Structural identification of the lipopolysaccharide O-antigen produced by Yersinia enterocolitica serotype O:28.

The structure of the O-antigenic polysaccharide (O-PS) component of the lipopolysaccharide produced by Yersinia enterocolitica serotype O:28 has been elucidated. From chemical methods involving glycose analysis, periodate oxidation, methylation and the use of one- and two-dimensional NMR spectroscopy, the O-PS was found to be a polymer of repeating branched hexasaccharide units composed of L-rhamnose (four parts), 2-acetamido-2-deoxy-D-glucose (one part), and 2-acetamido-2-deoxy-D-galacturonic acid (one part) having the following structure:     

Structure of the capsular polysaccharide of Haemophilus pleuropneumoniae serotype 3

The capsular polysaccharide of Haemophilus pleuropneumoniae serotype 3 (ATCC 27090) is composed of D-galactose (one part), 2-acetamido-2-deoxy-D-glucose (one part), glycerol (one part), and phosphate (one part). From hydrolysis, dephosphorylation, methylation, and 1H and 13C nuclear magnetic resonance studies, the polysaccharide was found to be a high molecular weight polymer of a repeating trisaccharide unit, joined through monophosphate diester linkages and having the following structure: (formula; see text).     

A kinetic study on the suicide inactivation of peroxidase by hydrogen peroxide

In the absence of reductant substrates, and with excess H2O2, peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) shows the kinetic behaviour of a suicide inactivation, H2O2 being the suicide substrate. From the complex (compound I-H2O2), a competition is established between two catalytic pathways (the catalase pathway and the compound III-forming pathway), and the suicide inactivation pathway (formation of inactive enzyme). A kinetic analysis of this system allows us to obtain a value for the inactivation constant, ki = (3.92 +/- 0.06) x 10(-3) x s-1. Two partition ratios (r), defined as the number of turnovers given by one mol of enzyme before its inactivation, can be calculated: (a) one for the catalase pathway, rc = 449 +/- 47; (b) the other for the compound III-forming pathway, rCoIII = 2.00 +/- 0.07. Thus, the catalase activity of the enzyme and, also, the protective role of compound III against an H2O2-dependent peroxidase inactivation are both shown to be important.     

Structural studies of the capsular polysaccharide from Haemophilus pleuropneumoniae serotype 2

The capsular polysaccharide of Haemophilus pleuropneumoniae serotype 2 (ATCC 27089) is composed of D-glucose (two parts), D-galactose (one part), glycerol (one part), and phosphate (one part). Hydrolysis, dephosphorylation, methylation, enzymic studies, and 1H and 13C nuclear magnetic resonance experiments showed that the polysaccharide is a high molecular weight polymer of a tetrasaccharide repeating units, linked by monophosphate diester and having the following structure: (Formula: see text).     

Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8

The Y. enterocolitica O:8 (YeO8) O-antigen repeat units consist of five sugar residues: N-acetyl- d -galactosamine (GalNAc), d -galactose (Gal), d -mannose (Man), l -fucose (Fuc), and 6-deoxy- d -gulose (6d-Gul). The nucleotide sequence of the O-antigen gene cluster of the YeO8 strain 8081-c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O-antigen biosynthesis. We previously characterized the 3'-end of the O-antigen gene cluster and identified four genes: two for GDP-Man biosynthesis, one for UDP-Gal biosynthesis, and one for O-antigen polymerase. Based on sequence similarity, Tn 5 -insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP-6d-Gul and two in GDP-Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene product was similar to Wzx, the O-antigen flippase. Two genes remained unassigned. By genetic complementation we also showed that YeO8 O-antigen biosynthesis was dependent on N-acetyl-glucosaminyl:undecaprenylphosphate transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical-composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction-deficient derivative of Y. enterocolitica O:8 strain 8081, a rough mutant, designated 8081-R2, was isolated. 8081-R2 was complemented in trans with a cloned O-antigen gene cluster restoring surface O-antigen expression. The virulence of the wild-type strain and that of the complemented strain were significantly higher (approx. 100-fold) than that of the rough mutant in an orally infected mouse model, showing that YeO8 O-antigen is a virulence factor.     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Partition coefficient of a surfactant between aggregates and solution: application to the micelle-vesicle transition of egg phosphatidylcholine and octyl beta-D-glucopyranoside.

