Isolation and characterization of high protein and phycocyanin producing mutants of Arthrospira platensis.

Cyanobacteria are known to exhibit their efficiency in producing high concentrations of compounds of commercial value. Arthrospira is one such cyanobacterium which is considered as important source of protein (65%) and other nutrients. In present study, chemical mutagenesis using N-methyl-N-Nitro-nitrosoguanidine (NTG), a proven potent mutagen for cyanobacteria was used to bring stable and desirable alteration in Arthrospira platensis ARM 730. Three morphological mutants (G-1, G-2, and SF) were selected and characterized. The G-1 and G-2 were helical, more bluish in pigmentation than the wild type strain where G-1 also showed enlarged cell size. The SF mutant was an altered straight-filament having maximum biomass. Among three mutants, higher protein and phycocyanin contents were observed in G-1 and G-2 mutants whereas chlorophyll was less in these mutants as compared to wild type strain indicating change in the pigment ratio. Carotenoid content was higher in SF mutant as compared to wild type and other mutants. Variation in total sugar content was not observed in comparison to wild type strain. The analysis of amino acid spectrum of all the mutants and wild type showed significant increase in proline content. Overall, it is revealed from the results that G-1 and G-2 mutants showed higher biomass, phycocyanin, and protein contents in comparison to wild type which indicated their great potential to be used in food and pharmaceutical industries.

Energy adaptive response during parthanatos is enhanced by PD98059 and involves mitochondrial function but not autophagy induction.

Parthanatos is a programmed necrotic demise characteristic of ATP (adenosine triphosphate) consumption due to NAD+ (nicotinamide adenine dinucleotide) depletion by poly(ADP-ribose) polymerase 1 (PARP1)-dependent poly(ADP-ribosyl)ation on target proteins. However, how the bioenergetics is adaptively regulated during parthanatos, especially under the condition of macroautophagy deficiency, remains poorly characterized. Here, we demonstrated that the parthanatic inducer N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggered ATP depletion followed by recovery in mouse embryonic fibroblasts (MEFs). Notably, Atg5-/- MEFs showed great susceptibility to MNNG with disabled ATP-producing capacity. Moreover, the differential energy-adaptive responses in wild-type (WT) and Atg5-/- MEFs were unequivocally worsened by inhibition ofAMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and mitochondrial activity. Importantly, Atg5-/- MEFs disclosed diminished SIRT1 and mitochondrial activity essential to the energy restoration during parthanatos. Strikingly, however, parthanatos cannot be exasperated by bafilomycin A1 and MNNG neither provokes microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and p62 elimination, suggesting that parthanatos does not induce autophagic flux. Intriguingly, we reported unexpectedly that PD98059, even at low concentration insufficient to inhibit MEK, can promote mitochondrial activity and facilitate energy-restoring process during parthanatos, without modulating DNA damage responses as evidenced by PARP1 activity, p53 expression, and gammaH2AX (H2A histone family, member X (H2AX), phosphorylated on Serine 139) induction. Therefore, we propose that Atg5 deficiency confers an infirmity to overcome the energy crisis during parthanatos and further underscore the deficits in mitochondrial quality control, but not incapability of autophagy induction, that explain the vulnerability in Atg5-deficient cells. Collectively, our results provide a comprehensive energy perspective for an improved treatment to alleviate parthanatos-related tissue necrosis and disease progression and also provide a future direction for drug development on the basis of PD98059 as an efficacious compound against parthanatos.

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011.

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.

Dexmedetomidine Inhibits Parthanatos in Cardiomyocytes and in Aortic Banded Mice by the ROS-Mediated NLRP3 Inflammasome Activation.

