Rutaecarpine Ameliorates Murine N-Methyl-N'-Nitro-N-Nitrosoguanidine-Induced Chronic Atrophic Gastritis by Sonic Hedgehog Pathway.

CAG is a burdensome and progressive disease. Numerous studies have shown the effectiveness of RUT in digestive system diseases. The therapeutic effects of RUT on MNNG-induced CAG and the potential mechanisms were probed. MNNG administration was employed to establish a CAG model. The HE and ELISA methods were applied to detect the treatment effects. WB, qRT-PCR, immunohistochemistry, TUNEL, and GES-1 cell flow cytometry approaches were employed to probe the mechanisms. The CAG model was successfully established. The ELISA and HE staining data showed that the RUT treatment effects on CAG rats were reflected by the amelioration of histological damage. The qRT-PCR and WB analyses indicated that the protective effect of RUT is related to the upregulation of the SHH pathway and downregulation of the downstream of apoptosis to improve gastric cellular survival. Our data suggest that RUT induces a gastroprotective effect by upregulating the SHH signaling pathway and stimulating anti-apoptosis downstream.

ARTP and NTG compound mutations improved Cry protein production and virulence of Bacillus thuringiensis X023.

A high production mutated strain Bacillus thuringiensis X023PN (BtX023PN) was screened from the wild strain Bacillus thuringiensis X023 (BtX023) after atmospheric and room temperature plasma (ARTP) and nitrosoguanidine (NTG) mutation. BtX023PN grows faster than the wild strain, and its lysis of mother cell was 6 h ahead BtX023, but the ability of sporulation was significantly reduced. Bioassay indicated that compared with the wild type strain, the virulence of BtX023PN against Plutella xylostella (P. xylostella) and Mythimna seperata (M. seperata) increased to 2.33-fold and 2.13-fold respectively. qRT-PCR and SDS-PAGE demonstrated that the production of Cry1Ac increased by 61%. Resequence indicated that the mutated sites enriched on the key carbohydrate metabolism and amino acid metabolism. This study provides a new strain resource for the development of Bt insecticides and a feasible technical strategy for the breeding of Bt. KEY POINTS: • Atmospheric and room temperature plasma used in breeding of Bacillus thuringiensis. • Less stationary phase time with more ICP production. • Semi-lethal concentration against Plutella xylostella reduced by about 57.

Breast cancer drugs perturb fundamental vascular functions of endothelial cells by attenuating protein S-nitrosylation.

Cardiovascular side effects of broadly used chemotherapeutic drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) among cancer survivors are well established. Nitric oxide (NO) is known to protect cardiovascular tissues under conditions of stress. NO can act through cyclic guanosine monophosphate (cGMP)-dependent and -independent pathways. Particularly, the S-nitrosylation of SH-groups in a protein by NO falls under cGMP-independent effects of NO. TC, CP, and EP are hypothesized as interfering with cellular protein S-nitrosylation, which, in turn, may lead to endothelial dysfunctions. The results show that all three drugs attenuate nitrosylated proteins in endothelial cells. A significant reduction in endogenous S-nitrosylated proteins was revealed by Saville-Griess assay, immunofluorescence and western blot. Incubation with the drugs causes a reduction in endothelial migration, vasodilation and tube formation, while the addition of S-nitrosoglutathione (GSNO) has a reversal of this effect. In conclusion, results indicate the possibility of decreased cellular nitrosothiols as being one of the reasons for endothelial dysfunctions under TC, CP and EP treatment. Identification of the down-regulated S-nitrosylated proteins so as to correlate their implications on fundamental vascular functions could be an interesting phenomenon.

Amelioration of spinal cord injury in rats by blocking peroxynitrite/calpain activity.

