nitro-N-nitrosoguanidine Research Articles

Potential preventive effects of Chelidonium majis L. (Papaveraceae) herb extract on glandular stomach tumor development in rats treated with N-methyl-N'-nitro-N nitrosoguanidine (MNNG) and hypertonic sodium chloride.

The modifying effects of Chelidonium majis L. (Papaveraceae) herb extract (CH), an analgesic traditionally prescribed for gastric and duodenal ulcer patients, on gastric tumor development were studied in rats given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Sixty-four male 6-week-old Wistar rats were used. Group 1 rats were initially given MNNG (200 mg/kg b.w.) by gavage at days 0 and 14 as well as saturated sodium chloride solution (S-NaCl, 1 ml per rat) every 3 days during weeks 0-3 (six times), and then placed on basal diet containing 0.1 or 0.2% CH for 16 weeks from week 4. Rats of Group 2 and 3 were treated with MNNG together with S-NaCl or saline (0.9% NaCl, 1 ml per rat), respectively, timed as in Group 1 but without further treatment. All surviving animals were killed at week 20 and histopathologically investigated. In the glandular stomach, the number of preneoplastic pepsinogen 1 altered pyloric glands (PAPGs) in the MNNG + S-NaCl-->CH (0.1%) group (Group 1) was significantly smaller than in the MNNG + S-NaCl group (Group 2) (P < 0.02). The incidences of forestomach neoplastic lesions (papillomas and squamous cell carcinomas) also showed a tendency to decrease with the CH treatment. The results thus indicate that CH exerts inhibitory effects on glandular stomach carcinogenesis in the rat, so that it may have potential as a chemopreventive agent for stomach cancer in man.

The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe.

Plants and the fission yeast Schizosaccharomyces pombe synthesize small cadmium-binding peptides, called phytochelatins, in response to cadmium. Derived from glutathione (GSH: gamma-Glu-Cys-Gly), they have the general structure (gamma-Glu-Cys)nGly, where n is 2-11. In order to study the biosynthesis of phytochelatins, we used the mutagen N-methyl-N'-nitro-N nitrosoguanidine (MNNG) to select mutants with a lowered GSH content. GSH-deficient mutants show a Cd-sensitive phenotype, whereas resistance to Cu is only slightly influenced. These Cd-sensitive mutants contain 2-15% of the wild-type GSH level. For three mutants a lowered activity of gamma-glutamylcysteine synthetase was measured. One of the mutants was transformed to Cd-resistance and the complementing fragment was analyzed further. The complementing fragment hybridized with chromosome III. In the transformants, GSH content was restored up to wild-type levels, whereas the activity of gamma-glutamylcysteine synthetase was significantly increased compared with the wild-type. Possible mechanisms for Cd-resistance in the transformants are discussed.

The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe

Plants and the fission yeast Schizosaccharomyces pombe synthesize small cadmium-binding peptides, called phytochelatins, in response to cadmium. Derived from glutathione (GSH: λ-Glu-Cys-Gly), they have the general structure (λ-Glu-Cys)nGly, where n is 2–11. In order to study the biosynthesis of phytochelatins, we used the mutagen N-methyl-N′-nitro-N nitrosoguanidine (MNNG) to select mutants with a lowered GSH content. GSH-deficient mutants show a Cd-sensitive phenotype, whereas resistance to Cu is only slightly influenced. These Cd-sensitive mutants contain 2–15% of the wild-type GSH level. For three mutants a lowered activity of λ-glutamylcysteine synthetase was measured. One of the mutants was transformed to Cd-resistance and the complementing fragment was analyzed further. The complementing fragment hybridized with chromosome III. In the transformants, GSH content was restored up to wild-type levels, whereas the activity of λ-glutamylcysteine synthetase was significantly increased compared with the wild-type. Possible mechanisms for Cd-resistance in the transformants are discussed.

