Diethyl Sulfate Research Articles

Diethyl sulfate-induced cell cycle arrest and apoptosis in human bronchial epithelial 16HBE cells

In this study, we investigated the effects of diethyl sulfate (DES) on cell proliferation, cell cycle progression and apoptosis in human bronchial epithelial 16HBE cells. Cells were treated with various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0mM) for 12, 24 or 36h. Cell proliferation and apoptosis were determined by MTT assay and flow cytometer, respectively. The results showed that DES inhibited cell proliferation in a dose- and time-dependent manner, and induced significant apoptosis in 16HBE cells. Apoptosis related proteins measurement results revealed that DES-induced apoptosis was concurrent with the increasing of Bax and cleavage fragment caspase-3 and the decreasing of Bcl-2 and full length procaspase-3. When cells were incubated with 2.0mM of DES for several time intervals, S and G2/M phase accumulation was observed. Further analysis indicated that both DES-induced G1/S transition acceleration and S arrest resulted in S phase accumulation, and that DES-induced G2/M arrest resulted in G2/M phase accumulation. Western blotting results demonstrated that after DES treatment p-chk1 (Ser345) and p-chk2 (Thr68) levels decreased in G1 cells, and increased in S and G2/M cells. In addition, the increasing of chk1 and chk2 were also induced by DES treatment. With the increase in the dose of DES, p53 levels first increased (0.5–4.0mM) and then decreased (8.0mM). Down-regulation of p53 by RNA interference increased 4.0mM of DES-induced apoptosis but did not affect 2.0mM DES-induced cell cycle arrest. In conclusion, DES inhibits 16HBE cells proliferation in a dose- and time-dependent behavior. Within the sublethal dose, DES induces S and G2/M arrest through activating DNA damage checkpoints. Within the lethal dose, DES induces apoptosis through evoking apoptosis programs. p53 might play an important role in the transition between evoking cell cycle arrest/pro-survival and apoptosis programs upon DES exposure.

설파디아진 은으로부터 항균 산성염료 및 분산염색용 항균 첨가제의 합성과 이들의 특성 분석

Two acid dyes having excellent antimicrobial activity were synthesized by using silver sulfadiazine, a well-known antibiotic, and 8-amino-1-naphthol-3,6-disulfonic acid (H-acid). The diazonium salt of silver sulfadiazine (S1) and the acetylated H-acid (H1) were prepared first, and then an antimicrobial red acid dye (Acid-Red) was synthesized via a coupling reaction of S1 and H1. An antimicrobial blue acid dye (Acid-Blue) was also synthesized by the successive coupling reactions of H-acid with the diazonium salt of aniline-2,5-disulfonic acid (B1) and the diazonium salt of sliver sulfadiazine (S1). Two colorless antimicrobial additives for disperse dyeing, Disperse-ES and Disperse-AS, were also synthesized by reacting silver sulfadiazine with diethyl sulfate and acetic anhydride, respectively. The synthesized antimicrobial dyes and additives were characterized by Fourier transform infrared (FT-IR), UV-vis, and energy dispersive X-ray (EDX) analyses. Although the nylon fabric dyed with either Acid-Red or Acid-Blue showed poor light fastness, it had good washing and crocking fastness. The nylon fabrics dyed with Acid-Red and Acid-Blue and polyethylene terephthalate (PET) fabrics treated with Disperse-ES or Disperse-AS showed excellent antimicrobial activities after 10 repeated launderings.

Antimutagenic activity of vitamin‐E (α‐ tocopherol) and gallic acid (polyphenol) on the callus raised from mutagenized seeds of Trigonella foenum‐graecum L.

Antimutagenicity of vitamin‐E (α‐ tocopherol) and gallic acid (polyphenol) has been determined in callus raised from diethyl sulphate (DES; pH 7.41) treated seeds of Trigonella foenum‐graecum L. using toxic effects on the callus growth as the criterion. In case of calli raised from seeds treated with the DES (mutagenic agent) alone, maximum reduction in the growth index (GI), i.e., 0.85 was observed in 0.75% DES concentration treated series. However, approximately 50% lethality in GI was observed in the 0.50% DES treated series as compared to control where GI was 3.87. Calli raised from DES mutagenized seeds of T. foenum‐graecum post‐treated with two concentrations of vitamin‐E (0.25% and 0.50%) and gallic acid (0.0010M and 0.0020M) exhibited antimutagenic activity by way of promotory effects on the callus growth.Vitamin‐E was found to be more potent as antimutagen (antitoxic) than gallic acid.

