Synthesis of thiaazaheterocycle nucleoside analogues.

The syntheses of thiazinone, thiazinedione and thiazolinone base modified nucleoside analogues have been discussed in both the deoxy- and ribosyl series. Both inter- and intramolecular N-glycosylations were evaluated.

Synthesis of 3'-O2-(azaheterocycle)-thymidines.

The synthesis of 3'-O2-(azaheterocycle)-thymidines is presented from 1-thia-3-aza- 1,3-butadiene precursors (N-thioacylamidines). A variety of heterocycles is accessible using the dienic, the electrophilic or the nucleophilic reactivity of these thia-azabutadiene systems. 3'-O2-(azaheterocycle)-thymidine analogues are regarded as potential substrates to interfere with the DNA-polymerization process.

A novel synthesis of alpha-D-Galp-(1-->3)-beta-D-Galp-1-O-(CH2)3-NH2, its linkage to activated matrices and absorption of anti-alphaGal xenoantibodies by affinity columns.

Pig organs transplanted into primates are rapidly rejected because of the interaction between Gal alpha(1-->3)Gal epitopes carried by the graft and natural antibodies (anti-alphaGal antibodies) present in the blood of the recipient. This report describes a simplified synthesis of the xenogeneic disaccharide and its linkage to activated gel matrices. The digalactosides alpha-D-Galp-(1-->3)-alpha,beta-D-Galp-OAll were synthesized by the condensation of the trichloroacetimidoyl 2,3,4,6-tetra-O-benzyl-beta-D-galactopyranoside donor with the 3,4-unprotected allyl 2,6-di-O-benzyl-alpha- or beta-D-galactopyranoside acceptor precursor. Deallylation and hydrogenolysis led to the free digalactoside, whereas hydrogenolysis alone resulted in the 1-O-propyl digalactoside. Both products were tested by inhibition ELISA of natural anti-Gal alpha(1-->3)Gal antibodies. The alpha-D-Galp-(1-->3)-beta-D-Galp-OPr was found to be the best inhibitor. Thus, the allyl group of the partially benzylated alpha-D-Galp-(1-->3)-beta-D-Galp-OAll was engineered, via the hydroxy-, the tosyloxy- and the azidopropyl intermediates, into an aminopropyl group amenable to binding to N-hydroxysuccinimide-activated agarose gel matrices in order to obtain specific immunoabsorption columns. Columns made of gel substituted with 5 micromol of disaccharide per milliliter were found efficient for the immunoabsorption of anti-alphaGal antibodies from human plasma.

Effects of lorazepam on emotional reactivity, performance, and vigilance in subjects with high or low anxiety.

This study examined the hypothesis that low doses of lorazepam modify emotional response. In accord with the results of prior studies that suggest a differential effect of benzodiazepines according to the subjects' anxiety level, the authors tested the effect of lorazepam (0.5 mg twice daily) on 2 groups of 32 subjects: those with high anxiety (HA) and those with low anxiety (LA). These groups were formed a priori on the basis of their scores on the Cattell Anxiety Scale and the Hamilton Rating Scale for Anxiety. The two groups were evaluated for psychomotor function and vigilance (visual analog scales [VAS], digit-symbol substitution test [DSST], and choice reaction time [CRT]), as well as emotional reactivity. Six emotions (fear, anger, disgust, sadness, joy, and neutral state) were induced by the presentation of six movie excerpts, and subjects' emotional responses were measured using the Differential Emotions Scale. The results suggest that at the doses studied, lorazepam led to an increase in negative emotions and a decrease in positive emotions, compared with placebo. This shift of emotional reactivity toward more negative emotions was slightly stronger with the HA than with the LA subjects. However, no reliable differences in the levels of performance and vigilance (CRT, DSST, and VAS) were observed as a function of either treatment or subject group. These findings suggest a possible relationship between benzodiazepine effects and subjects' anxiety level.

Naloxone potentiates the anxiolytic but not the amnestic action of chlordiazepoxide in C57BL/6 mice.

This study aimed to investigate the effects of the opiate antagonist naloxone (10 mg/kg) on the action of a subeffective dose of chlordiazepoxide. C57BL/6 mice were tested for either anxiety, using an elevated plus maze, emotional memory in a passive avoidance paradigm or spatial memory in a radial maze. In the elevated plus maze, chlordiazepoxide 2.5 mg/kg was ineffective per se but, when combined with naloxone, it increased the proportion of open-arm entries, as did a higher dose of chlordiazepoxide (5 mg/kg). Closed-arm entries were increased with chlordiazepoxide (2.5 mg/kg) injected alone but naloxone, alone or combined with chlordiazepoxide, did not modify this measure. In the passive avoidance test, chlordiazepoxide 2.5 mg/kg decreased the latency to enter a compartment associated with an electric foot-shock, while a dose of 1.25 mg/kg, alone or in combination with naloxone, was ineffective. Finally, in the radial arm maze, chlordiazepoxide (5 mg/kg) induced amnesia. However, a dose of 2.5 mg/kg, alone or with naloxone, had no effect on learning. Naloxone elicited no intrinsic action in any of these behavioural models. It can be concluded that naloxone potentiates the anxiolytic but not the amnestic action of chlordiazepoxide.

Evaluation of protective efficacy of an Actinobacillus pleuropneumoniae serotype 1 lipopolysaccharide-protein conjugate in mice.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The major adhesin of A. pleuropneumoniae has previously been identified as a lipopolysaccharide (LPS), and more recently, we demonstrated that high molecular mass LPS were involved in A. pleuropneumoniae adherence to porcine respiratory tract cells. We postulated that immunization with a LPS-based vaccine may confer a protective immunity. The high molecular mass O-polysaccharides obtained after acid hydrolysis and chromatographic separation were conjugated to bovine serum albumin (BSA) as a protein carrier. Groups of mice were injected twice with the following antigen preparations: whole-cell preparation, outer membrane preparation, O-polysaccharide-BSA conjugate, hydrolyzed LPS and phenol/water extracted LPS. A combination of different adjuvants was also used during these immunization procedures to induce a stronger immunological response to the polysaccharide antigen. Two weeks after the second injection, the mice were challenged intranasally with either homologous A. pleuropneumoniae serotype 1 strain or a serotype 5 strain. The highest survival rate, up to 80%, compared to the control groups (P < 0.05), was recorded when the mice were injected twice with 15 micrograms of carbohydrates of O-polysaccharide-BSA conjugate mixed with the saponin-derived adjuvant Quil A. Survival rates of between 60 and 70%, twice those observed in the control groups immunized with PBS, were recorded in mice injected with the O-polysaccharide-BSA conjugate mixed with other adjuvant preparations such as alhydrogel, peanut oil and Freund's incomplete adjuvant. However, the protection induced by the conjugate antigen preparation was serotype specific, because mice challenged with a serotype 5 strain were killed. Taken together, these results confirm the important role of A. pleuropneumoniae LPS in pathogenesis.

Examination of surface polysaccharides of Actinobacillus pleuropneumoniae serotype 1 grown under iron-restricted conditions.

We investigated the expression of important Actinobacillus pleuropneumoniae surface polysaccharides, namely, capsular polysaccharides (CPS) and lipopolysaccharides (LPS), after growth under iron-restricted conditions. Iron restriction did not seem to affect the production of CPS, as determined by labelling with a monoclonal antibody (mAb) against the serotype 1 K-antigen and flow cytometry analysis, and also as determined by electron microscopy. SDS-PAGE revealed that the LPS profiles of these cells were also unaffected by iron restriction. Using flow cytometry analysis, however, we observed that binding of mAb against serotype 1 O-antigen was altered in cells of A. pleuropneumoniae serotype 1 reference strain (4074) grown under iron-restricted conditions. This strain exhibited two subpopulations with distinct patterns of reactivity with the mAb against the O-antigen. When strain 4074 was grown under iron-restricted conditions, a shift from one cell subpopulation (moderately fluorescent) to another cell subpopulation (highly fluorescent, thus binding more antibodies) was observed. Our results indicate that growth of A. pleuropneumoniae serotype 1 under iron-restricted conditions did not seem to affect CPS production, but might alter, at least for the reference strain, the expression of LPS.

Purification and characterization of a 52-kilodalton immunoglobulin G-binding protein from Streptococcus suis capsular type 2.

