Profiling and identification of eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection

Background. Mongolian gerbils are frequently used to study Helicobacter pylori‐induced gastritis and its consequences. The presence of an indigenous bacterial flora with suppressive effect on H. pylori may cause difficulties with establishing this experimental model. Aim. The aim of the present study was to determine bacterial profiles in the stomach of Mongolian gerbils with and without (controls) H. pylori infection. Methods. Gastric tissue from H. pylori ATCC 43504 and CCUG 17874 infected and control animals were subjected to microbial culturing and histology. In addition, gastric mucosal samples from H. pylori ATCC 43504 infected and control animals were analyzed for bacterial profiling by temporal temperature gradient gel electrophoresis (TTGE), cloning and pyrosequencing of 16S rDNA variable V3 region derived PCR amplicons. Results. Oral administration of H. pylori ATCC 43504, but not CCUG 17874, induced colonization and gastric inflammation in the stomach of Mongolian gerbils. Temporal temperature gradient gel electrophoresis (TTGE) and partial 16S rDNA pyrosequencing revealed the presence of DNA representing a mixed bacterial flora in the stomach of both H. pylori ATCC 43504 infected and control animals. In both cases, lactobacilli appeared to be dominant. Conclusion. These findings suggest that indigenous bacteria, particularly lactobacilli, may have an impact on the colonization and growth of H. pylori strains in the stomach of Mongolian gerbils.     

Stereoselective single-dose kinetics of citalopram and its metabolites in rats

The single-dose kinetics of the enantiomers of citalopram (CIT) and its metabolites, demethylcitalopram (DCIT) and didemethylcitalopram (DDCIT), were investigated after administration of 10, 20, or 100 mg/kg (s.c.) rac-CIT to rats. Samples from serum and two brain regions were collected 1, 3, 10, or 20 h postdose for HPLC analysis. In the 100 mg/kg rats, the enantiomeric (S/R) serum concentration ratios of CIT decreased during the study period (0.93 at 1 h vs. 0.59 at 20 h; P < 0.001). In the 10 and 20 mg/kg rats, the decrease in serum S/R CIT ratios was not so evident as in the 100 mg/kg rats. In all three groups the S/R CIT ratio was almost the same in the brain as in serum, although both CIT enantiomer levels in the brain were found to be 5-10 times higher than the levels in serum. The serum and brain metabolite levels were low in the 10 and 20 mg/kg rats, whereas the levels increased during the study period in the 100 mg/kg rats. In conclusion, the CIT enantiomers were shown for the first time to be stereoselectively metabolized after single-dose administration to rats, as previously shown in steady-state dosing studies in humans and rats.     

5-Hydroxytryptamine2B receptors stimulate Ca2+ increases in cultured astrocytes from three different brain regions

The expression of 5-hydroxytryptamine-2B (5-HT2B) receptor mRNA has recently been shown in cultured astrocytes. Here the expression of functional 5-HT2B receptors has been studied in cultured astrocytes from rat cerebral cortex, hippocampus, and brain stem. Fluo-3- and fura-2-based microspectrofluorometry was used for measuring changes in intracellular free calcium concentrations ([Ca2+]i). The 5-HT2B agonist alpha-methyl 5-HT (40 nM) produced rapid transient increases in [Ca2+]i in astrocytes from all three brain regions studied, and these responses were blocked by the selective 5-HT2B antagonist rauwolscine (1 microM). The specificity of the responses to alpha-methyl 5-HT was further demonstrated by the failure of 4-(4-fluorobenzoyl)-1-(4-phenylbutyl)-piperidine oxalate (1 microM), a specific 5-HT2A/5-HT2C antagonist, to block these responses. The 5-HT2B-induced increases in [Ca2+]i persisted in Ca2+-free buffer, indicating that the increase in [Ca2+]i results from mobilization of intracellular Ca2+ stores. The expression of 5-HT2B receptors on astroglial cells was further verified immunohistochemically and by Western blot analysis. These results provide evidence of the existence of 5-HT2B receptors on astrocytes in primary culture.     

Site-selective dephosphorylation of the platelet-derived growth factor beta-receptor by the receptor-like protein-tyrosine phosphatase DEP-1

Ligand stimulation of PDGF beta-receptors leads to autophosphorylation of the regulatory tyrosine 857 and of tyrosine residues that in their phosphorylated form serve as docking sites for Src homology 2 domain-containing proteins. Regulation of the PDGF beta-receptor by protein-tyrosine phosphatases is poorly understood. We have investigated PDGF beta-receptor dephosphorylation by receptor-like protein-tyrosine phosphatase DEP-1 using a cell line with inducible DEP-1 expression and by characterizing in vitro dephosphorylation of the PDGF beta-receptor and of receptor-derived phosphopeptides by DEP-1. After DEP-1 induction PDGF beta-receptor.DEP-1 complexes and reduced receptor tyrosine phosphorylation were observed. Phosphopeptide analysis of the PDGF beta-receptors from DEP-1-expressing cells and of the receptors dephosphorylated in vitro by DEP-1 demonstrated that dephosphorylation of autophosphorylation sites of the receptor differed and revealed that the regulatory Tyr(P)(857) was not a preferred site for DEP-1 dephosphorylation. When dephosphorylation of synthetic receptor-derived peptides was analyzed, the selectivity was reproduced, indicating that amino acid sequence surrounding the phosphorylation sites is the major determinant of selectivity. This notion is supported by the observation that the poorly dephosphorylated Tyr(P)(562) and Tyr(P)(857), in contrast to other analyzed phosphorylation sites, are surrounded by basic amino acid residues at positions -4 and +3 relative to the tyrosine residue. Our study demonstrates that DEP-1 dephosphorylation of the PDGF beta-receptor is site-selective and may lead to modulation, rather than general attenuation, of signaling.     