Mutation breeding of acetoin high producing Bacillus subtilis blocked in 2,3-butanediol dehydrogenase

Bacillus subtilis mutants were obtained after the wild strain JNA 3-10 was mutagenized by UV irradiation coupled with diethyl sulfate. A visual filter assay was employed for the qualitative identification of 2,3-butanediol dehydrogenase (BDH) blocked B. subtilis. Selected mutants were tested for the activities of acetoin reductase (AR) and BDH. According to further batch fermentation, one mutant named JNA-UD-6 that produced 24.3% more acetoin than JNA 3-10 with the corresponding byproducts of 2,3-butanediol decreased by 39.8% was isolated. A nonsense mutation (p.Tyr118X) that precluded the synthesis of a full-length functional AR/BDH within the bdhA gene of JNA-UD-6 was detected. Acetoin production of JNA-UD-6 was further improved to about 53.9g/L in a 5-L fermentor with 150g/L glucose consumed. However,a small amount of 2,3-butanediol was found in late phase of JNA-UD-6 fermentation, and it was due to the existence of a putative gene that encoding a minor AR. This work proved a strategy to efficiently breeding an acetoin high producing strain by traditional mutation methods.

Genome shuffling of Lactobacillus plantarum for improving antifungal activity

Many species of lactic acid bacteria have the ability to inhibit the growth of spoilage fungi in food and feed products. Genome shuffling is a powerful process that could efficiently improve desirable industrial phenotypes of microbial strains through recursive protoplast fusion. In our study, the antifungal activity of Lactobacillus plantarum IMAU10014 was improved by genome shuffling. The starting populations were obtained via ultraviolet irradiation and diethyl sulphate mutagenesis and subjected to recursive protoplast fusion with Lactobacillus helveticus IMAU40097. The optimal conditions for protoplast formation and regeneration were found to be 15 mg/ml lysozyme and 5 μg/ml mutanolysin for 40 min at 37 °C. The protoplasts were inactivated under ultraviolet light for 30 min or heated at 60 °C for 120 min. After three rounds of genome shuffling, the inhibitory activity of strain F3C2 and F3A3 were improved to 192% and 200%, respectively, compared to the wild-type strain. The shuffled strains have a broad spectrum of antifungal activity and are promising candidates for bio-preservatives. It was also revealed that lactic acid has a positive correlation with antifungal activity. Antifungal activity was not related only to lactic acid, phenyllactic acid or any other single inhibitory substance, but rather to a combination of such substances.

Synthesis of 1,1-Bis(hydroxymethyl)cyclopropane as Intermediate for Montelukast Sodium

1,1-Bis(hydroxymethyl)cyclopropane was synthesized by reaction dibromoneopentyl glycol with zinc powder at mole ratio of nDG:nzinc=1:1.05 and 80oC (slightly reflux) for 7 hours. Under optimal conditions, yield and purity of the title compound were 87.3% and 98.67%, respectively. Dibromoneopentyl glycol was also prepared in 91.7% yield with pentaerythritol and hydrobromic acid as material and diethyl sulfate as catalyst under reflux for 10 hours. Structural characterization of 1,1-bis(hydroxymethyl)cyclopropane was conducted by IR and H-NMR.

Screening of a <i>Mortierella isabellina</i> Mutant for Producing Arachidonic Acid

Arachidonic acid (AA) is an essential component of structural lipids in the cellular membranes of mammals. To obtain efficient AA-producing microorganism, the strain of Mortierella isabellina 3410 was subject to mutagenesis by treating the wild-type spores exposed to ultraviolet or diethyl sulfate. A mutant of M. isabellina MS-1 was screened and chosen because of its higher AA production capability. Compared with the wild strain of M. isabellina 3410, the AA production by mutant of M. isabellina MS-1 was increased about 2.5 folds. Analysis by gas chromatography (GC) indicated that AA was the main fatty acid in the lipids extracted from the dried mycelia of the mutant of M. isabellina MS-1.

Reactions of 3-nitro-1,2,4-triazole derivatives with alkylating agents. 9.* Preparation of N(1)-, N(2)- and N(4)-ethyl-5-R-nitro-1,2,4-triazoles by the alkylation of 5-R-3-nitro-1,2,4-triazolate anions with diethyl sulfate

The product of substitution at the nitrogen atom N(4) is found along with main N(1)- and N(2)-substituted 5-R-3-nitro-1,2,4-triazoles among the products of 5-R-nitro-1,2,4-triazolate anion (R = H, Me, Et) reactions with diethyl sulfate in aqueous medium. The fraction of 5-R-4-ethyl-3-nitro-1,2,4-tri-azoles, which varies from 1.3 to 6.9%, depends on the temperature, ratio of the starting reagents, and the degree of the alkylating agent consumption.

