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Biomedical subjects

A W Murray

Publications and source records attributed to A W Murray.

At least 163 records · Page 9Linked to original sources
PubMed1984

Platelet Ca2+-activated, phospholipid-dependent protein kinase: evidence for proteolytic activation of the enzyme in cells treated with phospholipase C1.

Incubation of human platelets with C. perfringens phospholipase C caused an increase in soluble protein kinase activity assayed in the presence of EGTA, and a decrease in Ca2+/phospholipid-dependent protein kinase activity. Fractionation of extracts on DEAE-cellulose columns showed that phospholipase C treatment resulted in a new peak of protein kinase active in the presence of EGTA. On Sephadex G-100 chromatography this enzyme eluted as a single peak of protein kinase activity of MW about 50,000. An extract from untreated platelets eluted as a single peak of Ca2+/phospholipid-dependent protein kinase of MW about 77,000. It was concluded that phospholipase C treatment resulted in the proteolysis of this latter enzyme to the lower MW form.

Blood Platelets↗
PubMed1983

Pedigree analysis of plasmid segregation in yeast.

We have used pedigree analysis to investigate the mitotic segregation of circular and linear DNA plasmids in Saccharomyces cerevisae. Circular ARS plasmids, which bear putative chromosomal replication origins, have a high segregation frequency and a strong bias to segregate to the mother cell at mitosis. The segregation bias explains how the fraction of plasmid-bearing cells can be small despite the high average copy number of circular ARS plasmids. Linear ARS plasmids do not show strong segregation bias, nor does the 2 mu ori-containing plasmid YEp 13, when it is present in strains containing intact 2 mu circles. In the absence of endogenous 2 mu circles, YEp 13 behaves like an ARS plasmid, showing a strong maternal segregation bias. The presence of a centromere on circular ARS plasmids eliminates segregation bias. We discuss a model for plasmid segregation, which explains these findings and the possible biological significance of mother-daughter segregation bias.

DNA↗
PubMed1983

Decreased [20-3H]phorbol-12,13-dibutyrate binding to phospholipase C pretreated epidermal cells.

Incubation of mouse epidermal cells (HEL-37) with phospholipase C (Clostridium perfringens) caused about a 50% decrease in the specific binding of [20-3H]phorbol-12,13-dibutyrate. Phospholipase C caused a decrease in the apparent number of binding sites from 2.86 X 10(5) to 1.21 X 10(5) per cell with little effect on ligand affinity. The decrease was observed with purified phospholipase containing no detectable protease activity, and when cells were fixed with glutaraldehyde. The phorbol-12,13-dibutyrate binding capacity of treated cells was recovered within 4 h incubation in complete medium. The results suggest that not all phorbol ester binding sites are equivalent, with differences arising either from varying phospholipid requirements or from membrane localisation.

Animals↗
PubMed1983

Effects of a tumor promoter on phospholipid metabolism in HeLa cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a marked stimulation of inorganic [32P]orthophosphate incorporation into HeLa-cell phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylethanolamine. The increased incorporation of inorganic [32P]orthophosphate into PE and lysophosphatidylethanolamine in the presence of TPA was not associated with an increase in PE synthesis as detected by the incorporation of [3H]serine or [3H]ethanolamine. The PC-specific exchange protein from beef liver was used to insert PC labeled with [3H]choline, inorganic [32P]orthophosphate, or [14C]arachidonic acid plus [3H]palmitic acid into the outer monolayer of intact HeLa cell membranes. Radioactivity from the latter two compounds was rapidly incorporated into PE and lysophosphatidylethanolamine; the incorporation was stimulated by TPA. It was concluded that TPA stimulated the formation of PE by base exchange between ethanolamine and PC.

HeLa Cells↗
PubMed1983

Rapid and reversible inhibition of junctional communication by tumor promoters in a mouse cell line.

HEL-37 cells rapidly transferred microinjected fluorescein between contacting cells. This transfer was strongly inhibited by tumor-promoting phorbol esters and mezerein but not by non-promoting derivatives. The onset of the inhibition occurred after 5 to 10 min of exposure to the test compound and was complete within 20 min. Using a wash-off procedure, it was demonstrated that inhibition of dye transfer by phorbol-12, 13-dibutyrate was fully reversed by 100 min, after a lag of 40 to 60 min. The recovery of dye transfer competence was blocked by puromycin. Both 12-O-tetradecanoylphorbol-13-acetate and monensin caused elevated Na+ levels in HEL-37 cells, but the latter compound did not inhibit dye transfer. It was concluded that the promoter inhibition of transfer was not a consequence of Na+-induced increase in cytoplasmic Ca2+ concentration.

Animals↗
PubMed1982

Two-stage skin carcinogenesis in sensitive and resistant mouse strains.

Strains of laboratory mice vary markedly in their susceptibility to two-stage skin carcinogenesis using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating carcinogen and croton oil as promoter. This study has been undertaken in order to clarify the basis of the strain differences. LACA mice were used as the susceptible strain and BALB/c mice as the resistant strain. DMBA was a more effective complete carcinogen in LACA mice than in BALB/c mice. However, dose-response studies with respect to DMBA in the two strains in two-stage carcinogenesis suggested that metabolic activation of DMBA to the active carcinogen was not limiting in the resistant strain. The observed strain differences in response to DMBA in one and two stage carcinogenesis may reflect the ability of DMBA to also act as a promoter in the two strains. The possibility that the strains vary in their ability to repair damaged DNA has, however, not been eliminated. Unlike polycyclic aromatic hydrocarbon carcinogens such as DMBA, phorbol ester promoters (the active components in croton oil) do not appear to require metabolic activation. However, they are degraded and inactivated by epidermal and other cells. Experiments in which the dose and frequency of application of croton oil were increased, and the unrelated promoter anthralin was substituted for croton oil, failed to produce any evidence that differences in promoter degradation contributed to the differences in susceptibility between strains.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1982

Skin carcinogenesis and promoter binding characteristics in different mouse strains.

