Bibliothek

Notizen: Background. Helicobacter pullorum, first detected in the liver and intestinal contents of poultry, was defined as a new species in 1994. This organism has since been isolated from humans with gastroenteritis. Phenotypic as well as genotypic methods have been used to identify H. pullorum associated with cases of human disease.Materials and Methods. Clinical isolates were submitted for identification to the National Laboratory for Enteric Pathogens by Provincial Public Health Laboratories within Canada. Phenotypic characterization was conducted using a variety of growth and biochemical tests including oxidase, catalase, indoxyl acetate, H2S production in triple sugar iron (TSI) agar, antimicrobial susceptibility testing, and fatty acid analysis. Genotypic identification was performed using a polymerase chain reaction–restriction fragment–length polymorphism (PCR-RFLP) analysis of a 1-kb fragment of the Helicobacter 16S rRNA gene.Results. During the last 7 years (1993–1999) a total of 11 isolates of H. pullorum were detected from patients with gastroenteritis for inclusion in this study. Typically, these isolates were oxidase and catalase positive, produced optimal growth at 42°C, and produced H2S in TSI. Of these 11 isolates, 1 showed DNase activity, while another did not produce H2S in TSI, and only 2 showed tolerance to 1% bile. Antimicrobial susceptibility assays indicated that 6 of the 11 strains were resistant to nalidixic acid. The fatty acid profiles of the isolates were similar to each other and provided a distinguishing profile from the other related species. Genetically identical and distinct species-specific restriction fragment–length polymorphism (RFLP) patterns were produced using the restriction enzymes Bsr I and Dde I.Conclusion. Phenotypic and genotypic procedures were used to identify H. pullorum. Interspecies phenotypic variability was apparent and supported the use of a polyphasic approach for identification. Similarities to the more prominent human pathogens Campylobacter coli and C. lari were also noted. The use of a combination of phenotypic and, in particular, genotypic markers for H. pullorum should prove valuable both for epidemiological investigations and for the diagnosis of disease related to this emerging human pathogen.

Notizen: Abstract: In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2, Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1–1,000 μM), with a maximum response of 22 × 2% at approximately 100 μM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 μM The effect of carbachol (100 μM) on [Ca2+], was antagonised by atropine, but not by hexamethonium (10 μM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridinesensitive calcium channels.

Notizen: Abstract: We have determined the ultrastructure of 5-hydroxytryptamine3 (5-HT3) serotonin receptors purified from NG108-15 mouse neuroblastoma × rat glioma cells by electron microscopic examination of receptor particles embedded in uranyl acetate stain and metal replicas of rapidly frozen receptors. The 5-HT3 receptor can be modelled as a cylinder 11 nm in length and 8 nm in diameter with a closed end and a central cavity 3 nm in diameter. Analysis of the rotational symmetry of single receptor particles indicates that the 5-HT3 receptor is composed of five subunits arranged symmetrically around a central cavity. Together with evidence obtained for related proteins in other studies using ultrastructural, biochemical, or electrophysiological methods, our observations suggest that all members of the ligand-gated ion channel superfamily may possess a pentameric quaternary structure.

Notizen: Abstract: 5-Hydroxytryptamine3 (5-HT3) receptor-type binding sites were solubilised from NG108-15 mouse neuro-blastoma X rat glioma hybrid cells using five different detergents (n-octyl-β-D-glucoside, Triton X-100, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), sodium cholate, and deoxycholate) and the solubilisation efficiencies compared. The equilibrium binding, kinetic properties, and pharmacological profile of solubilised binding sites were similar to those of 5-HT3 receptor-type binding sites (5-HT3R) in membrane preparations determined using [3H]GR65630. The solubilised binding sites were purified using an affinity column constructed by coupling the high-affinity antagonist GR1 19566X to an Affi-Gel 15 resin. The affinity of purified 5-HT3R for [3H]GR65630 was reduced threefold compared to the crude soluble preparation, but the pharmacological profile was similar. The sedimentation coefficient of the purified protein (US, detergent: CHAPS) was determined by sucrose density gradient centrifugation. The apparent molecular mass of the detergent/binding site complex (370 kDa) was determined by size exclusion chromatography in the presence of n-dodecyl-β-D-maltoside. Gel electrophoresis of the purified protein revealed bands at apparent molecular masses of 36, 40, 50, and 76 kDa. Electron microscopy of the negatively stained purified protein showed the presence of round particles of 8–9 nm diameter with a 2-nm stained pit in the centre, closely resembling the doughnut shapes described for nicotinic acetylcholine receptors.

Notizen: Abstract: We have examined the ligand binding site of the serotonin 5-HT6 receptor using site-directed mutagenesis. Replacing the highly conserved Asp106 in transmembrane region III by asparagine eliminated d-[3H]lysergic acid diethylamide ([3H]LSD) binding to the mutant receptor transiently expressed in HEK293 cells. The potency of 5-HT and LSD to stimulate adenylyl cyclase was reduced by 3,600- and 500-fold, respectively, suggesting that an ionic interaction between the positively charged amino group of 5-HT and D106 is essential for high-affinity binding and important for receptor activation. In addition, basal cyclic AMP levels in cells expressing this mutant were increased. Mutation of a tryptophan residue one helix turn toward the extracellular side of transmembrane region III (Trp102) to phenylalanine produced significant changes in the binding affinity and potency of several ligands, consistent with a role of this residue in the formation of the ligand binding site. The exchange of two neighboring residues in the carboxy-terminal half of transmembrane region VI (Ala287 and Asn288) for leucine and serine resulted in a mutant receptor with increased affinities (seven- to 30-fold) for sumatriptan and several ergopeptine ligands. The identification of these interactions will help to improve models of the 5-HT6 receptor ligand binding site.