Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

Effect of procaine hydrochloride on DNA repair in Escherichia coli

Liquid-holding recovery and rejoining of γ-radiation-induced DNA singlestrand scissions in Escherichia coli could be effectively inhibited by procaine hydrochloride at the concentration of 20 m M. At this concentration, the drug also reversibly altered cellular permeability barrier as evidenced from the uptake of acriflavin by bacterial cells.     

Stimulation by Bradykinin, Angiotensin II, and Carbachol of the Accumulation of Inositol Phosphates in PC-12 Pheochromocytoma Cells: Differential Effects of Lithium Ions on Inositol Mono-and Polyphosphates

Rat PC-12 pheochromocytoma cells respond to stimulation with bradykinin, angiotensin II, and carbachol with an increased formation of labeled inositol phosphates after preincubation of the cells with [3H]inositol. Li+ potentiates greatly the agonist-induced increase in amount of inositol mono-, bis-, and trisphosphate but not the increase in amount of inositol tetrakisphosphate. Separation of the isomers of inositol trisphosphate shows that the lithium-induced increase in amount of inositol trisphosphate is due to potentiation evoked by lithium of the accumulation of inositol-1,3,4-trisphosphate.     

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.     

Increased Diacylglycerol Levels Inhibit [20-3H]Phorbol 12, 13-Dibutyrate Binding and the Glucocorticoid-Mediated Increase in Glycerol Phosphate Dehydrogenase Levels in C6 Rat Glioma Cells

I examined whether the phorbol ester-mediated inhibition of glycerol 3-phosphate dehydrogenase (GPDH) induction could be mimicked by raising the cellular diacylglycerol levels. Phorbol ester tumor promoters and diacylglycerols activate protein kinase C. An increase in radiolabeled diacylglycerol levels in C6 rat glioma cells was observed when cells were prelabeled overnight with [3H]arachidonic acid and treated with either phospholipase C (Clostridium perfringens) or 2-bromooctanoate. The increase was dose dependent. The diacylglycerols competed with [20-3H]phorbol 12,13-dibutyrate in binding to the phorbol ester receptor. A Scatchard analysis of the binding of cells treated with 0.1 unit/ml of phospholipase C demonstrated that the inhibition was mainly due to a decrease in binding affinity and not in the total number of binding sites. 2-Bromooctanoate and phospholipase C, but not the synthetic diacylglycerol 1-oleoyl 2-acetyl glycerol, inhibited the glucocorticoid induction of GPDH levels. Boiled phospholipase C, phospholipase A2, or phospholipase D was ineffective in inhibiting induction, a result suggesting that the inhibition was not due to nonspecific membrane perturbation. Thus, inhibition of the glucocorticoid-mediated increase in GPDH induction is most likely mediated by protein kinase C, and not by an alternate phorbol ester receptor.     

Fate of Cyclic Nucleotides in PC12 Cell Cultures: Uptake, Metabolism, and Effects of Metabolites on Nerve Growth Factor-Induced Neurite Outgrowth

The fate of cyclic AMP (cAMP), dibutyryl-cAMP (Bt2-cAMP), and the (Sp)-isomer of adenosine 3',5'-monophosphorothioate [(Sp)-cAMPS] was studied in the PC12 culture medium by means of HPLC. In the absence of PC12 cells, cAMP and Bt2-cAMP were rapidly degraded by nonspecific esterases and cyclic nucleotide phosphodiesterase both originating from the serum commonly used as a culture medium ingredient, whereas (Sp)-cAMPS was completely stable. Since 5'-AMP, adenosine, inosine, and hypoxanthine appeared in the culture medium after incubation with cAMP or Bt2-cAMP, we have determined their effect on nerve growth factor (NGF)-induced neurite outgrowth. 5'-AMP, adenosine, and inosine were indeed potent agents in producing a potentiating effect on NGF-induced early neurite outgrowth at a concentration of 1 mM. Thus, cAMP metabolites had the capacity to induce an effect that has been described as cAMP-specific. In serum-free culture medium and in the presence of cells, all cyclic nucleotides were taken up by PC12 cells. Uptake was highly correlated with the hydrophobic nature of the compounds, and was accompanied by a simultaneous excretion of metabolites. On incubation with cAMP, NGF had a pronounced effect on the metabolic pattern found in the culture medium. In particular, dephosphorylation of 5'-AMP was specifically enhanced. This effect of NGF on the degradation of cAMP was also apparent when cAMP metabolites were incubated with PC12 cells. Whereas 5'-AMP degradation was greatly increased, NGF had no effect on the metabolism of the other purine compounds.     

Nerve Growth Factor and Gangliosides Stimulate the Release of Glycoproteins from PC 12 Pheochromocytoma Cells

PC12 pheochromocytoma cells in monolayer cultures secrete increased amounts of glycoproteins into the medium following the addition of nerve growth factor (NGF) or of brain gangliosides. After a 48-h incubation with 50 ng/ml NGF there is approximately a twofold increase in the total [14C]glucosamine-labeled, ethanol-precipitable cellular material released into the medium. Between 30 and 50% of the radioactivity is associated with a glycoprotein (Gpl) of molecular weight of 52,000; the remaining radioactivity is distributed between five and six major bands. Only a small amount (10%) is associated with a glycoprotein of Mr greater than 200,000 which might correspond to the NGF-induced large external glycoprotein. A substantial increase in the release of the glycoproteins is also seen on the addition of a variety of gangliosides including asialo GMl. This increase is independent of the presence of NGF. GMl and GDlb/GTlb but not GDla stimulate release above the levels seen in the presence of NGF. Addition of GDla (2 micrograms/ml) enhances selectively the release of various glycoproteins between 2.6- and 8-fold. The pattern of glycoprotein secretion is similar to that seen with NGF, although Gp2 (Mr 78,000) is more abundant. Stimulation of release by GDla is not accompanied by neurite outgrowth, suggesting that the glycoproteins are not directly associated with neuritogenesis. The release of these glycoproteins following the addition of NGF or gangliosides may relate to the neurotrophic properties that these two entirely different ligands exert on PC12 cells.     

Siderophore production and outer membrane proteins of selected Vibrio vulnificus strains under conditions of iron limitation

Abstract The tonB gene product is necessary for the energy-dependent transport of ferric chelates and vitamin B₁₂ across the Escherichia coli outer membrane. When carried on multicopy plasmids, the cloned tonB gene complemented tonB hosts, restoring transport of ferri-siderophone complexes and vitamin B₁₂, and susceptibility to the group B colicins and phage ф80. The levels of these activities were all markedly lower than when the tonB ⁺ gene was present in single copy. This depression of TonB function occurred even when the chromosome carried the normal tonB ⁺ allele, but plasmids carrying only a portion of the tonB gene, including the 5'-regulatory region, were not inhibitory.