The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327–334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca²⁺ influx. In the presence of 1 mM Ca²⁺, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca²⁺ (EGTA 1 mM). Furthermore, thrombin-induced Mn²⁺ influx but not intracellular Ca²⁺ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca²⁺ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca²⁺ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca²⁺ channel of human platelets.     

Comparison of S-alleles and S-glycoproteins between two wild populations of Brassica campestris in Turkey and Japan

Summary The number of identical S-alleles between two wild populations of B. campestris, one in Turkey, the other in Japan, that have been independent of one another for a long time was investigated. Diallel pollination tests between 38 S-allele homozygotes, i.e., 16 S-allele homozygotes from Turkey and 22 from Japan, revealed that these were 29 different S-alleles only 4 common ones. These S-alleles were differentiated by the iso-electric focusing (IEF) analysis of S-locus glycoproteins (SLGs) stained with an antiserum against SLG⁸. All identical S-alleles had the major SLG band at the same pI value without exception, even though they were collected from different populations. However, the number of minor bands of SLGs varied between the two populations; the S-alleles in Balcesme had generally fewer minor bands than those in Oguni. The 29 independent S-alleles were numbered from S ²¹ to S ⁴⁹ according to the pI value of the major SLG band. The major bands whose pI values were 7.5–8.5 were most common. Blot-hybridization patterns of genomic DNA hybridized with SLG ⁸ cDNA were not always the same among the strains of identical S-alleles obtained from different populations. Because about 20% of the S-alleles were shared between the two populations, it can be inferred that more than hundreds of S-alleles have been accumulated by mutation in B. campestris throughout the world.     

Heme-biosynthetic enzyme activities and porphyrin accumulation in normal liver and hepatoma cell lines of rat

The activities of four heme-biosynthetic enzymes, -aminolevulinic acid (ALA) synthase, ALA dehydratase, porphobilogen (PBG) dearninase, and ferrochelatase, were studied in five epithelial cell lines of normal rat liver origin (Re, REC-10, RLC-24, M, Culb-TC) and five cell lines derived from Yoshida ascites hepatoma (JTC-1, JTC-2, JTC-15, JTC-16, JTC-24). The JTC series of hepatoma-derived cell lines exhibited decreased ALA synthase activity and increased ALA dehydratase activity, although the activities of all four enzymes and the Km values for their respective substrates varied widely from one cell line to another, a finding suggesting that specific regulatory mechanisms for porphyrin metabolism might operate in each cell type. M cells, which were transformed by 4-dimethylaminoazobenzene in vitro, gave the most abnormal Km values of heme-biosynthetic enzymes among all the cell lines studies, and were found to accumu2ate hematoporphyrin derivative (HpD).Abbreviations ALA o-aminolevulinic acid - DAB 4-dimethyl aminoazobenzene - HpD hematoporphyrin derivative - 4NQO 4-nitroquinoline 1-oxide - PBG porphobilinogen     

In vivo Treatments That Modulate PPi-Dependent H+ Transport Activity of Tonoplast-Enriched Membrane Vesicles from Barley Roots

The PP_i-dependent H⁺ transport activity of tonoplast-enrichedmembrane vesicles prepared from barley roots was greatly reducedwhen the plants were grown for 4 or 5 days with an additional3 raM KC1 in growth medium that contained only 0.1 mM CaCl₂in water. To characterize the mechanism of this reduction inactivity, we attempted to treat barley roots with K⁺ ions, Cl^-ions(or acetate), and A23187 [GenBank] (with or without Ca²⁺ ions), whichmight be expected to cause alkalization, acidification and mobilizationof Ca²⁺ ions in the cytoplasm, respectively. One-day treatmentof barley roots with K⁺ ions significantly decreased PP_i--dependentH⁺ transport activity of prepared tonoplast-enriched membranevesicles, while treatment with Cl^- ions or acetate significantlyincreased the activity. A similar increase in the activity alsooccurred by treatment with Ca²⁺ ions alone or in combinationwith A23187 [GenBank] . Determination of the PP_i-hydrolyzing activity ofmembrane vesicles showed that changes in this activity by thevarious treatments were similar to those in the PP_i-dependentH⁺ transport activity. The changes in ATP-dependent H⁺ transportactivity of membrane vesicles caused by these treatments weresmall. These results indicate that the in vivo treatments hadsignificant effects on the H⁺ transport activity of H⁺-PP_i-ase,one of the two active vacuolar H⁺-pumps (H⁺-PP_iase and H⁺-ATPase).In addition, these results suggest the possibility that changesin levels of cytoplasmic H⁺ or Ca²⁺ ions may be involved inmodulation of the H⁺ transport activity of the vacuolar H⁺-PP_iaseduring plant growth. (Received September 14, 1992; Accepted March 1, 1993)     

Crystallization and preliminary crystallographic analysis of a 2,3-dihydroxybiphenyl dioxygenase from Pseudomonas sp. strain KKS102 having polychlorinated biphenyl (PCB)-degrading activity

Crystals have been obtained for a 2,3-dihydroxybiphenyl dioxygenase (conventionally called BphC) from a polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain KKS1O2. The crystals were grown using both ammonium sulfate and MPD as the precipitating agents. The crystals belonged to a tetragonal space group (I422) and diffracted to 2.5 Å. © 1995 Wiley-Liss, Inc.     

