Enhanced Production Of Streptokinase By Chemical Mutagenesis Of Streptococcus agalactiae EBL-20

Abstract Streptokinase (SK) is an enzyme that is used for the treatment of cardiovascular diseases. The current study focused on the enhanced production of SK by inducing mutation in Streptococcus agalactiae EBL-20 and optimization of medium components and culture conditions for the maximum growth of mutant derived strain. S. agalactiae EBL-32 was selected as a potent mutant after exposure of S. agalactiae EBL-20 to EMS for 180 minutes. SK activity obtained from mutant derived strain was found to be 1.6 fold higher as compared to the activity achieved by wild strain. Nutritional requirements of the mutated strain were optimized by single factor analysis method suggesting glucose as the optimum carbon source; yeast extract and peptone as a suitable nitrogen sources and corn steep liquor (CSL) as an appropriate substrate for the maximum SK production. The culture conditions determined by response surface methodology (RSM) suggested that a temperature value of 37.5⁰C and pH 7 of the fermentation medium with 2.50 mL inoculum size for 36 hours of incubation was optimum for maximum yield of SK. Hence the optimization studies resulted into 1.92 fold increase in the yield of SK suggesting the new isolate suitable for commercial scale production of SK.

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

Inducible protective processes in animal systems XIV: Cytogenetic adaptive response induced by EMS or MMS in bone marrow cells of diabetic Mouse

Background: Adaptive response has been well studied by employing physical and chemical agents in normal test systems, whereas in diseased conditions very little data are available

Aim of the study: To know the presence or absence of adaptive response in diseased condition, alkylating agents such as EMS or MMS have been employed in diabetic mouse

Material and methods: To induce diabetes, mice were injected with 180 mg/kg body weight of Stz. Diabetic mice were treated with conditioning [100 mg/kg body weight of EMS or 40 mg/kg body weight of MMS], challenging [300 mg/kg body weight of EMS or 160 mg/kg body weight of MMS] and combined doses of EMS or MMS with 8 h time lag. Parallelly controls were maintained. Mice were sacrificed at 24 or 48 or 72 h RTs. Bone marrow was extracted and slides were prepared by a routine air dry technique by Evans et al. [1964] to analyze the chromosomal aberrations

Results: The results show that both the alkylating agents induced exclusively chromatid type of aberrations in both diabetic and non diabetic mice, but it is to be underlined that MMS is a more potent inducer of aberrations than EMS. Eventhough, combined treatment of EMS or MMS induced significantly less chromosomal breaks compared to challenging treatment [p< 0.05] in diabetic mice, EMS induced 40% reduction of breaks, compared to 51.74% by MMS at 24 h RT. This is true to other tested RTs

Conclusion:[1] Methylating agents are a more effective inducer of adaptive response than ethylating agents in diabetic mouse. [2] Further, it is interesting to note that the percentage reduction of chromosomal breaks in diabetics is comparatively much less than in non diabetic mouse, inferring that there is variation in adaptive response between diseased and non diseased condition

Enhanced alkaline cellulases production by the thermohalophilic Aspergillus terreus AUMC 10138 mutated by physical and chemical mutagens using corn stover as substrate

Abstract A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and β-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0–11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for β-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and β-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.

EMS mutation of suspension cells and selection of high temperature tolerant mutants from Pinellia ternata / 中国中药杂志

<p><b>OBJECTIVE</b>To determine the optimal concentration and processing time of EMS mutation for suspension cells from Pinellia ternata.</p><p><b>METHOD</b>Under four EMS concentration gradients (0.1% , 0.2%, 0.4%, 0.6%) and three processing time gradients (0.5, 1.0, 2.0 h), the suspension cells of P. ternata were mutagenized.</p><p><b>RESULT AND CONCLUSION</b>The results showed that the survival rate was significantly different under the different concentrations of EMS and the different processing time. In the same processing time, the EMS concentrations were increased, but the suspension cells survival rate decreased gradually. The optimum EMS concentration for the mutagenesis was 0.4% and the best processing time was 1 hour.</p>

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.

Efeito residual do extrato acetato de etila de Trichilia pallida Swartz ( Meliaceae ) para lagartas de diferentes idades de Spodoptera frugiperda ( J. E. Smith, 1797 ) ( Lepidoptera: Noctuidae ) / Residual effect of ethyl acetate extract of Trichilia pallida Swartz ( Meliaceae ) for Spodoptera frugiperda ( J. E. Smith, 1797 ) ( Lepidoptera: Noctuidae ) larvae of diferents ages

Inseticidas de origem vegetal mostram limitada persistência em condições ambientais já que são degradados pela temperatura, por raios ultravioletas e por outros fatores ambientais. Com o objetivo de avaliar o efeito residual do extrato acetato de etila de Trichilia pallida, plantas de milho cultivadas em casa de vegetação foram pulverizadas com o referido extrato na concentração de 2 por cento, e as suas folhas utilizadas para alimentação de lagartas de Spodoptera frugiperda, em laboratório. As folhas foram oferecidas às lagartas aos 1, 3 e 7 dias após a pulverização, correspondendo cada época a um tratamento. Foram utilizados dois grupos de lagartas: recém-eclodidas e com 10 dias de idade. As folhas foram trocadas diariamente e, 10 dias após o início do experimento, foram avaliados a mortalidade, a largura da cápsula cefálica e o peso das lagartas. Verificou-se que, nas três épocas, o extrato reduziu a sobrevivência e prolongou o desenvolvimento larval. A intensidade destes efeitos foi maior quanto menor o intervalo entre a aplicação do extrato e o início do fornecimento das folhas aos insetos. Lagartas alimentadas com folhas tratadas com o extrato desde a eclosão foram mais afetadas que aquelas alimentadas a partir dos 10 dias.

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

Assessment of EMS-induced genotoxicity in the Indian climbing perch, Anabas testudineus: cytogenetical vis-à-vis protein endpoints.

Genotoxic effects of EMS have been assessed in fish, A. testudineus, using widely accepted cytogenetic protocols like chromosome aberrations, nuclear anomalies in red blood cells and abnormal sperm head morphology. In addition, gel electrophoretic protein profiles and total protein contents in nine selected tissues were analysed for evaluating their utility as potential indicators of genotoxicity. EMS not only caused chromosomal aberrations in somatic cells, nuclear anomalies in red blood cells, and increased incidence of sperm with abnormal head morphology, but also altered significantly both protein profiles and total protein contents in all tissues tested vis-à-vis suitable controls, indicating relevance of protein data in genotoxicity assessment.

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes.

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.

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.

Mutagen sensitivity of an insecticide-resistant strain of Drosophila melanogaster to ethyl methanssulfonate: comparison with a standard strain

Duas cepas de Drosophila melanogaster, uma resistente a vários inseticidas (MRA) e a outra sensível (BK), foram analisadas quanto à mutabilidade face ao conhecido agente alquilante metanosulfato de etila(EMS). Os resultados relativos à induçäo de letales recessivos ligados ao sexo indicam que, embora a cepa padräo seja mais sensível aos efeitos mutagênicos do EMS, com uma taxa de mutaçäo induzida que é aproximadamente o dobro do valor obtido para a cepa MRA, ambas as cepas säo suficientemente sensíveis, apresentando um incremento da mutabilidade em células germinais masculinas pós-meióticas dependente de dose

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