Estrogens, androgens, and EGF receptor expression in gastric carcinoma induced byN-methyl-N'-nitro-N-nitrosoguanidine

Complex and conflicting relationships between epidermal growth factor (EGF), estrogens (E), androgens (A), and related receptors (EGF-R, E-R, A-R) have been reported in different biological situations associated with cell proliferation. There is also evidence that EGF and sex hormone receptors may be involved in normal and neoplastic growth of the gastrointestinal mucosa. In this study, we investigated the behavior of EGF receptors and sex hormone and related receptors, during N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced gastric carcinogenesis in Sprague-Dawley male rats. Four groups of 15 rats each (10 NG-treated and five controls) were sacrificed after 1, 20, 30, and 40 weeks of treatment. Gastric tissue from each rat was processed for receptor status (number and affinity) and proliferative activity. A significant and progressive decrease of A-R and EGF-R was observed starting from the 20th week, while no change of E-R occurred throughout the experiment. Cell proliferation in the gastric mucosa of NG-treated rats increased after 30 weeks of treatment. These data indicate that NG treatment is able to modify the receptor status of gastric mucosa in rats.

A Positive Selection of Methylotrophic YeastHansenula PolymorphaContainingURA 3Auxotrophic Mutation

ABSTRACTMethylotrophic yeast Hansenula polymorpha is an interesting eukaryotic system used in biotechnology. Yeast cells have been mutagenized with NTG (N-methyl-N'-nitro-N-nitrosoguanidine) to obtain strains defective in URA genes. The mutated cells were enriched with uracil auxotrophs by 5-FOA (S-Fluoro-orotic acid). From all H. polymorpha uracil auxotrophs 20.5% ura37minus; and ura5− mutant strains H. polymorpha were obtained. URA− mutants were transformed with shuttle vector YepBU36 containing the URA3 gene of S. cerevisiae as a selective marker in yeast, ARS of pL36 (a plasmid design for propagation in H. polymorpha leu2 arg4 strains) for rapid identification of ura3 mutants. 11 ura strains were transformed with YepBU36, 5 of them showed appearance of the uracil prototrofity, identifying them as ura3− mutants.

Chrymutasins: novel-aglycone antitumor antibiotics from a mutant of Streptomyces chartreusis. I. Taxonomy, mutation, fermentation, isolation and biological activities.

Three novel antibiotics, named chrymutasins A, B and C, were isolated from the fermentation products of a mutant strain obtained by NTG (N-methyl-N'-nitro-N-nitrosoguanidine) treatment. The mutant strain produced the chrymutasins, which differed in the aglycone moiety from chartreusin, and related compounds. The production of these compounds needed a characteristically long fermentation period. The antitumor activity of chrymutasin A is better in vivo than that of chartreusin, the cytotoxic activity against cell lines in vitro is equivalent, and the antimicrobial spectrum is narrower.

Physiological characterization of chlorate-resistant mutants of the cyanobacteriumSpirulina major

Chlorate-resistant (ChlR)mutants SM11 and SM25 of the cyanobacterium Spirulina major were produced by N-methyl-N'-nitro-N-nitrosoguanidine (NTG)1) mutagenesis. Strain SM11 showed nitrate reductase (NR) and glutamine synthetase (GS) activities equal to the SMP (parent) whereas SM25 had about 45% NR and 78% GS as compared to SMP. However, the uptake of nitrate or ammonia was almost equal in SMP, SM11 and SM25. ChlR strains differed physiologically between themselves, SM11 being blocked in uptake system of chlorate (Chl up−) and was not defective in NR or GS, while SM25 showed chlorate uptake (Chl up−) and was defective in NR (NRdef) and GS (GSdef). Thus strain SM11 resembles SMP except that the latter is chlorate sensitive. This is the first report of defective NR in any filamentous, non-heterocystous, non-nitrogen fixing cyanobacterium. Defective chlorate uptake not accompanied by defective NO uptake has also been observed in strain SM11.

Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from Pseudomonas fluorescens and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease

A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit R. solani and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by R. solani were generated by exposure to N-methyl-N' -nitro-N-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other P. fluorescens strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by R. solani was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two P. fluorescens strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress R. solani-induced damping-off of cotton. These results indicate that we have isolated from P. fluorescens BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by R. solani.

