MRE11A: a novel negative regulator of human DNA mismatch repair.

BACKGROUND: DNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of cancers. Although well characterized, MMR factors remain to be identified. As a 3'-5' exonuclease and endonuclease, meiotic recombination 11 homolog A (MRE11A) is implicated in multiple DNA repair pathways. However, the role of MRE11A in MMR is unclear. METHODS: Initially, short-term and long-term survival assays were used to measure the cells' sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Meanwhile, the level of apoptosis was also determined by flow cytometry after MNNG treatment. Western blotting and immunofluorescence assays were used to evaluate the DNA damage within one cell cycle after MNNG treatment. Next, a GFP-heteroduplex repair assay and microsatellite stability test were used to measure the MMR activities in cells. To investigate the mechanisms, western blotting, the GFP-heteroduplex repair assay, and chromatin immunoprecipitation were used. RESULTS: We show that knockdown of MRE11A increased the sensitivity of HeLa cells to MNNG treatment, as well as the MNNG-induced DNA damage and apoptosis, implying a potential role of MRE11 in MMR. Moreover, we found that MRE11A was largely recruited to chromatin and negatively regulated the DNA damage signals within the first cell cycle after MNNG treatment. We also showed that knockdown of MRE11A increased, while overexpressing MRE11A decreased, MMR activity in HeLa cells, suggesting that MRE11A negatively regulates MMR activity. Furthermore, we show that recruitment of MRE11A to chromatin requires MLH1 and that MRE11A competes with PMS2 for binding to MLH1. This decreases PMS2 levels in whole cells and on chromatin, and consequently comprises MMR activity. CONCLUSIONS: Our findings reveal that MRE11A is a negative regulator of human MMR.

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.

PARP-1 activation leads to cytosolic accumulation of TDP-43 in neurons.

Oxidative stress in neurodegenerative disease leads to poly(ADP-ribose) polymerase 1 (PARP-1) overactivation and subsequent cell death via excessive generation of Poly(ADP-ribose) polymer (PAR). PAR binds to neurodegenerative disease linked protein TAR DNA binding protein of 43 kDa (TDP-43). However, the consequence of this interaction is not yet fully understood. TDP-43 translocates from the nucleus to the cytoplasm in response to oxidative stress, but the mechanism of stress-induced translocation remains unknown. We used N-methyl-N-nitroso-N'-nitroguanidine (MNNG) and oxygen-glucose deprivation (OGD) in mouse neuronal cultures to activate PARP-1 and observed that pharmacological inhibition of PARP-1 blocked the cytosolic translocation of TDP-43. PARP-1 inhibition is also neuroprotective against both MNNG and OGD, suggesting that PARP inhibitors could play a role in the neuroprotective role in neurodegenerative diseases involving TDP-43. Together, these data present the novel finding that TDP-43 translocation depends on PARP-1 activation and set a ground for future research of how PARP-1 activation or PAR binding to TDP-43 may facilitate its cytosolic accumulation.

Synergistic Carcinogenesis of HPV18 and MNNG in Het-1A Cells through p62-KEAP1-NRF2 and PI3K/AKT/mTOR Pathway.

N-methyl-N´-nitro-N-nitrosoguanidine is a clear carcinogen, increasing evidence that indicates an etiological role of human papillomavirus in esophageal carcinoma. Studies have reported the synergistic effect on environmental carcinogens and viruses in recent years. On the basis of establishing the malignant transformation model of Het-1A cells induced by synergistic of HPV18 and MNNG, this study was to explore the synergistic carcinogenesis of MNNG and HPV. Our research indicated that HPV&MNNG led to a significant increase in the protein-expression levels of c-Myc, cyclinD1, BCL-2, BAX, E-cadherin, N-cadherin, mTOR, LC3II, and p62, with concomitant decreases in p21 and LC3I. HPV18 and MNNG induced accumulation of p62 and its interaction with KEAP1, which promoted NRF2 nuclear translocation. p62 loss prevents growth and increases autophagy of malignant cells by activating KEAP1/NRF2-dependent antioxidative response. In addition, PI3K and p-AKT were stimulated by HPV&MNNG, and PI3K/AKT/mTOR is positively associated with cell proliferation, migration, invasion, and autophagy during malignant transformation. Taken together, MNNG&HPV regulates autophagy and further accelerates cell appreciation by activating the p62/KEAP1/NRF2 and PI3K/AKT/mTOR pathway. MNNG&HPV may improve Het-1A cell autophagy to contribute to excessive cell proliferation, reduced apoptosis, and protection from oxidative damage, thus accelerating the process of cell malignant transformation and leading to cancerous cells.

