Cloning, sequencing and expression of a 5-lipoxygenase from Syrian hamster embryo fibroblasts.

The hamster ortholog of human and rat 5-lipoxygenase (5-LO) was cloned from a Syrian hamster embryo (SHE) cell line. A combination of polymerase chain reaction (PCR) and 5' and 3' RACE (rapid amplification of cDNA ends) was used to isolate the complete cDNA for this gene. The cDNA sequence demonstrates the extreme sequence conservation found in this gene family, with a deduced amino acid sequence 95% identical to the rat 5-LO, and 90% identical to the human enzyme. The hamster 5-LO was expressed in E. coli. The expressed protein was detected by an antibody to human 5-LO, and had an apparent molecular weight of 75-80 kD. The products of the action of this enzyme on arachidonic acid are 5-HETE and the diHETEs resulting from the breakdown of LTA4, in a pattern similar to that produced by the recombinant human 5-LO. No oxidation of linoleic acid by this enzyme was detected.

Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column.

An immunoaffinity column is described that facilitates the analysis of oxidative damage products of DNA and RNA in urine, blood plasma, and medium isolated from cultures of Escherichia coli. In intact animals, lesions (adducts) excised from DNA are transported from the cell through the circulation and excreted in urine. In bacteria, DNA adducts are excreted directly into the medium. In either case, the adducts can be assayed as a measure of oxidative damage to DNA. A monoclonal antibody that recognizes 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG;8-hydroxy-2'-deoxyguanosine), a bio-marker of oxidative damage to DNA, has been isolated, and its substrate binding properties have been characterized. The relative binding affinities of this monoclonal antibody for oxo8dG, unmodified nucleosides, or derivatives of Gua made it suitable for the preparation of immunoaffinity columns that greatly facilitate the isolation of oxo8dG, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroguanosine from various biological fluids. Quantitative analysis of these adducts in urine of rats fed a nucleic acid-free diet and in the medium from cultures of E. coli suggests that oxo8-7,8-dihydroguanine is the principal repair product from oxo8-dG in DNA of both eukaryotes and prokaryotes. The results support our previous estimate of about 10(5) oxidative lesions to DNA being formed and excised in an average rat cell per day.

Effects of paraquat on Escherichia coli: differences between B and K-12 strains.

Escherichia coli B and K-12 are equally susceptible to the bacteriostatic effects of aerobic paraquat, but they differed strikingly when the lethality of paraquat was evaluated. E. coli B suffered an apparent loss of viability when briefly exposed to paraquat, whereas E. coli K-12 did not. This difference depended on the ability of the B strain, but not the K-12 strain, to retain internalized paraquat; the B strain was killed on aerobic tryptic soy-yeast extract plates during the incubation which preceded the counting of colonies. This difference in retention of paraquat between strains was demonstrated by delayed loss of viability, by growth inhibition, and by cyanide-resistant respiration after brief exposure to paraquat, washing, and testing in fresh medium. This difference was also shown by using [14C]paraquat. This previously unrecognized difference between E. coli B and K-12 has been the cause of apparently contradictory reports and should lead to some reevaluation of the pertinent literature.

Effects of pH, glucose, and chelating agents on lethality of paraquat to Escherichia coli.

Retention of paraquat by Escherichia coli B was greatest after exposure at pH 9.0 and was progressively less after exposure at pH 7.0 and 5.0, respectively. This retained paraquat was capable of persistent growth inhibition. Uptake and retention of paraquat by E. coli B was dependent upon a carbon source, such as glucose. Under comparable conditions E. coli K-12 did not retain paraquat. The lethality of paraquat was seen in TSY medium but not in VB medium. The addition of Soytone, tryptone, or yeast extract, to the VB medium allowed the lethality of paraquat to be seen. A variety of chelating agents, including EDTA, 8-hydroxyquinoline, and o-phenanthroline, prevented the lethal effect of paraquat in TSY medium. Although EDTA protected against the lethality of paraquat, it did not protect against its bacteriostatic effect.

An activity stain for proteins containing noncovalently bound flavin adenine dinucleotide.

A procedure has been developed to allow the visualization of FAD-containing proteins on polyacrylamide gels. The technique is based on the reconstitution of apo-D-amino acid oxidase with FAD and is thus specific for this cofactor. The stain is sensitive enough to detect 5 pmol of FAD and is therefore useful for the detection of flavoproteins in systems as complex as crude tissue or bacterial extracts.