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Shiga-toxin-producing Escherichia coli (STEC) is an important food-borne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. Since no vaccines are available and antibiotic treatment is not recommended because promotes the appearance of HUS symptoms, the control of STEC intestinal colonization in cows, which is an important environmental reservoir, is crucial to control this zoonosis. Here, we evaluated the adaptation of an attenuated strain of Salmonella enterica serovar Typhimurium (ΔaroA mutant) as a vaccine platform for preventing STEC intestinal colonization that was studied in a mouse model. A chimeric antigen formed by the combination of the STEC peptides EspA

The SLAPTAG is a novel molecular TAG derived from a protein domain present in the sequence of Lactobacillus acidophilus SlpA (SlpA284–444). Proteins from different biological sources, with different molecular weights or biochemical functions, can be fused in frame to the SLAPTAG and efficiently purified by the specific binding to a bacterial-derived chromatographic matrix named here Bio-Matrix (BM). Different binding and elution conditions were evaluated to set an optimized protocol for the SLAPTAG-based affinity chromatography (SAC). The binding equilibrium between SLAPTAG and BM was reached after a few minutes at 4oC, being the apparent dissociation constant (KD) of 4.3 uM, a value similar to the one determined for other S-layer proteins and their respective bacterial cell walls. A reporter protein was generated (H6-GFP-SLAPTAG ) to compare the efficiency of SAC against a commercial system based on a Ni2+-charged agarose matrix, observing no differences in the H6-GFP-SLAPTAG purification performance. The stability and reusability of the BM were evaluated, and it was determined that the matrix was stable for more than a year, being possible to reuse it five times without a significant loss in the efficiency for protein purification. Alternatively, we explored the recovery of bound SLAP-tagged proteins by proteolysis using the SLAPASE (a SLAP-tagged version of the HRV-3c protease) that released a tag-less GFP (SLAPTAG-less). Additionally, iron nanoparticles were linked to the BM and the resulting BMmag was successfully adapted for a magnetic SAC, a technique that can be potentially applied for high-throughput-out protein production and purification.

El presente estudio se establece mediante el objetivo que permite identificar la influencia de la musicoterapia como apoyo socioemocional en jóvenes con discapacidad intelectual. La metodología de esta investigación está basada en una revisión bibliográfica con análisis sistemático y enfoque cualitativo no experimental, obteniéndose como resultado que los métodos particularmente aplicados en este tipo de casos como apoyo socioemocional son empleados mediante técnicas activas, pasivas o receptivas, identificando cómo la musicoterapia optimiza los procesos cognitivos interviniendo holísticamente, estableciendo una relación en la técnica analítica que detecta, mejora y previene problemas socioemocionales en jóvenes con discapacidad intelectual, concluyendo que la musicoterapia generó cambios notables aplicando métodos receptivo y recreativo que influyen en la parte emocional, cognitiva y social.

The SLAPTAGis a novel molecular TAG derived from a protein domain present in the sequence ofLactobacillus acidophilusSlpA (SlpA284–444). Proteins from different biological sources, with different molecular weights or biochemical functions, can be fused in frame to the SLAPTAGand efficiently purified by the specific binding to a bacterial-derived chromatographic matrix named here Bio-Matrix (BM). Different binding and elution conditions were evaluated to set an optimized protocol for the SLAPTAG-based affinity chromatography (SAC). The binding equilibrium between SLAPTAGand BM was reached after a few minutes at 4°C, being the apparent dissociation constant (KD) of 4.3 µM, a value which is similar to different Kd determined for other S-layer proteins and their respective bacterial cell walls. A reporter protein was generated (H6-GFP-SLAPTAG) to compare the efficiency of the SAC against a commercial system based on a Ni2+-charged agarose matrix, observing no differences in the H6-GFP-SLAPTAGpurification performance. The stability and reusability of the BM were evaluated, and it was determined that the matrix was stable for more than a year, being possible to reuse it five times without a significant loss in the efficiency for protein purification. Alternatively, we explored the recovery of bound SLAP-tagged proteins by proteolysis using the SLAPASE(a SLAP-tagged version of the HRV-3c protease) that released a tag-less GFP (SLAPTAG-less). Additionally, iron nanoparticles were linked to the BM and the resulting BMmagwas successfully adapted for a magnetic SAC, a technique that can be potentially applied for high-throughput-out protein production and purification.

La autoestima es un conjunto de percepciones, pensamientos, evaluaciones, sentimientos y tendencias de comportamientos dirigidos hacia uno mismo, según Maslow es la que se manifiesta "en el respeto que le merecemos a otros, más que el renombre, la celebridad y la adulación”. Por otro lado, el acoso es un comportamiento agresivo y no deseado en niños, adolescentes en etapas escolares que involucra un desequilibrio de poder que se repite con el tiempo tanto en acosados como acosadores. Se efectúa esta investigación para determinar si existen diferencias significativas entre el nivel de autoestima y el acoso escolar en estudiantes del 5to año de secundaria de Lima Metropolitana según la edad cronológica, sexo e institución educativa pública o privada. El método utilizado fue hipotético, analítico y deductivo, con un tipo de investigación básica, diseño no experimental, nivel descriptivo correlacional, enfoque cuantitativo, y corte transversal. La muestra estuvo conformada por 300 estudiantes, con una muestra censal divida en 75 estudiantes para cada una de las 4 instituciones educativas. Se aplicaron dos instrumentos, Inventario de Autoestima de Coopersmith (1967) adaptado por Llerena (1995) y la Evaluación del Bullying (INSEBULL) de Avilés y Elices (2007), con una alta confiabilidad Alfa de Cronbach de 0.90 y 0.95, respectivamente obteniendo como resultado. Los estudiantes en un 75% de alguna manera se ven afectados con acoso y el 25% un nivel medio de afectación de acoso y baja autoestima, siendo esta investigación una herramienta útil para la vigilancia en cuanto a prevención de estas dificultades en estudiantes. Palabras claves: Acoso escolar, adolescentes, autoestima, instituciones educativas, secundarias.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening hemolytic-uremic syndrome (HUS). Despite the magnitude of the social and economic problems caused by HUS, no licensed vaccine or effective therapy is currently available for human use. Prevention of STEC infections continues being the most important measure to reduce HUS incidence. This is especially true for Argentina where HUS incidence among children is extremely high and shows an endemic pattern. The aim of this work was to investigate serologically adult staff of kindergartens in Buenos Aires city and suburban areas in order to detect possible carriers, and to educate personnel about good practices to reduce HUS transmission. We also assessed the microbiological quality of water and meal samples from the same kindergartens. We tested 67 healthy adults, 13 water supplies and 6 meals belonging to 6 public kindergartens. We analysed hand swabs for isolation of STEC and serum samples for the presence of antibodies against Stx and lipopolysaccharide (LPS) of O157 serogroup. We identified 46 Stx2-positive individuals, but only 7 for O157 LPS. No presence of STEC pathogens was detected in hands of staff, water or meal samples

Mesorhizobium loti MAFF303099 is a rhizobial strain that nodulates Lotus spp. A M. loti MAFF303099 mutant strain affected in the tatC gene was generated. This strain presented an altered protein secretion level to the culture supernatant and also a higher sensitivity to SDS. Its nodulation phenotype on Lotus showed the induction of small and colorless nodules, and in a larger number than those induced by the wild-type strain. In addition, these nodules presented defects in the degree of occupation by rhizobia. Two Rieske Fe/S proteins, encoded by the mll2707 and mlr0970 genes, were predicted as potential Tat substrates in M. loti MAFF303099. The transcriptional expression of mll2707 and mlr0970 genes was analyzed under different oxygen growth conditions. The mll2707 gene was expressed constitutively, while the expression of the mlr0970 gene was only detected under anaerobic and microaerophilic in vitro conditions. Both genes were down-regulated in the tatC mutant strain. mll2707 and mlr0970 mRNAs from the wild-type strain were detected in nodules. Using a translational reporter peptide fusion, we found that the Mll2707 protein was only detectable in the wild-type strain. On the other hand, although Mlr0970 protein was detected in wild-type and tatC mutant strains, its association with the membrane was favored in the wild-type strain. The tatC and the mll2707 mutant strains were affected in the cytochrome c oxidase activity. These results confirm that Mll2707 is required for cytochrome c-dependent respiration and that Tat functionality is required for the correct activity of Mll2707. The mll2707 mutant strain showed a nodulation phenotype similar to the tatC mutant strain, although it presented only a slight difference in comparison with wild-type strain in terms of nodule occupation. No defective phenotype was observed in the nodulation with the mlr0970 mutant strain. These results indicate that, of the two Rieske Fe/S proteins coded by M. loti MAFF303099, only Mll2707 expression is required for the induction of effective nodules, and that the functionality of the Tat system is necessary not only for the correct function of this protein, but also for some other protein required in an earlier stage of the nodulation process. Fil: Basile, Laura Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Zalguizuri, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Lepek, Viviana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH7) from STEC, was constructed and expressed in B. abortus δpgm vaccine strain (Babδpgm). Mice orally immunized with Babδpgm(EITH7) elicited an immune response with the induction of anti-EITH7 antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t=4 days) than mice immunized with Babδpgm carrier strain (t=12 days). As expected, mice immunized with Babδpgm(EITH7) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles-the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC. Fil: Iannino, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Herrmann, Claudia Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Roset, Mara Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Brucella ssp. is a facultative intracellular pathogen that causes brucellosis, a worldwide zoonosis that affects a wide range of mammals including humans. A critical step for the establishment of a successful Brucella infection is its ability to survive within macrophages. To further understand the mechanisms that Brucella utilizes to adapt to an intracellular lifestyle, a differential proteomic study was performed for the identification of intracellular modulated proteins. Our results demonstrated that at 48 hours post-infection Brucella adjusts its metabolism in order to survive intracellularly by modulating central carbon metabolism. Remarkably, low iron concentration is likely the dominant trigger for reprogramming the protein expression profile. Up-regulation of proteins dedicated to reduce the concentration of reactive oxygen species, protein chaperones that prevent misfolding of proteins, and proteases that degrade toxic protein aggregates, suggest that Brucella protects itself from damage likely due to oxidative burst. This proteomic analysis of B. abortus provides novel insights into the mechanisms utilized by Brucella to establish an intracellular persistent infection and will aid in the development of new control strategies and novel targets for antimicrobial therapy. Fil: Roset, Mara Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Alefantis, T. G.. Vital Probes; Estados Unidos Fil: Delvecchio, V. G.. Vital Probes; Estados Unidos Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Most pathogens infect through mucosal surfaces, and parenteral immunization typically fails to induce effective immune responses at these sites. Development of oral-administered vaccines capable of inducing mucosal as well as systemic immunity while bypassing the issues of antigen degradation and immune tolerance could be crucial for the control of enteropathogens. This study demonstrates that U-Omp19, a bacterial protease inhibitor with immunostimulatory features, coadministered with Salmonella antigens by the oral route, enhances mucosal and systemic immune responses in mice. U-Omp19 was able to increase antigen-specific production of IFN-γ and IL-17 and mucosal (IgA) antibody response. Finally, oral vaccination with U-Omp19 plus Salmonella antigens conferred protection against virulent challenge with Salmonella Typhimurium, with a significant reduction in bacterial loads. These findings prove the efficacy of this novel adjuvant in the Salmonella infection model and support the potential of U-Omp19 as a suitable adjuvant in oral vaccine formulations against mucosal pathogens requiring T helper (Th)1-Th17 protective immune responses. Fil: Risso, Gabriela Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Carabajal, Marianela Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Bruno, Laura Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Ibañez, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Coria, Mirta Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Pasquevich, Karina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Lee, Seung Joo. University of California at Davis; Estados Unidos Fil: McSorley, Stephen J.. University of California at Davis; Estados Unidos Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Cassataro, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Type III protein secretion systems, which are organelles with the capacity to deliver bacterial proteins into host cells, have been adapted to deliver heterologous antigens for vaccine development. A limitation of these antigen delivery systems is that some proteins are not amenable to secretion through this pathway. We show here that proteins from the simian and human immunodeficiency viruses that are not permissive for secretion through aSalmonella entericaserovar Typhimurium type III secretion system can be modified to travel this secretion pathway by introduction of discrete mutations. Proteins optimized for secretion were presented more efficiently via the major histocompatibility complex class I pathway and were able to induce a better immune response.

Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimurium vaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium-NY-ESO-1) through a type III protein secretion system. The S. typhimurium-NY-ESO-1 construct elicited NY-ESO-1-specific CD8+ and CD4+ T cells from peripheral blood lymphocytes of cancer patients in vitro. Oral administration of S. typhimurium-NY-ESO-1 to mice resulted in the regression of established NY-ESO-1-expressing tumors. Intratu-moral inoculation of S. typhimurium-NY-ESO-1 to NY-ESO-1-negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1-spedific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimurium type III secretion system is a promising novel strategy for cancer vaccine development.

Brucella abortus cyclic glucan synthase (Cgs) is a 320-kDa (2868-amino acid) polytopic integral inner membrane protein responsible for the synthesis of the virulence factor cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. Cgs functions as an inverting processive beta-1,2-autoglucosyltransferase and has the three enzymatic activities required for the synthesis of the cyclic glucan: initiation, elongation, and cyclization. To gain further insight into the protein domains that are essential for the enzymatic activity, we have compared the Cgs sequence with other glycosyltransferases (GTs). This procedure allowed us to identify in the Cgs region (475-818) the widely spaced D, DxD, E/D, (Q/R)xxRW motif that is highly conserved in the active site of numerous GTs. By site-directed mutagenesis and in vitro and in vivo activity assays, we have demonstrated that most of the amino acid residues of this motif are essential for Cgs activity. These sequence and site-directed mutagenesis analyses also indicate that Cgs should be considered a bi-functional modular GT, with an N-terminal GT domain belonging to a new GT family related to GT-2 (GT-84) followed by a GH-94 glycoside hydrolase C-terminal domain. Furthermore, over-expression of inactive mutants results in wild-type (WT) production of cyclic glucan when bacteria co-express the mutant and the WT form, indicating that Cgs may function in the membrane as a monomeric enzyme. Together, these results are compatible with a single addition model by which Cgs acts in the membrane as a monomer and uses the identified motif to form a single center for substrate binding and glycosyl-transfer reaction.

Central to the study of type III secretion systems is the availability of reporter systems to monitor bacterial protein translocation into host cells. We report here the development of a bacteriophage P1 Cre recombinase-based system to monitor the translocation of bacterial proteins into mammalian cells. Bacteriophage P1 Cre recombinase fused to the secretion and translocation signals of Salmonella enterica serovar Typhimurium of the type III secreted protein SopE was secreted in a type III secretion system-dependent fashion. More importantly, the SopE-Cre chimera was translocated into host cells via the type III secretion system and activated the expression of luciferase and green fluorescent protein reporters of Cre recombinase activity.

An indirect enzyme-linked immunosorbent assay (IELISA), a competitive ELISA (CELISA), and a fluorescence polarization assay (FPA) for the presumptive serological diagnosis of swine brucellosis were evaluated using two populations of swine sera: sera from brucellosis-free Canadian herds and sera from Argentina selected based on positive reactions in the buffered antigen plate agglutination test (BPAT) and the 2-mercaptoethanol (2-ME) test. In addition, sera from adult swine from which Brucella suis was isolated at least once for each farm of origin were evaluated. The IELISA, CELISA, and FPA specificity values were 99.9, 99.5, and 98.3%, respectively, and the IELISA, CELISA, and FPA sensitivity values relative to the BPAT and the 2-ME test were 98.9, 96.6, and 93.8%, respectively. Actual sensitivity was assessed by using 37 sera from individual pigs from which B. suis was cultured, and the values obtained were as follows: BPAT, 86.5%; 2-ME test, 81.1%; IELISA, 86.5%; CELISA, 78.5%; and FPA, 80.0%.

The animal pathogen Brucella abortus contains a gene, cgs , that complemented a Rhizobium meliloti nodule development ( ndvB ) mutant and an Agrobacterium tumefaciens chromosomal virulence ( chvB ) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens , and nitrogen fixation in R. meliloti ; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn 3 -HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium , are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn 3 -HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor in Brucella infection.

An indirect enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Brucella ovis infection was developed. The assay uses a mouse monoclonal antibody to bovine IgG 1 horseradish peroxidase (HRPO) conjugate that cross-reacts with immunoglobulin from sheep and a purified antigen from Brucella ovis . The ELISA data were read and analyzed according to a targeting procedure. The ELISA results were compared with a cold complement fixation test (CFT). Sera from 675 rams from three uninfected flocks were used to determine the ELISA cut-off value (O.D. 405 nm: 0.095) and the diagnostic specificity of the ELISA (100%) and the CFT (99.69% ± 0.42). The ELISA cut-off value was corroborated by receiver operating characteristic (ROC) analysis. Six hundred and forty semen and serum samples from 419 rams from two naturally infected flocks were collected before and after mating-time during two consecutive years. All semen samples were cultured and Brucella ovis was isolated from 28 samples. Sera from the 28 rams with positive semen were used to determine the diagnostic sensitivity of the ELISA (96.43% ± 6.8) and of the CFT (including suspected positive samples with titers of 1:5; 88.89% ± 11.85). Considering the CFT suspicious and the anti-complementary reactions as positive resulted in a diagnostic sensitivity value of 89.28% ± 11.46. Six hundred and ten serum samples from the 640 sera were used to determine relative sensitivity (excluding sera with 1:5) at: ELISA/CFT 97.26% ± 3.74 and CFT/ELISA was 71.72% ± 8.87. The percent agreement, beyond chance measured by the Kappa index was 79.7. Relative sensitivity ELISA/CFT (including 1:5 titers in the CFT as positive) was 94.9% ± 4.83 and CFT/ELISA was 72.84% ± 8.59. The Kappa index was 79.4.

DRESS syndrome (Drug Reaction with Eosinophilia and Systemic Symptoms) or reaction to drugs with eosinophilia and systemic symptoms is a serious drug reaction associated with the use of aromatic anticonvulsants and allopurinol. At least 44 drugs have been associated with DRESS. The aim was to present the case of a patient with DRESS syndrome associated with phenytoin.a 20 year old woman, with a history of seizures since childhood, presented generalised tonic-clonic seizures for the last three months. Therefore, she began treatment with 100 mg of phenytoin, administered orally, every 8 hours. Three weeks later, she developed fever up to 42 degrees, papules in the hands extending to trunk and extremities, generalized rubicund, pruritus, pain while urinating, adding hyperoxia, dysphagia and dry cough. Consequently, she went to the emergency room.the diagnosis is clinical and it is set according to the criteria of the scale of RegiSCAR. As the initial manifestations are unspecific, the diagnosis and treatment could be delayed. The importance of recognizing this syndrome is an early treatment to get better prognostics. The mortality is up to 10 %.Introducción: el síndrome de DRESS (Drug Reaction with Eosinophilia and Systemic Symptoms) o la reacción a fármacos con eosinofilia y síntomas sistémicos es una reacción medicamentosa grave, asociada al uso de anticonvulsivos aromáticos y alopurinol. Se han descrito por lo menos 44 fármacos asociados a DRESS. El propósito es presentar el caso clínico de una paciente con síndrome de DRESS asociado a fenitoína. Caso clínico: paciente femenina de 20 años, con antecedente de crisis convulsivas desde la infancia, durante tres meses previos presentó crisis convulsivas tónico-clónicas generalizadas, por lo que inició tratamiento con fenitoína: 100 mg vía oral cada 8 horas. Tres semanas después presentó fiebre de hasta 42 grados, pápulas en manos con extensión a tronco y extremidades, rubicundez generalizada, prurito, dolor al orinar, además de hiporexia, disfagia y tos seca, por lo que acudió al servicio de urgencias. Discusión: el diagnóstico es clínico y se establece según los criterios de la escala de RegiSCAR. Debido a que las manifestaciones iniciales son poco específicas, el diagnóstico y el tratamiento definitivo pueden retrasarse. La importancia del reconocimiento y tratamiento temprano de esta entidad radica en la incidencia de mortalidad de hasta 10 %.

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella . To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus Δ cypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus Δ cypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus Δ cypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells.

Conservative treatment in severe preeclampsia has been documented by several authors citing significant improvement in neonatal outcome lacking a significant increase in maternal complications.Our objective was to inform of our preliminary results using protocolized conservative management in women with preeclampsia-eclampsia, favoring better neonate conditions.We included 34 patients with average age of 28.2 years with documented severe preeclampsia-eclampsia complicating a 36-weeks or less pregnancy, admitted in the obstetric intensive care unit (OICU) between October 2001 and February 2002. Patients received protocolized management consisting of intravascular colume expansion, anti-hypertensive control, target organ protection, monitoring, and clinical observation. We considered conservative management as a 24 or more period offered to patients with satisfactory response to medical treatment and no evidence of binomial compromise.Of our group, 85% corresponded to severe preeclampsia, 9% to eclampsia, 3% to imminence of eclampsia, and 3% to HELLP syndrome. Average stay in OICU was 5.5 days with 3.5 days average management before pregnancy was interrupted. These patients presented mean gestational age of 32.8 weeks during which we observed anemia, low platelets, D dimmer increments, MAP average of 112.8, PCOc 18.6, and BI 0.15. We obtained 36 live newborns of whom 12% four died, two were extremely immatures (510 g and 600 g, respectively); one 980-g newborn presented intraventricular hemorrhage, and a 1,450-g newborn had multiple organ failure. Conservative treatment in patients with severe preeclampsia-eclampsia is a feasible alternative in hospitals with an ICU. Conservative management can improve neonatal survival and prognosis in preterm newborns.

Salmonella typhimurium engineered to deliver cancer/testis antigen NY-ESO-1 through type III secretion (S typhimurium–NY-ESO-1) was shown to be an efficient cancer vaccine construct in mice and to stimulate NY-ESO-1–specific CD8+/CD4+ T cells in vitro in patients with cancer with NY-ESO-1 spontaneous immunity. We also showed that individuals without spontaneous immunity to NY-ESO-1 had specific CD4+ T-cell precursors with high avidity to NY-ESO-1 under tight control by CD4+CD25+ regulatory T (Treg) cells. We now found that in healthy donors and patients with melanoma without NY-ESO-1 spontaneous immunity, S typhimurium–NY-ESO-1 elicits CD4+ T helper 1 (Th1) cells in vitro recognizing naturally processed antigen from these high-avidity NY-ESO-1–specific naive precursors. In contrast to peptide stimulation, induction of specific Th1 cells with S typhimurium–NY-ESO-1 did not require in vitro depletion of CD4+CD25+ Treg cells, and this prevailing effect was partially blocked by disruption of interleukin-6 or glucocorticoid-induced TNF receptor (GITR) signals. Furthermore, S typhimurium–induced Th1 cells had higher GITR expression than peptide-induced Th1 cells and were resistant to suppression by CD4+CD25+ Treg cells in a GITR-dependent fashion. We propose that S typhimurium–NY-ESO-1 induces antigen-specific T-cell responses that are resistant to suppression by CD4+CD25+ Treg cells.

The prevalence of acute renal failure (ARF) in obstetric patients in our country is estimated to be between 3 and 42.8%. The most important causes are preeclampsia, especially when associated with thrombotic microangiopathy and hemolysis and less frequently to hemorrhagic shock. Early peritoneal dialysis (EPD) is the temporary treatment. For these patients, 100 % recovery in renal function was observed. When ARF is associated with multiple organ failure (MOF), the reported mortality ranges between 0 and 20 %.To describe clinical features and medical outcomes of patients treated with early peritoneal dialysis in pregnancy complicated by ARF.A case series was conducted at the Research Unit of the Instituto Materno Infantil del Estado de México. We reviewed the cases of patients admitted to the ICU matching the criteria for ARF. They were divided into two groups: those who received EPD vs. those who did not require EPD. The most important national series were included describing the association with preeclampsia and thrombotic microangiopathy with hemolysis.In a 5-year period, 1272 patients were admitted to the ICU; in 38 patients ARF was documented requiring peritoneal dialysis. In nine cases ARF was associated with thrombotic microangiopathy with hemolysis, two cases of stillbirth, and one case of mortality with MOF. A 100% recovery in renal function was observed in all cases, using 1.5% solution with an average of 34 dialysis treatments.The early use of peritoneal dialysis in obstetric patients with ARF has a good prognosis.

Pathogenic brucella bacteria have developed strategies to persist for prolonged periods of time in host cells, avoiding innate immune responses. Here we show that the cyclic beta-1,2-glucans (CbetaG) synthesized by brucella is important for circumventing host cell defenses. CbetaG acted in lipid rafts found on host cell membranes. CbetaG-deficient mutants failed to prevent phagosome-lysosome fusion and could not replicate. However, when treated with purified CbetaG or synthetic methyl-beta-cyclodextrin, the mutants were able to control vacuole maturation by avoiding lysosome fusion, and this allowed intracellular brucella to survive and reach the endoplasmic reticulum. Fusion between the endoplasmic reticulum and the brucella-containing vacuole depended on the brucella virulence type IV secretion system but not on CbetaG. Brucella CbetaG is thus a virulence factor that interacts with lipid rafts and contributes to pathogen survival.

Idiopathic spontaneous bladder rupture is a low incidence clinical entity. We perform a literature review and report a recurrent case, with special attention to its physiopathology mainly if associated with alcohol abuse.We report the case of a patient with recurrent spontaneous bladder rupture associated with alcohol abuse. The presentation of this case is uncommon.53 year old male with history of chronic alcohol abuse who presented with abdominal pain and renal failure.Spontaneous bladder rupture is the cause of acute abdomen and acute renal failure. Idiopathic rupture associated with substance abuse has proper clinical characteristics that make it to be considered a different entity than other causes of spontaneous bladder rupture.

To report an additional case of liposarcoma of the spermatic cord.A 69-year-old male presented with an indolent left inguinal mass that he had noted one month earlier and had gradually increased in size. Two irregular mobile nodular left inguinal lesions were detected on physical examination. Analytical tests were normal. An ultrasound showed three nodular lesions in the left spermatic cord. CT attenuation values indicated predominantly fat tissue in the lesion. The patient underwent a radical left orchidectomy and funiculectomy.The histopathological analysis of the surgical specimen showed a well-differentiated liposarcoma of the spermatic cord.Liposarcoma of the spermatic cord is uncommon. Some imaging techniques are useful in making the diagnosis. Like other authors, we believe that orchifuniculectomy is the treatment of choice in these patients.

Null cyclic β-1,2-glucan synthetase mutants ( cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain ( B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.

