Inducible protective processes in animal systems XV: Hyperthermia enhances the Ethyl methanesulfonate induced adaptive response in meiotic cells of grasshopper Poecilocerus pictus

Purpose: To understand the role of hyperthermia in adaptive response, Ethyl methanesulfonate [EMS] an anticarcinogenic agent, adapted meiotic cells of Poecilocerus pictus was used

Materials and methods: Based on the pilot toxicity study, the effective higher temperatures of 40[degree sign]C and 45[degree sign]C for 15 or 30 min were chosen. P. pictus were treated with conditioning [L] or challenging [H] doses of EMS and 2 h time lag [TL] between these doses [L-2 h-H] was employed. Different treatment schedules were used to analyze the influence of hyperthermia on EMS induced adaptive response namely [i] pre treatment; [ii] inter treatment; [iii] post treatment and [iv] cross adaptation. After each treatment schedule, animals were sacrificed at 12, 24, 36 and 48 h recovery times, testes were processed for meiotic chromosome preparations and anomalies were analyzed

Results: The frequencies of anomalies induced by both conditioning and challenging doses of EMS were significantly higher [p< 0.05] compared to those of the control and hyperthermia groups. The combined treatments resulted in 44-50% reduction compared to additive effect of EMS. The pre, inter, post and cross adaptation treatments with hyperthermia significantly reduced the frequencies of chromosomal anomalies compared to the challenge and combined treatments with EMS at all recovery times [p< 0.05] tested

Conclusion: There is a protection against EMS induced anomalies by hyperthermia in in vivo P. pictus. As far as our knowledge is concerned, this is the first report to demonstrate that hyperthermia enhances the EMS induced adaptive response in in vivo meiotic cells

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70 percent increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Isolation of 4-aminopyridine resistant mutants affecting alkali-insoluble glucan content of cell walls in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells when grown on synthetic medium plates containing 10 mM of 4-aminopyridine (4-AP) undergo cell lysis. Using an ethylmethane sulfonate mutagenesis (EMS) screen, 4-AP resistant mutants (apr) were isolated which could grow on inhibitory concentration of 4-AP. Eighty mutants were obtained that were recessive, monogenic and formed two complementation groups. To identify genes, whose products might be interacting with the apr loci, extragenic suppressors were isolated, which reverted 4-AP resistance phenotype of apr mutants. The suppressors, when genetically characterized, were found to be recessive and represented two loci with overlapping functions. Representative alleles from apr mutants were analyzed for cell wall composition. They were found to have a higher amount of alkali-insoluble glucan signifying the role of alkali-insoluble glucan in cell wall maintenance.

RAPD analysis of herbicide-resistant Brasilian rice lines produced via mutagenesis

Over the last two decades, mutational techniques have become one of the most important tools available to progressive rice- breeding programs. In a mutation-breeding program initiated in 1999 at the Instituto Agronômico of Campinas, SP, Brazil, a rice line, IAC103, was selected for mutational studies with gamma radiation and ethyl methyl sulfonate mutagenesis, with the aim of developing a herbicide-resistant crop. After mutagenesis, surviving plants were exposed to glufosinate to check for herbicide resistance, which was examined up to the second generation. A detailed RAPD analysis was made of the resistant plants. Eighty Operon technology primers were tested and 10 were selected for a detailed study of RAPD markers that could tag herbicide resistance genes. Resistant and susceptible lines produced variation in the RAPD patterns and certain bands were found only in certain lines. These results suggest genetic ligation that will be confirmed through a genetic segregation study

Chromosomal break points in irradiated and ethyl methane sulphonate treated leucocytes of patients with Down syndrome.

Frequencies of chromosomal damage in the peripheral leucocytes of patients with Down syndrome, on exposure to gamma rays (2Gy) or ethyl methane sulphonate (EMS, 1x 10(-4) M), were assessed. Analysis of break points in the chromosomes of irradiated cells revealed a non-random occurrence. Six of the break points observed in EMS-treated cells were found to overlap with those recorded in irradiated cells. Thirteen break points observed were found to correlate with the location of cancer-specific break points and four of these coincided with the bands where oncogenes have been located. Two break points were localised to the same bands as that of known heritable fragile sites.

Ethyl methanesulfonate-induce mutations in Phaseoulus vulgaris L.

Os alcalóides furoquinoleínico esquimianina e benzofenantridínico celeritrina, extraídos de uma espécie da família Rutaceae foram testados quanto ao aspecto tóxico-genético através do cromoteste-SOS. Nos testes realizados na ausência de metabolizaçäo, ambos alcalóides näo mostraram atividade genotóxica, sendo que a esquimianina apresentou um efeito citotóxico nas concentraçöes mais elevadas. Na presença de mistura de ativaçäo metabólica, a esquimianina mostrou-se genotóxica sendo que este efeito foi mais acentuado quando se empregou fraçäo microssomal induzida com Aroclor 1254 em relaçäo aquela induzida com 3-metilcolantreno

Combined effect of ethylmethane sulfonate and 60CO gamma radiation on the induction of lethal mutations in Drosophila melanogaster

Duas linhagens de Drosophila melanogaster foram tratadas com doses independentes e conjuntas de etilmetanosulfonato (EMS) e radiaçäo gama de 60CO. As linhagens Columbia (CO3) e Riverside, Califórnia (RC1) foram tratadas com três doses de EMS (0.003; 0.006 e 0.012 M), ou com quatro doses de radiaçäo (2,5; 5; 10 e 15 KR) ou, ainda, com três doses conjuntas dos dois mutagênicos (0.002M + 10 KR; 0.003 + 5 KR e 0.004M + 2.5 KR). Foram estudadas diferenças de sensibilidade entre as duas linhagens, quanto à induçäo de letais recessivos ligados ao sexo, e determinadas as relaçöes existentes entre altas doses de mutagênico e freqüência da mutaçäo induzida. A relaçäo entre dose de EMS e produçäo de letais se distribuiu dentro de uma curva parabólica. Tal comportamento decorreu ou da eliminaçäo seletiva das células mais sensíveis sob o ponto de vista da mutabilidade, ou da saturaçäo na capacidade de absorçäo do mutagênico, quando administrado em altas doses. Na dose de 0.006 M de EMS, a percentagem de letais obtida indicou a existência na linhagem CO3, de resistência à açäo deste mutagênico. No tratamento conjunto com EMS e radiaçäo, a linhagem CO3 apresentou um efeito sinergístico, enquanto a RC1 mostrou um efeito aditivo. Dados anteriores sugerem que a linhagem CO3 apresenta um mecanismo de reparo mais eficiente, já que o tratamento conjunto parece inibir as enzimas de reparo, a linhagem CO3 é mais atingida por este tipo de inibiçäo