Isolation and Expression of a Gene Cluster Responsible for Biosynthesis of the Glycopeptidolipid Antigens of \u3cem\u3eMicobacterium avium\u3c/em\u3e

Bacteria within the Mycobacterium avium complex are prominent in the environment and are a source of serious disseminated infections in patients with AIDS. Serovars of the M. avium complex are distinguished from all other mycobacteria and from one another by the presence of highly antigenic glycolipids, the glycopeptidolipids, on their surfaces. A genomic library of DNA from serovar 2 of the M. avium complex was constructed in the Escherichia coli-Mycobacterium shuttle cosmid, pYUB18, and used to clone and express in Mycobacterium smegmatis the genes responsible for the biosynthesis of the oligosaccharide segment of the M. avium serovar 2-specific glycopeptidolipid. The responsible gene cluster was mapped to a 22- to 27-kb functional region of the M. avium genome. The recombinant glycolipid was also isolated by high-pressure liquid chromatography and chemically characterized, by gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry, to demonstrate that the lipopeptide core originated in M. smegmatis, whereas the oligosaccharide segment arose from the cloned M. avium genes. This first-time demonstration of the cloning and expression, in a nonpathogenic mycobacterium, of the genes encoding complex cell wall glycoconjugates from a pathogenic mycobacterium presents a new approach for studying the role of such products in disease processes

Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant

BACKGROUND: Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium. METHODS: To be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain. RESULTS: In this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium. CONCLUSION: We report the second allelic exchange system for M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug resistance can be studied in a consistent manner

Intestinal fungi contribute to development of alcoholic liver disease

This study was supported in part by NIH grants R01 AA020703, U01 AA021856 and by Award Number I01BX002213 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development (to B.S.). K.H. was supported by a DFG (Deutsche Forschungsgemeinschaft) fellowship (HO/ 5690/1-1). S.B. was supported by a grant from the Swiss National Science Foundation (P2SKP3_158649). G.G. received funding from the Yale Liver Center NIH P30 DK34989 and R.B. from NIAAA grant U01 AA021908. A.K. received support from NIH grants RC2 AA019405, R01 AA020216 and R01 AA023417. G.D.B. is supported by funds from the Wellcome Trust. We acknowledge the Human Tissue and Cell Research (HTCR) Foundation for making human tissue available for research and Hepacult GmbH (Munich, Germany) for providing primary human hepatocytes for in vitro analyses. We thank Dr. Chien-Yu Lin Department of Medicine, Fu-Jen Catholic University, Taiwan for statistical analysis.Peer reviewedPublisher PD

Chemopreventive potential of β-Sitosterol in experimental colon cancer model - an In vitro and In vivo study

<p>Abstract</p> <p>Background</p> <p><it>Asclepias curassavica </it>Linn. is a traditional medicinal plant used by tribal people in the western ghats, India, to treat piles, gonorrhoea, roundworm infestation and abdominal tumours. We have determined the protective effect of β-sitosterol isolated from <it>A. curassavica </it>in colon cancer, using <it>in vitro </it>and <it>in vivo </it>models.</p> <p>Methods</p> <p>The active molecule was isolated, based upon bioassay guided fractionation, and identified as β-sitosterol on spectral evidence. The ability to induce apoptosis was determined by its <it>in vitro </it>antiradical activity, cytotoxic studies using human colon adenocarcinoma and normal monkey kidney cell lines, and the expression of β-catenin and proliferating cell nuclear antigen (PCNA) in human colon cancer cell lines (COLO 320 DM). The chemopreventive potential of β-sitosterol in colon carcinogenesis was assessed by injecting 1,2-dimethylhydrazine (DMH, 20 mg/kg b.w.) into male Wistar rats and supplementing this with β-sitosterol throughout the experimental period of 16 weeks at 5, 10, and 20 mg/kg b.w.</p> <p>Results</p> <p>β-sitosterol induced significant dose-dependent growth inhibition of COLO 320 DM cells (IC₅₀266.2 μM), induced apoptosis by scavenging reactive oxygen species, and suppressed the expression of β-catenin and PCNA antigens in human colon cancer cells. β-sitosterol supplementation reduced the number of aberrant crypt and crypt multiplicity in DMH-initiated rats in a dose-dependent manner with no toxic effects.</p> <p>Conclusion</p> <p>We found doses of 10-20 mg/kg b.w. β-sitosterol to be effective for future <it>in vivo </it>studies. β-sitosterol had chemopreventive potential by virtue of its radical quenching ability <it>in vitro</it>, with minimal toxicity to normal cells. It also attenuated β-catenin and PCNA expression, making it a potential anticancer drug for colon carcinogenesis.</p

B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells

Bcl-6 expression in CD4+ T cells is required to generate extrafollicular antibody responses

