Phenotypic characterization and description of two major O-serotypes in Tenacibaculum maritimum strains from marine fishes

Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes. The development of effective preventive measures (i.e. vaccination) requires biochemical, serological and genetic knowledge of the pathogen. With this aim, the biochemical and antigenic characteristics of T. maritimum strains isolated from sole, turbot and gilthead sea bream were analysed. Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains. The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated. All bacteria studied were biochemically identical to the T. maritimum reference strains. The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed. However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated. This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2. These 2 serotypes seem to be host-specfic. In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2. Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.     

Study of acute vulvovaginitis in sexually active adult women, with special reference to candidosis, in patients of the Francisco J. Muñiz Infectious Diseases Hospital

The results of microbiological vaginal secretions samples obtained from 749 women (from July 2001 to July 2002) were studied in the Bacteriology Unit of the Francisco Javier Mu?iz Hospital from Buenos Aires. All patients suffered acute vulvovaginitis were child bearing and sexually active women, 334 of them were HIV-positive. The following are the results of the microbiological studies: Lactobacillus spp 50.6%, Gardnerella vaginalis 25.6%, Candida spp 17.4%, Trichomonas vaginalis 5.3%, Neisseria gonorrhoeae 0.3% and B group Streptococcus 0.8%. Candida vaginitis was significantly more frequent in HIV-positive patients, (21.6% vs 14%; p = 0.0086); meanwhile, trichomoniasis was less common although the difference was not statistically significant (3.6 vs 6.7%, p = 0.0810). The following Candida species were isolated in this study: Candida albicans 76.8%, Candida glabrata 15.6%, Candida parapsilosis 2.9%, Candida tropicalis 1.5% and Candida krusei 0.7%. Eight cases (6.2%) of vaginitis were produced by two Candida species (C. albicans and C. glabrata), and in three cases (2.17%) Saccharomyces cerevisiae were isolated. Five women suffering acute vaginitis with Candida spp presented another etiologic agent of vaginal infection, three cases T. vaginalis and two cases G. vaginalis. The following are some of the most important findings of this study: 1) Half of the patients presented a normal microbial biota; 2) Candida spp vaginitis was significantly more frequent among HIV-positive women; 3) we observed a high incidence of Candida glabrata infections (15.9%), 4) 6.2% of vaginal candidiasis were caused by more than one Candida species and, 5) the susceptibility pattern of C. albicans and C. glabrata isolates against fluconazole was similar to the one observed in other studies. The majority of C. albicans isolates were susceptible to fluconazole (MIC90 = 0.5 microg/ml) meanwhile C. glabrata strains were much less susceptible to this drug (MIC50 and MIC90 = 32 microg/ml).     

The STATs in cell stress-type responses

In the early 1990's, a new cell signaling pathway was described. This new paradigm, now known as the JAK/STAT pathway, has been extensively investigated in immune-type cells in response to interferons and interleukins. However, recent evidence suggests that the JAK/STAT pathway also mediates diverse cellular responses to various forms of biological stress including hypoxia/reperfusion, endotoxin, ultraviolet light, and hyperosmolarity. The current literature describing the JAK/STAT pathway's role in cellular stress responses has been reviewed herein, but it is clear that our knowledge in this area is far from complete.     

The dimerization domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: a biophysical characterization

The type 1 HIV presents a conical capsid formed by approximately 1500 units of the capsid protein, CA. Homodimerization of CA via its C-terminal domain, CA-C, constitutes a key step in virion assembly. CA-C dimerization is largely mediated by reciprocal interactions between residues of its second alpha-helix. Here, we show that an N-terminal-acetylated and C-terminal-amidated peptide, CAC1, comprising the sequence of the CA-C dimerization helix plus three flanking residues at each side, is able to form a complex with the entire CA-C domain. Thermal denaturation measurements followed by circular dichroism (CD), NMR, and size-exclusion chromatography provided evidence of the interaction between CAC1 and CA-C. The apparent dissociation constant of the heterocomplex formed by CA-C and CAC1 was determined by several biophysical techniques, namely, fluorescence (using an anthraniloyl-labeled peptide), affinity chromatography, and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant, in the order of 50 microM. This apparent dissociation constant was only five times higher than was the dissociation constant of both CA-C and the intact capsid protein homodimers (10 microM).     

