A Specific Transduction Mechanism for the Glutamate Action on Phosphoinositide Metabolism via the Quisqualate Metabotropic Receptor in Rat Brain Synaptoneurosomes: II. Calcium Dependency, Cadmium Inhibition

In this article, we demonstrate that an increase in intracellular Ca2+ concentration may represent a specific common step(s) in the mechanism(s) of action of glutamate (Glu) and depolarizing agents on formation of inositol phosphates (IPs) in 8-day-old rat forebrain synaptoneurosomes. In fact, A23187, a Ca2+ ionophore, induces a dose-dependent accumulation of IPs, which is not additive with that evoked by Glu and K+ but is slightly synergistic with that induced by carbachol. In addition, Glu and K+ augment the intracellular Ca2+ concentration in synaptoneurosome preparations as measured by the fura-2 assay. The absence of external Ca2+ decreases basal and Glu-, and K(+)-stimulated formation of IPs. Cd2+ (100 microM) fully inhibits both Glu- and K(+)-evoked formation of IPs without affecting the carbachol-elicited response of IPs. Zn2+ inhibits Glu- and K(+)-stimulated accumulation of IPs (IC50 approximately 0.4 mM) but with a lower affinity than Cd2+ (IC50 approximately 0.035 mM). The organic Ca2+ channel blockers verapamil (10 microM), nifedipine (10 microM), omega-conotoxin (2 microM), and amiloride (10 microM) as well as the inorganic blockers Co2+ (100 microM) and La3+ (100 microM) block neither Glu- nor K(+)-evoked formation of IPs, a result suggesting that the opening of the L-, T-, N-, or P-type Ca2+ channels does not participate in these responses. All these data suggest that an increase in intracellular Ca2+ concentration resulting from an influx of Ca2+, sensitive to Cd2+ but not to other classical Ca2+ antagonists, may play a key role in the transduction mechanism activated by Glu or depolarizing agents.     

Revue rapide: 381 sujets impuissants,explores en 2 ans,dont 207 avec Prostaglandine E1

During the last two years, 381 impotent patients have been tested, 174 with papaverine and 207 with prostaglandin E1 (PGE1: Alprostadil). More positive responses were obtained using PGE1 (76.8%) than papaverine (44.8%), and some patients whose response to an initial PGE1 test was negative demonstrated a positive effect after a second injection. Two non-responders to PGE1 showed a positive response to papaverine+phentolamine. Tolerance of PGE1 was extremely good, provoking fewer prolonged erections than papaverine. Pain following intracavernosal injection of PGE1 was distressing in 10% of cases, all young men with psychogenic impotence. A therapeutic trial of repeated injections, carried out in 49 selected patients with mild organic dysfunction but strong psychogenic inhibition, typically showed a rapid clinical improvement. Moreover, 16 patients with cavernosal sclerosis and vascular impotence who were treated by injections two or three months apart (instead of having a prosthetic implant) showed a recovery of sexual activity. Consequently, during these two years only 35 patients (9.1%) were treated surgically, 21 by the implantation of hydraulic prostheses. The significant benefits of PGE1, especially the ease of self-administration, demonstrate the need for new therapeutic trials.     

Approach to the analysis of diuretics and masking agents by high-performance liquid chromatography—mass spectrometry in doping control

The application of thermospray and plasmaspray high-performance liquid chromatography—mass spectrometry to the analysis of diuretics and probenecid has been investigated. The latter method gave better ionization efficiency than the former, and its response was optimized by altering the solvent composition: best results were obtained with water—methanol—acetonitrile—trifluoroacetic acid. Using different proportions of these solvents, three isocratic systems were developed to separate the compounds under study. The principal characteristic of plasmaspray positive-ion mass spectra was a protonated molecular ion and very little fragmentation was evident. In the negative ionization mode, the plasmaspray method gave mass spectra showing more fragmentation, which resulted in additional structural information. The ability of trifluoroacetic acid to form negative cluster ions precluded its use as a mobile phase component. The minimum detectable amounts determined by the analysis in the positive-ion mode was compound-dependent, but generally ca. 10–150 ng. In many cases the compounds could be detected in urine extracts.     

Physical map of the Listeria monocytogenes chromosome.

The circular physical map of the pathogenic bacterium Listeria monocytogenes LO28 (serovar 1/2c) was established by using pulsed-field gel electrophoresis. The L. monocytogenes chromosome contains eight NotI fragments of 1,100, 940, 400, 335, 280, 45, 30, and 20 kb in size and eight Sse8387I fragments of 860, 680, 680, 370, 335, 130, 70, and 25 kb. Therefore, the total length of the genome is 3,150 kb. To order the NotI fragments on the chromosome, we used a strategy which can be of general use. We first cloned chromosomal HindIII or EcoRI fragments in pBR322. DNA extracted from the total libraries was digested by NotI and ligated to a NotI-kanamycin resistance cassette obtained by cutting Tn5 with NotI. After transformation in Escherichia coli, kanamycin-resistant clones originating from NotI-containing EcoRI or HindIII fragments were isolated. The two EcoRI-NotI or HindIII-NotI fragments of each recombinant plasmid were isolated and used as probes on Southern blot hybridizations to identify and link the corresponding NotI fragments. Seven NotI fragments were ordered in this way. The last junction was demonstrated by partial digest analysis. All L. monocytogenes genes identified so far as well as the six rRNA operons were localized on the NotI map. Regions homologous to genes from closely related bacteria were also detected and localized. Southern blot analysis of simple Sse8387I digests or double Sse8387I-NotI digests probed with the various NotI probes allowed us to align the Sse8387I fragments and localize the single SfiI site, resulting in the establishment of the first genetic and physical map of the L. monocytogenes chromosome.     

Biosynthesis of bacillomycin D by Bacillus subtilis. Evidence for amino acid-activating enzymes by the use of affinity chromatography.

Bacillomycin D is an antifungal lipopeptide produced by B. subtilis. The formation of the peptidyl bonds of bacillomycin D occurs non-ribosomally, as demonstrated by the use of chloramphenicol, an inhibitor of protein biosynthesis. Amino acid-activating enzymes were found in B. subtilis cell lysates purified by affinity chromatography on a gel containing L-Pro, an amino acid of bacillomycin D. Presence of ATP during this purification increases the binding of enzymatic proteins and their activity. An enzyme, with an apparent molecular weight of 230 kDa, catalyzed ATP-PPi exchange reactions, which were mediated by specific amino acids, corresponding to a partial sequence of bacillomycin D.     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

Interactions of surfactin,a biosurfactant fromBacillus subtilis,with inorganic cations

Summary The properties of surfactin, a biosurfactant lipopeptide, were highly modified in the presence of inorganic cations. The micellization of surfactin was favoured by monovalent and, especially by divalent cations with a modification of the molecular area at the air-water interface. Haemolysis of erythrocytes by surfactin was enhanced by low concentrations of divalent cations with an increase of the binding of the lipopeptide to membrane. Inorganic ions induced conformational rearrangements probably due to ion-surfactin associations which modify the surface-active properties.