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Sample records for dna methyltransferase 3b

  1. DNA (cytosine-5-methyltransferase 3B (DNMT 3B polymorphism and risk of Down syndrome offspring

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    Cláudia Melo de Moura

    2018-01-01

    Full Text Available Down syndrome (DS is the most common form of human genetic mental retardation. Several polymorphisms in genes coding folic acid cycle enzymes have been associated to the risk of bearing a DS child; however, the results are controversial. S-adenosyl-l-methionine (SAM is an important intermediate of folic acid pathway and acts as methyl donor and substrate for DNA (cytosine-5-methyltransferase 3B (DNMT3B – EC 2.1.1.37 de novo methylation processes during embryogenesis. Recent studies suggest that a functional polymorphism of DNMT 3B in maternal genotype may be associated with a decreased risk of having a DS child. We herein investigate the association of this polymorphism with the occurrence of DS in a Brazilian population. We have genotyped 111 mothers of DS infants (MDS and 212 control mothers (CM through PCR-RFLP. The observed genotypic frequencies were CC = 0.22; CT = 0.49 and TT = 0.29 in CM, and CC = 0.30; CT = 0.52 and TT = 0.18 in MDS. Allelic frequencies were C = 0.47 and T = 0.53 in CM and C = 0.56 and T = 0.44 in MDS. No deviation of HWE was observed, and both DNMT 3B rs2424913 genotype (χ2 = 4.53; DF = 1; P = 0.03 and allelic (χ2 = 4.90; DF = 1; P = 0.03 frequencies show significant differences between MDS and CM. The presence of the mutant DNMT 3B T allele decreases 30% the risk of bearing a DS child (OR = 0.69; 95% CI: 0.50–0.96; P = 0.03, and the risk is diminished up to 45% in association with the homozygous genotype (OR = 0.54; 95% CI: 0.31–0.96; P = 0.04. Our results suggest that women harboring the single nucleotide polymorphism DNMT 3B rs2424913 have a decreased risk of a DS pregnancy, and further studies are necessary to confirm this protective effect.

  2. DNA methyltransferase 3b is dispensable for mouse neural crest development.

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    Bridget T Jacques-Fricke

    Full Text Available The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined. Using a conditional DNMT3b allele crossed to the neural crest cre drivers Wnt1-cre and Sox10-cre, neural crest DNMT3b mutants were generated. In both neural crest-specific and fully DNMT3b-mutant embryos, cranial neural crest cells exhibited only subtle migration defects, with increased numbers of dispersed cells trailing organized streams in the head. In spite of this, the resulting cranial ganglia, craniofacial skeleton, and heart developed normally when neural crest cells lacked DNMT3b. This indicates that DNTM3b is not necessary in cranial neural crest cells for their development. We conclude that defects in neural crest derivatives in DNMT3b mutant mice reflect a requirement for DNMT3b in lineages such as the branchial arch mesendoderm or the cardiac mesoderm that interact with neural crest cells during formation of these structures.

  3. [Characteristic and clinical significance of DNA methyltransferase 3B overexpression in endometrial carcinoma].

    Science.gov (United States)

    Dong, Y; Zhou, M; Ba, X J; Si, J W; Li, W T; Wang, Y; Li, D; Li, T

    2016-10-18

    To determine the clinicopathological significance of the DNA methyltransferase 3B (DNMT3B) overexpression in endometrial carcinomas and to evaluate its correlation with hormone receptor status. Immunohistochemistry was performed to assess the expression of DNMT3B and hormone receptors in 104 endometrial carcinomas. DNMT3B overexpression occurred frequently in endometrioid carcinoma (EC, 54.8%) more than in nonendometrioid carcinoma (NEC, 30.0%) with statistical significance (P=0.028). Furthermore, there was a trend that EC with worse clinico-pathological variables and shorter survival had a higher DNMT3B expression, and the correlation between DNMT3B and tumor grade reached statistical significance (P=0.019).A negative correlation between DNMT3B and estrogen receptor (ER) or progesterone receptor (PR) expression was found in EC. NMT3B overexpression occurred frequently in the ER or PR negative subgroups (78.9%, 86.7%) more than in the positive subgroups (47.7%, 47.8%) with statistical significance (P=0.016, P=0.006). In addition, the DNMT3B overexpression increased in tumors with both ER and PR negative expression (92.9%, P=0.002). However, no such correlation was found in NEC (P>0.05). Sequence analyses demonstrated multiple ER and PR binding sites in the promoter regions of DNMT3B gene. This study showed that the expression of DNMT3B in EC and NEC was different. DNMT3B overexpression in EC was associated with the worse clinicopathological variables and might have predictive value. The methylation status of EC and NEC maybe different. In addition, in EC, DNMT3B overexpression negatively correlated with ER or PR expression. In NEC, the correlation between DNMT3B and ER or PR status was not present.

  4. DNA Methyltransferase 3B Gene Promoter and Interleukin-1 Receptor Antagonist Polymorphisms in Childhood Immune Thrombocytopenia

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    Margarita Pesmatzoglou

    2012-01-01

    Full Text Available Primary immune thrombocytopenia (ITP is one of the most common blood diseases as well as the commonest acquired bleeding disorder in childhood. Although the etiology of ITP is unclear, in the pathogenesis of the disease, both environmental and genetic factors including polymorphisms of TNF-a, IL-10, and IL-4 genes have been suggested to be involved. In this study, we investigated the rs2424913 single-nucleotide polymorphism (SNP (C46359T in DNA methyltransferase 3B (DNMT3B gene promoter and the VNTR polymorphism of IL-1 receptor antagonist (IL-1 Ra intron-2 in 32 children (17 boys with the diagnosis of ITP and 64 healthy individuals. No significant differences were found in the genotype distribution of DNMT3B polymorphism between the children with ITP and the control group, whereas the frequency of allele T appeared significantly increased in children with ITP (P = 0.03, OR = 2, 95% CI: 1.06–3.94. In case of IL-1 Ra polymorphism, children with ITP had a significantly higher frequency of genotype I/II, compared to control group (P = 0.043, OR = 2.60, 95% CI: 1.02–6.50. Moreover, genotype I/I as well as allele I was overrepresented in the control group, suggesting that allele I may have a decreased risk for development of ITP. Our findings suggest that rs2424913 DNMT3B SNP as well as IL-1 Ra VNTR polymorphism may contribute to the susceptibility to ITP.

  5. Zinc-fingers and homeoboxes 1 (ZHX1) binds DNA methyltransferase (DNMT) 3B to enhance DNMT3B-mediated transcriptional repression

    International Nuclear Information System (INIS)

    Kim, Sung-Hak; Park, Jinah; Choi, Moon-Chang; Kim, Hwang-Phill; Park, Jung-Hyun; Jung, Yeonjoo; Lee, Ju-Hee; Oh, Do-Youn; Im, Seock-Ah; Bang, Yung-Jue; Kim, Tae-You

    2007-01-01

    DNA methyltransferases (DNMT) 3B is a de novo DNMT that represses transcription independent of DNMT activity. In order to gain a better insight into DNMT3B-mediated transcriptional repression, we performed a yeast two-hybrid analysis using DNMT3B as a bait. Of the various binding candidates, ZHX1, a member of zinc-finger and homeobox protein, was found to interact with DNMT3B in vivo and in vitro. N-terminal PWWP domain of DNMT3B was required for its interaction with homeobox motifs of ZHX1. ZHX1 contains nuclear localization signal at C-terminal homeobox motif, and both ZHX1 and DNMT3B were co-localized in nucleus. Furthermore, we found that ZHX1 enhanced the transcriptional repression mediated by DNMT3B when DNMT3B is directly targeted to DNA. These results showed for First the direct linkage between DNMT and zinc-fingers homeoboxes protein, leading to enhanced gene silencing by DNMT3B

  6. Association of DNA methyltransferases 3A and 3B polymorphisms, and plasma folate levels with the risk of urothelial carcinoma.

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    Chi-Jung Chung

    Full Text Available Interindividual genetic variations of human DNA methyltransferases (DNMTs, which involve the methyl donor from the folate-related one-carbon metabolism pathway, are hypothesized as a risk factor for urothelial carcinoma (UC. Therefore, we evaluated the role of gene-environment interaction in UC carcinogenesis.A hospital-based case-control study was conducted by recruiting 192 patients with UC and 381 controls. Their plasma folate levels were measured using a competitive immunoassay kit. In addition, DNMT3A -448A>G and DNMT3B -579G>T genotyping was evaluated using a polymerase chain reaction-restriction fragment length polymorphism technique. Multivariate logistic regression and 95% confidence intervals (CIs were applied to estimate the UC risk.We observed that patients with UC exhibited a higher prevalence rate of folate insufficiency (folate levels ≤6 ng/mL compared with the controls (35.94% and 18.37%, respectively. Furthermore, folate levels were higher in the prevalent UC patients than in the incident UC patients. However, folate insufficiency was similarly associated with a nearly two-fold increase in the risk of UC regardless of the UC patient group. In addition, the frequencies of the variant alleles for DNMT3A and DNMT3B were 0.80 and 0.92, respectively, and no association was observed with UC risk. However, participants with a variant homozygous genotype of DNMT3B -579G>T and folate insufficiency or with high cumulative cigarette smoking exhibited an increased risk of UC.Overall, environmental factors may contribute more significantly to UC carcinogenesis compared with genetic susceptibility. Future studies should investigate other polymorphisms of DNMT3A and DNMT3B to determine genetic susceptibility.

  7. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

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    Sharma Shikhar

    2012-01-01

    Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

  8. DNA-methyltransferase 3B 39179 G > T polymorphism and risk of sporadic colorectal cancer in a subset of Iranian population

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    Abdolreza Daraei

    2011-01-01

    Full Text Available Background: Epigenetic event is a biological regulation that influences the expression of various genes involved in cancer. DNA methylation is established by DNA methyltransferases, particularly DNAmethyltransferase 3B (DNMT3B. It seems to play an oncogenic role in the creation of abnormal methylation during tumorigenesis. The polymorphisms of the DNMT3B gene may influence DNMT3B activity in DNA methylation and increase the susceptibility to several cancers. These genetic polymorphisms have been studied in several cancers in different populations. Methods: In this study, we performed a case-control study with 125 colorectal cancer patients and 135 cancer-free controls to evaluate the association between DNMT3B G39179T polymorphism (rs1569686 in the promoter region and the risk of sporadic colorectal cancer. Up to now, few studies have investigated the role of this gene variant in sporadic colorectal cancer with no familial history. The genotypes of DNMT3B G39179T polymorphism was analyzed by PCR-RFLP. Results: We found that compared with G allele carriers, statistically the DNMT3B TT genotype (%34 was significantly associated with increased risk of colorectal cancer (adjusted OR, 3.993, 95% CI, 1.726-9.238, P = 0.001. Compared with DNMT3B TT genotype, the GT and GG genotypes had lower risk of developing sporadic colorectal cancer (OR = 0.848, 95% CI = 0.436-1.650. Conclusions: Our findings were consistent with that of previously reported case-control studies with colorectal cancer. These results suggest that the DNMT3B G39179T polymorphism influences DNMT3B expression, thus contributing to the genetic susceptibility to colorectal cancer. Further mechanistic studies are needed to unravel the causal molecular mechanisms.

  9. MiR-29c regulates the expression of miR-34c and miR-449a by targeting DNA methyltransferase 3a and 3b in nasopharyngeal carcinoma

    International Nuclear Information System (INIS)

    Niu, Man; Gao, Dan; Wen, Qiuyuan; Wei, Pingpin; Pan, Suming; Shuai, Cijun; Ma, Huiling; Xiang, Juanjuan; Li, Zheng; Fan, Songqing; Li, Guiyuan; Peng, Shuping

    2016-01-01

    Nasopharyngeal carcinoma (NPC) is prevalent in South East Asia and Southern China particularly, despite the reported 5-year survival ratio is relative higher than other deadly cancers such as liver, renal, pancreas cancer, the lethality is characterized by high metastatic potential in the early stage and high recurrence rate after radiation treatment. MicroRNA-29c was found to be down-regulated in the serum as well as in the tissue of nasopharyngeal carcinoma tissue. In this study, we found accidentally that the transfection of pre-miR-29c or miR-29c mimics significantly increases the expression level of miR-34c and miR-449a but doesn’t affect that of miR-222 using real-time quantitative PCR in nasopharyngeal carcinoma cell lines. To explore the molecular mechanism of the regulatory role, the cells are treated with 5-Aza-2-deoxycytidine (5-Aza-CdR) treatment and the level of miR-34c and miR-449a but not miR-222 accumulated by the treatment. DNA methyltransferase 3a, 3b were down-regulated by the 5-Aza-CdR treatment with western blot and real-time quantitative PCR. We found that pre-miR-29c or miR-29c mimics significantly increases the expression level of miR-34c and miR-449a. We further found DNA methyltransferase 3a and 3b are the target gene of miR-29c. Restoration of miR-29c in NPC cells down-regulated DNA methyltransferase 3a, 3b, but not DNA methyltransferase T1. The regulation of miR-29c/DNMTs/miR-34c/449a is an important molecular axis of NPC development and targeting DNMTs or restoring of miR-29c might be a promising therapy strategy for the prevention of NPC

  10. Interactions within the mammalian DNA methyltransferase family

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    Ehrenhofer-Murray Ann E

    2003-05-01

    Full Text Available Abstract Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase.

  11. Interactions within the mammalian DNA methyltransferase family

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    Margot, Jean B; Ehrenhofer-Murray, Ann E; Leonhardt, Heinrich

    2003-01-01

    Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase. PMID:12777184

  12. Recruitment of DNA methyltransferase I to DNA repair sites

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    Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M. Cristina; Leonhardt, Heinrich

    2005-01-01

    In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair. PMID:15956212

  13. Roles of DNA methyltransferases in Arabidopsis development ...

    African Journals Online (AJOL)

    Mutations that cause severe loss of DNA methylation often leads to abnormal development. In the present review, we summarized recent findings of the three major DNA methyltransferases mutants playing vital role in development of Arabidopsis thaliana. Keywords: DNA methylation, epigenetics, methyltransferase, mutant ...

  14. An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

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    Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

  15. Isolation of DNA methyltransferase from plants

    International Nuclear Information System (INIS)

    Ehrlich, K.; Malbroue, C.

    1987-01-01

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from [ 3 H]AdoMet incorporated into acid precipitable material per h at 30 0 ). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30 0

  16. Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic Activity and Gene Expression?

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    Bayraktar, Gonca; Kreutz, Michael R

    2018-04-01

    DNMT3A and 3B are the main de novo DNA methyltransferases (DNMTs) in the brain that introduce new methylation marks to non-methylated DNA in postmitotic neurons. DNA methylation is a key epigenetic mark that is known to regulate important cellular processes in neuronal development and brain plasticity. Accumulating evidence disclosed rapid and dynamic changes in DNA methylation of plasticity-relevant genes that are important for learning and memory formation. To understand how DNMTs contribute to brain function and how they are regulated by neuronal activity is a prerequisite for a deeper appreciation of activity-dependent gene expression in health and disease. This review discusses the functional role of de novo methyltransferases and in particular DNMT3A1 in the adult brain with special emphasis on synaptic plasticity, memory formation, and brain disorders.

  17. Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells.

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    Cai, Yi; Tsai, Hsing-Chen; Yen, Ray-Whay Chiu; Zhang, Yang W; Kong, Xiangqian; Wang, Wei; Xia, Limin; Baylin, Stephen B

    2017-04-01

    Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential. © 2017 Cai et al.; Published by Cold Spring Harbor Laboratory Press.

  18. Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  19. Development of fluorescent methods for DNA methyltransferase assay

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    Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

    2017-03-01

    DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

  20. A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding

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    Frauer, Carina; Leonhardt, Heinrich

    2009-01-01

    We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1C1229W catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity. PMID:19129216

  1. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

  2. DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

    International Nuclear Information System (INIS)

    Kim, Hak Jae; Kim, Jin Ho; Chie, Eui Kyu; Da Young, Park; Kim, In Ah; Kim, Il Han

    2012-01-01

    Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair

  3. Potential advantages of DNA methyltransferase 1 (DNMT1)-targeted inhibition for cancer therapy.

    Science.gov (United States)

    Jung, Yeonjoo; Park, Jinah; Kim, Tai Young; Park, Jung-Hyun; Jong, Hyun-Soon; Im, Seock-Ah; Robertson, Keith D; Bang, Yung-Jue; Kim, Tae-You

    2007-10-01

    The deoxyribonucleic acid (DNA) methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) has been used as a drug in a part of cancer therapy. However, because of its incorporation into DNA during DNA synthesis, 5-aza-dC can cause DNA damage, mutagenesis, and cytotoxicity. In view of the adverse effects of 5-aza-dC, DNMT-targeted inhibition may be a more effective approach than treatment with 5-aza-dC. To address the possibility of DNMT-targeted cancer therapy, we compared the effects of treatment with small interfering ribonucleic acids (siRNAs) specific for DNMT1 or DNMT3b and treatment with 5-aza-dC on transcription, cell growth, and DNA damage in gastric cancer cells. We found that DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CDKN2A, RASSF1A, HTLF, RUNX3, and AKAP12B) out of seven genes examined, and 5-aza-dC reactivated and demethylated all seven genes. In contrast, DNMT3b siRNAs did not show any effect. Furthermore, the double knockdown of DNMT1 and DNMT3b did not show a synergistic effect on gene re-expression and demethylation. In addition, DNMT1 siRNAs showed an inhibitory effect of cell proliferation in the cancer cells and the induction of cell death without evidence of DNA damage, whereas treatment with 5-aza-dC caused DNA damage as demonstrated by the comet assay. These results provide a rationale for the development of a DNMT1-targeted strategy as an effective epigenetic cancer therapy.

  4. ∆DNMT3B4-del Contributes to Aberrant DNA Methylation Patterns in Lung Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Mark Z. Ma

    2015-10-01

    Full Text Available Aberrant DNA methylation is a hallmark of cancer but mechanisms contributing to the abnormality remain elusive. We have previously shown that ∆DNMT3B is the predominantly expressed form of DNMT3B. In this study, we found that most of the lung cancer cell lines tested predominantly expressed DNMT3B isoforms without exons 21, 22 or both 21 and 22 (a region corresponding to the enzymatic domain of DNMT3B termed DNMT3B/∆DNMT3B-del. In normal bronchial epithelial cells, DNMT3B/ΔDNMT3B and DNMT3B/∆DNMT3B-del displayed equal levels of expression. In contrast, in patients with non-small cell lung cancer NSCLC, 111 (93% of the 119 tumors predominantly expressed DNMT3B/ΔDNMT3B-del, including 47 (39% tumors with no detectable DNMT3B/∆DNMT3B. Using a transgenic mouse model, we further demonstrated the biological impact of ∆DNMT3B4-del, the ∆DNMT3B-del isoform most abundantly expressed in NSCLC, in global DNA methylation patterns and lung tumorigenesis. Expression of ∆DNMT3B4-del in the mouse lungs resulted in an increased global DNA hypomethylation, focal DNA hypermethylation, epithelial hyperplastia and tumor formation when challenged with a tobacco carcinogen. Our results demonstrate ∆DNMT3B4-del as a critical factor in developing aberrant DNA methylation patterns during lung tumorigenesis and suggest that ∆DNMT3B4-del may be a target for lung cancer prevention.

  5. In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases.

    Directory of Open Access Journals (Sweden)

    Julia Arand

    2012-06-01

    Full Text Available The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites. The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM to calculate the relative contribution of DNA methyltransferases (Dnmts for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.

  6. DNA Electrochemistry Shows DNMT1 Methyltransferase Hyperactivity in Colorectal Tumors.

    Science.gov (United States)

    Furst, Ariel L; Barton, Jacqueline K

    2015-07-23

    DNMT1, the most abundant human methyltransferase, is responsible for translating the correct methylation pattern during DNA replication, and aberrant methylation by DNMT1 has been linked to tumorigenesis. We have developed a sensitive signal-on electrochemical assay for the measurement of DNMT1 activity in crude tissue lysates. We have further analyzed ten tumor sets and have found a direct correlation between DNMT1 hyperactivity and tumorous tissue. In the majority of samples analyzed, the tumorous tissue has significantly higher DNMT1 activity than the healthy adjacent tissue. No such correlation is observed in measurements of DNMT1 expression by qPCR, DNMT1 protein abundance by western blotting, or DNMT1 activity using a radiometric DNA labeling assay. DNMT1 hyperactivity can result from both protein overexpression and enzyme hyperactivity. DNMT1 activity measured electrochemically provides a direct measure of activity in cell lysates and, as a result, provides a sensitive and early indication of cancerous transformation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    International Nuclear Information System (INIS)

    Chen, Zirong; Jin, Guorong; Lin, Shuibin; Lin, Xiumei; Gu, Yumei; Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong; Wu, Lizi; Shen, Huangxuan

    2012-01-01

    Highlights: ► CDA-II inhibits myogenic differentiation in a dose-dependent manner. ► CDA-II repressed expression of muscle transcription factors and structural proteins. ► CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  8. Crystal structure of Mycobacterium tuberculosis O-6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA

    Czech Academy of Sciences Publication Activity Database

    Miggiano, R.; Perugino, G.; Ciaramella, M.; Serpe, M.; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, S.; Lahiri, S.; Rizzi, M.; Rossi, F.

    2016-01-01

    Roč. 473, č. 2 (2016), s. 123-133 ISSN 0264-6021 EU Projects: European Commission(XE) 241587 - SYSTEMTB Institutional support: RVO:61388963 Keywords : DNA repair * DNA-binding protein * Mycobacterium tuberculosis * O-6-methylguanine-DNA methyltransferase * co-operativity * crystal structure Subject RIV: CE - Biochemistry Impact factor: 3.797, year: 2016

  9. DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

    NARCIS (Netherlands)

    B.J. Glassner (Brian); G. Weeda (Geert); J.M. Allan (James); J.L.M. Broekhof (Jose'); N.H.E. Carls (Nick); I. Donker (Ingrid); B.P. Engelward (Bevin); R.J. Hampson (Richard); R. Hersmus (Remko); M.J. Hickman (Mark); R.B. Roth (Richard); H.B. Warren (Henry); M.M. Wu (Mavis); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1999-01-01

    textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA

  10. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-01-01

    Roč. 24, č. 6 (2016), s. 1268-1276 ISSN 0968-0896 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : nucleosides * nucleotides * pyrimidines * DNA methyltransferases * DNA polymerases Subject RIV: CC - Organic Chemistry Impact factor: 2.930, year: 2016

  11. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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    Monica K Akre

    Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

  12. Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

    Directory of Open Access Journals (Sweden)

    Rohini Garg

    Full Text Available DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases, namely Methyltransferase (MET, Chromomethylase (CMT and Domains Rearranged Methyltransferase (DRM, which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2 subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

  13. miR-125b targets DNMT3b and mediates p53 DNA methylation involving in the vascular smooth muscle cells proliferation induced by homocysteine

    International Nuclear Information System (INIS)

    Cao, ChengJian; Zhang, HuiPing; Zhao, Li; Zhou, Longxia; Zhang, Minghao; Xu, Hua; Han, Xuebo; Li, Guizhong; Yang, Xiaoling; Jiang, YiDeng

    2016-01-01

    MicroRNAs (miRNAs) are short non-coding RNA and play crucial roles in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Our previous studies found that homocysteine(Hcy) can stimulate the proliferation of vascular smooth muscle cells (VSMCs), however, the underlying mechanisms were not fully elucidated. Here, we found proliferation of VSMCs induced by Hcy was of correspondence to the miR-125b expression reduced both in vitro and in the ApoE knockout mice, the hypermethylation of p53, its decreased expression, and DNA (cytosine-5)-methyltransferase 3b (DNMT3b) up-regulated. And, we found DNMT3b is a target of miR-125b, which was verified by the Dual-Luciferase reporter assay and western blotting. Besides, the siRNA interference for DNMT3b significantly decreased the methylation level of p53, which unveiled the causative role of DNMT3b in p53 hypermethylation. miR-125b transfection further confirmed its regulative roles on p53 gene methylation status and the VSMCs proliferation. Our data suggested that a miR-125b-DNMT3b-p53 signal pathway may exist in the VSMCs proliferation induced by Hcy.

  14. miR-125b targets DNMT3b and mediates p53 DNA methylation involving in the vascular smooth muscle cells proliferation induced by homocysteine

    Energy Technology Data Exchange (ETDEWEB)

    Cao, ChengJian [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Zhang, HuiPing [Department of Prenatal Diagnosis Center, General Hospital of Ningxia Medical University, Yinchuan (China); Zhao, Li [Department of Medical Laboratory, Ningxia Medical University, Yinchuan (China); Zhou, Longxia [Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Zhang, Minghao; Xu, Hua [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Han, Xuebo [Department of Medical Laboratory, Ningxia Medical University, Yinchuan (China); Li, Guizhong; Yang, Xiaoling [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Jiang, YiDeng, E-mail: jyjcyxy@yeah.net [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China)

    2016-09-10

    MicroRNAs (miRNAs) are short non-coding RNA and play crucial roles in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Our previous studies found that homocysteine(Hcy) can stimulate the proliferation of vascular smooth muscle cells (VSMCs), however, the underlying mechanisms were not fully elucidated. Here, we found proliferation of VSMCs induced by Hcy was of correspondence to the miR-125b expression reduced both in vitro and in the ApoE knockout mice, the hypermethylation of p53, its decreased expression, and DNA (cytosine-5)-methyltransferase 3b (DNMT3b) up-regulated. And, we found DNMT3b is a target of miR-125b, which was verified by the Dual-Luciferase reporter assay and western blotting. Besides, the siRNA interference for DNMT3b significantly decreased the methylation level of p53, which unveiled the causative role of DNMT3b in p53 hypermethylation. miR-125b transfection further confirmed its regulative roles on p53 gene methylation status and the VSMCs proliferation. Our data suggested that a miR-125b-DNMT3b-p53 signal pathway may exist in the VSMCs proliferation induced by Hcy.

  15. DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

    Directory of Open Access Journals (Sweden)

    Alessandra eMaresca

    2015-03-01

    Full Text Available Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is

  16. Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

    OpenAIRE

    Jiang Li; Caili Li; Shanfa Lu

    2018-01-01

    Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, ei...

  17. Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase.

    Science.gov (United States)

    Shiraishi, A; Sakumi, K; Sekiguchi, M

    2000-10-01

    O(6)-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the MGMT: gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. MGMT(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD(50) values of MGMT(-/-) and MGMT(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of MGMT(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of MGMT(+/+) and MGMT(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O(6)-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. MGMT(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are MGMT(+/+) mice. Thus, the level of O(6)-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.

  18. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-05-15

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  19. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    International Nuclear Information System (INIS)

    Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong

    2012-01-01

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  20. Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Robert J Wood

    Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

  1. O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Mitra, S.; Pal, B.C.; Foote, R.S.

    1982-01-01

    O 6 -Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3 H-labeled O 6 -methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase

  2. Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B

    Science.gov (United States)

    2013-09-01

    Future work is needed to understand A3B regulation and the potential interaction with other oncogenes and tumour suppressors . For example, although...Limited. All rights reserved©2013 METHODS SUMMARY Flash -frozen tissues were obtained from the University of Minnesota Tissue Procurement Facility...used for RNA isolation, cDNA synthesis and qPCR as described14. Tissue RNA was from 100mg flash -frozen tissue disrupted by a 2-h water bath sonication in

  3. Investigating the potential role of genetic and epigenetic variation of DNA methyltransferase genes in hyperplastic polyposis syndrome.

    Directory of Open Access Journals (Sweden)

    Musa Drini

    2011-02-01

    Full Text Available Hyperplastic Polyposis Syndrome (HPS is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC. At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP, potentially linked to aberrant DNA methyltransferase (DNMT activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT, and negative regulators of Wnt signaling (such as WIF1. In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.We utilized high resolution melting (HRM analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene was over-represented in cases with HPS (p<0.01 compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053. BRAF mutations were common (11 out of 21 polyps, whilst KRAS mutations were identified in 4 of 21 polyps.Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.

  4. The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

    Science.gov (United States)

    Gianoglio, Silvia; Moglia, Andrea; Acquadro, Alberto; Comino, Cinzia; Portis, Ezio

    2017-01-01

    Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

  5. The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

    Directory of Open Access Journals (Sweden)

    Silvia Gianoglio

    Full Text Available Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1, CMT (chromomethyltransferases and DRM (domains rearranged methyltransferases. Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

  6. Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition.

    Science.gov (United States)

    Woodcock, Clayton B; Yakubov, Aziz B; Reich, Norbert O

    2017-08-01

    Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K m , k cat , k methylation , and K d for single-stranded and hemimethylated substrates, revealing discrimination of 10 7 -fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

  7. The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing

    International Nuclear Information System (INIS)

    Sato, Noriko; Kondo, Mitsumasa; Arai, Ken-ichi

    2006-01-01

    Somatic DNA methylation patterns are determined in part by the de novo methylation that occurs after early embryonic demethylation. Oct-3/4, a pluripotency gene, is unmethylated in the blastocyst, but undergoes de novo methylation and silencing during gastrulation. Here we show that the transcriptional repressor GCNF recruits DNA methyltransferase to the Oct-3/4 promoter and facilitates its methylation. Although acetylation of histone H3 at lysine 9 (K9) and/or 14 (K14) and methylation of H3 at lysine 4 (K4) decrease during this period, as do Oct-3/4 transcript levels, H3K9 and H3K27 methylation levels remain constant, indicating that DNA methylation does not require repressive histone modifications. We found that GCNF interacts directly with Dnmt3 molecule(s) and verified that this interaction induces the methylation of the Oct-3/4 promoter. Our finding suggests a model in which differentiation-induced GCNF recruits de novo DNA methyltransferase and facilitates the silencing of a pluripotency gene

  8. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: lizhean@lzu.edu.cn [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: tianl@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)

    2010-05-28

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  9. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    International Nuclear Information System (INIS)

    Song, Yuan; Wu, Keqiang; Dhaubhadel, Sangeeta; An, Lizhe; Tian, Lining

    2010-01-01

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  10. Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.

    Science.gov (United States)

    Miggiano, Riccardo; Perugino, Giuseppe; Ciaramella, Maria; Serpe, Mario; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, Silvia; Lahiri, Samarpita; Rizzi, Menico; Rossi, Franca

    2016-01-15

    Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair. © 2016 Authors; published by Portland Press Limited.

  11. Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy.

    Science.gov (United States)

    Zhang, Linqun; Liu, Yuanjian; Li, Ying; Zhao, Yuewu; Wei, Wei; Liu, Songqin

    2016-08-24

    A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6](3+), RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL(-1) to 10 U mL(-1), with a detection limit down to 0.03 U mL(-1). Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

    Directory of Open Access Journals (Sweden)

    Yu F

    2018-01-01

    Full Text Available Fei Yu,1,* Ya-min Xiong,1,* Song-cheng Yu,1 Lei-liang He,1 Shan-shan Niu,1 Yu-ming Wu,1 Jie Liu,1 Ling-bo Qu,2 Li-e Liu,1 Yong-jun Wu1 1College of Public Health, 2College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China *These authors contributed equally to this work Background: DNA methyltransferase 1 (DNMT1, a dominant enzyme responsible for the transfer of a methyl group from the universal methyl donor to the 5-position of cytosine residues in DNA, is essential for mammalian development and closely related to cancer and a variety of age-related chronic diseases. DNMT1 has become a useful biomarker in early disease diagnosis and a potential therapeutic target in cancer therapy and drug development. However, till now, most of the studies on DNA methyltransferase (MTase detection have focused on the prokaryote MTase and its activity.Methods: A magnetic fluorescence-linked immunosorbent assay (FLISA using CdSe/ZnS quantum dots as fluorescent probes was proposed for the rapid and sensitive detection of the DNMT1 level in this study. Key factors that affect the precision and accuracy of the determination of DNMT1 were optimized.Results: Under the optimal conditions, the limit of detection was 0.1 ng/mL, the linear range was 0.1–1,500 ng/mL, the recovery was 91.67%–106.50%, and the relative standard deviations of intra- and inter-assays were respectively 5.45%–11.29% and 7.03%–11.25%. The cross-reactivity rates with DNA methyltransferases 3a and 3b were only 4.0% and 9.4%, respectively. Furthermore, FLISA was successfully used to detect the levels of DNMT1 in human serum samples, and compared with commercial enzyme-linked immunosorbent assay (ELISA kits. The results revealed that there was a good correlation between FLISA and commercial ELISA kits (correlation coefficient r=0.866, p=0.001. The linear scope of FLISA was broader than ELISA, and the measurement time was much shorter

  13. Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

    Science.gov (United States)

    Takeshita, Kohei; Suetake, Isao; Yamashita, Eiki; Suga, Michihiro; Narita, Hirotaka; Nakagawa, Atsushi; Tajima, Shoji

    2011-01-01

    Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291–1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes. PMID:21518897

  14. Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio)

    International Nuclear Information System (INIS)

    Aluru, Neelakanteswar; Kuo, Elaine; Helfrich, Lily W.; Karchner, Sibel I.; Linney, Elwood A.; Pais, June E.; Franks, Diana G.

    2015-01-01

    DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5 nM TCDD for 1 h from 4 to 5 h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns. - Highlights: • TCDD altered the dnmt expression in a gene and developmental time-specific manner. • TCDD hypermethylated ahrra and hypomethylated c-fos proximal promoter regions. • Functional analysis suggests that ahr2 can regulate dnmt3a1, 3a2, and 3b2 expression. • Dnmt

  15. Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio)

    Energy Technology Data Exchange (ETDEWEB)

    Aluru, Neelakanteswar, E-mail: naluru@whoi.edu [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Kuo, Elaine [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Stanford University, 450 Serra Mall, Stanford, CA 94305 (United States); Helfrich, Lily W. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Northwestern University, 633 Clark St, Evanston, IL 60208 (United States); Karchner, Sibel I. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Linney, Elwood A. [Department of Molecular Genetics and Microbiology, Duke University Medical Center, Box 3020, Durham, NC 27710 (United States); Pais, June E. [New England Biolabs, 240 County Road, Ipswich, MA 01938 (United States); Franks, Diana G. [Biology Department and Woods Hole Center for Oceans and Human Health, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

    2015-04-15

    DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5 nM TCDD for 1 h from 4 to 5 h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns. - Highlights: • TCDD altered the dnmt expression in a gene and developmental time-specific manner. • TCDD hypermethylated ahrra and hypomethylated c-fos proximal promoter regions. • Functional analysis suggests that ahr2 can regulate dnmt3a1, 3a2, and 3b2 expression. • Dnmt

  16. Genome-wide identification and comparative analysis of cytosine-5 DNA methyltransferases and demethylase families in wild and cultivated peanut

    Directory of Open Access Journals (Sweden)

    Pengfei eWang

    2016-02-01

    Full Text Available AbstractDNA methylation plays important roles in genome protection, regulation of gene expression and was associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferases and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequence, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases and demethylase in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in known MET, CMT and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 numbers didn’t contain UBA domain which was different from other plants such as Arabidopsis, maize, soybean. Five DNA demethylase were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTases gene mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferases and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or drought stress could influence the expression level of C5-MTases and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut.

  17. Rationalization of activity cliffs of a sulfonamide inhibitor of DNA methyltransferases with induced-fit docking.

    Science.gov (United States)

    Medina-Franco, José L; Méndez-Lucio, Oscar; Yoo, Jakyung

    2014-02-21

    Inhibitors of human DNA methyltransferases (DNMT) are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure-activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of 'activity cliffs', e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

  18. Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts.

    Science.gov (United States)

    Green, M H; Karran, P; Lowe, J E; Priestley, A; Arlett, C F; Mayne, L

    1990-01-01

    We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

  19. Rationalization of Activity Cliffs of a Sulfonamide Inhibitor of DNA Methyltransferases with Induced-Fit Docking

    Directory of Open Access Journals (Sweden)

    José L. Medina-Franco

    2014-02-01

    Full Text Available Inhibitors of human DNA methyltransferases (DNMT are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure–activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of ‘activity cliffs’, e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

  20. Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1.

    Directory of Open Access Journals (Sweden)

    Samuel Brocklehurst

    Full Text Available Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1 for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles.

