Genotoxicity assessment of three reference chemicals in rat hepatocytes by alkaline comet assay / 上海预防医学

Objective:To establish a comet test method for detection of genotoxicity of three reference chemicals in rat liver cells. Methods:6-10 week old Sprague Dawley rats were randomly divided into 4 groups， with normal saline （0.9% NaCl solution） as negative control group. Animals in three test groups were treated， respectively， with ethyl methanesulfonate （EMS） 200 mg/kg， N-methyl-N-nitrosourea （MNU） 50 mg/kg， and D-mannitol 2 000 mg/kg. There were 10 animals in each group， 5 males and 5 females. The animals received two times （21 h interval） of test compounds through intragastric administration， and their clinical symptoms and body weight changes were recorded during the experiment. The rats were sacrificed 3 h after the last exposure. The liver was weighed， then used to prepare single-cell suspensions for the alkaline comet test which determines the average tail DNA content percentage （DNA%） of hepatocytes and other comet indicators. Results:（1） D-mannitol， EMS and MNU did not show significant toxicity in the whole animal. （2） The mean values of tail DNA content percentage （DNA%） of rat hepatocytes in EMS ［（60.07±24.69）%］ and MNU ［（41.66±22.35）%］ groups were higher than that in the negative control group ［（2.32±1.39）%］ and the difference was statistically significant （P<0.05）. The difference between D-mannitol group ［（3.06±3.30）%］ and the negative control group was not significant （P>0.05）. Conclusion:This laboratory has established a comet test method using hepatocytes from treated rats. Among three testing chemicals， EMS and MNU have displayed genotoxicity by this assay， but no genotoxicity was observed in D-mannitol treated animals.

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.

Lipopeptide production by Bacillus subtilis R1 and its possible applications

Abstract The possible application of a bacterial strain - Bacillus subtilis R1, isolated from an oil contaminated desert site in India, as biocontrol agent and its biosurfactant in microbial enhanced oil recovery are discussed. The biosurfactant production in minimal medium was carried out at different temperatures and salt concentrations, where it produced an efficient biosurfactant at 30-45 °C and in presence of up to 7% salt. It significantly reduced the surface tension from 66 ± 1.25 mN/m to 29 ± 0.85 mN/m within 24 h. In order to enhance the biosurfactant production, random mutagenesis of B. subtilis R1 was performed using chemical mutagen - ethyl methanesulfonate. Majority of the isolated 42 mutants showed biosurfactant production, but the difference was statistically insignificant as compared with parent strain R1. Therefore none of the mutants were selected for further study, and only parent strain R1 was studied. The biosurfactant was quite stable under harsh conditions for up to 10 days. The biosurfactant was extracted and characterized as similar to the lipopeptide group - surfactins and fengycin. The crude oil displacement experiments using biosurfactant broth in sand pack glass columns showed 33 ± 1.25% additional oil recovery. The strain also showed inhibition of various plant pathogenic fungi on potato dextrose agar medium.

Determination of the Genotoxicity Impurity in Mesylate Nafamostat Raw Materia by GC-MS / 中国药房

OBJECTIVE:To establish a method for determination the genotoxicity impurities (methyl methanesulfonate,ethyl methanesulfonate and isopropyl methanesulfonate) in mesylate nafamostat raw materia. METHODS:GC-MS was conducted,and the genotoxicity impurities were extracted by dichloromethane. The column was DB-5 capillary column by programmed tempera-ture,the inlet temperature was 240 ℃,column flow was 3.0 ml/min,purge flow was 6.0 ml/min,sample mode splitless injection, carrier gas was high purity helium,detector is a mass spectrometer detector,ion source temperature was 230 ℃,the interface tem-perature was 230 ℃,the delay time of solvent was 2.5 min,ionization mode was electron impact,detector voltage was respect to the tuning results,scanning(detection)method was selective ion monitoring,electron energy was 70 eV,and the injection volume was 1.0μl. RESULTS:The separation degree of 3 impurities were greater than 2.0;the linear range of 3 impurities were 0.10-20μg/ml (r≥0.999 5);RSDs of precision,stability and reproducibility tests were lower than 2%;recoveries were 97.7%-104.8%(RSD=2.8%, n=9),102.5%-110.7%(RSD=2.6%,n=9)and 103.0%-107.6%(RSD=1.6%,n=9). CONCLUSIONS:The method is simple, accurate,sensitive and rapid,and can be used for the genotoxicity impurities in mesylate nafamostat raw materia.

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.