Dexmedetomidine (DEX) is clinically used for sedation of patients in intensive care, which also has been shown to have a strong anti-inflammatory effect on a variety of diseases. Parthanatos is a newly discovered form of programmed cell death. Here, we aimed to explore whether DEX protects cardiomyocytes from parthanatos in chronic heart failure (CHF). The levels of malondialdehyde (MAD), total superoxide dismutase (SOD), and adenosine triphosphate (ATP) were measured by corresponding detection kits. CHF mice model was established by transverse aortic constriction (TAC). PARP-1 expression in cardiac tissues of wild-type CHF mice was evaluated by immunohistochemistry. Flow cytometry was used to detect the effect of N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) on cell death. Masson trichrome staining and hematoxylin and eosin staining were conducted in cardiac tissues to evaluate the histological changes. TUNEL and caspase-1 double-staining and caspase-1 and NLRP3 double-staining were conducted in cardiac tissues to evaluate the effect of DEX on cell death in vivo. The relative expression of parthanatos and NLRP3 inflammasome-related proteins was evaluated by western blotting. MNNG induced parthanatos in mouse HL-1 cardiomyocytes. MNNG-induced parthanatos was promoted by ROS production and NLRP3 inflammasome activation. DEX treatment suppressed MNNG-induced parthanatos via NLRP3 inflammasome activation mediated by ROS. Importantly, DEX inhibited pathological changes and parthanatos in CHF mice. DEX suppressed the ROS/NLRP3 signaling pathway in CHF mice. DEX inhibited parthanatos in cardiomyocytes and in CHF mice by regulating the ROS-mediated NLRP3 inflammasome activation. The PARP-1 activation and NLRP3 inflammasome activation induced by MNNG was inhibited by DEX treatment, thus the generation of ROS was further inhibited, suggesting the inhibitory effect of DEX treatment on parthanatos in cardiomyocytes.

Novel pathway of centrosome amplification that does not require DNA lesions.

Centrosome amplification (also known as centrosome overduplication) is common in cancer cells and can be induced by DNA damaging agents. However, the mechanism and significance of centrosome amplification during carcinogenesis or after DNA damage are not clear. Previously, we showed that centrosome amplification could be induced by 3-aminobenzamide (3-AB), an inhibitor of poly(ADP-ribose) polymerases (PARPs) in mouse embryonic fibroblasts. In this paper, we determined if the effect of 3-AB on centrosome amplification was dependent on DNA damage in CHO-K1 cells. We used the well-known mutagen/carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Ten micromolar MNNG and 10 mM 3-AB induced significant centrosome amplification in 18.1 ± 1.1% and 19.4 ± 1.8% of CHO-K1 cells, respectively, compared to 7.0 ± 0.5% of untreated CHO-K1 cells. AG14361, another potent inhibitor of PARPs, also induced centrosome amplification. We then used γ-H2AX analysis and alkaline comet assays to show that 10 µM MNNG induced a significant number of DNA lesions and cell cycle arrest at the G(2) /M phase. However, 10 mM 3-AB neither induced DNA lesions nor altered cell cycle progression. In the umu test, 10 µM MNNG was mutagenic, but 10 mM 3-AB was not. In addition, 10 µM MNNG induced significant accumulation of ataxia telangiectasia mutated protein in the nuclei, but 10 mM 3-AB did not. Thus, we found no association between apparent DNA lesions and centrosome amplification after 3-AB treatment. Therefore, we propose the presence of a novel pathway for centrosome amplification that does not necessarily require DNA lesions but involves regulation of epigenetic changes or post-translational modifications including polyADP-ribosylation.

Screening and characteristics of a butanol-tolerant strain and butanol production from enzymatic hydrolysate of NaOH-pretreated corn stover.

As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hydroscopicity. However, solvent production appeared limited by butanol toxicity. The strain of Clostridium acetobutylicum was subjected to mutation by mutagen of N-methyl-N'-nitro-N-nitrosoguanidine for 0.5 h. Screening of mutants was done according to the individual resistance to butanol. A selected butanol-resistant mutant, strain 206, produced 50 % higher solvent concentrations than the wild-type strain when 60 g glucose/l was employed as substrate. The strain was also able to produce solvents of 23.47 g/l in 80 g/l glucose P2 medium after 70 h fermentation, including 5.41 g acetone/l, 15.05 g butanol/l and 3.02 g ethanol/l, resulting in an ABE yield and productivity of 0.32 g/g and 0.34 g/(l h). Subsequently, Acetone-butanol-ethanol (ABE) production from enzymatic hydrolysate of NaOH-pretreated corn stover was investigated in this study. An ABE yield of 0.41 and a productivity of 0.21 g/(l h) was obtained, compared to the yield of 0.33 and the productivity of 0.20 g/(l h) in the control medium containing 52.47 mixed sugars. However, it is important to note that although strain 206 was able to utilize all the glucose rapidly in the hydrolysate, only 32.9 % xylose in the hydrolysate was used after fermentation stopped compared to 91.4 % xylose in the control medium. Strain 206 was shown to be a robust strain for ABE production from lignocellulosic materials and has a great potential for industrial application.

