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Biomedical subjects

R Keogh

Publications and source records attributed to R Keogh.

6 recordsLinked to original sources
PubMed1994

The role of protein kinase C in arachidonic acid release and prostaglandin E production from CHO cells transfected with EGF receptors.

Arachidonic acid release and prostaglandin production are stimulated by both phorbol esters and growth factors in various cell types. Whereas phorbol esters activate and transmit a signal via protein kinase C, this pathway is not necessarily involved in growth factor signal transduction. We investigated the involvement of protein kinase C in the pathway of arachidonic acid metabolism by CHO cells transfected with full-length EGF receptor (CHOwt). Two isoforms of protein kinase C were identified in CHOwt cells, alpha and zeta. On downregulation, the parallel loss of phorbol ester-stimulated arachidonic acid release and the alpha-isoform suggests a possible involvement of this isoform in phospholipase A2 activation in these cells. In addition, we propose that the zeta-isoform may be separately involved in prostaglandin production as residual phorbol ester-stimulation of PGE production occurs in downregulated cells where PKC zeta is the sole remaining isoform. EGF stimulation of arachidonic acid release, as a measure of phospholipase A2 activation, and subsequent prostaglandin production are unaffected by inhibition of protein kinase C in CHOwt cells. Indeed one such inhibitor, staurosporine, augmented the EGF effect. These results suggest that PKC is not required for EGF activation of phospholipase A2 in these cells.

Alkaloids↗
PubMed1993

Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product.

The alginate lyase-encoding gene (algL) of Pseudomonas aeruginosa was localized to a 1.7-kb EcoRI-XbaI fragment within the alginate biosynthetic gene cluster at 34 minutes on the chromosome. The nucleotide sequence of this DNA fragment revealed an ORF encoding a protein of M(r) 40,885 which is transcribed in the same orientation as the other alg genes within the biosynthetic gene cluster. The predicted protein has a potential N-terminal signal peptide which is consistent with its proposed periplasmic location. The AlgL protein was overproduced in Escherichia coli and purified. The purified protein was shown to have alginate lyase activity. In addition, an algL insertion mutant of the mucoid P. aeruginosa 8830 was constructed. This mutant (alm1) had a nonmucoid phenotype due to a polar effect on the transcription of an essential alg gene, algA. Thus, the algL gene is located within a region of the alginate biosynthetic gene cluster that appears to be non-essential for alginate production.

Alginates↗
PubMed1993

Fibronectin-induced increase in mesangial cell prostaglandin release. Effect of hyperglycemia and PKC inhibition.

Glomerular accumulation of extracellular matrix in diabetes is a potential regulator of mesangial cell-matrix interactions through transmembrane matrix receptors. We now provide evidence that PG production from rat glomerular mesangial cells is increased by Fn. An increase in PG (measured as PGE) was demonstrated in mesangial cell-enriched glomerular cores after 1-h exposure (149 +/- 8% of timed control) and was sustained over a 24-h period (214 +/- 7%). Increased PG production followed exposure to a chymotryptic fragment (120,000 M(r)) of Fn and occurred concomitant with an increase in particulate PKC activity. A tetrapeptide (Arg-Gly-Asp-Ser) with the Arg-Gly-Asp sequence, contained in Fn and the chymotryptic fragment and recognized by specific membrane receptors (integrin matrix-binding proteins), also raised PG levels. As has been shown previously, exposure to high glucose concentration can increase mesangial cell PGE production (from 677 +/- 61 pg.mg protein-1.2 h-1 at 5.6 mM glucose to 1561 +/- 132 pg.mg protein-1.2 h-1 at 50 mM glucose, P < 0.001). The response to the chymotryptic fragment of Fn also was enhanced by concurrent exposure to high glucose concentration (from 2560 +/- 199 pg.mg protein-1.2 h-1 at 5.6 mM glucose to 4672 +/- 358 pg.mg protein-1.2 h at 50 mM glucose, P < 0.001). Coincubation with H-7, an inhibitor of PKC, abolished the PG response to glucose and the chymotryptic fragment. Involvement of PKC was supported further by abrogation of the effect of chymotryptic fragment in mesangial cells cultured for a prior prolonged period with phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Regulation of cyclic AMP levels by calcium in bovine adrenal medullary cells.

Both nicotine and histamine have been reported to increase cyclic AMP levels in chromaffin cells by Ca(2+)-dependent mechanisms. The present study investigated whether Ca2+ was an adequate and sufficient signal for increasing cyclic AMP in cultured bovine adrenal medullary cells. Depolarization with 50 mM K+ caused a two- to three-fold increase in cellular cyclic AMP levels over 5 min, with no change in extracellular cyclic AMP. This response was abolished by omission of extracellular Ca2+ and by 100 microM methoxyverapamil, and was unaffected by 1 microM tetrodotoxin and by 1 mM isobutylmethylxanthine. Veratridine (40 microM) also increased cellular cyclic AMP levels by two- to fourfold. This response was abolished by either methoxyverapamil or tetrodotoxin. The Ca2+ ionophore A23187 (10-50 microM) had little or no effect on cellular cyclic AMP levels. When the concentration of K+ used to depolarize the cells was reduced to 12-15 mM, the catecholamine release was similar to that induced by 50 microM A23187, and the cyclic AMP response was almost abolished. The results suggest that Ca2+ entry into chromaffin cells is a sufficient stimulus for increasing cellular cyclic AMP production. The possible involvement of a Ca2+/calmodulin-dependent isozyme of adenylate cyclase is discussed.

1-Methyl-3-isobutylxanthine↗