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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Enteropathogenic Escherichia coli (EPEC) secretes several Esps (E. coli-secreted proteins) that are required for full virulence. Insertion of the bacterial protein Tir into the host epithelial cell membrane is facilitated by a type III secretion apparatus, and at least EspA and EspB are required for Tir translocation. An EPEC outer membrane protein, intimin, interacts with Tir on the host membrane to establish intimate attachment and formation of a pedestal-like structure. In this study, we identified a Tir chaperone, CesT, whose gene is located between tir and eae (which encodes intimin). A mutation in cesT abolished Tir secretion into culture supernatants and significantly decreased the amount of Tir in the bacterial cytoplasm. In contrast, this mutation did not affect the secretion of the Esp proteins. The level of tir mRNA was not affected by the cesT mutation, indicating that CesT acts at the post-transcriptional level. The cesT mutant could not induce host cytoskeletal rearrangements, and displayed the same phenotype as the tir mutant. Gel overlay and GST pulldown assays demonstrated that CesT specifically interacts with Tir, but not with other Esp proteins. Furthermore, by using a series of Tir deletion derivatives, we determined that the CesT binding domain is located within the first 100 amino-terminal residues of Tir, and that the pool of Tir in the bacterial cytoplasm was greatly reduced when this domain was disrupted. Interestingly, this domain was not sufficient for Tir secretion, and at least the first 200 residues of Tir were required for efficient secretion. Gel filtration studies showed that Tir–CesT forms a large multimeric complex. Collectively, these results indicate that CesT is a Tir chaperone that may act as an anti-degradation factor by specifically binding to its amino-terminus, forming a multimeric stabilized complex.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 129 (1995), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The transcript of the spvR gene on the virulence plasmid, pKDSC5O, of Salmonella choleraesuis serovar Choleraesuis was detected for the first time by Northern blot analysis, and the transcriptional regulation of the spvR gene was investigated. The transcription of the spuR was negatively regulated by spvA and spvB, and enhanced at stationary phase under control of a sigma factor RpoS (σ38). The spvR transcript was 2.4 kilonucleotides in Salmonella cells, and deduced to encode SpvR and SpvA, suggesting that SpvA but not SpvB is the functioning repressor in spv operon. The promoter sequence analysis revealed that spvR was transcribed from a single promoter and the 5′ end of the transcript was located at 18 bp upstream from the start codon of spvR. Sequential similarity between the promoter of spvR and other σ38-controlled genes was not found, but the consensus sequence was found in − 10 to −35 region of spvR and spvA, which may correlate to our previous data indicating that both genes were positively regulated by the SpvR protein.
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  • 3
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract We have developed a reverse dot-blot hybridization assay for detection of Salmonella using Salmonella-specific o oligonucleotide probes designed from the base sequence of the 16S rRNA gene (rDNA). The target fragment of 16S rDNA was amplified, and labelled with biotin by the polymerase chain reaction. The amplified fragment was hybridized with the membrane-immobilized probe and the hybridization was detected by chemiluminescence. Amplified fragments from 24 different serovars of Salmonella hybridized with the probes, whereas those of species of Enterobacteriaceae, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus failed to hybridize. By this assay, it was possible to detect in the order of 104 bacteria in fish meat homogenate in 10 h.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-069X
    Keywords: Key words Atopic dermatitis ; Serum cytokine levels ; Plasma cytokine levels ; Calcium ion ; Protease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Changes in the levels of cytokines in the circulating blood and skin have been reported in patients with atopic dermatitis (AD). We determined IFN-γ, IL-2, IL-4, IL-5 and IL-10 in both the serum and plasma of 45 AD patients and 20 healthy donors. Since differences in the levels of these cytokines between serum and plasma were found, the roles of Ca2+ and proteolytic enzymes were examined. Levels of IL-2 and IL-10 were measured in citrated plasma to which various amounts of CaCl2, protease inhibitors, and proteases had been added. All cytokine determinations were carried out using a standard ELISA. The plasma levels of IFN-γ, IL-2, and IL-5 were significantly elevated, but the serum levels of these cytokines were not significantly changed. The levels of IL-2 in the plasma of the AD patients averaged 4.25-fold higher than in the serum of the AD patients, and 2.5-fold higher than in the plasma of healthy controls (P 〈 0.001). CaCl2 produced a dose-dependent decrease in IL-2 and IL-10 in citrated plasma. The protease inhibitors PMSF, aprotinin and leupeptin produced a dose-dependent increase in measurable levels of IL-2 and IL-10 in plasma. A decrease in IL-2 levels was also seen in CaCl2-supplemented serum-free medium, and this was accentuated by the addition of the proteases thrombin, trypsin, chymotrypsin and elastase. These findings suggest that although significant changes in cytokine levels have been reported not to occur in circulating blood but have been reported to occur in the skin of AD patients both in vivo and in vitro, cytokines can indeed also be found to be elevated in circulating blood when assessed carefully by statistically valid methods. Further, it is suggested that during the preparation of serum, some circulating cytokines are degraded by calcium-dependent proteases, and that Ca2+ itself can also affect the measurement of cytokines. The measurement of circulating cytokines needs to be carefully reassessed.
