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1.
Detection of lipid hydroperoxides and hydrogen peroxide at picomole levels by an HPLC and isoluminol chemiluminescence assay 总被引:3,自引:0,他引:3
An isoluminol assay is utilized for the detection of hydrogen peroxide and lipid hydroperoxides in biological samples. The combination of this assay as a post-column detection for HPLC avoids interference of antioxidants and enables characterization of hydroperoxides at picomole levels. Two useful HPLC conditions for the separation of hydrogen peroxide, lipid hydroperoxides, antioxidants, and unoxidized lipids are described. 相似文献
2.
An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp 总被引:21,自引:12,他引:9
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G Storz F S Jacobson L A Tartaglia R W Morgan L A Silveira B N Ames 《Journal of bacteriology》1989,171(4):2049-2055
The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonella typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-1 and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus. 相似文献
3.
Levels of 5'-adenyl dinucleotides, measured as diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A), were found to accumulate in cultured human fibroblasts following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the radiomimetic drug bleomycin, and nitroquinoline-1-oxide (NQO) or UV-irradiation in the presence of cytosine arabinofuranoside (araC). In contrast, cells derived from patients with xeroderma pigmentosum complementation group A (XP-A) did not demonstrate an increase in DNA-strand breaks following UV irradiation or NQO in the presence of araC nor an increase in Ap4A levels. Ap4A accumulation did occur in XP-A cells following treatment with MNNG. Cells derived from patients characterized as XP variants, which are incision repair-proficient, accumulated 5'-dinucleotides following bleomycin, MNNG and UV or NQO in the presence of araC. Taken together, these data suggest that Ap4A accumulates as a response to DNA-strand breaks. 相似文献
4.
Effects of drought on host and endophyte development in mycorrhizal soybeans in relation to water use and phosphate uptake 总被引:4,自引:0,他引:4
Gabor J. Bethlenfalvay Milford S. Brown Robert N. Ames Richard S. Thomas 《Physiologia plantarum》1988,72(3):565-571
Bethlenfalvay, G. J., Brown, M. S., Ames, R. N. and Thomas, R. S. 1988. Effects of drought on host and endophyte development in mycorrhizal soybeans in relation to water use and phosphate uptake. - Physiol. Plant. 72: 565–571.
Soybean [ Glycine max (L.) Merr.] plants were grown in pot cultures and inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or provided with P fertilizer (non-VAM plants). After an initial growth period (21 days), plants were exposed to cycles of severe, moderate or no drought stress over a subsequent 28-day period by rewatering at soil water potentials of -1.0, -0.3 or -0.05 MPa. Dry weights of VAM plants were greater at severe stress and smaller at no stress than those of non-VAM plants. Phosphorus fertilization was applied to produce VAM and non-VAM plants of the same size at moderate stress. Root and leaf P concentrations were higher in non-VAM plants at all stress levels. All plants were stressed to permanent wilting prior to harvest. VAM plants had lower soil moisture content at harvest than non-VAM plants. Colonization of roots by G. mosseae did not vary with stress, but the biomass and length of the extraradical mycelium was greater in severely stressed than in non-stressed plants. Growth enhancement of VAM plants relative to P-fertilized non-VAM plants under severe stress was attributed to increased uptake of water as well as to more efficient P uptake. The ability of VAM plants to deplete soil water to a greater extent than non-VAM plants suggests lower permanent wilting potentials for the former. 相似文献
Soybean [ Glycine max (L.) Merr.] plants were grown in pot cultures and inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or provided with P fertilizer (non-VAM plants). After an initial growth period (21 days), plants were exposed to cycles of severe, moderate or no drought stress over a subsequent 28-day period by rewatering at soil water potentials of -1.0, -0.3 or -0.05 MPa. Dry weights of VAM plants were greater at severe stress and smaller at no stress than those of non-VAM plants. Phosphorus fertilization was applied to produce VAM and non-VAM plants of the same size at moderate stress. Root and leaf P concentrations were higher in non-VAM plants at all stress levels. All plants were stressed to permanent wilting prior to harvest. VAM plants had lower soil moisture content at harvest than non-VAM plants. Colonization of roots by G. mosseae did not vary with stress, but the biomass and length of the extraradical mycelium was greater in severely stressed than in non-stressed plants. Growth enhancement of VAM plants relative to P-fertilized non-VAM plants under severe stress was attributed to increased uptake of water as well as to more efficient P uptake. The ability of VAM plants to deplete soil water to a greater extent than non-VAM plants suggests lower permanent wilting potentials for the former. 相似文献
5.