The mechanism of the solubilization of egg phosphatidylcholine containing 10% (M/M) of egg phosphatidic acid unilamellar vesicles by the nonionic detergent, octyl beta-D-glucopyranoside, has been investigated at both molecular and supramolecular levels by using fluorescence and turbidity measurements. In the lamellar region of the transition, the solubilization process has been shown to be first a function of the initial size before reaching an equilibrium aggregation state at the end of this region (the onset of the micellization process). The analysis during the solubilization process of the evolution of both the fluorescence energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (Rho-PE) and the fluorescence of 6-dodecanoyl-2-dimethylaminoaphtalene (Laurdan) has allowed us to determine the evolution of the detergent partitioning between the aqueous and the lipidic phases, i.e., the evolution of the molar fraction of OG in the aggregates (XOG/Lip) with its monomeric detergent concentration in equilibrium ([OG]H2O), throughout the vesicle-to-micelle transition without isolating the aqueous medium from the aggregates. The curve described by XOG/Lip versus [OG]H2O shows that the partition coefficient of OG is changing throughout the solubilization process. From this curve, which tends to a value of 1/(critical micellar concentration), five different domains have been delimited: two in the lamellar part of the transition (for 0 < [OG]H2O < 15.6 mM), one in the micellization part, and finally two in the pure micellar region (for 16.5 < [OG]H2O < 21 mM). The first domain in the lamellar part of the transition is characterized by a continuous variation of the partition coefficient. In the second domain, a linear relation relates XOG/Lip and [OG]H2O, indicating the existence of a biphasic domain for which the detergent presents a constant partition coefficient of 18.2 M-1. From the onset to the end of the solubilization process (domain 3), the evolution of (XOG/Lip) with [OG]H2O can be fitted by a model corresponding to the coexistence of detergent-saturated lamellar phase with lipid-saturated mixed micelles, both in equilibrium with an aqueous phase, i.e., a three-phase domain. The micellar region is characterized first by a small two-phase domain (domain 4) with a constant partition coefficient of 21 M-1, followed by a one-phase mixed-micellar domain for which XOG/Lip no longer linearly depends on [OG]H2O. The results are discussed in terms of a phase diagram.     

Protonated nucleobase ligands: synthesis, structure and characterization of 9-methyladeninium hexachloroplatinate and pentachloro(9-methyladeninium)platinum(IV)

Na(2)[PtCl(6)] was found to react with (9-MeAH)Cl(.)H(2)O (2) (9-MeA=9-methyladenine) in aqueous solution yielding (9-MeAH)(2)[PtCl(6)](.)2H(2)O (3). The same compound was obtained from hexachloroplatinic acid and 9-methyladenine. Performing this reaction at 60 degrees C, complex formation took place yielding the 9-methyladeninium complex [PtCl(5)(9-MeAH)](.)2H(2)O (4a). An analogous complex, [PtCl(5)(9-MeAH)](.)1/2(18C6)(.)H(2)O (4b, 18C6=crown ether 18-crown-6), was formed in the reaction of aquapentachloroplatinic acid (H(3)O)[PtCl(5)(H(2)O)](.)2(18C6)(.)6H(2)O (1) with 9-methyladenine in 1:1 ratio. All complexes were isolated in moderate to good yields as yellow powder (4b) and crystals (3, 4a), respectively. They were fully characterized by microanalysis, IR and NMR ((1)H, (13)C, (195)Pt) spectroscopies, and in part (2, 3, 4a) also by single-crystal X-ray diffraction analysis. Molecular structure of complex 4a exhibited that the 9-methyladeninium ligand is N1 protonated and coordinated through N7 to platinum(IV).     