The Escherichia coli alkylation response protein AidB is a redox partner of flavodoxin and binds RNA and acyl carrier protein.

Microorganisms are exposed to a wide variety of exogenous and endogenous chemical agents that alkylate DNA. Escherichia coli cells exhibit an adaptive response that recognizes and repairs alkylated DNA lesions using Ada, AlkA, and AlkB enzymes. Another alkylation response protein, the DNA-binding flavoprotein AidB, was proposed to repair DNA or protect it from chemical alkylating agents, but direct evidence for its role is lacking. Here, AidB was shown to form tight complexes with both flavodoxin and acyl carrier protein. In addition, electron transfer between 1-electron and 2-electron reduced flavodoxin to oxidized AidB was observed, although with very small rate constants. AidB was found to bind to RNA, raising the prospect that the protein may have a role in protection of RNA from chemical alkylation. Finally, the reagent N-methyl-N'-nitro-N-nitrosoguanidine was eliminated as a direct substrate of the enzyme.

In vitro mutagen binding and antimutagenic activity of human Lactobacillus rhamnosus 231.

In vitro mutagen binding ability of human Lactobacillus rhamnosus 231 (Lr 231) was evaluated against acridine orange (AO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-amino-3, 8-dimethylimidazo-[4,5-f]-quinoxaline (MeIQx) and 4-nitro-o-phenylenediamine (NPD). Binding of AO by Lr 231 is due to adsorption, thereby leading to removal of mutagen in solution and is instantaneous, pH- and concentration-dependent. Whereas, binding of MNNG and MeIQx by Lr 231 results into biotransformation leading to detoxification with subsequent loss of mutagenicity as determined by spectral analysis, thin layer chromatography and Ames test. Binding of mutagen by Lr 231 was dependent on culture age and optimum binding of AO, MNNG and MeIQx was observed to occur with 24 h old culture. Cells of Lr 231 were subjected to different chemical treatments prior to binding studies. Results indicated cell wall component such as cell wall polysaccharide, peptidoglycan, carbohydrates and proteins plays an important role in adsorption of AO, also involving hydrophilic and ionic interactions. Binding, biotransformation and detoxification of MNNG and MeIQx by Lr 231 was dependent on cell surface characteristics mainly involving carbohydrates, proteins, teichoic acid/lipoteichoic acid, hydrophobic interaction and presence of thiol group. L. rhamnosus 231 bound MNNG instantaneously. More than 96 (p < 0.01) and 70% (p < 0.05) cells remained viable after mutagen binding and various pretreatments respectively. This study shows Lr 231 exhibits ability to bind and detoxify potent mutagens, and this property can be useful in formulating fermented foods for removal of potent mutagens.

Synergism of HPV and MNNG repress miR-218 promoting Het-1A cell malignant transformation by targeting GAB2.

Dysregulation of microRNAs (miRNAs) is induced during tumorigenesis. Our previous research suggested that HPV and MNNG led to malignant transformation of esophageal epithelial cells. To investigate the regulation and function of miR-218(miR-218-5p) during the malignant transformation of esophageal epithelial cells, we found miR-218 was inhibited synergistically by HPV and MNNG, suppressing cell proliferation, migration and invasion by up-regulating 3' untranslated region (3'UTR) GAB2 in Het-1A-HPV-MNNG cells (malignant Het-1A cells induced by HPV and MNNG). A negative correlation was found between miR-218 and GAB2 mRNA expression in esophageal cancer patients and control people. GAB2 was up-regulated in Het-1A-HPV-MNNG cells. Further, down-expression of GAB2 reversed HPV&MNNG-mediated activation of migration and invasion and repressed SHP2/ERK and Akt/mTOR pathway signaling. In conclusion, miR-218 partially accounts for the prevention effect during malignant transformation of normal esophageal epithelial cells, which targets GAB2, which supplies the potential treatment in cancer therapy.

Enhanced DNA accessibility and increased DNA damage induced by the absence of poly(ADP-ribose) hydrolysis.

Poly(ADP-ribose) (PAR) is a therapeutic target primarily identified through inhibiting its synthesis by PAR polymerase-1 (PARP-1). However, inhibiting its hydrolysis by PAR glycohydrolase (PARG) has therapeutic potential in cancer. Unknown is the effect of elevated PAR levels on cellular processes and if this effect can enhance the therapeutic value of PARG. Here, we demonstrate in PARG null embryonic trophoblast stem (TS) cells that the absence of PAR hydrolysis led to PAR-modified histones H1, H2A, and H2B. To determine if this led to the differential vulnerability of DNA to modification, TS cells were treated with DNA-modifying agents. The results demonstrate increased DNA laddering by micrococcal nuclease and an increased amount of DNA intercalation by acridine orange in PARG null-TS cells. This increased access to PARG null-TS cell DNA was further verified by the detection of increased DNA damage following treatment with UV radiation and a minimal dose of the DNA-alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Further, this DNA damage was predominantly unrepaired 12 h after treatment in PARG null-TS cells. Finally, TS cells were treated with DNA-modifying chemotherapeutic agents. The results demonstrate up to 4-fold increases in cell death in PARG null-TS cells after treatment with epirubicin or sub-IC(50) doses of cisplatin and cyclophosphamide. Taken together, we provide compelling evidence that increased DNA access induced by the absence of PARG enhances the efficacy of DNA-modifying agents. Thus, this study demonstrates that greater DNA accessibility, increased DNA damage, and increased cell death all contribute to the PARG null cell phenotype in response to genotoxic stress.

Poly(ADP-ribose) polymerase-1 inhibits mitochondrial respiration by suppressing PGC-1α activity in neurons.

Poly(ADP-ribose) polymerase-1 (PARP1) is a ubiquitous nuclear enzyme that regulates DNA repair and genomic stability. In oxidative genotoxic conditions, PARP1 activity is enhanced significantly, leading to excessive depletion of nicotinamide adenine dinucleotide (NAD+) and mitochondrial dysfunction. We hypothesized that PARP1-induced NAD+ depletion inhibits NAD+-dependent sirtuin deacetylase activity, thereby interfering with the mitochondrial regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). The DNA alkylator, N'-Nitro-N-nitroso-N-methylguanidine (MNNG), induced NAD+ depletion, inhibited sirtuin deacetylase activity and enhanced acetylation of PGC-1α. This was associated with reduced interaction between PGC-1α and nuclear respiratory factor 1 (NRF-1), which is a nuclear transcription factor that drives mitochondrial replication by regulating mitochondrial transcription factor A (TFAM). MNNG also reduced binding of NRF-1 to the tfam upstream promoter region and reduced TFAM mRNA, mitochondrial DNA copy number and respiratory function. MNNG effects were mitigated by PARP1 inhibition and genetic loss of function, by enhancing intracellular NAD+ levels, and with sirtuin (SIRT1) gain of function, supporting a mechanism dependent on PARP1 activity, NAD+-depletion and SIRT1 inhibition. This and other work from our group supports a destructive sequelae of events related to PARP1-induced sirtuin inhibition and sirtuin-mediated regulation of transcription.

Homologous recombination rescues mismatch-repair-dependent cytotoxicity of S(N)1-type methylating agents in S. cerevisiae.

Resistance of mammalian cells to S(N)1-type methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) generally arises through increased expression of methylguanine methyltransferase (MGMT), which reverts the cytotoxic O(6)-methylguanine ((Me)G) to guanine, or through inactivation of the mismatch repair (MMR) system, which triggers cell death through aberrant processing of (Me)G/T mispairs generated during DNA replication when MGMT capacity is exceeded. Given that MMR and (Me)G-detoxifying proteins are functionally conserved through evolution, and that MMR-deficient Escherichia coli dam(-) strains are also resistant to MNNG, the finding that MMR status did not affect the sensitivity of Saccharomyces cerevisiae to MNNG was unexpected. Because (Me)G residues in DNA trigger homologous recombination (HR), we wondered whether the efficient HR in S. cerevisiae might alleviate the cytotoxic effects of (Me)G processing. We now show that HR inactivation sensitizes S. cerevisiae to MNNG and that, as in human cells, defects in the MMR genes MLH1 and MSH2 rescue this sensitivity. Inactivation of the EXO1 gene, which encodes the only exonuclease implicated in MMR to date, failed to rescue the hypersensitivity, which implies that scExo1 is not involved in the processing of (Me)G residues by the S. cerevisiae MMR system.

Neoplastic transformation and induction of H+,K+ -adenosine triphosphatase by N-methyl-N'-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line.

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H(+),K(+)-adenosine triphosphatase (H(+),K(+)-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.

Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin.

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.

Denitrosation as a determinant of nitrosocimetidine in vivo activity.