Alkylative damage of mRNA leads to ribosome stalling and rescue by trans translation in bacteria.

Similar to DNA replication, translation of the genetic code by the ribosome is hypothesized to be exceptionally sensitive to small chemical changes to its template mRNA. Here we show that the addition of common alkylating agents to growing cultures of Escherichia coli leads to the accumulation of several adducts within RNA, including N(1)-methyladenosine (m1A). As expected, the introduction of m1A to model mRNAs was found to reduce the rate of peptide bond formation by three orders of magnitude in a well-defined in vitro system. These observations suggest that alkylative stress is likely to stall translation in vivo and necessitates the activation of ribosome-rescue pathways. Indeed, the addition of alkylation agents was found to robustly activate the transfer-messenger RNA system, even when transcription was inhibited. Our findings suggest that bacteria carefully monitor the chemical integrity of their mRNA and they evolved rescue pathways to cope with its effect on translation.

Leptin enhances N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced tumour growth in gastric mucosa of male Sprague-Dawley rats.

Individuals who are obese are at a greater risk of developing gastric cancer. They are however also hyperleptinaemic. Chronic leptin treatment has been shown to upregulate numerous cancer-causing genes in the stomach of male Sprague-Dawley rats. It is however unclear if leptin enhances the effect of gastric carcinogens in vivo. This study was therefore done to investigate the effect of leptin on gastric carcinogenesis in rats treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Twenty-four, 6-week old male Sprague-Dawley rats were divided equally into three groups: G1 served as age-matched controls; G2 was treated with MNNG in drinking water ad libitum (200 mg L-1); G3 was given leptin and MNNG. Rats were euthanized after 40 weeks of treatment and their stomachs were removed for histopathology, microarray, and RT-qPCR analysis. Fisher's exact test and one-way ANOVA were used to analyse the data. Fifty percent of the MNNG-treated rats developed gastric hyperplasia (p < 0.05), but there was no significant change in any carcinogenic genes. Concurrent MNNG and leptin treatment however induced hyperplasia, dysplasia, hypertrophy, and adenocarcinoma in 75% (6/8) of the rats; with upregulation of microRNAs, olfactory receptors, Hey2 (transcription factor), Tmed2 (vesicular trafficking), and Lcn11 (cell proliferation) genes. It appears that leptin enhances MNNG- induced tumour growth in stomachs of Sprague-Dawley rats and its role in gastric cancer requires further scrutiny.

The gastric mucosal protective effects of astragaloside IV in mnng-induced GPL rats.

Gastric Cancer is one of the most common types of cancer. And the occurrence of gastric carcinoma is an evolutionary histopathological stage. As a result, further research of GPL, which is a borderline of gastric cancer, is indispensable for preventing the formation and development of gastric carcinoma. Several studies have demonstrated a correlation between the expression of autophagy, apoptosis and Gastric cancer (GC). However, the effects of autophagy and apoptosis on human gastric cancer progression, particularly on gastric precancerous lesions (GPL), have not totally been investigated. At present, Astragaloside IV(AS-IV) is a saponin purified from Astragalus membranaceous Bge, a traditional Chinese herb that has been widely used for more than 2000 y in the treatment of cancer, cardiovascular and immune disorders. This study was designed to investigate the mechanism of AS-IV protecting gastric mucosa in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced GPL rats. The lesions of GIM and GED were significantly ameliorated compared with the model rats, especially crowded tubular glandular and back-to-back tubular structure, which were the dangerous borderline between GPL and GC. Western Blot analysis showed that the ratio of Bcl-2/Bax and the protein expression of Bcl-XL, p53, Beclin1, p62, ATG5 and ATG12 were decreased and the level of Caspase3 was increased in the group of AS-IV compared with the model group; RT-PCR analysis showed that the gene expression Ambra1, Beclin1, ATG5, LC3 and p62 were decreased in the group of AS-IV compared with the model group. This research manifested that the occurrence of gastric cancer was preceded by a prolonged precancerous stage, which could be ameliorated by the AS-IV. Meanwhile, the mild and moderate stage of precancerous lesions is similar with gastric adenocarcinoma in critical biological processes, including inflammation, cell proliferation, differentiation. But this lesion is very different from cancer, because it does not appear obvious invasion and malignant lesions in this pathologic stag. Further, AS-IV could regulate p53 expression to activate the Ambra1/Beclin1 complex in GPL, and it will protect the gastric mucosal injury, prevent and cure gastric mucosal atrophy, intestinal metaplasia and atypical hyperplastic lesions. It provided a potential therapeutic strategy in reversing intestinal metaplasia and dysplasia of gastric precancerous lesions and protecting the gastric mucosa in GPL rats.