  1. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  2. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    International Nuclear Information System (INIS)

    Pang, Jinsong; Dong, Mingyue; Li, Ning; Zhao, Yanli; Liu, Bao

    2013-01-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  3. DNA methyltransferase 3A gene polymorphism contributes to daily life stress susceptibility

    Directory of Open Access Journals (Sweden)

    Barliana MI

    2017-12-01

    Full Text Available Melisa I Barliana,1,2 Shintya N Amalya,1 Ivan S Pradipta,3 Sofa D Alfian,3 Arif SW Kusuma,1,2 Tiana Milanda,1,4 Rizky Abdulah3,4 1Department of Biological Pharmacy, Biotechnology Pharmacy Laboratory, 2Pharmacy Services Development Research Center, 3Department of Pharmacology and Clinical Pharmacy, Clinical Pharmacy Laboratory, 4Center for Drug Discovery and Product Development, Faculty of Pharmacy, Universitas Padjadjaran, Jatinangor, West Java, Indonesia Abstract: Daily life stress markedly affects the response toward stressful stimuli. DNA methy­lation is one of the factors that regulate this response, and is a normal mechanism of somatic cell growth, but its regulatory gene variations may cause alterations in the stress response. The aim of the present study was to investigate genotypic variants of the DNA methyltransferase 3A (DNMT3A gene in 129 healthy subjects and evaluate its association with daily life stress. Blood samples were collected, and genomic DNA was isolated. DNA was amplified using specific tetra primers for DNMT3A (C/T rs11683424 and visualized following 2% agarose gel electrophoresis. The association of DNMT3A genetic variants with daily life stress was analyzed using the Kessler Psychological Distress Scale (K10. We observed that the distribution of subjects with genotype CC (wild type, CT (heteromutant, and TT (homomutant was 13.95%, 81.4%, and 4.65%, respectively. Genetic variations significantly affected the daily life stress condition (p=0.04 in Indonesian healthy subjects, but most of the subjects with the CT phenotype were classified in a stress condition. Keywords: daily life stressor, DNA methylation, epigenetic, Kessler Psychological Distress Scale (K10, rs11683424, DNMT3A

  4. Association of low race performance with mtDNA haplogroup L3b of Australian thoroughbred horses.

    Science.gov (United States)

    Lin, Xiang; Zheng, Hong-Xiang; Davie, Allan; Zhou, Shi; Wen, Li; Meng, Jun; Zhang, Yong; Aladaer, Qimude; Liu, Bin; Liu, Wu-Jun; Yao, Xin-Kui

    2018-03-01

    Mitochondrial DNA (mtDNA) encodes the genes for respiratory chain sub-units that determine the efficiency of oxidative phosphorylation in mitochondria. The aim of this study was to determine if there were any haplogroups and variants in mtDNA that could be associated with athletic performance of Thoroughbred horses. The whole mitochondrial genomes of 53 maternally unrelated Australian Thoroughbred horses were sequenced and an association study was performed with the competition histories of 1123 horses within their maternal lineages. A horse mtDNA phylogenetic tree was constructed based on a total of 195 sequences (including 142 from previous reports). The association analysis showed that the sample groups with poor racing performance history were enriched in haplogroup L3b (p = .0003) and its sub-haplogroup L3b1a (p = .0007), while those that had elite performance appeared to be not significantly associated with haplogroups G2 and L3a1a1a (p > .05). Haplogroup L3b and L3b1a bear two and five specific variants of which variant T1458C (site 345 in 16s rRNA) is the only potential functional variant. Furthermore, secondary reconstruction of 16s RNA showed considerable differences between two types of 16s RNA molecules (with and without T1458C), indicating a potential functional effect. The results suggested that haplogroup L3b, could have a negative association with elite performance. The T1458C mutation harboured in haplogroup L3b could have a functional effect that is related to poor athletic performance.

  5. The ada operon of Mycobacterium tuberculosis encodes two DNA methyltransferases for inducible repair of DNA alkylation damage.

    Science.gov (United States)

    Yang, Mingyi; Aamodt, Randi M; Dalhus, Bjørn; Balasingham, Seetha; Helle, Ina; Andersen, Pernille; Tønjum, Tone; Alseth, Ingrun; Rognes, Torbjørn; Bjørås, Magnar

    2011-06-10

    The ada operon of Mycobacterium tuberculosis, which encodes a composite protein of AdaA and AlkA and a separate AdaB/Ogt protein, was characterized. M. tuberculosis treated with N-methyl-N'-nitro-N-nitrosoguanidine induced transcription of the adaA-alkA and adaB genes, suggesting that M. tuberculosis mount an inducible response to methylating agents. Survival assays of the methyltransferase defective Escherichia coli mutant KT233 (ada ogt), showed that expression of the adaB gene rescued the alkylation sensitivity. Further, adaB but not adaA-alkA complemented the hypermutator phenotype of KT233. Purified AdaA-AlkA and AdaB possessed methyltransferase activity. These data suggested that AdaB counteract the cytotoxic and mutagenic effect of O(6)-methylguanine, while AdaA-AlkA most likely transfers methyl groups from innocuous methylphosphotriesters. AdaA-AlkA did not possess alkylbase DNA glycosylase activity nor rescue the alkylation sensitivity of the E. coli mutant BK2118 (tag alkA). We propose that AdaA-AlkA is a positive regulator of the adaptive response in M. tuberculosis. It thus appears that the ada operon of M. tuberculosis suppresses the mutagenic effect of alkylation but not the cytotoxic effect of lesions such as 3-methylpurines. Collectively, these data indicate that M. tuberculosis hypermutator strains with defective adaptive response genes might sustain robustness to cytotoxic alkylation DNA damage and confer a selective advantage contributing to host adaptation. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    International Nuclear Information System (INIS)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-01-01

    Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  7. Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.

    Science.gov (United States)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

    2014-04-01

    Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

  8. Contrasting roles for DNA methyltransferases and histone deacetylases in single-item and associative recognition memory.

    Science.gov (United States)

    Scott, Hannah; Smith, Anna E; Barker, Gareth R; Uney, James B; Warburton, E Clea

    2017-03-01

    Recognition memory enables us to judge whether we have encountered a stimulus before and to recall associated information, including where the stimulus was encountered. The perirhinal cortex (PRh) is required for judgment of stimulus familiarity, while hippocampus (HPC) and medial prefrontal cortex (mPFC) are additionally involved when spatial information associated with a stimulus needs to be remembered. While gene expression is known to be essential for the consolidation of long-term recognition memory, the underlying regulatory mechanisms are not fully understood. Here we investigated the roles of two epigenetic mechanisms, DNA methylation and histone deacetylation, in recognition memory. Infusion of DNA methyltransferase inhibitors into PRh impaired performance in novel object recognition and object-in-place tasks while infusions into HPC or mPFC impaired object-in-place performance only. In contrast, inhibition of histone deacetylases in PRh, but not mPFC, enhanced recognition memory. These results support the emerging role of epigenetic processes in learning and memory.

  9. Contrasting roles for DNA methyltransferases and histone deacetylases in single-item and associative recognition memory

    Directory of Open Access Journals (Sweden)

    Hannah Scott

    2017-03-01

    Full Text Available Recognition memory enables us to judge whether we have encountered a stimulus before and to recall associated information, including where the stimulus was encountered. The perirhinal cortex (PRh is required for judgment of stimulus familiarity, while hippocampus (HPC and medial prefrontal cortex (mPFC are additionally involved when spatial information associated with a stimulus needs to be remembered. While gene expression is known to be essential for the consolidation of long-term recognition memory, the underlying regulatory mechanisms are not fully understood. Here we investigated the roles of two epigenetic mechanisms, DNA methylation and histone deacetylation, in recognition memory. Infusion of DNA methyltransferase inhibitors into PRh impaired performance in novel object recognition and object-in-place tasks while infusions into HPC or mPFC impaired object-in-place performance only. In contrast, inhibition of histone deacetylases in PRh, but not mPFC, enhanced recognition memory. These results support the emerging role of epigenetic processes in learning and memory.

  10. O6-methylguanine DNA methyltransferase in human fetal tissues: fetal and maternal factors

    International Nuclear Information System (INIS)

    D'Ambrosio, S.M.; Samuel, M.J.; Dutta-Choudhury, T.A.; Wani, A.A.

    1986-01-01

    O 6 -Methylguanine methyltransferase (O 6 -MT) was measured and compared in extracts of 7 human fetal tissues obtained from 21 different fetal specimens as a function of fetal age and race, and maternal smoking and drug usage. Activity was determined from the proteinase-K solubilized radioactivity transferred from the DNA to the O 6 -MT. S9 homogenates were incubated with a heat depurinated [ 3 H]-methylnitrosourea alkylated DNA. Liver exhibited the highest activity followed by kidney, lung, small intestine, large intestine, skin and brain. Each of the tissues exhibited a 3- to 5-fold level of interindividual variation of O 6 -MT. There did not appear to be any significant difference of O 6 -MT in the tissues obtained from mothers who smoked cigarettes during pregnancy. Also, fetal race and age did not appear to account for the level of variation of O 6 -MT. The fetal tissues obtained from an individual using phenobarbital and smoking exhibited 4-fold increases in O 6 -MT activity. The tissues obtained from another individual on kidney dialysis were 2- to 3-fold higher than the normal population. These data suggest that the variation in human O 6 -MT can not be explained by racial or smoking factors, but may be modulated by certain drugs

  11. DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA.

    Science.gov (United States)

    Govindaraju, Gayathri; Jabeena, C A; Sethumadhavan, Devadathan Valiyamangalath; Rajaram, Nivethika; Rajavelu, Arumugam

    2017-10-01

    In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xiangbo; Zhang, Lu; Ding, Nianhua; Yang, Xinghui; Zhang, Jin; He, Jiang; Li, Zhi; Sun, Lun-Quan, E-mail: lunquansun@csu.edu.cn

    2015-05-29

    Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possess efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy. - Highlights: • Identified DNMT1-targeted DNAzymes by multiplex selection system. • Biochemically characterized a lead DNAzyme with high kinetic efficiency. • Validated DNMT1-targeted DNAzyme in its enzymatic and cellular activities.

  13. Atomic Insight into the Altered O6-Methylguanine-DNA Methyltransferase Protein Architecture in Gastric Cancer.

    Directory of Open Access Journals (Sweden)

    Naveed Anjum Chikan

    Full Text Available O6-methylguanine-DNA methyltransferase (MGMT is one of the major DNA repair protein that counteracts the alkalyting agent-induced DNA damage by replacing O6-methylguanine (mutagenic lesion back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations in the form of nucleotide polymorphism may result in altered protein structure that in turn can lead to the loss of function. In the present study, we focused on the population feared for high exposure to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon151 resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS. The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA-Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical-functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein by either eliminating the stabilizing salt bridges in cluster C3, C4, and C5 or by locally destabilizing the

  14. DNA Methyltransferases Modulate Hepatogenic Lineage Plasticity of Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Chien-Wei Lee

    2017-07-01

    Full Text Available The irreversibility of developmental processes in mammalian cells has been challenged by rising evidence that de-differentiation of hepatocytes occurs in adult liver. However, whether reversibility exists in mesenchymal stromal cell (MSC-derived hepatocytes (dHeps remains elusive. In this study, we find that hepatogenic differentiation (HD of MSCs is a reversible process and is modulated by DNA methyltransferases (DNMTs. DNMTs are regulated by transforming growth factor β1 (TGFβ1, which in turn controls hepatogenic differentiation and de-differentiation. In addition, a stepwise reduction in TGFβ1 concentrations in culture media increases DNMT1 and decreases DNMT3 in primary hepatocytes (Heps and confers Heps with multi-differentiation potentials similarly to MSCs. Hepatic lineage reversibility of MSCs and lineage conversion of Heps are regulated by DNMTs in response to TGFβ1. This previously unrecognized TGFβ1-DNMTs-MSC-HD axis may further increase the understanding the normal and pathological processes in the liver, as well as functions of MSCs after transplantation to treat liver diseases.

  15. Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

    Science.gov (United States)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O’Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

    2014-01-01

    Overgrowth disorders are a heterogeneous group of conditions characterised by increased growth parameters and variable other clinical features, such as intellectual disability and facial dysmorphism1. To identify novel causes of human overgrowth we performed exome sequencing in 10 proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations through DNMT3A sequencing of a further 142 individuals with overgrowth. The mutations were all located in functional DNMT3A domains and protein modelling suggests they interfere with domain-domain interactions and histone binding. No similar mutations were present in 1000 UK population controls (13/152 vs 0/1000; P<0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and increased height. DNMT3A encodes a key methyltransferase essential for establishing the methylation imprint in embryogenesis and is commonly somatically mutated in acute myeloid leukaemia2-4. Thus DNMT3A joins an emerging group of epigenetic DNA and histone modifying genes associated with both developmental growth disorders and haematological malignancies5. PMID:24614070

  16. Reconsolidation of a cocaine associated memory requires DNA methyltransferase activity in the basolateral amygdala

    Science.gov (United States)

    Shi, Hai-Shui; Luo, Yi-Xiao; Yin, Xi; Wu, Hong-Hai; Xue, Gai; Geng, Xu-Hong; Hou, Yan-Ning

    2015-01-01

    Drug addiction is considered an aberrant form of learning, and drug-associated memories evoked by the presence of associated stimuli (drug context or drug-related cues) contribute to recurrent craving and reinstatement. Epigenetic changes mediated by DNA methyltransferase (DNMT) have been implicated in the reconsolidation of fear memory. Here, we investigated the role of DNMT activity in the reconsolidation of cocaine-associated memories. Rats were trained over 10 days to intravenously self-administer cocaine by nosepokes. Each injection was paired with a light/tone conditioned stimulus (CS). After acquisition of stable self-administration behaviour, rats underwent nosepoke extinction (10 d) followed by cue-induced reactivation and subsequent cue-induced and cocaine-priming + cue-induced reinstatement tests or subsequently tested to assess the strength of the cocaine-associated cue as a conditioned reinforcer to drive cocaine seeking behaviour. Bilateral intra-basolateral amygdala (BLA) infusion of the DNMT inhibitor5-azacytidine (5-AZA, 1 μg per side) immediately following reactivation decreased subsequent reinstatement induced by cues or cocaine priming as well as cue-maintained cocaine-seeking behaviour. In contrast, delayed intra-BLA infusion of 5-AZA 6 h after reactivation or 5-AZA infusion without reactivation had no effect on subsequent cue-induced reinstatement. These findings indicate that memory reconsolidation for a cocaine-paired stimulus depends critically on DNMT activity in the BLA. PMID:26289919

  17. DNA methyltransferase mediates dose-dependent stimulation of neural stem cell proliferation by folate.

    Science.gov (United States)

    Li, Wen; Yu, Min; Luo, Suhui; Liu, Huan; Gao, Yuxia; Wilson, John X; Huang, Guowei

    2013-07-01

    The proliferative response of neural stem cells (NSCs) to folate may play a critical role in the development, function and repair of the central nervous system. It is important to determine the dose-dependent effects of folate in NSC cultures that are potential sources of transplantable cells for therapies for neurodegenerative diseases. To determine the optimal concentration and mechanism of action of folate for stimulation of NSC proliferation in vitro, NSCs were exposed to folic acid or 5-methyltetrahydrofolate (5-MTHF) (0-200 μmol/L) for 24, 48 or 72 h. Immunocytochemistry and methyl thiazolyl tetrazolium assay showed that the optimal concentration of folic acid for NSC proliferation was 20-40 μmol/L. Stimulation of NSC proliferation by folic acid was associated with DNA methyltransferase (DNMT) activation and was attenuated by the DNMT inhibitor zebularine, which implies that folate dose-dependently stimulates NSC proliferation through a DNMT-dependent mechanism. Based on these new findings and previously published evidence, we have identified a mechanism by which folate stimulates NSC growth. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Egg-specific expression of protein with DNA methyltransferase activity in the biocarcinogenic liver fluke Clonorchis sinensis.

    Science.gov (United States)

    Kim, Seon-Hee; Cho, Hye-Jeong; Sohn, Woon-Mok; Ahn, Chun-Seob; Kong, Yoon; Yang, Hyun-Jong; Bae, Young-An

    2015-08-01

    Despite recent reports regarding the biology of cytosine methylation in Schistosoma mansoni, the impact of the regulatory machinery remains unclear in diverse platyhelminthes. This ambiguity is reinforced by discoveries of DNA methyltransferase 2 (DNMT2)-only organisms and the substrate specificity of DNMT2 preferential to RNA molecules. Here, we characterized a novel DNA methyltransferase, named CsDNMT2, in a liver fluke Clonorchis sinensis. The protein exhibited structural properties conserved in other members of the DNMT2 family. The native and recombinant CsDNMT2 exhibited considerable enzymatic activity on DNA. The spatiotemporal expression of CsDNMT2 mirrored that of 5-methylcytosine (5 mC), both of which were elevated in the C. sinensis eggs. However, CsDNMT2 and 5 mC were marginally detected in other histological regions of C. sinensis adults including ovaries and seminal receptacle. The methylation site seemed not related to genomic loci occupied by progenies of an active long-terminal-repeat retrotransposon. Taken together, our data strongly suggest that C. sinensis has preserved the functional DNA methylation machinery and that DNMT2 acts as a genuine alternative to DNMT1/DNMT3 to methylate DNA in the DNMT2-only organism. The epigenetic regulation would target functional genes primarily involved in the formation and/or maturation of eggs, rather than retrotransposons.

  19. Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

    Energy Technology Data Exchange (ETDEWEB)

    Sontag, Ryan L.; Weber, Thomas J.

    2012-05-04

    In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

  20. O6-Methylguanine-DNA methyltransferase status in neuroendocrine tumours: prognostic relevance and association with response to alkylating agents.

    Science.gov (United States)

    Walter, T; van Brakel, B; Vercherat, C; Hervieu, V; Forestier, J; Chayvialle, J-A; Molin, Y; Lombard-Bohas, C; Joly, M-O; Scoazec, J-Y

    2015-02-03

    O(6)-Methylguanine-DNA methyltransferase (MGMT) loss of expression has been suggested to be predictive of response to temozolomide in neuroendocrine tumours (NETs), but so far, only limited data are available. We evaluated the prognostic and predictive value of MGMT status, assessed by two molecular methods and immunohistochemistry, in a large series of NETs of different origins. A total of 107 patients, including 53 treated by alkylants (temozolomide, dacarbazine or streptozotocin), were retrospectively studied. In each case, we used methyl-specific PCR (MS-PCR) and pyrosequencing for evaluation of promoter methylation and immunohistochemistry for evaluation of protein status. MGMT promoter methylation was detected in 12 out of 99 (12%) interpretable cases by MS-PCR and in 24 out of 99 (24%) by pyrosequencing. O(6)-Methylguanine-DNA methyltransferase loss of expression was observed in 29 out of 89 (33%) interpretable cases. Status of MGMT was not correlated with overall survival (OS) from diagnosis. Progression-free survival and OS from first alkylant use (temozolomide, dacarbazine and streptozotocin) were higher in patients with MGMT protein loss (respectively, 20.2 vs 7.6 months, Palkylant-based chemotherapy in NETs.

  1. DNA methyltransferase 1-targeting miRNA-148aof dairymilk: apotential bioactive modifier of thehumanepigenome

    Directory of Open Access Journals (Sweden)

    Bodo C. Melnik

    2017-09-01

    Full Text Available Background: The perception of milk has changed from a “simple food” to a more sophisticated bioactive functional signaling system that promotes mTORC1-driven postnatal anabolism, growth, and development of the newborn infant. Accumulating evidence supports the view that milk´s miRNAs significantly contribute to these processes. The most abundant miRNA of milk found in milk fat and milk exosomes is miRNA-148a, which targets DNA methyltransferase 1 (DNMT1, a pivotal epigenetic regulator that suppresses transcription. Furthermore, milk-derived miRNA-125b, miRNA-30d, and miRNA-25 target TP53, the guardian of the genome that interacts with DNMT1 and regulates metabolism, cell kinetics, and apoptosis. Thus, the question arose whether cow´s milk-derived miRNAs may modify epigenetic regulation of the human milk consumer. Methods: To understand the potential impact of dairy milk consumption on human epigenetics, we have analyzed all relevant research-based bioinformatics data related to milk, milk miRNAs, epigenetic regulation, and lactation performance with special attention to bovine miRNAs that modify gene expression of DNA methyltransferase 1 (DNMT1 and p53 (TP53, the two guardians of the mammalian genome. By means of translational research and comparative functional genomics, we investigated the potential impact of cow´s milk miRNAs on epigenetic regulation of human DNMT1, TP53, FOXP3, and FTO, which are critically involved in immunologic and metabolic programming respectively. miRNA sequences have been obtained from mirbase.org. miRNA-target site prediction has been performed using TargetScan release 7.0. Results: The most abundant miRNA of cow´s milk is miRNA-148a, which represents more than 10% of all miRNAs of cow´s milk, survives pasteurization and refrigerated storage. The seed sequence of human and bovine miRNA-148a-3p is identical. Furthermore, human and bovine DNMT1 mRNA share 88% identity. The miRNA-148a 7mer seed is conserved in

  2. Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents.

    Science.gov (United States)

    Cai, Shanbao; Xu, Yi; Cooper, Ryan J; Ferkowicz, Michael J; Hartwell, Jennifer R; Pollok, Karen E; Kelley, Mark R

    2005-04-15

    DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

  3. DNA methyltransferase 3A promoter polymorphism is associated with the risk of human spontaneous abortion after assisted reproduction techniques and natural conception.

    Science.gov (United States)

    Liu, Yudong; Zheng, Haiyan; Guo, Pingping; Feng, Shuxian; Zhou, Xingyu; Ye, Desheng; Chen, Xin; Chen, Shiling

    2017-02-01

    The aim of this study was to explore the association of the DNA-methyltransferase (DNMT)-3A and DNMT3B promoter polymorphisms with the risk of human spontaneous abortion after assisted reproduction techniques (ARTs) and natural conception. We collected tissues from women who underwent abortion procedures: (a) chorionic villus samples (CVS) and muscle samples (MS) from spontaneous abortions conceived by ART and natural cycle (study group), n = 152; and (b) CVS and MS from normal early pregnancy and second trimester (control group), n = 155. The single-nucleotide polymorphism (SNP) -448A > G in the DNMT3A promoter region and -149C/T polymorphism of DNMT3B were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and confirmed by sequencing. The allele frequency of -448A among pregnancy loss group and control group was 34.2 % vs. 16.5 %, respectively. Compared with GG carriers, the DNMT3A -448AA homozygotes had an about 16-fold increased risk of spontaneous abortion [odds ratio (OR) = 16.130, 95 % confidence interval (CI), 3.665-70.984], and AG heterozygotes had an OR of 2.027 (95 % CI, 1.247-3.293). However, the distribution of -448A > G in individuals derived from ART pregnancies was not statistically significantly compared with those derived from spontaneous pregnancies (P = 0.661). For DNMT3B, we observed genotype frequencies of 100 % (TT) in the study group and the control group. The DNMT3A -448A > G polymorphism may be a novel functional SNP and contribute to its genetic susceptibility to spontaneous abortion in Chinese women, and ART may not affect the distribution of -448A > G in pregnancy loss and normal pregnancy. The observed TT genotype of DMNT3B suggests that this is the predominant genotype of this population. The findings provide new insights into the etiology of human spontaneous abortion.

  4. Expression of DNA methyltransferases is influenced by growth hormone in the long-living Ames dwarf mouse in vivo and in vitro.

    Science.gov (United States)

    Armstrong, Vanessa L; Rakoczy, Sharlene; Rojanathammanee, Lalida; Brown-Borg, Holly M

    2014-08-01

    Methyltransferase expression and DNA methylation are linked to aging and age-related disease. We utilized 3-, 12-, and 24-month-old Ames dwarf and their wild-type siblings to examine the genotype and age-related differences in the expression of methyltransferase enzymes related to DNA methylation in the liver, glycine-N-methyltransferase and DNA methyltransferase (DNMT). We found that DNMT proteins and transcripts are differentially expressed in dwarf mice compared with wild-type siblings that can be attributed to age and/or genotype. However, DNMT1 protein expression is drastically reduced compared with wild-type controls at every age. DNMT3a protein levels coincide with differences observed in DNMT activity. Growth hormone appears to modulate expression of DNMT1 and 3a in dwarf liver tissue and primary hepatocytes. Therefore, growth hormone may contribute to age-related processes, DNA methylation, and, ultimately, longevity. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Caught in the act: visualization of an intermediate in the DNA base-flipping pathway induced by HhaI methyltransferase | Center for Cancer Research

    Science.gov (United States)

    HHAI methyltransferase (blue ribbon) bound to oligonucleotide (strands with bonds colored yellow and green) containing a pseudorotationally constrained sugar analogue at the target position (orange bonds with cyan atoms). The south-constrained pseudosugar is rotated about its flanking phosphodiester bonds, 90° from its initial position in B-form DNA, but short of a completely

  6. Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

    Science.gov (United States)

    Hassler, Melanie R.; Klisaroska, Aleksandra; Kollmann, Karoline; Steiner, Irene; Bilban, Martin; Schiefer, Ana-Iris; Sexl, Veronika; Egger, Gerda

    2012-01-01

    DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use. PMID:22687603

  7. DNA (Cytosine-C5) Methyltransferase Inhibition by Oligodeoxyribonucleotides Containing 2-(1H)-Pyrimidinone (Zebularine Aglycon) at the Enzymatic Target Site

    OpenAIRE

    van Bemmel, Dana M.; Brank, Adam S.; Eritja, Ramon; Marquez, Victor E.; Christman, Judith K.

    2009-01-01

    Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferas...

  8. DNA methyltransferases contribute to the fungal development, stress tolerance and virulence of the entomopathogenic fungus Metarhizium robertsii.

    Science.gov (United States)

    Wang, Yulong; Wang, Tiantian; Qiao, Lintao; Zhu, Jianyu; Fan, Jinrui; Zhang, Tingting; Wang, Zhang-Xun; Li, Wanzhen; Chen, Anhui; Huang, Bo

    2017-05-01

    DNA methylation is an important epigenetic mark in mammals, plants, and fungi and depends on multiple genetic pathways involving de novo and maintenance DNA methyltransferases (DNMTases). Metarhizium robertsii, a model system for investigating insect-fungus interactions, has been used as an environmentally friendly alternative to chemical insecticides. However, little is known concerning the molecular basis for DNA methylation. Here, we report on the roles of two DNMTases (MrRID and MrDIM-2) by characterizing ΔMrRID, ΔMrDIM-2, and ΔRID/ΔDIM-2 mutants. The results showed that approximately 71, 10, and 8% of m C sites remained in the ΔMrRID, ΔMrDIM-2, and ΔRID/ΔDIM-2 strains, respectively, compared with the wild-type (WT) strain. Further analysis showed that MrRID regulates the specificity of DNA methylation and MrDIM-2 is responsible for most DNA methylation, implying an interaction or cooperation between MrRID and MrDIM-2 for DNA methylation. Moreover, the ΔMrDIM-2 and ΔRID/ΔDIM-2 strains showed more defects in radial growth and conidial production compared to the WT. Under ultraviolet (UV) irradiation or heat stress, an obvious reduction in spore viability was observed for all the mutant strains compared to the WT. The spore median lethal times (LT 50 s) for the ΔMrDIM-2 and ΔRID/ΔDIM-2 strains in the greater wax moth, Galleria mellonella, were decreased by 47.7 and 65.9%, respectively, which showed that MrDIM-2 is required for full fungal virulence. Our data advances the understanding of the function of DNMTase in entomopathogenic fungi, which should contribute to future epigenetic investigations in fungi.

  9. 5-aza-2′-deoxycytidine impairs mouse spermatogenesis at multiple stages through different usage of DNA methyltransferases

    International Nuclear Information System (INIS)

    Song, Ning; Endo, Daisuke; Song, Bin; Shibata, Yasuaki; Koji, Takehiko

    2016-01-01

    Mammalian spermatogenesis is a progressive process comprising spermatogonial proliferation, spermatocytic meiosis, and later spermiogenesis, which is considered to be under the regulation of epigenetic parameters. To gain insights into the significance of DNA methylation in early spermatogenesis, 5-azadC was used as a molecular biological tool to mimic the level of DNA methylation in vivo. Since the drug is incorporated into DNA during the S-phase, spermatogonia and spermatocytes would be affected primarily in mouse spermatogenesis. Adult male ICR mice were intraperitoneally injected with 5-azadC at a dose of 0.25 mg/kg/day for 10 consecutive days, allowing us to examine its maximum effect on the kinetics of spermatogonia and spermatocytes. In this short-term protocol, 5-azadC induced significant histological abnormalities, such as a marked increase in apoptosis of spermatogonia and spermatocytes, followed by severe loss of spermatids, while after termination of 5-azadC treatment, normal histology was restored in the testis within 35 days. Quantification of the methylation level of CCGG sites as well as whole DNA showed spermatogonial hypomethylation, which correlated with increased apoptosis of spermatogonia. Interestingly, the hypomethylated cells were simultaneously positive for tri-methylated histone H3 at K4. On the other hand, no changes in methylation level were found in spermatocytes, but PCNA staining clearly showed disordered accumulation of S-phase spermatocytes, which increased their apoptosis in stage XII. In addition, different immunohistochemical staining pattern was found for DNA methyltransferases (DNMTs); DNMT1was expressed in the majority of all germ cells, but DNMT3a and b were only expressed in spermatogonia. Our results indicate that 5-azadC caused DNA hypomethylation in spermatogonia, but induced prolongation of S-phase in spermatocytes, resulting in the induction of apoptosis in both cases. Thus, 5-azadC affects spermatogenesis at more than

  10. Increased cytosine DNA-methyltransferase activity in A/J mouse lung cells following carcinogen exposure and during tumor progression

    International Nuclear Information System (INIS)

    Belinsky, S.A.; Issa, J.-P.J.; Baylin, S.B.

    1994-01-01

    Considerable evidence has accumulated that 5-methylcytosine modification of mammalian DNA, both in exons and CpG rich islands located in promoter regions, is important in gene regulation. For example, a decrease of 5-methylcytosine in 5' flanking regions or exons of genes has been associated with increased gene transcription. In addition, hypermethylation at specific regions of chromosomes 17p and 3p have also been observed in lung and colon cancer. During colon cancer development, these hypermethylation changes precede allelic loss. In addition, the activity of the enzyme which maintains the methylation status at CpG dinucleotides, DNA methyltransferase (MT), has been shown to increase during colon cancer progression. These observations suggest changes in methylation patterns within specific genes could result in either inappropriate gene expression or gene deletion, both of which would contribute to the establishment of the malignant phenotype. The purpose of this investigation was to determine if DNA MT activity is elevated in target (alveolar type II), but not in nontarget (Clara, endothelial, macrophage) lung cells isolated from the A/J mouse following exposure to nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). In addition, the activity of this enzyme during tumor progression was examined

  11. Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats

    Directory of Open Access Journals (Sweden)

    Bianca Genenncher

    2018-02-01

    Full Text Available The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5 RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.

  12. Rapid, large-scale purification and characterization of Ada protein (O sup 6 methylguanine-DNA methyltransferase) of E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharyya, D.; Tano, K.; Bunick, G.J.; Uberbacher, E.C.; Mitra, S. (Oak Ridge National Laboratory, TN (USA)); Behnke, W.D. (Univ. of Cincinnati College of Medicine, OH (USA))

    1988-07-25

    The E. coli Ada protein (O{sup 6}-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E{sup 280nm}{sub 1%}) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from O{sup 6}-methylguanine in DNA. Its reaction with O{sup 6}-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 {times} 10{sup 9} M{sup {minus}1} min{sup {minus}1} at 0{degree}C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low {alpha}-helical content and the radius of gyration of 23 {angstrom} indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.

  13. Dysregulated DNA Methyltransferase 3A Upregulates IGFBP5 to Suppress Trophoblast Cell Migration and Invasion in Preeclampsia.

    Science.gov (United States)

    Jia, Yuanhui; Li, Ting; Huang, Xiaojie; Xu, Xianghong; Zhou, Xinyao; Jia, Linyan; Zhu, Jingping; Xie, Dandan; Wang, Kai; Zhou, Qian; Jin, Liping; Zhang, Jiqin; Duan, Tao

    2017-02-01

    Preeclampsia is a unique multiple system disorder during human pregnancy, which affects ≈5% to 8% of pregnancies. Its risks and complications have become the major causes of maternal and fetal morbidity and mortality. Although abnormal placentation to which DNA methylation dysregulation is always linked is speculated to be one of the reasons causing preeclampsia, the underlying mechanisms still remain elusive to date. Here we revealed that aberrant DNA methyltransferase 3A (DNMT3A) plays a critical role in preeclampsia. Our results show that the expression and localization of DNMT3A are dysregulated in preeclamptic placenta. Moreover, knockdown of DNMT3A obviously inhibits trophoblast cell migration and invasion. Mechanistically, IGFBP5 (insulin-like growth factor-binding protein 5), known as a suppressor, is upregulated by decreased DNMT3A because of promoter hypomethylation. Importantly, IGFBP5 downregulation can rescue the defects caused by DNMT3A knockdown, thereby, consolidating the significance of IGFBP5 in the downstream of DNMT3A in trophoblast. Furthermore, we detected low promoter methylation and high protein expression of IGFBP5 in the clinical samples of preeclamptic placenta. Collectively, our study suggests that dysregulation of DNMT3A and IGFBP5 is relevant to preeclampsia. Thus, we propose that DNMT3A and IGFBP5 can serve as potential markers and targets for the clinical diagnosis and therapy of preeclampsia. © 2017 American Heart Association, Inc.

  14. Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow

    Directory of Open Access Journals (Sweden)

    A. M. Luciano

    2009-12-01

    Full Text Available DNA methyltransferase-1 (Dnmt1 is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation.We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM. RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals.

  15. The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma.

    Science.gov (United States)

    Andrade, Augusto Faria; Borges, Kleiton Silva; Suazo, Veridiana Kiill; Geron, Lenisa; Corrêa, Carolina Alves Pereira; Castro-Gamero, Angel Mauricio; de Vasconcelos, Elton José Rosas; de Oliveira, Ricardo Santos; Neder, Luciano; Yunes, José Andres; Dos Santos Aguiar, Simone; Scrideli, Carlos Alberto; Tone, Luiz Gonzaga

    2017-02-01

    Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

  16. O6-Methylguanine DNA Methyltransferase Status Does Not Predict Response or Resistance to Alkylating Agents in Well-Differentiated Pancreatic Neuroendocrine Tumors.

    Science.gov (United States)

    Raj, Nitya; Klimstra, David S; Horvat, Natally; Zhang, Liying; Chou, Joanne F; Capanu, Marinela; Basturk, Olca; Do, Richard Kinh Gian; Allen, Peter J; Reidy-Lagunes, Diane

    2017-07-01

    Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown. We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing. Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact. We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

  17. The Prognostic Roles of Gender and O6-Methylguanine-DNA Methyltransferase Methylation Status in Glioblastoma Patients: The Female Power.

    Science.gov (United States)

    Franceschi, Enrico; Tosoni, Alicia; Minichillo, Santino; Depenni, Roberta; Paccapelo, Alexandro; Bartolini, Stefania; Michiara, Maria; Pavesi, Giacomo; Urbini, Benedetta; Crisi, Girolamo; Cavallo, Michele A; Tosatto, Luigino; Dazzi, Claudio; Biasini, Claudia; Pasini, Giuseppe; Balestrini, Damiano; Zanelli, Francesca; Ramponi, Vania; Fioravanti, Antonio; Giombelli, Ermanno; De Biase, Dario; Baruzzi, Agostino; Brandes, Alba A

    2018-04-01

    Clinical and molecular factors are essential to define the prognosis in patients with glioblastoma (GBM). O6-methylguanine-DNA methyltransferase (MGMT) methylation status, age, Karnofsky Performance Status (KPS), and extent of surgical resection are the most relevant prognostic factors. Our investigation of the role of gender in predicting prognosis shows a slight survival advantage for female patients. We performed a prospective evaluation of the Project of Emilia Romagna on Neuro-Oncology (PERNO) registry to identify prognostic factors in patients with GBM who received standard treatment. A total of 169 patients (99 males [58.6%] and 70 females [41.4%]) were evaluated prospectively. MGMT methylation was evaluable in 140 patients. Among the male patients, 36 were MGMT methylated (25.7%) and 47 were unmethylated (33.6%); among the female patients, 32 were methylated (22.9%) and 25 were unmethylated (17.9%). Survival was longer in the methylated females compared with the methylated males (P = 0.028) but was not significantly different between the unmethylated females and the unmethylated males (P = 0.395). In multivariate analysis, gender and MGMT methylation status considered together (methylated females vs. methylated males; hazard ratio [HR], 0.459; 95% confidence interval [CI], 0.242-0.827; P = 0.017), age (HR, 1.025; 95% CI, 1.002-1.049; P = 0.032), and KPS (HR, 0.965; 95% CI, 0.948-0.982; P factor. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Achaete-scute complex homolog-1 promotes DNA repair in the lung carcinogenesis through matrix metalloproteinase-7 and O(6-methylguanine-DNA methyltransferase.

    Directory of Open Access Journals (Sweden)

    Xiao-Yang Wang

    Full Text Available Lung cancer is the leading cause of cancer-related deaths in the world. Achaete-scute complex homolog-1 (Ascl1 is a member of the basic helix-loop-helix (bHLH transcription factor family that has multiple functions in the normal and neoplastic lung such as the regulation of neuroendocrine differentiation, prevention of apoptosis and promotion of tumor-initiating cells. We now show that Ascl1 directly regulates matrix metalloproteinase-7 (MMP-7 and O(6-methylguanine-DNA methyltransferase (MGMT. Loss- and gain-of-function experiments in human bronchial epithelial and lung carcinoma cell lines revealed that Ascl1, MMP-7 and MGMT are able to protect cells from the tobacco-specific nitrosamine NNK-induced DNA damage and the alkylating agent cisplatin-induced apoptosis. We also examined the role of Ascl1 in NNK-induced lung tumorigenesis in vivo. Using transgenic mice which constitutively expressed human Ascl1 in airway lining cells, we found that there was a delay in lung tumorigenesis. We conclude that Ascl1 potentially enhances DNA repair through activation of MMP-7 and MGMT which may impact lung carcinogenesis and chemoresistance. The study has uncovered a novel and unexpected function of Ascl1 which will contribute to better understanding of lung carcinogenesis and the broad implications of transcription factors in tobacco-related carcinogenesis.