Enhancement of methionine production by methionine analogue ethionine resistant mutants of Brevibacterium heali

AbstractIn order to improve the methionine yield of the isolate B. heali, attempts were made to isolate mutants resistant to the methionine analogue DL‐ethionine after mutagenesis with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (NTG). The minimum inhibitory concentration (MIC) of ethionine for B. heali was found to be 2 mM. After mutagenesis and screening, five mutants resistant to 50 mM of ethionine were isolated. The yield of the best ethionine resistant mutant, B. heali Br EthR, was 13 mg/l of methionine medium under optimum cultivation conditions.

A Novel Δ5-Desaturase-Defective Mutant of Mortierella alpina 1S-4 and Its Dihomo-γ-Linolenic Acid Productivity

A novel Delta5-desaturase-defective mutant was derived from an arachidonic acid-producing fungus, Mortierella alpina 1S-4, after treating the parental spores with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant produced only a trace (about 1%) amount of arachidonic acid, and the ratio of dihomo-gamma-linolenic acid (DGLA) to total fatty acids in each lipid class was markedly high, accounting for as much as 60% in phosphatidylcholine. Under submerged batch culture conditions, the mutant produced 2.4 g of DGLA per liter (43.3% of total fatty acids) when grown at 28 degrees C for 7 days in a 5-liter jar fermentor. The other major (more that 1%) fatty acids were palmitic acid (21.2%), stearic acid (9.6%), oleic acid (14.3%), linoleic acid (4.4%), and gamma-linolenic acid (5.8%). About 80 mol% of the DGLA produced was found in triacylglycerol.

Selection of antibiotic-resistant mutants with enhanced isoamylase activity in Pseudomonas amyloderamosa

Isoamylase-hyperproducing strains of Pseudomonas amyloderamosa were bred by mutagenesis with UV light and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). The selection criterion for such strains was based on the formation of large turbid zones around the bacterial colonies in agar medium containing antibiotics and 1% waxy corn starch. Mutant WN6410 was obtained by treating P. amyloderamosa JD210 with five cycles of 1 × 104 μJ UV light and one cycle of NTG. P. amyloderamosa WN6410 had 22-fold increase in isoamylase activity when compared to wild-type strain SB15 and the maximal enzyme activity, 5,100 U/ml, could be achieved within 48 h in 2.5 L fed-batch fermentation.

PRODUCTION of FLAVOR BYAMUTANT of YEAST

ABSTRACT. Strain CCU-N16-18143 was derived with N-methyl-N‘-nitro-N-nitrosoguanidine (NTG) treatment three times from a wild strain of yeast CCU-16 as a mutant to produce more delicious and intense flavors for nutrient beverage from glucose medium. This mutant was identified by computer system as Saccharomyces sp. the optimal culture medium for the production of flavor is one liter of medium containing 100 g glucose, 12 g ammonium nitrate, 3 g yeast extract, 1 g magnesium sulfate, 1 g ammonium sulfate, 2 g potassium dihydrogen phosphate, pH: 3.5. the optimal culture conditions are: temperature: 30C; agitation: 150 rpm., 50 ml medium in 500-ml Hinton flask; incubation time: 96 h. the volatile flavor compounds in the culture medium were analyzed by capillary gas chromatography and capillary gas chromatography-mass spectrometry. It was found that more volatile compounds were produced by mutant strain than wild strain. the content of isoamyl alcohol increased from 7.76 to 61.64%, whereas ethyl alcohol decreased from 85.25 to 5.77%.

3] High-level secretion of heterologous proteins Bacillus brevis

Publisher Summary Among various host-vector systems for the production of foreign proteins in microorganisms, the use of Bacillus brevis ( B. brevis )as a host offers the advantage that proteins are secreted directly into the culture medium, where they are accumulated at high levels in a relatively pure state. B. brevis is isolated from soil as a protein-hyperproducing bacterium and is found to show little extracellular protease activity. Even when positive transformants of B. brevis are found, these cells sometimes cannot maintain the correct plasmid. Deletions of the foreign gene or cwp promoter are frequently found. Serial single-colony isolation of positive clones is helpful. The mutagenesis of B . brevis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) prior to transformation is also helpful to obtain clones that can maintain the plasmid with the correct structure. When clones maintaining the plasmid can be obtained but the amount of foreign proteins produced is not large, mutants producing the protein with improved yields can be isolated by the mutagenesis of the clones with NTG.

