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OppA, the Substrate-Binding Subunit of the Oligopeptide Permease, Is the Major Ecto-ATPase of Mycoplasma hominis. Miriam Hopfe, 2004.Most ATPases, involved in energy-driven processes, act in the cytoplasm . However, external membrane-bound ATPases have also been described in parasites and eukaryotic cells . In Mycoplasma hominis, a bacterium lacking a cell wall, the surface-exposed substrate-binding protein OppA of an oligopeptide permease (Opp) contains an ATP binding P-loop structure in the C-terminal region . With ATP affinity chromatography and tryptic digestion in the presence or absence of ATP, the functionality of the Mg2+-dependent ATP binding site is demonstrated . In addition to ATP, ADP also could bind to OppA . The presence of an ATPase activity on the surface of M . hominis is indicated by the inactivation of ATP hydrolyzing activity of intact mycoplasma cells by the impermeable ATPase inhibitor 4',4'-diisothiocyanostilbene-2',2'-disulfonic acid and influenced by the ATP analog 5'-fluorosulfonyl-benzoyladenosine . Comparing equimolar amounts of OppA in intact mycoplasma cells and in the purified form indicated that more than 80% of the surface-localized ATPase activity is derived from OppA, implying that OppA is the main ATPase on the surface of mycoplasma cells . Together, these data present the first evidence that the cytoadhesive substrate binding protein OppA of the oligopeptide permease also functions as an ecto-ATPase in Mycoplasma hominis . Emergence and Persistence of Macrolide Resistance in Oropharyngeal Flora and Elimination of Nasal Carriage of Staphylococcus aureus after Therapy with Slow-Release Clarithromycin: a Randomized, Double-Blind, Placebo-Controlled Study. Hans F. Berg, 2004.To investigate the effect of slow-release (SR) clarithromycin on colonization and the development of resistance in oropharyngeal and nasal flora, a double-blind, randomized, placebo-controlled trial was performed with 8 weeks of follow-up . A total of 296 patients with documented coronary artery disease were randomized in the preoperative outpatient clinic to receive a daily dose of SR clarithromycin (500 mg) (CL group) or placebo tablets (PB group) until the day of surgery . Nose and throat swabs were taken before the start of therapy, directly after the end of therapy, and 8 weeks later . The presence of potential pathogenic bacteria was determined, and if they were isolated, MIC testing was performed . Quantitative culture on media with and without macrolides was performed for the indigenous oropharyngeal flora . In addition, analysis of the mechanism of resistance was performed with the macrolide-resistant indigenous flora . Basic patient characteristics were comparable in the two treatment groups . The average number of tablets taken was 15 (standard deviation = 6.4) . From the throat swabs, Haemophilus parainfluenzae was isolated and carriage was not affected in either of the treatment groups . Nasal carriage of Staphylococcus aureus, however, was significantly reduced in the CL group (from 35.3 to 4.3%) compared to the PB group (from 32.4 to 30.3%) (P < 0.0001; relative risk [RR], 7.0; 95% confidence interval [CI], 3.1 to 16.0) . Resistance to clarithromycin was present significantly more frequently in H . parainfluenzae in the CL group after treatment (P = 0.007; RR, 1.6; 95% CI, 1.1 to 2.3); also, the percentage of patients with resistance to macrolides in the indigenous flora after treatment was significantly higher in the CL group (31 to 69%) (P < 0.0001; RR, 1.9; 95% CI, 1.4 to 2.5) . This persisted for at least 8 weeks . This study shows that besides the effective elimination of nasal carriage of S . aureus, treatment with SR clarithromycin for approximately 2 weeks has a marked and sustained effect on the development of resistance in the oropharyngeal flora for at least 8 weeks . Combination Therapy Using Sodium Antimony Gluconate in Stearylamine-Bearing Liposomes against Established and Chronic Leishmania donovani Infection in BALB/c Mice. Swati Pal, 2004.In this work we report the activity seen with combination therapy using sodium antimony gluconate in liposomes composed of egg phosphatidyl choline and stearylamine for elimination of Leishmania donovani parasites from the liver and spleen of BALB/c mice with established and chronic infections . Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes. René Ullrich, 2004.Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids . The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) . A . aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal . Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography . The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H2O2 (2 mM) for optimum activity . The apparent Km values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H2O2 were 37, 298, 1,001, 2,367 and 1,313 µM, respectively . The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago . This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO . The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils . The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains. Anne Marie Di Guilmi, 2003.The bacterial peptidoglycan consists of glycan chains of repeating ß-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains . The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs) . The ß-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria . In this context, the GT catalytic domain represents a potential target in the antibacterial fight . In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented . Dansylated lipid II was used as the substrate, and the kinetic parameters Km and kcat/Km were measured at 40.6 µM (± 15.5) and 1 x 10-3 M-1 s-1, respectively . The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin . Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding . In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor . The characterization of the catalytically active GT domain of S . pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal . Attachment Organelle Formation Represented by Localization of Cytadherence Proteins and Formation of the Electron-Dense Core in Wild-Type and Mutant Strains of Mycoplasma pneumoniae. Shintaro Seto, 2003.Cytadherence proteins of Mycoplasma pneumoniae are localized at the attachment organelle, which is involved in adhesion, gliding motility, and cell division . The localization of these proteins in cytadherence-deficient mutants was examined by immunofluorescence microscopy . In the class I-2 mutant, which has a frameshift mutation in the hmw2 gene, fluorescent foci for HMW1 and HMW3 were found with reduced intensity, and P1 adhesin showed reduced focusing . However, foci for P90, P40, P30, and P65 were not observed in this mutant . In the class IV-22 mutant, which lacks expression of P1, P90, and P40, the other cytadherence proteins (HMW1, HMW3, P30, and P65) were focused . In a mutant lacking HMW1, signals for HMW3, P90, P40, P30, and P65 were not found, and P1 was distributed throughout the cell . These results suggest that HMW1 is essential for the localization of all other cytadherence proteins, while HMW2 is essential for the localization of P90, P40, P30, and P65 . The electron-dense core in cytadherence mutants was observed by thin-section electron microscopy, suggesting that its formation depends on HMW1 and HMW2 and that P1 localization occurs independent of the formation of the electron-dense core . Doubly stained preparations visualized by immunofluorescence microscopy showed that the P1 adhesin, P90, and P40 colocalized to a subregion of the attachment organelle in the wild-type strain . HMW1 and HMW3 also colocalized to a different subregion of the attachment organelle, while P30 and P65 localized at more distal ends of cell poles than HMW1 and HMW3 . These differences were more pronounced in cytadherence mutants . These results suggest that there are three distinct subcellular protein localization sites in the attachment organelle, which were represented by HMW1-HMW3, P1-P90-P40, and P30-P65 . Diversity, Frequency, and Persistence of Escherichia coli O157 Strains from Range Cattle Environments. David G. Renter, 2003.Genetic diversity, isolation frequency, and persistence were determined for Escherichia coli O157 strains from range cattle production environments . Over the 11-month study, analysis of 9,122 cattle fecal samples, 4,083 water source samples, and 521 wildlife fecal samples resulted in 263 isolates from 107 samples presumptively considered E . coli O157 as determined by culture and latex agglutination . Most isolates (90.1%) were confirmed to be E . coli O157 by PCR detection of intimin and Shiga toxin genes . Pulsed-field gel electrophoresis (PFGE) of XbaI-digested preparations revealed 79 unique patterns (XbaI-PFGE subtypes) from 235 typeable isolates confirmed to be E . coli O157 . By analyzing up to three isolates per positive sample, we detected an average of 1.80 XbaI-PFGE subtypes per sample . Most XbaI-PFGE subtypes (54 subtypes) were identified only once, yet the seven most frequently isolated subtypes represented over one-half of the E . coli O157 isolates (124 of 235 isolates) . Recurring XbaI-PFGE subtypes were recovered from samples on up to 10 sampling occasions and up to 10 months apart . Seven XbaI-PFGE subtypes were isolated from both cattle feces and water sources, and one of these also was isolated from the feces of a wild opossum (Didelphis sp.) . The number of XbaI-PFGE subtypes, the variable frequency and persistence of subtypes, and the presence of identical subtypes in cattle feces, free-flowing water sources, and wildlife feces indicate that the complex molecular epidemiology of E . coli O157 previously described for confined cattle operations is also evident in extensively managed range cattle environments .
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