  19. DNA methyltransferases and stress-related genes expression in zebrafish larvae after exposure to heat and copper during reprogramming of DNA methylation.

    Science.gov (United States)

    Dorts, Jennifer; Falisse, Elodie; Schoofs, Emilie; Flamion, Enora; Kestemont, Patrick; Silvestre, Frédéric

    2016-10-12

    DNA methylation, a well-studied epigenetic mark, is important for gene regulation in adulthood and for development. Using genetic and epigenetic approaches, the present study aimed at evaluating the effects of heat stress and copper exposure during zebrafish early embryogenesis when patterns of DNA methylation are being established, a process called reprogramming. Embryos were exposed to 325 μg Cu/L from fertilization (<1 h post fertilization - hpf) to 4 hpf at either 26.5 °C or 34 °C, followed by incubation in clean water at 26.5 °C till 96 hpf. Significant increased mortality rates and delayed hatching were observed following exposure to combined high temperature and Cu. Secondly, both stressors, alone or in combination, significantly upregulated the expression of de novo DNA methyltransferase genes (dnmt3) along with no differences in global cytosine methylation level. Finally, Cu exposure significantly increased the expression of metallothionein (mt2) and heat shock protein (hsp70), the latter being also increased following exposure to high temperature. These results highlighted the sensitivity of early embryogenesis and more precisely of the reprogramming period to environmental challenges, in a realistic situation of combined stressors.

  20. The impact of SF3B1 mutations in CLL on the DNA-damage response

    DEFF Research Database (Denmark)

    Te Raa, G D; Derks, I A M; Navrkalova, V

    2015-01-01

    Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part assoc...

  1. The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states

    Directory of Open Access Journals (Sweden)

    Tochiki Keri K

    2012-02-01

    Full Text Available Abstract Background DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2 and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated. Results Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury. Conclusion Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

  2. Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription.

    Science.gov (United States)

    Pai, Chen-Chun; Kishkevich, Anastasiya; Deegan, Rachel S; Keszthelyi, Andrea; Folkes, Lisa; Kearsey, Stephen E; De León, Nagore; Soriano, Ignacio; de Bruin, Robertus Antonius Maria; Carr, Antony M; Humphrey, Timothy C

    2017-09-12

    Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription

    Directory of Open Access Journals (Sweden)

    Chen-Chun Pai

    2017-09-01

    Full Text Available Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB binding factor (MBF-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR expression, reduced deoxyribonucleoside triphosphate (dNTP synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast.

  4. O6-methylguanine-DNA methyltransferase in equine sarcoids: molecular and epigenetic analysis

    Directory of Open Access Journals (Sweden)

    Altamura Gennaro

    2012-11-01

    Full Text Available Abstract Background Bovine papillomaviruses (BPVs types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to the loss of function of a number of oncosuppressor genes. In this study the putative role of O6-methylguanine-DNA methyltrasferase (MGMT was investigated for sarcoids. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissues. In addition, the DNA methylation profile was analysed to assess the role of epigenetic mechanism in regulation of MGMT expression. Results A group of 15 equine sarcoids and two primary sarcoid cell lines (fibroblasts were analyzed for the expression of MGMT protein by immunohistochemistry, immunofluorescence and Western blotting techniques. The sarcoid cell line EqSO4b and the tumour samples showed a reduction or absence of MGMT expression. To investigate the causes of deregulated MGMT expression, ten samples were analyzed for the DNA methylation profile of the CpG island associated to the MGMT promoter. The analysis of 73 CpGs encompassing the region of interest showed in 1 out of 10 (10% sarcoids a pronouncedly altered methylation profile when compared to the control epidermal sample. Similarily the EqSO4b cell line showed an altered MGMT methylation pattern in comparison to normal fibroblasts. Conclusion As previously demonstrated for the oncosuppressor gene FHIT, analysis of MGMT expression in sarcoid tissues and a sarcoid-derived fibroblast cell line further suggests that

  5. Post-implantation mortality of in vitro produced embryos is associated with DNA methyltransferase 1 dysfunction in sheep placenta.

    Science.gov (United States)

    Ptak, Grazyna Ewa; D'Agostino, Antonella; Toschi, Paola; Fidanza, Antonella; Zacchini, Federica; Czernik, Marta; Monaco, Federica; Loi, Pasqualino

    2013-02-01

    Is DNA methyltransferase 1 (DNMT1) dysfunction involved in epigenetic deregulation of placentae from embryos obtained by assisted reproduction technologies (ARTs)? DNMT1 expression in growing placentae of in vitro produced (IVP) embryos is compromised and associated with pregnancy loss. DNMT1 maintains the methylation profile of genes during cell division. The methylation status of genes involved in placenta development is altered in embryos obtained in vitro. Disturbances in the epigenetic regulation of gene expression during placentogenesis could be involved in the frequent developmental arrest and loss of IVP embryos. Forty sheep were naturally mated (Group 1, CTR). IVP blastocysts (2-4 per ewe) were surgically transferred to the remaining 46 recipient sheep 6 days after oestrus (Group 2). Twenty-one recipients from Group 1 and 27 recipients from Group 2 were allowed to deliver in order to compare embryo survival in both groups at term (150 days). From the remaining recipients (n = 38), fetuses and placentae of both groups were recovered by paramedian laparotomy at Days 20, 22, 24, 26 and 28 of gestation. Immediately after collection, early placental tissues (chorion-allantois) were snap frozen in liquid nitrogen and DNMT1 expression and activity was evaluated. mRNA levels (for DNMT1, HDAC2, PCNA, DMAP1, MEST, IGF2, CDKN1C, H19) and the methylation status of H19 were also analyzed. Furthermore, embryo size and survival rate were measured. Our study shows that DNMT1 expression was reduced in early placentae from sheep IVP embryos. This reduction was associated with growth arrest and subsequent death of the sheep embryos. Conversely, normal levels of DNMT1 and its cofactors were observed in placentae from IVP embryos that survived this developmental bottleneck. Although DNA methylation machinery was severely compromised in IVP placentae only up to Day 24, the low DNMT1 enzymatic activity that persisted after this stage in IVP placentae was not lethal for the

  6. Combined effects of DNA methyltransferase 1 and 3A polymorphisms and urinary total arsenic levels on the risk for clear cell renal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Shu-Mei [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Huang, Chao-Yuan [Department of Urology, National Taiwan University Hospital, College of Medicine National Taiwan University, Taipei, Taiwan (China); Shiue, Horng-Sheng [Department of Chinese Medicine, Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Pu, Yeong-Shiau [Department of Urology, National Taiwan University Hospital, College of Medicine National Taiwan University, Taipei, Taiwan (China); Hsieh, Yi-Hsun; Chen, Wei-Jen [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Lin, Ying-Chin [Department of Family Medicine, Shung Ho Hospital, Taipei Medical University, Taipei, Taiwan (China); Department of Health Examination, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan (China); Division of Family Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan (China); Hsueh, Yu-Mei, E-mail: ymhsueh@tmu.edu.tw [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China)

    2016-08-15

    Our previous study showed that high urinary total arsenic levels were associated with higher odds ratio (OR) for renal cell carcinoma (RCC). Single nucleotide polymorphisms (SNPs) of DNA methyltransferases (DNMTs) might influence DNMT enzyme activity associated with tumorigenesis. In this study, we investigated the association of five SNPs from DNMT1 (rs8101626 and rs2228611), DNMT3A (rs34048824 and rs1550117), and DNMT3B (rs1569686) with the risk of clear cell renal cell carcinoma (ccRCC). We also examined the combined effects of DNMT genotypes and urinary arsenic levels on ccRCC risk. We conducted a hospital-based case-control study, which included 293 subjects with ccRCC and 293 age- and gender-matched controls. The urinary arsenic species were determined by a high performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. Genotypes were investigated using polymerase chain reaction and restriction fragment length polymorphism analyses. We observed that the DNMT1 rs8101626 G/G genotype was significantly associated with reduced odds ratio (OR) of ccRCC [OR = 0.38, 95% confidence interval (CI) 0.14–0.99]. Subjects with concurrent DNMT1 rs8101626 A/A + A/G and DNMT3A rs34048824 T/T + T/C genotypes had significantly higher OR for ccRCC [OR = 2.88, 95% CI 1.44–5.77]. Participants with the high-risk genotype of DNMT1 rs8101626 and DNMT3A rs34048824 with concurrently high urinary total arsenic levels had even higher OR of ccRCC in a dose-response manner. This is the first study to evaluate variant DNMT1 rs8101626 and DNMT3A rs34048824 genotypes that modify the arsenic-related ccRCC risk in a geographic area without significant arsenic exposure in Taiwan. - Highlights: • High urinary total arsenic level or polymorphism of DNMT1 increased the OR of ccRCC. • High risk genotypes of combination of DNMT1 and DNMT3A increased the OR of ccRCC. • A joint effect of urinary total arsenic level and DNMTs genotypes may affect ccRCC.

  7. Human C6orf211 Encodes Armt1, a Protein Carboxyl Methyltransferase that Targets PCNA and Is Linked to the DNA Damage Response

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    J. Jefferson P. Perry

    2015-03-01

    Full Text Available Recent evidence supports the presence of an L-glutamyl methyltransferase(s in eukaryotic cells, but this enzyme class has been defined only in certain prokaryotic species. Here, we characterize the human C6orf211 gene product as “acidic residue methyltransferase-1” (Armt1, an enzyme that specifically targets proliferating cell nuclear antigen (PCNA in breast cancer cells, predominately methylating glutamate side chains. Armt1 homologs share structural similarities with the SAM-dependent methyltransferases, and negative regulation of activity by automethylation indicates a means for cellular control. Notably, shRNA-based knockdown of Armt1 expression in two breast cancer cell lines altered survival in response to genotoxic stress. Increased sensitivity to UV, adriamycin, and MMS was observed in SK-Br-3 cells, while in contrast, increased resistance to these agents was observed in MCF7 cells. Together, these results lay the foundation for defining the mechanism by which this post-translational modification operates in the DNA damage response (DDR.

  8. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Science.gov (United States)

    Henderson, Morgan L; Kreuzer, Kenneth N

    2015-01-01

    Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  9. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Directory of Open Access Journals (Sweden)

    Morgan L Henderson

    Full Text Available Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs, located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs. Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation: RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA. In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  10. SIRT3 restricts HBV transcription and replication via epigenetic regulation of cccDNA involving SUV39H1 and SETD1A histone methyltransferases.

    Science.gov (United States)

    Ren, Ji-Hua; Hu, Jie-Li; Cheng, Sheng-Tao; Yu, Hai-Bo; Wong, Vincent Kam Wai; Law, Betty Yuen Kwan; Yang, Yong-Feng; Huang, Ying; Liu, Yi; Chen, Wei-Xian; Cai, Xue-Fei; Tang, Hua; Hu, Yuan; Zhang, Wen-Lu; Liu, Xiang; Long, Quan-Xin; Zhou, Li; Tao, Na-Na; Zhou, Hong-Zhong; Yang, Qiu-Xia; Ren, Fang; He, Lin; Gong, Rui; Huang, Ai-Long; Chen, Juan

    2018-04-06

    Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA) which serves as a template for HBV RNA transcription is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified SIRT3 as a host factor restricting HBV transcription and replication by screening seven members of Sirtuin family which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA as well as replicative intermediate DNA in HBV-infected HepG2-NTCP cells and PHH. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. Mechanistic study found nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9 (H3K9). Importantly, occupancy of SIRT3 onto cccDNA could increase the recruitment of histone methyltransferase SUV39H1 to cccDNA and decrease recruitment of SETD1A, leading to a marked increase of H3K9me3 and a decrease of H3K4me3 on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor YY1 to cccDNA. Finally, viral protein HBx could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. SIRT3 is a novel host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase. These data provided a rational for the use of SIRT3 activators in the prevention or treatment of HBV infection. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

  11. TGF-β regulates DNA methyltransferase expression in prostate cancer, correlates with aggressive capabilities, and predicts disease recurrence.

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    Qiang Zhang

    Full Text Available DNA methyltransferase (DNMT is one of the major factors mediating the methylation of cancer related genes such as TGF-β receptors (TβRs. This in turn may result in a loss of sensitivity to physiologic levels of TGF-β in aggressive prostate cancer (CaP. The specific mechanisms of DNMT's role in CaP remain undetermined. In this study, we describe the mechanism of TGF-β-mediated DNMT in CaP and its association with clinical outcomes following radical prostatectomy.We used human CaP cell lines with varying degrees of invasive capability to describe how TGF-β mediates the expression of DNMT in CaP, and its effects on methylation status of TGF-β receptors and the invasive capability of CaP in vitro and in vivo. Furthermore, we determined the association between DNMT expression and clinical outcome after radical prostatectomy. We found that more aggressive CaP cells had significantly higher TGF-β levels, increased expression of DNMT, but reduced TβRs when compared to benign prostate cells and less aggressive prostate cancer cells. Blockade of TGF-β signaling or ERK activation (p-ERK was associated with a dramatic decrease in the expression of DNMT, which results in a coincident increase in the expression of TβRs. Blockade of either TGF-β signaling or DNMT dramatically decreased the invasive capabilities of CaP. Inhibition of TGF-β in an TRAMP-C2 CaP model in C57BL/6 mice using 1D11 was associated with downregulation of DNMTs and p-ERK and impairment in tumor growth. Finally, independent of Gleason grade, increased DNMT1 expression was associated with biochemical recurrence following surgical treatment for prostate cancer.Our findings demonstrate that CaP derived TGF-β may induce the expression of DNMTs in CaP which is associated with methylation of its receptors and the aggressive potential of CaP. In addition, DNMTs is an independent predictor for disease recurrence after prostatectomy, and may have clinical implications for Ca

  12. Polymorphisms of the DNA methyltransferase 1 associated with reduced risks of Helicobacter pylori infection and increased risks of gastric atrophy.

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    Jing Jiang

    Full Text Available INTRODUCTION: DNA methyltransferase-1(DNMT1 is an important enzyme in determining genomic methylation patterns in mammalian cells. We investigated the associations between SNPs in the DNMT1 gene and risks of developing H. pylori seropositivity, gastric atrophy and gastric cancer in the Chinese population. METHODS: The study consisted of 447 patients with gastric cancer; 111 patients with gastric atrophy; and 961 healthy controls. Five SNPs, rs10420321, rs16999593, rs8101866, rs8111085 and rs2288349 of the DNMT1 gene were genotyped. Anti-H.pylori IgG was detected by ELISA. Gastric atrophy was screened by the level of serum pepsinogen Ι and II and then confirmed by endoscopy and histopatholgical examinations. RESULTS: The age- and sex-adjusted OR of H. pylori seropositivity was 0.67 (95%CI: 0.51-0.87 for rs8111085 TC/CC genotypes, significantly lower than the TT genotype in healthy controls. The adjusted OR of H.pylori seropositivity was 0.68 (95%CI: 0.52-0.89 for rs10420321 AG/GG genotypes. In addition, patients carrying rs2228349 AA genotype have a significantly increased risk for H.pylori seropositivity (OR=1.67; 95%CI: 1.02-2.75. Further haplotype analyses also showed that the ATTTG and ATCTA are significantly associated with increased risks in H.pylori infection compared to the GTCCG haplotype (OR=1.38, 95%CI: 1.08-1.77; OR=1.40, 95% CI: 1.09-1.80. The adjusted ORs of gastric atrophy were 1.66 (95%CI: 1.06-2.61 for rs10420321 GG genotype, and 1.67 (95%CI 1.06-2.63, P=0.03 for rs8111085 CC genotype, but no association was found between SNPs in the DNMT1 gene and risk of developing gastric cancer. CONCLUSIONS: Individuals with rs10420321 GG and rs8111085 CC genotype of the DNMT1 gene were associated with reduced risks for H.pylori infection. On the other hand, higher risks of gastric atrophy were found in the carriers with these two genotypes compared to other genotypes. Our results suggested that SNPs of DNMT1 could be used as genotypic

  13. Syntheses of DNA adducts of two heterocyclic amines, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) and identification of DNA adducts in organs from rats dosed with MeA alpha C

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Frandsen, Henrik Lauritz; Pfau, W.

    2004-01-01

    2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (AalphaC) are mutagenic and carcinogenic heterocyclic amines formed during ordinary cooking. MeAalphaC and AalphaC are activated to mutagenic metabolites by cytochrome P450-mediated N-oxidation...... by reaction of the parent amines with acetylated guanine N3-oxide. N-2-OH-MeAalphaC and N-2-OH-AalphaC reacted with calf thymus DNA after addition of acetic anhydride. P-32-postlabelling analysis of modified DNA showed one major adduct co-migrating with N-2-(3',5'-diphospho-2'-deoxyguanosin-8-yl...

  14. Methylation by a unique α-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725.

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    Daehwan Chung

    Full Text Available Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.

  15. The Helicobacter pylori HpyAXII restriction–modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

    Science.gov (United States)

    Humbert, Olivier; Salama, Nina R.

    2008-01-01

    The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model. PMID:18978016

  16. O6-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C

    International Nuclear Information System (INIS)

    Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

    2006-01-01

    Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O 6 -methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC 5 values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N 7 guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism

  17. O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

    Science.gov (United States)

    Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

    2006-03-01

    Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

  18. Molecular mechanisms of adaptive response to alkylating agents in Escherichia coli and some remarks on O(6)-methylguanine DNA-methyltransferase in other organisms.

    Science.gov (United States)

    Kleibl, Karol

    2002-09-01

    Alkylating agents are environmental genotoxic agents with mutagenic and carcinogenic potential, however, their properties are also exploited in the treatment of malignant diseases. O(6)-Methylguanine is an important adduct formed by methylating agents that, if not repaired, can lead to mutations and death. Its repair is carried out by O(6)-methylguanine DNA-methyltransferase (MTase) in an unique reaction in which methyl groups are transferred to the cysteine acceptor site of the protein itself. Exposure of Escherichia coli cells to sublethal concentrations of methylating agents triggers the expression of a set of genes, which allows the cells to tolerate DNA lesions, and this kind of inducible repair is called the adaptive response. The MTase of E. coli, encoded by the ada gene was the first MTase to be discovered and one of best characterised. Its repair and regulatory mechanisms are understood in considerable detail and this bacterial protein played a key role in identification of its counterparts in other living organisms. This review summarises the nature of alkylation damage in DNA and our current knowledge about the adaptive response in E. coli. I also include a brief mention of MTases from other organisms with the emphasis on the human MTase, which could play a crucial role in both cancer prevention and cancer treatment.

  19. Distribution of the type III DNA methyltransferases modA, modB and modD among Neisseria meningitidis genotypes: implications for gene regulation and virulence.

    Science.gov (United States)

    Tan, Aimee; Hill, Dorothea M C; Harrison, Odile B; Srikhanta, Yogitha N; Jennings, Michael P; Maiden, Martin C J; Seib, Kate L

    2016-02-12

    Neisseria meningitidis is a human-specific bacterium that varies in invasive potential. All meningococci are carried in the nasopharynx, and most genotypes are very infrequently associated with invasive meningococcal disease; however, those belonging to the 'hyperinvasive lineages' are more frequently associated with sepsis or meningitis. Genome content is highly conserved between carriage and disease isolates, and differential gene expression has been proposed as a major determinant of the hyperinvasive phenotype. Three phase variable DNA methyltransferases (ModA, ModB and ModD), which mediate epigenetic regulation of distinct phase variable regulons (phasevarions), have been identified in N. meningitidis. Each mod gene has distinct alleles, defined by their Mod DNA recognition domain, and these target and methylate different DNA sequences, thereby regulating distinct gene sets. Here 211 meningococcal carriage and >1,400 disease isolates were surveyed for the distribution of meningococcal mod alleles. While modA11-12 and modB1-2 were found in most isolates, rarer alleles (e.g., modA15, modB4, modD1-6) were specific to particular genotypes as defined by clonal complex. This suggests that phase variable Mod proteins may be associated with distinct phenotypes and hence invasive potential of N. meningitidis strains.

  20. Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density

    DEFF Research Database (Denmark)

    Barres, Romain; Osler, Megan E; Yan, Jie

    2009-01-01

    -CpG nucleotides. Non-CpG methylation was acutely increased in human myotubes by exposure to tumor necrosis factor-alpha (TNF-alpha) or free fatty acids, but not insulin or glucose. Selective silencing of the DNA methyltransferase 3B (DNMT3B), but not DNMT1 or DNMT3A, prevented palmitate-induced non......-CpG methylation of PGC-1alpha and decreased mtDNA and PGC-1alpha mRNA. We provide evidence for PGC-1alpha hypermethylation, concomitant with reduced mitochondrial content in type 2 diabetic patients, and link DNMT3B to the acute fatty-acid-induced non-CpG methylation of PGC-1alpha promoter....

  1. Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase.

    Science.gov (United States)

    Gao, Yuanyuan; Fotovati, Abbas; Lee, Cathy; Wang, Michelle; Cote, Gilbert; Guns, Emma; Toyota, Brian; Faury, Damien; Jabado, Nada; Dunn, Sandra E

    2009-12-01

    Glioblastoma multiforme (GBM) is an aggressive type of brain tumor where 5 years. In adults, GBM is the most common type of brain tumor. It is rarer in children, where it constitutes approximately 15% of all brain tumors diagnosed. These tumors are often invasive, making surgical resection difficult. Further, they can be refractory to current therapies such as temozolomide. The current dogma is that temozolomide resistance rests on the expression of O6-methylguanine-DNA methyltransferase (MGMT) because it cleaves methylated DNA adducts formed by the drug. Our laboratory recently reported that another drug resistance gene known as the Y-box binding protein-1 (YB-1) is highly expressed in primary GBM but not in normal brain tissues based on the evaluation of primary tumors. We therefore questioned whether GBM depend on YB-1 for growth and/or response to temozolomide. Herein, we report that YB-1 inhibition reduced tumor cell invasion and growth in monolayer as well as in soft agar. Moreover, blocking this protein ultimately delayed tumor onset in mice. Importantly, inhibiting YB-1 enhanced temozolomide sensitivity in a manner that was independent of MGMT in models of adult and pediatric GBM. In conclusion, inhibiting YB-1 may be a novel way to improve the treatment of GBM.

  2. DNA methyltransferase expressions in Japanese rice fish (Oryzias latipes) embryogenesis is developmentally regulated and modulated by ethanol and 5-azacytidine

    Science.gov (United States)

    We aimed to investigate the impact of the epigenome in inducting fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish embryogenesis. One of the significant events in epigenome is DNA methylation which is catalyzed by DNA methyl transferase (DNMT) enzymes. We analyzed DNMT enzyme m...

  3. Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

    Science.gov (United States)

    Xiong, Hua; Chen, Zhao-Fei; Liang, Qin-Chuan; Du, Wan; Chen, Hui-Min; Su, Wen-Yu; Chen, Guo-Qiang; Han, Ze-Guang; Fang, Jing-Yuan

    2009-09-01

    DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

  4. Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones.

    Science.gov (United States)

    Jansen, M; Bardenheuer, W; Sorg, U R; Seeber, S; Flasshove, M; Moritz, T

    2001-07-01

    Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

  5. Polysomnographic and neurometabolic features may mark preclinical autosomal dominant cerebellar ataxia, deafness, and narcolepsy due to a mutation in the DNA (cytosine-5-)-methyltransferase gene, DNMT1.

    Science.gov (United States)

    Moghadam, Keivan Kaveh; Pizza, Fabio; Tonon, Caterina; Lodi, Raffaele; Carelli, Valerio; Poli, Francesca; Franceschini, Christian; Barboni, Piero; Seri, Marco; Ferrari, Simona; La Morgia, Chiara; Testa, Claudia; Cornelio, Ferdinando; Liguori, Rocco; Winkelmann, Juliane; Lin, Ling; Mignot, Emmanuel; Plazzi, Giuseppe

    2014-05-01

    We aimed to report the clinical picture of two asymptomatic daughters of a patient with autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN) due to a mutation in the DNA (cytosine-5-)-methyltransferase gene, DNMT1. Clinical assessment based on history, neurologic examination, sleep recordings, neurophysiologic neuroimaging, and genetic tests was performed. History and neurologic examination in both subjects were unremarkable. Genetic analysis disclosed in both the paternally-inherited heterozygous point mutation in the DNMT1 gene. Sleep recordings found sleep-onset rapid eye movement periods (SOREMPs) and proton magnetic resonance spectroscopy (MRS) revealed increased cerebellar myoinositol (mI) in both subjects. Auditory and ophthalmologic investigations as well as structural brain magnetic resonance imaging (MRI) scans revealed no abnormalities. The two asymptomatic carriers of the heterozygous DNMT1 mutation for ADCA-DN, a late-onset neurodegenerative disease, presented with SOREMPs associated with an increase of mI in the brain, a marker of glial cell activity and density characteristic of early stages of neurodegenerative diseases. Therefore, SOREMPs may precede the clinical picture of ADCA-DN as an early polysomnographic marker of central nervous system involvement detected by MRS. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. In vitro induction of O6-methylguanine-DNA methyltransferase in C3H/10T1/2 cells by X-rays is inhibited by nitrogen

    International Nuclear Information System (INIS)

    Hofe, E. von; Kennedy, A.R.

    1988-01-01

    Ionizing radiation is one of the most potent inducers of O 6 -methylguanine-DNA methyltransferase (MT) in rat liver in vivo. In this study we show that MT is readily induced in C3H/10T1/2 cells in culture, which provides a system more amenable to determining the molecular events involved in the induction of this repair enzyme. Maximal induction was observed in logarithmically growing cells 48 h after a dose of 200 rad, similar to the optimal induction time seen in rat liver in vivo. The absolute level of MT observed in C3H/10T1/2 cells which had been at confluence for 24 h was less than in cells in log growth but was still inducible by X-rays, exhibiting an ∼ 2-fold increase over unirradiated cells similar to MT induction in logarithmically growing cells. Irradiating cells under anaerobic conditions abolished MT induction by 100 rad. Cells irradiated with 200 rad under anaerobic conditions exhibited ∼ 70% inhibition of induction compared with aerobically irradiated cells. The possibility that MT may be partially inactivated by interaction with radicals produced by ionizing radiation is discussed. (author)

  7. Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

    Science.gov (United States)

    Tanaka, Satoshi; Oka, Hidehiro; Fujii, Kiyotaka; Watanabe, Kaoru; Nagao, Kumi; Kakimoto, Atsushi

    2005-09-01

    1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

  8. MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

    Directory of Open Access Journals (Sweden)

    Ya-Ling Yang

    2017-01-01

    Full Text Available MicroRNA-29 (miR-29 is found to modulate hepatic stellate cells’ (HSCs activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice and wild-type littermates were subjected to bile duct-ligation (BDL to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

  9. Synthesis, DNA binding and cytotoxic activity of pyrimido[4',5':4,5]thieno(2,3-b)quinoline with 9-hydroxy-4-(3-diethylaminopropylamino) and 8-methoxy-4-(3-diethylaminopropylamino) substitutions.

    Science.gov (United States)

    KiranKumar, Hulihalli N; RohitKumar, Heggodu G; Advirao, Gopal M

    2018-01-01

    Two new derivatives of pyrimido[4',5';4,5]thieno(2,3-b)quinoline (PTQ), 9-hydroxy-4-(3-diethylaminopropylamino)pyrimido[4',5';4,5]thieno(2,3-b)quinoline (Hydroxy-DPTQ) and 8-methoxy-4-(3-diethylaminopropylamino)pyrimido[4',5';4,5]thieno(2,3-b)quinoline (Methoxy-DPTQ) were synthesized and their DNA binding ability was analyzed using spectroscopy (UV-visible, fluorescence and circular dichroism), ethidium bromide dye displacement assay, melting temperature (T m ) analysis and computational docking studies. The hypochromism in UV-visible spectrum and increased fluorescence emission of Hydroxy-DPTQ and Methoxy-DPTQ in the presence of DNA suggested the molecule-DNA interaction. The association constants calculated from UV-visible and spectral titrations were of the order 10 4 to 10 6 M -1 . Circular dichroism studies corroborated the induced conformational changes in DNA upon addition of molecules. The change in the ellipticity was observed both in negative and positive peak of DNA, thus, suggesting the intercalation of molecules. The observed displacement of ethidium bromide from the DNA and increased T m , upon addition of DNA confirmed the intercalative mode of binding. This was further validated by computational docking, which showed clear intercalation of molecules into the d(GpC)-d(CpG) site of the receptor DNA. Anticancer activities of these molecules are evaluated by using MTT assay. Both molecules showed antiproliferative activity against all the three cancer cells studied, with Hydroxy-DPTQ being more potential molecule among the two. IC 50 value of Hydroxy-DPTQ and Methoxy-DPTQ were in the range of 3-5μM and 130-250μM, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct-acting) alkylating agents: breaking a "salt-link".

    Science.gov (United States)

    Oh, H K; Teo, A K; Ali, R B; Lim, A; Ayi, T C; Yarosh, D B; Li, B F

    1996-09-24

    Human O6-methylguanine-DNA methyltransferase (MGMT) repairs DNA by transferring alkyl (R-) adducts from O6-alkylguanine (6RG) in DNA to its own cysteine residue at codon 145 (formation of R-MGMT). We show here that R-MGMT in cell extracts, which is sensitive to protease V8 cleavage at the glutamic acid residues at codons 30 (E30) and 172 (E172), can be specifically immunoprecipitated with an MGMT monoclonal antibody, Mab.3C7. This Mab recognizes an epitope of human MGMT including the lysine 107 (K107) which is within the most basic region that is highly conserved among mammalian MGMTs. Surprisingly, the K107L mutant protein is repair-deficient and readily cleaved by protease V8 similar to R-MGMT. We propose that R-MGMT adopted an altered conformation which exposed the Mab.3C7 epitope and rendered that protein sensitive to protease V8 attack. This proposal could be explained by the disruption of a structural "salt-link" within the molecule based on the available structural and biochemical data. The specific binding of Mab.3C7 to R-MGMT has been compared with the protease V8 method in the detection of R-MGMT in extracts of cells treated with low dosages of methyliodide (SN2) and O6-benzylguanine. Their identical behaviors in producing protease V8 sensitive R-MGMT and Mab.3C7 immunoprecipitates suggest that probably methyl iodide (an ineffective agent in producing 6RG in DNA) can directly alkylate the active site of cellular MGMT similar to O6-benzylguanine. The effectiveness of MeI in producing R-MGMT, i.e., inactivation of cellular MGMT, indicates that this agent can increase the effectiveness of environmental and endogenously produced alkylating carcinogens in producing the mutagenic O6-alkylguanine residues in DNA in vivo.

  11. O6-methylguanine-DNA methyltransferase activity is associated with response to alkylating agent therapy and with MGMT promoter methylation in glioblastoma and anaplastic glioma

    Science.gov (United States)

    Bobola, Michael S.; Alnoor, Mohammad; Chen, John Y.-S.; Kolstoe, Douglas D.; Silbergeld, Daniel L.; Rostomily, Robert C.; Blank, A.; Chamberlain, Marc C.; Silber, John R.

    2014-01-01

    Background CpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. Methods We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). Results Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P ≤ 0.001) and continuous (P ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P ≤ 0.005) and longer PFS (P ≤ 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P ≤ 0.005), and among unmethylated tumors with less than median activity (P ≤ 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs. Conclusions Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas. General significance MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status. PMID:25558448

  12. Is the prognostic significance of O6-methylguanine- DNA methyltransferase promoter methylation equally important in glioblastomas of patients from different continents? A systematic review with meta-analysis.

    Science.gov (United States)

    Meng, Wei; Jiang, Yangyang; Ma, Jie

    2017-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) is an independent predictor of therapeutic response and potential prognosis in patients with glioblastoma multiforme (GBM). However, its significance of clinical prognosis in different continents still needs to be explored. To explore the effects of MGMT promoter methylation on both progression-free survival (PFS) and overall survival (OS) among GBM patients from different continents, a systematic review of published studies was conducted. A total of 5103 patients from 53 studies were involved in the systematic review and the total percentage of MGMT promoter methylation was 45.53%. Of these studies, 16 studies performed univariate analyses and 17 performed multivariate analyses of MGMT promoter methylation on PFS. The pooled hazard ratio (HR) estimated for PFS was 0.55 (95% CI 0.50, 0.60) by univariate analysis and 0.43 (95% CI 0.38, 0.48) by multivariate analysis. The effect of MGMT promoter methylation on OS was explored in 30 studies by univariate analysis and in 30 studies by multivariate analysis. The combined HR was 0.48 (95% CI 0.44, 0.52) and 0.42 (95% CI 0.38, 0.45), respectively. In each subgroup divided by areas, the prognostic significance still remained highly significant. The proportion of methylation in each group was in inverse proportion to the corresponding HR in the univariate and multivariate analyses of PFS. However, from the perspective of OS, compared with data from Europe and the US, higher methylation rates in Asia did not bring better returns.

  13. Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

    Science.gov (United States)

    Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

    2016-06-01

    We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  14. Crystal structure of MboIIA methyltransferase

    OpenAIRE

    Osipiuk, Jerzy; Walsh, Martin A.; Joachimiak, Andrzej

    2003-01-01

    DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-l-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 Å resolution the crystal structure of a β-class DNA MTase MboIIA (M·MboIIA) from the bacterium Moraxella bovis,...

  15. Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions.

    Science.gov (United States)

    Juhas, Mario; Ajioka, James W

    2015-07-01

    The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  16. Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis.

    Science.gov (United States)

    Esteller, M; Risques, R A; Toyota, M; Capella, G; Moreno, V; Peinado, M A; Baylin, S B; Herman, J G

    2001-06-15

    Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.

  17. DNMT1 and DNMT3b silencing sensitizes human hepatoma cells to TRAIL-mediated apoptosis via up-regulation of TRAIL-R2/DR5 and caspase-8.

    Science.gov (United States)

    Kurita, Satoshi; Higuchi, Hajime; Saito, Yoshimasa; Nakamoto, Nobuhiro; Takaishi, Hiromasa; Tada, Shinichiro; Saito, Hidetsugu; Gores, Gregory J; Hibi, Toshifumi

    2010-06-01

    DNA methylation plays a critical role in chromatin remodeling and gene expression. DNA methyltransferases (DNMTs) are hypothesized to mediate cellular DNA methylation status and gene expression during mammalian development and in malignant diseases. In this study, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b in cell proliferation and survival of hepatocellular carcinoma (HCC) cells. Gene silencing of both DNMT1 and DNMT3b by targeted siRNA knockdown reduces cell proliferation and sensitizes the cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death. The proapoptotic protein caspase-8 demonstrated promoter hypermethylation in HCC cells and was up-regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. In addition, death receptor TRAIL-R2/DR5 (TRAIL receptor 2/death receptor 5) did not exhibit promoter hypermethylation in HCC cells but was also up-regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. Consistent with this observation, the combined transfection of DNMT1-siRNA plus DNMT3b-siRNA enhanced formation of the TRAIL-death-inducing signaling complex formation in HCC cells. In conclusion, our data suggest that DNA methylation of specific genomic regions maintained by DNMT1 and DNMT3b plays a critical role in survival of HCC cells, and a simultaneous knockdown of both DNMT1 and DNMT3b may be a novel anticancer strategy for the treatment of HCC.

  18. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

    Science.gov (United States)

    Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

    2000-05-01

    O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

  19. Two siblings with immunodeficiency, facial abnormalities and chromosomal instability without mutation in DNMT3B gene but liability towards malignancy; a new chromatin disorder delineation?

    Directory of Open Access Journals (Sweden)

    Neitzel Heidemarie

    2010-03-01

    Full Text Available Abstract Background ICF syndrome (standing for Immunodeficiency, Centromere instability and Facial anomalies syndrome is a very rare autosomal recessive immune disorder caused by mutations of the gene de novo DNA-methyltransferase 3B (DNMT3B. However, in the literature similar clinical cases without such mutations are reported, as well. Results We report on a family in which the unrelated spouses had two female siblings sharing similar phenotypic features resembling ICF-syndrome, i.e. congenital abnormalities, immunodeficiency, developmental delay and high level of chromosomal instability, including high frequency of centromeric/pericentromeric rearrangements and breaks, chromosomal fragments despiralization or pulverization. However, mutations in DNMT3B could not be detected. Conclusion The discovery of a new so-called 'chromatin disorder' is suggested. Clinical, molecular genetic and cytogenetic characteristics are reported and compared to other 'chromatin disorders'.

  20. Two siblings with immunodeficiency, facial abnormalities and chromosomal instability without mutation in DNMT3B gene but liability towards malignancy; a new chromatin disorder delineation?