Anaerobic c(1) metabolism of the o-methyl-C-labeled substituent of vanillate

The O-methyl substituents of aromatic compounds constitute a C(1) growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C(1) substrate was determined by C radiotracer techniques. O-[methyl-C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C(1) substrate. The data showed that for every O-methyl carbon converted to [C]acetate, two were oxidized to CO(2). Quantitation of the carbon recovered in the two products, acetate and CO(2), indicated that acetate was formed in part by the fixation of unlabeled CO(2). The specific activity of C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO(2) and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-C]vanillate by strain TH-001 can be described as follows: 3CH(3)OC(7)H(5)O(3) + CO(2) + 4H(2)O --> CH(3)COOH + 2CO(2) + 10H + 10e + 3HOC(7)H(5)O(3).     

Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).

The lipopolysaccharide (LPS) of Klebsiella serotype O2 is antigenically heterogeneous; some strains express multiple antigenic factors. To study this heterogeneity, we determined the structure of the O-antigen polysaccharides in isolates belonging to serotypes O2(2a), O2(2a,2b), and O2(2a,2c), by using composition analysis, methylation analysis, and both 1H and 13C nuclear magnetic resonance spectroscopy. The repeating unit structure of the 2a polysaccharide was identified as the disaccharide [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] and was identical to D-galactan I, one of two O polysaccharides present in the LPS of Klebsiella pneumoniae serotype O1 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). LPS from serotype O2(2a,2b) also contained D-galactan I as the only O polysaccharide, suggesting that the 2b antigen is not an O antigen. The LPS of serotype O2(2a,2c) contained a mixture of two structurally distinct O polysaccharides and provides a second example of this phenomenon in Klebsiella spp. One polymer was identical to D-galactan I, and the other polysaccharide, the 2c antigen, was a polymer with a disaccharide repeating unit structure, [----3)-beta-D-GlcpNAc-(1----5)-beta-D-Galf-(1----]. The 2c structure does not resemble previously reported O polysaccharides from Klebsiella spp. Periodate oxidation confirmed that D-galactan I and the 2c polysaccharide are distinct glycans, rather than representing domains within a single polysaccharide chain. Monoclonal antibodies against the 2c antigen indicated that only LPS molecules with the longest O-polysaccharide chains contained the 2c epitope.     

Diffusion and consumption of oxygen in the resting frog sartorius muscle

Adaptations of the method of Takahashi et al. (1966. J. Gen. Physiol. 50:317-333) were used to test the validity of the one-dimensional diffusion equation for O2 in the resting excised frog sartorius muscle. This equation is: (formula: see text) where x is the distance perpendicular to the muscle surface. t is time, P(x, t) is the partial pressure of O2,D and alpha are the diffusion coefficient and solubility for O2 in the tissue, and Q is the rate of O2 consumption. P(O, t), the time-course of PO2 at one muscle surface, was measured by a micro-oxygen electrode. Transients in the PO2 profile of the muscle were induced by two methods: (a) after an equilibration period, one surface was sealed off by a disc in which the O2 electrode was embedded; (b) when PO2 at this surface reached a steady state, a step change was made in the PO2 at the other surface. With either method, the agreement between the measured P(O, t) and that predicted by the diffusion equation was excellent, making possible the calculation of D and Q. These two methods yielded statistically indistinguishable results, with the following pooled means (+/- SEM): (formula: see text) At each temperature, D was independent of muscle thickness (range, 0.67-1.34 mm). The activation energy (EA) for diffusion of oxygen in muscle was -3.85 kcal/mol, which closely matches the corresponding value in water. Together with absolute values of D in water taken from the literature, the present data imply that (Dmuscle/DH2O) is in the range 0.59-0.69. This value, and that of EA, are in agreement with the theory of Wang (1954, J. Am. Chem. Soc. 76:4755-4763), suggesting that with respects to the diffusion of O2, to a useful approximation, frog skeletal muscle may be considered simply as a homogeneous protein solution.     