Changes in the Activity of Digestive Enzymes in Response to Chemical Mutagen Diethyl Sulfate in the Silkworm Bombyx mori L. (Lepidoptera: Bombycidae)

The silkworm Bombyx mori L. is a economic sericigenous insect show a high degree of sensitivity towards chemical mutagens and have drastic physiological and biochemical effects during its larval stages. In the present study a bivoltine race of silkworm NB 4D 2 was treated with chemical Mutagen Diethyl Sulfate (DES). The larvae were used for the treatment with different concentrations of DES orally as well as injected through body wall. In F 1 generation, the larvae of 4th and 5th instar were used to study the activity of prominent digestive enzymes amylase and protease for evaluation of the mutagenic response in haemolymph and midgut tissues. The enzyme activity was found to be higher in treated sets, particularly in 8 mM injection sets. Further, maximum enzyme activity was noticed during middle part of both the instars with a few variations among the treated sets. The data were statistically analysed using One-way ANOVA and discussed. © 2012 Knowledgia Review, Malaysia.

Effect of Chemical Mutagens on Vegetative Growth and Flowering in Calendula Officinalis L. (Cv. Calypso Yellow

The experiments were carried out to study the effect of different concentrations of two chemical mutagens; Sodium Azide (SA) and Diethyl Sulphate (DES); on some morphological traits in the M1-and M2-generations of Calendula officinalis (L.) c.v. Calypso Yellow. Seeds were treated with different SA and DES concentrations : 0 ( distilled water), 1000, 2000, 3000, 4000 and 5000 ppm. Germination percentage, plant height, number of leaves, number of branches, fresh weight, dry weight, number of inflorescences, inflorescences diameter, flowering date, leaf total chlorophyll, and root length were recorded. Both chemical mutagens was found to have significant effects in M1- generation with respect to dry weight, inflorescences diameter, flowering date, total leaf chlorophyll. The results clarified that DES significantly increased dry weight, total leaf chlorophyll. The different concentrations had significant effect in M1– generation with respect to seed germination percentage, plant height, number of leaves, fresh weight, flowering date, inflorescences diameter, total leaf chlorophyll content. While both chemical mutagens have significant effect the M2- generation with respect to number of leaves, leaf area, fresh weight, number of inflorescences, inflorescences diameter, flowering date and root length. The interaction between the two chemical mutagens and the concentrations treatments exerted significant effects for most of the studied parameters.

Purpurogemutantin and Purpurogemutantidin, New Drimenyl Cyclohexenone Derivatives Produced by a Mutant Obtained by Diethyl Sulfate Mutagenesis of a Marine-Derived Penicillium purpurogenum G59

Two new drimenyl cyclohexenone derivatives, named purpurogemutantin (1) and purpurogemutantidin (2), and the known macrophorin A (3) were isolated from a bioactive mutant BD-1-6 obtained by random diethyl sulfate (DES) mutagenesis of a marine-derived Penicillium purpurogenum G59. Structures and absolute configurations of 1 and 2 were determined by extensive spectroscopic methods, especially 2D NMR and electronic circular dichroism (ECD) analysis. Possible biosynthetic pathways for 1–3 were also proposed and discussed. Compounds 1 and 2 significantly inhibited human cancer K562, HL-60, HeLa, BGC-823 and MCF-7 cells, and compound 3 also inhibited the K562 and HL-60 cells. Both bioassay and chemical analysis (HPLC, LC-ESIMS) demonstrated that the parent strain G59 did not produce 1–3, and that DES-induced mutation(s) in the mutant BD-1-6 activated some silent biosynthetic pathways in the parent strain G59, including one set for 1–3 production.

SYNTHESIS AND ANTIPLASMODIAL ACTIVITY TESTING OF (1)-N-ALKYL- AND (1)-N-BENZYL-6-NITRO-1,10-PHENANTHROLINIUM SALTS AS NEW POTENTIAL ANTIMALARIAL AGENTS