Genomic evidence that the live Chlamydia abortus vaccine strain 1B is not attenuated and has the potential to cause disease.

BACKGROUND: The live, temperature-attenuated vaccine strain 1B of Chlamydia abortus, the aetiological agent of ovine enzootic abortion (OEA), has been implicated in cases of vaccine breakdown. The aim of this study was to understand the nature of this attenuation through sequencing of the vaccine parent strain (AB7) and the derived mutant strains 1B and 1H, as well as to clarify the role of the vaccine strain in causing disease through comparative whole genome analysis. METHODS: Whole genome sequencing was performed on: vaccine parent strain AB7; N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced temperature attenuated mutant strain 1B grown from the commercial live vaccines Cevac Chlamydia and Enzovax; strain 1H a reverted NTG mutant; and 5 strains isolated from cases of OEA originating from animals from the original vaccine safety trial (2 strains) or from vaccinated ewes or ewes exposed to vaccinated animals (3 strains). RESULTS: We confirmed that AB7 is in a different lineage from the reference strain S26/3. The genome of vaccine strain 1B contains ten single nucleotide polymorphisms (SNPs) created by the NTG treatment, which are identical to those found in strain 1H. The strains from OEA cases also cluster phylogenetically very tightly with these vaccine strains. CONCLUSIONS: The results show that C. abortus vaccine strain 1B has an identical genome sequence to the non-attenuated "reverted mutant" strain 1H. Thus, the protection of the 1B vaccine is unlikely to be due to the NTG induced SNPs and is more likely caused by the administration of high doses of C. abortus elementary bodies that stimulate protective immunity. Vaccine-identical strains were also isolated from cases of disease, as well as strains which had acquired 1-3 SNPs, including an animal that had not been vaccinated with either of the commercial live OEA vaccines, indicating that the 1B vaccine strain may be circulating and causing disease.

Lead facilitates foci formation in a Balb/c-3T3 two-step cell transformation model: role of Ape1 function.

Several possible mechanisms have been examined to gain an understanding on the carcinogenic properties of lead, which include among others, mitogenesis, alteration of gene expression, oxidative damage, and inhibition of DNA repair. The aim of the present study was to explore if low concentrations of lead, relevant for human exposure, interfere with Ape1 function, a base excision repair enzyme, and its role in cell transformation in Balb/c-3T3. Lead acetate 5 and 30 µM induced APE1 mRNA and upregulation of protein expression. This increase in mRNA expression is consistent throughout the chronic exposure. Additionally, we also found an impaired function of Ape1 through molecular beacon-based assay. To evaluate the impact of lead on foci formation, a Balb/c-3T3 two-step transformation model was used. Balb/c-3T3 cells were pretreated 1 week with low concentrations of lead before induction of transformation with n-methyl-n-nitrosoguanidine (MNNG) (0.5 µg/mL) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1 µg/mL) (a classical two-step protocol). Morphological cell transformation increased in response to lead pretreatment that was paralleled with an increase in Ape1 mRNA and protein overexpression and an impairment of Ape1 activity and correlating with foci number. In addition, we found that lead pretreatment and MNNG (transformation initiator) increased DNA damage, determined by comet assay. Our data suggest that low lead concentrations (5, 30 µM) could play a facilitating role in cellular transformation, probably through the impaired function of housekeeping genes such as Ape1, leading to DNA damage accumulation and chromosomal instability, one of the most important hallmarks of cancer induced by chronic exposures.

ATR-Chk1 activation mitigates replication stress caused by mismatch repair-dependent processing of DNA damage.

