Formation of reactive oxygen species in rat epithelial cells upon stimulation with fly ash.

Fly ash was used as a model for ambient particulate matter which is under suspicion to cause adverse pulmonary health effects. The fly ash was pre-sized and contained only particles < 20 microm including an ultrafine fraction (< 100 nm) that contributed 31% to the particle number. In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the formation of ROS with regard to the mass of particles applied. Lipopolysaccharide (LPS) added as a co-stimulus did not increase the formation of ROS induced by fly ash. Furthermore, in LPS (0.1 microg/ml) and tumour necrosis factor-alpha (TNF-alpha; 1 ng/ml) pre-treated cells no increase in reactive oxygen species comparable to fly ash alone is observable. In presence of the metal chelator, desferrioxamine (DFO), ROS formation can be significantly reduced. Neither fly ash nor LPS induced a significant NO release in RLE-6TN cells.

DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication.

A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-microns plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.

Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity.

Mutants of the yeast, Saccharomyces cerevisiae, deficient in DNA topoisomerase I activity have been identified. One mutant has normal topoisomerase I activity when assayed at 25 degrees C and about 20% of normal activity when assayed at 36 degrees C. Strains with this mutation grow normally at all temperatures tested. The mutation has been mapped to MAK1, a gene required for maintenance of killer RNA. Three previously isolated mak1 mutants exhibit less than 1% of normal topoisomerase I activity in our assay, but yet they grow normally. The implications of these results for the role of DNA topoisomerase I in the cell are discussed.

Reduced expression of the isoleucine and valine enzymes in integration host factor mutants of Escherichia coli.

The level of the isoleucine and valine (Ilv) enzymes specified by the ilvB and ilvGEDA operons is reduced in integration host factor mutants (himA and himD) of Escherichia coli K-12. Growth inhibition of these strains in minimal medium can be explained by the decreased amounts of one of the Ilv enzymes, acetohydroxy acid synthase I (AHASI). No growth inhibition, or reduction in AHASI activity, was found in a himA derivative of a mutant strain containing high constitutive levels of AHASI. A strong correlation was observed in himA strains between the reduced amount of the Ilv enzymes and of Ilv-specific messenger RNA. These data suggest that integration host factor may be a positive effector for transcription of the ilvB and ilvGEDA operons.

Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes.

Escherichia coli deletion mutants lacking DNA topoisomerase I have been identified previously and shown to grow at a normal rate. We show that such strains grow normally only because of spontaneously arising mutations that compensate for the topoisomerase I defect. Several of these compensatory mutations have been found to map at or near the genes encoding DNA gyrase, gyrA and gyrB. DNA gyrase assays of crude extracts show that strains carrying the mutations have lower gyrase activity. Thus the mutations are in the gyrase structural genes or in nearby regulatory sequences. These results, in conjunction with DNA supercoiling measurements of others, indicate that in vivo DNA superhelicity is a result of a balance between topoisomerase I and gyrase activities. An excess of negative supercoils due to an absence of topoisomerase I is deleterious to the cell, but a moderate gyrase deficiency is not harmful.

Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition.

Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome, near cysB. Strains carrying deletions of the top gene are viable. The top mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerase I have a more rapid rate of induction and a higher level of catabolite-sensitive enzymes including tryptophanase and beta-galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished. The frequency of transposition of Tn5, a transposon carrying kanamycin resistance, is decreased by a factor of 40 or more in top mutants. A direct or indirect role of the topoisomerase in transposition is discussed. The transposition frequency of Tn3, however, is not dependent on top. Based on the studies of the E. coli top mutants, it appears that the supX gene, which was originally studied in Salmonella typhimurium [Dubnau, E. & Margolin, P. (1972) Mol. Gen. Genet. 117, 91-112] is likely to be the structural gene for DNA topoisomerase I.