Interphasic nucleolar organizer regions expression and cell kinetics evaluation during gastric carcinogenesis induced by nitrosoguanidine in the rat.

An increased number of interphasic nucleolar organizer regions containing ribosomal cistrons associated with argyrophilic proteins (AgNORs) has been described in human malignant tumor cells. In this study variations in AgNOR numbers have been compared with changes of cell kinetics, evaluated by the mitotic count (MC) and bromodeoxyuridine labeling index (BrdU LI), during gastric carcinogenesis induced with N-methyl-N'-nitro-N-nitrosoguanidine (NG) in rats. Significant differences (2 P < 0.005) in AgNOR mean numbers, evaluated in the antral isthmic cells, in MC mean values and BrdU LI, evaluated in the whole antral cellular population, were found when comparing areas of acute gastritis, atrophy and hyperplasia in NG-treated rats with the normal mucosa in controls. No differences were observed in MC and BrdU LI between normal antrum and carcinoma cells which showed an AgNORs mean number lower than in the isthmic cells of controls (2 P < 0.005). Moreover, significant correlations were found comparing changes in AgNOR numbers with MC (r = 0.89, P < 0.001) and BrdU LI (r = 0.66, P < 0.001) in different lesions. These data show that evaluation of AgNOR numbers does not allow the identification of malignant cells in NG-induced gastric carcinoma. However AgNOR quantification seems to be a reliable index of cell kinetics and related well with the cellular dividing fraction.

Elimination of by-products in 11 beta-hydroxylation of substance S using Curvularia lunata clones regenerated from NTG-treated protoplasts.

Stable mutants showing improved 11-hydroxylation of Substance S were isolated, following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and regeneration of uninucleate protoplasts of the appropriate fungal strains. This procedure was especially suitable for obtaining more directed 11 beta-hydroxylation of Substance S with Curvularia lunata IM 2901. Apart from producing cortisol (11 beta-hydroxy-S), the parent strain formed several by-products that significantly lowered the yield of the desired 11 beta-hydroxyderivative. Isolated mutants of this microorganism carried out directed 11 beta-hydroxylation with only a small amount of one of the by-products, which resulted in a much higher yield of cortisol.

Isolation and characterization of furanomycin nonproducing Streptomyces threomyceticus mutants.

Stable mutants that are blocked in the production of the polyketide antibiotic furanomycin were generated by treatment of Streptomyces threomyceticus (ATCC 15795) with a combination of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ultraviolet light. On the basis of their cosynthetic properties in mixed culture, the mutants were grouped into eleven phenotypic classes. The polarity of the cosynthetic reactions for seven classes of mutants was revealed by filtrate feeding experiments. This allowed them to be arranged in the most probable linear sequence of metabolic blocks. One of the remaining groups of mutants exhibited no cosynthetic behavior and is believed to be blocked in a gene that regulates furanomycin biosynthesis.

Relationship between Pigment Productivity and Some Other Bacteriological Properties in the Mutant Strains of Pseudomonas glumae Induced by Nitrosoguanidine-treatment.

Pseudomonas glumae, the causal agent of the grain rot and seedling rot of rice, is known to show antibacterial activities against other plant pathogenic bacteria and caused rotting of potato tuber slices. In this study, 50 mutant strains were raised from a strain Kyu82-34-2, which produced water soluble, yellowish-green pigment on YPDA or in YPD medium, by NTG (N-methyl-N'-nitro-N-nitrosoguanidine)-treatment. The mutant strains were compared each other in pigment productivity, antibacterial activities to several plant pathogenic bacteria, potato rotting activity and virulence to rice seedlings. All of the 28 pigment productive strains were virulent to rice seedlings and potato tuber slices. Almost all of pigment non-productive strains were avirulent to rice seedlings with only exception of two strains, NT49 and NT106. Pigment productive strains showed wider antibacterial spectra with higher activities as compared to pigment non-productive strains. About a half of the mutant strains lost both pigment productivity and potato rotting activity.