    Science.gov (United States)

    Polityko, Anna; Khurs, Olga; Rumyantseva, Natalia; Naumchik, Irina; Kosyakova, Nadezda; Tönnies, Holger; Sperling, Karl; Neitzel, Heidemarie; Weise, Anja; Liehr, Thomas

    2010-03-08

    ICF syndrome (standing for Immunodeficiency, Centromere instability and Facial anomalies syndrome) is a very rare autosomal recessive immune disorder caused by mutations of the gene de novo DNA-methyltransferase 3B (DNMT3B). However, in the literature similar clinical cases without such mutations are reported, as well. We report on a family in which the unrelated spouses had two female siblings sharing similar phenotypic features resembling ICF-syndrome, i.e. congenital abnormalities, immunodeficiency, developmental delay and high level of chromosomal instability, including high frequency of centromeric/pericentromeric rearrangements and breaks, chromosomal fragments despiralization or pulverization. However, mutations in DNMT3B could not be detected. The discovery of a new so-called 'chromatin disorder' is suggested. Clinical, molecular genetic and cytogenetic characteristics are reported and compared to other 'chromatin disorders'.

  1. Mutagenicity, stable DNA adducts, and abasic sites induced in Salmonella by phenanthro[3,4-b]- and phenanthro[4,3-b]thiophenes, sulfur analogs of benzo[c]phenanthrene

    Energy Technology Data Exchange (ETDEWEB)

    Swartz, Carol D. [Department of Environmental Science and Engineering, University of North Carolina, Chapel Hill, NC 27599 (United States); King, Leon C.; Nesnow, Stephen [Environmental Carcinogenesis Division, US Environmental Protection Agency, Research Triangle Park, NC, 27711 (United States); Umbach, David M. [Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC 27709 (United States); Kumar, Subodh [Environmental Toxicology and Chemistry Laboratory, Great Lakes Center, State University of New York College at Buffalo, Buffalo, NY 14222 (United States); DeMarini, David M. [Environmental Carcinogenesis Division, US Environmental Protection Agency, Research Triangle Park, NC, 27711 (United States)], E-mail: demarini.david@epa.gov

    2009-02-10

    Sulfur-containing polycyclic aromatic hydrocarbons (thia-PAHs or thiaarenes) are common constituents of air pollution and cigarette smoke, but only a few have been studied for health effects. We evaluated the mutagenicity in Salmonella TA98, TA100, and TA104 of two sulfur-containing derivatives of benzo[c]phenanthrene, phenanthro[3,4-b]thiophene (P[3,4-b]T), and phenanthro[4,3-b]thiophene (P[4,3-b]T) as well as their dihydrodiol and sulfone derivatives. In addition, we assessed levels of stable DNA adducts (by {sup 32}P-postlabeling) as well as abasic sites (by an aldehydic-site assay) produced by six of these compounds in TA100. P[3,4-b]T and its 6,7- and 8,9-diols, P[3,4-b]T sulfone, P[4,3-b]T, and its 8,9-diol were mutagenic in TA100. P[3,4-b]T sulfone, the most potent mutagen, was approximately twice as potent as benzo[a]pyrene in both TA98 and TA100. Benzo-ring dihydrodiols were much more potent than K-region dihydrodiols, which had little or no mutagenic activity in any strain. P[3,4-b]T sulfone produced abasic sites and not stable DNA adducts; the other five compounds examined, B[c]P, B[c]P 3,4-diol, P[3,4-b]T, P[3,4-b]T 8,9-diol, and P[4,3-b]T 8,9-diol, produced only stable DNA adducts. P[3,4-b]T sulfone was the only compound that produced significant levels of frameshift mutagenicity and induced mutations primarily at GC sites. In contrast, B[c]P, its 3,4-diol, and the 8,9 diols of the phenanthrothiophenes induced mutations primarily at AT sites. P[3,4-b]T was not mutagenic in TA104, whereas P[3,4-b]T sulfone was. The two isomeric forms (P[3,4-b]T and P[4,3-b]T) are apparently activated differently, with the latter, but not the former, involving a diol pathway. This study is the first illustrating the potential importance of abasic sites in the mutagenicity of thia-PAHs.

  2. Reduced Self-Diploidization and Improved Survival of Semi-cloned Mice Produced from Androgenetic Haploid Embryonic Stem Cells through Overexpression of Dnmt3b

    Directory of Open Access Journals (Sweden)

    Wenteng He

    2018-02-01

    Full Text Available Summary: Androgenetic haploid embryonic stem cells (AG-haESCs hold great promise for exploring gene functions and generating gene-edited semi-cloned (SC mice. However, the high incidence of self-diploidization and low efficiency of SC mouse production are major obstacles preventing widespread use of these cells. Moreover, although SC mice generation could be greatly improved by knocking out the differentially methylated regions of two imprinted genes, 50% of the SC mice did not survive into adulthood. Here, we found that the genome-wide DNA methylation level in AG-haESCs is extremely low. Subsequently, downregulation of both de novo methyltransferase Dnmt3b and other methylation-related genes was determined to be responsible for DNA hypomethylation. We further demonstrated that ectopic expression of Dnmt3b in AG-haESCs could effectively improve DNA methylation level, and the high incidence of self-diploidization could be markedly rescued. More importantly, the developmental potential of SC embryos was improved, and most SC mice could survive into adulthood. : Ectopic expression of Dnmt3b could rescue DNA methylation level in repetitive sequences of hypomethylated AG-haESCs, suppress high incidence of self-diploidization, and promote developmental potential of SC embryos, and most SC mice could survive into adulthood. Keywords: androgenetic haploid embryonic stem cells, self-diploidization, semi-cloned mice, DNA methylation, Dnmt3b

  3. Histone methyltransferases in cancer

    DEFF Research Database (Denmark)

    Albert, Mareike; Helin, Kristian

    2009-01-01

    Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic...... regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some...

  4. A DNMT3B alternatively spliced exon and encoded peptide are novel biomarkers of human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Sailesh Gopalakrishna-Pillai

    Full Text Available A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell research. In this study, several genes encoding proteins involved in important signaling pathways were screened to detect alternatively spliced transcripts that exhibited differential expression in pluripotent stem cells (PSCs relative to spontaneously differentiated cells (SDCs. Transcripts containing the alternatively spliced exon 10 of the de novo DNA methyltransferase gene, DNMT3B, were identified that are expressed in PSCs. To demonstrate the utility and superiority of splice variant specific reagents for stem cell research, a peptide encoded by DNMT3B exon 10 was used to generate an antibody, SG1. The SG1 antibody detects a single DNMT3B protein isoform that is expressed only in PSCs but not in SDCs. The SG1 antibody is also demonstrably superior to other antibodies at distinguishing PSCs from SDCs in mixed cultures containing both pluripotent stem cells and partially differentiated derivatives. The tightly controlled down regulation of DNMT3B exon 10 containing transcripts (and exon 10 encoded peptide upon spontaneous differentiation of PSCs suggests that this DNMT3B splice isoform is characteristic of the pluripotent state. Alternatively spliced exons, and the proteins they encode, represent a vast untapped reservoir of novel biomarkers that can be used to develop superior reagents for stem cell research and to gain further insight into mechanisms controlling stem cell pluripotency.

  5. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

    International Nuclear Information System (INIS)

    Brell, Marta; Ibáñez, Javier; Tortosa, Avelina

    2011-01-01

    The DNA repair protein O 6 -Methylguanine-DNA methyltransferase (MGMT) confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP) the most commonly used for promoter methylation study, while immunohistochemistry (IHC) has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. A computer-aided search of MEDLINE (1950-October 2009), EBSCO (1966-October 2009) and EMBASE (1974-October 2009) was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Of 254 studies identified as eligible for full-text review, 52 (20.5%) met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others) was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably

  6. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Ibáñez Javier

    2011-01-01

    Full Text Available Abstract Background The DNA repair protein O6-Methylguanine-DNA methyltransferase (MGMT confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP the most commonly used for promoter methylation study, while immunohistochemistry (IHC has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. Methods A computer-aided search of MEDLINE (1950-October 2009, EBSCO (1966-October 2009 and EMBASE (1974-October 2009 was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Results Of 254 studies identified as eligible for full-text review, 52 (20.5% met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Conclusions Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably.

  7. O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro.

    Science.gov (United States)

    Liu, Y; Egyhazi, S; Hansson, J; Bhide, S V; Kulkarni, P S; Grafström, R C

    1997-10-01

    Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.

  8. Type III restriction endonucleases are heterotrimeric: comprising one helicase–nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Science.gov (United States)

    Butterer, Annika; Pernstich, Christian; Smith, Rachel M.; Sobott, Frank; Szczelkun, Mark D.; Tóth, Júlia

    2014-01-01

    Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism. PMID:24510100

  9. Crystal structure of MboIIA methyltransferase.

    Science.gov (United States)

    Osipiuk, Jerzy; Walsh, Martin A; Joachimiak, Andrzej

    2003-09-15

    DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.

  10. An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

    International Nuclear Information System (INIS)

    Meister, Glenna E.; Chandrasegaran, Srinivasan; Ostermeier, Marc

    2008-01-01

    The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency

  11. DNA methyl transferase (DNMT gene polymorphisms could be a primary event in epigenetic susceptibility to schizophrenia.

    Directory of Open Access Journals (Sweden)

    Koramannil Radha Saradalekshmi

    Full Text Available DNA methylation has been implicated in the etiopathology of various complex disorders. DNA methyltransferases are involved in maintaining and establishing new methylation patterns. The aim of the present study was to investigate the inherent genetic variations within DNA methyltransferase genes in predisposing to susceptibility to schizophrenia. We screened for polymorphisms in DNA methyltransferases, DNMT1, DNMT3A, DNMT3B and DNMT3L in 330 schizophrenia patients and 302 healthy controls for association with Schizophrenia in south Indian population. These polymorphisms were also tested for subgroup analysis with patient's gender, age of onset and family history. DNMT1 rs2114724 (genotype P = .004, allele P = 0.022 and rs2228611 (genotype P = 0.004, allele P = 0.022 were found to be significantly associated at genotypic and allelic level with Schizophrenia in South Indian population. DNMT3B rs2424932 genotype (P = 0.023 and allele (P = 0.0063 increased the risk of developing schizophrenia in males but not in females. DNMT3B rs1569686 (genotype P = 0.027, allele P = 0.033 was found to be associated with early onset of schizophrenia and also with family history and early onset (genotype P = 0.009. DNMT3L rs2070565 (genotype P = 0.007, allele P = 0.0026 confers an increased risk of developing schizophrenia at an early age in individuals with family history. In-silico prediction indicated functional relevance of these SNPs in regulating the gene. These observations might be crucial in addressing and understanding the genetic control of methylation level differences from ethnic viewpoint. Functional significance of genotype variations within the DNMTs indeed suggest that the genetic nature of methyltransferases should be considered while addressing epigenetic events mediated by methylation in Schizophrenia.

  12. Study of the Role of siRNA Mediated Promoter Methylation in DNMT3B Knockdown and Alteration of Promoter Methylation of CDH1, GSTP1 Genes in MDA-MB -453 Cell Line.

    Science.gov (United States)

    Naghitorabi, Mojgan; Mir Mohammad Sadeghi, Hamid; Mohammadi Asl, Javad; Rabbani, Mohammad; Jafarian-Dehkordi, Abbas

    2017-01-01

    Promoter methylation is one of the main epigenetic mechanisms that leads to the inactivation of tumor suppressor genes during carcinogenesis. Due to the reversible nature of DNA methylation, many studies have been performed to correct theses epigenetic defects by inhibiting DNA methyltransferases (DNMTs). In this case novel therapeutics especially siRNA oligonucleotides have been used to specifically knock down the DNMTs at mRNA level. Also many studies have focused on transcriptional gene silencing in mammalian cells via siRNA mediated promoter methylation. The present study was designed to assess the role of siRNA mediated promoter methylation in DNMT3B knockdown and alteration of promoter methylation of Cadherin-1 (CDH1), Glutathione S-Transferase Pi 1(GSTP1), and DNMT3B genes in MDA-MB-453 cell line. MDA-MB-453 cells were transfected with siDNMT targeting DNMT3B promoter and harvested at 24 and 48 h post transfection to monitor gene silencing and promoter methylation respectively. DNMT3B expression was monitored by quantitative RT-PCR method. Promoter methylation was quantitatively evaluated using differential high resolution melting analysis. A non-significant 20% reduction in DNMT3B mRNA level was shown only after first transfection with siDNMT, which was not reproducible. Promoter methylation levels of DNMT3B, CDH1, and GSTP1 were detected at about 15%, 70% and 10% respectively, in the MDA-MB-453 cell line, with no significant change after transfection. Our results indicated that siDNMT sequence were not able to affect promoter methylation and silencing of DNMT3B in MDA-MB-453 cells. However, quantitation of methylation confirmed a hypermethylated phenotype at CDH1 and GSTP1 promoters as well as a differential methylation pattern at DNMT3B promoter in breast cancer.

  13. Time-Qualified Patterns of Variation of PPARγ, DNMT1, and DNMT3B Expression in Pancreatic Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Valerio Pazienza

    2012-01-01

    Full Text Available Carcinogenesis is related to the loss of homeostatic control of cellular processes regulated by transcriptional circuits and epigenetic mechanisms. Among these, the activities of peroxisome proliferator-activated receptors (PPARs and DNA methyltransferases (DNMTs are crucial and intertwined. PPARγ is a key regulator of cell fate, linking nutrient sensing to transcription processes, and its expression oscillates with circadian rhythmicity. Aim of our study was to assess the periodicity of PPARγ and DNMTs in pancreatic cancer (PC. We investigated the time-related patterns of PPARG, DNMT1, and DNMT3B expression monitoring their mRNA levels by qRT-PCR at different time points over a 28-hour span in BxPC-3, CFPAC-1, PANC-1, and MIAPaCa-2 PC cells after synchronization with serum shock. PPARG and DNMT1 expression in PANC-1 cells and PPARG expression in MIAPaCa-2 cells were characterized by a 24 h period oscillation, and a borderline significant rhythm was observed for the PPARG, DNMT1, and DNMT3B expression profiles in the other cell lines. The time-qualified profiles of gene expression showed different shapes and phase relationships in the PC cell lines examined. In conclusion, PPARG and DNMTs expression is characterized by different time-qualified patterns in cell lines derived from human PC, and this heterogeneity could influence cell phenotype and human disease behaviour.

  14. S-phase Specific Downregulation of Human O6-Methylguanine DNA Methyltransferase (MGMT and its Serendipitous Interactions with PCNA and p21cip1 Proteins in Glioma Cells

    Directory of Open Access Journals (Sweden)

    AGM Mostofa

    2018-04-01

    Full Text Available Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21cip1 in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4Cdt2 ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process.

  15. Effect of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine on radiosensitivity of the human lung cancer cells in three-dimensional culture

    International Nuclear Information System (INIS)

    Pan Dong; Xue Gang; Li Xiaoman; Chen Yaxiong; Ren Zhenxin; Du Yarong; Hu Burong

    2014-01-01

    5-Aza-CdR is a specific inhibitor of DNMTs which could suppress tumor growth by demethylation of genomic DNA. There have only few studies thus far concerning it as radiosensitizers in three-dimensional (3D) cells. The principal aim of this study is to evaluate the effects of 5-Aza-CdR on the radiosensitivity of A549 cells in monolayer (2D) and 3D cultures in an attempt to find out a new combination treatments with radiotherapy. The cell proliferation was detected by MTT assay after pretreated with different doses of 5-Aza-CdR for 72 h. A549 cells were treated with or without 5-Aza-CdR (2, 5 μmol/L) for 72 h before be exposed to X-rays of 1, 2, 4, 6 Gy, respectively. The DNA damage was evaluated by micronucleus assay and clonogenic assays. Pretreatment with 5-Aza-CdR inhibited the A549 cell proliferation significantly. More micronucleus were observed after irradiation in 3D cells pretreated with 2 and 5 μmol/L concentration of drug than those without treatment. The survival fractions of cells pretreated by both 2 and 5 μmol/L drug reduced significantly in 3D cultures after irradiation. These significances, however, were found in 2D cells pretreated by only 5 μmol/L drug. Our results suggest that 5-Aza-CdR can inhibit the A549 cells proliferation and apparently enhance the radiosensitivity of cells in 3D cultures. Using of the low dose 5-Aza-CdR in clinical radiotherapy may reduce side effects and enhance effectively the cancer target therapy. (authors)

  16. Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2 in human oral cancer cell line.

    Directory of Open Access Journals (Sweden)

    Daisuke Yamamoto

    2010-09-01

    Full Text Available Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM, which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails.Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2. Protein level of DNA methyltransferase 3B (DNMT3B and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant.OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.

  17. Coffee consumption protects human lymphocytes against oxidative and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) induced DNA-damage: Results of an experimental study with human volunteers

    Czech Academy of Sciences Publication Activity Database

    Bichler, J.; Cavin, C.; Simic, T.; Chakraborty, A.; Ferk, F.; Hoelzl, C.; Schulte-Hermann, R.; Kundi, M.; Haidinger, G.; Angelis, Karel; Knasmüller, S.

    2007-01-01

    Roč. 45, č. 8 (2007), s. 1428-1436 ISSN 0278-6915 Institutional research plan: CEZ:AV0Z50380511 Keywords : coffee * comet assay * DNA-damage Subject RIV: ED - Physiology Impact factor: 2.186, year: 2007

  18. Comparative DNA adduct formation and induction of colonic aberrant crypt foci in mice exposed to 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and azoxymethane

    Science.gov (United States)

    Kim, Sangyub; Guo, Jingshu; O’Sullivan, M. Gerald; Gallaher, Daniel D.; Turesky, Robert J.

    2015-01-01

    Considerable evidence suggests that environmental factors, including diet and cigarette smoke, are involved in the pathogenesis of colon cancer. Carcinogenic nitroso compounds (NOC), such as N-nitrosodimethylamine (NDMA), are present in tobacco and processed red meat, and NOC have been implicated in colon cancer. Azoxymethane (AOM), commonly used for experimental colon carcinogenesis, is an isomer of NDMA, and it produces the same DNA adducts as does NDMA. Heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and high-temperature cooking of meats are also associated with an elevated risk of colon cancer. The most abundant carcinogenic HAA formed in tobacco smoke is 2-amino-9H-pyrido[2,3-b]indole (AαC), whereas 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is the most potent carcinogenic HAA formed during the cooking of meat and fish. However, the comparative tumor-initiating potential of AαC, MeIQ, and AOM is unknown. In this report, we evaluate the formation of DNA adducts as a measure of genotoxicity, and the induction of colonic aberrant crypt foci (ACF) and dysplastic ACF, as an early measure of carcinogenic potency of these compounds in the colon of male A/J mice. Both AαC and AOM induced a greater number of DNA adducts than MeIQ in the liver and colon. AOM induced a greater number of ACF and dysplastic ACF than either AαC or MeIQ. Conversely, based on adduct levels, MeIQ-DNA adducts were more potent than AαC- and AOM-DNA adducts at inducing ACF. Long-term feeding studies are required to relate levels of DNA adducts, induction of ACF, and colon cancer by these colon genotoxicants. PMID:26734915

  19. 18 CFR 3b.5 - Legal guardians.

    Science.gov (United States)

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Legal guardians. 3b.5... INFORMATION General § 3b.5 Legal guardians. For the purposes of this part, the parent of any minor, or the legal guardian of any individual who has been declared to be incompetent due to physical or mental...

  20. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome....... The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact...

  1. Involvement of methyltransferases enzymes during the energy

    African Journals Online (AJOL)

    Mgina

    INVOLVEMENT OF METHYLTRANSFERASES ENZYMES DURING THE. ENERGY METABOLISM OF ..... cell extract still exhibited relatively high methanogenesis with methanol (Fig ... product CH3-CoM into methane (see Fig. 1). The HS-CoM ...

  2. Epigenetic editing of DNA Methylation and DeRewriting DNA Methylation Signatures at Will : The Curable Genome Within Reach?

    NARCIS (Netherlands)

    Stolzenburg, Sabine; Goubert, Désirée; Rots, Marianne

    2016-01-01

    DNA methyltransferases are important enzymes in a broad range of organisms. Dysfunction of DNA methyltransferases in humans leads to many severe diseases, including cancer. This book focuses on the biochemical properties of these enzymes, describing their structures and mechanisms in bacteria,

  3. 18 CFR 3b.223 - Fees.

    Science.gov (United States)

    2010-04-01

    ... INFORMATION Rules for Disclosure of Records § 3b.223 Fees. (a) Fees will be charged for the direct cost of... practicable, self-service duplication of requested documents may also be made on duplicating machines by the... to determine if they fall within the disclosure requirements of this part; and (3) When the system...

  4. [Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis].

    Science.gov (United States)

    Yu, Xiao-Dan; Jiang, Chao; Huang, Lu-Qi; Qin, Shuang-Shuang; Zeng, Xiang-Mei; Chen, Ping; Yuan, Yuan

    2013-08-01

    To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

  5. Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

    Energy Technology Data Exchange (ETDEWEB)

    Duangtum, Natapol [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Junking, Mutita; Sawasdee, Nunghathai [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Cheunsuchon, Boonyarit [Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai, E-mail: limjindaporn@yahoo.com [Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Medical Molecular Biology Unit, Office for Research and Development Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

    2011-09-16

    Highlights: {yields} Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). {yields} The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. {yields} The co-localization between kAE and KIF3B was detected in human kidney tissues. {yields} A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. {yields} KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of {alpha}-intercalated cells of the kidney collecting duct leads to the defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange and the failure of proton (H{sup +}) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney {alpha}-intercalated cells.

  6. Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

    International Nuclear Information System (INIS)

    Duangtum, Natapol; Junking, Mutita; Sawasdee, Nunghathai; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2011-01-01

    Highlights: → Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). → The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. → The co-localization between kAE and KIF3B was detected in human kidney tissues. → A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. → KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl - /HCO 3 - exchange and the failure of proton (H + ) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells.

  7. LAND/R3B DAQ developments

    Energy Technology Data Exchange (ETDEWEB)

    Toernqvist, Hans; Aumann, Thomas; Loeher, Bastian [Technische Universitaet Darmstadt, Darmstadt (Germany); Simon, Haik [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, Darmstadt (Germany); Johansson, Haakan [Chalmers Institute of Technology, Goeteborg (Sweden); Collaboration: R3B-Collaboration

    2015-07-01

    Existing experimental setups aim to exploit most of the improved capabilities and specifications of the upcoming FAIR facility at GSI. Their DAQ designs will require some re-evaluation and upgrades. This presentation summarizes the R3B experimental campaigns in 2014, where the R3B DAQ was subject to testing of several new features that will aid researchers in using larger and more complicated experimental setups in the future. It also acted as part of a small testing ground for the NUSTAR DAQ infrastructure. In order to allow to extract correlations between several experimental sites, new suggested triggering and timestamping implementations were tested over significant distances. Also, with growing experimental complexity comes a greater risk of problems that may be difficult to characterize and solve. To this end, essential remote monitoring and debugging tools have been used successfully.

  8. Chemical Probes of Histone Lysine Methyltransferases

    Science.gov (United States)

    2015-01-01

    Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

  9. Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

    Science.gov (United States)

    We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

  10. APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Manikandan Periyasamy

    2015-10-01

    Full Text Available Estrogen receptor α (ERα is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER and to repair by non-homologous end-joining (NHEJ pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.

  11. R3B time projection chamber

    Directory of Open Access Journals (Sweden)

    Legou P.

    2012-07-01

    Full Text Available Within the international FAIR project, the R3B collaboration (Reaction studies with Radioactive Relativistic beams will be in charge of the physics program with secondary beams of energy between 200 and 1500MeV per nucleon. Central to the R3B set-up will be a large-aperture superconducting magnet under construction at CEA-Saclay. An international collaboration has been formed to work on the design of a large time-projection chamber (TPC to be installed behind this magnet to cover the full phase-space of the charged fragments produced in the target. Within this collaboration, tests of detector prototypes were performed with the heavy-ions beams at GSI Darmstadt. These prototypes were equipped with a gaseous Micromegas detector. Two amplification technologies were tested, either conventional or resistive Micromegas and two construction concepts, bulk-Micromegas or micro-meshes screwed on the PCB. We will present the results of the last beam and the 1m TPC station which will be dedicated to make tests using the real drift distance and compare the performances of micromegas and GEM.

  12. Flavivirus methyltransferase as target for virus treatment

    Czech Academy of Sciences Publication Activity Database

    Krafčíková, Petra; Chalupská, Dominika; Hercík, Kamil; Nencka, Radim; Bouřa, Evžen

    2017-01-01

    Roč. 284, Suppl 1 (2017), s. 216-217 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : flavivirus methyltransferase * antivirals Subject RIV: CE - Biochemistry

  13. The Role of Semaphorin 3B (SEMA3B) in the Pathogenesis of Breast Cancer

    Science.gov (United States)

    2006-04-01

    apoptotic and anti-proliferative effect on cancer lines it is in part by the inhibition of Akt pathway. In conclusion, we hypothesize that VEGF165...autocrine activity and by inhibiting the Akt pathway. 15. SUBJECT TERMS tumor suppressor gene, breast cancer and apoptosis 16. SECURITY...TGFβ TGFR2 Smad4 M D A M B A 54 9 H 12 99 H el a H 46 0 M C F7 ZR -7 5 H 15 7 2 31 GAPDH TGFR1 B. C 2H 24H 48H 72H SEMA3B SEMA3B

  14. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    International Nuclear Information System (INIS)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P.

    1991-01-01

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A) + RNA

  15. Nicotinamide -Methyltransferase in Health and Cancer

    Directory of Open Access Journals (Sweden)

    David B Ramsden

    2017-06-01

    Full Text Available Over the past decade, the roles of nicotinamide N -methyltransferase and its product 1-methyl nicotinamide have emerged from playing merely minor roles in phase 2 xenobiotic metabolism as actors in some of the most important scenes of human life. In this review, the structures of the gene, messenger RNA, and protein are discussed, together with the role of the enzyme in many of the common cancers that afflict people today.

  16. The dynamics of DNA methylation and hydroxymethylation during amelogenesis.

    Science.gov (United States)

    Yoshioka, Hirotaka; Minamizaki, Tomoko; Yoshiko, Yuji

    2015-11-01

    Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear. Here, we have uncovered the spatial distributions of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) to determine epigenetic events in the mandibular incisors of mice. Immunohistochemistry and dot blotting showed that 5-hmC in ameloblasts increased from the secretory stage to the later maturation stage. We also demonstrated the distribution of 5-mC-positive ameloblasts with punctate nuclear labeling from sometime after the initiation of the secretory stage to the later maturation stage; however, dot blotting failed to detect this change. No obvious alteration of 5-mC/5-hmC staining in odontoblasts and dental pulp cells was observed. Concomitant with quantitative expression data, immunohistochemistry showed that maintenance DNA methyltransferase DNMT1 was highly expressed in immature dental epithelial cells and subsequently decreased at later stages of development. Meanwhile, de novo DNA methyltransferase Dnmt3a and Dnmt3b and DNA demethylase Tet family genes were universally expressed, except Tet1 that was highly expressed in immature dental epithelial cells. Thus, DNMT1 may sustain the undifferentiated status of dental epithelial cells through the maintenance of DNA methylation, while the hydroxylation of 5-mC may occur through the whole differentiation process by TET activity. Taken together, these data indicate that the dynamic changes of 5-mC and 5-hmC may be critical for the regulation of amelogenesis.

  17. Structural Chemistry of Human RNA Methyltransferases.

    Science.gov (United States)

    Schapira, Matthieu

    2016-03-18

    RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

  18. Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

    Science.gov (United States)

    Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

    2000-12-01

    Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

  19. Methyltransferases mediate cell memory of a genotoxic insult.

    Science.gov (United States)

    Rugo, R E; Mutamba, J T; Mohan, K N; Yee, T; Chaillet, J R; Greenberger, J S; Engelward, B P

    2011-02-10

    Characterization of the direct effects of DNA-damaging agents shows how DNA lesions lead to specific mutations. Yet, serum from Hiroshima survivors, Chernobyl liquidators and radiotherapy patients can induce a clastogenic effect on naive cells, showing indirect induction of genomic instability that persists years after exposure. Such indirect effects are not restricted to ionizing radiation, as chemical genotoxins also induce heritable and transmissible genomic instability phenotypes. Although such indirect induction of genomic instability is well described, the underlying mechanism has remained enigmatic. Here, we show that mouse embryonic stem cells exposed to γ-radiation bear the effects of the insult for weeks. Specifically, conditioned media from the progeny of exposed cells can induce DNA damage and homologous recombination in naive cells. Notably, cells exposed to conditioned media also elicit a genome-destabilizing effect on their neighbouring cells, thus demonstrating transmission of genomic instability. Moreover, we show that the underlying basis for the memory of an insult is completely dependent on two of the major DNA cytosine methyltransferases, Dnmt1 and Dnmt3a. Targeted disruption of these genes in exposed cells completely eliminates transmission of genomic instability. Furthermore, transient inactivation of Dnmt1, using a tet-suppressible allele, clears the memory of the insult, thus protecting neighbouring cells from indirect induction of genomic instability. We have thus demonstrated that a single exposure can lead to long-term, genome-destabilizing effects that spread from cell to cell, and we provide a specific molecular mechanism for these persistent bystander effects. Collectively, our results impact the current understanding of risks from toxin exposures and suggest modes of intervention for suppressing genomic instability in people exposed to carcinogenic genotoxins.

  20. In-silico investigations into natural products as nonnucleoside DNA ...

    African Journals Online (AJOL)

    nucleoside DNA methyltransferase 1 (DNMT1) inhibitor of epimutation in gastric cancer. Methods: A dataset of reported non-nucleoside DNMT1 inhibitors was used to target the active site of crystallized DNMT1 protein. Molecular docking simulations ...

  1. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

    Science.gov (United States)

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. nucleoside DNA methyltransferase 1 inhibitors for treating epi

    African Journals Online (AJOL)

    Li et al. Trop J Pharm Res, February 2017; 16(2): 445. Tropical Journal of ... 1Department of Radiotherapy, Hebei Cixian Cancer Hospital, Cixian 056500, Hebei Province, 2Handan City Kang Ye Biological ..... Verstovsek S, Talpaz M, Garcia-Manero G, Kantarjian. HM. ... Medina-Franco JL, López-Vallejo F, Kuck D, Lyko F.

  3. Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8

    Energy Technology Data Exchange (ETDEWEB)

    Judge, Russell A.; Zhu, Haizhong; Upadhyay, Anup K.; Bodelle, Pierre M.; Hutchins, Charles W.; Torrent, Maricel; Marin, Violeta L.; Yu, Wenyu; Vedadi, Masoud; Li, Fengling; Brown, Peter J.; Pappano, William N.; Sun, Chaohong; Petros, Andrew M.

    2016-12-08

    SETD8 is a histone H4–K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent (Ki 50 nM, IC50 0.33 μM) and selective norleucine containing peptide inhibitor has been obtained.

  4. Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies

    International Nuclear Information System (INIS)

    Lerch, Thomas F.; Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.

    2009-01-01

    Crystals of adeno-associated virus serotype 3b, a human DNA virus with promise as a vector for gene therapy, have been grown, diffract X-rays to ∼2.6 Å resolution and are suitable for structure determination in spite of twinning. Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication

  5. Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

    DEFF Research Database (Denmark)

    Atkinson, Gemma C; Hansen, Lykke H; Tenson, Tanel

    2013-01-01

    The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the Rlm......N methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine....... The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed....

  6. 18 CFR 3b.226 - Accounting of disclosures.

    Science.gov (United States)

    2010-04-01

    ... accounting of disclosures is not a system of records under the definition in § 3b.2(e) and no accounting will... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Accounting of... IDENTIFIABLE PERSONAL INFORMATION Rules for Disclosure of Records § 3b.226 Accounting of disclosures. (a) The...

  7. Development and improvement for MCNP-3B interactive plotter

    International Nuclear Information System (INIS)

    Gao Yanfeng

    1996-01-01

    The author briefly explains the development and improvement for the MCNP-3B interactive plotter. It describes the functions of geometry visualization and tally result plot, and introduces the progresses in user interface, process display and surface matching. The construction of MCNP-3B/PC is given

  8. Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

    Science.gov (United States)

    Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang; Jian, Yap Li; Chao, Alexander Theodore; Lescar, Julien; Yin, Zheng; Vedananda, T. R.; Keller, Thomas H.; Shi, Pei-Yong

    2011-01-01

    Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome. PMID:21147775

  9. Monolignol 4-O-methyltransferases and uses thereof

    Science.gov (United States)

    Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

    2014-11-18

    Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

  10. Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

    OpenAIRE

    Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang

    2010-01-01

    Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crysta...

  11. Magnetic and Moessbauer studies on GdCo3B2 and DyCo3B2

    International Nuclear Information System (INIS)

    Malik, S.K.; Umarji, A.M.; Shenoy, G.K.

    1984-10-01

    Magnetization and Moessbauer studies have been carried out on GdCo 3 B 2 and DyCo 3 B 2 . These compounds are magnetically ordered with Curie temperatures of 56 0 and 21 0 K respectively. The Co atoms are either nonmagnetic or carry a small moment in these compounds. The saturation moment of DyCo 3 B 2 at 5 0 K is smaller than the Dy 3+ free-ion value. From 161 Dy Moessbauer studies, the measured hyperfine magnetic field at the Dy site is also observed to be smaller than the free-ion value. 155 Gd Moessbauer measurements in GdCo 3 B 2 reveal the presence of large crystalline electric fields at the rare earth site. This causes the moment and the hyperfine field at the Dy site in DyCo 3 B 2 to be reduced from its free-ion value

  12. Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

    Science.gov (United States)

    DeVry, C G; Tsai, W; Clarke, S

    1996-11-15

    The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

  13. Molecular characterization of a KIF3B-like kinesin gene in the testis of Octopus tankahkeei (Cephalopoda, Octopus).

    Science.gov (United States)

    Dang, Ran; Zhu, Jun-Quan; Tan, Fu-Qing; Wang, Wei; Zhou, Hong; Yang, Wan-Xi

    2012-05-01

    KIF3B is known for maintaining and assembling cilia and flagellum. To date, the function of KIF3B and its relationship with KIF3A during spermiogenesis in the cephalopod Octopus tankahkeei remains unknown. In the present study, we characterized a gene encoding a homologue of rat KIF3B in the O. tankahkeei testis and examined its temporal and spatial expression pattern during spermiogenesis. The cDNA of KIF3B was obtained with degenerate and RACE PCR and the distribution pattern of ot-kif3b were observed with RT-PCR. The morphological development during spermiogenesis was illustrated by histological and transmission electron microscopy and mRNA expression of ot-kif3b was observed by in situ hybridization. The 2,365 nucleotides cDNA consisted of a 102 bp 5' untranslated region (UTR), a 2,208 bp open reading frame (ORF) encoding a protein of 736 amino acids, and a 55 bp 3' UTR. Multiple alignments revealed that the putative Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with that of Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively, along with high identities with Ot-KIF3A in fundamental structures. Ot-kif3b transcripts appeared gradually in early spermatids, increased in intermediate spermatids and maximized in drastically remodeled and final spermatids. The kif3b gene is identified and its expression pattern is demonstrated for the first time in O. tankahkeei. Compared to ot-kif3a reported by our laboratory before, our data suggested that the putative heterodimeric motor proteins Ot-KIF3A/B may be involved in intraspermatic transport and might contribute to structural changes during spermiogenesis.

  14. A pressure study of CePt{sub 3}B

    Energy Technology Data Exchange (ETDEWEB)

    Rauch, Daniela; Suellow, Stefan [Institute of Condensed Matter Physics, University of Technology Braunschweig, Braunschweig (Germany); Hartwig, Steffen [Institute of Condensed Matter Physics, University of Technology Braunschweig, Braunschweig (Germany); BENSC, Helmholtz Zentrum Berlin, Berlin (Germany); Hidaka, Hiroyuki; Yamazaki, Seigo; Amitsuka, Hiroshi [Department of Physics, Hokkaido University, Sapporo (Japan); Bauer, Ernst [Institute of Solid State Physics, Vienna University of Technology, Vienna (Austria)

    2013-07-01

    CePt{sub 3}B is isostructural to the non-centro symmetric heavy-fermion superconductor CePt{sub 3}Si. In contrast to the latter system, CePt{sub 3}B exhibits a complex magnetically ordered state at low temperatures, with an antiferromagnetic phase below T{sub N}=7.8 K and a weakly ferromagnetic transition below T{sub C}∼5 K. CePt{sub 3}B can be understand as a low pressure variant of CePt{sub 3}Si. Here we report a study of CePt{sub 3}B by means of high pressure magnetization measurements, this way in particular accessing the pressure evolution of the ferromagnetic transition temperature T{sub C}. From our investigation up to about 40 kbar we observe an almost constant transition temperature T{sub C} with pressure. This behavior we discuss in the context of alloying studies on this material.

  15. Histone H3 lysine 36 methyltransferase mobilizes NER factors to regulate tolerance against alkylation damage in fission yeast.