The widely used drug cimetidine (Tagamet) can be nitrosated in the presence of nitrite and under mild acid conditions to form a compound, nitrosocimetidine (NC), which has a chemical structure very similar to those of the mutagens and laboratory carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU). NC has given positive indications in several short-term tests for possible carcinogenic activity and is capable of methylating DNA in vitro and in cultured cells in a manner identical to that of MNNG and MNU. Nevertheless, NC has been found to be a weak carcinogen or a noncarcinogen and to be very poor at modifying DNA in vivo when administered p.o. We have found that NC, like MNNG, decomposes very rapidly when incubated with thiol compound in neutral pH buffer. Much of this decomposition is denitrosation. In the presence of excess reduced glutathione, about 35% of the degradation results in denitrosation to produce cimetidine, and in the presence of excess cysteine about 65% results in denitrosation to produce cimetidine. The compound also rapidly decomposes in whole blood isolated from rats; about 70% of this decomposition produces cimetidine. In solution with purified rat hemoglobin, approximately 90% of the NC degradation proceeds via a denitrosation pathway; hemoglobin cysteine residues have been implicated in the denitrosation reaction. In parallel experiments with MNNG, it has been found that, although a fraction of the decomposition of this agent in the presence of thiol compound, in isolated whole blood, and in solution with purified hemoglobin generates the denitrosated derivative, denitrosation is in the range of one-third to one-half of that found for NC. Denitrosation and degradation to form a methylating species appear to be the major NC and MNNG decomposition pathways in vitro. There is no indication that MNU degradation is sensitive to thiols, nor is the compound susceptible to denitrosation at neutral pH. Molar-equivalent doses of methyl group-radiolabeled NC, MNNG, and MNU were administered via the tail vein to groups of F344 rats, and the DNA methylation yields in lung, liver, kidney, and brain tissue were assessed. Of the organs considered, DNA methylation was greatest in the lungs of MNNG-treated animals, followed by kidney (25% of the lung value). Methylation of lung tissue DNA in MNU-treated animals was about 50% of that observed in the MNNG experiments; DNA methylation in the other organs was about equivalent to that found in the lung.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct study of alkylating agent--RNA interaction by 13C nuclear magnetic resonance spectroscopy.

The interactions of Torula yeast RNA with four biologically important methylating agents (methyl methanesulfonate, dimethyl sulfate, 1-methyl-1-nitrosourea, and 1-methyl-3-nitro-1-nitrosoguanidine) have been studied by 13C nuclear magnetic resonance spectroscopy. This direct stable isotope method eliminated all tedious and questionable degradation processes for determining the reactive sites and product distribution. Based on the integration curves, two types of product distribution can be distinctly observed according to the biological potency of the methylating agents [(a) weakly mutagenic and carcinogenic, methyl methanesulfonate and dimethyl sulfate and (b) strongly mutagenic and carcinogenic, 1-methyl-1-nitrosourea and 1-methyl-3-nitro-1-nitrosoguanidine]. With 90% 13C-enriched methylating agent, it significantly increases the specificity and sensitivity and provides better quantitative results.

Nucleocytoplasmic functionality of metallothionein.

Appropriate nucleocytoplasmic partitioning of proteins can direct diverse cellular processes. Metallothioneins (MTs) are thiol-rich, stress-inducible proteins that can afford protection against oxidants, mutagens, and anticancer drugs. MTs display discrete nucleocytoplasmic sequestration patterns despite their small size (Mr 6,000). We demonstrate subcellular location-specific functionality of MT using a regulated expression system that restricts MT expression to the nucleus or the cytoplasm in MT-null fibroblasts. Specifically, we found that cytoplasmic but not nuclear expression of MT decreases the level of intracellular reactive oxygen species and is more cytoprotective against the prototypic oxidizing agent tert-butyl hydroperoxide. Cytoplasmic MT expression also protects against the cytotoxicity of the heavy metal CdCl2, whereas nuclear expression protects against the cytotoxicity of the mutagenic agent N-methyl-N'-nitro-N-nitrosoguanidine. These data support the hypothesis that essential cytotoxic targets of both oxidants and heavy metals reside in the cytoplasm and establish the importance of nucleocytoplasmic partitioning for the function of small protective proteins such as MTs.

Rat, mouse and hamster isozyme specificity in the glutathione transferase-mediated denitrosation of nitrosoguanidinium compounds.

