Adhesion of Acinetobacter spp. to para-xylene

Hydrophobicity of 200 of Acinetobacter spp. strains was defined by the BATH (Bacterial Adhesion to Hydrocarbon Test) method of Rosenberg et al. 48.0% strains of Acinetobacter spp. adhered to para-xylene. 40.0% of strains showed waek hydrophobicity, 7.5% moderate and one strain strongly adhered to para-xylene. A. baumannii strains adhered in 52.2%, A. junii in 42.1% and 20.0% in A. haemolyticus. Significantly we find weak adhesion to para-xylene than mild and strong. Mostly adhesion was discovered in A. baumannii and A. junii than in A. haemolyticus. Results show that Acinetobacter spp. has adhesive properties to para-xylene.     

Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence.

Much has been learned about the mechanisms underlying cellular senescence. The pathways leading to senescence appear to vary, depending on the cell type and cell culture conditions. In this respect, little is known about senescence of human peritoneal mesothelial cells (HPMC). Previous studies have significantly differed in the reported proliferative lifespan of HPMC. Therefore, in the present study, we have examined how HPMC enter state of senescence under conditions typically used for HPMC culture. HPMC were isolated from omentum and grown into senescence. The cultures were assessed for the growth rate, the presence of senescence markers, activation of cell-cycle inhibitors, and the oxidative stress. HPMC were found to reach, on average, six population doublings before senescence. The terminal growth arrest was associated with decreased expression of Ki67 antigen, increased percentage of cells in the G1 phase, reduced early population doubling level cDNA-1 mRNA expression, and the presence of senescence-associated beta-galactosidase. Compared with early-passage cells, the late-passage HPMC exhibited increased expression of p16INK4a but not of p21Cip1. In addition, these cells generated more reactive oxygen species and displayed increased presence of oxidatively modified DNA (8-hydroxy-2'-deoxyguanosine). These results demonstrate that early onset of senescence in omentum-derived HPMC may be associated with oxidative stress-induced upregulation of p16INK4a.     

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.     

A Functional Kinase Homology Domain Is Essential for the Activity of Photoreceptor Guanylate Cyclase 1

Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the second messenger of phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg²⁺ binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.     

An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase

Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys¹⁶¹ residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis.     

The influence of the two different light intensities duringthe daytime on the colon motility in patients with irritable bowel syndrome (IBS): a preliminary report

Irritable bowel syndrome (IBS) is the most frequent functional disorder of the gastrointestinal tract, with great economical impact. The etiology and pathogenesis of the disease are unclear. Among patients seeking medical attention for IBS, 70 - 90% have psychiatric co-morbidity, most commonly major depression. In this study we test the influence of bright light on colon motility and subjectively felt symptoms. Eight IBS patients participated in the experiment. The passage rate was evaluated twice for every person: (1) after three days of exposure to bright light -3000 lux (from 8:00 to 18:00), (2) after three days exposure to dim light -100 lux (from 8:00 to 18:00). The comparison of colonic time in IBS patients point to differences in the elimination of markers, depending on experimental light conditions. After bright light conditions a tendency is observed to slower elimination of markers in the IBS-diarrhoea patients, and to a quicker elimination in the IBS-constipated patients. According to subjective feeling, all IBS patients reported an improvement in bowel function and relief of pain after bright but not after dim light application. Results of this preliminary study suggest that phototherapy might be a valuable addition to the conventional treatment of IBS.     

Mutation in the Sp1 motif of the bovine leptin gene affects its expression

Leptin is expressed mainly by adipocytes and plays a crucial role in the regulation of energy expenditure, food intake, and adiposity. Using PCR-heteroduplex analysis and sequencing, we investigated a C/G substitution in the promoter region of the bovine leptin gene. Application of the electrophoretic mobility shift assay showed that the C→G transversion decreased the leptin gene promoter binding capacity for nuclear proteins. With real-time PCR and Western blotting, we showed that the leptin expression level was higher in cattle with the CC than with the GG genotype.     

T-antigen of the human polyomavirus JC attenuates faithful DNA repair by forcing nuclear interaction between IRS-1 and Rad51

JC polyomavirus (JCV), which infects 90% of the human population, is detectable in human tumors. Its early protein, JCV T-antigen, transforms cells in vitro and is tumorigenic in experimental animals. Although T-antigen-mediated transformation involves genetic alterations of the affected cells, the mechanism underlying this genomic instability is not known. We show that JCV T-antigen inhibits homologous recombination DNA repair (HRR), which results in an accumulation of mutations. T-antigen does not operate directly but utilizes a cytosolic molecule, insulin receptor substrate 1 (IRS-1). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with artificial nuclear localization signal. Our observations define a new mechanism by which viral protein utilizes cytosolic molecule to inhibit faithful DNA repair, and suggest how polyomaviruses could compromise stability of the genome. (c) 2005 Wiley-Liss, Inc.     

Mutations in the amino terminus of the cystic fibrosis transmembrane conductance regulator enhance endocytosis

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype.