Characterization of the ligand binding of PGRP-L in half-smooth tongue sole (Cynoglossus semilaevis) by molecular dynamics and free energy calculation

Background: Peptidoglycan (PGN) recognition proteins (PGRPs) are important pattern recognition receptors of the host innate immune system that are involved in the immune defense against bacterial pathogens. PGRPs have been characterized in several fish species. The PGN-binding ability is important for the function of PGRPs. However, the PGRP-PGN interaction mechanism in fish remains unclear. In the present study, the 3-D model of a long PGRP of half-smooth tongue sole (Cynoglossus semilaevis) (csPGRP-L), a marine teleost with great economic value, was constructed through the comparative modeling method, and the key amino acids involved in the interaction with Lys-type PGNs and Dap-type PGNs were analyzed by molecular dynamics and molecular docking methods. Results: csPGRP-L possessed a typical PGRP structure, consisting of five ß-sheets and four α-helices. Molecular docking showed that the van der Waals forces had a slightly larger contribution than Coulombic interaction in the csPGRP-L-PGN complex. Moreover, the binding energies of csPGRP-L-PGNs computed by MM-PBSA method revealed that csPGRP-L might selectively bind both types of MTP-PGNs and MPP-PGNs. In addition, the binding energy of each residue of csPGRP-L was also calculated, revealing that the residues involved in the interaction with Lys-type PGNs were different from that with Dap-type PGNs. Conclusions: The 3-D structure of csPGRP-L possessed typical PGRP structure and might selectively bind both types of MTP- and MPP-PGNs, which provided useful insights to understanding the functions of fish PGRPs.

Oleanolic acid and ursolic acid inhibit peptidoglycan biosynthesis in Streptococcus mutans UA159

In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.

Association between the Brazilian Breastfeeding Network implementation and breastfeeding indicators / Associação entre a implantação da Rede Amamenta Brasil e indicadores de aleitamento materno

OBJECTIVE: To estimate the association between the implementation of the Brazilian Breastfeeding Network and prevalence of breastfeeding in a medium-size city in southern Brazil. METHODS: This was a cross-sectional study involving 405 children under 1 year who participated in the second phase of the multivaccination campaign in 2012. Children's consumption of food on the day before the interview was obtained through interviews with mothers or guardians. The manager and one health professional from every health facility that joined the Network were interviewed in order to investigate the process of implementation of this initiative. The association between prevalence of breastfeeding and exclusive breastfeeding and adherence to the Network implementation process was tested using Poisson regression with robust variance. RESULTS: Multivariate analysis revealed that among the children assisted by health facilities who joined the Network and those attending services that did not adhere to this strategy, the prevalence of breastfeeding (74% and 70.4% among children under 1 year, respectively) and exclusive breastfeeding (43.3% and 38.1% among children under 6 months, respectively) did not differ significantly. Difficulties in implementing the Network, such as high turnover of professionals, not meeting the criteria for accreditation, and insufficient participation of tutors in the process were identified. CONCLUSION: Contrary to the hypothesis of this study, there was no significant association between the implementation of the Brazilian Breastfeeding Network and prevalence of breastfeeding and exclusive breastfeeding in the studied city. It is possible that the difficulties found in implementing the Network in this city have influenced this result. .

OBJETIVO: Estimar a associação entre a implementação da Rede Amamenta Brasil e as prevalências de aleitamento materno (AM) em um município de médio porte do sul do Brasil. MÉTODOS: Estudo transversal que envolveu 405 crianças menores de um ano que participaram da segunda fase da campanha de multivacinação de 2012. O consumo de alimentos pela criança no dia anterior à entrevista foi obtido mediante entrevistas com as mães ou os responsáveis. Para investigar o processo de implementação da rede foram entrevistados o gerente e um profissional de saúde de cada unidade que aderiu a esse processo. A associação entre as prevalências de AM e AM exclusivo e a adesão ao processo de implementação da rede foi testada com a regressão de Poisson com variância robusta. RESULTADOS: A análise multivariada revelou que entre as crianças assistidas por unidades que aderiram ao processo de implementação da rede e as que frequentam serviços que não aderiram a essa estratégia as prevalências de AM (74% e 70,4% em menores de um ano, respectivamente) e AME (43,3% e 38,1% em menores de seis meses, respectivamente) não diferiram significativamente. Foram identificadas dificuldades na implementação da rede, tais como alta rotatividade dos profissionais, não cumprimento dos critérios para certificação e acompanhamento insuficiente das unidades pelos tutores da rede. CONCLUSÃO: Contrariando a nossa hipótese, não houve associação significativa entre a implementação da Rede Amamenta Brasil e as prevalências de AM e AME no município estudado. É possível que as dificuldades encontradas na implementação da rede nesse município tenham influenciado esse resultado. .

Peptidoglycan from Staphylococcus aureus induces the overexpression of TRLs 1-8 mRNA in corneal fibroblasts, but its lipoteichoic acid and muramyl dipeptide only induced the overexpression of TLR5 or TRL9

Lipopolysaccharide induces TLR-1-8 mRNAs over-expression in corneal fibroblast. Analyzing if other TLR-ligands can do the same, we found that peptidoglycan does, but not muramyldipeptide, lipoteichoic acid and polyI:C. This suggests that the recognition of lipopolysaccharide and peptidoglycan is enough to alert these cells against microorganisms through the over-expression of the majority TLRs.

Curtobacterium flaccumfaciens pv. flaccumfaciens detection in bean seeds using a semi-selective culture medium

The bacterial wilt caused by Curtobacterium flaccumfaciens pv. flaccumfaciens is currently considered one of the most important bacterial bean disease in Brazil. One of the most effective control methods against this disease is the use of healthy seeds. However, no methods are known that could be routinely used to detect this bacterium in bean seeds under Brazilian condition. The aim of this work was to evaluate qualitative and quantitative detection methods for Curtobacterium flaccumfaciens pv. flaccumfaciens in naturally-infected bean seeds, and the detection of this pathogen in thirty bean seed samples, by sowing onto a semi-selective culture medium the leachate obtained from soaked bean seeds. Both the qualitative and quantitative methods were effective for detecting the presence of the bacteria in the seeds samples analysed. The qualitative method proved more practical for rotine use; of the thirty bean seed samples analyzed by this method, fifty percent were infected with Curtobacterium flaccumfaciens pv. flaccumfaciens.

Atualmente, a murcha-de-curtobacterium, causada por Curtobacterium flaccumfaciens pv. flaccumfaciens, é considerada uma das principais doenças baterianas da cultura do feijoeiro, no Brasil. Um dos métodos eficazes de controle desta doença é o emprego de sementes sadias. Entretanto, não se tem conhecimento de métodos que possam ser utilizados em rotina para o isolamento desta bactéria em sementes de feijoeiro. O presente trabalho teve por objetivo avaliar os métodos qualitativos e quantitativos de isolamento de Curtobacterium flaccumfaciens pv. flaccumfaciens em sementes de feijoeiro naturalmente infectadas e a detecção deste patógeno em trinta amostras de sementes de feijoeiro, pela semeadura do líquido de maceração das sementes em meio de cultura semi-seletivo. Tanto o método quantitativo quanto o quantitativo foram eficazes em detectar a presença da bactéria nas amostras de sementes analisadas. O método qualitativo mostrou-se mais prático para o uso em rotina e das trinta amostras de sementes de feijoeiro analisadas por este método cinqüenta porcento delas estavam infectadas com Curtobacterium flaccumfaciens pv. flaccumfaciens.

El kininógeno de alto peso molecular: su participación en la respuesta inflamatoria y en la angiogénesis. Propiedades y posible aplicación terapéutica / High molecular weight kininogen in inflammation and angiogenesis: a review of its properties and therapeutic applications

The plasma kallikrein-kinin system (KKS) participates in the pathogenesis of inflammatory reactions involved in cellular injury, coagulation, fibrinolysis, kinin formation, complement activation, cytokine secretion and release of proteases. It has been shown that KKS activation in the systemic inflammatory response syndrome results in decrease of its component plasma proteins. Similar changes have been documented in diabetes, sepsis, children with vasculitis, allograft rejection, disseminated intravascular coagulation, patients with recurrent pregnancy losses, hereditary angioedema, adult respiratory distress syndrome and coronary artery disease. Direct involvement of the KKS in the pathogenesis of experimental acute arthritis and acute and chronic enterocolitis has been documented by previous studies from our laboratory using experimental animal models. It has been found that in HK deficient Lewis rats, experimental IBD was much less severe. We showed a genetic difference in kininogen structure between resistant Buffalo and susceptible Lewis rats, which results in accelerated cleavage of HK and it is responsible for the susceptibility to the inflammatory process in the Lewis rats. It has been demostrated that therapy with a specific plasma kallikrein inhibitor (P8720) modulated the experimental enterocolitis, arthritis and systemic inflammation. Furthermore, it has been shown that a bradykinin 2 receptor (B2R) antagonist attenuates the inflammatory changes in the same animal model. We have showed that a monoclonal antibody targeting HK decreases angiogénesis and arrests tumor growth in a syngeneic animal model. In summary, these results indicate that the plasma KKS plays a central role in the pathogenesis of chronic intestinal inflammation, arthritis and angiogenesis.

Se ha demostrado la participación del sistema plasmático de kalikreína-kininas (KKS) en el proceso inflamatorio, el cual incluye reacciones de daño celular, coagulación y fibrinólisis, formación de kininas, activación del complemento, secreción de citoquinas y liberación de proteasas. El KKS se encuentra activado en el síndrome de respuesta inflamatoria sistémica con una disminución en la concentración plasmática de las proteínas que lo constituyen. También se ha demostrado una activación similar en la diabetes, choque séptico, vasculitis en infantes, enfermedad injerto-huésped, coagulación intravascular diseminada, pacientes con abortos de repetición, angioedema hereditario, el síndrome de estrés respiratorio del adulto y enfermedad coronaria arterial. Mediante el uso de modelos animales experimentales, nuestro laboratorio ha demostrado una participación directa del KKS en la patogénesis de la artritis experimental aguda y la enterocolitis aguda y crónica. Se ha demostrado que en la rata tipo Lewis, cuando es deficiente de kininógeno de alto peso molecular (HK), la enfermedad inflamatoria intestinal es menos severa comparada con la presentada en ratas con niveles normales de HK como la Buffalo. Nosotros mostramos una diferencia entre el gene que codifica la molécula del kininógeno de la rata tipo Buffalo (resistentes) y Lewis (susceptibles), que resulta en un incremento de la actividad proteolítica de kalikreína sobre su substrato HK, lo cual predispone a las ratas Lewis al desarrollo de la enfermedad inflamatoria crónica. Se ha demostrado una disminución en las manifestaciones inflamatorias sistémicas de la enterocolitis y artritis experimental mediante el uso de un inhibidor específico de la kalikreína (P8720). Además, el antagonista del receptor 2 de la bradikinina (BR2) atenuó los cambios inflamatorios en el mismo modelo animal. Asimismo, se ha demostrado que las ratas Lewis deficientes de kininógeno desarrollaron inflamación intestinal sistémica menos severa. Mediante el uso del anticuerpo monoclonal C11C1 contra HK se logró una disminución de la angiogenesis y, consecuentemente, el crecimiento tumoral. En conclusión, los resultados demuestran que el sistema plasmático de KKS desempeña un papel preponderante en la patogénesis de la artritis reumatoide, la enfermedad intestinal crónica y en el proceso angiogénico.

Principales protagonistas de la respuesta inflamatoria a la infección / Main agents of the inflammatory response to infection

Se hace una revisión de los principales elementos implicados en la respuesta inflamatoria del individuo a la infección, por el beneficio que este conocimiento reporta al mejor manejo terapéutico de los pacientes. Se revisan los principales protagonistas de la inflamación, desde los detonantes como: lipopolisacárido, peptidoglicano, ácidos lipoteicoico y murámico, hasta los principales mediadores implicados. Se discute el proceso de activación de monocitos y células endoteliales y la repercusión de ello, y se hace un análisis dinámico del proceso de activación en función del tiempo trancurrido desde el comienzo de la infección, y sus etapas fisiopatológicas. Se evidencia el papel protagónico que tiene el propio organismo en el daño producido por la respuesta a la infección, además de la importancia del adecuado equilibrio entre proinflamación y contrarregulación para el pronóstico del enfermo, lo que obliga a tenerlo en cuenta a la hora del tratamiento

Structural features of LPPG and related compounds isolated from Trypanosoma cruzi trypomastigotes

A hydrophobic fraction isolated from trypomastigotes of Trypanosoma cruzi is being characterized using immunological and chemical techniques. The lipopeptidophosphoglycan (LPPG) was identified in this fraction since it gave a positive reaction with anti-LPPG rabbit serum and had similar structural features such as the presence of ceramide as the lipid moiety, furanoic galactose, and a glycan moiety consistent with that obtained from an authentic sample of epimastigote LPPG, as judged by thin-layer chromatography. Furthermore, the hydrophobic fraction contained other glycolipids with different structural features. The lipid moiety of these compounds is alkylglycerol rather than a ceramide, the carbohydrate chain appears to be less complex than that in LPPG and no reactivity was observed towards an anti-LPPG serum

Free and protein-linked glycoinositolphospholipids in Trypanosoma cruzi

Two glycoinositol phospholipids (GIPL A and GIPL B) have been purified from epimastigotes of Trypanosoma cruzi at the logarithmic phase of growth (2 days). The GIPLs differ mainly in the lipid moiety and are similar to the lipopeptidophosphoglycan (LPPG) previously isolated from epimastigotes at the stationary phase (4-5 days). [3H]-palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in GIPL A and into a sphinganine ceramide with palmitic acid and lignoceric acid as the fatty acids in GIPL B. The lipids could be released by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) or glycosylphosphatidylinositol phospholipase D (GPI-PLD) from rat serum. The oligosaccharides share the common core structure of the glycosylphosphatidilinositol (GPI) membrane anchors. Microheterogeneity was demonstrated, as well as substitution by galactose, which is mainly in the furanose configuration as was previously described for the LPPG. However, methylation analysis indicated that 20 percent of the galactose is present as terminal pyranose units. In infective trypomastigotes, [3H]-palmitic acid was incorporated into the anchor of the Tc-85 glycoprotein. The lipid cleaved by phospholipase C digestion was identified as 1-O-hexadecylglycerol and the main oligosaccharide has the structure of the conserved core of all GPI anchors. [3H]-palmitic acid-labelled Tc-85 released into the culture medium as membrane vesicles showed 80 percent resistance to the action of PI-PLC. However, after mild alkaline hydrolysis, part of the radioactivity was released by the enzyme

Molecular approaches to diagnosis of Chagas' disease: use of defined antigens and a target ribosomal RNA sequence

We evaluated the performance of two defined antigens in the serological diagnosis of Chagas' disease. One of them is a recombinant protein named B13 isolated from a genomic library of Trypanosoma cruzi in the expression vector lambda gtll. We show that the gene corresponding to B13 is conserved in the evolutive stages of the two ®polar® strains of T. cruzi. The protein epitopes cloned in B13 are represented in 140 kDa, 116 kDa and 35 kDa polypeptides of trypomastigotes. The other antigen chosen for serodiagnosis is a lipopeptidophosphoglycan (LPPG). This glycoconjugate is also widely distributed in T. cruzi strains. The use of a rabbit serum to LPPG allowed the demonstration that this molecule bears epitopes in common to LPPG-like components and to 80-90 kDa glycoproteins of trypomastigotes. Both B13 and LPPG were evaluated in serodiagnosis by ELISA and RIA using a panel of normal human, Chagasic and Leishmaniasis sera. It was observed that B13 presents high sensitivity and specificity for Chagasic sera. For LPPG it was also concluded that this reagent discriminates between individuals infected and non-infected with T. cruzi. A heterogeneity in the level of antibodies to LPPG in Chagasic patients was detected. No correlation was found between the clinical form of Chagas' disease and the preferential reactivity to B13 or LPPG. We also report preliminary studies towards the characterization of a 100 bp sequence of the 24S alpha rRNA as a target for DNA-based detection systems for diagnosis. We show that polymerase chain reaction of total DNA of different trypanosomatids lead to the specific amplification of a 100 bp fragment only for T. cruzi. Northern blots confirmed the presence of the target region in the mature 24S alpha rRNA. Titration experiments based on the direct amplification of RNA with Taq DNA polymerase allowed the detection of 50 parasites. Studies are in progress to increase the sensitivity of the proposed system

Evidence for the presence of ß-D-galactofuranosyl residues (1->3)-linked to a-D-mannopyranose on the cell membrane in all developmental stages of Trypanosoma cruzi

The lipopeptidophosphoglycan (LPPG) component isolated from epimastigote forms of Trypanosoma cruzi has antigenic properties. Using a rabbit antiserum aginst LPPG, specific for ß-D-galactofuranosyl residues (1-3)-linked to alfa-Dmannopyranose, we identified, by indirect immunofluorescence, the presence of epitopes containing ß-D-galactofuranosyl structures on the cell membrane of epimastigote, amastigote and trypomastigote forms of the parasite. The results demonstrade that all developmental stages of T. cruzi contain similar antigenic determinants on their cell body and flagellar membrane

Inmunoquimica de una lipopeptidofosfoglicana extraida de trofozoitos de Entamoeba histolytica cepa HK-9 cultivados en medio axenico, utilizando el metodo de fenol-agua. / Immunochemistry of a lipopeptidophosphoglycan extracted from trophozoites of Entamoeba histolytica strain HK-9 cultured in axenic medium, using the phenol-water method

El proposito de este trabajo fue el de extraer moleculas polisacaridicas de trofozoitos de la cepa HK-9 de Entamoeba histolytica por el metodo de fenol-agua (Westphal-Jann). La molecula extraida fue estudiada por metodos quimicos e inmunologicos. Los metodos quimicos demostraron que se trataba de una lipopeptidofosfoglicana. La lipopeptidofosfoglicana fue inmunogena para conejos y reaccion por contrainmunoelectroforesis y hemolisis pasiva con el suero de pacientes diagnosticados de absceso hepatico amibiano

Localizacion en los trofozoitos de Entamoeba histolytica de una lipopeptidofosfoglicana extraida por fenol-agua de la cepa HK-9. / Identification in the trophozoites of Entamoeba histolytica of a lipopeptidophosphoglycan extracted by phenol-water from the HK-9 strain

Una lipopeptidofosfoglicana (LPFG) de trofozoitos de la cepa HK-9 de Entamoeba histolytica fue extraida por el metodo de fenol-agua Los anticuerpos contra esta LPFG fueron producidos inmunizando a conejos con la molecula y el titulo de los mismos fueron medidos por hemolisis pasiva. Posteriormente, los anticuerpos fueron purificados precipitando el suero con sulfato de amonio y pasandolos a traves de una columna de DEAE-celulosa. Los anticuerpos IgG anti-LPFG se utilizaron como primer anticuerpo para que se fijaran a las moleculas de lipopeptidofosfoglicana presentes en los trofozoitos de E. histolytica; posteriormente se agrego un suero de cabra anti-IgG de conejo marcando con fluoresceina para hacer visible el sitio de reaccion del primer anticuerpo. Los resultados de inmunofluorescencia indirecta demostraron que la LPFG esta presente en la superficie de los trofozoitos