Bevantolol hydrochloride (nc-1400): Absolute configuration and direct separation of the enantiomers

Synthesis of (?)-bevantolol hydrochloride from 3,4-dimethoxyphenethylamine and (S)-(+)-m-tolyl glycidyl ether derived from (R)-(?)-epichlorohydrin established the absolute configuration of the (+) and (?) enantiomer as R and S, respectively. The purity of the enantiomers was determines using a chiral cellulose column (CHIRALCEL OD®) which allowed direct separation of the enantiomers. A separation factor (α) of 4.20 and a resolution factor (Rs) of 9.21 were obtained. © 1995 Wiley-Liss, Inc.     

Laboratory-induced development of the ice-ice disease of the farmed red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The most common perception of unfavorable environmental factors causing the ice-ice disease in the farmed seaweeds, Kappaphycus and Eucheuma, was demonstrated in this study for the first time using stressful conditions of abiotic factors in a continuous culture system. Light intensity of less than 50 mol photon m^–2 s^–1 and salinity of 20% or less induced ice-ice whitening characterized by short segments at midbranches which were similar to those observed in the Philippine seaweed farms, while temperatures of up to 33–35 °C resulted in wide-scale whitening leading to complete damage of the branches. These effects were preceded by slow growth rates from an optimum of 3.7% d^–1 to almost –2.0% d^–1. Mechanical stress by wound injury did not result to ice-ice whitening similar to the above. Environmental factors observed to trigger ice-ice in the laboratory, although may not necessarily parallel those in the field, may act synergestically to produce similar effects.     

Stereochemistry of protected ornithine side chains in gramicidin S derivatives and their resistance to N-methylation

Derivatives of gramicidin S (GS) and its mono- and di-d-cyclohexylalanine (d-Cha) analogs possessing various protecting groups on Orn side chains were prepared. ¹H NMR spectra of the unsymmetrically protected analogs [Orn(X)²,Orn(X)²,d-Cha⁴]GS were similar to the composites of the spectra of the symmetrical derivatives [Orn(X)²,2,d-Cha⁴,4]GS and [Orn(X)²,2]GS, revealing the proximity of the protecting groups of NH of Orn residues at the 2 and 2 positions to the side chains of d-Phe (or d-Cha) residues at the 4 and 4 positions, respectively. The results indicated the presence of H-bonds between the NH of Orn and the carbonyl of d-Phe residues in the i i + 2 sense and not in i i – 3, which was also supported by the ROESY analysis. The substantially strong H-bonds can explain the observed resistance of the urethane NH of the Orn side chains in the GS derivatives to the N-methylation with CH³I–Ag²O in DMF.     

Physical Linkage of the B29/Ig-β (CD79B) Gene to the Skeletal Muscle, Sodium-Channel, and Growth Hormone Genes in Rat and Human

In the region between the polyadenylation site of the rat skeletal muscle (SkM) Na-channel gene and the 5′ end of the growth hormone (GH) gene, a gene coding for B-cell-specific membrane protein B29/Ig-β was found and noted to have the same orientation as the Na-channel and GH genes. Rat B29/Ig-β gene was 3.1 kb in length with six exons and was separated by 3.3 and 9.3 kb from Na-channel and GH genes, respectively. Rat B29/Ig-β protein comprised 228 amino acids, and its amino acid sequence was 85 and 69% identical with the mouse and human counterparts, respectively. With the long-area PCR method, genomic DNA connecting human SkM Na-channel (SCN4A) and B29/Ig-β (CD79B) genes and CD79B and GH (GH1) genes was amplified, and the physical linkage of SCN4A/CD79B/GH1 genes in the human genome was established. The human CD79B gene was separated by 6.3 and 10.5 kb from the SCN4A and GH1 genes, respectively.     

Radioadaptive response: An implication for the biological consequences of low dose-rate exposure to radiations

Radioadaptive response provides a considerable impact on the risk assessment of low-dose and low dose-rate exposures to ionizing radiations. The cells previously exposed to low doses of radiations become resistant to the induction of mutations, chromosome aberrations and cell killing by the subsequent doses but more susceptible to malignant transformation. The reaction kinetics of radioadaptive response were incorporated into a modelling of biological consequences of protracted low dose-rate exposures to radiations. The model is also consistent with the low dose-rate effects on spermatogonial mutations and translocations in the experimental animals and inverse dose-rate effects of morphological transformation in cultured cells.