    Science.gov (United States)

    Lim, Kim Kiat; Nguyen, Thi Thuy Trang; Li, Adelicia Yongling; Yeo, Yee Phan; Chen, Ee Sin

    2018-04-09

    The Set2 methyltransferase and its target, histone H3 lysine 36 (H3K36), affect chromatin architecture during the transcription and repair of DNA double-stranded breaks. Set2 also confers resistance against the alkylating agent, methyl methanesulfonate (MMS), through an unknown mechanism. Here, we show that Schizosaccharomyces pombe (S. pombe) exhibit MMS hypersensitivity when expressing a set2 mutant lacking the catalytic histone methyltransferase domain or a H3K36R mutant (reminiscent of a set2-null mutant). Set2 acts synergistically with base excision repair factors but epistatically with nucleotide excision repair (NER) factors, and determines the timely nuclear accumulation of the NER initiator, Rhp23, in response to MMS. Set2 facilitates Rhp23 recruitment to chromatin at the brc1+ locus, presumably to repair alkylating damage and regulate the expression of brc1+ in response to MMS. Set2 also show epistasis with DNA damage checkpoint proteins; regulates the activation of Chk1, a DNA damage response effector kinase; and acts in a similar functional group as proteins involved in homologous recombination. Consistently, Set2 and H3K36 ensure the dynamicity of Rhp54 in DNA repair foci formation after MMS treatment. Overall, our results indicate a novel role for Set2/H3K36me in coordinating the recruitment of DNA repair machineries to timely manage alkylating damage.

  16. The histone H3 lysine 9 methyltransferase DIM-5 modifies chromatin at frequency and represses light-activated gene expression.

    Science.gov (United States)

    Ruesch, Catherine E; Ramakrishnan, Mukund; Park, Jinhee; Li, Na; Chong, Hin S; Zaman, Riasat; Joska, Tammy M; Belden, William J

    2014-11-25

    The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output. Copyright © 2015 Ruesch et al.

  17. Epigenetic modulation upon exposure of lung fibroblasts to TiO2 and ZnO nanoparticles: alterations in DNA methylation

    Directory of Open Access Journals (Sweden)

    Patil NA

    2016-09-01

    Full Text Available Nayana A Patil,1,2 WN Gade,2 Deepti D Deobagkar1 1Department of Zoology, Molecular Biology Research Laboratory, Centre of Advanced Studies, 2Department of Biotechnology, Proteomic Research Laboratory, Savitribai Phule Pune University, Pune, India Abstract: Titanium dioxide (TiO2 and zinc oxide (ZnO nanoparticles (NPs are promising candidates for numerous applications in consumer products. This will lead to increased human exposure, thus posing a threat to human health. Both these types of NPs have been studied for their cell toxicity, immunotoxicity, and genotoxicity. However, effects of these NPs on epigenetic modulations have not been studied. Epigenetics is an important link in the genotype and phenotype modulation and misregulation can often lead to lifestyle diseases. In this study, we have evaluated the DNA methylation-based epigenetic changes upon exposure to various concentrations of NPs. The investigation was designed to evaluate global DNA methylation, estimating the corresponding methyltransferase activity and expression of Dnmt gene using lung fibroblast (MRC5 cell line as lungs are the primary route of entry and target of occupational exposure to TiO2 and ZnO NPs. Enzyme-linked immunosorbent assay-based immunochemical assay revealed dose-related decrease in global DNA methylation and DNA methyltransferase activity. We also found direct correlation between the concentration of NPs, global methylation levels, and expression levels of Dnmt1, 3A, and 3B genes upon exposure. This is the first study to investigate effect of exposure to TiO2 and ZnO on DNA methylation levels in MRC5 cells. Epigenetic processes are known to play an important role in reprogramming and adaptation ability of an organism and can have long-term consequences. We suggest that changes in DNA methylation can serve as good biomarkers for early exposure to NPs since they occur at concentrations well below the sublethal levels. Our results demonstrate a clear

  18. Cross-talk between the NR3B and NR4A families of orphan nuclear receptors

    International Nuclear Information System (INIS)

    Lammi, Johanna; Rajalin, Ann-Marie; Huppunen, Johanna; Aarnisalo, Piia

    2007-01-01

    Estrogen-related receptors (NR3B family) and Nurr1, NGFI-B, and Nor1 (NR4A family) are orphan nuclear receptors lacking identified natural ligands. The mechanisms regulating their transcriptional activities have remained elusive. We have previously observed that the members of NR3B and NR4A families are coexpressed in certain cell types such as osteoblasts and that the ability of Nurr1 to transactivate the osteopontin promoter is repressed by ERRs. We have now studied the cross-talk between NR3B and NR4A receptors. We show that NR3B and NR4A receptors mutually repress each others' transcriptional activity. The repression involves intact DNA-binding domains and dimerization interfaces but does not result from competition for DNA binding or from heterodimerization. The activation functions of NR3B and NR4A receptors are dispensable for the cross-talk. In conclusion, we report that cross-talk between NR3B and NR4A receptors is a mechanism modulating the transcriptional activities of these orphan nuclear receptors

  19. Stable knockdown of PASG enhances DNA demethylation but does not accelerate cellular senescence in TIG-7 human fibroblasts.

    Science.gov (United States)

    Suzuki, Toshikazu; Farrar, Jason E; Yegnasubramanian, Srinivasan; Zahed, Muhammed; Suzuki, Nobuo; Arceci, Robert J

    2008-09-01

    Demethylation of 5-methylcytosine in genomic DNA is believed to be one of the mechanisms underlying replicative life-span of mammalian cells. Both proliferation associated SNF2-like gene (PASG, also termed Lsh) and DNA methyltransferase 3B (Dnmt3b) knockout mice result in embryonic genomic hypomethylation and a replicative senescent phenotype. However, it is unclear whether gradual demethylation of DNA during somatic cell division is directly involved in senescence. In this study, we retrovirally transduced TIG-7 human fibroblasts with a shRNA against PASG and compared the rate of change in DNA methylation as well as the replicative life-span to control cells under low (3%) and ambient (20%) oxygen. Expression of PASG protein was decreased by approximately 80% compared to control cells following transduction of PASG shRNA gene. The rate of cell growth was the same in both control and PASG-suppressed cells. The rate of demethylation of DNA was significantly increased in PASG-suppressed cells as compared control cells. However, decreased PASG expression did not shorten the replicative life-span of TIG-7 cells. Culture under low oxygen extended the life-span of TIG-7 cells but did not alter the rate of DNA demethylation. While knockout of PASG during development results in genomic hypomethylation and premature senescence, our results show that while downregulation of PASG expression in a somatic cell also leads to DNA hypomethylation, there is no associated senescent phenotype. These results suggest differences in cellular consequences of hypomethylation mediated by PASG during development compared to that in somatic cells.

  20. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

    Energy Technology Data Exchange (ETDEWEB)

    Haruta, Mayumi [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Nishiyama, Atsuya; Johmura, Yoshikazu [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Le Tallec, Benoît; Debatisse, Michelle [Institut Curie, Centre de Recherche, 26 rue d’Ulm, CNRS UMR 3244, 75248 ParisCedex 05 (France); Nakanishi, Makoto, E-mail: mkt-naka@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

  1. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

    International Nuclear Information System (INIS)

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-01

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

  2. Involvement of DNA methylation in memory processing in the honey bee.

    Science.gov (United States)

    Lockett, Gabrielle A; Helliwell, Paul; Maleszka, Ryszard

    2010-08-23

    DNA methylation, an important and evolutionarily conserved epigenetic mechanism, is implicated in learning and memory processes in vertebrates, but its role in behaviour in invertebrates is unknown. We examined the role of DNA methylation in memory in the honey bee using an appetitive Pavlovian olfactory discrimination task, and by assessing the expression of DNA methyltransferase3, a key driver of epigenetic reprogramming. Here we report that DNA methyltransferase inhibition reduces acquisition retention and alters the extinction depending on treatment time, and DNA methyltransferase3 is upregulated after training. Our findings add to the understanding of epigenetic mechanisms in learning and memory, extending known roles of DNA methylation to appetitive and extinction memory, and for the first time implicate DNA methylation in memory in invertebrates.

  3. Radiographic findings in type 3 b Gaucher disease

    International Nuclear Information System (INIS)

    Hill, S.C.; Damaska, B.M.; Tsokos, M.; Kreps, C.; Brady, R.O.; Barton, N.W.

    1996-01-01

    The purpose of this paper is to describe the radiographic findings in type 3 b Gaucher disease, a chronic neuronopathic form of the illness with severe systemic manifestations. Between 1980 and 1985 17 consecutive patients were evaluated with radiography of the chest, long bones and spine, CT of the head and chest, abdominal sonography, and MRI of the head, abdomen and spine. Clinical manifestations were severe, and led to death from hepatic, pulmonary or cardiac failure in nine patients. Type 3 b Gaucher disease shares the same spectrum of radiographic findings observed in type 1 disease, but the systemic manifestations are more severe. Pulmonary infiltrates, thoracic lymph node enlargement, vertebral compression fractures and osteonecrosis of the long bones occur much more frequently in patients with type 3 b disease. (orig.). With 7 figs., 2 tabs

  4. Crystal structure of inactive form of Rab3B

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wei [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China); Shen, Yang [Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, Ontario, Canada M5G 1L7 (Canada); Jiao, Ronghong [Department of Function Inspection, Hebei Provincial People' s Hospital, Shijiazhuang 050051 (China); Liu, Yanli; Deng, Lingfu [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China); Qi, Chao, E-mail: qichao@mail.ccnu.edu.cn [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer This is the first structural information of human Rab3B. Black-Right-Pointing-Pointer To provides a structural basis for the GDP/GTP switch in controlling the activity of Rab3. Black-Right-Pointing-Pointer The charge distribution of Rab3B indicates its unique roles in vesicular trafficking. -- Abstract: Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 A resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

  5. Crystal structure of inactive form of Rab3B

    International Nuclear Information System (INIS)

    Zhang, Wei; Shen, Yang; Jiao, Ronghong; Liu, Yanli; Deng, Lingfu; Qi, Chao

    2012-01-01

    Highlights: ► This is the first structural information of human Rab3B. ► To provides a structural basis for the GDP/GTP switch in controlling the activity of Rab3. ► The charge distribution of Rab3B indicates its unique roles in vesicular trafficking. -- Abstract: Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

  6. Identification and characterization of new molecular partners for the protein arginine methyltransferase 6 (PRMT6.

    Directory of Open Access Journals (Sweden)

    Alessandra Lo Sardo

    Full Text Available PRMT6 is a protein arginine methyltransferase that has been implicated in transcriptional regulation, DNA repair, and human immunodeficiency virus pathogenesis. Only few substrates of this enzyme are known and therefore its cellular role is not well understood. To identify in an unbiased manner substrates and potential regulators of PRMT6 we have used a yeast two-hybrid approach. We identified 36 new putative partners for PRMT6 and we validated the interaction in vivo for 7 of them. In addition, using invitro methylation assay we identified 4 new substrates for PRMT6, extending the involvement of this enzyme to other cellular processes beyond its well-established role in gene expression regulation. Holistic approaches create molecular connections that allow to test functional hypotheses. The assembly of PRMT6 protein network allowed us to formulate functional hypotheses which led to the discovery of new molecular partners for the architectural transcription factor HMGA1a, a known substrate for PRMT6, and to provide evidences for a modulatory role of HMGA1a on the methyltransferase activity of PRMT6.

  7. Detector production for the R3B Si-tracker

    Energy Technology Data Exchange (ETDEWEB)

    Borri, M., E-mail: marcello.borri@liverpool.ac.uk [University of Liverpool, Department of Physics, Oxford Street, Liverpool L69 7ZE (United Kingdom); Lemmon, R. [STFC Daresbury Laboratory, Daresbury, Warrington WA4 4 CE (United Kingdom); Thornhill, J.; Bate, R.; Chartier, M. [University of Liverpool, Department of Physics, Oxford Street, Liverpool L69 7ZE (United Kingdom); Clague, N. [STFC Daresbury Laboratory, Daresbury, Warrington WA4 4 CE (United Kingdom); Herzberg, R.-D. [University of Liverpool, Department of Physics, Oxford Street, Liverpool L69 7ZE (United Kingdom); Labiche, M. [STFC Daresbury Laboratory, Daresbury, Warrington WA4 4 CE (United Kingdom); Lindsay, S.; Nolan, P.; Pearce, F.; Powell, W.; Wells, D. [University of Liverpool, Department of Physics, Oxford Street, Liverpool L69 7ZE (United Kingdom)

    2016-11-11

    R3B is a fixed target experiment which will study reactions with relativistic radioactive beams at FAIR. Its Si-tracker will surround the target volume and it will detect light charged-particles like protons. The detector technology in use consists of double-sided silicon strip sensors wire bonded to the custom made R3B-ASIC. The tracker allows for a maximum of two outer layers and one inner layer. This paper reports on the production of detectors necessary to build the minimum tracking configuration: one inner layer and one outer layer.

  8. Protein Arginine Methyltransferase 5 Inhibition Upregulates Foxp3+ Regulatory T Cells Frequency and Function during the Ulcerative Colitis

    Directory of Open Access Journals (Sweden)

    Yingxia Zheng

    2017-05-01

    Full Text Available Ulcerative colitis (UC pathogenesis is related to imbalance of immune responses, and the equilibrium between inflammatory T cells and Foxp3+ regulatory T cells (Tregs plays an important role in the intestinal homeostasis. Protein arginine methyltransferases (PRMTs regulate chromatin remodeling and gene expression. Here, we investigated whether inhibition of PRMTs affects colitis pathogenesis in mice and inflammatory bowel disease patients and further explored the underlying mechanisms. In this study, we found that protein arginine N-methyltransferase inhibitor 1 (AMI-1 treatments increased Tregs frequency, function, and reduced colitis incidence. Adoptive transfer of AMI-1-treated Tregs could reduce the colitis incidence. Colitis was associated with increased local PRMT5 expression, which was inhibited by AMI-1 treatment. Additionally, PRMT5 knockdown T cells produced a better response to TGFβ and promoted Tregs differentiation through decreased DNA methyltransferase 1 (DNMT1 expression. PRMT5 also enhanced H3K27me3 and DNMT1 binding to Foxp3 promoter, which restricted Tregs differentiation. Furthermore, PRMT5 knockdown led to decreased Foxp3 promoter methylation during Tregs induction. PRMT5 expression had a negative relationship with Tregs in UC patients, knockdown of PRMT5 expression increased Tregs frequency and decreased TNFα, IL-6, and IL-13 levels. Our study outlines a novel regulation of PRMT5 on Tregs development and function. Strategies to decrease PRMT5 expression might have therapeutic potential to control UC.

  9. Cerebellar oxidative DNA damage and altered DNA methylation in the BTBR T+tf/J mouse model of autism and similarities with human post mortem cerebellum.

    Directory of Open Access Journals (Sweden)

    Svitlana Shpyleva

    Full Text Available The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG, 5-methylcytosine (5mC, and 5-hydroxymethylcytosine (5hmC. The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1 and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b. Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1 and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism.

  10. Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression

    NARCIS (Netherlands)

    van der Wijst, Monique G. P.; van Tilburg, Amanda Y.; Ruiters, Marcel H. J.; Rots, Marianne G.

    2017-01-01

    Like the nucleus, mitochondria contain their own DNA and recent reports provide accumulating evidence that also the mitochondrial DNA (mtDNA) is subjective to DNA methylation. This evidence includes the demonstration of mitochondria-localised DNA methyltransferases and demethylases, and the

  11. The in-beam tracking detectors for R3B

    Energy Technology Data Exchange (ETDEWEB)

    Paschalis, Stefanos; Johansen, Jacob; Scheit, Heiko [Institut fuer Kernphysik, Technische Universitaet, D 64289 Darmstadt (Germany); Heil, Michael [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Aumann, Thomas [Institut fuer Kernphysik, Technische Universitaet, D 64289 Darmstadt (Germany); GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Krivshich, Anatoly [PNPI St. Petersburg, 188300 Gatchina (Russian Federation); Collaboration: R3B-Collaboration

    2015-07-01

    The R3B experiment is part of the NUSTAR pillar at FAIR. One of the great strengths of the R3B experiment is the kinematically complete measurement of reactions with exotic ions with energies of up to 1 AGeV. Key components of the R3B experiment are the neutron detector NeuLAND, the γ and charge-particle calorimeter CALIFA, the Si Tracker and the in-beam tracking detectors. A cornerstone instrument of the setup is the new dipole magnet (GLAD) which bends and momentum analyses the high-rigidity beams. A precise tracking of the charged particles through the magnetic field is crucial to resolve the masses of heavy ions and measure the momentum of the fragments with high resolution. In this contribution we present the technical design details of the in-beam tracking detectors that will be used in the R3B experiment together with recent results obtained from in-beam prototype testing. In particular, we discuss Si detectors, detectors based on plastic-scintillator fibers and paddles, straw-tube gas detectors and the overall performance of the system.

  12. Itinerant magnetism in CeRh3B2

    DEFF Research Database (Denmark)

    Eriksson, Olle; Johansson, Börje; Brooks, M. S. S.

    1989-01-01

    Spin-polarized energy-band calculations, including spin-orbit coupling in the band Hamiltonian, have been performed on CeRh3B2. Good agreement is obtained between theory and experiment concerning the magnetic moment. It is also found that the magnetic moment varies strongly with volume and from...

  13. Characterization of a Vitis vinifera cv. Cabernet Sauvignon 3',5'-O-methyltransferase showing strong preference for anthocyanins and glycosylated flavonols.

    Science.gov (United States)

    Lücker, Joost; Martens, Stefan; Lund, Steven T

    2010-09-01

    At ripening initiation in red grapevine (Vitis vinifera) berries, the exocarp turns color from green to red and then to purple due to the accumulation and extent of methylation of anthocyanins. The accumulation of transcripts encoding an O-methyltransferase was recently shown to be closely correlated with the onset of ripening and the degree of blue/purple pigmentation in grapevine berries; however, the biochemical function of this gene has remained uncharacterized. In this study, an O-methyltransferase cDNA that showed a distinct expression pattern when compared to closely related sequences was expressed in Escherichia coli and enzyme assays were carried out with a broad array of anthocyanin and other flavonoid substrates. We demonstrate that this enzyme carries out 3',5'-O-methylation of anthocyanins and flavonol compounds in vitro, which are known to be present in grape berries, with a preference for glycosylated substrates. The highest relative specific activity for the enzyme was found with delphinidin 3-O-glucoside as substrate. The enzyme is not able to methylate flavan type skeletons with chiral centers, such as either catechins or dihydroquercetin. The enzyme showed negligible specific activity for caffeoyl-CoA, compared to flavonol and anthocyanin substrates. Phylogenetic analysis of the O-methyltransferase suggests that it may be a member of a distinct subclass of Type 2 bivalent metal-dependent S-adenosyl-methionine O-methyltransferases. (c) 2010. Published by Elsevier Ltd. All rights reserved.

  14. Minimal traumatic brain injury causes persistent changes in DNA methylation at BDNF gene promoters in rat amygdala: A possible role in anxiety-like behaviors.

    Science.gov (United States)

    Sagarkar, Sneha; Bhamburkar, Tanmayi; Shelkar, Gajanan; Choudhary, Amit; Kokare, Dadasaheb M; Sakharkar, Amul J

    2017-10-01

    Minimal traumatic brain injury (MTBI) often transforms into chronic neuropsychiatric conditions including anxiety, the underlying mechanisms of which are largely unknown. In the present study, we employed the closed-head injury paradigm to induce MTBI in rats and examined whether DNA methylation can explain long-term changes in the expression of the brain-derived neurotrophic factor (BDNF) in the amygdala as well as trauma-induced anxiety-like behaviors. The MTBI caused anxiety-like behaviors and altered the expression of DNA methyltransferase (DNMT) isoforms (DNMT1, DNMT3a, and DNMT3b) and factors involved in DNA demethylation such as the growth arrest and DNA damage 45 (GADD45a and GADD45b). After 30days of MTBI, the over-expression of DNMT3a and DNMT3b corresponded to heightened DNMT activity, whereas the mRNA levels of GADD45a and GADD45b were declined. The methylated cytosine levels at the BDNF promoters (Ip, IVp and IXp) were increased in the amygdala of the trauma-induced animals; these coincided negatively with the mRNA levels of exon IV and IXa, but not of exon I. Interestingly, treatment with 5-azacytidine, a pan DNMT inhibitor, normalized the MTBI-induced DNMT activity and DNA hypermethylation at exon IVp and IXp. Furthermore, 5-azacytidine also corrected the deficits in the expression of exons IV and IXa and reduced the anxiety-like behaviors. These results suggest that the DNMT-mediated DNA methylation at the BDNF IVp and IXp might be involved in the regulation of BDNF gene expression in the amygdala. Further, it could also be related to MTBI-induced anxiety-like behaviors via the regulation of synaptic plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

    Science.gov (United States)

    Kong, Guanghui; Zhao, Yao; Jing, Maofeng; Huang, Jie; Yang, Jin; Xia, Yeqiang; Kong, Liang; Ye, Wenwu; Xiong, Qin; Qiao, Yongli; Dong, Suomeng; Ma, Wenbo; Wang, Yuanchao

    2015-08-01

    Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

  16. Cocoa Consumption Alters the Global DNA Methylation of Peripheral Leukocytes in Humans with Cardiovascular Disease Risk Factors: A Randomized Controlled Trial.

    Science.gov (United States)

    Crescenti, Anna; Solà, Rosa; Valls, Rosa M; Caimari, Antoni; Del Bas, Josep M; Anguera, Anna; Anglés, Neus; Arola, Lluís

    2013-01-01

    DNA methylation regulates gene expression and can be modified by different bioactive compounds in foods, such as polyphenols. Cocoa is a rich source of polyphenols, but its role in DNA methylation is still unknown. The objective was to assess the effect of cocoa consumption on DNA methylation and to determine whether the enzymes involved in the DNA methylation process participate in the mechanisms by which cocoa exerts these effects in humans. The global DNA methylation levels in the peripheral blood were evaluated in 214 volunteers who were pre-hypertensive, stage-1 hypertensive or hypercholesterolemic. The volunteers were divided into two groups: 110 subjects who consumed cocoa (6 g/d) for two weeks and 104 control subjects. In addition, the peripheral blood mononuclear cells (PBMCs) from six subjects were treated with a cocoa extract to analyze the mRNA levels of the DNA methyltransferases (DNMTs), methylenetetrahydrofolate reductase (MTHFR), and methionine synthase reductase (MTRR) genes. Cocoa consumption significantly reduced the DNA methylation levels (2.991±0.366 vs. 3.909±0.380, pcocoa effects on DNA methylation and three polymorphisms located in the MTHFR, MTRR, and DNMT3B genes. Furthermore, in PBMCs, the cocoa extract significantly lowered the mRNA levels of the DNMTs, MTHFR, and MTRR. Our study demonstrates for the first time that the consumption of cocoa decreases the global DNA methylation of peripheral leukocytes in humans with cardiovascular risk factors. In vitro experiments with PBMCs suggest that cocoa may exert this effect partially via the down-regulation of DNMTs, MTHFR and MTRR, which are key genes involved in this epigenetic process. Clinicaltrials.govNCT00511420 and NCT00502047.

  17. Cocoa Consumption Alters the Global DNA Methylation of Peripheral Leukocytes in Humans with Cardiovascular Disease Risk Factors: A Randomized Controlled Trial.

    Directory of Open Access Journals (Sweden)

    Anna Crescenti

    Full Text Available DNA methylation regulates gene expression and can be modified by different bioactive compounds in foods, such as polyphenols. Cocoa is a rich source of polyphenols, but its role in DNA methylation is still unknown. The objective was to assess the effect of cocoa consumption on DNA methylation and to determine whether the enzymes involved in the DNA methylation process participate in the mechanisms by which cocoa exerts these effects in humans. The global DNA methylation levels in the peripheral blood were evaluated in 214 volunteers who were pre-hypertensive, stage-1 hypertensive or hypercholesterolemic. The volunteers were divided into two groups: 110 subjects who consumed cocoa (6 g/d for two weeks and 104 control subjects. In addition, the peripheral blood mononuclear cells (PBMCs from six subjects were treated with a cocoa extract to analyze the mRNA levels of the DNA methyltransferases (DNMTs, methylenetetrahydrofolate reductase (MTHFR, and methionine synthase reductase (MTRR genes. Cocoa consumption significantly reduced the DNA methylation levels (2.991±0.366 vs. 3.909±0.380, p<0.001. Additionally, we found an association between the cocoa effects on DNA methylation and three polymorphisms located in the MTHFR, MTRR, and DNMT3B genes. Furthermore, in PBMCs, the cocoa extract significantly lowered the mRNA levels of the DNMTs, MTHFR, and MTRR. Our study demonstrates for the first time that the consumption of cocoa decreases the global DNA methylation of peripheral leukocytes in humans with cardiovascular risk factors. In vitro experiments with PBMCs suggest that cocoa may exert this effect partially via the down-regulation of DNMTs, MTHFR and MTRR, which are key genes involved in this epigenetic process.Clinicaltrials.govNCT00511420 and NCT00502047.

  18. The role of DNA methylation during anoxia tolerance in a freshwater turtle (Trachemys scripta elegans).

    Science.gov (United States)

    Wijenayake, Sanoji; Storey, Kenneth B

    2016-04-01

    Oxygen deprivation is a lethal stress that only a few animals can tolerate for extended periods. This study focuses on analyzing the role of DNA methylation in aiding natural anoxia tolerance in a champion vertebrate anaerobe, the red-eared slider turtle (Trachemys scripta elegans). We examined the relative expression and total enzymatic activity of four DNA methyltransferases (DNMT1, DNMT2, DNMT3a and DNMT3b), two methyl-binding domain proteins (MBD1 and MBD2), and relative genomic levels of 5-methylcytosine under control, 5 h anoxic, and 20 h anoxic conditions in liver, heart, and white skeletal muscle (n = 4, p < 0.05). In liver, protein expression of DNMT1, DNMT2, MBD1, and MBD2 rose significantly by two- to fourfold after 5 h anoxic submergence compared to normoxic-control conditions. In heart, 5 h anoxia submergence resulted in a 1.4-fold increase in DNMT3a levels and a significant decrease in MBD1 and MBD2 levels to ~30 % of control values. In white muscle, DNMT3a and DNMT3b increased threefold and MBD1 levels increased by 50 % in response to 5 h anoxia. Total DNMT activity rose by 0.6-2.0-fold in liver and white muscle and likewise global 5mC levels significantly increased in liver and white muscle under 5 and 20 h anoxia. The results demonstrate an overall increase in DNA methylation, DNMT protein expression and enzymatic activity in response to 5 and 20 h anoxia in liver and white muscle indicating a potential downregulation of gene expression via this epigenetic mechanism during oxygen deprivation.

  19. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    International Nuclear Information System (INIS)

    Kanno, Yuichiro; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-01-01

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR

  20. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    Energy Technology Data Exchange (ETDEWEB)

    Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  1. Plant isoflavone and isoflavanone O-methyltransferase genes

    Science.gov (United States)

    Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

    2014-08-19

    The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

  2. Structural characterization of the mitomycin 7-O-methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shanteri; Chang, Aram; Goff, Randal D.; Bingman, Craig A.; Grüschow, Sabine; Sherman, David H.; Phillips, Jr., George N.; Thorson, Jon S. (Michigan); (UW)

    2014-10-02

    Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.

  3. Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing

    DEFF Research Database (Denmark)

    Lee, Seungmin; Rose, Simon; Metodiev, Metodi D

    2015-01-01

    maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment of multiple cellular processes. In contrast, it was recently reported that signaling induced......Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus...... by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce...

  4. A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

    Science.gov (United States)

    Li, L; Popko, J L; Zhang, X H; Osakabe, K; Tsai, C J; Joshi, C P; Chiang, V L

    1997-05-13

    S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

  5. A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

    Science.gov (United States)

    Li, Laigeng; Popko, Jacqueline L.; Zhang, Xing-Hai; Osakabe, Keishi; Tsai, Chung-Jui; Joshi, Chandrashekhar P.; Chiang, Vincent L.

    1997-01-01

    S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. PMID:9144260

  6. Biochemical and structural studies of the Mycobacterium tuberculosis O6-methylguanine methyltransferase and mutated variants.

    Science.gov (United States)

    Miggiano, Riccardo; Casazza, Valentina; Garavaglia, Silvia; Ciaramella, Maria; Perugino, Giuseppe; Rizzi, Menico; Rossi, Franca

    2013-06-01

    Mycobacterium tuberculosis displays remarkable genetic stability despite continuous exposure to the hostile environment represented by the host's infected macrophages. Similarly to other organisms, M. tuberculosis possesses multiple systems to counteract the harmful potential of DNA alkylation. In particular, the suicidal enzyme O(6)-methylguanine-DNA methyltransferase (OGT) is responsible for the direct repair of O(6)-alkylguanine in double-stranded DNA and is therefore supposed to play a central role in protecting the mycobacterial genome from the risk of G · C-to-A · T transition mutations. Notably, a number of geographically widely distributed M. tuberculosis strains shows nonsynonymous single-nucleotide polymorphisms in their OGT-encoding gene, leading to amino acid substitutions at position 15 (T15S) or position 37 (R37L) of the N-terminal domain of the corresponding protein. However, the role of these mutations in M. tuberculosis pathogenesis is unknown. We describe here the in vitro characterization of M. tuberculosis OGT (MtOGT) and of two point-mutated versions of the protein mimicking the naturally occurring ones, revealing that both mutated proteins are impaired in their activity as a consequence of their lower affinity for alkylated DNA than the wild-type protein. The analysis of the crystal structures of MtOGT and MtOGT-R37L confirms the high level of structural conservation of members of this protein family and provides clues to an understanding of the molecular bases for the reduced affinity for the natural substrate displayed by mutated MtOGT. Our in vitro results could contribute to validate the inferred participation of mutated OGTs in M. tuberculosis phylogeny and biology.

  7. Scintillator concept of NeuLAND at R3B

    Energy Technology Data Exchange (ETDEWEB)

    Aumann, Thomas; Ignatov, Alexander [Technische Universitaet Darmstadt (Germany); Boretzky, Konstanze; Heil, Michael; Simon, Haik [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); Maroussov, Vassili [Institut fuer Kernphysik, Universitaet zu Koeln (Germany); GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); Zilges, Andreas [Institut fuer Kernphysik, Universitaet zu Koeln (Germany); Collaboration: R3B-Collaboration

    2011-07-01

    For the R3B experiment at FAIR a detection system for fast neutrons, NeuLAND (new Large Area Neutron Detector), is foreseen. Besides a time resolution of {sigma}{sub t}{approx_equal} 100 ps, spatial resolutions of {sigma}{sub x,y,z}{approx_equal} 1 cm, the detection efficiency of above 90% for neutrons of 0.2-1 GeV and a dedicated multi-neutron recognition capability are demanded. Using the FLUKA Monte Carlo code we studied a NeuLAND detector concept relying entirely on bars of a plastic scintillator (BC408). With a detector depth of 2 m the required efficiency is reached and the fraction of incident neutrons detected within resolution requirements varies from {proportional_to}70% to 80% in the desired energy range. Simulations have verified that the introduction of an inactive converter like iron deteriorates the timing performance. Due to the low density of the scintillator secondary protons typically cross several modules, thus allowing the tracking of secondaries. The status of the multi-hit recognition algorithm using the tracking information is presented along with the latest results for the scintillator prototypes for NeuLAND. Using the same framework a competing concept for NeuLAND based on MRPCs was studied as well and is contrasted to the scintillator concept.

  8. Evolution of DNA repair defects during malignant progression of low-grade gliomas after temozolomide treatment

    NARCIS (Netherlands)

    van Thuijl, Hinke F.; Mazor, Tali; Johnson, Brett E.; Fouse, Shaun D.; Aihara, Koki; Hong, Chibo; Malmström, Annika; Hallbeck, Martin; Heimans, Jan J.; Kloezeman, Jenneke J.; Stenmark-Askmalm, Marie; Lamfers, Martine L M; Saito, Nobuhito; Aburatani, Hiroyuki; Mukasa, Akitake; Berger, Mitchell S.; Söderkvist, Peter; Taylor, Barry S.; Molinaro, Annette M.; Wesseling, Pieter; Reijneveld, Jaap C.; Chang, Susan M.; Ylstra, Bauke; Costello, Joseph F.

    2015-01-01

    Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with an improved

  9. Evolution of DNA repair defects during malignant progression of low-grade gliomas after temozolomide treatment

    NARCIS (Netherlands)

    van Thuijl, Hinke F.; Mazor, Tali; Johnson, Brett E.; Fouse, Shaun D.; Aihara, Koki; Hong, Chibo; Malmström, Annika; Hallbeck, Martin; Heimans, Jan J.; Kloezeman, Jenneke J.; Stenmark-Askmalm, Marie; Lamfers, Martine L. M.; Saito, Nobuhito; Aburatani, Hiroyuki; Mukasa, Akitake; Berger, Mitchell S.; Söderkvist, Peter; Taylor, Barry S.; Molinaro, Annette M.; Wesseling, Pieter; Reijneveld, Jaap C.; Chang, Susan M.; Ylstra, Bauke; Costello, Joseph F.

    2015-01-01

    Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O (6) -methylguanine-DNA methyltransferase (MGMT) promoter is associated with an

  10. The gene expressions of DNA methylation/demethylation enzymes ...

    African Journals Online (AJOL)

    user

    2011-01-31

    Jan 31, 2011 ... A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases.

  11. The gene expressions of DNA methylation/demethylation enzymes ...

    African Journals Online (AJOL)

    A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases (Dnmts), DNA ...

  12. Fingersomatotopy in area 3b: an fMRI-study

    Directory of Open Access Journals (Sweden)

    Rosén Birgitta

    2004-08-01

    Full Text Available Abstract Background The primary sensory cortex (S1 in the postcentral gyrus is comprised of four areas that each contain a body map, where the representation of the hand is located with the thumb most laterally, anteriorly and inferiorly and the little finger most medially, posteriorly and superiorly. Previous studies on somatotopy using functional MRI have either used low field strength, have included a small number of subjects or failed to attribute activations to any area within S1. In the present study we included twenty subjects, who were investigated at 3 Tesla (T. We focused specifically on Brodmann area 3b, which neurons have discrete receptive fields with a potentially more clearcut somatotopic organisation. The spatial distribution for all fingers' peak activation was determined and group as well as individual analysis was performed. Results Activation maps from 18 subjects were of adequate quality; in 17 subjects activations were present for all fingers and these data were further analysed. In the group analysis the thumb was located most laterally, anteriorly and inferiorly with the other fingers sequentially positioned more medially, posteriorly and superiorly. At the individual level this somatotopic relationship was present for the thumb and little finger, with a higher variability for the fingers in between. The Euclidian distance between the first and fifth finger was 17.2 mm, between the first and second finger 10.6 mm and between the remaining fingers on average 6.3 mm. Conclusion Results from the group analysis, that is both the location of the fingers and the Euclidian distances, are well comparable to results from previous studies using a wide range of modalities. On the subject level the spatial localisation of the fingers showed a less stringent somatotopic order so that the location of a finger in a single subject cannot be predicted from the group result.

  13. Inefficient binding of IgM immune complexes to erythrocyte C3b-C4b receptors (CR1) and weak incorporation of C3b-iC3b into the complexes

    DEFF Research Database (Denmark)

    Kávai, M; Rasmussen, J M; Baatrup, G

    1988-01-01

    , but the binding was low (2-3%) when compared to the binding of the corresponding IgG-IC (50-60%). Solid phase IC were prepared by coating microwells with heat-aggregated bovine serum albumin (BSA) followed by incubation with rabbit IgM anti-BSA antibody. The IC were reacted with human serum at 37 degrees C....... The binding of C3b-iC3b was determined by use of biotinylated F(ab')2 antibodies to C3b-C3c and avidin-coupled alkaline phosphatase. The incorporation of C3b-iC3b into solid-phase IgM-IC increased when increasing amounts of IgM antibody were reacted with the antigen. The binding reaction was slow, reaching...

  14. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly

  15. TMPA Products 3B42RT & 3B42V6: Evaluation and Application in Qinghai-Tibet Plateau

    Science.gov (United States)

    Hao, Z.; Sun, L.; Wang, J.

