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Altering the Substrate Specificity of Polyhydroxyalkanoate Synthase 1 Derived from Pseudomonas putida GPo1 by Localized Semirandom Mutagenesis. Der-Shyan Sheu, 2004.The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Pp, class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis . The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR . According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA– as the host . The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB–4 supplemented with octanoate . Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%) . Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R . eutropha PHB–4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates . The 3-HA and MCL 3-HA units of the PHA produced by R . eutropha PHB–4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum . Dual-Action Mechanism of Viramidine Functioning as a Prodrug and as a Catabolic Inhibitor for Ribavirin. Jim Zhen Wu, 2004.An investigational nucleoside analogue drug, viramidine, has recently emerged as a potentially safer alternative to ribavirin for the treatment of hepatitis C viral infection . We have reported that viramidine mainly functions as a prodrug of ribavirin that is enriched in the liver . This in vitro study further explores viramidine's activity against nucleoside phosphorylase, a host enzyme that is responsible for phosphorolysis of ribavirin in vivo . Our experiments show that viramidine inhibits ribavirin phosphorolysis with a Ki of 2.5 µM . This result suggests that viramidine may act through a dual-action mechanism by serving as a prodrug of ribavirin and concomitantly as an inhibitor for nucleoside phosphorylase catabolism of ribavirin . A Mucoadhesive Polymer Extracted from Tamarind Seed Improves the Intraocular Penetration and Efficacy of Rufloxacin in Topical Treatment of Experimental Bacterial Keratitis. Emilia Ghelardi, 2004.Bacterial keratitis is a serious infectious ocular disease requiring prompt treatment to prevent frequent and severe visual disabilities . Standard treatment of bacterial keratitis includes topical administration of concentrated antibiotic solutions repeated at frequent intervals in order to reach sufficiently high drug levels in the corneal tissue to inhibit bacterial growth . However, this regimen has been associated with toxicity to the corneal epithelium and requires patient hospitalization . In the present study, a mucoadhesive polymer extracted from tamarind seeds was used for ocular delivery of 0.3% rufloxacin in the treatment of experimental Pseudomonas aeruginosa and Staphylococcus aureus keratitis in rabbits . The polysaccharide significantly increased the intra-aqueous penetration of rufloxacin in both infected and uninfected eyes . Rufloxacin delivered by the polysaccharide reduced P . aeruginosa and S . aureus in the cornea at a higher rate than that obtained by rufloxacin alone . In particular, use of the polysaccharide allowed a substantial reduction of S . aureus in the cornea to be achieved even when the time interval between drug administrations was extended . These results suggest that the tamarind seed polysaccharide prolongs the precorneal residence times of antibiotics and enhances drug accumulation in the cornea, probably by reducing the washout of topically administered drugs . The tamarind seed polysaccharide appears to be a promising candidate as a vehicle for the topical treatment of bacterial keratitis . Genotyping of Cryptosporidium Isolates from Chamelea gallina Clams in Italy. Donato Traversa, 2004. Cleavage of Treponema denticola PrcA Polypeptide To Yield Protease Complex-Associated Proteins Prca1 and Prca2 Is Dependent on PrtP. Si Young Lee, 2002.Analysis of potential virulence factors of oral spirochetes focuses on surface and secreted proteins . The Treponema denticola chymotrypsin-like protease (CTLP) is implicated in degradation of host cell molecules and contributes to tissue invasion . The CTLP complex, composed of the 72-kDa PrtP protein and two auxiliary proteins with molecular masses of approximately 40 and 30 kDa, is also involved in localization and oligomerization of the T . denticola major surface protein (Msp) . The larger auxiliary protein was reported to be encoded by an open reading frame (ORF2) directly upstream of prtP . The deduced 39-kDa translation product of ORF2 contains a sequence matching the N-terminal sequence determined from one of the CTLP complex proteins . No proteins with significant homology are known, nor was information available on the third protein of the complex . DNA sequence analysis showed that ORF2 extended an additional 852 bp upstream of the reported sequence . The complete gene, designated prcA, encodes a predicted N-terminally-acylated polypeptide of approximately 70 kDa . Isogenic mutants with mutations in prtP, prcA, and prcA-prtP all lacked CTLP protease activity . The prcA mutant lacked all three CTLP proteins . The prcA-prtP mutant