BACKGROUND: Spinal cord injury (SCI) is one of the leading causes of disability and chronic pain. In SCI-induced pathology, homeostasis of the nitric oxide (NO) metabolome is lost. Major NO metabolites such as S-nitrosoglutathione (GSNO) and peroxynitrite are reported to play pivotal roles in regulating the activities of key cysteine proteases, calpains. While peroxynitrite (a metabolite of NO and superoxide) up regulates the activities of calpains leading to neurodegeneration, GSNO (a metabolite of NO and glutathione) down regulates the activities of calpains leading to neuroprotection. In this study, effect of GSNO on locomotor function and pain threshold and their relationship with the levels of peroxynitrite and the activity of calpain in the injured spinal cord were investigated using a 2-week rat model of contusion SCI. RESULTS: SCI animals were initially treated with GSNO at 2 h after the injury followed by a once daily dose of GSNO for 14 days. Locomotor function was evaluated by "Basso Beattie and Bresnahan (BBB) locomotor rating scale" and pain by mechanical allodynia. Peroxynitrite level, as expression of 3-nitrotyrosine (3-NT), calpain activity, as the degradation products of calpain substrate alpha II spectrin, and nNOS activity, as the expression phospho nNOS, were measured by western blot analysis. Treatment with GSNO improved locomotor function and mitigated pain. The treatment also reduced the levels of peroxynitrite (3-NT) and decreased activity of calpains. Reduced levels of peroxynitrite resulted from the GSNO-mediated inhibition of aberrant activity of neuronal nitric oxide synthase (nNOS). CONCLUSIONS: The data indicates that higher levels of 3-NT and aberrant activities of nNOS and calpains correlated with SCI pathology and functional deficits. Treatment with GSNO improved locomotor function and mitigated mechanical allodynia acutely post-injury. Because GSNO shows potential to ameliorate experimental SCI, we discuss implications for GSNO therapy in clinical SCI research.

Enhancing the Production of D-Mannitol by an Artificial Mutant of Penicillium sp. T2-M10.

D-Mannitol belongs to a linear polyol with six-carbon and has indispensable usage in medicine and industry. In order to obtain more efficient D-mannitol producer, this study has screened out a stable mutant Penicillium sp. T2-M10 that was isolated from the initial D-mannitol-produced strain Penicillium sp.T2-8 via UV irradiation as well as nitrosoguanidine (NTG) induction. The mutant had a considerable enhancement in yield of D-mannitol based on optimizing fermentation. The production condition was optimized as the PDB medium with 24 g/L glucose for 9 days. The results showed that the production of D-mannitol from the mutant strain T2-M10 increased 125% in contrast with the parental strain. Meanwhile, the fact that D-mannitol is the main product in the mutant simplified the process of purification. Our finding revealed the potential value of the mutant strain Penicillium sp. T2-M10 to be a D-mannitol-producing strain.

Antimutagenic activity of vitamin B1 against damages induced by chemical and physical mutagens in Salmonella typhimurium and Escherichia coli.

Thiamine (vitamin B1) is an essential nutrient acting mainly as an enzymatic cofactor on diverse cell processes. It has been reported that vitamin B1 has a significant role in the signaling pathways related to the response to adverse environmental conditions (chemical and physical). The objectives of this study were to evaluate the antimutagenic potential of vitamin B1 in front of DNA-alkylating agents in the presence/absence of ogt and ada repairing genes in Salmonella typhimurium strains and against damage induced by ultraviolet light type C in Escherichia coli strains mutated at the uvrABC system and recBCD enzymes. For S. typhimurium, an antimutagenesis test (Ames test) was performed using strains deficient in one or both genes (YG7100 ada-/ogt+, YG7104 ada+/ogt-, YG7108 ada-/ogt-). For E. coli, mutated strains (K-12 derived strains Hfr H180 uvrB+/recA+, W3110 uvrB+/recA- and ATCC®8739 uvrB-/recA+) were exposed to UV-C light at different time intervals, with and without vitamin B1. Our results showed that thiamine is an antimutagen against methyl-N-nitro-N-nitrosoguanidine or ethyl-N-nitro-N-nitrosoguanidine only when the ogt gene is present. While for E. coli, the presence of vitamin B1 increased the survival rate, implying an antimutagenesis independent of uvrABC repairing system and recBCD enzymes.

Enhanced butanol production from cassava with Clostridium acetobutylicum by genome shuffling.