The major isozymes from affinity column-purified glutathione transferases isolated from Sprague-Dawley rat liver, kidney, and testis cytosol and also from BALB/c mouse and Syrian golden hamster liver cytosol have been resolved by chromatofocusing and tested for their ability to denitrosate and thus detoxicate the DNA-methylating agents and potential carcinogens nitrosocimetidine and 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG). The isozymes have been kinetically characterized using a battery of substrates permitting, in the rat and mouse cases, subunit composition identification. It has been found that the rat and mouse isozymes belonging to the mu class are uniquely and highly active in the denitrosation of nitrosocimetidine and CyanoDMNG. A specific set of hamster glutathione transferase isozymes were also found to be active in these reactions. We have identified the reaction products produced by the rat liver 3-4 isozyme activity. The glutathione transferase-mediated degradations of 1-methyl-2-nitro-1-nitrosoguanidine and CyanoDMNG generate one molecule of S-nitrosoglutathione per molecule of denitrosated guanidinium compound produced. In the CyanoDMNG incubations essentially all degradation was via denitrosation; nitrite and glutathione disulfide were minor products. In the 1-methyl-2-nitro-1-nitrosoguanidine case nonenzymic degradation of the nitroso compound in the presence of reduced glutathione was evident but little of this decomposition produced S-nitrosoglutathione or 1-methyl-2-nitroguanidine. In the presence of rat transferase 3-4 isozyme, glutathione-dependent 1-methyl-2-nitro-1-nitrosoguanidine degradation was shifted markedly towards denitrosation with the concomitant production of S-nitrosoglutathione.

Mechanisms of inhibition by ascorbate of microbial mutagenesis induced by N-nitroso compounds.

Mutagenesis induced by N-methyl-N-nitrosoguanidine (MNNG) and dimethylnitrosamine (DMN) in Salmonella TA 1530 was inhibited by ascorbate. Inhibition of MNNG-induced mutagenesis resulted from a reaction between ascorbate and MNNG that led to consumption of MNNG. The rate of this reaction was considerably enhanced by catalytic amounts of Cu(II) and Fe(III). No direct reaction between DMN and ascorbate was detectable, but relatively high concentrations of Cu(II) enchanced inhibition of DMN-induced mutagenesis by ascorbate. Added protein reduced the effectiveness of Cu(II) as a catalyst of the reaction between ascorbate and MNNG, which suggested that the microsomal protein necessary to activate DMN, may reduce the concentration of free Cu(II) and thereby lower its catalytic efficiency. Mutagenesis by N-methyl-N-nitrosourea was not inhibited by ascorbate.

Chemopreventive Activity of MGN-3/Biobran Against Chemical Induction of Glandular Stomach Carcinogenesis in Rats and Its Apoptotic Effect in Gastric Cancer Cells.

In the current study, we investigated the chemopreventive activity of arabinoxylan rice bran, MGN-3/Biobran, against chemical induction of glandular stomach carcinogenesis in rats. Gastric cancer was induced by carcinogen methylnitronitrosoguanidine (MNNG), and rats received MNNG alone or MNNG plus Biobran (40 mg/kg body weight) for a total of 8 months. Averaged results from 2 separate readings showed that exposure to MNNG plus Biobran caused gastric dysplasia and cancer (adenocarcinoma) in 4.5/12 rats (9/24 readings, 37.5%), with 3.5/12 rats (7/24 readings, 29.2%) showing dysplasia and 1/12 rats (8.3%) developing adenocarcinoma. In contrast, in rats treated with MNNG alone, 8/10 (80%) developed dysplasia and adenocarcinoma, with 6/10 rats (60%) showing dysplasia and 2/10 rats (20%) developing adenocarcinoma. The effect of combining both agents was also associated with significant suppression of the expression of the tumor marker Ki-67 and remarkable induction in the apoptotic gastric cancer cells via mitochondrial-dependent pathway as indicated by the upregulation in p53 expression, Bax expression, downregulation in Bcl-2 expression, an increase in Bax/Bcl-2 ratio, and an activation of caspase-3. In addition, Biobran treatment induced cell-cycle arrest in the subG1 phase, where the hypodiploid cell population was markedly increased. Moreover, Biobran treatment protected rats against MNNG-induced significant decrease in lymphocyte levels. We conclude that Biobran provides protection against chemical induction of glandular stomach carcinogenesis in rats and may be useful for the treatment of human patients with gastric cancer.