The mismatch repair pathway (MMR) is essential for removing DNA polymerase errors, thereby maintaining genomic stability. Loss of MMR function increases mutation frequency and is associated with tumorigenesis. However, how MMR is executed at active DNA replication forks is unclear. This has important implications for understanding how MMR repairs O6-methylguanine/thymidine (MeG/T) mismatches created upon exposure to DNA alkylating agents. If MeG/T lesion recognition by MMR initiates mismatch excision, the reinsertion of a mismatched thymidine during resynthesis could initiate futile repair cycles. One consequence of futile repair cycles might be a disruption of overall DNA replication in the affected cell. Herein, we show that in MMR-proficient HeLa cancer cells, treatment with a DNA alkylating agent slows S phase progression, yet cells still progress into the next cell cycle. In the first S phase following treatment, they activate ataxia telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) signaling, which limits DNA damage, while inhibition of ATR kinase activity accelerates DNA damage accumulation and sensitivity to the DNA alkylating agent. We also observed that exposure of human embryonic stem cells to alkylation damage severely compromised DNA replication in a MMR-dependent manner. These cells fail to activate the ATR-Chk1 signaling axis, which may limit their ability to handle replication stress. Accordingly, they accumulate double-strand breaks and undergo immediate apoptosis. Our findings implicate the MMR-directed response to alkylation damage as a replication stress inducer, suggesting that repeated MMR processing of mismatches may occur that can disrupt S phase progression.

Mutants of Yarrowia lipolytica NCIM 3589 grown on waste cooking oil as a biofactory for biodiesel production.

BACKGROUND: Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here. RESULTS: In this study, chemical mutagenesis using the alkylating agent, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L-1 h-1) as compared to the wild type (0.033 g L-1 h-1). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and higher heating values than the wild type strain. CONCLUSION: The chemical mutagenesis strategy adopted in this study resulted in the successful isolation of three stable high SCO yielding mutants. The mutants, namely, YlB6, YlC7 and YlE1 exhibited a 1.22, 1.33 and 1.49-fold increase in lipid contents when grown on 100 g L-1 waste cooking oil than the parental yeast strain. The fatty acid methyl ester (FAME) profiles of all the three mutants was determined to be suitable for biodiesel suggesting their potential applicability while simultaneously addressing the management of waste cooking oil.

Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro.

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display a mild mutator phenotype, while nuclear extracts of these cells exhibit reduced MMR activity in vitro, and these defects are complemented by overexpression or addition of exogenous human Exonuclease 1 (EXO1). By contrast, another proofreading-deficient mutant, PolδD515V, which has a weaker strand displacement activity, does not decrease the MMR activity as significantly as PolδD316A;E318A. In addition, PolδD515V does not increase the mutation frequency in MMR-proficient cells. Based on our findings, we propose that the proofreading activity restricts the strand displacement activity of Polδ in MMR. This contributes to maintain the nicks required for EXO1 entry, and in this manner ensures the dominance of the EXO1-dependent MMR pathway.

Effect of acetic acid on ethanol production by Zymomonas mobilis mutant strains through continuous adaptation.

BACKGROUND: Acetic acid is a predominant by-product of lignocellulosic biofuel process, which inhibits microbial biocatalysts. Development of bacterial strains that are tolerant to acetic acid is challenging due to poor understanding of the underlying molecular mechanisms. RESULTS: In this study, we generated and characterized two acetic acid-tolerant strains of Zymomonas mobilis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-acetate adaptive breeding. Two mutants, ZMA-142 and ZMA-167, were obtained, showing a significant growth rate at a concentration of 244 mM sodium acetate, while the growth of Z. mobilis ATCC 31823 were completely inhibited in presence of 195 mM sodium acetate. Our data showed that acetate-tolerance of ZMA-167 was attributed to a co-transcription of nhaA from ZMO0117, whereas the co-transcription was absent in ATCC 31823 and ZMA-142. Moreover, ZMA-142 and ZMA-167 exhibited a converstion rate (practical ethanol yield to theorical ethanol yield) of 90.16% and 86% at 195 mM acetate-pH 5 stress condition, respectively. We showed that acid adaptation of ZMA-142 and ZMA-167 to 146 mM acetate increased ZMA-142 and ZMA-167 resulted in an increase in ethanol yield by 32.21% and 21.16% under 195 mM acetate-pH 5 stress condition, respectively. CONCLUSION: The results indicate the acetate-adaptive seed culture of acetate-tolerant strains, ZMA-142 and ZMA-167, could enhance the ethanol production during fermentation.

Down-regulation of PARP1 by miR-891b sensitizes human breast cancer cells to alkylating chemotherapeutic drugs.