Composition, structure, and biological properties of lipopolysaccharides from different strains of Pseudomonas syringae pv. atrofaciens

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C10:0, C12:0, 2-HO-C12:0, 3-HO-C12:0, C16:0, C16:1, C18:0, and C18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L- (two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substitute. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.     

The structure of the glycopeptides from the fish pathogen Flavobacterium columnare

Proteolytic digestion of the phenol-water extraction product of the fish pathogen Flavobacterium columnare afforded a mixture of glycopeptides in which the oligosaccharide moiety was an unusual hexasaccharide composed of 4-O-methyl-2-acetamido-2-deoxy-D-glucuronic acid (GlcNAcA), D-glucuronic acid (D-GlcA), 2,3-di-O-acetyl-D-xylose (D-Xyl), 2-O-methyl-D-glucuronic acid (D-GlcA), D-mannose (D-Man), and 2-O-methyl-L-rhamnose (L-Rha). By the application of high-resolution 1D and 2D NMR, mass spectrometry, and chemical analysis, the hexasaccharide structure was determined to be: [carbohydrate structure--see text] where all monosaccharides have the D-configuration except for 2-O-methyl-L-rhamnose; and were in the pyranose form. Only one carbohydrate structure was found. The peptide part was represented by tri- to hepta-peptides with a minimal common tripeptide fragment Asp-Ser-Ala, extended with Ala and Val.     

Hypoxic reperfusion of the ischemic heart and oxygen radical generation

Postischemic myocardial contractile dysfunction is in part mediated by the burst of reactive oxygen species (ROS), which occurs with the reintroduction of oxygen. We hypothesized that tissue oxygen tension modulates this ROS burst at reperfusion. After 20 min of global ischemia, isolated rat hearts were reperfused with temperature-controlled (37.4 degrees C) Krebs-Henseleit buffer saturated with one of three different O2 concentrations (95, 20, or 2%) for the first 5 min of reperfusion and then changed to 95% O2. Additional hearts were loaded with 1) allopurinol (1 mM), a xanthine oxidase inhibitor, 2) diphenyleneiodonium (DPI; 1 microM), an NAD(P)H oxidase inhibitor, or 3) Tiron (10 mM), a superoxide scavenger, and were then reperfused with either 95 or 2% O2 for the first 5 min. ROS production and tissue oxygen tension were quantitated using electron paramagnetic resonance spectroscopy. Tissue oxygen tension was significantly higher in the 95% O2 group. However, the largest radical burst occurred in the 2% O2 reperfusion group (P < 0.001). Recovery of left ventricular (LV) contractile function and aconitase activity during reperfusion were inversely related to the burst of radical production and were significantly higher in hearts initially reperfused with 95% O2 (P < 0.001). Allopurinol, DPI, and Tiron reduced the burst of radical formation in the 2% O2 reperfusion groups (P < 0.05). Hypoxic reperfusion generates an increased ROS burst originating from multiple pathways. Recovery of LV function during reperfusion is inversely related to this oxygen radical burst, highlighting the importance of myocardial oxygen tension during initial reperfusion.     

Trends in the Epidemiology of Pandemic and Non-pandemic Strains of Vibrio parahaemolyticus Isolated from Diarrheal Patients in Kolkata,India

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.     

A comparative study of the effects of molsidomine and 3-morpholinosydnonimine on the redox status of rat erythrocytes and reticulocytes