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  • 5
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Analysis of a Japanese population by oligonucleotide genotyping revealed that one Japanese HLA-DRw14 allele had a DRB1 genotype different from that of the known HLA-DRw14-related alleles, DRB1 * 1401 (DRw14-Dw9) and DRB1 * 1402 (DRw14-Dw16). The second exon of the DRB1 gene of the novel DRw14 allele (designated DRB1-14c) was amplified enzymatically and sequenced after cloning intto a plasmid vector. The amino acid sequence of the first domain in the DRβ1 chain encoded in the DRB1-14c allele was more similar to that of the DRB1 * 1401 allele (three amino acid substitutions).than to that of the DRB1 * 1402 allele (six amino acid substitutions). No polymorphic amino acid residue that could explain the common serologic HLA-DRw14 specificity was identified among the sequences of the three DRw14-related alleles. Sequence-specific oligonucleotides (SSOs) were synthesized on the basis of the DRB1-14c nucleotide sequence and used for genotyping of the Japanese population. These SSOs served as useful probes for identifying the DRB1-14c allele in a wide range of donors.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-1211
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We determined the DNA sequence of the enzymatically amplified second exon of theDRB1 gene of theDRw12 haplotypes derived from three Japanese donors and found two distinct subtypes of theDRw12 haplotype. The two subtypes, designatedDRw12a andDrw12b, had single-base substitutions that predicted one amino acid change at residue number 67. The sequence of theDrw12a andDRw12b subtypes differed from those of the otherDR haplotypes, but in the first hypervariable region of theDRB1 gene the sequences were identical to those of theDRw8(Dw8.1) andDRw8(Dw8.3) haplotypes. TheDRw12a andDRw12b subtypes were detected in a wide range of Japanse donors by genotyping with sequence-specific oligonucleotide probes synthesized according to the DNA sequence of the two subtypes. Results of this study demonstrated that theDRw12 haplotypes in the Japanese population are genetically diverse, as many otherDR haplotypes are.
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  • 7
    ISSN: 1617-4623
    Keywords: Salmonella serovar Choleraesuis ; Virulence plasmid ; Positive regulator ; Northern blot hybridization ; DNA binding protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The regulation of mouse bacteremia genes (mba genes) encoded by a 6.4 kb region on the 50 kb virulence plasmid (pKDSC50) of Salmonella serovar Choleraesuis was analyzed. The genes mba1, mba2, mba3, and mba4, are arranged in this order, and form a cluster located in the 6.4 kb mba region. We prepared four antibodies, each specific for an individual Mba protein, using synthetic peptides as antigens. Their amino acid sequences were deduced from the DNA sequence of the corresponding mba genes. Each Mba peptide antiserum was able to recognize the corresponding Mba protein produced by Escherichia coli carrying a recombinant plasmid containing individual mba genes. When the recombinant plasmid contained all four mba genes (pMKD601), three Mba proteins (Mba2, Mba3, and Mba4) were identified by Western blotting analysis using Mba antisera. These proteins could not be detected when the recombinant plasmid lacked mba1 (pMKD201). Three species of mRNA for mba2, mba3, and mba4 with different chain length were detected from pMKD601 by Northern blot hybridization, and two start sites were identified by primer extension assay. Gel mobility shift assays demonstrated that Mbal specifically bound to a fragment containing the start sites of mRNAs. The amino acid sequence of Mbal had significant homology to the LysR family of DNA binding proteins, possessing a characteristic helix-turn-helix DNA binding motif. The present study provides clear evidence to show that the Mba1 protein binds to the promoter region of mba2, and positively regulates the expression of mba2, mba3, and mba4 genes.
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  • 8
    Publication Date: 2015-02-13
    Description: Three category 3 (organic) particles (RB-QD04-0001, RB-QD04-0047-02, and RA-QD02-0120) and so-called ?white object? found in the sample container have been examined by micro-Raman and IR spectroscopy. In addition, several artificial substances that could occur as possible contaminants, and chondritic IOM prepared from the Murchison CM2 chondrite were analyzed. The Raman spectra of the particles show broad G-band and weak D-band. The G-band parameters plot in the disordered region and close to the artifact produced from a Viton glove after laser exposure rather than chondritic IOM. The particles were therefore originally at low maturity level, suggesting they have not experienced strong heating, and are therefore not related to the LL4-6 parent body. IR spectra are not similar to that of chondritic IOM. Furthermore, the particles cannot be identified as some artificial carbonaceous substances, including the white object, which are the possible contaminants, examined in this investigation. Although it cannot be determined exactly whether the three category 3 particles are extraterrestrial, the limited IR and Raman results in this investigation strongly suggest their terrestrial origin. Although they could not be directly related to the artificial contaminants examined in this investigation, they may yet be reaction products from similar substances that flew on the mission. In particular, RB-QD04-0047-02 shows several infrared spectral absorption bands in common with the ?white object?. This may relate to the degradation of a polyimide/polyamide resin.
    Print ISSN: 1343-8832
    Electronic ISSN: 1880-5981
    Topics: Geosciences
    Published by SpringerOpen
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