PURPOSE: Breast cancer is the most common invasive type of cancer among women. Role of different microRNAs (miRNAs) and poly(ADP-ribose) polymerases (PARPs) in breast cancer has been well established. This study aimed to explore the effects of miR-891b on sensitizing breast cancer cells to alkylating chemotherapeutic drugs through PARPs. METHODS: The expression of miR-891b and PARP1 in human breast cancer cells HCC1806 was overexpressed by transfection with their mimics or expressing vector. Then, the transfected cells were exposed to 40 µM N-methyl-N-nitro-N-nitrosoguanidine (MNNG) for 1 h. The correlation between miR-891b and PARP1 was detected by RT-qPCR, western blot, and dual-luciferase reporter assay. Besides, MTT assay and Annexin V assay were done to measure cell proliferation and apoptosis, respectively. RESULTS: PARP1 was a target of miR-891b, and it was negatively regulated by miR-891b. MiR-891b increased the sensitivity of the HCC1806 cells to the cytotoxic effects of MNNG through suppressing cell proliferation and increasing the percentage of apoptotic cells. Restoration of PARP1 activity in the HCC1806 cells led to loss of miR-891b mediated sensitivity of the HCC1806 cells to MNNG. CONCLUSION: MiR-891b increases the sensitivity of the breast cancer cells (HCC1806) to the cytotoxic effects of the chemotherapeutic agent MNNG by suppressing the expression of PARP1.

Inactivation of genes involved in base excision repair of Corynebacterium glutamicum and survival of the mutants in presence of various mutagens.

Base Excision Repair (BER) is considered as the most active DNA repair pathway in vivo, which is initiated by recognition of the nucleotide lesions and excision of the damaged DNA base. The genome of Corynebacterium glutamicum ATCC 13032 contains various DNA glycosylases encoding genes (ung, fpg/mutM, tagI, alkA, mutY), two AP-endonuclease encoding genes (nei and nth) and an exonuclease encoding gene xth. To investigate the role of these genes during DNA repair in C. glutamicum, mutants with deletions of one or more genes in BER pathway were created. After treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C (MMC), zeocin and UV-light, we characterised the function of the different BER genes by determination of the survival capability. DNA lesions caused by MNNG strongly reduced survival of the tagI, mutY and alkA mutants but had a negligible effect on the ung and mutM mutants. The endonucleases Nth and Nei turned out to be essential for the repair of base modifications caused by MMC while UV-light and zeocin did not seem to address the BER. So far, BER in C. glutamicum appears to be very similar to that in E. coli.

Approaches towards the enhanced production of Rapamycin by Streptomyces hygroscopicus MTCC 4003 through mutagenesis and optimization of process parameters by Taguchi orthogonal array methodology.

The present research was conducted to define the approaches for enhanced production of rapamycin (Rap) by Streptomyces hygroscopicus microbial type culture collection (MTCC) 4003. Both physical mutagenesis by ultraviolet ray (UV) and chemical mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine (NTG) have been applied successfully for the improvement of Rap production. Enhancing Rap yield by novel sequential UV mutagenesis technique followed by fermentation gives a significant difference in getting economically scalable amount of this industrially important macrolide compound. Mutant obtained through NTG mutagenesis (NTG-30-27) was found to be superior to others as it initially produced 67% higher Rap than wild type. Statistical optimization of nutritional and physiochemical parameters was carried out to find out most influential factors responsible for enhanced Rap yield by NTG-30-27 which was performed using Taguchi orthogonal array approach. Around 72% enhanced production was achieved with nutritional factors at their assigned level at 23 °C, 120 rpm and pH 7.6. Results were analysed in triplicate basis where validation and purification was carried out using high performance liquid chromatography. Stability study and potency of extracted Rap was supported by turbidimetric assay taking Candida albicans MTCC 227 as test organism.

DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in Pseudomonas aeruginosa and Pseudomonas putida.

Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of imuCdinB double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis.

Augmentation of poly(ADP-ribose) polymerase-dependent neuronal cell death by acidosis.