The synthesis of 5-nitro-1,10-phenanthroline hydrate 2 derivatives from 1,10-phenanthroline monohydrate as starting material has been carried out. The 5-nitro-1,10-phenanthroline hydrate 2 was obtained through nitration reaction using H2SO4 and HNO3 as catalyst and reagent, respectively. Synthesis of (1)-N-alkyl-6-nitro- and (1)-N-benzyl-6-nitro-1,10-phenanthrolinium have been prepared using dimethyl sulphate (DMS), diethyl sulphate (DES), benzyl chloride, benzyl bromine, and benzyl iodide. The reagents of benzyl bromine, and benzyl iodide were synthesized from benzyl chloride using NaBr in ethanol absolute and NaI in acetone, respectively. The five compounds of 5-nitro-1,10-phenanthroline hydrate 2 derivatives were conducted to evaluate the in vitro antiplasmodial activity. The in vitro antiplasmodial was evaluated on strains of Plasmodium falciparum FCR-3 resistant chloroquine and D10 sensitive chloroquine. The 50% inhibition concentration (IC50) of the five compounds ranged from 2.41±1.41 to 0.07±0.01 μM. The results showed that the (1)-N-benzyl-6-nitro-1,10-phenanthrolinium iodide had highest antiplasmodial activity.

Improvement and optimization of the media of Saccharomyces cerevisiae strain for high tolerance and high yield of ethanol

Saccharomyces cerevisiae, ATCC 76740 was mutated after treatment with diethyl sulfate (DES) in the paper. Successive mutants showed high tolerance against both higher concentrations of ethanol and furfural and higher temperature. In addition, the mutant 4D-6 showed 2.2 fold in ethanol yield to the wild type at 37°C in shake culture. Furthermore, in order to improve the ethanol production of 4D-6, the sequential statistical approach was applied to optimize compositions of the medium, which the optimal medium was comprised 29 g/L glucose, 10 g/L yeast extract, 20 g/L peptone, 1.5 g/L KH2PO4, 1.81 g/L KNO3 and 0.078 g/L ZnCl2. In a word, the maximum ethanol production of fermentation 4D-6 in optimal medium was 11.2 g/L, which was about 1.6-fold to the original medium and 3.6-fold to the wild type stain in the original medium. The relative error between expected value and validation experimental value was 2.3%. Key words: High tolerance, high yield of ethanol, mutation breeding, Plackett-Burman experimental design, artificial neural network, genetic algorithm.

Assessment of mutagenicity induced by MMS and DES in Capsicum annuum L.

Seeds of Capsicum annuum L. var. G4 were subjected to different concentrations of methyl methane sulphonate (MMS) and diethyl sulphate (DES). The effects of different mutagenic treatments on meiosis, chiasma frequency, and pollen fertility have been studied in M1 generation. Various types of meiotic aberrations such as univalent, multivalent, stickiness, bridge, laggards, cytomixis etc. were observed in all the treatments. However, the MMS treatments proved to be more effective in inducing meiotic aberrations as compared to DES. Moreover, the frequency of meiotic aberrations was at its maximum at metaphase followed by anaphase and telophase stages. As the concentrations increase, reduction in chiasma frequency and pollen fertility was observed in all the treatments and, MMS again was found to be more effective than DES treatments.

Improvement of Coenzyme Q10 Production: Mutagenesis Induced by High Hydrostatic Pressure Treatment and Optimization of Fermentation Conditions

Coenzyme Q10 (CoQ10, ubiquinone), a potent antioxidative dietary supplement, was produced by submerged fermentation using Agrobacterium tumefaciens instead of chemical synthesis or solvent extraction. Agrobacterium tumefaciens 1.2554 was subjected to mutagenesis using a series of treatments including high hydrostatic pressure (HHP) treatment, UV irradiation, and diethyl sulfate (DES) treatment to obtain mutant strains showing higher CoQ10 production than wild-type strains. A mutant strain PK38 with four genetic markers was isolated: the specific CoQ10 content of the mutant strain increased by 52.83% compared with the original strain. Effects of carbon and nitrogen sources on CoQ10 production with PK38 were studied. Sucrose at concentration of 30 g/l was tested as the best carbon source, and yeast extract at concentration of 30 g/l supplemented with 10 g/l of ammonium sulfate was identified to be the most favorable for CoQ10 production using PK38. Fed-batch culture strategy was then used for increasing production of CoQ10 in 5-l fermentor. Using the exponential feeding fed-batch culture of sucrose, cell growth and CoQ10 formation were significantly improved. With this strategy, the final cell biomass, CoQ10 production, and specific CoQ10 production increased by 126.11, 173.12, and 22.76%, respectively, compared to those of batch culture.