Stereoselective inhibition of glutamate carboxypeptidase by organophosphorus derivatives of glutamic acid

A series of alkyl and aryl phosphonyl, thiophosphonyl, and dithiophosphonyl derivatives of (S)- and (R)-glutamic acid were prepared and examined for inhibitory potency against glutamate carboxypeptidase (carboxypeptidase G). The acquisition of the phosphonamidodithioic acids and the individual phosphonamidothioic acid diastereomers was achieved through a common phosphonamidothiolate precursor, which also allowed for the chromatographic resolution of the chiral phosphorus center of the phosphonamidothioic acids. The most potent inhibitor of the series was the n-butylphosphonamidate derivative of the natural isomer of glutamic acid. Although each diastereomeric pair of three phosphonamidothionates exhibited stereoselective inhibition consistent with the configuration of the chiral phosphorus center, this effect was generally not remarkable. More important, was the effect of carbon stereochemistry upon glutamate carboxypeptidase inhibition as exemplified by a limited series of enantiomeric pairs of phosphonamidate and phosphonamidodithionate derivatives of glutamic acid. The phosphonamidate analogs derived from the unnatural stereoisomer of glutamic acid were devoid of inhibitory potency in contrast to their enantiomers. Surprisingly, the phosphonamidodithionates derived from the unnatural stereoisomer of glutamic acid demonstrated greater inhibitory potency than their naturally-derived antipodes.     

Modulation of airway inflammation and bacterial clearance by epithelial cell ICAM-1

Many cell types in the airway express the adhesive glycoprotein for leukocytes intercellular adhesion molecule-1 (ICAM-1) constitutively and/or in response to inflammatory stimuli. In this study, we identified functions of ICAM-1 on airway epithelial cells in defense against infection with Haemophilus influenzae. Initial experiments using a mouse model of airway infection in which the bacterial inoculum was mixed with agar beads that localize inflammation in airways demonstrated that ICAM-1 expression was required for efficient clearance of H. influenzae. Airway epithelial cell ICAM-1 expression required few or no leukocytes, suggesting that epithelial cells could be activated directly by interaction with bacteria. Specific inhibition of ICAM-1 function on epithelial cells by orotracheal injection of blocking antibodies resulted in decreased leukocyte recruitment and H. influenzae clearance in the airway. Inhibition of endothelial cell ICAM-1 resulted in a similar decrease in leukocyte recruitment but did not affect bacterial clearance, indicating that epithelial cell ICAM-1 had an additional contribution to airway defense independent of effects on leukocyte migration. To assess this possibility, we used an in vitro model of neutrophil phagocytosis of bacteria and observed significantly greater engulfment of bacteria by neutrophils adherent to epithelial cells expressing ICAM-1 compared with nonadherent neutrophils. Furthermore, bacterial phagocytosis and killing by neutrophils after interaction with epithelial cells were decreased when a blocking antibody inhibited ICAM-1 function. The results indicate that epithelial cell ICAM-1 participates in neutrophil recruitment into the airway, but its most important role in clearance of H. influenzae may be assistance with neutrophil-dependent bacterial killing.     

CD1d function is regulated by microsomal triglyceride transfer protein

CD1d is a major histocompatibility complex (MHC) class I-related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-alpha chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell-mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.     

Arginine/serine repeats are sufficient to constitute a splicing activation domain

SR proteins are essential pre-mRNA splicing factors that have been shown to bind a number of exonic splicing enhancers where they function to stimulate the splicing of adjacent introns. Members of the SR protein family contain one or two N-terminal RNA binding domains, as well as a C-terminal arginine–serine (RS) rich domain. The RS domains mediate protein–protein interactions with other RS domain containing proteins and are essential for many, but not all, SR protein functions. Hybrid proteins containing an RS domain fused to the bacteriophage MS2 coat protein are sufficient to activate enhancer-dependent splicing in HeLa cell nuclear extract when bound to the pre-mRNA. Here we report progress towards determining the protein sequence requirements for RS domain function. We show that the RS domains from non-SR proteins can also function as splicing activation domains when tethered to the pre-mRNA. Truncation experiments with the RS domain of the human SR protein 9G8 identified a 29 amino acid segment, containing 26 arginine or serine residues, that is sufficient to activate splicing when fused to MS2. We also show that synthetic domains composed solely of RS dipeptides are capable of activating splicing, although their potency is proportional to their size.