    2012-04-01

    Hydrological researchers in Qinghai-Tibet Plateau tend to be haunted by deficiency of station gauged precipitation data for the sparse and uneven distribution of local meteorological stations. Fortunately, alternative data can be obtained from TRMM (Tropic Rainfall Measurement Mission) satellite. Preliminary evaluation and necessary correction of TRMM satellite rainfall products is required for the sake of reliability and suitability considering that TRMM precipitation is unconventional and natural condition in Qinghai-Tibet Plateau is unusually complicated. 3B42RT and 3B42V6 products from TRMM Multisatellite Precipitation Analysis(TMPA) are evaluated in northeast Qinghai-Tibet Plateau with 50 stations quality-controlled gauged daily precipitation as the benchmark precipitation set. It is found that the RT data overestimates the actual precipitation greatly while V6 only overestimates it slightly. RT data shows different seasonal and inter-annual accuracies. Summer and autumn see better accuracies than winter and spring and wet years see higher accuracies than dry years. Latitude is believed to be an important factor that influences the accuracy of satellite precipitation. Both RT and V6 can reflect the general pattern of the spatial distribution of precipitation even though RT overestimates the quantity greatly. A new parameter, accumulated precipitation weight point (APWP), was introduced to describe the temporal-spatial pattern evolution of precipitation. The APWP of both RT and V6 were moving from south to north in the past decade, but they are all in the west of station gauged precipitation APWP(s).V6 APWP track fit gauged precipitation perfectly while RT APWP track has over-exaggerated legs, indicating that spatial distribution of RT precipitation experienced unreasonable sharp changes. A practical and operational procedure to correct satellite precipitation data is developed. For RT, there are two steps. Step 1, the downscaling, original daily precipitation

  16. Crystal structure of arginine methyltransferase 6 from Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Chongyuan Wang

    Full Text Available Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6 is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH. The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.

  17. Extended Reconstructed Sea Surface Temperature (ERSST) Monthly Analysis, Version 3b

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Version 3b (v3b) of the Extended Reconstructed Sea Surface Temperature (ERSST) dataset is a monthly SST analysis on a 2-degree global grid based on the International...

  18. Association of low-affinity FC gamma receptor 3B (FCGR3B) copy number variation with rheumatoid arthritis in Caucasian subjects

    NARCIS (Netherlands)

    Merriman, T.R.; Fanciulli, M.; Merriman, M.E.; Alizadeh, B.Z.; Koeleman, B.P.C.; Dalbeth, N.; Gow, P.; Harrison, A.A.; Highton, J.; Jones, P.B.; Stamp, L.K.; Steer, S.; Barrera, P.; Coenen, M.J.H.; Franke, B.; Vyse, T.; Aitman, T.; Radstake, T.; McKinney, C.

    2009-01-01

    Aim: There is increasing evidence that gene copy-number variation influences phenotypic variation. The low-affinity Fc receptor 3B (FCGR3B) is a copy-number polymorphic gene involved in the recruitment to sites of inflammation and activation of polymorphonuclear neutrophils (PMN). Given the

  19. Mitochondrial DNA Hypomethylation Is a Biomarker Associated with Induced Senescence in Human Fetal Heart Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Dehai Yu

    2017-01-01

    Full Text Available Background. Fetal heart can regenerate to restore its normal anatomy and function in response to injury, but this regenerative capacity is lost within the first week of postnatal life. Although the specific molecular mechanisms remain to be defined, it is presumed that aging of cardiac stem or progenitor cells may contribute to the loss of regenerative potential. Methods. To study this aging-related dysfunction, we cultured mesenchymal stem cells (MSCs from human fetal heart tissues. Senescence was induced by exposing cells to chronic oxidative stress/low serum. Mitochondrial DNA methylation was examined during the period of senescence. Results. Senescent MSCs exhibited flattened and enlarged morphology and were positive for the senescence-associated beta-galactosidase (SA-β-Gal. By scanning the entire mitochondrial genome, we found that four CpG islands were hypomethylated in close association with senescence in MSCs. The mitochondrial COX1 gene, which encodes the main subunit of the cytochrome c oxidase complex and contains the differentially methylated CpG island 4, was upregulated in MSCs in parallel with the onset of senescence. Knockdown of DNA methyltransferases (DNMT1, DNMT3a, and DNMT3B also upregulated COX1 expression and induced cellular senescence in MSCs. Conclusions. This study demonstrates that mitochondrial CpG hypomethylation may serve as a critical biomarker associated with cellular senescence induced by chronic oxidative stress.

  20. 17 CFR 240.3b-13 - Definition of eligible OTC derivative instrument.

    Science.gov (United States)

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Definition of eligible OTC derivative instrument. 240.3b-13 Section 240.3b-13 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... Under the Securities Exchange Act of 1934 Definitions § 240.3b-13 Definition of eligible OTC derivative...

  1. RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2.

    Directory of Open Access Journals (Sweden)

    Jafar Kiani

    2013-05-01

    Full Text Available RNA-mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2(-/- mice and that the Sox9 paramutation was also not established in Dnmt2(-/- embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA-mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA-mediated epigenetic heredity.

  2. Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a

    International Nuclear Information System (INIS)

    Miranda, Tina Branscombe; Webb, Kristofor J.; Edberg, Dale D.; Reeves, Raymond; Clarke, Steven

    2005-01-01

    The HMGA family proteins HMGA1a and HMGA1b are nuclear nonhistone species implicated in a wide range of cellular processes including inducible gene transcription, modulation of chromosome structure through nucleosome and chromosome remodeling, and neoplastic transformation. HMGA proteins are highly modified, and changes in their phosphorylation states have been correlated with the phase of the cell cycle and changes in their transcriptional activity. HMGA1a is also methylated in the first DNA-binding AT-hook at Arg25 and other sites, although the enzyme or enzymes responsible have not been identified. We demonstrate here that a GST fusion of protein arginine methyltransferase 6 (PRMT6) specifically methylates full-length recombinant HMGA1a protein in vitro. Although GST fusions of PRMT1 and PRMT3 were also capable of methylating the full-length HMGA1a polypeptide, they recognize its proteolytic degradation products much better. GST fusions of PRMT4 or PRMT7 were unable to methylate the full-length protein or its degradation products. We conclude that PRMT6 is a good candidate for the endogenous enzyme responsible for HGMA1a methylation

  3. Molecular Cloning and Characterization of O-Methyltransferase from Mango Fruit (Mangifera indica cv. Alphonso).

    Science.gov (United States)

    Chidley, Hemangi G; Oak, Pranjali S; Deshpande, Ashish B; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

    2016-05-01

    Flavour of ripe Alphonso mango is invariably dominated by the de novo appearance of lactones and furanones during ripening. Of these, furanones comprising furaneol (4-hydroxy-2,5-dimethyl-3(2H)-furanone) and mesifuran (2,5-dimethyl-4-methoxy-3(2H)-furanone) are of particular importance due to their sweet, fruity caramel-like flavour characters and low odour detection thresholds. We isolated a 1056 bp complete open reading frame of a cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase from Alphonso mango. The recombinantly expressed enzyme, MiOMTS showed substrate specificity towards furaneol and protocatechuic aldehyde synthesizing mesifuran and vanillin, respectively, in an in vitro assay reaction. A semi-quantitative PCR analysis showed fruit-specific expression of MiOMTS transcripts. Quantitative real-time PCR displayed ripening-related expression pattern of MiOMTS in both pulp and skin of Alphonso mango. Also, early and significantly enhanced accumulation of its transcripts was detected in pulp and skin of ethylene-treated fruits. Ripening-related and fruit-specific expression profile of MiOMTS and substrate specificity towards furaneol is a suggestive of its involvement in the synthesis of mesifuran in Alphonso mango. Moreover, a significant trigger in the expression of MiOMTS transcripts in ethylene-treated fruits point towards the transcriptional regulation of mesifuran biosynthesis by ethylene.

  4. Characterization of a mimivirus RNA cap guanine-N2 methyltransferase.

    Science.gov (United States)

    Benarroch, Delphine; Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

    2009-04-01

    A 2,2,7-trimethylguanosine (TMG) cap is a signature feature of eukaryal snRNAs, telomerase RNAs, and trans-spliced nematode mRNAs. TMG and 2,7-dimethylguanosine (DMG) caps are also present on mRNAs of two species of alphaviruses (positive strand RNA viruses of the Togaviridae family). It is presently not known how viral mRNAs might acquire a hypermethylated cap. Mimivirus, a giant DNA virus that infects amoeba, encodes many putative enzymes and proteins implicated in RNA transactions, including the synthesis and capping of viral mRNAs and the promotion of cap-dependent translation. Here we report the identification, purification, and characterization of a mimivirus cap-specific guanine-N2 methyltransferase (MimiTgs), a monomeric enzyme that catalyzes a single round of methyl transfer from AdoMet to an m(7)G cap substrate to form a DMG cap product. MimiTgs, is apparently unable to convert a DMG cap to a TMG cap, and is thereby distinguished from the structurally homologous yeast and human Tgs1 enzymes. Nonetheless, we show genetically that MimiTgs is a true ortholog of Saccharomyces cerevisiae Tgs1. Our results hint that DMG caps can satisfy many of the functions of TMG caps in vivo. We speculate that DMG capping of mimivirus mRNAs might favor viral protein synthesis in the infected host.

  5. DNA Damage Induced by Alkylating Agents and Repair Pathways

    OpenAIRE

    Natsuko Kondo; Akihisa Takahashi; Koji Ono; Takeo Ohnishi

    2010-01-01

    The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O 6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O 6-methylguanine-DNA methyltransferase, and O 6MeG:T mispairs are recognized...

  6. In vivo and in vitro studies suggest a possible involvement of HPV infection in the early stage of breast carcinogenesis via APOBEC3B induction.

    Directory of Open Access Journals (Sweden)

    Kenji Ohba

    Full Text Available High prevalence of infection with high-risk human papilloma virus (HPV ranging from 25 to 100% (average 31% was observed in breast cancer (BC patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.

  7. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    OpenAIRE

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand brea...

  8. Highly purified, multi-wall carbon nanotubes induce light-chain 3B expression in human lung cells

    International Nuclear Information System (INIS)

    Tsukahara, Tamotsu; Matsuda, Yoshikazu; Usui, Yuki; Haniu, Hisao

    2013-01-01

    Highlights: •HTT2800-treated BEAS-2B cells induced LC3B in a time-dependent manner. •HTT2800-treated BEAS-2B cells showed decreased cell proliferation that was both time- and dose-dependent. •Addition of 3-MA, LC3B-II protein and mRNA levels were significantly decreased. •3-MA and E64-d + pepstatin A, but not brefeldin A, provided protection against HTT2800-induced cell death. •These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells. -- Abstract: Bronchial epithelial cells are targets of inhalation and play a critical role in the maintenance of mucosal integrity as mechanical barriers against various particles. Our previous result suggest that vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. Increasing evidence suggests that autophagy may critically influence vital cellular processes such as apoptosis, cell proliferation and inflammation and thereby may play a critical role in pulmonary diseases. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles. In this study, the authors focus on the autophagic responses of HTT2800 exposure. The HTT2800-exposed cells induced LC3B expression and induced cell growth inhibition

  9. Highly purified, multi-wall carbon nanotubes induce light-chain 3B expression in human lung cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@kanazawa-med.ac.jp [Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 (Japan); Matsuda, Yoshikazu [Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama 362-0806 (Japan); Usui, Yuki [Research Center for Exotic Nanocarbons, Shinshu University, 4-17-1 Wakasato, Nagano-shi, Nagano 380-8553 (Japan); Haniu, Hisao [Department of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

    2013-10-18

    Highlights: •HTT2800-treated BEAS-2B cells induced LC3B in a time-dependent manner. •HTT2800-treated BEAS-2B cells showed decreased cell proliferation that was both time- and dose-dependent. •Addition of 3-MA, LC3B-II protein and mRNA levels were significantly decreased. •3-MA and E64-d + pepstatin A, but not brefeldin A, provided protection against HTT2800-induced cell death. •These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells. -- Abstract: Bronchial epithelial cells are targets of inhalation and play a critical role in the maintenance of mucosal integrity as mechanical barriers against various particles. Our previous result suggest that vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. Increasing evidence suggests that autophagy may critically influence vital cellular processes such as apoptosis, cell proliferation and inflammation and thereby may play a critical role in pulmonary diseases. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles. In this study, the authors focus on the autophagic responses of HTT2800 exposure. The HTT2800-exposed cells induced LC3B expression and induced cell growth inhibition.

  10. Hepatic protein phosphatase 1 regulatory subunit 3B (Ppp1r3b) promotes hepatic glycogen synthesis and thereby regulates fasting energy homeostasis.

    Science.gov (United States)

    Mehta, Minal B; Shewale, Swapnil V; Sequeira, Raymond N; Millar, John S; Hand, Nicholas J; Rader, Daniel J

    2017-06-23

    Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (G L ) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion ( Ppp1r3b Δ hep ) or liver-specific overexpression of Ppp1r3b The Ppp1r3b deletion significantly reduced glycogen synthase protein abundance, and the remaining protein was predominantly phosphorylated and inactive. As a consequence, glucose incorporation into hepatic glycogen was significantly impaired, total hepatic glycogen content was substantially decreased, and mice lacking hepatic Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3b Δ hep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12-36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3b Δ hep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation

    KAUST Repository

    Gao, Zhihuan; Liu, Hai-Liang; Daxinger, Lucia; Pontes, Olga; He, Xinjian; Qian, Weiqiang; Lin, Huixin; Xie, Mingtang; Lorkovic, Zdravko J.; Zhang, ShouDong; Miki, Daisuke; Zhan, Xianqiang; Pontier, Dominique; Lagrange, Thierry; Jin, Hailing; Matzke, Antonius J.; Matzke, Marjori; Pikaard, Craig S.; Zhu, Jian-Kang

    2010-01-01

    DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2

  12. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2

  13. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Directory of Open Access Journals (Sweden)

    Anna Schrader

    Full Text Available The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  14. Loss of the histone methyltransferase EZH2 induces resistance to multiple drugs in acute myeloid leukemia

    DEFF Research Database (Denmark)

    Göllner, Stefanie; Oellerich, Thomas; Agrawal-Singh, Shuchi

    2017-01-01

    In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction...

  15. Molecular Basis for the Regulation of the H3K4 Methyltransferase Activity of PRDM9

    Directory of Open Access Journals (Sweden)

    Hong Wu

    2013-10-01

    Full Text Available PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2 and S-adenosylhomocysteine (AdoHcy, which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4 and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

  16. Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

    International Nuclear Information System (INIS)

    Kimura, Shuhei; Sawatsubashi, Shun; Ito, Saya; Kouzmenko, Alexander; Suzuki, Eriko; Zhao, Yue; Yamagata, Kaoru; Tanabe, Masahiko; Ueda, Takashi; Fujiyama, Sari; Murata, Takuya; Matsukawa, Hiroyuki; Takeyama, Ken-ichi; Yaegashi, Nobuo

    2008-01-01

    Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressed EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly

  17. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

    Science.gov (United States)

    McGann, Patrick; Chahine, Sarah; Okafor, Darius; Ong, Ana C; Maybank, Rosslyn; Kwak, Yoon I; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2016-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    Science.gov (United States)

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Sulforaphane Reverses the Expression of Various Tumor Suppressor Genes by Targeting DNMT3B and HDAC1 in Human Cervical Cancer Cells

    Directory of Open Access Journals (Sweden)

    Munawwar Ali Khan

    2015-01-01

    Full Text Available Sulforaphane (SFN may hinder carcinogenesis by altering epigenetic events in the cells; however, its molecular mechanisms are unclear. The present study investigates the role of SFN in modifying epigenetic events in human cervical cancer cells, HeLa. HeLa cells were treated with SFN (2.5 µM for a period of 0, 24, 48, and 72 hours for all experiments. After treatment, expressions of DNMT3B, HDAC1, RARβ, CDH1, DAPK1, and GSTP1 were studied using RT-PCR while promoter DNA methylation of tumor suppressor genes (TSGs was studied using MS-PCR. Inhibition assays of DNA methyl transferases (DNMTs and histone deacetylases (HDACs were performed at varying time points. Molecular modeling and docking studies were performed to explore the possible interaction of SFN with HDAC1 and DNMT3B. Time-dependent exposure to SFN decreases the expression of DNMT3B and HDAC1 and significantly reduces the enzymatic activity of DNMTs and HDACs. Molecular modeling data suggests that SFN may interact directly with DNMT3B and HDAC1 which may explain the inhibitory action of SFN. Interestingly, time-dependent reactivation of the studied TSGs via reversal of methylation in SFN treated cells correlates well with its impact on the epigenetic alterations accumulated during cancer development. Thus, SFN may have significant implications for epigenetic based therapy.

  20. A novel role of KIF3b in the seminoma cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Hao-Qing; Xiao, Yu-Xi; She, Zhen-Yu [The Sperm Laboratory, College of Life Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058 (China); Tan, Fu-Qing [The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003 (China); Yang, Wan-Xi, E-mail: wxyang@spermlab.org [The Sperm Laboratory, College of Life Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058 (China)

    2017-03-01

    KIF3b is a protein of the kinesin-2 family which plays an important role in intraflagellar transport. Testis cancer is a common cancer among young men. Its diagnostic rate is increasing and over half of the cases are seminomas. Many aspects of the mechanism and gene expression background of this cancer remain unclear. Using western-blotting and semi-quantitative PCR we found high protein levels of KIF3b enrichment in seminoma tissue despite the mRNA levels remaining equivalent to that of normal testicular tissues. The distribution of KIF3b was mainly in cells with division potential. Wound-healing assays and cell counting kit assays showed that the knockdown of KIF3b significantly suppressed cell migration ability, viability and number in HeLa cells. Immunofluorescence images during the cell cycle revealed that KIF3b tended to gather at the spindles and was enriched at the central spindle. This indicated that KIF3b may also have direct impacts upon spindle formation and cytokinesis. By counting the numbers of nuclei, spindles and cells, we found that the rates of multipolar division and multi-nucleation were raised in KIF3b-knockdown cells. In this way we demonstrate that KIF3b functions importantly in mitosis and may be essential to seminoma cell division and proliferation as well as being necessary for normal cell division. - Highlights: • A significant upregulation of KIF3b is detected in seminoma. • Knockdown of KIF3b impacts on cell proliferation and migration. • KIF3b may have direct impacts upon spindle formation and cytokinesis.

  1. Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

    International Nuclear Information System (INIS)

    Som, S.; Friedman, S.

    1991-01-01

    DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet. The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase

  2. Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. Pt. 6

    International Nuclear Information System (INIS)

    De Serres, F.J.; Inoue, H.; Schuepbach, M.E.

    1983-01-01

    Genetic characterization of ad-3B mutants induced in wild-type and UV-sensitive strains has revealed qualitative differences between the spectra of genetic alterations at the molecular level. Ad-3B mutants induced in the two nucleotide excision-repair-deficient strains upr-1 and uvs-2 had significantly lower frequencies of nonpolarized complementation patterns and higher frequencies of noncomplementing mutants than ad-3B mutants induced in the wild-type strain in samples induced by either UV, #betta#-rays, 4NQO or MNNG. In these same samples ad-3B mutants induced in uvs-4, uvs-5 or uvs-6 did not differ significantly from those induced in the wild-type strain. After ICR-170 treatment, ad-3B mutants induced in the UV-sensitive strains did not differ significantly from those induced in wild-type. The comparisons in the present and previous studies demonstrate that the process of mutation-induction in the ad-3 region is under the control of other loci that not only alter mutant recovery quantitatively but also qualitatively. These data have important implications for comparative chemical mutagenesis, since the spectrum of genetic alterations produced by a given agent can be modified markedly as a result of defects in DNA repair. (orig./AJ)

  3. Korean Byungkyul - Citrus platymamma Hort.et Tanaka flavonoids induces cell cycle arrest and apoptosis, regulating MMP protein expression in Hep3B hepatocellular carcinoma cells.

    Science.gov (United States)

    Hong, Gyeong Eun; Lee, Ho Jeong; Kim, Jin A; Yumnam, Silvia; Raha, Suchismita; Venkatarame Gowda Saralamma, Venu; Heo, Jeong Doo; Lee, Sang Joon; Kim, Eun Hee; Won, Chun Kil; Kim, Gon Sup

    2017-02-01

    Citrus platymamma Hort.et Tanaka is an indigenous fruit of Jeju island in Korea. In this study the bioactivity of C. platymamma flavonoids were evaluated on human hepatoma Hep3B cell lines. Eleven flavonoids were identified from the peels of C. platymamma Hort.et Tanaka through high-performance liquid chromatography-Tandem mass spectrometry and the anticancer effect of these C. platymamma flavonoids on human hepatoma Hep3B were studied. Chromatin condensation was observed in Hep3B cells treated with C. platymamma flavonoids. DNA fragmentation was confirmed through agarose gel electrophoresis and TUNEL assay. An increase in the total apoptotic cells and G2/M cell cycle arrest with decreased protein expression of CDC25C, CDK1, cyclin B1 and p21 were observed in Hep3B cells treated with flavonoids of C. platymamma. Further, protein expression of Bcl-XL, Bax, caspase-3 and -9 were also modulated by C. platymamma flavonoids treatment indicating that cell death is through intrinsic apoptotic pathway. Moreover, C. platymamma flavonoids also regulated the phosphorylation of MAPKs, PI3K, and Akt in Hep3B cells. Relevant to inhibiting metastasis, C. platymamma treatment reduced wound closure of Hep3B cells and the protein expression of matrix metalloproteinase-2 and -9 were reduced in C. platymamma treated cells. The results show that C. platymamma flavonoids induce cell cycle arrest and apoptosis following activation of MAPKs and suppression of PI3K/Akt pathway which eventually inhibits cell migration in Hep3B cells. The finding provides evidence on biochemical activities of C. platymamma Hort.et Tanaka, which would be an essential agent for hepatocellular carcinoma (HCC) treatment.

  4. Cloning, functional characterization and catalytic mechanism of a bergaptol O-methyltransferase from Peucedanum praeruptorum Dunn

    Directory of Open Access Journals (Sweden)

    Yucheng eZhao

    2016-05-01

    Full Text Available Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum transcriptome database using the nucleotide sequence of Ammi majus bergaptol O-methyltransferase (AmBMT, GenBank accession No: AY443006 as a template. As a result, a 1335 bp full-length of cDNA sequence which contains an open reading frame of 1080 bp encoding a BMT polypeptide of 359 amino acids was obtained. The recombinant protein was functionally expressed in Escherichia coli and displayed an observed activity to bergaptol. In vitro experiments show that the protein has narrow substrate specificity for bergaptol. Expression profile indicated that the cloned gene had a higher expression level in roots and can be induced by methyl jasmonate (MeJA. Subcellular localization analysis showed that the BMT protein was located in cytoplasm in planta. Homology modeling and docking based site-directed mutagenesis have been employed to investigate the amino acid residues in BMT required for substrate binding and catalysis. Conservative amino acid substitutions at residue H264 affected BMT catalysis, whereas substitutions at residues F171, M175, D226 and L312 affected substrate binding. The systemic study summarized here will enlarge our knowledge on OMTs and provide useful information in investigating the coumarins biosynthesis mechanism in P. praeruptorum.

  5. Protein arginine methyltransferase 5 regulates multiple signaling pathways to promote lung cancer cell proliferation

    International Nuclear Information System (INIS)

    Sheng, Xiumei; Wang, Zhengxin

    2016-01-01

    Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of symmetrical dimethylation of arginine residues in proteins. WD repeat domain 77 (WDR77), also known as p44, MEP50, or WD45, forms a stoichiometric complex with PRMT5. The PRMT5/p44 complex is required for cellular proliferation of lung and prostate epithelial cells during earlier stages of development and is re-activated during prostate and lung tumorigenesis. The molecular mechanisms by which PRMT5 and p44 promote cellular proliferation are unknown. Expression of PRMT5 and p44 in lung and prostate cancer cells was silenced and their target genes were identified. The regulation of target genes was validated in various cancer cells during lung development and tumorigenesis. Altered expression of target genes was achieved by ectopic cDNA expression and shRNA-mediated silencing. PRMT5 and p44 regulate expression of a specific set of genes encoding growth and anti-growth factors, including receptor tyrosine kinases and antiproliferative proteins. Genes whose expression was suppressed by PRMT5 and p44 encoded anti-growth factors and inhibited cell growth when ectopically expressed. In contrast, genes whose expression was enhanced by PRMT5 and p44 encoded growth factors and increased cell growth when expressed. Altered expression of target genes is associated with re-activation of PRMT5 and p44 during lung tumorigenesis. Our data provide the molecular basis by which PRMT5 and p44 regulate cell growth and lay a foundation for further investigation of their role in lung tumor initiation. The online version of this article (doi:10.1186/s12885-016-2632-3) contains supplementary material, which is available to authorized users

  6. Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

    Directory of Open Access Journals (Sweden)

    Yoda Satoshi

    2008-11-01

    Full Text Available Abstract Background Alterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis. Results We observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1 that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest. Conclusion Our results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

  7. 17 CFR 240.3b-15 - Definition of ancillary portfolio management securities activities.

    Science.gov (United States)

    2010-04-01

    ... governing body of the dealer and included in the internal risk management control system for the dealer... of incidental trading activities for portfolio management purposes; and (3) Are limited to risk... portfolio management securities activities. 240.3b-15 Section 240.3b-15 Commodity and Securities Exchanges...

  8. Evidence that deletion at FCGR3B is a risk factor for systemic sclerosis

    NARCIS (Netherlands)

    McKinney, C.; Broen, J.C.A.; Vonk, M.C.; Beretta, L.; Hesselstrand, R.; Hunzelmann, N.; Riemekasten, G.; Scorza, R.; Simeon, C.P.; Fonollosa, V.; Carreira, P.E.; Ortego-Centeno, N.; Gonzalez-Gay, M.A.; Airo, P.; Coenen, M.J.; Martin, J.; Radstake, T.R.D.J.; Merriman, T.R.

    2012-01-01

    There is increasing evidence that gene copy number (CN) variation influences clinical phenotype. The low-affinity Fc receptor 3B (FCGR3B) located in the FCGR gene cluster is a CN polymorphic gene involved in the recruitment of polymorphonuclear neutrophils to sites of inflammation and their

  9. A Revised Mechanism for the Activation of Complement C3 to C3b

    Science.gov (United States)

    Rodriguez, Elizabeth; Nan, Ruodan; Li, Keying; Gor, Jayesh; Perkins, Stephen J.

    2015-01-01

    The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b. C3b cleavage results in C3c and C3d (thioester-containing domain; TED). To resolve functional questions in relation to C3b and C3u, analytical ultracentrifugation and x-ray and neutron scattering studies were used with C3, C3b, C3u, C3c, and C3d, using the wild-type allotype with Arg102. In 50 mm NaCl buffer, atomistic scattering modeling showed that both C3b and C3u adopted a compact structure, similar to the C3b crystal structure in which its TED and macroglobulin 1 (MG1) domains were connected through the Arg102–Glu1032 salt bridge. In physiological 137 mm NaCl, scattering modeling showed that C3b and C3u were both extended in structure, with the TED and MG1 domains now separated by up to 6 nm. The importance of the Arg102–Glu1032 salt bridge was determined using surface plasmon resonance to monitor the binding of wild-type C3d(E1032) and mutant C3d(A1032) to immobilized C3c. The mutant did not bind, whereas the wild-type form did. The high conformational variability of TED in C3b in physiological buffer showed that C3b is more reactive than previously thought. Because the Arg102-Glu1032 salt bridge is essential for the C3b-Factor H complex during the regulatory control of C3b, the known clinical associations of the major C3S (Arg102) and disease-linked C3F (Gly102) allotypes of C3b were experimentally explained for the first time. PMID:25488663

  10. RAMONA-3B/MINET composite representation of BWR thermal-hydraulic systems

    International Nuclear Information System (INIS)

    Van Tuyle, G.J.; Slovik, G.; Cazzoli, E.G.; Nepsee, T.C.; Guppy, J.G.

    1985-01-01

    The modification and interfacing of two computer codes, RAMONA-3B and MINET, for the thermal hydraulic transient analysis of a Boiling Water Reactor nuclear steam supply system, is described. The RAMONA-3B code provides for multi-channel thermal hydraulics and three-dimensional (or one-dimensional) neutron kinetics analysis of a boiling water reactor core. The RAMONA-3B system representation terminates at the end of the steam line and at the junction of the feedwater line at the vessel inlet. By interfacing RAMONA-3B with MINET, a generic balance-of-plant systems analysis code, a complete BWR systems code with detailed core modeling was obtained. The result is a code of particular importance to the analysis of transients such as ATWS. A comparison between the 3-D and 1-D neutronics representation is provided, along with a test case utilizing the composite RAMONA-3B/MINET code

  11. The Role of Protein Arginine Methyltransferases in Inflammatory Responses

    Directory of Open Access Journals (Sweden)

    Ji Hye Kim

    2016-01-01

    Full Text Available Protein arginine methyltransferases (PRMTs mediate the methylation of a number of protein substrates of arginine residues and serve critical functions in many cellular responses, including cancer development, progression, and aggressiveness, T-lymphocyte activation, and hepatic gluconeogenesis. There are nine members of the PRMT family, which are divided into 4 types (types I–IV. Although most PRMTs do not require posttranslational modification (PTM to be activated, fine-tuning modifications, such as interactions between cofactor proteins, subcellular compartmentalization, and regulation of RNA, via micro-RNAs, seem to be required. Inflammation is an essential defense reaction of the body to eliminate harmful stimuli, including damaged cells, irritants, or pathogens. However, chronic inflammation can eventually cause several types of diseases, including some cancers, atherosclerosis, rheumatoid arthritis, and periodontitis. Therefore, inflammation responses should be well modulated. In this review, we briefly discuss the role of PRMTs in the control of inflammation. More specifically, we review the roles of four PRMTs (CARM1, PRMT1, PRMT5, and PRMT6 in modulating inflammation responses, particularly in terms of modulating the transcriptional factors or cofactors related to inflammation. Based on the regulatory roles known so far, we propose that PRMTs should be considered one of the target molecule groups that modulate inflammatory responses.

  12. Active site labeling of the guanine-7-methyltransferase

    International Nuclear Information System (INIS)

    Streaker, E.; Sitz, T.O.

    1992-01-01

    Studies on the guanine-7-methyltransferase have defined three domains in the active site: the S-adenosylmethionine (SAM) region, the cap region (GpppG), and the RNA binding domain (--NpNpNpNpNp---). The authors attempted to label the SAM binding domain by a photoaffinity label using 8-azido-SAM and another method using 3 H-SAM and long exposures to uv-light. Neither method was successful. The next approach was to attempt to label the cap-RNA binding domain (GpppGpNpNpNpNpN) by synthesizing RNA containing 8-azido-Ap using an in vitro transcription system and T7 RNA polymerase. The 8-azido-ATP inhibited the T7 RNA polymerase preventing the synthesis of RNA. As they were unable to synthesize the photoaffinity label, they next tried to synthesize an end labeled RNA and directly label by long exposures to uv-light. When the enzyme was incubated with 32 P-labeled RNA for 15 min at 37 degrees and then exposed to a germicidal lamp for various times at O degrees, optimal labeling occurred after 45 min. Various enzyme preparations were labeled by this method and two polypeptides were found to specifically bind the non-methylated mRNA analog. This labeling method should allow characterization of the subunit structure and generate information about the nature of the RNA binding domain

  13. Characterization of the relationship between APOBEC3B deletion and ACE Alu insertion.

    Directory of Open Access Journals (Sweden)

    Kang Wang

    Full Text Available The insertion/deletion (I/D polymorphism of the angiotensin converting enzyme (ACE, commonly associated with many diseases, is believed to have affected human adaptation to environmental changes during the out-of-Africa expansion. APOBEC3B (A3B, a member of the cytidine deaminase family APOBEC3s, also exhibits a variable gene insertion/deletion polymorphism across world populations. Using data available from published reports, we examined the global geographic distribution of ACE and A3B genotypes. In tracking the modern human dispersal routes of these two genes, we found that the variation trends of the two I/D polymorphisms were directly correlated. We observed that the frequencies of ACE insertion and A3B deletion rose in parallel along the expansion route. To investigate the presence of a correlation between the two polymorphisms and the effect of their interaction on human health, we analyzed 1199 unrelated Chinese adults to determine their genotypes and other important clinical characteristics. We discovered a significant difference between the ACE genotype/allele distribution in the A3B DD and A3B II/ID groups (P = 0.045 and 0.015, respectively, indicating that the ACE Alu I allele frequency in the former group was higher than in the latter group. No specific clinical phenotype could be associated with the interaction between the ACE and A3B I/D polymorphisms. A3B has been identified as a powerful inhibitor of Alu retrotransposition, and primate A3 genes have undergone strong positive selection (and expansion for restricting the mobility of endogenous retrotransposons during evolution. Based on these findings, we suggest that the ACE Alu insertion was enabled (facilitated by the A3B deletion and that functional loss of A3B provided an opportunity for enhanced human adaptability and survival in response to the environmental and climate challenges arising during the migration from Africa.

  14. SF3B1-initiating mutations in MDS-RSs target lymphomyeloid hematopoietic stem cells.

    Science.gov (United States)

    Mortera-Blanco, Teresa; Dimitriou, Marios; Woll, Petter S; Karimi, Mohsen; Elvarsdottir, Edda; Conte, Simona; Tobiasson, Magnus; Jansson, Monika; Douagi, Iyadh; Moarii, Matahi; Saft, Leonie; Papaemmanuil, Elli; Jacobsen, Sten Eirik W; Hellström-Lindberg, Eva

    2017-08-17

    Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal ( SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS. © 2017 by The American Society of Hematology.

  15. Zinc-induced Self-association of Complement C3b and Factor H

    Science.gov (United States)

    Nan, Ruodan; Tetchner, Stuart; Rodriguez, Elizabeth; Pao, Po-Jung; Gor, Jayesh; Lengyel, Imre; Perkins, Stephen J.

    2013-01-01

    The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100 μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions showed that putative weak zinc binding sites with different capacities exist in all five proteins, in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much reduced at 60 μm zinc and even more so at >100 μm zinc. The removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced age-related macular degeneration in higher risk patients. PMID:23661701

  16. Stress-related methylation of the catechol-O-methyltransferase Val 158 allele predicts human prefrontal cognition and activity.

    Science.gov (United States)

    Ursini, Gianluca; Bollati, Valentina; Fazio, Leonardo; Porcelli, Annamaria; Iacovelli, Luisa; Catalani, Assia; Sinibaldi, Lorenzo; Gelao, Barbara; Romano, Raffaella; Rampino, Antonio; Taurisano, Paolo; Mancini, Marina; Di Giorgio, Annabella; Popolizio, Teresa; Baccarelli, Andrea; De Blasi, Antonio; Blasi, Giuseppe; Bertolino, Alessandro

    2011-05-04

    DNA methylation at CpG dinucleotides is associated with gene silencing, stress, and memory. The catechol-O-methyltransferase (COMT) Val(158) allele in rs4680 is associated with differential enzyme activity, stress responsivity, and prefrontal activity during working memory (WM), and it creates a CpG dinucleotide. We report that methylation of the Val(158) allele measured from peripheral blood mononuclear cells (PBMCs) of Val/Val humans is associated negatively with lifetime stress and positively with WM performance; it interacts with stress to modulate prefrontal activity during WM, such that greater stress and lower methylation are related to reduced cortical efficiency; and it is inversely related to mRNA expression and protein levels, potentially explaining the in vivo effects. Finally, methylation of COMT in prefrontal cortex and that in PBMCs of rats are correlated. The relationship of methylation of the COMT Val(158) allele with stress, gene expression, WM performance, and related brain activity suggests that stress-related methylation is associated with silencing of the gene, which partially compensates the physiological role of the high-activity Val allele in prefrontal cognition and activity. Moreover, these results demonstrate how stress-related DNA methylation of specific functional alleles impacts directly on human brain physiology beyond sequence variation.

  17. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  18. What do unicellular organisms teach us about DNA methylation?

    Science.gov (United States)

    Harony, Hala; Ankri, Serge

    2008-05-01

    DNA methylation is an epigenetic hallmark that has been studied intensively in mammals and plants. However, knowledge of this phenomenon in unicellular organisms is scanty. Examining epigenetic regulation, and more specifically DNA methylation, in these organisms represents a unique opportunity to better understand their biology. The determination of their methylation status is often complicated by the presence of several differentiation stages in their life cycle. This article focuses on some recent advances that have revealed the unexpected nature of the epigenetic determinants present in protozoa. The role of the enigmatic DNA methyltransferase Dnmt2 in unicellular organisms is discussed.

  19. DNA Tumor Virus Regulation of Host DNA Methylation and Its Implications for Immune Evasion and Oncogenesis.

    Science.gov (United States)

    Kuss-Duerkop, Sharon K; Westrich, Joseph A; Pyeon, Dohun

    2018-02-13

    Viruses have evolved various mechanisms to evade host immunity and ensure efficient viral replication and persistence. Several DNA tumor viruses modulate host DNA methyltransferases for epigenetic dysregulation of immune-related gene expression in host cells. The host immune responses suppressed by virus-induced aberrant DNA methylation are also frequently involved in antitumor immune responses. Here, we describe viral mechanisms and virus-host interactions by which DNA tumor viruses regulate host DNA methylation to evade antiviral immunity, which may contribute to the generation of an immunosuppressive microenvironment during cancer development. Recent trials of immunotherapies have shown promising results to treat multiple cancers; however, a significant number of non-responders necessitate identifying additional targets for cancer immunotherapies. Thus, understanding immune evasion mechanisms of cancer-causing viruses may provide great insights for reversing immune suppression to prevent and treat associated cancers.

