Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.     

A killer factor produced by the cellular slime mold Polysphondylium pallidum

A killer strain was discovered in cellular slime molds. The wild isolate CK-8 of Polysphondylium pallidum kills all other strains in Polysphondylium and Dictyostelium, as far as could be determined, except strain CK-8 itself and its complementary mating type strain CK-9. Growth-phase cells of CK-8 excrete a killer factor which is sensitive to heat, above 60°C for 5 min, and trypsin. The apparent molecular mass of the factor was determined at 10 000–12 000.Abbreviations BSS Bonner's salt solution - CM conditioned medium     

Studies on peptides CLVIII. Model experiments for the synthesis of open-chain unsymmetrical cystine-peptides

Treatment of a mixture of Cys(R)(O) and Cys(R) with an acid was found to generate cystine in fairly good yields, when suitable R, R, and an acid were selected. An unsymmetrical cystine peptide was prepared by treatment of a mixture of Z(OMe)-Cys(R) (0)-Ala-NH₂ (R=Acm or MBzl) and Z(OMe)-Cys(MBzl)-Gly-OBzl with TFA or 1 M TFMSA/TFA.³ Oxytocin was obtained in an excellent yield by TFA treatment of the protected peptide containing Cys(Acm)(0) and Cys(MBzl). Thus, formation of the disulfide bond was found feasible at the position of Cys(R) (0).The following abbreviations are used Boc t-butyloxycarbonyl - Z(OMe) p-methoxybenzyloxycarbonyl - MBzl p-methoxybenzyl - Acm acetamidomethyl - Bzl benzyl - Ad l-adamantyl - tBu t-butyl - TFA trifluoroacetic acid - TFMSA trifluoromethanesulfonic acid - TMSOTf trimethylsilyl trifluoromethane sulfonate     

Chronopharmacology of trichlormethiazide in rats; (II). Examination in aged rats

We have previously demonstrated a time-dependent variability in the diuretic effects of trichlormethiazide, a thiazide diuretic agent, in young rats. The study suggested that the time-dependent variations in urinary trichlormethiazide and susceptibility of renal tissues to the agent might be involved in this phenomenon. The present study was undertaken to test a hypothesis that such a daily variation in the effects of trichlormethiazide is blunted by age. Trichlormethiazide (0.5 and 2.0 mg/kg) was given orally at 1200 hrs (day trial) or at 2400 hrs (night trial) in young (10-11 week old) and aged (23-24 month old) Wistar rats. Urine was collected for 8 hours after the agent and urinary excretions of sodium, chloride and trichlormethiazide were determined. Urine volume and urinary excretions of sodium, chloride and trichlormethiazide following the agent were significantly greater at 1200 hrs than at 2400 hrs in the young rats. However these administration time-dependent changes in the effects of trichlormethiazide and its urinary amount diminished in the aged rats. In the day and night trials, there were significant correlations between urinary trichlormethiazide and its effects (urine volume, urinary sodium and chloride) in both groups of rats. The regression lines in each parameter of two trials differed in the young, but not in the aged group of rats. These data indicate that the mode of the time-dependent changes in the effects of trichlormethiazide is altered in aged Wistar rats. Dampening of the time-dependent variations in urinary trichlormethiazide and susceptibility to the agent might be involved in these chronopharmacological alterations in aged rats.     

Determination of 9α,11β-prostaglandin F2 by stereospecific antibody in various rat tissues

In view of the recent finding that prostaglandin D₂ is stereospecifically converted to 9α,11β-prostaglandin F₂, an isomer of prostaglandin F₂α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F₂ was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F₂ were raised in rabbits immunized with the bovine serum albumin conjugate, and [³H]9α,11β-prostaglandin F₂ was enzymatically prepared from [³H]prostaglandin D₂. The assay detected 9α,11β-prostaglandin F₂ over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F₂α, prostaglandin F₂β and 9β,11β-prostaglandin F₂. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F₂ was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D₂ (ca. 180 ng/g wet weight) and prostaglandin F₂α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F₂ was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F₂ is a minor pathway, if one at all, of prostaglandin D₂ metabolism in the rat brain.     

Identification of bile alcohols in rat bile

Bile alcohols in rat bile were analyzed by gas-liquid chromatography-mass spectrometry. Six bile alcohols were newly identified as minor constituents in addition to 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, major bile alcohol of rat bile. The bile alcohols newly identified were 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, and 5 beta-cholestane-3 alpha,6 beta,7 beta,25,26-pentol. The biliary bile alcohols of the rat occurred mainly as the sulfuric acid esters and, in lesser amounts, as glucuronoconjugated and unconjugated forms. The amount of total bile alcohols was about 27.9 nmol in 1 ml of bile.     

A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non-hybridized probes

We report a simplified and reliable method for non-radioactive in situ hybridization to whole Drosophila embryos. In the previous method (Tautz and Pfeifle, 1989) the post-hybridization wash, or the procedure for washing non-hybridized probe away from embryos depends simply on diffusion. We modified the method with application of electrophoresis to the wash. After hybridized with RNA probe, embryos were transferred to a small well where an electric charge was given to drive non-hybridized probe away from the embryos. This procedure enables us to acquire a much higher signal-to-noise ratio than that obtained from a conventional method. Furthermore, this is a time-saving method. We propose a term "electro-wash" for this procedure.     

A new snailfish of the genus Careproctus (Cottoidei: Liparidae) from the Pacific coast of southern Japan

Ichthyological Research - A new snailfish, Careproctus tomiyamai, is described on the basis of four specimens collected from Suruga Bay, Tosa Bay, and the Hyuga-nada Sea, southern Japan...     

Relationship between hormone content and autonomy in various autonomous tobacco cells cultured in suspension

Various autonomous cultured tobacco cells including crown gallwere examined for their contents of growth regulators by meansof Avena curvature test, cell-division induction test, and tobaccopith callus test. The crown gall cells derived from cv. Hicks produced auxin andcytokinin in the high levels of 300–500 µg IAA equivalentsand 40–80 µg kinetin equivalents per kg, respectively.The major auxin was identified as indole-3-acetic acid basedon mass spectrometry and gas chromatography. These cells alsoproduced methyl indole-3-acetate as a minor component. One ofthe cytokinins was identified as ribosyl-trans-zeatin by meansof both gas chromatography-mass spectrometry and high performanceliquid chromatography. Auxin and cytokinin activities were not detected in the followingthree suspension cultured tobacco cells: cells requiring neithercytokinin nor auxin derived from the callus of N. tabacum cv.Bright Yellow and cells requiring auxin but not cytokinin derivedfrom the calluses of cv. Bright Yellow and cv. Hicks. Theirauxin and cytokinin contents per kg were less than 1 µgIAA equivalent and less than 0.1 µg kinetin equivalent,respectively. The results obtained in this study indicate that enhanced hormonalcontent is not the only reason for autonomous growth. (Received August 16, 1979; )