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Effect of a Combination of Clevudine and Emtricitabine with Adenovirus-Mediated Delivery of Gamma Interferon in the Woodchuck Model of Hepatitis B Virus Infection. A. C. Jacquard, 2004.Our aim was to evaluate the antiviral effect of a combination of two nucleoside reverse transcriptase inhibitors, emtricitabine (FTC) and clevudine (L-FMAU), with the addition of an adenovirus-driven delivery of recombinant gamma interferon (IFN-  ) in the woodchuck model of hepatitis B virus infection . Six woodchuck hepatitis virus (WHV)-infected woodchucks received L-FMAU (10 mg/kg) plus FTC (30 mg/kg) intraperitoneally for 8 weeks; six other animals received in addition an intravenous injection of a recombinant adenovirus vector expressing woodchuck IFN- (Ad-IFN) at weeks 4 and 8 . In the control group, two animals received Ad-IFN alone, two received adenovirus vector expressing the green fluorescent protein reporter gene, and one remained untreated . In less than 2 weeks, all woodchucks that received L-FMAU plus FTC showed a rapid and marked inhibition of viral replication, with a 4-log10 drop in serum WHV DNA . In two animals, viremia remained suppressed for several months after the end of treatment . Similarly, a dramatic decrease in intrahepatic replicative intermediates of viral DNA was observed in the L-FMAU/FTC-treated groups . The additional administration of Ad-IFN led to increased inflammation in the liver but did not enhance the antiviral effect of the L-FMAU/FTC combination . In conclusion, therapies combining L-FMAU and FTC in WHV-infected woodchucks resulted in a potent and sustained antihepadnaviral effect both in the liver and in the blood circulation . However, no extra benefit of adding IFN- gene transduction to the L-FMAU/FTC combination could be detected .
The Positive Regulator, TraJ, of the Escherichia coli F Plasmid Is Unstable in a cpxA* Background. Michael J. Gubbins, 2002.The Cpx (conjugative plasmid expression) stress response of Escherichia coli is induced in response to extracytoplasmic signals generated in the cell envelope, such as misfolded proteins in the periplasm . Detection of stress is mediated by the membrane-bound histidine kinase, CpxA . Signaling of the response regulator CpxR by activated CpxA results in the expression of several factors required for responding to cell envelope stress . CpxA was originally thought to be required for the expression of the positive regulator of the F plasmid transfer (tra) operon, TraJ . It was later determined that constitutive gain-of-function mutations in cpxA led to activation of the Cpx envelope stress response and decreased TraJ expression . In order to determine the nature of the downregulation of TraJ, the level of expression of TraJ, TraM, and TraY, the F-encoded regulatory proteins of the F tra region, was determined both in a cpxA* background and in a wild-type background in which the Cpx stress response was induced by overexpression of the outer membrane lipoprotein, NlpE . Our results suggest that TraJ downregulation is controlled by a posttranscriptional mechanism that operates in the cytoplasm in response to upregulation of the Cpx stress response by both the cpxA* gain-of-function mutation and the overexpression of NlpE . A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides. Xiaohua Zeng, 2003.A new operon (designated the puc2BA operon) displaying a high degree of similarity to the original pucBA genes of Rhodobacter sphaeroides 2.4.1 (designated puc1) was identified and studied genetically and biochemically . The puc2B-encoded polypeptide is predicted to exhibit 94% identity with the original ß-apoprotein . The puc2A-encoded polypeptide is predicted to be much larger (263 amino acids) than the 54-amino-acid puc1A-encoded polypeptide . In the first 48 amino acids of the puc2A-encoded polypeptide there is 58% amino acid sequence identity to the original puc1A-encoded polypeptide . We found that puc2BA is expressed, and DNA sequence data suggested that puc2BA is regulated by the PpsR/AppA repressor-antirepressor and FnrL . Employing genetic and biochemical approaches, we obtained evidence that the puc2B-encoded polypeptide is able to enter into LH2 complex formation, but neither the full-length puc2A-encoded polypeptide nor its N-terminal 48-amino-acid derivative is able to enter into LH2 complex formation . Thus, the sole source of  -polypeptides for the LH2 complex is puc1A . The role of the puc1C-encoded polypeptide was also determined . We found that the presence of this polypeptide is essential for normal levels of transcription and translation of the puc1 operon but not for transcription and translation of the puc2 operon . Thus, the puc1C gene product appears to have both transcriptional and posttranscriptional roles in LH2 formation . Finally, the absence of any LH2 complex when puc1B was deleted in frame was surprising since we know that in the presence of functional puc2BA, approximately 30% of the LH2 complexes normally observed contain a puc2B-encoded ß-polypeptide .
Natural Variation in the Microcystin Synthetase Operon mcyABC and Impact on Microcystin Production in Microcystis Strains. Bjørg Mikalsen, 2003.Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known . This has been attributed to relaxed substrate specificity of the adenylation domain . Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes . Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains . Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC . Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry . Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes . Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting . Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon . Surface Display of the Receptor-Binding Region of the Lactobacillus brevis S-Layer Protein in Lactococcus lactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells. Silja Åvall-Jääskeläinen, 2003.Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector . The N-terminal region of the S-layer protein (SlpA) of L . brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro . In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor . The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000 . Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer . Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface . In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L . lactis NZ9000 . Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene . The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system . These results show that, with the aid of the receptor-binding region of the L . brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium .
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