We previously reported that group D streptococci exhibited immunoglobulin G (IgG)-binding activity and that a 52-kDa IgG-binding protein was present in all Streptococcus suis strains examined (B. Serhir, R. Higgins, B. Foiry, and M. Jacques, J. Gen. Microbiol. 139:2953-2958, 1993). The objective of the present study was to purify and characterize this protein. Pig IgG were immobilized through their Fab fragments to ECH-Sepharose 4B, and the protein was purified by affinity chromatography. Electron microscopy observations of the purified material showed filamentous structures with a diameter of approximately 4 nm; these structures were not observed when the material was treated with either urea or ethanolamine. Electrophoretic and Western immunoblot analyses showed that the 52-kDa protein constituted the bulk of the recovered material. This protein was stained with either Coomassie brilliant blue or silver nitrate; it reacted with a large variety of mammalian IgG, human IgG (Fc) fragments, human IgA, and other human plasma proteins. The 52-kDa protein exhibited lower IgG-binding affinities than protein A and protein G. However, it was able to compete with protein A and protein G for binding to human IgG. In addition, it bound chicken IgG with high affinity. This last property differentiated the 52-kDa protein of S. suis from the six IgG-binding proteins described to date. The 52-kDa protein displayed similar affinities for untreated and deglycosylated pig IgG. The N-terminal amino acid sequence (SIITDVYAXEVLDSXGNPTLEV) revealed no homology with any bacterial proteins in the Swiss-Prot database. Its isoelectric point of approximately 4.6 and its amino acid composition, rich in aspartic and glutamic acids, showed that it had some similarities with other IgG-binding proteins. In this report, we have purified and characterized a 52-kDa IgG-binding protein from S. suis capsular type 2. Although this protein shares some similarities with other IgG- and/or IgA-binding proteins, it is unique in reacting with chicken IgG.

High-molecular-mass lipopolysaccharides are involved in Actinobacillus pleuropneumoniae adherence to porcine respiratory tract cells.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The major adhesin of A. pleuropneumoniae has been identified as the lipopolysaccharides (LPSs) (M. Bélanger, D. Dubreuil, J. Harel, C. Girard, and M. Jacques, Infect. Immun. 58:3523-3530, 1990). Using immunoelectron microscopy and flow cytometry, we showed in the present study that LPSs were well exposed at the surface of this encapsulated microorganism. Immunolocalization with porcine lung and tracheal frozen sections showed that extracted LPS bound to the lung mesenchyme and vascular endothelium and to the tracheal epithelium, respectively. Inhibition of adherence of A. pleuropneumoniae with extracted LPS was also performed with lung and tracheal frozen sections. Acid hydrolysis of LPS revealed that the active component of LPS was not lipid A but the polysaccharides. LPSs from A. pleuropneumoniae serotypes 1 and 2 were separated by chromatography on Sephacryl S-300 SF, in the presence of sodium deoxycholate, according to their molecular masses. The adherence-inhibitory activity was found in the high-molecular-mass fractions. These high-molecular-mass fractions contained 2-keto-3-deoxyoctulosonic acid and neutral sugars, and they were recognized by a monoclonal antibody directed against A. pleuropneumoniae O antigen but not recognized by a monoclonal antibody against capsular antigen.

Chromosomal analysis of gregarious oviposition by Drosophila melanogaster.

The contribution of different chromosomes to variation in the gregarious oviposition behavior of Drosophila melanogaster females was elucidate by a chromosome substitution analysis. Lines of D. melanogaster previously selected for more than 190 generations for high or low levels of gregarious oviposition were crossed to a tester strain having genetically marked inversion-containing chromosomes. Genes influencing gregarious oviposition behavior are distributed over chromosome II and III. Interactions among the chromosomes were negligible as measured in this experiment. The differences in the gregarious oviposition performance of these two selected lines is due mainly to the accumulation of factors for high gregarious oviposition on chromosomes II and factors for low gregarious oviposition on chromosome III.

Proteins found within porcine respiratory tract secretions bind lipopolysaccharides of Actinobacillus pleuropneumoniae.

