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Effects of Maribavir and Selected Indolocarbazoles on Epstein-Barr Virus Protein Kinase BGLF4 and on Viral Lytic Replication. Edward Gershburg, 2004.The human cytomegalovirus (HCMV) homolog of the Epstein-Barr virus (EBV) protein kinase (PK), UL97, is inhibited by maribavir (1263W94) and selected indolocarbazoles . Here we show that only one of these indolocarbazoles (K252a), but not maribavir, inhibits autophosphorylation of the EBV PK, BGLF4 . However, maribavir and another indolocarbazole, NGIC-I, do inhibit EBV DNA synthesis, suggesting that although these last compounds inhibit both HCMV and EBV, they seem to operate through differ-ent pathways . Impact of Soil Drying-Rewetting Stress on Microbial Communities and Activities and on Degradation of Two Crop Protection Products. Manuel Pesaro, 2004.Prior to registration of crop protection products (CPPs) their persistence in soil has to be determined under defined conditions . For this purpose, soils are collected in the field and stored for up to 3 months prior to the tests . During storage, stresses like drying may induce changes in microbiological soil characteristics (MSCs) and thus may influence CPP degradation rates . We investigated the influence of soil storage-related stress on the resistance and resilience of different MSCs by assessing the impact of a single severe drying-rewetting cycle and by monitoring recovery from this event for 34 days . The degradation and mineralization of the fungicide metalaxyl-M and the insecticide lufenuron were delayed by factors of 1.5 to 5.4 in the dried and rewetted soil compared to the degradation and mineralization in an undisturbed reference . The microbial biomass, as estimated by direct cell counting and from the soil DNA content, decreased on average by 51 and 24%, respectively . The bulk microbial activities, as determined by measuring substrate-induced respiration and fluorescein diacetate hydrolysis, increased after rewetting and recovered completely within 6 days after reequilibration . The effects on Bacteria, Archaea, and Pseudomonas were investigated by performing PCR amplification of 16S rRNA genes and reverse-transcribed 16S rRNA, followed by restriction fragment length polymorphism (RFLP) and terminal RFLP (T-RFLP) fingerprinting . Statistical analyses of RFLP and T-RFLP profiles indicated that specific groups in the microbial community were sensitive to the stress . In addition, evaluation of rRNA genes and rRNA as markers for monitoring the stress responses of microbial communities revealed overall similar sensitivities . We concluded that various structural and functional MSCs were not resistant to drying-rewetting stress and that resilience depended strongly on the parameter investigated . The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis. Johan Palmfeldt, 2004.Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate . Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate . In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells . Two of the four lactococcal strains investigated with maltose, L . lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L . lactis ATCC 19435 or IL-1403 . In resting cell experiments all four strains exhibited homolactic fermentation . In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of Pi was decreased compared with the concentrations in growing cells . Addition of an ionophore (monensin or valinomycin) to resting cultures of L . lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations . ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro . Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells . This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells . A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L . lactis is discussed . Fe2+-Tetracycline-Mediated Cleavage of the Tn10 Tetracycline Efflux Protein TetA Reveals a Substrate Binding Site near Glutamine 225 in Transmembrane Helix 7. Laura M. McMurry, 2002.TetA specified by Tn10 is a class B member of a group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline . A tetracycline-divalent metal cation complex is expelled from the cell in exchange for a entering proton . The site(s) where tetracycline binds to this export pump is not known . We found that, when chelated to tetracycline, Fe2+ cleaved the backbone of TetA predominantly at a single position, glutamine 225 in transmembrane helix 7 . The related class D TetA protein from plasmid RA1 was cut at exactly the same position . There was no cleavage with glycylcycline, an analog of tetracycline that does not bind to TetA . The Fe2+-tetracycline complex was not detectably transported by TetA . However, cleavage products of the same size as with Fe2+ occurred with Co2+, known to be cotransported with tetracycline . The known substrate Mg 2+-tetracycline interfered with cleavage by Fe2+ . These findings suggest that cleavage results from binding at a substrate-specific site . Fe2+ is known to be able to cleave amide bonds in proteins at distances up to approximately 12 Å . We conclude that the  carbon of glutamine 225 is probably within 12 Å of the position of the Fe2+ ion in the Fe2+-tetracycline complex bound to the protein .
Glucose-Resistant Sporulation in Bacillus subtilis crsA47 Mutants Does Not Depend on Promoter Switching at the spo0A Gene. Laurie G. Dixon, 2002.We have found that sporulation in Bacillus subtilis crsA47 mutants does not require the  H-dependent promoter of the spo0A gene . This implies that the glucose-resistant sporulation phenotype of this strain is not related to the switch from the vegetative-stage
A-dependent promoter to the
H-dependent promoter at the spo0A gene .
Domain Architectures of  54-Dependent Transcriptional Activators.David J. Studholme, 2003. Microplate Bioassay for Nisin in Foods, Based on Nisin-Induced Green Fluorescent Protein Fluorescence. J. Reunanen, 2003.A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter . The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614 . The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence . The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily . By using this strain, an assay for quantification of nisin was developed . With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 µg of nisin in cheese, and 1 µg of nisin per ml in salad dressings .
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