Analysis of genetic relationships and antimicrobial susceptibility of Escherichia coli isolated from Clethrionomys glareolus

Eleven strains of Escherichia coli were isolated from 54 bank voles living in the LomZa Landscape Park of the Narew River Valley, indicating that E. coli is not common in the alimentary tract of these mammals. On the basis of pulsed-field gel electrophoresis and computer-assisted analysis, the isolates were grouped into six genotypes at similarities of 39%. Chromosome length of E. coli under study differed by as much as 900 kb, ranging 2.7-3.6 Mb. All strains were susceptible to amikacin and ciprofloxacin, whereas, for tetracycline, streptomycin, ampicillin, and cefonicid, different results were noted. No differences were detected among the plasmid complements of eight strains (73%), for which plasmid profiles revealed the presence of two plasmidic bands. One, three and four plasmids were observed in a plasmid pattern of single isolates. The observation from the study indicated the high genetic polymorphism among the isolates recovered from the animals of one species living in the same environment.     

A novel mutation A1270G of the EDA1 gene causing Tyr343Cys substitution in ectodysplasin-A in a family with anhidrotic ectodermal dysplasia

The structure of the EDA1 gene was investigated in a patient with anhidrotic ectodermal dysplasia. Sequence analysis revealed a novel A1270G transition in exon 9 of the EDA1 gene in the patient and his uncle, whereas the patient's mother and grandmother were heterozygotes. This mutation resulted in Tyr343Cys substitution in the extracellular domain of the EDA1 gene product - ectodysplasin-A. The additional Cys343 was located between Cys332 and Cys346 and formed with Cys352 a cluster of four closely situated residues that could potentially form disulfide bonds. This mutation might affect the tertiary structure of the receptor-binding domain of ectodysplasin-A and precipitate the clinical symptoms of anhidrotic ectodermal dysplasia.     

Fluorescent in situ sequencing on polymerase colonies

Integration of DNA isolation, amplification, and sequencing can be achieved by the use of polymerase colonies (polonies) and cycles of fluorescent dNTP incorporation. In this paper, we present four advances that bring us closer to sequencing genomes cost-effectively using the polony technology. First, a polymerase trapping technique enables efficient nucleotide extension by DNA polymerase in a polyacrylamide matrix and eliminates loss of enzyme during sequencing cycles. Next, we present two novel types of reversibly dye-labeled nucleotide analogues, show that DNA polymerase can incorporate these analogues, and demonstrate that the dyes can be removed by thiol reduction or light exposure. Using these nucleotides, we have sequenced multiple polonies in parallel. In addition, we have found that a high density of polonies can be achieved with minimal overlap between adjacent polonies by limiting the concentration of free primer in the polony amplification reactions. Finally, we have developed software for automated image alignment and sequence calling.     

Isolation and characterization of unsaturated fatty acids as natural ligands for the retinoid-X receptor

The retinoid-X receptor (RXR) is a ligand activated nuclear receptor that is the heterodimer partner for many class II nuclear receptors. Previously identified natural ligands for this receptor include 9-cis retinoic acid (9cRA), docosahexaenoic acid, and phytanic acid. Our studies were performed to determine if there are any unidentified, physiologically important RXR ligands. Agonists for RXR were purified from rat heart and testes lipid extracts with the use of a cell-based reporter assay to monitor RXR activation. Purified active fractions contained a variety of unsaturated fatty acids and components were quantified by gas-liquid chromatography of derivatized samples. The corresponding fatty acid standards elicited a similar response in the reporter cell assay. Competition binding analysis revealed that the active fatty acids compete with [3H]9cRA for binding to RXR. Non-esterified fatty acids were analyzed from lipid extracts of isolated heart and testes nuclei and endogenous concentrations were found to be within the range of their determined binding affinities. Our studies reveal tissue dependent profiles of RXR agonists and support the idea of unsaturated fatty acids as physiological ligands of RXR.     

Substitution pattern of 3-deoxy-D-manno-oct-2-ulosonic acid in bacterial lipopolysaccharides investigated by methylation analysis of whole LPS

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) is a constituent of the inner core part of bacterial lipopolysaccharides (LPS). This sugar may contribute to biological activities of the LPS, the type of substitution of Kdo is thus of importance and this work is aimed at the evaluation of a method for monitoring the substitution of Kdo in LPS. The procedure consists of three steps, namely permethylation of the lipopolysaccharide, with iodomethane and sodium methylsulfinylmethanide or NaOH in Me(2)SO, or with methyl triflate, then the product is methanolysed with HCl in MeOH and acetylated with acetic anhydride in pyridine. The resulting partially methylated acetates of Kdo methyl glycosides were analyzed by gas-liquid chromatography-electron impact ionization mass spectrometry (GLC-MS). For several derivatives of Kdo, specific GLC retention times and MS fragmentation patterns were determined. Lipopolysaccharides from several bacterial strains were isolated and analyzed with three different methods of methylation. The complete solubilization of the LPS in the acid form allows diminishing possible undermethylation. Sodium methylsulfinylmethanide is the most efficient agent in the permethylation of the whole LPS, of all the tested procedures. Methylation with methyl triflate allows the detection of base labile substituents on Kdo residues.     

Structure of a new 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii PCM 1542

The O-specific polysaccharide of Citrobacter gillenii PCM 1542 from serotype O-12a,12 b is composed of one residue each of D-glucose, D-GlcNAc, 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose (D-GlcNAcyl) and two GalNAc residues. On the basis of sugar and methylation analyses of the intact and Smith degraded polysaccharides, along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched pentasaccharide repeating unit of the O-specific polysaccharide was established:This structure differs significantly from that of the O-specific polysaccharide of C. gillenii PCM 1544 from the same serotype O-12a,12 b, which has been established earlier (Kübler-Kielz.shtsls;b, J. et al. Carbohydr. Res. 2001, 331, 331-336). Serological studies confirmed that the two O-antigens are not related and suggested that strains PCM 1542 and 1544 should be classified into different O-serogroups.     

Inhibition of cathepsin G by 2-amino-3,1-benzoxazin-4-ones: kinetic investigations and docking studies

A series of benzoxazinones was used to investigate the interaction of human cathepsin G with acyl-enzyme inhibitors. With respect to the primary specificity of cathepsin G, inhibitors with hydrophobic or basic residues at position 2 were included in the study. Parameters of the enzyme acylation and deacylation were determined by slow-binding kinetics in the presence of a chromogenic substrate. For selected inhibitors, the time course of the enzyme-catalyzed conversion of the inhibitors was followed. This approach was suitable to elucidate a rate-determining deacylation step. Docking simulations of the noncovalent enzyme-inhibitor complexes were performed and several clusters were analyzed for each inhibitor. The amino acids of the active site that participate in the binding of the inhibitors were determined. The arrangements in several clusters of an inhibitor were not uniform with respect to the orientation by which the inhibitor was bound in the S(1) pocket. Docking of the basic piperazino derivatives 6 and 10 indicated an interaction with Glu 226 at the bottom of the S(1) specificity pocket. The (N-methyl)benzylamino derivative 1 showed the strongest acylation rate (k(on)=1200 M(-1) s(-1)), which was attributed to a high extent of pseudo-productive orientations of the noncovalent preassociation complex.     

PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-DeltaTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-DeltaTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from approximately 24 to approximately 13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-DeltaTRL did not differ. However, endocytic recycling of CFTR-DeltaTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.