The high-affinity histidine permease of Salmonella typhimurium is encoded by a four-gene operon containing a large intercistronic region located between the first gene (hisJ) and the three distal genes (hisQ, hisM, hisP). The level of expression of hisJ is 30-fold greater than that of hisP. In order to investigate the role of the intercistronic region in intra-operonic control of gene expression, we have isolated MudII-mediated lacZ gene fusions to hisQ, hisM and hisP. We have used these fusions to isolate and analyse mutants that have altered levels of expression of the hisQ gene, the first gene downstream from the intercistronic region. The results indicate that intra-operonic regulation is due to a combination of factors including efficiency of translational initiation, mRNA degradation, and retroregulation of hisJ expression. They also suggest that the REP (Repetitive Extragenic Palindromic) sequences, which are located in the hisJ-hisQ intercistronic region, may interfere with translation of the hisQ gene and affect upstream messenger RNA stability by protecting it from 3' to 5' nuclease degradation (in agreement with data presented by Newbury et al., 1987). 相似文献
6.
ATP-dependent bacterial transporters and cystic fibrosis: analogy between channels and transporters.
The traffic ATPases superfamily includes known transporters, both prokaryotic and eukaryotic, including the medically important proteins, P-glycoprotein, and the cystic fibrosis gene product (CFTR), which is known to be a Cl- channel. The structure and mechanism of action of the best-studied members of the superfamily, the periplasmic permeases, are described and related to that of CFTR and eukaryotic traffic ATPases in general. The contention is put forward that the distinction between the architecture and mechanisms of action of channels and transporters is blurred. 相似文献
7.
Light-induced increases in cGMP metabolic flux correspond with electrical responses of photoreceptors 总被引:6,自引:0,他引:6
A Ames T F Walseth R A Heyman M Barad R M Graeff N D Goldberg 《The Journal of biological chemistry》1986,261(28):13034-13042
The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction. 相似文献
8.
A high-performance liquid chromatography (HPLC) method has been developed to quantify the major and modified nucleoside composition of total, unfractionated transfer RNA. The method is rapid and sensitive and offers a high degree of chromatographic resolution suitable for quantifying both stable and unstable modified nucleosides. It is nondestructive and allows the recovery of nucleosides for further characterization. We apply the method in the analysis of the 29 modified nucleosides in tRNA from Salmonella typhimurium (and Escherichia coli) and show it to be useful in examining changes in the modified nucleoside content of tRNA. Such changes may be important in regulation. 相似文献
9.
Sensitive fluorographic detection of 3H and 14C on chromatograms using methyl anthranilate as a scintillant 总被引:1,自引:0,他引:1
Methyl anthranilate is a simple, sensitive, and inexpensive liquid scintillant for fluorographic detection of weak beta-emitting isotopes on chromatograms. Detection of tritium is enhanced 1000-fold compared to autoradiography in a 24-h exposure. Since methyl anthranilate is a viscous liquid, it is easily applied as an even coating which subsequently solidifies at the low temperature (-80 degrees C) used for fluorography. Of several liquid scintillants tested, methyl anthranilate was most effective, followed by 9-ethyl fluorene, methyl salicylate, and 1-methyl naphthalene. The efficiency of 1-methyl naphthalene could be raised to the level of methyl anthranilate by the addition of a small amount (0.5%) of 2,5-diphenyloxazole (PPO). 相似文献
10.
Revised methods for the Salmonella mutagenicity test 总被引:77,自引:0,他引:77
The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed. 相似文献