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Activities of Benzimidazole D- and L-Ribonucleosides in Animal Models of Cytomegalovirus Infections. Earl R. Kern, 2004.Since human cytomegalovirus (HCMV) does not infect or replicate in nonhuman cells and tissues, there are few animal models currently available for evaluation of antiviral therapies for these infections . In the present studies, we utilized two different models in which severe combined immunodeficient (SCID) mice were implanted with human fetal tissue that was subsequently infected with HCMV . In one model, human fetal retinal tissue was implanted into the anterior chamber of the SCID mouse eye, and in the second, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule . After the implants were established, they were infected with 2,000 to 9,000 PFU of HCMV . To determine the efficacy of three benzimidazole nucleosides, 2-bromo-5,6-dichloro-(1-ß-D-ribofuranosyl)benzimidazole (BDCRB), GW275175X (175X), and GW257406X (1263W94, maribavir [MBV]) treatment was initiated 24 h after infection of the implants and continued for 28 days . Treatment consisted of either placebo, 25 mg of ganciclovir (GCV)/kg of body weight administered intraperitoneally (i.p.) twice daily, 33 or 100 mg of BDCRB/kg administered i.p . twice daily, or 75 mg of either MBV or 175X/kg administered orally twice daily . GCV was effective in both models, inhibiting HCMV infection by 5- to 3,000-fold . In the retinal tissue model, MBV and BDCRB reduced HCMV replication about fourfold through 21 days postinfection compared with results for the vehicle control . In the thy/liv tissue model, all three benzimidazole nucleosides were effective in inhibiting HCMV replication by approximately 30- to 3,000-fold in comparison to the vehicle control . These data indicate that the benzimidazole nucleosides were efficacious in these animal models and suggest that this class of compounds should be active against the various HCMV infections that occur in the immunocompromised host . The Flavoprotein MrsD Catalyzes the Oxidative Decarboxylation Reaction Involved in Formation of the Peptidoglycan Biosynthesis Inhibitor Mersacidin. Florian Majer, 2002.The lantibiotic mersacidin inhibits peptidoglycan biosynthesis by binding to the peptidoglycan precursor lipid II . Mersacidin contains an unsaturated thioether bridge, which is proposed to be synthesized by posttranslational modifications of threonine residue +15 and the COOH-terminal cysteine residue of the mersacidin precursor peptide MrsA . We show that the flavoprotein MrsD catalyzes the oxidative decarboxylation of the COOH-terminal cysteine residue of MrsA to an aminoenethiol residue . MrsD belongs to the recently described family of homo-oligomeric flavin-containing Cys decarboxylases (i.e., the HFCD protein family) . Members of this protein family include the bacterial Dfp proteins (which are involved in coenzyme A biosynthesis), eukaryotic salt tolerance proteins, and further oxidative decarboxylases such as EpiD . In contrast to EpiD and Dfp, MrsD is a FAD and not an FMN-dependent flavoprotein . HFCD enzymes are characterized by a conserved His residue which is part of the active site . Exchange of this His residue for Asn led to inactivation of MrsD . The lantibiotic-synthesizing enzymes EpiD and MrsD have different substrate specificities . Efficient Turnover of Chlorocatechols Is Essential for Growth of Ralstonia eutropha JMP134(pJP4) in 3-Chlorobenzoic Acid. D. Pérez-Pantoja, 2003.Ralstonia eutropha JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters, tfdCIDIEIFI and tfdDIICIIEIIFII . Introduction of several copies of each gene cluster into R . eutropha JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate . However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB . The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway . Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability . This toxicity effect was not observed with strain JMP222 harboring multiple copies of the tfdCI gene, and the derivative of strain JMP222 containing tfdCIDIEIFI plus multiple copies of the tfdCI gene could efficiently grow in 3-CB . In addition, tfdCI and tfdCII gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively . The introduction into strain JMP134 of the xylS-xylXYZL genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB . These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB . This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions . Quantifying Mold Biomass on Gypsum Board: Comparison of Ergosterol and Beta-N-Acetylhexosaminidase as Mold Biomass Parameters. M. Reeslev, 2003.Two mold species, Stachybotrys chartarum and Aspergillus versicolor, were inoculated onto agar overlaid with cellophane, allowing determination of a direct measurement of biomass density by weighing . Biomass density, ergosterol content, and beta-N-acetylhexosaminidase (3.2.1.52) activity were monitored from inoculation to stationary phase . Regression analysis showed a good linear correlation to biomass density for both ergosterol content and beta-N-acetylhexosaminidase activity . The same two mold species were inoculated onto wallpapered gypsum board, from which a direct biomass measurement was not possible . Growth was measured as an increase in ergosterol content and beta-N-acetylhexosaminidase activity . A good linear correlation was seen between ergosterol content and beta-N-acetylhexosaminidase activity . From the experiments performed on agar medium, conversion factors (CFs) for estimating biomass density from ergosterol content and beta-N-acetylhexosaminidase activity were determined . The CFs were used to estimate the biomass density of the molds grown on gypsum board . The biomass densities estimated from ergosterol content and beta-N-acetylhexosaminidase activity data gave similar results, showing significantly slower growth and lower stationary-phase biomass density on gypsum board than on agar .
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