Affinity for porcine respiratory tract secretions was found in some isolates of Actinobacillus pleuropneumoniae and involved lipopolysaccharides (LPS) (M. Bélanger, S. Rioux, B. Foiry, and M. Jacques, FEMS Microbiol. Lett. 97:119-126, 1992). In the present study, the affinity for a crude preparation of porcine respiratory tract mucus of isolates of the Pasteurellaceae family, i.e., Actinobacillus, Haemophilus, and Pasteurella spp., and of some unrelated gram-negative bacteria was examined. Affinity for crude porcine respiratory tract mucus was not a property shared by all Pasteurellaceae isolates tested. Furthermore, affinity for the porcine crude mucus preparation was not unique to the Pasteurellaceae group and did not seem to be restricted to bacteria originating from pigs. Different surface properties of A. pleuropneumoniae isolates in relation to their adherence to crude mucus were examined. The capsular layer seemed to mask the adhesin and interfered with adherence to crude mucus. Two poorly capsulated isolates, which had a more hydrophobic surface and bound Congo red, were also heavily labeled by gold particles coated with polymyxin, which is known to interact with the lipid A-core region of LPS, and adhered strongly to respiratory tract secretions. Tetramethylurea, charged polymers, divalent cations, chelators, monosaccharides and amino sugars, or lectins were unable to inhibit adherence of A. pleuropneumoniae to the crude mucus preparation. To identify the receptor(s) recognized by the lipopolysaccharidic adhesin of A. pleuropneumoniae, affinity chromatography was used. Two bands, which were proteinaceous in nature, of 10 and 11 kDa were recovered. Our results suggest that two low-molecular-mass proteins present in porcine respiratory tract secretions bind A. pleuropneumoniae LPS.

A species-specific periplasmic flagellar protein of Serpulina (Treponema) hyodysenteriae.

We have previously reported that a 46-kDa protein present in an outer membrane protein preparation seemed to be a species-specific antigen of Serpulina hyodysenteriae (Z. S. Li, N. S. Jensen, M. Bélanger, M.-C. L'Espérance, and M. Jacques, J. Clin. Microbiol. 30:2941-2947, 1992). The objective of this study was to further characterize this antigen. A Western blot (immunoblot) analysis and immunogold labeling with a monospecific antiserum against this protein confirmed that the protein was present in all S. hyodysenteriae reference strains but not in the nonpathogenic organism Serpulina innocens. The immunogold labeling results also indicated that the protein was associated with the periplasmic flagella of S. hyodysenteriae. N-terminal amino acid sequencing confirmed that the protein was in fact a periplasmic flagellar sheath protein. The molecular mass of this protein, first estimated to be 46 kDa by Western blotting, was determined to be 44 kDa when the protein was evaluated more precisely by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was glycosylated, as determined by glycoprotein staining and also by N-glycosidase F treatment. Five other periplasmic flagellar proteins of S. hyodysenteriae, which may have been the core proteins and had molecular masses of 39, 35, 32, 30, and 29 kDa, were antigenically related and cross-reacted with the periplasmic flagellar proteins of S. innocens. Finally, serum from a pig experimentally infected with S. hyodysenteriae recognized the 44-kDa periplasmic flagellar sheath protein. Our results suggest that the 44-kDa periplasmic flagellar sheath protein of S. hyodysenteriae is a species-specific glycoprotein antigen.

Identification of partial agonists with low intrinsic activity at the inositol-1,4,5-trisphosphate receptor.

The interactions of synthetic L-chiro-inositol-2,3,5-trisphosphorothioate [L-ch-Ins(2,3,5)PS3] and D-6-deoxy-myo-inositol-1,4,5-trisphosphorothioate [D-6-deoxy-Ins(1,4,5)PS3] with D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptors have been examined using radioligand binding assays and Ca2+ mobilization from permeabilized SH-SY5Y cells. The ability of these analogues to compete with [3H]Ins(1,4,5)P3 for specific sites on adrenal cortical membranes indicated that, although weaker than Ins(1,4,5)P3, both ligands competed fully for these sites [L-ch-Ins(2,3,5)PS3,Ki = 0.5 microM; D-6-deoxy-Ins(1,4,5)PS3,Ki = 5.3 microM]. However, in assays examining the amount of Ca2+ mobilized from the stores of permeabilized SH-SY5Y cells, both of these synthetic analogues displayed low intrinsic activity [L-ch-Ins(2,3,5)PS3, 34%; D-6-deoxy-Ins(1,4,5)PS3, 42% of that of Ins(1,4,5)P3]. Moreover, L-ch-Ins(2,3,5)PS3 and D-6-deoxy-Ins(1,4,5)PS3 were able to inhibit the response to Ins(1,4,5)P3 with Ki values (6 microM and 33 microM, respectively) virtually identical to their EC50 values for Ca2+ release. This is consistent with partial agonist behavior, because these compounds exhibit low maximal responses when the extent of Ca2+ release is examined. These compounds represent the first examples of inositol-based analogues with low intrinsic activity and may point the way towards the design of selective antagonists at Ins(1,4,5)P3 receptors. It also seems probable that these may represent the first true affinity values of inositol phosphates at the active receptor.

A diallel analysis of gregarious oviposition in Drosophila melanogaster.

