

Amino Acid Sequence↗
PubMed1992

Importance of substrate conformation in the phosphorylation of chromatin-associated proteins by exogenous protein kinase C.

Protein kinase C (PKC)-mediated phosphorylation of chromatin-associated proteins was studied in vitro. HL-60 and HeLa nuclear proteins were notably unresponsive to exogenously added brain PKC. In contrast, 3T3 fibroblasts and lymphocytes from primary cultures exhibited PKC-dependent phosphorylation of chromatin-associated proteins when chromatin was induced to expand. Unexpanded nuclei in all cell lines were unresponsive. Responsiveness was particularly obvious in the decondensed chromatin of primary lymphocytes, where a large number of proteins were phosphorylated in response to exogenous PKC. DNAase-I and micrococcal nuclease strongly modulated these phosphorylation patterns indicating that the substrates were DNA-associated. It was concluded that although substrate conformation, i.e. condensation state, was the primary determining factor in control of PKC-dependent nuclear protein phosphorylation, different cell lines greatly differ in their overall responsiveness to exogenous PKC.

Animals↗
PubMed1992

Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase.

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.

Anaphase↗
PubMed1992

Tumour promoting phorbol esters activate phospholipase D in mouse skin.

Phospholipase D catalyses a transphosphatidylation reaction in the presence of primary alcohols, resulting in the formation of phosphatidylalcohol derivatives. In the present work we show that application of phorbol esters and butanol to mouse skin causes the rapid accumulation of phosphatidylbutanol (PBol), indicating the activation of phospholipase D. A similar accumulation of PBol was observed when the skin was treated with phorbol esters in vivo and skin pieces incubated with butanol in vitro. PBol formation was stimulated by the active tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein and phorbol-12,13-didecanoate (PDD) but not by the inactive promoter 4 alpha-PDD. Accumulation of PBol was not observed 24 h after application of TPA, a treatment which has been shown to deplete epidermal protein kinase C activity.

Animals↗
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↗