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

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

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

Genomic organization, sequence analysis and expression of all five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato

We have cloned and sequenced all five members of the gene family for the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from tomato, Lycopersicon esculentum cv. VFNT LA 1221 cherry line. Two of the five genes, designated Rbcs-1 and Rbcs-2 , are present as single genes at individual loci. Three genes, designated Rbcs-3A, Rbcs-3B and Rbcs-3C , are organized in a tandem array within 10 kb at a third independent locus. The Rbcs-2 gene contains three introns; all the other members of the tomato gene family contain two introns. The coding sequence of Rbcs-1 differs by 14.0% from that of Rbcs-2 and by 13.3% from that of Rbcs-3 genes. Rbcs-2 shows 10.4% divergence from Rbcs-3 . The exon and intron sequences of Rbcs-3A are identical to those of Rbcs-3C , and differ by 1.9% from those of Rbcs-3B . Nucleotide sequence analysis suggests that the five rbcS genes encode four different precursors, and three different mature polypeptides. S 1 nuclease mapping of the 5′ end of rbcS mRNAs revealed that the mRNA leader sequences vary in length from 8 to 75 nucleotides. Northern analysis using gene-specific oligonucleotide probes from the 3′ non-coding region of each gene reveals a four to five-fold difference among the five genes in maximal steady-state mRNA levels in leaves.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47566/1/438_2004_Article_BF00329650.pd

Movable genetic elements and antibiotic resistance in enterococci

The enterococci possess genetic elements able to move from one strain to another via conjugation. Certain enterococcal plasmids exhibit a broad host range among gram-positive bacteria, but only when matings are performed on solid surfaces. Other plasmids are more specific to enterococci, transfer efficiently in broth, and encode a response to recipient-produced sex phermones. Transmissible non-plasmid elements, the conjugative transposons, are widespread among the enterococci and determine their own fertility properties. Drug resistance, hemolysin, and bacteriocin determinants are commonly found on the various transmissible enterococcal elements. Examples of the different systems are discussed in this review.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47900/1/10096_2005_Article_BF01963632.pd