Drosophila females exhibit gregarious oviposition. This behaviour was analysed by a diallel cross. The trait shows considerable additive genetic variation and a significant dominant effect. A variance and covariance analysis suggests that ebony and taxi strains contain the most dominant alleles. Some maternal effect may also be present. This genetical architecture is in line with previous studies of selection and hybridization of the lines selected for high and low gregarious oviposition in Drosophila melanogaster females.

A sandwich enzyme-linked immunosorbent assay for the detection of Streptococcus suis.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and the identification of Streptococcus suis capsular types 1, 2, 1/2, 3 and 22. The specificity of this test was first evaluated using reference strains of S. suis capsular types 1 to 28 and 1/2 as well as 15 different bacterial species susceptible to be isolated from swine. The ELISA developed was very specific for capsular types 1, 3 and 22 but it could not discriminate between capsular types 2 and 1/2. In a second study, S. suis isolates from 328, 493, 368 and 76 diseased pigs were used to detect capsular types 1, 2 or 1/2, 3 and 22 respectively. The relative specificity and sensitivity varied between 98% and 100%. The ELISA results were in excellent agreement with the standard techniques (biochemical tests, coagglutination and capsular reaction tests) in detecting both positive and negative strains. Kappa values were 0.80, 0.99, 0.97 and 1.00 for detecting S. suis capsular types 1, 2 or 1/2, 3, and 22 respectively. To evaluate the relative-sensitivity of the test, primary cultures from 73 diseased pigs and tissue samples from 67 diseased pigs were used directly for detecting these capsular types. With primary cultures, the relative specificity and sensitivity (95.9% and 91.6% respectively) remained high and the test was very suitable (Kappa = 0.87). The ELISA using tissue samples gave a good specificity (97.6%), a moderate sensitivity (62.5%) and a low agreement with standard tests (Kappa = 0.64).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli.

The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165). The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787.

Production and characterization of two Streptococcus suis capsular type 2 mutants.

Two avirulent mutants of Streptococcus suis capsular type 2 (M2 and M42) were produced from a highly virulent strain. Mutant M2, obtained after serial subcultures of the parent strain in the presence of rabbit anti-capsular type 2 serum, no longer possessed the type-specific capsular antigen, as demonstrated by serotyping methods and immunoelectron microscopy. The Lancefield group D antigen could not be detected on the cell surface of this mutant using the immunogold labelling technique. SDS-PAGE of lysozyme treated cells demonstrated that a 44 kDa protein which was present in the parent strain, was absent in mutant M2. Immunoblotting using rabbit whole cell homologous anti-serum revealed that the protein was strongly immunogenic. Mutant M2 was totally avirulent in mice, and the homologous antiserum completely failed to protect mice against challenge with the parent strain. However, mutant M42, obtained after passages of the parent strain at 42 degrees C, remained capsulated but lacked the same 44 kDa protein as mutant M2. The quantity of sialic acid present in the capsule was similar to that of the parent strain. Despite the presence of antibodies against the capsule, antiserum prepared against M42 only partially protected mice against a challenge with the parent strain. The 44 kDa cell wall protein could act as a virulence factor as well as an important immunogen of S. suis capsular type 2.

Detection and characterization of leptospiral antigens using a biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot.

A biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of Leptospira interrogans serovars in experimentally inoculated bovine urine samples was evaluated. Immunoglobulin G (IgG) from rabbits immunized with L. interrogans serovar hardjo type hardjobovis sonicated, whole cell, and formalinized-heated antigen preparations were purified by a protein A-superose column coupled to fast protein liquid chromatography, and evaluated for species specificity in the ELISA. The ELISA using each specific IgG detected as few as 10(4) leptospires of the homologous serovar hardjo diluted in phosphate-buffered saline solution with Tween 20 (PBSS-Tween 20). On immunoblot analysis of proteinase-K-digested whole cell leptospiral preparations, each IgG revealed the presence of bands specific to serovar hardjo, suggesting the presence of serovar-specific epitopes on the lipopolysaccharide molecules. The minimum number of cells of heterologous serovars pomona, grippotyphosa, bratislava, icterohaemorrhagiae and copenhageni detected by each ELISA was greater, ranging from 10(6) to 10(7). The common antigenic determinants observed on immunoblot analysis were different for each specific IgG, except for a major cross-reacting, possibly flagellar, protein doublet at approximately 36-36.5 kDa. Leptospires were equally well detected by the ELISA in both bovine urine and PBSS-Tween 20.