To obtain strains exhibiting high levels of solvent tolerance and butanol production, wild type strains of Clostridium acetobutylicum butanol-producing strain GX01 and Lactobacillus mucosae butanol-tolerant strain M26 were subjected to mutagenesis combining N-methyl-N-nitro-N-nitrosoguanidine induction with genome shuffling. After four successive rounds of genome shuffling, the C. acetobutylicum shuffled strain GS4-3 showing greater levels of fermentation performances (such as secreting a higher level of amylase, improving the thermal stability, and possessing greater environmental robustness) compared to the wild type strains was isolated. As a result, after optimization of culture conditions, mutant GS4-3 produced 32.6 g/L of total solvent, 20.1 g/L of butanol production, and 0.35 g/L/h of butanol productivity, which were, respectively, increased by 23.5, 23.3, and 40.0 % than the wild-type strain GX01, in a 10 L bioreactor. The enhanced production of butanol and tolerance of solvent of mutant associated with GS4-3 make it promising for acetone/butanol/ethanol fermentation from cassava (Manihot esculenta).

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function.

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500 nmol/mg protein-approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.

Chemical and UV Mutagenesis.

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

The NanoString-based multigene assay as a novel platform to screen EGFR, HER2, and MET in patients with advanced gastric cancer

Introduction: Molecular targets are emerging rapidly and the development of clinical tests that simultaneously screen for multiple targets has become especially important. We assessed the gene expression levels of three known targets in advanced gastric cancer, epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), and N-methyl-N-nitrosoguanidine human osteosarcoma transforming gene (MET), using the nCounter assay (NanoString Technologies, Seattle, WA, USA) and compared these results with protein overexpression, detected by immunohistochemistry, to evaluate the performance of this new technology. Methods: We investigated 42 formalin-fixed, paraffinembedded tumor samples from patients with gastric cancer. A NanoString-based assay containing a 522 kinase gene panel was investigated. We analyzed the correlations between immunohistochemical findings and kinase gene expression levels of EGFR, HER2 and MET to validate this assay. Results EGFR, HER2, and MET overexpression were observed in 7 (16.6 %), 5 (11.9 %), and 3 (7.1 %) cases, respectively. For EGFR, HER2, and MET, the concordance rates between the NanoString-based assay results and the immunohistochemistry methods were 83.3, 97.6, and 100 %, respectively. Relative to immunohistochemistry findings, the NanoString-based assay sensitivities and specificities were 85.7 and 82.8 % for EGFR, 100 and 97.2 % for HER2, and 100 and 100 % for MET, respectively. Conclusions: We found a high concordance between immunohistochemistry- and nCounter-based assessments of EGFR, HER2, and MET in advanced gastric cancer. Judged against immunohistochemistry results, the NanoString assay had high sensitivities and high specificities. These results suggest that the nCounter assay provides a reliable, high-throughput assay to simultaneously screen for the overexpression of several target proteins (AU)

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Endogenic oxidative stress response contributes to glutathione over-accumulation in mutant Saccharomyces cerevisiae Y518.

Mechanisms of glutathione (GSH) over-accumulation in mutant Saccharomyces cerevisiae Y518 screened by ultraviolet and nitrosoguanidine-induced random mutagenesis were studied. Y518 accumulated higher levels of GSH and L-cysteine than its wild-type strain. RNA-Seq and pathway enrichment analysis indicated a difference in the expression of key genes involved in cysteine production, the GSH biosynthesis pathway, and antioxidation processes. GSH1, MET17, CYS4, GPX2, CTT1, TRX2, and SOD1 and the transcriptional activators SKN7 and YAP1 were up-regulated in the mutant. Moreover, Y518 showed a dysfunctional respiratory chain resulting from dramatically weakened activity of complex III and significant elevation of intracellular reactive oxygen species (ROS) levels. The supplementation of antimycin A in the culture of the parent strain showed equivalent changes of ROS and GSH level. This study indicates that defective complex III prompts abundant endogenic ROS generation, which triggers an oxidative stress response and upregulation of gene expression associated with GSH biosynthesis. This finding may be helpful for developing new strategies for GSH fermentation process optimization or metabolic engineering.

Improvement of microbial strain and fermentation process of rapamycin biosynthesis.

The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) and N-methyl-N'-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain.

Acetone, butanol, and ethanol production from cane molasses using Clostridium beijerinckii mutant obtained by combined low-energy ion beam implantation and N-methyl-N-nitro-N-nitrosoguanidine induction.