Identification and purification of vitamin K-dependent proteins and peptides with monoclonal antibodies specific for gamma -carboxyglutamyl (Gla) residues

Novel monoclonal antibodies that specifically recognize gamma-carboxyglutamyl (Gla) residues in proteins and peptides have been produced. As demonstrated by Western blot and time-resolved immunofluorescence assays the antibodies are pan-specific for most or all of the Gla-containing proteins tested (factors VII, IX, and X, prothrombin, protein C, protein S, growth arrest-specific protein 6, bone Gla protein, conantokin G from a cone snail, and factor Xa-like proteins from snake venom). Only the Gla-containing light chain of the two-chain proteins was bound. Decarboxylation destroyed the epitope(s) on prothrombin fragment 1, and Ca(2+) strongly inhibited binding to prothrombin. In Western blot, immunofluorescence, and surface plasmon resonance assays the antibodies bound peptides conjugated to bovine serum albumin that contained either a single Gla or a tandem pair of Gla residues. Binding was maintained when the sequence surrounding the Gla residue(s) was altered. Replacement of Gla with glutamic acid resulted in a complete loss of the epitope. The utility of the antibodies was demonstrated in immunochemical methods for detecting Gla-containing proteins and in the immunopurification of a factor Xa-like protein from tiger snake venom. The amino acid sequences of the Gla domain and portions of the heavy chain of the snake protein were determined.     

Functional characteristics of urinary tract smooth muscles in mice lacking cGMP protein kinase type I

Nitric oxide (NO)-mediated smooth muscle relaxation is mediated by cGMP through activation of cGMP-dependent protein kinase I (cGKI). We studied the importance of cGKI for lower urinary tract function in mice lacking the gene for cGKI (cGKI-/-) and in litter-matched wild-type mice (cGKI+/+) in vitro and in vivo. cGKI deficiency did not result in any changes in bladder gross morphology or weight. Urethral strips from cGKI-/- mice showed an impaired relaxant response to nerve-derived NO. The cGMP analog 8-bromo-cGMP (8-BrcGMP) and the NO-donor SIN-1 relaxed the wild-type urethra (50-60%) but had only marginal effects in the cGKI-deficient urethra. Bladder strips from cGKI-/- mice responded normally to electrical field stimulation and to carbachol but not to 8-BrcGMP. In vivo, the cGKI-deficient mice showed bladder hyperactivity characterized by decreased intercontraction intervals and nonvoiding bladder contractions. Loss of cGKI abolishes NO-cGMP-dependent relaxations of urethral smooth muscle and results in hyperactive voiding. These data suggest that certain voiding disturbances may be associated with impaired NO-cGKI signaling.     

Molecular analysis of two genes, DD9A and B, which are expressed during the postmolt stage in the decapod crustacean Penaeus japonicus

In decapod crustaceans, deposition of calcium carbonate crystals (calcification) in the exoskeleton takes place during the postmolt phase of the molt cycle. In an attempt to identify proteins which regulate the calcification process, the differential display technique was used to identify genes which were specifically expressed in the integument during the postmolt stage in the penaeid prawn Penaeus japonicus. One of the genes thus identified, named DD9A, was expressed in the epithelial cells of the tail fan. DD9A encoded a putative precursor of a secreted protein of 113 amino acids which exhibited sequence similarities to a group of crustacean and insect cuticular proteins, suggesting that DD9A was a protein component of the exoskeleton. Another gene, DD9B, which was also transcribed specifically during the postmolt period was identified based on its sequence similarity to DD9A. Potential roles of the DD9A protein in the calcification of the exoskeleton will be discussed.     

Centromere cleavage is a mechanism underlying isochromosome formation in skin and head and neck carcinomas

Centromeric rearrangements, in the form of isochromosomes or whole-arm translocations, are the most common recurrent changes in head and neck and skin carcinomas. Little is known about the mechanisms behind the origin of these chromosome rearrangements. In the present study, one basal cell carcinoma and two squamous cell carcinomas of the head and neck were thoroughly studied by cytogenetic and fluorescence in situ hybridization techniques. All tumors showed intratumor heterogeneity in the form of cytogenetically related subclones (in all tumors) and unrelated clones (in one tumor). Assessment of karyotypic evolution in these tumors suggests that centromeric cleavage is a mechanism giving rise to isochromosomes. A similar mechanism may also be involved in the formation of whole-arm translocations.