Valores de referência para plumbemia em população urbana

INTRODUCTION: The lead reference values for blood used in Brazil come from studies conducted in other countries, where socioeconomic, clinical, nutritional and occupational conditions are significantly different. In order to guarantee an accurate biomonitoring of the population which is occupationally exposed to lead, a major health concern of the studied community, reference values for individuals who are not occupationally exposed and who live in the southern region of the city were established. MATERIAL AND METHOD: The sample was composed of 206 subjects of at least 15 years of age. Various strategies were employed to assure good-quality sampling. Subjects who presented values outside clinical or laboratory norms were excluded, as well as those whose specific activities might interfere with the results. RESULTS: Lead reference values for blood were found to be from 2.40 to 16.6 µg.dL-1, obtained by the interval ; ± 2s (where ; is the mean and s is the standard deviation form observed values) and the median was 7.9 µg.dL-1.INTRODUÇÃO: Os valores de referência utilizados no Brasil, para chumbo em sangue, advêm de estudos realizados em outros países onde as condições socioeconômicas, clínicas, nutricionais e ocupacionais diferem bastante das brasileiras. Para garantir uma correta biomonitorização da população ocupacionalmente exposta ao chumbo, um dos principais problemas identificados no município estudado, foram estabelecidos valores de referência na população não exposta ocupacionalmente da região sul do município. MATERIAL E MÉTODO: Diferentes estratégias foram utilizadas para assegurar a qualidade de amostragem, que foi dimensionada em 206 sujeitos acima de 15 anos. Sujeitos que apresentaram valores clínicos e laboratoriais fora da faixa de normalidade foram excluídos, bem como os que apresentaram atividades específicas que pudessem interferir nos valores de plumbemia. RESULTADOS: Foram encontrados valores de referência para chumbo em sangue de 2,4 a 16,6 mg.dL-1, obtidos através do intervalo ; ± 2s (onde ; é o valor médio e s é o desvio-padrão dos valores observados) e mediana = 7,9 µg.dL-1

Presence of Mycoplasma fermentans in the bloodstream of Mexican patients with rheumatoid arthritis and IgM and IgG antibodies against whole microorganism

<p>Abstract</p> <p>Background</p> <p>Increasing evidence incriminates bacteria, especially <it>Mycoplasma fermentans</it>, as possible arthritogenic agents in humans. The purpose of this study was to investigate <it>M. fermentans </it>in the bloodstream of patients with rheumatoid arthritis.</p> <p>Methods</p> <p>Two hundred and nineteen blood samples from patients with rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome, and healthy individuals were screened by bacterial culture and direct PCR in order to detect mycoplasmas; IgM and IgG against <it>M. fermentans </it>PG18 were also detected by ELISA and Immunoblotting assays in patients with rheumatoid arthritis and healthy individuals.</p> <p>Results</p> <p>Blood samples from patients with antiphospholipid syndrome and healthy individuals were negative for mycoplasma by culture or direct PCR. In blood samples from patients with systemic lupus erythematosus were detected by direct PCR <it>M. fermentans </it>in 2/50 (2%), <it>M. hominis </it>in 2/50 (2%) and <it>U. urealyticum </it>in 1/50 (0.5%). In patients with RA <it>M. fermentans </it>was detected by culture in 13/87 blood samples and in 13/87 by direct PCR, however, there was only concordance between culture and direct PCR in six samples, so <it>M. fermentans </it>was detected in 20/87(23%) of the blood samples from patients with RA by either culture or PCR. Antibody-specific ELISA assay to <it>M. fermentans </it>PG18 was done, IgM was detected in sera from 40/87 patients with RA and in sera of 7/67 control individuals, IgG was detected in sera from 48/87 RA patients and in sera from 7/67 healthy individuals. Antibody-specific immunoblotting to <it>M. fermentans </it>PG18 showed IgM in sera from 35/87 patients with RA and in sera from 4/67 healthy individuals, IgG was detected in sera from 34/87 patients and in sera from 5/67 healthy individuals.</p> <p>Conclusion</p> <p>Our findings show that only <it>M. fermentans </it>produce bacteremia in a high percentage of patients with RA. This finding is similar to those reported in the literature. IgM and IgG against <it>M. fermentans </it>PG18 were more frequent in patients with RA than healthy individuals.</p

Distinct Roles of ComK1 and ComK2 in Gene Regulation in Bacillus cereus

The B. subtilis transcriptional factor ComK regulates a set of genes coding for DNA uptake from the environment and for its integration into the genome. In previous work we showed that Bacillus cereus expressing the B. subtilis ComK protein is able to take up DNA and integrate it into its own genome. To extend our knowledge on the effect of B. subtilis ComK overexpression in B. cereus we first determined which genes are significantly altered. Transcriptome analysis showed that only part of the competence gene cluster is significantly upregulated. Two ComK homologues can be identified in B. cereus that differ in their respective homologies to other ComK proteins. ComK1 is most similar, while ComK2 lacks the C-terminal region previously shown to be important for transcription activation by B. subtilis ComK. comK1 and comK2 overexpression and deletion studies using transcriptomics techniques showed that ComK1 enhances and ComK2 decreases expression of the comG operon, when B. subtilis ComK was overexpressed simultaneously