Adhesion of Macroconidia to the Plant Surface and Virulence of Nectria haematococca

To study spore attachment of the cucurbit pathogen Nectria haematococca (anamorph, Fusarium solani f. sp. cucurbitae), mutants with adhesion-deficient macroconidia were isolated. The adhesion-deficient mutants were selected after treatment with N-methyl-N' -nitro-N-nitrosoguanidine followed by repeated enrichment for macroconidia which did not attach to polystyrene. Two independently derived mutants produced macroconidia with an approximately 50% reduction in attachment to polystyrene and to zucchini fruits. When macroconidia were inoculated into wounded zucchini fruits, the adhesion-deficient mutants were as virulent as the wild-type strain. However, in disease assays in which macroconidia were deposited onto the surface of unwounded zucchini, the mutants were less virulent than the wild type. Thus, adhesion of N. haematococca macroconidia to its host surface appears to be a virulence factor.

Biosynthesis of anthracyclines: analysis of mutants of Streptomyces sp. strain C5 blocked in daunomycin biosynthesis

Summary: Streptomyces sp. strain C5, an organism that normally produces baumycins, daunomycin and ɛ-rhodomycinone, was treaed with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). Mutants blocked at arious points in daunomycin and baumycin production were isolated by screening for altered pigmentation and absnce of bioactivity against Staphylococcus aureus. Examination of the mutants by thin-layer chromatography of their accumulated anthracycline metabolites, by cosynthesis assays, and by extract feeding experiments allowed a classification into six groups. Theses were: dauA, strains that accumulated no anthracyclines but with other blocked mutants cosynthesized anthracyclines (polyketide-synthase-minus mutants); dauG, regulatory mutants that, either alone or mixed with other blocked mutants, accumulated no anthracyclines; dauC, mutants that accumulated aklanonic acid; dauE, mutants that accumulated maggiemycin; dauF, mutants that accumulated aklavinone; and dauH, mutants that accumulated only ɛ-rhodomycinone. Mutant SC5-24 (dauE), which accumulated that shunt product maggiemycin, was re-mutagenized with NTG to obtain blocked mutants in preceding biosynthetic steps; the three grops of double mutants obtained accumulated aklanonic acid (dauC,E), aklanonic acid methyl ester (dauD,E) and akalviketone (dauE,F).

The use of cellulases from a β‐glucosidase‐hyperproducing mutant of Trichoderma reesei in simultaneous saccharification and fermentation of wheat straw

Conidia of the cellulolytic strain Trichoderma reesei F522 were mutagenized with UV irradiation and N-methyl|-N'-nitro-N-nitrosoguanidine (NTG). A visual agar plate detection system was developed, using esculin and ferric ions, to identify mutants of T. reesei with increased beta-glucosidase activity. Selected mutants were tested for production of extracellular cellulases in shake flasks on autohydrolyzed wheat straw as carbon source. The most active mutant V-7 showed about 6-times higher activity of beta-glucosidase than the parent strain F-522, whereas the filter paper degrading and endo-1,4-beta-D-glucanase activities increased by 45% and by almost 31%, respectively. Cellulase preparations obtained from the parent and mutant strains were then used along with Kluyveromyces fragilis cells for ethanol production from ethanol-alkali pulped straw in the simultaneous saccharification and fermentation (SSF) process. From 10% (w/v) of straw pulp (dry matter), 2.5% (w/v) ethanol was obtained at 43 degrees C after 48 h using cellulase derived from the parent strain of T. reesei. When the beta-glucosidase-hyperproducing mutant V-7 was employed, the ethanol yield in the SSF process increased to 3.4% (w/v), the reaction time was shortened to 24 h and no cellobiose was detected in straw hydrolyzates.