  20. DNA Tumor Virus Regulation of Host DNA Methylation and Its Implications for Immune Evasion and Oncogenesis

    Directory of Open Access Journals (Sweden)

    Sharon K. Kuss-Duerkop

    2018-02-01

    Full Text Available Viruses have evolved various mechanisms to evade host immunity and ensure efficient viral replication and persistence. Several DNA tumor viruses modulate host DNA methyltransferases for epigenetic dysregulation of immune-related gene expression in host cells. The host immune responses suppressed by virus-induced aberrant DNA methylation are also frequently involved in antitumor immune responses. Here, we describe viral mechanisms and virus–host interactions by which DNA tumor viruses regulate host DNA methylation to evade antiviral immunity, which may contribute to the generation of an immunosuppressive microenvironment during cancer development. Recent trials of immunotherapies have shown promising results to treat multiple cancers; however, a significant number of non-responders necessitate identifying additional targets for cancer immunotherapies. Thus, understanding immune evasion mechanisms of cancer-causing viruses may provide great insights for reversing immune suppression to prevent and treat associated cancers.

  1. The O-methyltransferase PMT2 mediates methylation of pinosylvin in Scots pine.

    Science.gov (United States)

    Paasela, Tanja; Lim, Kean-Jin; Pietiäinen, Milla; Teeri, Teemu H

    2017-06-01

    Heartwood extractives are important determinants of the natural durability of pine heartwood. The most important phenolic compounds affecting durability are the stilbenes pinosylvin and its monomethylether, which in addition have important functions as phytoalexins in active defense. A substantial portion of the synthesized pinosylvin is 3-methoxylated but the O-methyltransferase responsible for this modification has not been correctly identified. We studied the expression of the stilbene pathway during heartwood development as well as in response to wounding of xylem and UV-C treatment of needles. We isolated and enzymatically characterized a novel O-methyltransferase, PMT2. The methylated product was verified as pinosylvin monomethylether using ultra performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography analyses. The PMT2 enzyme was highly specific for stilbenes as substrate, in contrast to caffeoyl-CoA O-methyltransferase (CCoAOMT) and PMT1 that were multifunctional. Expression profile and multifunctional activity of CCoAOMT suggest that it might have additional roles outside lignin biosynthesis. PMT1 is not involved in the stilbene pathway and its biological function remains an open question. We isolated a new specific O-methyltransferase responsible for 3-methoxylation of pinosylvin. Expression of PMT2 closely follows stilbene biosynthesis during developmental and stress induction. We propose that PMT2 is responsible for pinosylvin methylation in Scots pine (Pinus sylvestris), instead of the previously characterized methyltransferase, PMT1. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  2. Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration

    Directory of Open Access Journals (Sweden)

    Vítor Jorge MB

    2009-09-01

    Full Text Available Abstract Background Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.

  3. Comparative analysis of the CRDA using BNL-TWIGL and RAMONA-3B

    International Nuclear Information System (INIS)

    Neogy, P.; Carew, J.F.

    1983-06-01

    A comparative analysis of the BWR control rod drop accident (CRDA) using BNL-TWIGL and RAMONA-3B has been performed as part of the BNL/NRC evaluation of methods currently used to analyze BWR CRDA events. A principal objective of this analysis was to test the two-dimensional neutronics model used in BNL-TWIGL aganist the full three-dimensional model in RAMONA-3B. Additionally, the results of analyzing the identical transient with the two codes were expected to help evaluate other approximate models used, such as the coarse mesh nodal neutronics scheme in RAMONA-3B and the equilibrium bulk boiling model in BNL-TWIGL

  4. STAT3 induces transcription of the DNA methyltransferase 1 gene (DNMT1) in malignant T lymphocytes

    DEFF Research Database (Denmark)

    Zhang, Qian; Wang, Hong Y; Woetmann, Anders

    2006-01-01

    In this study, we demonstrated that STAT3, a well-characterized transcription factor expressed in continuously activated oncogenic form in the large spectrum of cancer types, induces in malignant T lymphocytes the expression of DNMT1, the key effector of epigenetic gene silencing. STAT3 binds in ...

  5. Magnetic bead-based electrochemical assay for determination of DNA methyltransferase activity

    Czech Academy of Sciences Publication Activity Database

    Dudová, Zdenka; Bartošík, M.; Fojta, Miroslav

    2017-01-01

    Roč. 231, MAR2017 (2017), s. 575-581 ISSN 0013-4686 R&D Projects: GA ČR GBP206/12/G151; GA ČR(CZ) GA17-08971S Institutional support: RVO:68081707 Keywords : promoter hypermethylation * gene-expression * cell-lines Subject RIV: CG - Electrochemistry OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis) Impact factor: 4.798, year: 2016

  6. Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability

    DEFF Research Database (Denmark)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise

    2014-01-01

    Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent tr......Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband...

  7. The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100

    International Nuclear Information System (INIS)

    Olsson, Ida; Berrez, Jean-Marc; Leipus, Arunas; Ostlund, Cecilia; Mutvei, Ann

    2007-01-01

    Arginine methylation is a post-translational modification of proteins implicated in RNA processing, protein compartmentalization, signal transduction, transcriptional regulation and DNA repair. In a screen for proteins associated with the nuclear envelope in the yeast Saccharomyces cerevisiae, we have identified the arginine methyltransferase Rmt2, previously shown to methylate the ribosomal protein L12. By indirect immunofluorescence and subcellular fractionations we demonstrate here that Rmt2 has nuclear and cytoplasmic localizations. Biochemical analysis of a fraction enriched in nuclei reveals that nuclear Rmt2 is resistant to extractions with salt and detergent, indicating an association with structural components. This was supported by affinity purification experiments with TAP-tagged Rmt2. Rmt2 was found to co-purify with the nucleoporins Nup49, Nup57 and Nup100, revealing a novel link between arginine methyltransferases and the nuclear pore complex. In addition, a genome-wide transcription study of the rmt2Δ mutant shows significant downregulation of the transcription of MYO1, encoding the Type II myosin heavy chain required for cytokinesis and cell separation

  8. Cloning of a protein arginine methyltransferase PRMT1 homologue from Schistosoma mansoni: Evidence for roles in nuclear receptor signaling and RNA metabolism

    International Nuclear Information System (INIS)

    Mansure, Jose Joao; Furtado, Daniel Rodrigues; Bastos de Oliveira, Francisco Meirelles; Rumjanek, Franklin David; Franco, Gloria Regina; Fantappie, Marcelo Rosado

    2005-01-01

    The most studied arginine methyltransferase is the type I enzyme, which catalyzes the transfer of an S-adenosyl-L-methionine to a broad spectrum of substrates, including histones, RNA-transporting proteins, and nuclear hormone receptor coactivators. We cloned a cDNA encoding a protein arginine methyltransferase in Schistosoma mansoni (SmPRMT1). SmPRMT1 is highly homologous to the vertebrate PRMT1 enzyme. In vitro methylation assays showed that SmPRMT1 recombinant protein was able to specifically methylate histone H4. Two schistosome proteins likely to be involved in RNA metabolism, SMYB1 and SmSmD3, that display a number of RGG motifs, were strongly methylated by SmPRMT1. In vitro GST pull-down assays showed that SMYB1 and SmSmD3 physically interacted with SmPRMT1. Additional GST pull-down assay suggested the occurrence of a ternary complex including SmPRMT1, SmRXR1 nuclear receptor, and the p160 (SRC-1) nuclear receptor coactivator. Together, these data suggest a mechanism by which SmPRMT1 plays a role in nuclear receptor-mediated chromatin remodeling and RNA transactions

  9. Crystallization and preliminary X-ray crystallographic characterization of TrmFO, a folate-dependent tRNA methyltransferase from Thermotoga maritima

    International Nuclear Information System (INIS)

    Cicmil, Nenad

    2008-01-01

    T. maritima TrmFO was overexpressed, purified and crystallized. A diffraction data set was collected to a resolution of 2.6 Å. TrmFO, previously classified as GID, is a methyltransferase that catalyzes the formation of 5-methyluridine or ribothymidine (T) at position 54 in tRNA in some Gram-positive bacteria. To date, TrmFO is the only characterized tRNA methyltransferase that does not use S-adenosylmethionine as the methyl-group donor. Instead, the donor of the methyl group is N 5 ,N 10 -methylenetetrahydrofolate. The crystallization and preliminary X-ray crystallographic studies of TrmFO are reported here. The recombinant protein, cloned from Thermotoga maritima genomic DNA, was overproduced in Esherichia coli and crystallized in 25%(v/v) PEG 4000, 100 mM NaCl and sodium citrate buffer pH 5.0 at 291 K using the hanging-drop vapor-diffusion method. The plate-shaped crystals diffracted to 2.6 Å and belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 79.94, b = 92.46, c = 127.20 Å

  10. Crystallization and preliminary X-ray crystallographic characterization of TrmFO, a folate-dependent tRNA methyltransferase from Thermotoga maritima

    Energy Technology Data Exchange (ETDEWEB)

    Cicmil, Nenad, E-mail: cicmil@uiuc.edu [Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)

    2008-03-01

    T. maritima TrmFO was overexpressed, purified and crystallized. A diffraction data set was collected to a resolution of 2.6 Å. TrmFO, previously classified as GID, is a methyltransferase that catalyzes the formation of 5-methyluridine or ribothymidine (T) at position 54 in tRNA in some Gram-positive bacteria. To date, TrmFO is the only characterized tRNA methyltransferase that does not use S-adenosylmethionine as the methyl-group donor. Instead, the donor of the methyl group is N{sup 5},N{sup 10}-methylenetetrahydrofolate. The crystallization and preliminary X-ray crystallographic studies of TrmFO are reported here. The recombinant protein, cloned from Thermotoga maritima genomic DNA, was overproduced in Esherichia coli and crystallized in 25%(v/v) PEG 4000, 100 mM NaCl and sodium citrate buffer pH 5.0 at 291 K using the hanging-drop vapor-diffusion method. The plate-shaped crystals diffracted to 2.6 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.94, b = 92.46, c = 127.20 Å.

  11. Coactivator-associated arginine methyltransferase 1 enhances transcriptional activity of the human T-cell lymphotropic virus type 1 long terminal repeat through direct interaction with Tax.

    Science.gov (United States)

    Jeong, Soo-Jin; Lu, Hanxin; Cho, Won-Kyung; Park, Hyeon Ung; Pise-Masison, Cynthia; Brady, John N

    2006-10-01

    In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).

  12. Lysine methyltransferase SMYD2 promotes triple negative breast cancer progression.

    Science.gov (United States)

    Li, Linda Xiaoyan; Zhou, Julie Xia; Calvet, James P; Godwin, Andrew K; Jensen, Roy A; Li, Xiaogang

    2018-02-27

    We identified SMYD2, a SMYD (SET and MYND domain) family protein with lysine methyltransferase activity, as a novel breast cancer oncogene. SMYD2 was expressed at significantly higher levels in breast cancer cell lines and in breast tumor tissues. Silencing of SMYD2 by RNAi in triple-negative breast cancer (TNBC) cell lines or inhibition of SMYD2 with its specific inhibitor, AZ505, significantly reduced tumor growth in vivo. SMYD2 executes this activity via methylation and activation of its novel non-histone substrates, including STAT3 and the p65 subunit of NF-κB, leading to increased TNBC cell proliferation and survival. There are cross-talk and synergistic effects among SMYD2, STAT3, and NF-κB in TNBC cells, in that STAT3 can contribute to the modification of NF-κB p65 subunit post-translationally by recruitment of SMYD2, whereas the p65 subunit of NF-κB can also contribute to the modification of STAT3 post-translationally by recruitment of SMYD2, leading to methylation and activation of STAT3 and p65 in these cells. The expression of SMYD2 can be upregulated by IL-6-STAT3 and TNFα-NF-κB signaling, which integrates epigenetic regulation to inflammation in TNBC development. In addition, we have identified a novel SMYD2 transcriptional target gene, PTPN13, which links SMYD2 to other known breast cancer associated signaling pathways, including ERK, mTOR, and Akt signaling via PTPN13 mediated phosphorylation.

  13. Structural Basis of Substrate Recognition in Thiopurine S-Methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yi; Feng, Qiping; Wilk, Dennis; Adjei, Araba A.; Salavaggione, Oreste E.; Weinshilboum, Richard M.; Yee, Vivien C. (Case Western); (MCCM)

    2008-09-23

    Thiopurine S-methyltransferase (TPMT) modulates the cytotoxic effects of thiopurine prodrugs such as 6-mercaptopurine by methylating them in a reaction using S-adenosyl-l-methionine as the donor. Patients with TPMT variant allozymes exhibit diminished levels of protein and/or enzyme activity and are at risk for thiopurine drug-induced toxicity. We have determined two crystal structures of murine TPMT, as a binary complex with the product S-adenosyl-l-homocysteine and as a ternary complex with S-adenosyl-l-homocysteine and the substrate 6-mercaptopurine, to 1.8 and 2.0 {angstrom} resolution, respectively. Comparison of the structures reveals that an active site loop becomes ordered upon 6-mercaptopurine binding. The positions of the two ligands are consistent with the expected S{sub N}2 reaction mechanism. Arg147 and Arg221, the only polar amino acids near 6-mercaptopurine, are highlighted as possible participants in substrate deprotonation. To probe whether these residues are important for catalysis, point mutants were prepared in the human enzyme. Substitution of Arg152 (Arg147 in murine TPMT) with glutamic acid decreases V{sub max} and increases K{sub m} for 6-mercaptopurine but not K{sub m} for S-adenosyl-l-methionine. Substitution at this position with alanine or histidine and similar substitutions of Arg226 (Arg221 in murine TPMT) result in no effect on enzyme activity. The double mutant Arg152Ala/Arg226Ala exhibits a decreased V{sub max} and increased K{sub m} for 6-mercaptopurine. These observations suggest that either Arg152 or Arg226 may participate in some fashion in the TPMT reaction, with one residue compensating when the other is altered, and that Arg152 may interact with substrate more directly than Arg226, consistent with observations in the murine TPMT crystal structure.

  14. A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase

    NARCIS (Netherlands)

    van Haren, Matthijs J; Sastre Torano, Javier; Sartini, Davide; Emanuelli, Monica; Parsons, Richard B; Martin, Nathaniel I

    2016-01-01

    Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine

  15. Assessment of TRMM 3B43 product for drought monitoring in Singapore

    Science.gov (United States)

    Tan, Mou Leong; Chua, Vivien P.; Tan, Kok Chooi; Brindha, K.

    2017-10-01

    Drought is one of the most hazardous natural disasters for human beings and the environment. Using only rain gauge is insufficient to monitor the drought pattern effectively as it impacts large areas. This situation is more critical on small island countries, with limited rain gauges for monitoring drought pattern over the ocean regions. This study aims to assess the capability of Tropical Rainfall Measuring Mission (TRMM) Multisatellite Precipitation Analysis (TMPA) 3B43 product in monitoring drought in Singapore from 1998 to 2014. The Standardized Precipitation Index (SPI) at various time-scales is used for identifying drought patterns. Results show moderate to good correlations between TMPA- 3B43 and rain gauges in the SPI estimations. Besides that, TMPA-3B43 exhibits a similar temporal drought behavior as the rain gauges. These findings indicate the TMPA 3B43 product as a very useful tool to study drought pattern over Singapore.

  16. NUSTAR and the status of the R3B project at FAIR

    Indian Academy of Sciences (India)

    elementary building blocks of matter and the fundamental forces acting between them on an ... The above general reasoning has let the German Government to approve a com- .... At the high-energy branch, the R3B concept builds upon.

  17. Synthesis of pyrrolo(2,3-b)quinolines by palladium-catalyzed heteroannulation

    International Nuclear Information System (INIS)

    Gee, Moon Bae; Lee, Won Jung; Yum, Eul Kgun

    2003-01-01

    Palladium-catalyzed heteroannulation of 2-amino-3-iodoquinoline derivatives and 1-trimethylsilyl internal alkynes provided highly regioselective pyrrolo(2,3-b)quinolines with trimethylsilyl group next to the nitrogen atom in the pyrrole ring

  18. Neuropsychology and neuropharmacology of P3a and P3b.

    Science.gov (United States)

    Polich, John; Criado, José R

    2006-05-01

    Perspectives on the P300 event-related brain potential (ERP) are reviewed by outlining the distinction between the P3a and P3b subcomponents. The critical factor for eliciting P3a is how target/standard discrimination difficulty rather than novelty modulates task processing. The neural loci of P3a and P3b generation are sketched and a theoretical model is developed. P3a originates from stimulus-driven disruption of frontal attention engagement during task processing. P3b originates when temporal-parietal mechanisms process the stimulus information for memory storage. The neuropharmacological implications of this view are then outlined by evaluating how acute and chronic use of ethanol, marijuana, and nicotine affect P3a and P3b. The findings suggest that the circuit underlying ERP generation is influenced in a different ways for acute intake and varies between chronic use levels across drugs. Theoretical implications are assessed.

  19. Studies on N5-methyltetrahydrofolate-homocystein methyltransferase in normal and leukemia leukocytes.

    Science.gov (United States)

    Peytremann, R; Thorndike, J; Beck, W S

    1975-11-01

    A cobalamin-dependent N5-methyltetra-hydrofolate-homocysteine methyltransferase (methyl-transferase) was demonstrated in unfractioned extracts of human normal and leukemia leukocytes. Activity was substantially reduced in the absence of an added cobalamin derivative. Presumably, this residual activity reflects the endogeneous level of holoenzyme. Enzyme activity was notably higher in lymphoid cells than in myeloid cells. Thus, mean specific activities (+/-SD) were: chronic lymphocytic leukemia lymphocytes, 2.15+/-1.16; normal lymphocytes, 0.91+/-0.59; normal mature granulocytes, 0.15+/-0.10; chronic myelocytic leukemia granulocytes, barely detectable activity. Properties of leukocytes enzymes resembled those of methyltransferases previously studied in bacteria and other animal cells. Granulocytes and chronic myelocytic leukemia cells contain a factor or factors that inhibits Escherichia coli enzyme. The data suggest that the prominence of this cobalamin-dependent enzyme in lymphocytes and other mononuclear cell types may be related to their potential for cell division.

  20. Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase from Halorhodospira halochoris

    International Nuclear Information System (INIS)

    Kallio, Juha Pekka; Jänis, Janne; Nyyssölä, Antti; Hakulinen, Nina; Rouvinen, Juha

    2009-01-01

    The crystallization and preliminary X-ray diffraction analysis of sarcosine dimethylglycine methyltransferase from H. halochoris is reported. Sarcosine dimethylglycine methyltransferase (EC 2.1.1.157) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 Å resolution and the orthorhombic crystal diffracted to 1.8 Å resolution

  1. Human T-Cell Leukemia Virus Type I-Mediated Repression of PDZ-LIM Domain-Containing Protein 2 Involves DNA Methylation But Independent of the Viral Oncoprotein Tax

    Directory of Open Access Journals (Sweden)

    Pengrong Yan

    2009-10-01

    Full Text Available Human T-cell leukemia virus type I (HTLV-I is the etiological agent of adult T-cell leukemia (ATL. Our recent studies have shown that one important mechanism of HTLV-I-Mediated tumorigenesis is through PDZ-LIM domain-containing protein 2 (PDLIM2 repression, although the involved mechanism remains unknown. Here, we further report that HTLV-I-Mediated PDLIM2 repression was a pathophysiological event and the PDLIM2 repression involved DNA methylation. Whereas DNA methyltransferases 1 and 3b but not 3a were upregulated in HTLV-I-transformed T cells, the hypomethylating agent 5-aza-2′-deoxycytidine (5-aza-dC restored PDLIM2 expression and induced death of these malignant cells. Notably, the PDLIM2 repression was independent of the viral regulatory protein Tax because neither short-term induction nor long-term stable expression of Tax could downregulate PDLIM2 expression. These studies provide important insights into PDLIM2 regulation, HTLV-I leukemogenicity, long latency, and cancer health disparities. Given the efficient antitumor activity with no obvious toxicity of 5-aza-dC, these studies also suggest potential therapeutic strategies for ATL.

  2. Germline recombination in a novel Cre transgenic line, Prl3b1-Cre mouse.

    Science.gov (United States)

    Al-Soudy, Al-Sayed; Nakanishi, Tsuyoshi; Mizuno, Seiya; Hasegawa, Yoshikazu; Shawki, Hossam H; Katoh, Megumi C; Basha, Walaa A; Ibrahim, Abdelaziz E; El-Shemy, Hany A; Iseki, Hiroyoshi; Yoshiki, Atsushi; Hiromori, Youhei; Nagase, Hisamitsu; Takahashi, Satoru; Oishi, Hisashi; Sugiyama, Fumihiro

    2016-07-01

    Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1-cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL-2) protein in placenta along with increased expression toward the end of pregnancy. PL-2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1-cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1-cre;R26GRR mice revealed that tdsRed-positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1-cre;R26GRR testes suggested that Cre-mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1-cre mice line provides a unique resource to understand testicular germ-cell development. genesis 54:389-397, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Myelotoxicity after high-dose methotrexate in childhood acute leukemia is influenced by 6-mercaptopurine dosing but not by intermediate thiopurine methyltransferase activity

    DEFF Research Database (Denmark)

    Levinsen, Mette; Rosthøj, Susanne; Nygaard, Ulrikka

    2015-01-01

    Purpose: Through enhancement of 6-mercaptopurine (6MP) bioavailability and inhibition of purine de novo synthesis, high-dose methotrexate (HD-MTX) may increase incorporation into DNA of 6-thioguanine nucleotides, the cytotoxic metabolites of 6MP. Patients with intermediate activity of thiopurine...... methyltransferase (TPMTIA) have higher cytosol 6-thioguanine nucleotide levels. We investigated toxicity following HD-MTX during MTX/6MP maintenance therapy in relation to 6MP and TPMT. Methods: Using linear mixed models, we explored myelo- and hepatotoxicity in relation to 6MP dosage and TPMT phenotype following 1......,749 HD-MTX courses to 411 children with acute lymphoblastic leukemia on maintenance therapy. Results: The degree of myelosuppression following HD-MTX was similar for patients with TPMTIA and patients with high TPMT activity (TPMTHA), when HD-MTX started with same blood counts and 6MP doses. However...

  4. Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine

    Directory of Open Access Journals (Sweden)

    Benitez-Bribiesca Luis

    2006-08-01

    Full Text Available Abstract Background The development of resistance to cytotoxic chemotherapy continues to be a major obstacle for successful anticancer therapy. It has been shown that cells exposed to toxic concentrations of commonly used cancer chemotherapy agents develop DNA hypermetylation. Hence, demethylating agents could play a role in overcoming drug resistance. Methods MCF-7 cells were rendered adriamycin-resistant by weekly treatment with adriamycin. Wild-type and the resulting MCF-7/Adr cells were analyzed for global DNA methylation. DNA methyltransferase activity and DNA methyltransferase (dnmt gene expression were also determined. MCF-7/Adr cells were then subjected to antisense targeting of dnmt1, -3a, and -b genes and to treatment with the DNA methylation inhibitor hydralazine to investigate whether DNA demethylation restores sensitivity to adriamycin. Results MCF-7/Adr cells exhibited the multi-drug resistant phenotype as demonstrated by adriamycin resistance, mdr1 gene over-expression, decreased intracellular accumulation of adriamycin, and cross-resistance to paclitaxel. The mdr phenotype was accompanied by global DNA hypermetylation, over-expression of dnmt genes, and increased DNA methyltransferase activity as compared with wild-type MCF-7 cells. DNA demethylation through antisense targeting of dnmts or hydralazine restored adriamycin sensitivity of MCF-7/Adr cells to a greater extent than verapamil, a known inhibitor of mdr protein, suggesting that DNA demethylation interferes with the epigenetic reprogramming that participates in the drug-resistant phenotype. Conclusion We provide evidence that DNA hypermethylation is at least partly responsible for development of the multidrug-resistant phenotype in the MCF-7/Adr model and that hydralazine, a known DNA demethylating agent, can revert the resistant phenotype.

  5. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation.

    Directory of Open Access Journals (Sweden)

    Si-Yang Liu

    Full Text Available Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase of species with validated DNA methylation. The lack of repeat contents of the A. flavus' genome and the high RIP-index of the small amount of remanent repeat potentially support our speculation that DNA methylation may be absent in A. flavus or that it may possess de novo DNA methylation which occurs very transiently during the obscure sexual stage of this fungal species. This work contributes to our understanding on the DNA methylation status of A. flavus, as well as reinforces our views on the DNA methylation in fungal species. In addition, our strategy of applying bisulfite sequencing to DNA methylation detection in species with low DNA methylation may serve as a reference for later scientific investigations in other hypomethylated species.

  6. Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability.

    Science.gov (United States)

    Heidari, Abolfazl; Tongsook, Chanakan; Najafipour, Reza; Musante, Luciana; Vasli, Nasim; Garshasbi, Masoud; Hu, Hao; Mittal, Kirti; McNaughton, Amy J M; Sritharan, Kumudesh; Hudson, Melissa; Stehr, Henning; Talebi, Saeid; Moradi, Mohammad; Darvish, Hossein; Arshad Rafiq, Muhammad; Mozhdehipanah, Hossein; Rashidinejad, Ali; Samiei, Shahram; Ghadami, Mohsen; Windpassinger, Christian; Gillessen-Kaesbach, Gabriele; Tzschach, Andreas; Ahmed, Iltaf; Mikhailov, Anna; Stavropoulos, D James; Carter, Melissa T; Keshavarz, Soraya; Ayub, Muhammad; Najmabadi, Hossein; Liu, Xudong; Ropers, Hans Hilger; Macheroux, Peter; Vincent, John B

    2015-10-15

    Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability

    Science.gov (United States)

    Heidari, Abolfazl; Tongsook, Chanakan; Najafipour, Reza; Musante, Luciana; Vasli, Nasim; Garshasbi, Masoud; Hu, Hao; Mittal, Kirti; McNaughton, Amy J. M.; Sritharan, Kumudesh; Hudson, Melissa; Stehr, Henning; Talebi, Saeid; Moradi, Mohammad; Darvish, Hossein; Arshad Rafiq, Muhammad; Mozhdehipanah, Hossein; Rashidinejad, Ali; Samiei, Shahram; Ghadami, Mohsen; Windpassinger, Christian; Gillessen-Kaesbach, Gabriele; Tzschach, Andreas; Ahmed, Iltaf; Mikhailov, Anna; Stavropoulos, D. James; Carter, Melissa T.; Keshavarz, Soraya; Ayub, Muhammad; Najmabadi, Hossein; Liu, Xudong; Ropers, Hans Hilger; Macheroux, Peter; Vincent, John B.

    2015-01-01

    Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability. PMID:26206890

  8. Evolution of DNA Methylation across Insects.

    Science.gov (United States)

    Bewick, Adam J; Vogel, Kevin J; Moore, Allen J; Schmitz, Robert J

    2017-03-01

    DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Current use of pharmacogenetic testing: a national survey of thiopurine methyltransferase testing prior to azathioprine prescription.

    Science.gov (United States)

    Fargher, E A; Tricker, K; Newman, W; Elliott, R; Roberts, S A; Shaffer, J L; Bruce, I; Payne, K

    2007-04-01

    Azathioprine is an immunosuppressant prescribed for the treatment of inflammatory conditions and after organ transplantation. Risk of neutropaenia has limited the effective use of azathioprine (AZA) and driven requirements for careful monitoring and blood tests. Thiopurine methyltransferase (TPMT) is a genetically moderated key enzyme involved in the metabolism of AZA that can be used to stratify individuals into different levels of risk of developing neutropaenia. Two techniques can be used to measure TPMT status: enzyme-level testing (phenotype testing) and DNA based testing (genotype testing). To identify the current uptake of TPMT enzyme-level testing, TPMT genotype testing, and, the role of guidelines; to inform the prescribing and monitoring of AZA. A survey was mailed to a consultant dermatologist, gastroenterologist, and rheumatologist at every NHS Hospital Trust in England. The survey comprised mainly closed questions exploring: use of AZA and monitoring; use of TPMT enzyme-level testing and genotype testing; and, the role of guidelines to guide prescribing practice. A 70% (n=287) response rate was obtained. The majority of respondents reported prescribing AZA (99%, n=283). Prescribing and monitoring patterns differed between individual respondents and between the three disciplines. TPMT enzyme-level testing was reportedly used by 67% (n=189) of respondents, but this differed by discipline (dermatologists 94%, gastroenterologists 60%, rheumatologists 47%). In 91% of cases enzyme-level testing was carried out prior to prescribing AZA. Genotype testing is not typically available to NHS clinicians but 15 clinicians (six dermatologists, six gastroenterologists, three rheumatologists) reported using it. Most consultants (82%) reported using guidelines to inform their AZA prescribing and monitoring (dermatologists 81%, gastroenterologists 75%, rheumatologists 94%). Two-thirds of the consultants surveyed in England are using TPMT enzyme-level testing, prior to

  10. Unique Features and Anti-microbial Targeting of Folate- and Flavin-Dependent Methyltransferases Required for Accurate Maintenance of Genetic Information

    Directory of Open Access Journals (Sweden)

    Hannu Myllykallio

    2018-05-01

    Full Text Available Comparative genome analyses have led to the discovery and characterization of novel flavin- and folate-dependent methyltransferases that mainly function in DNA precursor synthesis and post-transcriptional RNA modification by forming (ribo thymidylate and its derivatives. Here we discuss the recent literature on the novel mechanistic features of these enzymes sometimes referred to as “uracil methyltransferases,” albeit we prefer to refer to them as (ribo thymidylate synthases. These enzyme families attest to the convergent evolution of nucleic acid methylation. Special focus is given to describing the unique characteristics of these flavin- and folate-dependent enzymes that have emerged as new models for studying the non-canonical roles of reduced flavin co-factors (FADH2 in relaying carbon atoms between enzyme substrates. This ancient enzymatic methylation mechanism with a very wide phylogenetic distribution may be more commonly used for biological methylation reactions than previously anticipated. This notion is exemplified by the recent discovery of additional substrates for these enzymes. Moreover, similar reaction mechanisms can be reversed by demethylases, which remove methyl groups e.g., from human histones. Future work is now required to address whether the use of different methyl donors facilitates the regulation of distinct methylation reactions in the cell. It will also be of great interest to address whether the low activity flavin-dependent thymidylate synthases ThyX represent ancestral enzymes that were eventually replaced by the more active thymidylate synthases of the ThyA family to facilitate the maintenance of larger genomes in fast-growing microbes. Moreover, we discuss the recent efforts from several laboratories to identify selective anti-microbial compounds that target flavin-dependent thymidylate synthase ThyX. Altogether we underline how the discovery of the alternative flavoproteins required for methylation of DNA and

  11. A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

    Science.gov (United States)

    A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver CytosolShan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas S-adenosyl-L-methionine (AdoMet): ar...

  12. Global developmental delay in guanidionacetate methyltransferase deficiency : differences in formal testing and clinical observation

    NARCIS (Netherlands)

    Verbruggen, Krijn T.; Knijff, Wilma A.; Soorani-Lunsing, Roelineke J.; Sijens, Paul E.; Verhoeven, Nanda M.; Salomons, Gajja S.; Goorhuis-Brouwer, Siena M.; van Spronsen, Francjan J.

    Guanidinoacetate N-methyltransferase (GAMT) deficiency is a defect in the biosynthesis of creatine (Cr). So far, reports have not focused on the description of developmental abilities in this disorder. Here, we present the result of formal testing of developmental abilities in a GAMT-deficient

  13. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases

    NARCIS (Netherlands)

    T.B. van Dijk (Thamar); N. Gillemans (Nynke); C. Stein (Claudia); P. Fanis (Pavlos); J.A.A. Demmers (Jeroen); M.P.C. van de Corput (Mariëtte); J. Essers (Jeroen); F.G. Grosveld (Frank); U.M. Bauer (Uta-Maria); J.N.J. Philipsen (Sjaak)

    2010-01-01

    textabstractWe describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified

  14. Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline

    Science.gov (United States)

    Wu, Qian; Caine, Joanne M.; Thomson, Stuart A.; Slavica, Meri; Grunewald, Gary L.

    2009-01-01

    Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT. PMID:19171483

  15. Tyrosine 87 is vital for the activity of human protein arginine methyltransferase 3 (PRMT3)

    Czech Academy of Sciences Publication Activity Database

    Handrková, H.; Petrák, J.; Halada, Petr; Pospíšilová, D.; Čmejla, R.

    2011-01-01

    Roč. 1814, č. 2 (2011), s. 277-282 ISSN 1570-9639 R&D Projects: GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510 Keywords : DIAMOND-BLACKFAN ANEMIA * SUBSTRATE -SPECIFICITY * N-METHYLTRANSFERASE Subject RIV: CE - Biochemistry Impact factor: 3.635, year: 2011

  16. Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Madsen, Ch. T.; Jakobsen, L.; Buriánková, Karolína; Doucet-Populaire, F.; Perdonet, J. L.; Douthwaite, S.

    2005-01-01

    Roč. 280, č. 47 (2005), s. 38942-38947 ISSN 0021-9258 R&D Projects: GA ČR GA310/03/0292 Institutional research plan: CEZ:AV0Z50200510 Keywords : methyltransferase erm * mycobacterium tuberculosis * rRNA Subject RIV: EE - Microbiology, Virology Impact factor: 5.854, year: 2005

  17. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    Science.gov (United States)

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

  18. A mouse speciation gene encodes a meiotic histone H3 methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Mihola, Ondřej; Trachtulec, Zdeněk; Vlček, Čestmír; Schimenti, J.C.; Forejt, Jiří

    2009-01-01

    Roč. 323, č. 5912 (2009), s. 373-375 ISSN 0036-8075 Institutional research plan: CEZ:AV0Z50520514 Keywords : hybrid sterility * histone H3K4 methyltransferase * Prdm9 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 29.747, year: 2009

  19. The histone methyltransferase SET8 is required for S-phase progression

    DEFF Research Database (Denmark)

    Jørgensen, Stine; Elvers, Ingegerd; Trelle, Morten Beck

    2008-01-01

    Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show...

  20. A fluorescence resonance energy transfer-based method for histone methyltransferases

    DEFF Research Database (Denmark)

    Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla

    2015-01-01

    A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye...

  1. The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Santoni-Rugiu, Eric; Simon, Ronald

    2011-01-01

    Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involved...

  2. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Jensen, Michael R

    2004-01-01

    SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show...

  3. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing

    DEFF Research Database (Denmark)

    Relling, M V; Gardner, E E; Sandborn, W J

    2011-01-01

    Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TP...

  4. Catechol-O-methyltransferase gene methylation and substance use in adolescents : the TRAILS study

    NARCIS (Netherlands)

    van der Knaap, L. J.; Schaefer, J. M.; Franken, I. H. A.; Verhulst, F. C.; van Oort, F. V. A.; Riese, H.

    Substance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val(108/158)Met polymorphism modulates COMT activity

  5. Catechol-O-methyltransferase gene methylation and substance use in adolescents: The TRAILS study

    NARCIS (Netherlands)

    L.J. van der Knaap (Lisette); J.M. Schäfer (Johanna); I.H.A. Franken (Ingmar); F.C. Verhulst (Frank); F.V.A. van Oort (Floor); H. Riese (Harriëtte)

    2014-01-01

    textabstractSubstance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val108/158Met polymorphism

  6. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

    Science.gov (United States)

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

  7. Photometric investigation of hot exoplanets: TrES-3b and Qatar-1b

    Science.gov (United States)

    Püsküllü, Ç.; Soydugan, F.; Erdem, A.; Budding, E.

    2017-08-01

    New photometric follow-up observations of transitting 'hot Jupiters' TrES-3b and Qatar-1b are presented. Weighted mean values of the solutions of light curves in R-filter for both planetary systems are reported and compared with the previous results. The transit light curves were analysed using the WINFITTER code. The physical properties of the planets were estimated. The planet radii are found to be Rp = 1.381 ± 0.033RJ for TrES-3b and Rp = 1.142 ± 0.025RJ for Qatar-1b. Transit times and their uncertainties were also determined and a new linear ephemeris was computed for both systems. Analysis of transit times showed that a significant signal could not be determined for TrES-3b, while weak evidence was found for Qatar-1b, which might be tested using more precise future transit times.

  8. Crystallization and preliminary diffraction studies of CBM3b of cellobiohydrolase 9A from Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Jindou, Sadanari [Department of Molecular Microbiology and Biotechnology, Tel Aviv University 69978 (Israel); Petkun, Svetlana [Department of Molecular Microbiology and Biotechnology, Tel Aviv University 69978 (Israel); The Daniella Rich Institute for Structural Biology, Tel Aviv University 69978 (Israel); Shimon, Linda [Department of Chemical Research Support, The Weizmann Institute of Science, Rehovot 76100 (Israel); Bayer, Edward A. [Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100 (Israel); Lamed, Raphael; Frolow, Felix, E-mail: mbfrolow@post.tau.ac.il [Department of Molecular Microbiology and Biotechnology, Tel Aviv University 69978 (Israel); The Daniella Rich Institute for Structural Biology, Tel Aviv University 69978 (Israel)

    2007-12-01

    The cloning, expression, purification and crystallization of the CBM3b module of cellobiohydrolase 9A from C. thermocellum is described. The crystals diffract to 2.68 Å. Family 3 carbohydrate-binding modules (CBM3s) are associated with the scaffoldin subunit of the multi-enzyme cellulosome complex and with the family 9 glycoside hydrolases, which are multimodular enzymes that act on plant cell-wall polysaccharides, notably cellulose. Here, the crystallization of CBM3b from cellobiohydrolase 9A is reported. The crystals are tetragonal and belong to space group P4{sub 1} or P4{sub 3}. X-ray diffraction data for CBM3b have been collected to 2.68 Å resolution on beamline ID14-4 at the ESRF.