After enzymic biotransformation, molsidomine (MO) acts via the metabolite 3-morpholinosydnonimine (SIN-1) through spontaneous liberation of nitric oxide (NO) and superoxide (O(2)(.-)). The aim of this study was to compare the effects of MO and its active metabolite SIN-1 on the redox status of rat erythrocytes and reticulocytes. Rat erythrocyte as well as reticulocyte-rich red blood cell (RBC) suspensions were aerobically incubated (2 h, 37 degrees C) without (control) or in the presence of different concentrations of MO or SIN-1. In rat erythrocytes, biotransformation of MO resulted in the production of NO and nitroxyl (NO(-)). Endogenous superoxide anion (O(2)(.-)) participated in peroxynitrite generation. SIN-1 simultaneously liberated NO and O(2)(.-), which formed peroxynitrite (at least in part), but the liberated NO predominantly reacted with haemoglobin, forming methaemoglobin in erythrocytes. In reticulocytes, MO and SIN-1 caused an increase in the levels of both nitrite and 3-nitrotyrosine (an indicator of peroxynitrite), whereas they decreased the level of O(2)(.-). In reticulocytes, MO was metabolized into SIN-1 which led to the generation of NO, which reacted with O(2)(.-) (endogenous or exogenous) forming reactive nitrogen species. In conclusion, there are two metabolic pathways for MO biotransformation: one causing NO and NO(-) generation predominantly in erythrocytes and the other, via SIN-1 metabolism, in reticulocytes. The main difference between the action of MO and SIN-1 was that the latter caused oxidative damage in RBCs.     

Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.     

Structural determination of the O-antigenic polysaccharide from Escherichia coli O166

The O-antigen of the lipopolysaccharide from Escherichia coli O166 has been determined by component analysis together with 1D and 2D NMR spectroscopy techniques. The polysaccharide has pentasaccharide repeating units consisting of D-glucose (1), D-galactose (2) and N-acetyl-D-galactosamine (2) with the following structure: [STRUCTURE: SEE TEXT]. In the 1H NMR, spectrum resonances of low intensity were observed. Further analysis of these showed that they originate from the terminal part of the polysaccharide, thereby revealing that the repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.     

Preliminary structure determination of the capsular polysaccharide of Vibrio cholerae O139 Bengal Al1837.

Vibrio cholerae O139 Bengal has recently been identified as a cause of epidemic cholera in Asia. In contrast to V. cholerae O1, V. cholerae O139 Bengal has a polysaccharide capsule. As determined by high-performance anion-exchange chromatography and 1H nuclear magnetic resonance analysis, the capsular polysaccharide of V. cholerae O139 Bengal strain Al1837 has six residues in the repeating subunit; this includes one residue each of N-acetylglucosamine, N-acetylquinovosamine (QuiNAc), galacturonic acid (GalA), and galactose and two residues of 3,6-dideoxyxylohexose (Xylhex). The proposed structure is [formula: see text]     

Sequences from myeloblastosis-associated virus MAV-2(O) and UR2AV involved in the formation of plaques and the induction of osteopetrosis, anemia, and ataxia.

Recombinant viruses were made between myeloblastosis-associated virus MAV-2(O) and UR2AV to examine the relationship between regions of the MAV-2(O) genome and disease induction. The env-long terminal repeat (LTR) portion of MAV-2(O), when substituted into UR2AV, was sufficient to induce osteopetrosis identical to that caused by the parent MAV-2(O). When this region was reduced to the gp37 and LTR of MAV-2(O), osteopetrosis more severe than that caused by the parent virus was induced. Recombinant viruses that contained all or part of the MAV-2(O) env gene in the absence of the MAV-2(O) LTR induced a severe, chronic anemia and late-onset osteopetrosis, leading to the conclusion that the MAV-2(O) LTR, in addition to env, was required for rapid induction of osteopetrosis. A viral recombinant, pEU, which contained the gp85 segment of UR2AV substituted into MAV-2(O), induced an ataxia/cerebellar dysfunction not seen during infection with the other chimeric or parent viruses. In vitro studies of the parent and recombinant viruses demonstrated that the ability to form plaques on chicken embryo fibroblasts correlated with the presence of the MAV-2(O) gp37 and LTR except for construct pEU. When the viruses were inoculated into 10-day-old chickens, chimeras containing the env-LTR of gp37-LTR region of MAV-2(O) induced severe regenerative anemia similar to that induced by MAV-2(O). pEU was the exception, suggesting that the unique configuration of this chimera is responsible for its unusual pathogenic properties.