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81.
Basar T Havlícek V Bezousková S Hackett M Sebo P 《The Journal of biological chemistry》2001,276(1):348-354
The capacity of adenylate cyclase toxin (ACT) to penetrate into target cells depends on post-translational fatty-acylation by the acyltransferase CyaC, which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, the in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic serine 20 and histidine 33 residues ablated acylating activity of CyaC. Conservative replacements of alanine 140 by glycine (A140G) and valine (A140V) residues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi- and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983, or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost exclusively at Lys-983. The respective proportion of toxin molecules acylated at Lys-983 correlated well with the cell-invasive activity of both ACT mixtures, which was about half of that of ACT fully acylated on Lys-983 by intact CyaC. These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary and sufficient. 相似文献
82.
Comamonas terrigena N3H is a gram-negative rod-shaped bacterium that was isolated from contaminated soil in Slovakia. This bacterium showed remarkable biodegradation properties. We investigated the expression and functioning of two catalase isozymes in this bacterium. The typical catalase could be induced by cadmium ions, whereas the catalase-peroxidase enzyme was constitutively expressed. Since C. terrigena lacks the key enzyme for complete degradation of phenols (phenolhydroxylase), we analysed the possible removal of phenol by the two catalases of this bacterium. Addition of phenol to the culture medium led to increased expression of the catalase-peroxidase. Applying oxidative stress prior to phenol administration markedly induced the expression of the typical catalase, irrespective of the nature of the added agent. Thus, the rate of phenol degradation is rather reduced under these conditions, while growth of the cells is not impaired. We concluded that phenol peroxidation in C. terrigena can be largely attributed to the action of a catalase-peroxidase. The potential application of this enzyme in the removal of phenol from the environment is discussed. 相似文献
83.
The intensity of adult diapause in Pyrrhocoris apterus was measured in two series of experiments as the duration of pre-oviposition period at a constant temperature of 25 degrees C after transfer from short (12L:12D) to long day conditions (18L:6D). Higher diapause intensity was induced with a thermoperiod than at constant temperatures. After the induction throughout larval instars 3-5 and during 4 weeks of adult life at short days and a thermoperiod of 25/15 degrees C the pre-oviposition period was 30+/-4 and 26+/-3 days. After induction at constant 25 degrees C the pre-oviposition period was 22+/-3 and 23+/-4 days, while after induction at constant 20 degrees C it was 17+/-4 and 19+/-4 days. Induction at a lower constant temperature of 20 degrees C was thus followed by a less intense diapause than the induction at a higher constant temperature of 25 degrees C. These counterintuitive results are discussed. The oxygen consumption rate measured at experimental temperatures prior to transfer from short to long days was higher at thermoperiodic conditions than at constant temperatures and it was similar at constant 20 and 25 degrees C. Thus, the oxygen consumption rate measured prior to the transfer was highest (indication of the least intense diapause) in the insects that showed later, after the transfer to long days, the longest pre-oviposition period (indication of the most intense diapause). Within the first two days after transfer to constant 25 degrees C, oxygen consumption rate measured at 25 degrees C decreased in the thermoperiodic insects, while it transiently increased in insects from constant 20 degrees C. Two days and later after the transfer, oxygen consumption rate was similar in all groups. Cold hardiness was not correlated with diapause intensity. The low lethal temperature in diapausing insects was correlated with the night temperature during diapause induction. 相似文献
84.
Ihnatovych I Hejnová L Kostrnová A Mares P Svoboda P Novotný J 《Journal of neurochemistry》2001,79(1):88-97
Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12. 相似文献
85.
86.
Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans 总被引:5,自引:0,他引:5
Dusinská M Ficek A Horská A Raslová K Petrovská H Vallová B Drlicková M Wood SG Stupáková A Gasparovic J Bobek P Nagyová A Kováciková Z Blazícek P Liegebel U Collins AR 《Mutation research》2001,474(1-2):47-55
The fem-3 gene of Caenorhabditis elegans was employed to determine the mutation frequency as well as the nature of mutations induced by low earth orbit space radiation ambient to Space Shuttle flight STS-76. Recovered mutations were compared to those induced by accelerated iron ions generated by the AGS synchrotron accelerator at Brookhaven National Laboratory. For logistical reasons, dauer larvae were prepared at TCU, transported to either Kennedy Space Center or Brookhaven National Laboratory, flown in space or irradiated, returned to TCU and screened for mutants. A total of 25 fem-3 mutants were recovered after the shuttle flight and yielded a mutation frequency of 2.1x10(-5), roughly 3.3-fold higher than the spontaneous rate of 6.3x10(-6). Four of the mutations were homozygous inviable, suggesting that they were large deletions encompassing fem-3 as well as neighboring, essential genes. Southern blot analyses revealed that one of the 25 contained a polymorphism in fem-3, further evidence that space radiation can induce deletions. While no polymorphisms were detected among the iron ion-induced mutations, three of the 15 mutants were homozygous inviable, which is in keeping with previous observations that high LET iron particles generate deficiencies. These data provide evidence, albeit indirect, that an important mutagenic component of ambient space radiation is high LET charged particles such as iron ions. 相似文献
87.
Antioxidative and antimutagenic effect of yeast cell wall mannans, in particular, extracellular glucomannan (EC-GM) and glucomannan (GM-C.u.) both from Candida utilis, mannan from Saccharomyces cerevisiae (M-S.c.) and mannan from Candida albicans (M-C.a.) was evaluated. Luminol-dependent photochemical method using trolox as a standard showed that EC-GM, GM-C.u., M-S.c. and M-C.a. have relatively good antioxidative properties. EC-GM exhibited the highest antioxidative activity, followed by GM-C.u. and M-S.c. M-C.a. showed the least antioxidative activity. These mannans were experimentally confirmed to exhibit different, statistically significant antimutagenic activity in reducing damage of chloroplast DNA of the flagellate Euglena gracilis induced by ofloxacin and acridine orange (AO). We suggest that the antimutagenic effect of EC-GM, GM-C.u., M-S.c. and M-C.a. against ofloxacin is based on their ability to scavenge reactive oxygen radicals. With AO, the reduction of the chloroplast DNA lession could be a result of the absorptive capacity of the mannans. The important characteristics of mannans isolated from the yeast cell walls, such as good water solubility, relatively small molecular weight (15-30kDa), and antimutagenic effect exerted through different mode of action, appear to be a promising features for their prospective use as a natural protective (antimutagenic) agents. 相似文献
88.
Vargas VM Migliavacca SB de Melo AC Horn RC Guidobono RR de Sá Ferreira IC Pestana MH 《Mutation research》2001,490(2):141-158
The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present. 相似文献
89.
Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli. Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases. Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane. The molecular mechanism of how this pump operates and the manner by which it is linked to the oxygen chemistry at the active site of the enzyme are unknown. Several proposals have featured changes in the coordination of CuB during enzyme turnover that would result in sequential protonation or deprotonation events that are key to the functioning proton pump. This would imply lability of the ligands to CuB. In this work, the structure of the protein in the immediate vicinity of CuB, in both the fully oxidized and fully reduced forms of the enzyme, has been examined by Cu XAS, a technique that is particularly sensitive to changes in metal coordination. The results show that in the oxidized enzyme, CuB(II) is four-coordinate, consistent with three imidazoles and one hydroxyl (or water). Upon reduction of the enzyme, the coordination of CuB(I) is significantly altered, consistent with the loss of one of the histidine imidazole ligands in at least a substantial fraction of the population. These data add to the credibility that changes in the ligation of CuB might occur during catalytic turnover of the enzyme and, therefore, could, in principle, be part of the mechanism of proton pumping. 相似文献
90.
The kinetics of the production of fusaproliferin by Fusarium subglutinans ITEM 2404 in maize and rice cultures was investigated at various incubation temperatures. The growth rate of F. subglutinans was highest at 20 degrees C and 25 degrees C in maize cultures and at 15 degrees C in rice cultures. Although the growth rate was higher in rice than in maize, the maximal production of fusaproliferin was obtained in maize cultures, with a maximum yield (4309 microg g(-1)) at 20 degrees C for 6 weeks. In rice cultures the optimal incubation regimen was at 15 degrees C for 6 weeks, with a fusaproliferin level of 1557 microg g(-1). The production of fusaproliferin at 25 degrees C and 30 degrees C in both substrates was very low, with maximal yield at 25 degrees C of 979 microg g(-1) after 2 weeks and 143 microg g(-1) after 3 weeks in maize and rice cultures, respectively. 相似文献