Inhibition of Escherichia coli DNA polymerase-I by the anti-cancer drug cis-diaminedichloroplatinum(II): what roles do polymerases play in cis-platin-induced cytotoxicity?

Activities of Escherichia coli DNA polymerase-I were examined in the presence of the anti-tumor drug cis-diaminedichloroplatinum(II) and its inactive geometric isomer trans-diaminedichloroplatinum(II). The trans-isomer did not inhibit the enzyme activity. The anti-tumor drug, on the other hand, retarded the enzyme in its ability to extend the primer strand of DNA. Two alternative mechanisms of inhibition, covalent binding of cis-diaminedichloroplatinum(II) to the polymerase and to the template DNA, were explored. Selective preincubations of the platinum drug with the polymerase and DNA reveal that the inhibition is primarily due to covalent binding to the enzyme. The rates of inhibition were found to be first order in enzyme and zeroth order in platinum in the concentration range 0.05-3.0 mM. A mechanism that deals with the formation of an initial platinum-polymerase-I complex with a binding constant > 10(5) M(-1) followed by a further reaction to form an inhibitory complex is consistent with the kinetic data. The rate limiting first order rate constant for the formation of the inhibitory complex is comparable to that observed for the thiol coordination of peptides containing cysteine residues. Analyses of known structures and functions of catalytic domains of various polymerases point to the direction that the inhibition is perhaps due to the distortion of the DNA binding domain of the enzyme due to platinum coordination.     

Development of sporeless/low sporing strains of Pleurotus through mutation

Summary The sporophores of Pleurotus are gymnocarpous and continuously release spores in the atmosphere causing respiratory allergies like hay fever and farmer’s lung disease among workers. The allergy is caused by the antigens present on the walls of the spores. Apart from this, during commercial production, these spores settle on the fruit bodies, germinate and form a velvety film which gives an unpleasant appearance to the mushrooms. The spores emitted may include new genotypes likely to attack wood or trees. Spore allergy is one of the most important limiting factors for the large scale cultivation of this species. Different approaches are being adopted at IIHR for the production of commercial sporeless/low-sporing strains of Pleurotus to alleviate the spore allergy problem. Attempts were made during the present investigation to produce sporeless or low-sporing mutants through u.v. mutation. Mutation of the mycelium did not yield the desired results. Mutation of the spores of Pleurotus sajor-caju yielded an extremely low-sporing mutant after 75 min exposure. The character has been found to be stable for more than 10 generations of subculturing.     

Autogenous growth factor production by reticuloendotheliosis virus-transformed hematopoietic cells

Reticuloendotheliosis virus strain T (REV-T)-transformed cells gave rise spontaneously to variants which secrete a factor that forms a distinct visible ring of precipitation (halo) surrounding colonies grown in soft agar. An Mr 15,000 protein was produced at higher levels by halo variants than by nonhalo-producing cells. An assay designed to detect the formation of precipitates enabled purification of an Mr 15,000 protein, p15, from serum-free medium conditioned by the growth of REV-T-transformed hematopoietic cells. Fractions enriched in p15 permitted the growth of REV-T-transformed cells under conditions where they normally failed to proliferate.     

Inhalation toxicity of methylisocyanate: assessment of germ cell mutagenicity and reproductive effects in rats.

Adult male Wistar rats were exposed to methylisocyanate (MIC, 3.2 mg/l, single inhalation exposure for 8 min under static condition) or ethyl methanesulphonate (EMS, 150 mg/kg, single ip dose) for the assessment of germ cell mutagenicity and reproductive effects. Sequential matings of treated males with normal females on days 1-7, 8-14 and 15-21 post-exposure did not indicate any induction of dominant lethal mutation (increased frequency of preimplantation losses and early fetal deaths) by MIC but it was significantly induced by EMS as compared to respective controls. Males, necropsied after 21 days of exposure, showed no effect of MIC on epididymal sperm density and morphology. EMS also had no effect on sperm density but it significantly induced morphological abnormalities in sperm as compared to untreated controls. There was an acute and transitional reduction in reproductive performance (10-21%) of MIC-exposed males during days 1-14 post-exposure followed by recovery to the normal level during days 15-21 post-exposure. The progeny of MIC-exposed males was also normal in terms of litter size, litter weight, neonatal survival and body weight gain in litters up to 10 days post-partem. It is concluded with the evidence at hand that the observed failure of MIC to cause germ cell mutagenicity is related to its poor biodistribution to the target site(s) and a transient reduction in the reproductive performance of MIC-exposed males is a result of general stress and disconsolate copulation.     

Molecular cloning of Clostridium difficile toxin A gene fragment in lambda gt11

Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in lambda gt11. Approx. 35,000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, lambda cd19. Verification of the immunological identity of the isolated toxin A gene fragment in lambda cd19 was determined by affinity purifying toxin A antibodies specific for lambda cd19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in lambda cd19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in lambda cd19 was not cytotoxic for 3T3 mammalian tissue culture cells.     

G6PD Punjab,a dialysis sensitive variant of human glucose-6-phosphate dehydrogenase

Erythrocyte samples from 101 individuals, originally from Punjab and living at the time of investigation in England, were screened for glucose-6-phosphate dehydrogenase (G6PD) variants by Beutler’s fluorescent spot test and standard cellulose acetate gel (Cellogel) electrophoresis. All but 2 of the 40 males in the study were found to be indistinguishable from normal G6PD B. One of the variants had 2% of the normal activity and resembled G6PD Mediterranean in electrophoretic behaviour. The other variant showed 52% of the normal activity and migrated slower than G6PD B in Cellogel with about half of the normal band intensity. A set of physicochemical characteristics of the variant determined by conventional methods distinguished it from the variants reported so far. It was designated as G6PD Punjab, and the corresponding allele asG6PD PUN. The most striking feature of G6PD Punjab is a remarkable alteration in its electrophoretic behaviour after dialysis.     

UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli

Summary Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function.     

Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obtained by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.