In order to obtain mutant strains showing higher solvent tolerance and butanol production than those of wild-type strains, the butanol-producing strain Clostridium beijerinckii L175 was subjected to mutagenesis using a combined method of low-energy ion beam implantation and N-methyl-N-nitro-N-nitrosoguanidine induction. With this effort, mutant strain MUT3 was isolated. When it was used for butanol fermentation in P2 medium, the production of butanol was 15.8±0.7 g/L 46% higher than the wild-type strain. Furthermore, after optimization of butanol production from cane molasses with MUT3, the maximum butanol production of 14.9±0.5 g/L were obtained in crew-capped bottles. When ABE production by MUT3 was carried out in a bioreactor, the production of butanol and total solvent were 15.1±0.8 g/L and 22.1±0.9 g/L, respectively. The remarkable butanol production and solvent tolerance of MUT3 make it promising for butanol production from cane molasses.

Isolation of Streptomyces globisporus and Blakeslea trispora mutants with increased carotenoid content.

Hyperpigmented mutants of Streptomyces globisporus 1912-Hp7 and Blakeslea trispora 18(+), 184(-) were isolated by action of hydrogen peroxide and nitrosoguanidine, correspondingly, from initial strains S. globisporus 1912-4Lcp and B. trispora 72(-), 198(+). The carotenoids of dry biomass of obtained strains, rubbed thoroughly with glass powder by a pestle in porcelain mortar were extracted by acetone and purified by TLC. Identification of the individual carotenoids was performed by means of HPLC and LC/MS spectrometry. It was shown that strain S. globisporus 1912-4Crt produced beta-carotene/lycopene (6.91/3.24 mg/L), mutants 1912-4Lcp and 1912-7Hp synthesized only lycopene (26.05 and 50.9 mg/L, respectively), and strains B. trispora 18(+) and 184(-)-beta-carotene (6.2% in dry biomass or more 2.5 g/L) without illumination in shake flasks. It is the first example of high constitutive production of the carotenoids by the representative of genus Streptomyces without photoinduction or increased synthesis of sigma factor The improved strains of B. trispora 18(+) and 184(-) can be used for biotechnological production of beta-carotene in industrial conditions.

[Genetic variability of synthesis feature of carotenoids in Streptomyces globisporus 1912].

Seventeen spontaneous and induced mutants, that acquired a new characteristic--the synthesis of beta-carotene and lycopene, were obtained from strain Streptomyces globisporus 1912. It was found that spontaneous mutants inherited more stably the acquired carotenogenesis as compared to induced ones. Synthesis of carotenoids by all isolated Crt+ Lcp+ cultures is a constitutive feature. It was shown that Crt(+)-mutants (4Crt, 6Crt, 7Crt, RVCrt and R3Crt) synthesized beta-carotene and lycopene, while Lcp(+)-mutants (TpS16-1, TpS16-2, 4Lcp and R3Lcp)--only lycopene. The obtained mutants and transformants of S. globisporus 1912, synthesizing carotene were characterized by a simultaneous change of two or three phenotypic characteristics: synthesis of the antibiotic landomycin E. sporullation and carotenogenesis. It can be assumed that the high instability of this characteristic (carotenogenesis) in strain S. globisporus 1912 was caused by localization of the crt-genes cluster close to a TIR-element in a chromosome terminal region, frequent structural reorganization of DNA here were reported in the literature.

Enhanced ß-galactosidase production from whey powder by a mutant of the psychrotolerant yeast Guehomyces pullulans 17-1 for hydrolysis of lactose.