Synthesis, characterization and chemoprotective activity of polyoxovanadates against DNA alkylation

The alkylation of pUC19 plasmid DNA has been employed as a model reaction for the first studies on chemoprotective action by a mixed-valence (+IV/+V) polyoxovanadate. A new, non-hydrothermal route for the high yield preparation of the test compound is described. The deep green, microcrystalline solid A was isolated after a three-day reaction in water at 80°C and 1atm, while the reaction at 100°C gave green crystals of B. Both solids were structurally characterized by X-ray diffractometry and FTIR, EPR, NMR and Raman spectroscopies. Product A was identified as (NH4)2V3O8, while B corresponds to the spherical polyoxoanion [V15O36(Cl)]6−, isolated as the NMe4+ salt. The lack of solubility of A in water and buffers prevented its use in DNA interaction studies, which were then carried out with B. Complex B was also tested for its ability to react with DNA alkylating agents by incubation with diethylsulphate (DES) and dimethylsulphate (DMS) in both the absence and presence of pUC19. For DMS, the best results were obtained with 10mM of B (48% protection); with DES, this percentage increased to 70%. The direct reaction of B with increasing amounts of DMS in both buffered (PIPES 50mM) and non-buffered aqueous solutions revealed the sequential formation of several vanadium(IV), vanadium(V) and mixed-valence aggregates of different nuclearities, whose relevance to the DNA-protecting activity is discussed.

Screening of <i>Lactobacillus delbruekii</i> <i>Subsp. Bulgaricus</i> with Weak Post-Acidification Capacity by Natural and Induced Mutation

To derive new starter culture strains with weak post-acidification capacity,Lactobacillus delbruekii subsp. bulgaricusLY were screened by natural screening and inducing screening treated with ultraviolet and diethyl sulfate. Then treated strains were inoculated into low pH MRS medium and skim milk at 37°C for 24h, respectively. The mutants that could grow in skim milk but did not grow in low pH MRS medium were selected strains with weak post-acidification capacity. The mutants were preserved and their generation stabilities were tested. The natural screening, diethyl sulfate and ultraviolet treated mutants showed 5.1%, 5.9% and 11.9% lower post-acidification capacity than original strain at 25°C, respectively. The mutants showed genetically stable post-acidification capacity.

Heat Pipe-Mediated Control of Fast and Highly Exothermal Reactions

The synthesis of 1-ethyl-3-methylimidazolium ethyl-sulfate ([EMIM]EtSO4) from 1-methyl-imidazole and diethyl sulfate suffers from highly exothermal behavior. Once the activation energy barrier is reached (EA = 89 kJ mol–1), the bimolecular reaction accelerates with a high reaction enthalpy (ΔH = −130 kJ mol–1).(1-3) The excess of heat has to be concurrently dissipated to avoid hot spots or thermal runaways. Depending on the volume flow velocity of the reactants and the applied reactor temperature, the reaction zone can be shifted inside the reactor from the inlet to the outlet and vice versa. Therefore, a sophisticated thermal control, oscillating between providing activation energy and intense cooling of the reaction mixture, is required. Heat pipes intrinsically allow both fast dynamic bidirectional heating and cooling. A stainless steel chiplike setup equipped with an externally heated, modified heat pipe system was used.(4) Independently from the reactant volume flow, a colorless and 1H NMR clean product could be obtained by setting a self-controlled optimal operating temperature of about 100 °C.

Genome shuffling improves production of the low-temperature alkalophilic lipase by Acinetobacter johnsonii

The production of a low-temperature alkalophilic lipase from Acinetobacter johnsonii was improved using genome shuffling. The starting populations, obtained by UV irradiation and diethyl sulfate mutagenesis, were subjected to recursive protoplast fusion. The optimal conditions for protoplast formation and regeneration were 0.15 mg lysozyme/ml for 45 min at 37°C. The protoplasts were inactivated under UV for 20 min or heated at 60°C for 60 min and a fusant probability of ~98% was observed. The positive colonies were created by fusing the inactivated protoplasts. After two rounds of genome shuffling, one strain, F22, with a lipase activity of 7 U/ml was obtained.

Synthesis and electrochromic application of surfactants tailored WO 3 nanostructures

As WO 3 is excellent material for electrochromic displays more investigation is needed to find the good and low cost method for preparation of WO 3 nanostructures with uniform morphology and narrow distributions using a surfactant mediated method. In this study, the synthesis of WO 3 nanostructures was accomplished using various surfactants such as polyethylene glycol, sodium chloride and sodium dodecyl sulfate and sodium tungstate and diethyl sulfate as the inorganic precursors. All samples were characterized for their opto-structural and morphological studies by UV–Vis spectrophotometer, X-ray diffractometer and scanning electron microscopy techniques. The electrochromic performance of these samples was studied in LiClO 4/PC as electrolyte for Li + insertion/extraction. The use of surfactants has been employed to enhance the uniformity of WO 3 samples.