Tissue acidosis is a key component of cerebral ischemic injury, but its influence on cell death signaling pathways is not well defined. One such pathway is parthanatos, in which oxidative damage to DNA results in activation of poly(ADP-ribose) polymerase and generation of poly(ADP-ribose) polymers that trigger release of mitochondrial apoptosis-inducing factor. In primary neuronal cultures, we first investigated whether acidosis per sé is capable of augmenting parthanatos signaling initiated pharmacologically with the DNA alkylating agent, N-methyl- N'-nitro- N-nitrosoguanidine. Exposure of neurons to medium at pH 6.2 for 4 h after N-methyl- N'-nitro- N-nitrosoguanidine washout increased intracellular calcium and augmented the N-methyl- N'-nitro- N-nitrosoguanidine-evoked increase in poly(ADP-ribose) polymers, nuclear apoptosis-inducing factor , and cell death. The augmented nuclear apoptosis-inducing factor and cell death were blocked by the acid-sensitive ion channel-1a inhibitor, psalmotoxin. In vivo, acute hyperglycemia during transient focal cerebral ischemia augmented tissue acidosis, poly(ADP-ribose) polymers formation, and nuclear apoptosis-inducing factor , which was attenuated by a poly(ADP-ribose) polymerase inhibitor. Infarct volume from hyperglycemic ischemia was decreased in poly(ADP-ribose) polymerase 1-null mice. Collectively, these results demonstrate that acidosis can directly amplify neuronal parthanatos in the absence of ischemia through acid-sensitive ion channel-1a . The results further support parthanatos as one of the mechanisms by which ischemia-associated tissue acidosis augments cell death.

Pueraria mirifica Exerts Estrogenic Effects in the Mammary Gland and Uterus and Promotes Mammary Carcinogenesis in Donryu Rats.

Pueraria mirifica (PM), a plant whose dried and powdered tuberous roots are now widely used in rejuvenating preparations to promote youthfulness in both men and women, may have major estrogenic influence. In this study, we investigated modifying effects of PM at various doses on mammary and endometrial carcinogenesis in female Donryu rats. Firstly, PM administered to ovariectomized animals at doses of 0.03%, 0.3%, and 3% in a phytoestrogen-low diet for 2 weeks caused significant increase in uterus weight. Secondly, a 4 week PM application to non-operated rats at a dose of 3% after 7,12-dimethylbenz[a]anthracene (DMBA) initiation resulted in significant elevation of cell proliferation in the mammary glands. In a third experiment, postpubertal administration of 0.3% (200 mg/kg body weight (b.w.)/day) PM to 5-week-old non-operated animals for 36 weeks following initiation of mammary and endometrial carcinogenesis with DMBA and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), respectively, resulted in significant increase of mammary adenocarcinoma incidence. A significant increase of endometrial atypical hyperplasia multiplicity was also observed. Furthermore, PM at doses of 0.3%, and more pronouncedly, at 1% induced dilatation, hemorrhage and inflammation of the uterine wall. In conclusion, postpubertal long-term PM administration to Donryu rats exerts estrogenic effects in the mammary gland and uterus, and at a dose of 200 mg/kg b.w./day was found to promote mammary carcinogenesis initiated by DMBA.

The Major Replicative Histone Chaperone CAF-1 Suppresses the Activity of the DNA Mismatch Repair System in the Cytotoxic Response to a DNA-methylating Agent.

The DNA mismatch repair (MMR) system corrects DNA mismatches in the genome. It is also required for the cytotoxic response of O6-methylguanine-DNA methyltransferase (MGMT)-deficient mammalian cells and yeast mgt1Δ rad52Δ cells to treatment with Sn1-type methylating agents, which produce cytotoxic O6-methylguanine (O6-mG) DNA lesions. Specifically, an activity of the MMR system causes degradation of irreparable O6-mG-T mispair-containing DNA, triggering cell death; this process forms the basis of treatments of MGMT-deficient cancers with Sn1-type methylating drugs. Recent research supports the view that degradation of irreparable O6-mG-T mispair-containing DNA by the MMR system and CAF-1-dependent packaging of the newly replicated DNA into nucleosomes are two concomitant processes that interact with each other. Here, we studied whether CAF-1 modulates the activity of the MMR system in the cytotoxic response to Sn1-type methylating agents. We found that CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast mgt1Δ rad52Δ cells to the prototypic Sn1-type methylating agent N-methyl-N'-nitro-N-nitrosoguanidine. We also report evidence that in human MGMT-deficient cell-free extracts, CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system. Taken together, these findings suggest that CAF-1-dependent incorporation of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system, thereby defending the cell against killing by the Sn1-type methylating agent.