In order to isolate ß-galactosidase overproducers of the psychrotolerant yeast Guehomyces pullulans 17-1, its cells were mutated by using nitrosoguanidine (NTG). One mutant (NTG-133) with enhanced ß-galactosidase production was obtained. The mutant grown in the production medium with 30.0 g/l lactose and 2.0 g/l glucose could produce more ß-galactosidase than the same mutant grown in the production medium with only 30.0 g/l lactose while ß-galactosidase production by its wild type was sensitive to the presence of glucose in the medium. It was found that 40.0 g/l of the whey powder was the most suitable for ß-galactosidase production by the mutant. After optimization of the medium and cultivation conditions, the mutant could produce 29.2 U/ml of total ß-galactosidase activity within 132 h at the flask level while the mutant could produce 48.1 U/ml of total ß-galactosidase activity within 144 h in 2-l fermentor. Over 77.1% of lactose in the whey powder (5.0% w/v) was hydrolyzed in the presence of the ß-galactosidase activity of 280 U/g of lactose within 9 h while over 77.0% of lactose in the whey was hydrolyzed in the presence of ß-galactosidase activity of 280 U/g of lactose within 6 h. This was the first time to show that the ß-galactosidase produced by the psychrotolerant yeast could be used for hydrolysis of lactose in the whey powder and whey.

[Genoprotective activities of the oils from leaves and fruits of Fagus orientalis Lipsky].

The antimutagenic activities of the oils obtained from leaves and fruits of Fagus orientalis have been shown in experiments with spontaneous and mutagen- and ageing-induced variability. The aberrations of chromosomes in the meristematic cells of the Allium cepa L., Vicia faba L., Triticum aestivum L., and marrow cells of Vistar rats as well as Arabidopsis thaliana gene mutations have been mobilized as experimental tests.

Synergistic cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine and absence of poly(ADP-ribose) glycohydrolase involves chromatin decondensation.

DNA-alkylating agents in combination with poly (ADP-ribose) (PAR) synthesis inhibitors are a promising treatment for cancer. In search of other efficacious alternatives, we hypothesized that the absence of poly(ADP-ribose) glycohydrolase (PARG), which leads to the inhibition of PAR hydrolysis, would lead to increased DNA alkylation after treatment with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). At a sublethal dose, MNNG shows synergistic cytotoxicity in PARG-null embryonic trophoblast stem (TS) cells. The PAR modifications of histone H1 and histone H2B are much more pronounced in PARG null-TS cells exposed to MNNG, suggesting their relevance in the efficacy of this combination therapy. Because the PAR modification of these chromatin binding proteins leads to chromatin remodeling, a possible mechanism for the observed synergistic effects involves the subsequent decondensation of chromatin, which may cause the genomic DNA to be more accessible to MNNG alkylation. Further analysis demonstrated chromatin decondensation in PARG null-TS cells as visualized by electron microscopy. In addition, treatment with MNNG led to an increase in O6- methylguanine levels in PARG null-TS cells compared to wild-type, which demonstrates increased DNA alkylation in the absence of PARG. Taken together, we provide compelling evidence that the absence of PARG leads to chromatin decondensation, which in turn leads to increased amounts of DNA alkylation and cell death induced by low doses of MNNG. Therefore, combination therapy of PARG inhibition and a DNA- alkylating agent is a potential treatment to induce the death of cancer cells.

[Genetic improvement of technological characteristics of starters for fermented milk products].

Possibility for improvement of technological characteristics of lactobacilli using mutations of resistance to rifampicin (rif(r)) and streptomycin (str(r)) was studied. Using starter model of Narine Lactobacillus acidophilus INMIA-9602 Armenian diet milk product, it was showed that a possibility for selecting strains with increased rate of milk fermentation and acid production is higher in Rif(r) and Str(r) mutants induced by nitrosoguanidine than in cultures sensitive to antibiotics. The milk products obtained using Rif(r) and Str(r) strains had high viscosity, improved texture, increased amount of alive cells and good organoleptic features.

Multiple induced mutagenesis for improvement of ethanol production by Kluyveromyces marxianus.

Kluyveromyces marxianus GX-15 was mutated multiple times by alternately treatment with UV irradiation and NTG for two cycles. Four mutant strains with improved ethanol yield were obtained. The maximum ethanol concentration, ethanol yield coefficient and theoretical ethanol yield of the best mutant strain, GX-UN120, was 69 g/l, 0.46 g/g and 91%, respectively, when fermenting 150 g glucose/l at 40°C. The corresponding values for GX-15 were 58 g/l, 0.39 g/g and 76%, respectively. GX-UN120 grew well in 11% (v/v) of ethanol, while GX-15 could not grow when ethanol was greater than 8% (v/v).