Arachidonic acid-induced calcium influx in human platelets. Comparison with the effect of thrombin.

The effects of arachidonic acid and thrombin on calcium movements have been studied in fura-2-loaded platelets by a procedure which allows simultaneous monitoring of the uptake of manganese, a calcium surrogate for Ca2+ channels, and the release of Ca2+ from intracellular stores. Arachidonic acid induced both Ca2+ (Mn2+) entry through the plasma membrane and Ca2+ release from the intracellular stores. The release of Ca2+ was prevented by cyclo-oxygenase inhibitors and mimicked by the prostaglandin H2/thromboxane A2 receptor agonist U46619. Ca2+ (Mn2+) entry required higher concentrations of arachidonic acid and was not prevented by either cyclo-oxygenase or lipoxygenase inhibitors. Several polyunsaturated fatty acids reproduced the effect of arachidonic acid on Ca2+ (Mn2+) entry, but higher concentrations were required. The effects of maximal concentrations of arachidonic acid and thrombin on the uptake of Mn2+ were not additive. Both agonists induced the entry of Ca2+, Mn2+, Co2+ and Ba2+, but not Ni2+, which, in addition, blocked the entry of the other divalent cations. However, arachidonic acid, but not thrombin, increased a Ni2(+)-sensitive permeability to Mg2+. The effect of thrombin but not that of arachidonic acid was prevented either by pretreatment with phorbol ester or by an increase in cyclic-AMP levels. Arachidonic acid also accelerated the uptake of Mn2+ by human neutrophils, rat thymocytes and Ehrlich ascites-tumour cells.     

Biosynthesis of platelet-activating factor in glandular gastric mucosa. Evidence for the involvement of the ''de novo'' pathway and modulation by fatty acids.

The biosynthesis of platelet-activating factor (PAF), a phospholipid autocoid with potent ulcerogenic properties that is produced in secretory exocrine glands by physiological secretagogues, was assessed in microsomal preparations of glandular gastric mucosa. For this purpose, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67); the enzymes of the 'de novo' pathway: 1-O-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP):acetyl-CoA acetyltransferase and 1-O-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-choline cholinephosphotransferase (EC 2.7.8.16); and some enzymes involved in the catabolism of PAF and lyso-PAF were assayed. Only the enzymes of the 'de novo' pathway and small amounts of PAF acetylhydrolase, phospholipase A2 and a lysophospholipase D acting on either lipids could be detected in the gastric preparations, whereas lyso-PAF:acetyl-CoA acetyltransferase activity was undetectable. The specific activity of alkyl-lyso-GP:acetyl-CoA acetyltransferase in the gastric mucosa was about one-tenth of that found in spleen microsomes and its apparent Km for acetyl-CoA was 454 microM compared with 277 microM in spleen microsomes. Glandular mucosa homogenates contained preformed PAF at a concentration of 2.7 +/- 0.7 ng equivalents of PAF (hexadecyl)/mg of protein. When gastric microsomes were incubated with micromolar concentrations of fatty acids (arachidonic, palmitic and oleic) prior to the assay of dithiothreitol (DTT)-insensitive cholinephosphotransferase, a dose-dependent reduction in the formation of PAF was observed, arachidonic acid being the most potent inhibitor, followed by linoleic acid (only tested on spleen microsomes) and oleic acid. By contrast, 1,2-diolein and phosphatidylcholine (dipalmitoyl) showed no or little effect. These results indicate that glandular gastric mucosa can produce PAF through the 'de novo' pathway, and that fatty acids, especially unsaturated, can reduce that synthesis by modulating the expression of DTT-insensitive cholinephosphotransferase.     

流行性出血热病毒R22株cDNA克隆及其特异性鉴定

用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.     

An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus.

Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall. All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme. The rate of synthesis of the nuclease depended on the growth medium. In NBG medium, in which the enzyme is not produced, the size of lytic plaques of several actinophages was larger than that in GYM or GAE medium, in which synthesis of the nuclease takes place late in growth. In addition, one of the phages assayed, phi A6, showed a diminution of its efficiency of plating in GYM medium with respect to that in NBG medium; another phage, phi A9, grew in NBG medium but not in the other two media. It is postulated that the presence of the host nuclease, together with the capability of the particular phage to absorb on S. antibioticus of different growth phases, determines the efficiency of growth and the plaque size of the phages on productive media. This hypothesis was confirmed when the growth of phi A6 and phi A9 in a mutant of S. antibioticus lacking the endonuclease activity was analyzed. It is concluded that the enzyme can assume, under some circumstances, a role in in vivo restriction.     

Multicatalytic proteinase in fish muscle

Proteinase II, a high-molecular-mass proteinase previously identified in white croaker skeletal muscle, was purified to apparent homogeneity by DEAE-Sephacel, phenyl-Sepharose CL 4B, and Sephacryl S-300 chromatographies. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with Mr ranging from 18,000 to 26,000 and a large subunit with a Mr 60,000. The proteinase was able to hydrolyze N-terminal-blocked 4-methyl-7-coumarylamide substrates having either an aromatic amino acid (chymotrypsin-like activity) or an arginine residue (trypsin-like activity) adjacent to the fluorogenic group. The trypsin-like activity of the enzyme was inhibited by fatty acids and sodium dodecyl sulfate, whereas the chymotrypsin-like activity was stimulated by those compounds but inhibited by nonionic and cationic detergents. Several thiol reagents inhibited both proteinase II activities. However, leupeptin and Cu2+ strongly inhibited its trypsin-like activity but only slightly affected its chymotrypsin-like activity. Dithiothreitol stimulated both activities, but at different extents and in different concentration ranges. These results suggest that the enzyme is multicatalytic, having at least two different active sites.     

Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF)

The effects of fibroblast growth factor (FGF) on testicular aromatase activity has been studied using primary cultures of porcine Leydig cells. After culture for 3 days in the absence or presence of FGF, the ability of the cells to produce estrogen was examined in a 4h-test period in which either (a) hCG (10(-9) M) or (b) androstenedione (3 x 10(-6) M) was added to the medium. FGF produced a 3- to 20-fold increase in estrogen formation from endogenous or exogenous substrate during the test period, in spite of a marked decrease (approximately equal to 60%) in [125I]-hCG binding and no significant change in testosterone concentration. Stimulation of estrogen secretion by FGF was dose-(ED50 approximately equal to 2 ng/ml) and time-dependent, the first and maximal effects were observed after 12h and 48h, respectively. Preliminary tests with several other factors (insulin, EGF, TGF-beta, FSH and hCG) showed that hCG alone directly stimulated aromatase activity. From these findings a role is suggested for FGF as a paracrine/autocrine agent in the control of estrogen secretion by Leydig cells.     

A map of barley chromosome 2 using isozymic and morphological markers

The F₂ progeny of a cross between a chromosome 2 multiple marker stock and an adapted cultivar of barley were analyzed for four morphological markers and electrophoretic patterns of eight leaf isozymes. TheIdh-2 locus was linked to thePer-5 locus (27.96±5.07 cM) and to thee locus (10.26±3.13 cM). Also, thePer-5 ande loci were located on the short arm of chromosome 2. In additionIdh-2 was also located on barley chromosome 2 and was linked to thev locus (13.18±3.56 cM), which is located on the long arm of chromosome 2. Two other marker genes,li andwst,,B, were linked (26.50±5.24 cM) on chromosome 2 but segregate independently of the other loci evaluated. This project was supported by funds from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.