produced only a C-terminally-truncated 62-kDa PrcA protein . The prtP mutant produced a full-length 70-kDa PrcA . Immunoblot analysis of recombinant PrcA constructs confirmed that PrcA is cleaved to yield the two smaller proteins of the CTLP complex, designated PrcA1 and PrcA2 . These data indicate that PrtP is required for cleavage of PrcA and suggest that this cleavage may be required for formation or stability of outer membrane complexes . Identification by RNA Profiling and Mutational Analysis of the Novel Copper Resistance Determinants CrdA (HP1326), CrdB (HP1327), and CzcB (HP1328) in Helicobacter pylori. Barbara Waidner, 2002.Mechanisms involved in maintaining cytoplasmic metal ion homeostasis play a central role in the adaptation of Helicobacter pylori to the changing gastric environment . An investigation of the global regulatory responses to copper ions by using RNA profiling with a threshold factor of 4.0 revealed that copper induces transcription of 19 H . pylori genes and that only the ferritin gene pfr is repressed . The 57-fold copper induction identified the HP1326 gene encoding an H . pylori-specific protein as a candidate for a novel copper resistance determinant . The HP1326 gene is expressed as a monocistronic unit, and two small HP1326 mRNAs are copper induced . The HP1326 protein is secreted and is required for copper resistance maintained by cytoplasmic copper homeostasis, as H . pylori HP1326 mutants were copper sensitive and displayed increased copper induction of HP1326 transcription as well as elevated copper repression of ferritin synthesis . The clear copper-sensitive phenotype displayed by H . pylori HP1327 and HP1328 mutants provides strong evidence that the HP1326 protein, together with the signal peptide site of the H . pylori-specific protein HP1327, whose gene is located downstream from that encoding HP1326, and the CzcB and CzcA metal efflux system component homologs HP1328 and HP1329, constitutes a novel type of copper efflux pump, as discussed below . The HP1329 gene could not be inactivated, but the 14-fold transcriptional copper induction determined by RNA profiling points towards a function of the encoded CzcA homolog in copper resistance . In summary, results from RNA profiling identified the novel H . pylori-specific copper resistance determinants CrdA (HP1326) and CrdB (HP1327), which are required for adaptation to copper-rich environmental conditions . Mycotoxigenic Fusarium and Deoxynivalenol Production Repress Chitinase Gene Expression in the Biocontrol Agent Trichoderma atroviride P1. Matthias P. Lutz, 2003.Mycotoxin contamination associated with head blight of wheat and other grains caused by Fusarium culmorum and F . graminearum is a chronic threat to crop, human, and animal health throughout the world . One of the most important toxins in terms of human exposure is deoxynivalenol (DON) (formerly called vomitoxin), an inhibitor of protein synthesis with a broad spectrum of toxigenicity against animals . Certain Fusarium toxins have additional antimicrobial activity, and the phytotoxin fusaric acid has recently been shown to modulate fungus-bacterium interactions that affect plant health (Duffy and Défago, Phytopathology 87:1250-1257, 1997) . The potential impact of DON on Fusarium competition with other microorganisms has not been described previously . Any competitive advantage conferred by DON would complicate efforts to control Fusarium during its saprophytic growth on crop residues that are left after harvest and constitute the primary inoculum reservoir for outbreaks in subsequent plantings . We examined the effect of the DON mycotoxin on ecological interactions between pathogenic Fusarium and Trichoderma atroviride strain P1, a competitor fungus with biocontrol activity against a wide range of plant diseases . Expression of the Trichoderma chitinase genes, ech42 and nag1, which contribute to biocontrol activity, was monitored in vitro and on crop residues of two maize cultivars by using goxA reporter gene fusions . We found that DON-producing F . culmorum and F . graminearum strains repressed expression of nag1-gox . DON-negative wild-type Fusarium strains and a DON-negative mutant with an insertional disruption in the tricothecene biosynthetic gene, tri5, had no effect on antagonist gene expression . The role of DON as the principal repressor above other pathogen factors was confirmed . Exposure of Trichoderma to synthetic DON or to a non-DON-producing Fusarium mutant resulted in the same level of nag1-gox repression as the level observed with DON-producing Fusarium . DON repression was specific for nag1-gox and had no effect, either positive or negative, on expression of another key chitinase gene, ech42. This is the first demonstration that a target pathogen down-regulates genes in a fungal biocontrol agent, and our results provide evidence that mycotoxins have a novel ecological function as factors in Fusarium competitiveness .
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