PubMed · 4556357
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G W Foess. 1972. Aquatic sediments.. https://pubmed.ncbi.nlm.nih.gov/4556357/
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Neocarzinostatin chromophore (NCS-chrom) has been found previously to induce thiol-dependent abasic site formation and cleavage on the RNA strand of RNA-DNA hybrids. When a four-base nucleotide was deleted from the 5' end of the DNA strand, however, an unexpected product with slow mobility, potentially a drug adduct or interstrand cross-linked material, was observed instead. End-labeling of the RNA and DNA strands showed that only the RNA strand is present in the product. The product, isolated from a denaturing polyacrylamide gel, has a fluorescence spectrum similar to that of activated NCS-chrom, and thiols with different charges give rise to products with various mobilities on the gel, indicating that the product consists of a drug adduct with NCS-chrom and thiol attached to the RNA strand of the hybrid. Mass spectroscopic (MS) analysis shows that it is a monoadduct. Chemical sequencing of 5'- and 3'-end-labeled monoadduct and MS/MS data show that drug is attached only to the U9 of the RNA in the hybrid 5'-r(CACAGAAUU9CG)/5'-d(TTCTGTG). Unlike most other DNA adducts and cross-linked products found previously, this adduct can be formed readily in the presence of atmospheric oxygen. 2'-O-Methyl and 2'-H substitutions and nucleotide replacements on the 4-base RNA overhang affect monoadduct formation to different extents, reflecting the existence of a tight three-dimensional binding pocket for the activated drug. Stability of drug adduct to heat and aniline-HOAc treatment suggest that the drug is covalently bound to the ribose of U9. Since no deuterium isotope selection effect was observed for C-1' of U9, it is possible, although not required, that the drug abstracts H from another hydrogen source on the sugar of the targeted ribonucleotide.
In the present study we have analyzed protein oxidation on Escherichia coli when these cells were submitted to different stress conditions such as hydrogen peroxide, superoxide-generating compounds, and iron overloading. Carbonyl groups on oxidized cell proteins were examined by Western blot immunoassay. When anaerobically grown E. coli cells were exposed to hydrogen peroxide stress, alcohol dehydrogenase E, elongation factor G, the heat shock protein DNA K, oligopeptide-binding protein A, enolase, and the outer membrane protein A were identified as the major protein targets. A similar immunostained band pattern was found when cells were shifted from anaerobic to aerobic conditions in the presence of different concentrations of iron; it is relevant to note that oxidation of outer membrane protein C, not observed in peroxide stress conditions, was clearly detected as the concentration of iron was increased in the culture media. The hydrogen peroxide stress performed under aerobic conditions affected the beta-subunit of F0F1-ATPase; the rest of the oxidized protein pattern was very similar to that found for anaerobic conditions, with the exception of alcohol dehydrogenase E, a protein not synthesized aerobically. Cells submitted to superoxide stress using menadione showed a more specific pattern in which elongation factor G and the beta-subunit of F0F1-ATPase were affected significantly. When paraquat was used, although the degree of oxidative damage was lower, the same two modified proteins were detected, and DNA K was also clearly damaged. Cell viability was affected to different extents depending on the type of stress exerted. The results described in this paper provide data about the in vivo effects of oxidative stress on protein oxidation and give insights into understanding how such modifications can affect cellular functions.
Despite its reactivity with many biological molecules, formaldehyde can be commonly encountered by virtually all cells. The widespread existence of glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) in procaryotes and eucaryotes suggests this enzyme plays a central and universal role in biological formaldehyde oxidation. This work sought to determine the role of GSH-FDH in the facultative phototrophic bacterium Rhodobacter sphaeroides. Growth phenotypes of wild-type and mutant cells, changes in enzyme specific activities, and the pattern of 13C-labeled compounds detected by NMR spectroscopy cumulatively suggest that R. sphaeroides GSH-FDH can play a critical role in formaldehyde metabolism under both photosynthetic and aerobic respiratory conditions. In photosynthetic cells, the data indicate that GSH-FDH generates reducing power, in the form of NADH, and one-carbon skeletons that are oxidized to carbon dioxide for subsequent assimilation by the Calvin-Benson-Bassham cycle. For example, use of methanol as a sole photosynthetic carbon source increases the specific activities of GSH-FDH, an NAD-dependent formate dehydrogenase, and the key Calvin-Benson-Bassham cycle enzyme, ribulose-1,5-bisphosphate carboxylase. This role of GSH-FDH is also supported by the pattern of [13C]formaldehyde oxidation products that accumulate in photosynthetic cells and the inability of defined GSH-FDH or Calvin cycle mutants to use methanol as a sole carbon source. Our data also suggest that GSH-FDH acts in formaldehyde dissimilation when aerobic respiratory cultures cometabolize methanol and succinate.