The sensitivity of various mouse strains to skin carcinogenesis has been investigated in two ways; (i) by comparing carcinogenesis induced by treatments involving both the application of tumour promoters and the application of single or multiple doses of a carcinogen, and (ii) by comparing the binding characteristics of the tumour promoter [3H]phorbol-12,13-dibutyrate to particulate skin preparations. The mouse strains tested were Swiss albino, Balb/c, C3H, LACA and AKR/J. Although some differences were apparent, Swiss albino, LACA and C3H mice were generally sensitive to skin carcinogenesis by both benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene, either with or without promotion with croton oil. AKR/J mice were sensitive to both carcinogens with promotion but relatively insensitive in the absence of promotion. Balb/c mice had low sensitivity except when exposed to repeated low doses of benzo[a]pyrene. The [3H]phorbol-12,13-dibutyrate binding characteristics of particulate skin preparations from all strains were indistinguishable.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1982

Resistance of A-431 epidermoid carcinoma cells to early membrane changes induced by tumour promoters.

The A-431 human epidermoid carcinoma cell line was resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibition of epidermal growth factor binding, enhanced incorporation of [3H]choline into phospholipids and uptake of 86Rb an [3H]2-deoxyglucose. The cells were also resistant to TPA-stimulated release of radioactive choline derivatives and arachidonic acid from cells prelabelled with [3H]choline or [14C]arachidonic acid, respectively. The A-431 cells did not metabolise [3H]TPA. Despite their TPA-unresponsiveness, A-431 cells contained specific, high affinity binding sites for [3H]phorbol-12,13-dibutyrate with characteristics similar to other cultured cell lines.

Arachidonic Acid↗
PubMed1982

The effect of ischemia on cyclic adenosine 3',5'monophosphate accumulation in mouse skin.

The incubation of adult mouse skin pieces in a buffered salts medium at 37 degrees C led to a rapid accumulation of cyclic adenosine 3'5-monophosphate (cyclic AMP) in the tissue. In mouse skin maximum accumulation occurred after 2 min incubation; levels reverted to near control levels after a further 7 min incubation. the increase in cyclic AMP contents of the skin pieces was probably not due to the release of materials which activate adenylate cyclase after binding to cellular receptors. Thus, cyclic AMP accumulation was unaffected by the inclusion of alpha- or beta-adrenergic antagonists, or by the pretreatment of adult mouse skin with indomethacin (an inhibitor of prostaglandin synthetase). Furthermore, adenosine, a known activator of epidermal adenylate cyclase, could not be detected in the incubation medium. The functional integrity of epidermal adenylate cyclase was maintained during the cyclic AMP accumulation in response to ischemia. Thus, adenosine, histamine, isoproterenol and prostaglandin E2 (PGE2) augmented the cyclic AMP response. Cyclic AMP accumulation at 37 degrees C was not observed in newborn mouse skin; this lack of cyclic AMP accumulation was probably not due to increased activity of low affinity cyclic AMP phosphodiesterase in newborn mouse skin.

Adenosine↗
PubMed1982

Decreased accumulation of cyclic adenosine 3',5'-monophosphate in "ischemic" skin after 12-0-tetradecanoyl-phorbol-13-acetate treatment.

The effect of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on cyclic adenosine 3',5'-monophosphate (cyclic AMP) level in adult mouse skin in response to ischemia was examined. The incubation of skin pieces in a buffered salts medium at 37 degrees C resulted in a rapid accumulation of cyclic AMP. In mouse skin pieces maximum accumulation (about 6 times the basal level) occurred after 2 min incubation and was followed by a rapid decline in the cyclic AMP level. This "ischemic" rise in epidermal cyclic AMP was greatly reduced if skin was used 16 hr after a single application of 17 nmoles of TPA. The effect of TPA on cyclic AMP accumulation in response to ischemia was first observed at 1 hr after TPA treatment and was maximal at 4 hr. The lack of "ischemic" response in TPA-treated skin was not related to an increase in the activity of cyclic AMP phosphodiesterase after TPA application. In addition, the accumulation of cyclic AMP in skin in response to both ischemia and exposure to isoproterenol, adenosine, histamine, or prostaglandin E2 (PGE2) was not observed in skin treated with the tumor promoter TPA.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Tumor promoter stimulation of phosphatidylcholine turnover in HeLa cells.

Incubation of HeLa cells for 24 hr with [3H]choline resulted in extensive labeling of the phosphorylcholine and phosphatidylcholine pools. 12-O-Tetradecanoylphorbol-13-acetate (TPA), other phorbol ester tumor promoters, and mezerein stimulated the release of [3H]choline and [3H]phosphorylcholine from such prelabeled cells. The release was accompanied by decreased radioactivity in the phosphorylcholine pool, raising the possibility that the released materials were derived by leakage from this pool. However, TPA did not induce the release of radioactivity from cells containing a prelabeled nucleotide pool. Similarly, the TPA-stimulated release of radioactivity from prelabeled cells closely paralleled the label present in the phospholipid pool rather than the phosphocholine pool. Consequently, it is suggested that the primary source of the released material is phosphatidylcholine acting as a substrate for a phospholipase C enzyme. TPA also simulated the incorporation of [3H]choline into phospholipids, but a time-course study indicated that phospholipase C activation preceded this event. This was supported by the observation that incorporation of [3H]choline was also stimulated by exogenously added phospholipase C.

Choline↗