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Biomedical subjects

A W Murray

Publications and source records attributed to A W Murray.

At least 91 records · Page 5Linked to original sources
PubMed1994

Effect of exogenous fatty acids with greater than 22 carbon atoms (very long chain fatty acids) on superoxide production by human neutrophils.

The effects of exogenous long chain fatty acids (LCFA) and very long chain fatty acids (VLCFA) on superoxide production by human neutrophils were compared. Superoxide production was greater and more rapid in response to arachidonic (20:4 (n-6)), eicosapentanoic (20:5 (n-3)), and docosahexanoic (22:6 (n-3)) acids than for triacontatetranoic (30:4 (n-6)), dotriacontatetranoic (32:4 (n-6)), and tetratriacontahexanoic (34:6 (n-3)) acids, although all of these fatty acids gave responses larger than FMLP. A similar decline in activity with increasing carbon chain length was observed for the monoenoic VLCFA (22:1 (n-9) to 34:1 (n-9)). 32:4 (n-6) did not affect responses to a maximally stimulatory concentration of 20:4 (n-6). However, the simultaneous addition of 20:4 (n-6) and 30:4 (n-6) gave additive responses if suboptimal dosages of 20:4 (n-6) were used. This suggests that the LCFA and VLCFA may use the same signal transduction systems. In addition, 30:4 (n-6) was only 10% as effective as was 20:4 (n-6) at gaining access to the organic solvent extractable cellular fraction. This figure correlated with the relative biologic potency of 20:4 (n-6) and 30:4 (n-6), suggesting that the extent of association with the cell may regulate the biologic activity of the fatty acids. The saturates, arachidic (20:0) and cerotic (26:0) acids, were either inactive or poor activators in all assay systems examined. The failure of 20:0 to induce superoxide production and the lower responses to 30:4 (n-6) and 34:6 (n-3) were not because of extracellular Ca2+, because the biologic potency of these fatty acids was not greatly enhanced by removing Ca2+ from the extracellular medium. In contrast, 20:4 (n-6)- and 22:6 (n-3)-induced superoxide production was markedly increased under Ca(2+)-free conditions.

Calcium↗
PubMed1994

Mitosis. Kinetochores pass the IQ test.

Kinetochores oscillate to and fro on the mitotic spindle. The oscillations seem to be biased by the forces acting on the kinetochore, explaining the variety of chromosome movements seen at different stages of mitosis.

Animals↗
PubMed1994

Cyclin-dependent kinases: regulators of the cell cycle and more.

Cyclin-dependent kinases determine the timing of key events in the cell cycle, and may also regulate other important cellular functions. Although some of the effects of activating these kinases are clear, the mechanisms by which the effects are produced are not; several types of chemical probes that might be enlightening can be imagined.

Animals↗
PubMed1994

In vitro activation of rat brain protein kinase C by polyenoic very-long-chain fatty acids.

A variety of fatty acids including the cis-polyunsaturated very-long-chain fatty acids (VLCFA) (> 22 carbon atoms) common in retina, spermatozoa, and brain were examined for their ability to activate protein kinase C (PKC) purified from rat brain. Arachidonic [20:4(n-6)], eicosapentaenoic [20:5(n-3)], and docosahexaenoic [22:6(n-3)] acids as well as the VLCFA dotriacontatetraenoic [32:4(n-6)] and tetratriacontahexaenoic [34:6(n-3)] were equally capable of activating PKC in vitro with maximal activity being between 25 and 50 microM. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate further enhanced the in vitro activation of PKC when added to the protein kinase assay system with the fatty acids. The fully saturated arachidic acid (20:0) was inactive in both assay systems. The potential significance of the in vitro activation of PKC by the VLCFA is discussed.

Animals↗
PubMed1993

The mitotic feedback control gene MAD2 encodes the alpha-subunit of a prenyltransferase.

The mad2-1 mutation inactivates the cell-cycle feedback control that prevents budding yeast cells from leaving mitosis until spindle assembly is complete. The gene product of MAD2 shows significant sequence similarity to the alpha-subunit of prenyltransferases. Here we isolate a new temperature-sensitive mad2 mutant, mad2-2ts, and find that Mad2p is required for the membrane association of Ypt1p and Sec4p, two prenylated small GTP-binding proteins involved in protein trafficking. Extracts from mad2-2ts mutant cells fail to geranylgeranylate a number of substrates at the non-permissive temperature. mad2-2ts is synthetically lethal with bet2-1, a mutation in the gene that encodes for the beta-subunit of the Ypt1p and Sec4p geranylgeranyl transferase. Therefore MAD2 and BET2 gene products may physically interact to form a geranylgeranyl transferase complex. In addition, the difference between the phenotypes of mad2-1 and mad2-2ts suggests that MAD2 has distinct roles in protein transport and the mitotic feedback control.

Calcium-Binding Proteins↗
PubMed1993

Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor.

We have used frog egg extracts that assemble mitotic spindles to identify the event that triggers sister chromatid separation. Adding a nondegradable form of cyclin B prevents maturation-promoting factor (MPF) inactivation but does not block sister chromatid separation, showing that MPF inactivation is not needed to initiate anaphase. In contrast, adding an N-terminal fragment of cyclin, which acts as a specific competitor for cyclin degradation, produces a dose-dependent delay in MPF inactivation and sister chromatid separation. Methylated ubiquitin, which inhibits ubiquitin-mediated proteolysis, also delays sister chromatid separation, suggesting that ubiquitin-mediated proteolysis is necessary to initiate anaphase. The N-terminal cyclin fragment inhibits chromosome separation even in extracts that contain only nondegradable forms of cyclin, suggesting that proteins other than the known cyclins must be degraded to dissolve the linkage between sister chromatids.

Anaphase↗
PubMed1993

Epidermal growth factor inhibits gap junctional communication and stimulates serine-phosphorylation of connexin43 in WB cells by a protein kinase C-independent mechanism.

Epidermal growth factor (EGF) stimulated the phosphorylation of connexin43 (Cx43) in WB cells as evidenced by the formation of multiple immunoreactive Cx43 proteins of higher molecular mass which were abolished by treatment with alkaline phosphatase. Phosphorylation of Cx43 occurred within 10 min of EGF stimulation, was sustained for 1 h, and was associated with almost complete inhibition of gap junctional communication in these cells. EGF-induced phosphorylation and communication inhibition were retained in cells pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete protein kinase C. These results show that the EGF inhibition of communication is tightly linked to protein kinase C-independent phosphorylation of Cx43. Further, Cx43 phosphorylated in the presence of EGF did not react with phosphotyrosine antibodies and in 32Pi incorporation experiments was shown to contain only phosphoserine indicating that the tyrosine kinase activity of the EGF receptor was not directly involved.

Animals↗
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↗