Biosynthesis of periplasmic cyclic 1,2-β-glucans inBrucella ovisstrain REO198 andB. abortusstrain S19 was found to be carried out by membrane-bound enzymes that use UDP-glucose (UDP-Glc) as donor substrate. Contrary to what happens in species of the generaAgrobacteriumandRhizobium, the accumulation of the reaction products inBrucellaappeared not to be osmoticaliy regulated. Incubation of permeabilized cells with UDP-[14C]Glc led to the formation of soluble neutral cyclic 1,2-β-glucans and [14C]glucose-labelled glucoproteins. PAGE of pulse–chase experiments carried out with permeabilized cells showed that the molecular mass of the labelled protein was indistinguishable fromAgrobacterium tumefaciensA348 andRhizobium frediiUSDA191 glucoproteins known to be intermediates in the synthesis of cyclic glucans.Brucellatotal membrane preparations were less efficient than permeabilized cells in the formation of cyclic glucan; this was attributed to defective cyclization. Accumulation of protein intermediates having oligosaccharides of high molecular mass that were not released from the protein was observed after chase with 2 mM UDP-Glc. This defect was not observed when permeabilized cells were used as enzyme preparation, thus suggesting that inBrucellaa factor(s) that was lost or inactivated upon the preparation of membranes was required for the effective regulation between elongation and cyclization reactions.

Three wild type strains of Rhizobium fredii, USDA 191, USDA 257 and HH 303, do not synthesize in vivo or in vitro beta(1-3), beta(1-6) cyclic glucans, all strains form in vitro and in vivo cyclic beta(1-2) glucans. Approximately 80% of the recovered R. fredii cellular cyclic beta(1-2) glucans were anionic and the substituent was identified as phosphoglycerol. Inner membranes prepared from these R. fredii strains have a beta(1-2) glucan-intermediate-protein with apparent molecular mass undistinguishable from Agrobacterium tumefaciens beta(1-2) glucan intermediate protein. Studies of the degree of polymerization of the oligosaccharides recovered from the protein-intermediate after short pulse incubations with UDP-14C-glucose suggested that the rate limiting step in the biosynthesis of cyclic glucan is cyclization. Kinetic studies revealed that the K(m) for UDP-glucose was 0.33 mM. No difference was detected between the K(m) for initiation/elongation and cyclization reactions. Nodulation studies of a ndvB R. fredii mutant with Mc Call and Peking soybean cultivars, revealed that beta(1-2) glucans do not seem to be required for normal nodule invasion of these soybean cultivars. Fil:Ugalde, R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii. Fil: Ibañez, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Coria, Mirta Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Carabajal, Marianela Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Delpino, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Risso, Gabriela Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Gonzalez Cobiello, Paula Lucía. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Cátedra de Microbiología, Parasitología E Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Barrionuevo, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Bruno, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Frank, Fernanda Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones En Microbiología y Parasitología Medica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones En Microbiología y Parasitología Medica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Pasquevich, Karina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Cassataro, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "dr. Raul Alfonsin" (sede Chascomus) | Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina

In contrast to what happens in AGROBACTERIUM: tumefaciens and RHIZOBIUM: meliloti, synthesis of periplasmic cyclic 1,2-beta-glucan in BRUCELLA: spp. was not inhibited when bacteria were grown in media of high osmolarity. Studies performed with crude membrane preparations showed that cyclic 1,2-beta-glucan synthetase of BRUCELLA: spp. was not inhibited by 0.5 M KCl or potassium glutamate; concentrations that completely inhibit the osmosensitive enzymes of A. tumefaciens A348 or R. meliloti 102F34, respectively encoded by the chvB or ndvB genes. The BRUCELLA: abortus cyclic 1, 2-beta-glucan synthetase gene (cgs) was introduced into A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants. Synthesis of cyclic 1,2-beta-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (0.25 M NaCl or 0.5 M mannitol). On the other hand, when the A. tumefaciens cyclic 1, 2-beta-glucan synthetase gene was introduced into the R. meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-beta-glucan synthesis when grown in high-osmolarity media. However, the same gene introduced into a B. abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity. In vitro studies with crude membranes isolated from recombinant strains revealed that BRUCELLA: cyclic 1, 2-beta-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in AGROBACTERIUM: or RHIZOBIUM: backgrounds. It was concluded that the lack of effect of high osmolarity on 1,2-beta-glucan synthesis in BRUCELLA: is due to two convergent mechanisms: a) the presence of a cyclic 1, 2-beta-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1, 2-beta-glucan synthetase.