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Immunopharmacology of CpG Oligodeoxynucleotides and Ribavirin. Jörg Vollmer, 2004.To investigate their potential mechanisms of action, the nucleoside analogue ribavirin and a TLR9 agonist were compared . The CpG oligodeoxynucleotides (ODN) demonstrated strong TLR9-related Th1-type effects, and ribavirin appeared only to mediate signaling in TLR-transfected cells . CpG ODN represent a promising new type of therapeutic drug for hepatitis C or other infectious diseases . Bifidobacterium lactis DSM 10140: Identification of the atp (atpBEFHAGDC) Operon and Analysis of Its Genetic Structure, Characteristics, and Phylogeny. Marco Ventura, 2004.The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene . PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species . All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases . Genes encoding the subunits of the F1F0-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced . The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms . We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species . Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes . The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences . Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer . The acid inducibility of the atp operon of B . lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B . lactis DSM 10140 . The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B . lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step . Biogenesis of T Pili in Agrobacterium tumefaciens Requires Precise VirB2 Propilin Cleavage and Cyclization. Erh-Min Lai, 2002.VirB2 propilin is processed by the removal of a 47-amino-acid signal peptide to generate a 74-amino-acid peptide product in both Escherichia coli and Agrobacterium tumefaciens . The cleaved VirB2 protein is further cyclized to form the T pilin in A . tumefaciens but not in E . coli . Mutations in the signal peptidase cleavage sequence of VirB2 propilin cause the formation of aberrant T pilin and also severely attenuate virulence . No T pilus was observed in these mutants . The potential role of the exact VirB2 propilin cleavage and cyclization in T pilus biogenesis and virulence is discussed . Identification of a New Gene Essential for Germination of Bacillus subtilis Spores with Ca2+-Dipicolinate. Katerina Ragkousi, 2003.Bacillus subtilis spores can germinate with a 1:1 chelate of Ca2+ and dipicolinic acid (DPA), a compound present at high levels in the spore core . Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca2+-DPA, three mutations were identified . One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca2+-DPA . The other two were mapped to an open reading frame, ywdL, encoding a protein of unknown function. Analysis of ywdL expression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is  E dependent.
Functional characterization of YwdL demonstrated that it is a new spore
coat protein that is essential for the presence of CwlJ in the spore
coat . Assembly of YwdL itself into the spore coat is dependent on the
coat morphogenetic proteins CotE and SpoIVA . However, other than
lacking CwlJ, ywdL spores have no obvious defect in their
spore coat . Because of the role for YwdL in a part of the spore
germination process, we propose renaming ywdL as a spore
germination gene,
gerQ .
The Mode of Action of the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3A Differs from That of Cry1Ab  -Endotoxin.Mi Kyong Lee, 2003.The Vip3A protein, secreted by Bacillus spp . during the vegetative stage of growth, represents a new family of insecticidal proteins . In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G) . Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV) . Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors . In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M . sexta APN protein, or a cadherin Cry1Ab ecto-binding domain . In voltage clamping assays with dissected midgut from the susceptible insect, M . sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation . In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus) . In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A . Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity . The mode of action of Vip3A supports its use as a novel insecticidal agent .
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