  9. THERMAL EMISSION AND TIDAL HEATING OF THE HEAVY AND ECCENTRIC PLANET XO-3b

    International Nuclear Information System (INIS)

    Machalek, Pavel; Greene, Tom; McCullough, Peter R.; Burrows, Adam; Burke, Christopher J.; Hora, Joseph L.; Johns-Krull, Christopher M.; Deming, Drake L.

    2010-01-01

    We determined the flux ratios of the heavy and eccentric planet XO-3b to its parent star in the four Infrared Array Camera bands of the Spitzer Space Telescope: 0.101% ± 0.004% at 3.6 μm; 0.143% ± 0.006% at 4.5 μm; 0.134% ± 0.049% at 5.8 μm; and 0.150% ± 0.036% at 8.0 μm. The flux ratios are within [-2.2, 0.3, -0.8, and -1.7]σ of the model of XO-3b with a thermally inverted stratosphere in the 3.6 μm, 4.5 μm, 5.8 μm, and 8.0 μm channels, respectively. XO-3b has a high illumination from its parent star (F p ∼ (1.9-4.2) x 10 9 erg cm -2 s -1 ) and is thus expected to have a thermal inversion, which we indeed observe. When combined with existing data for other planets, the correlation between the presence of an atmospheric temperature inversion and the substellar flux is insufficient to explain why some high insolation planets like TrES-3 do not have stratospheric inversions and some low insolation planets like XO-1b do have inversions. Secondary factors such as sulfur chemistry, atmospheric metallicity, amounts of macroscopic mixing in the stratosphere, or even dynamical weather effects likely play a role. Using the secondary eclipse timing centroids, we determined the orbital eccentricity of XO-3b as e = 0.277 ± 0.009. The model radius-age trajectories for XO-3b imply that at least some amount of tidal heating is required to inflate the radius of XO-3b, and the tidal heating parameter of the planet is constrained to Q p ∼ 6 .

  10. Type III methyltransferase M.NgoAX from Neisseria gonorrhoeae FA1090 regulates biofilm formation and human cell invasion

    Directory of Open Access Journals (Sweden)

    Agnieszka eKwiatek

    2015-12-01

    Full Text Available Neisseria gonorrhoeae is the etiological factor of the sexually transmitted gonorrhea disease that may lead, under specific conditions, to systemic infections. The gonococcal genome encodes many Restriction Modification (RM systems, which main biological role is to defend the pathogen from potentially harmful foreign DNA. However, RM systems seem also to be involved in several other functions. In this study, we examined the effect of inactivation the N. gonorrhoeae FA1090 ngo0545 gene encoding M.NgoAX methyltransferase on the global gene expression, biofilm formation, interactions with human epithelial host cells and overall bacterial growth. Expression microarrays showed at least a two-fold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wt strain under standard grow conditions. As determined by the assay with crystal violet, the NgoAX knock-out strain formed a slightly larger biofilm biomass per cell than the wt strain (OD570/600 = 13.8  2.24 and 9.35  2.06, respectively. SCLM observations showed that the biofilm formed by the gonococcal ngo0545 gene mutant is more relaxed and dispersed than the one formed by the wt strain. Thickness of the biofilm formed by both strains was 48.3 (14.9 µm for the mutant and 28.6 (4.0 µm for the wt. This more relaxed feature of the biofilm in respect to adhesion and bacterial interactions seems advantageous for pathogenesis of the NgoAX-deficient gonococci at the stage of human epithelial cell invasion. Indeed, the overall adhesion of mutant bacterial cells to human cells was lower than adhesion of the wt gonococci (adhesion index = 0.672 ( 0.2 and 2.15 ( 1.53, respectively; yet, a higher number of mutant than wt bacteria were found inside the Hec-1-B epithelial cells (invasion index = 3.38 ( 0.93  105 for mutant and 4.67 ( 3.09  104 for the wt strain. These results indicate that NgoAX-deficient cells have lower ability to attach to human cells

  11. A 128-channel event driven readout ASIC for the R3B tracker

    International Nuclear Information System (INIS)

    Jones, L.; Bell, S.; Morrissey, Q.; Prydderch, M.; Church, I.; Lazarus, I.; Kogimtzis, M.; Pucknell, V.; Labiche, M.; Thornhill, J.; Borri, M.

    2016-01-01

    R 3 B is a detector with high efficiency, acceptance, and resolution for kinematically complete measurements of reactions with high-energy radioactive beams. Detectors track and identify radioactive beams into and out of a reaction target. Three layers of double-sided stereoscopic silicon strips form the tracker detector which must provide precise tracking and vertex determination and in addition include energy and multiplicity measurements. The R 3 B ASIC has been manufactured and is intended for processing and digitising signals generated by ionising particles passing through the tracker. The ASIC processes signals and provides spatial, energy and time measurements

  12. fMRI assessment of somatotopy in human Brodmann area 3b by electrical finger stimulation.

    Science.gov (United States)

    Kurth, R; Villringer, K; Mackert, B M; Schwiemann, J; Braun, J; Curio, G; Villringer, A; Wolf, K J

    1998-01-26

    Functional magnetic resonance imaging (fMRI) is capable of detecting focal brain activation induced by electrical stimulation of single fingers in human subjects. In eight subjects somatotopic arrangement of the second and fifth finger was found in Brodmann area 3b of the primary somatosensory cortex. In four subjects the representation area of the second finger was located lateral and inferior to the fifth finger; in one subject the somatotopy was reversed. In three subjects representation areas of the two fingers in Brodmann area 3b were found overlapping. Additional activated areas were found on the crown of ipsilateral and contralateral postcentral gyrus (Brodmann areas 1 and 2) and posterior parietal cortex.

  13. Analyzing the Effects of Nitrogen Deficiency on the PHB Production of Methylosinus trichosporium OB3b

    Science.gov (United States)

    Kyauk, E.

    2011-12-01

    Polyhydroxybutyrate (PHB) is a biodegradable thermoplastic that is produced by various microorganisms. Because of its potential to replace conventional plastics, it has been closely researched in the past few years. Methanotrophic bacteria, bacteria that consume methane, produce this bioplastic when it lacks certain nutrients. The utilization of methane to produce PHB shows much promise as methane is a cheap, plentiful gas. In this study, we observed the methanotroph, Methylosinus trichosporium OB3b , and its yield of PHB in the absence of nitrogen. The optical density of Methylosinus trichosporium OB3b was measured in order to observe cell growth and PHB production patterns over a 48 hour period.

  14. Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

    Science.gov (United States)

    Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

    2018-01-01

    Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

  15. Floral Benzenoid Carboxyl Methyltransferases: From in Vitro to in Planta Function

    Energy Technology Data Exchange (ETDEWEB)

    Effmert,U.; Saschenbrecker, S.; Ross, J.; Negre, F.; Fraser, C.; Noel, J.; Dudareva, N.; Piechulla, B.

    2005-01-01

    Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT's three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in plants depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses

  16. An 11bp region with stem formation potential is essential for de novo DNA methylation of the RPS element.

    Directory of Open Access Journals (Sweden)

    Matthew Gentry

    Full Text Available The initiation of DNA methylation in Arabidopsis is controlled by the RNA-directed DNA methylation (RdDM pathway that uses 24nt siRNAs to recruit de novo methyltransferase DRM2 to the target site. We previously described the REPETITIVE PETUNIA SEQUENCE (RPS fragment that acts as a hot spot for de novo methylation, for which it requires the cooperative activity of all three methyltransferases MET1, CMT3 and DRM2, but not the RdDM pathway. RPS contains two identical 11nt elements in inverted orientation, interrupted by a 18nt spacer, which resembles the features of a stemloop structure. The analysis of deletion/substitution derivatives of this region showed that deletion of one 11nt element RPS is sufficient to eliminate de novo methylation of RPS. In addition, deletion of a 10nt region directly adjacent to one of the 11nt elements, significantly reduced de novo methylation. When both 11nt regions were replaced by two 11nt elements with altered DNA sequence but unchanged inverted repeat homology, DNA methylation was not affected, indicating that de novo methylation was not targeted to a specific DNA sequence element. These data suggest that de novo DNA methylation is attracted by a secondary structure to which the two 11nt elements contribute, and that the adjacent 10nt region influences the stability of this structure. This resembles the recognition of structural features by DNA methyltransferases in animals and suggests that similar mechanisms exist in plants.

  17. O6-alkylguanine-DNA alkyltransferase activity correlates with the therapeutic response of human rhabdomyosarcoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea

    International Nuclear Information System (INIS)

    Brent, T.P.; Houghton, P.J.; Houghton, J.A.

    1985-01-01

    Immune-deprived female CBA/CaJ mice bearing xenografts of six different human rhabdomyosarcoma lines were treated with 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU). Tumor responses were compared with levels of O 6 -methylguanine-DNA methyltransferase activity because of evidence indicating that repair of DNA interstrand cross-link precursors, mediated by the transferase, may be an important determinant of MeCCNU cytotoxicity. Levels of methyltransferase in tumor extracts were measured by determining the loss of O 6 -methylguanine from 3 H-labeled methylated DNA. Five of the six tumor lines examined showed either no response to MeCCNU or regrowth after an incomplete response. In each instance, the extent of tumor regression correlated with the level of O 6 -methylguanine-DNA methyltransferase activity in tumor extracts. These results suggest that O 6 -methylguanine-DNA methyltransferase levels in human tumor cells may be a clinically useful predictor of sensitivity to the chloroethylnitrosoureas

  18. Conversion of chlorinated propanes by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

    NARCIS (Netherlands)

    Bosma, T.; Janssen, D.B.

    Chlorinated propanes are important pollutants that may show persistent behaviour in the environment. The biotransformation of 1-chloropropane, 1,2-dichloropropane, 1,3-dichloropropane and 1,2,3-trichloropropane was studied using resting cell suspensions of Methylosinus trichosporium OB3b expressing

  19. Data of evolutionary structure change: 1ADEA-1DJ3B [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1ADEA-1DJ3B 1ADE 1DJ3 A B -------GNNVVVLGTQWGDEGKGKIVDLLTERAKYVVR.../pdbID> A 1ADEA LVING...pdbChain>A 1ADEA ALDNA-REKAR A 1ADEA RGAKAIGTTGR A 1ADEA HNFQLVNYYKA

  20. Online / Offline reconstruction of trigger-less readout in the R3B experiment at FAIR

    International Nuclear Information System (INIS)

    Kresan, Dmytro; Al-Turany, Mohammad; Uhlig, Florian

    2015-01-01

    The R3B (Reactions with Rare Radioactive Beams) experiment is one of the planned experiments at the future FAIR facility at GSI Darmstadt. R3B will cover experimental reaction studies with exotic nuclei far off stability, thus enabling a broad physics program with rare-isotope beams with emphasis on nuclear structure and dynamics. Several different detection subsystems as well as sophisticated DAQ system and data-analysis software are being developed for this purpose. The data analysis software for R3B is based on FairRoot framework and called R3BRoot. R3BRoot is being used for simulation and detector design studies for the last few years. Recently, it was successfully used directly with the data acquisition and for the analysis of the R3B test beam-time in April 2014. For the future beam times the framework has to deal with the free streaming readout of the detectors. The implementation within R3BRoot to fulfil this trigger-less run mode will be discussed in this paper, as well as the set of tools developed for the online reconstruction and quality assurance of the data during the run. (paper)

  1. Degradation of Chlorinated Aliphatic Hydrocarbons by Methylosinus trichosporium OB3b Expressing Soluble Methane Monooxygenase

    NARCIS (Netherlands)

    Oldenhuis, R.; Vink, Ruud L.J.M.; Janssen, D. B.; Witholt, B.

    1989-01-01

    Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium

  2. Kinetics of Chlorinated Hydrocarbon Degradation by Methylosinus trichosporium OB3b and Toxicity of Trichloroethylene

    NARCIS (Netherlands)

    Oldenhuis, Roelof; Oedzes, Johannes Y.; Waarde, Jacob J. van der; Janssen, Dick B.

    The kinetics of the degradation of trichloroethylene (TCE) and seven other chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b were studied. All experiments were performed with cells grown under copper stress and thus expressing soluble methane monooxygenase. Compounds that were

  3. A superconducting battery material: Lithium gold boride (LiAu3B)

    Science.gov (United States)

    Aydin, Sezgin; Şimşek, Mehmet

    2018-04-01

    The superconducting and potential cathode material properties of ternary boride of LiAu3B have been investigated by density functional first principles. The Li-concentration effects on the actual electronic and structural properties, namely the properties of LixAu9B3 (x = 0, 1, 2) sub-systems are studied. It is remarkably shown that the existence of Li-atoms has no considerable effect on the structural properties of Au-B skeleton in LiAu3B. Then, it can be offered as a potential cathode material for Li-ion batteries with the very small volume deviation of 0.42%, and the suitable average open circuit voltage of ∼1.30 V. Furthermore, the vibrational and superconducting properties such as electron-phonon coupling constant (λ) and critical temperature (Tc) of LiAu3B are studied. The calculated results suggest that LiAu3B should be a superconductor with Tc ∼5.8 K, also.

  4. Effect of chlorinated ethene conversion on viability and activity of Methylosinus trichosporium OB3b

    NARCIS (Netherlands)

    van Hylckama Vlieg, Johan E.T.; Koning, Wim de; Janssen, Dick B.

    1997-01-01

    The effect of transformation of chlorinated ethenes on the cell viability of Methylosinus trichosporium OB3b was investigated. A comparison of the loss of viability with the decrease in transformation rates shelved that for the monooxygenase-mediated transformation of all chlorinated ethenes except

  5. Evaluation of IMERG and TRMM 3B43 Monthly Precipitation Products over Mainland China

    Directory of Open Access Journals (Sweden)

    Fengrui Chen

    2016-06-01

    Full Text Available As the successor of the Tropical Rainfall Measuring Mission (TRMM, the Global Precipitation Measurement (GPM mission significantly improves the spatial resolution of precipitation estimates from 0.25° to 0.1°. The present study analyzed the error structures of Integrated Multisatellite Retrievals for GPM (IMERG monthly precipitation products over Mainland China from March 2014 to February 2015 using gauge measurements at multiple spatiotemporal scales. Moreover, IMERG products were also compared with TRMM 3B43 products. The results show that: (1 overall, IMERG can capture the spatial patterns of precipitation over China well. It performs a little better than TRMM 3B43 at seasonal and monthly scales; (2 the performance of IMERG varies greatly spatially and temporally. IMERG performs better at low latitudes than at middle latitudes, and shows worse performance in winter than at other times; (3 compared with TRMM 3B43, IMERG significantly improves the estimation accuracy of precipitation over the Xinjiang region and the Qinghai-Tibetan Plateau, especially over the former where IMERG increases Pearson correlation coefficient by 0.18 and decreases root-mean-square error by 54.47 mm for annual precipitation estimates. However, most IMERG products over these areas are unreliable; and (4 IMERG shows poor performance in winter as TRMM 3B43 even if GPM improved its ability to sense frozen precipitation. Most of them over North China are unreliable during this period.

  6. 17 CFR 240.3b-2 - Definition of “officer”.

    Science.gov (United States)

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Definition of âofficerâ. 240... Securities Exchange Act of 1934 Definitions § 240.3b-2 Definition of “officer”. The term officer means a... accounting officer, and any person routinely performing corresponding functions with respect to any...

  7. Off-wing fleet maintenance study of a CFM56-3B turbofan engine ...

    African Journals Online (AJOL)

    An off wing fleet maintenance study of the CFM56-3B Turbofan engine that propels the Boeing 737-300 aircraft is presented. The engine performance and deteriorating behavior was modeled with a view to estimate the creep life consumption and operating severity. The predicted severity factor of each degradation was ...

  8. 17 CFR 240.3b-12 - Definition of OTC derivatives dealer.

    Science.gov (United States)

    2010-04-01

    ... to options, forwards, futures, swap agreements, or collars involving currencies, interest or other... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Definition of OTC derivatives... Securities Exchange Act of 1934 Definitions § 240.3b-12 Definition of OTC derivatives dealer. The term OTC...

  9. Zebrafish foxo3b negatively regulates canonical Wnt signaling to affect early embryogenesis.

    Directory of Open Access Journals (Sweden)

    Xun-wei Xie

    Full Text Available FOXO genes are involved in many aspects of development and vascular homeostasis by regulating cell apoptosis, proliferation, and the control of oxidative stress. In addition, FOXO genes have been showed to inhibit Wnt/β-catenin signaling by competing with T cell factor to bind to β-catenin. However, how important of this inhibition in vivo, particularly in embryogenesis is still unknown. To demonstrate the roles of FOXO genes in embryogenesis will help us to further understand their relevant physiological functions. Zebrafish foxo3b gene, an orthologue of mammalian FOXO3, was expressed maternally and distributed ubiquitously during early embryogenesis and later restricted to brain. After morpholino-mediated knockdown of foxo3b, the zebrafish embryos exhibited defects in axis and neuroectoderm formation, suggesting its critical role in early embryogenesis. The embryo-developmental marker gene staining at different stages, phenotype analysis and rescue assays revealed that foxo3b acted its role through negatively regulating both maternal and zygotic Wnt/β-catenin signaling. Moreover, we found that foxo3b could interact with zebrafish β-catenin1 and β-catenin2 to suppress their transactivation in vitro and in vivo, further confirming its role relevant to the inhibition of Wnt/β-catenin signaling. Taken together, we revealed that foxo3b played a very important role in embryogenesis and negatively regulated maternal and zygotic Wnt/β-catenin signaling by directly interacting with both β-catenin1 and β-catenin2. Our studies provide an in vivo model for illustrating function of FOXO transcription factors in embryogenesis.

  10. Development of a graphic interface for the Ramona-3B code; Desarrollo de una interfaz grafica para el codigo Ramona-3B

    Energy Technology Data Exchange (ETDEWEB)

    Maldonado D, D.; Santos O, M.A. [Laboratorio de Analisis de Ingenieria de Reactores Nucleares, Paseo Cuauhnahuac 8532, Jiutepec, Morelos (Mexico)]. e-mail: chalismx@hotmail.com

    2003-07-01

    In this work a graphic interface that interprets the data of the Ramona-3B code is presented. The Ramona-3B code it is a computer program, that it uses text files as input and its generate output also of this type. The quantity of generated information is so big that always it is necessary to process this information with graphic tools to be able to analyze the results of the simulations of nuclear centrals with boiling water reactors. When having a modern tool that it translates text in graphics in an automatic way and that it is of great versatility, one can obtain a graphic interface that facilitates the interpretation of how a BWR nuclear plant behaves. To achieve this tool the key it has been a program that it reads chains of previously indicated characters that keeps the data in a file for later to manipulate them in the creation of the graphic interface. It is used a software of easy access that resists the processing of a great one quantity of data and that later its have been able to graph. Another important function of this interface it is allowing the modification of the input file for Ramona using graphic unfolding and helps in it lines without necessarily to go to the file with input data. For the design of graphic interface it was decided first to show the more representative variables of a BWR type nuclear plant. It is used Mat lab as platform on several options, as PHP, Lab view or C{sup '}. The obtained graphs allow monitoring the plant and to have the control of selected variables. For the graphic interface only is necessary to indicate it the variable to simulate for to be able to interpret graphically the behavior of the BWR type nuclear plant. This tool is of great utility for the teaching of students that they are interested in this type of nuclear topics. (Author)

  11. Functional characterization of KanP, a methyltransferase from the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus.

    Science.gov (United States)

    Nepal, Keshav Kumar; Yoo, Jin Cheol; Sohng, Jae Kyung

    2010-09-20

    KanP, a putative methyltransferase, is located in the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus ATCC12853. Amino acid sequence analysis of KanP revealed the presence of S-adenosyl-L-methionine binding motifs, which are present in other O-methyltransferases. The kanP gene was expressed in Escherichia coli BL21 (DE3) to generate the E. coli KANP recombinant strain. The conversion of external quercetin to methylated quercetin in the culture extract of E. coli KANP proved the function of kanP as S-adenosyl-L-methionine-dependent methyltransferase. This is the first report concerning the identification of an O-methyltransferase gene from the kanamycin gene cluster. The resistant activity assay and RT-PCR analysis demonstrated the leeway for obtaining methylated kanamycin derivatives from the wild-type strain of kanamycin producer. 2009 Elsevier GmbH. All rights reserved.

  12. Carrier frequency of guanidinoacetate methyltransferase deficiency in the general population by functional characterization of missense variants in the GAMT gene

    NARCIS (Netherlands)

    Desroches, C.L.; Patel, J.; Wang, P.X.; Minassian, B.; Marshall, C.R.; Salomons, G.; Mercimek-Mahmutoglu, S.

    2015-01-01

    Guanidinoacetate methyltransferase (GAMT) deficiency is a neurodegenerative disease. Although no symptomatic patients on treatment achieved normal neurodevelopment, three asymptomatic newborns were reported with normal neurodevelopmental outcome on neonatal treatment. GAMT deficiency is therefore a

  13. Investigation of the effect of radiation of DNA methylation patterns

    International Nuclear Information System (INIS)

    Kalinich, J.F.

    1986-01-01

    Four lines of cultured mammalian cells were used in this project: V79A03, a Chinese hamster lung fibroblast; HeLa S-3, an epithelial cell line from a human cervix carcinoma; CHO K-1, an epithelial cell line from a Chinese hamster ovary; and C-1300 N1E-115, a mouse neuroblastoma line. The 5-methylcytosine levels in DNA following exposure to cobalt-60 gamma radiation were measured. Induction of metallothionein in V79A03 cells and acetylcholinesterase in C-1300 N1E-115 cells after irradiation was determined and the effect of radiation on cytoplasmic and nuclear levels of DNA methyltransferase was studied and nuclear demethylase activity assayed. This study showed gamma radiation resulted in a decrease of 5-methylcytosine levels in the DNA of cultured mammalian cells. This radiation-induced hypomethylation resulted in the induction of acetylcholinesterase in mouse neuroblastoma cells and metallothionein in Chinese hamster lung fibroblasts and was caused by a decrease in DNA methyltransferase activity in the nucleus after irradiation and not by the presence of DNA demethylase

  14. RNA-directed DNA methylation: Mechanisms and functions

    KAUST Repository

    Mahfouz, Magdy M.

    2010-07-01

    Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt- long siRNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental, and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives.

  15. Mitochondrial DNA: A Blind Spot in Neuroepigenetics.

    Science.gov (United States)

    Manev, Hari; Dzitoyeva, Svetlana; Chen, Hu

    2012-04-01

    Neuroepigenetics, which includes nuclear DNA modifications such as 5-methylcytosine and 5-hydoxymethylcytosine and modifications of nuclear proteins such as histones, is emerging as the leading field in molecular neuroscience. Historically, a functional role for epigenetic mechanisms, including in neuroepigenetics, has been sought in the area of the regulation of nuclear transcription. However, one important compartment of mammalian cell DNA, different from nuclear but equally important for physiological and pathological processes (including in the brain), mitochondrial DNA has for the most part not had a systematic epigenetic characterization. The importance of mitochondria and mitochondrial DNA (particularly its mutations) in central nervous system physiology and pathology has long been recognized. Only recently have mechanisms of mitochondrial DNA methylation and hydroxymethylation, including the discovery of mitochondrial DNA-methyltransferases and the presence and the functionality of 5-methylcytosine and 5-hydroxymethylcytosine in mitochondrial DNA (e.g., in modifying the transcription of mitochondrial genome), been unequivocally recognized as a part of mammalian mitochondrial physiology. Here we summarize for the first time evidence supporting the existence of these mechanisms and we propose the term "mitochondrial epigenetics" to be used when referring to them. Currently, neuroepigenetics does not include mitochondrial epigenetics - a gap that we expect to close in the near future.

  16. Methylation of the RASSF1A, RARβ2, and SEMA3B genes in epithelial breast and ovarian tumors, and in patients with polyneoplasia

    Directory of Open Access Journals (Sweden)

    T. P. Kazubskaya

    2012-01-01

    Full Text Available The methylation status of the tumor suppressor genes RASSF1A, RARβ2, and SEMA3B was studied in the samples of cancer and its histologically normal tissue of the breast and ovaries. The high rate of abnormal methylation of the CpG islet in the RASSF1A, RARβ2, and SEMA3B genes was found in the tumors of the breast (78% (32/41, 46% (26/56, and 35% (22/65, respectively and ovaries 73% (33/45, 30% (15/50, and 50% (25/51. Hypermethylation in the CpG islets belonging to the RASSF1A and RARβ2 genes was first ascertained in 90% of the patients with polyneoplasms involving the breast and ovaries. Abnormal methylation of the promotor region of the RASSF1A gene was shown to be detectable in preclinical-stage and anaplasia-degree breast and ovarian cancer. There was a correlation of the rate of methylation in the promoter regions of the RARβ2 and SEMA3B genes with clinical-stage and anaplasia-degree breast and ovarian cancer. Analysis of gene methylation in biological fluids provides considerable opportunity to use methylation of DNA as a marker in clinical practice.

  17. Global DNA Methylation in the Chestnut Blight Fungus Cryphonectria parasitica and Genome-Wide Changes in DNA Methylation Accompanied with Sectorization

    Directory of Open Access Journals (Sweden)

    Kum-Kang So

    2018-02-01

    Full Text Available Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase, demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

  18. The new silver borate Ag{sub 3}B{sub 5}O{sub 9}

    Energy Technology Data Exchange (ETDEWEB)

    Sohr, Gerhard; Falkowski, Viktoria; Huppertz, Hubert, E-mail: hubert.huppertz@uibk.ac.at

    2015-05-15

    Single crystals of Ag{sub 3}B{sub 5}O{sub 9} were obtained via high-pressure synthesis at 3 GPa and 600 °C, using a Walker-type multianvil high-pressure device. Ag{sub 3}B{sub 5}O{sub 9} crystalizes with a=674.7(2), b=943.5(2), c=1103.5(2) pm, V=0.7025(2) nm{sup 3}, and Z=4 in the noncentrosymmetric space group P2{sub 1}2{sub 1}2{sub 1} (no. 19). The orthorhombic structure was refined from 3740 independent reflections with R1=0.0496 and wR2=0.587 (all data). It is built up from infinite corner-sharing chains of BO{sub 4} tetrahedra along the a axis, which are interconnected by BO{sub 3} groups to form a network. In the structure, three crystallographically independent sites are occupied with Ag{sup +} cations exhibiting argentophillic interactions. The synthetic conditions as well as the results of the single crystal structure analysis are presented. - Graphical abstract: Noncentrosymmetric silver borate: During investigations in the system Ag–B–O, a new noncentrosymmetric silver borate Ag{sub 3}B{sub 5}O{sub 9} was discovered. The new structure type is built up from corner-sharing BO{sub 3} and BO{sub 4} groups, forming a network. Argentophillic interactions are clearly indicated by the Ag{sup +}⋯Ag{sup +} distances present in the structure. - Highlights: • A noncentrosymmetric borate Ag{sub 3}B{sub 5}O{sub 9} is accessible via high-pressure synthesis. • Ag{sub 3}B{sub 5}O{sub 9} is the second high-pressure silver borate. • Ag{sup +}⋯Ag{sup +} distances in Ag3B5O9 clearly indicate the presence of argentophillic interactions.

  19. BSRF-3B3 Medium Energy X-ray Beamline and Its Application for XAFS Research

    International Nuclear Information System (INIS)

    Ma Chenyan; Cui Mingqi; Zhou Kejin; Zhao Yidong; Tian Yulian; Wu Ziyu; Zheng Lei; Zhu Jie; Zhao Jia; Chen Kai; Sun Lijuan

    2007-01-01

    A new medium X-ray beamline 3B3 covering energy from 1.2 keV up to 6.0 keV was built at Beijing Synchrotron Radiation Facility (BSRF) in 2005. With perfect capability of high monochromaticity, good focus and low harmonics, it could be applied to study metrology, optic component characteristics and medium X-ray absorption spectroscopy (XAS). A simple XAFS apparatus has been set up and some measurements such as S, P, Cl, Ca, Al, Mg K-edge X-ray absorption fine structure (XAFS) in their compounds have also been carried out. The results show that it is feasible to do XAFS research at 3B3 beamline under present condition. The fabrication of a more delicate medium XAFS spectrometer is underway including transmission, fluorescence and electronic yield modes

  20. Microstructural analysis of aluminum oxide boron carbide (Al2 O3-B4 C)

    International Nuclear Information System (INIS)

    Oliveira, E.E.M.; Bressiani, Ana H.A.; Bressiani, J.C.

    1996-01-01

    The densification Al 2 O 3 -B 4 C of composite was accomplished under two conditions: I- tungsten resistance furnace in commercial argon atmosphere without gas treatment system.II- graphite resistance furnace in argon atmosphere with gas treatment for humidity removal. The sintering with gas treatment showed higher density and smaller loss of mass for all composition related to the sintering in tungsten resistance furnace without gas treatment system. Microstructural characterization also showed that grain growth of alumina matrix is greatly influenced by particle size and concentration of B 4 C. Samples sintered at temperatures higher than 1750 deg C without gas treatment presented the formation of phase Al 3 B O 6 which was identified by transmission electron microscopy. (author)

  1. Modeling amorphous Si3B3N7: Structure and elastic properties

    International Nuclear Information System (INIS)

    Hannemann, A.; Schoen, J.C.; Jansen, M.; Putz, H.; Lengauer, T.

    2004-01-01

    We investigate the structure and elastic properties of the amorphous high-temperature ceramic a-Si 3 B 3 N 7 . Several different structural models are generated and their properties such as the radial and angular distribution functions, the degree of local order, the density, the bulk modulus and the phonon spectrum, are calculated and compared with the experiment. The best structural agreement between model and experimental observations is found for models exhibiting a certain degree of local ( 3 B 3 N 7 has not been synthesized by cooling from the melt but via the polymerization and subsequent pyrolysis of molecular precursors. Furthermore, we suggest that, due to the synthesis process, stable nanoscale cavities (diameter 3 )

  2. Conversion of chlorinated propanes by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

    OpenAIRE

    Bosma, T.; Janssen, D.B.

    1998-01-01

    Chlorinated propanes are important pollutants that may show persistent behaviour in the environment. The biotransformation of 1-chloropropane, 1,2-dichloropropane, 1,3-dichloropropane and 1,2,3-trichloropropane was studied using resting cell suspensions of Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. The transformation followed first-order kinetics. The rate constants were in the order 1-chloropropane > 1,3-dichloropropane > 1,2-dichloropropane > 1,2,3-trichloropr...

  3. Association of GSK3B With Alzheimer Disease and Frontotemporal Dementia

    Science.gov (United States)

    Schaffer, Barbara A. J.; Bertram, Lars; Miller, Bruce L.; Mullin, Kristina; Weintraub, Sandra; Johnson, Nancy; Bigio, Eileen H.; Mesulam, Marsel; Wiedau-Pazos, Martina; Jackson, George R.; Cummings, Jeffrey L.; Cantor, Rita M.; Levey, Allan I.; Tanzi, Rudolph E.; Geschwind, Daniel H.

    2009-01-01

    Background Deposits of abnormally hyperphosphorylated tau are a hallmark of several dementias, including Alzheimer disease (AD), and about 10% of familial frontotemporal dementia (FTD) cases are caused by mutations in the tau gene. As a known tau kinase, GSK3B is a promising candidate gene in the remaining cases of FTD and in AD, for which tau mutations have not been found. Objective To examine the promoter of GSK3B and all 12 exons, including the surrounding intronic sequence, in patients with FTD, patients with AD, and aged healthy subjects to identify single-nucleotide polymorphisms associated with disease. Design, Setting, and Participants Single-nucleotide polymorphism frequency was examined in a case-control cohort of 48 patients with probable AD, 102 patients with FTD, 38 patients with primary progressive aphasia, and 85 aged healthy subjects. Results were followed up in 2 independent AD family samples consisting of 437 multiplex families with AD (National Institute of Mental Health Genetics Initiative AD Study) or 150 sibships discordant for AD (Consortium on Alzheimer’s Genetics Study). Results Several rare sequence variants in GSK3B were identified in the case-control study. An intronic polymorphism (IVS2−68G>A) occurred at more than twice the frequency among patients with FTD (10.8%) and patients with AD (14.6%) than in aged healthy subjects (4.1%). The polymorphism showed association with disease in both follow-up samples independently, although only the Consortium on Alzheimer’s Genetics sample showed the same direction of association as the case-control sample. Conclusions To our knowledge, this is the first evidence that a gene known to be involved in tau phosphorylation, GSK3B, is associated with risk for primary neurodegenerative dementias. This supports previous work in animal models suggesting that such genes are therapeutic targets. PMID:18852354

  4. Qatar Exoplanet Survey : Qatar-3b, Qatar-4b, and Qatar-5b

    Science.gov (United States)

    Alsubai, Khalid; Mislis, Dimitris; Tsvetanov, Zlatan I.; Latham, David W.; Bieryla, Allyson; Buchhave, Lars A.; Esquerdo, Gilbert A.; Bramich, D. M.; Pyrzas, Stylianos; Vilchez, Nicolas P. E.; Mancini, Luigi; Southworth, John; Evans, Daniel F.; Henning, Thomas; Ciceri, Simona

    2017-04-01

    We report the discovery of Qatar-3b, Qatar-4b, and Qatar-5b, three new transiting planets identified by the Qatar Exoplanet Survey. The three planets belong to the hot Jupiter family, with orbital periods of {P}{{Q}3{{b}}} = 2.50792 days, {P}{{Q}4{{b}}} = 1.80539 days, and {P}{{Q}5{{b}}} = 2.87923 days. Follow-up spectroscopic observations reveal the masses of the planets to be {M}{{Q}3{{b}}} = 4.31 ± 0.47 {M}{{J}}, {M}{{Q}4{{b}}} = 6.10 ± 0.54 {M}{{J}}, and {M}{{Q}5{{b}}} = 4.32 ± 0.18 {M}{{J}}, while model fits to the transit light curves yield radii of {R}{{Q}3{{b}}} = 1.096 ± 0.14 {R}{{J}}, {R}{{Q}4{{b}}} = 1.135 ± 0.11 {R}{{J}}, and {R}{{Q}5{{b}}} = 1.107 ± 0.064 {R}{{J}}. The host stars are low-mass main sequence stars with masses and radii M Q3 = 1.145 ± 0.064 M ⊙, M Q4 = 0.896 ± 0.048 M ⊙, M Q5 = 1.128 ± 0.056 M ⊙ and R Q3 = 1.272 ± 0.14 R ⊙, R Q4 = 0.849 ± 0.063 R ⊙, and R Q5 = 1.076 ± 0.051 R ⊙ for Qatar-3, 4, and 5 respectively. The V magnitudes of the three host stars are V Q3 = 12.88, V Q4 = 13.60, and V Q5 = 12.82. All three new planets can be classified as heavy hot Jupiters (M > 4 M J).

  5. Evaluation of TRMM 3B42V7 product on extreme precipitation measurements over peninsular Malaysia

    Science.gov (United States)

    Paska, Jacquoelyne; Lau, Alvin M. S.; Tan, Mou Leong; Tan, Kok Chooi

    2017-10-01

    Climate variability has become a matter worth our attention as this issue has unveiled to the extreme water-related disasters such as flood and drought. Increments in heavy precipitation have happened over the past century and future climate scenarios show that it may alter the recurrence, timing, force, and length of these occasions. Satellite precipitation products (SPPs) could be used as representation of precipitation over a large region. This could be useful for the monitoring of the precipitation pattern as well as extreme events. Nevertheless, application of these products in monitoring extreme precipitation is still limited because insufficiency of quality assessment. This study aims to evaluate the performance of the Tropical Rainfall Measuring Mission (TRMM) Multi-satellite Precipitation Analysis (TMPA) 3B42V7 product in capturing the behavior of extreme precipitation events over Peninsular Malaysia from 2000 to 2015. Four extreme precipitation indices, in two general categories of absolute threshold (R10mm, R20mm and R50mm) and maximum (Rx1d) indices that recommended by the Expert Team on Climate Change Detection and Indices (ETCCDI) were used. General evaluation has shown that the TRMM 3B42V7 product performed good on the measurements of monthly and annual precipitation. In the respect of extreme precipitation measurements, weak to moderate positive correlations were found between the TRMM 3B42 product and rain gauges over Peninsular Malaysia. The TRMM 3B42V7 product overestimated the R10mm and R20mm indices, while an underestimation was found for the R50mm and Rx1d indices.

  6. Intercomparison of the Extended Reconstructed Sea Surface Temperature v4 and v3b Datasets

    Science.gov (United States)

    Wang, Jinping; Chen, Xianyao

    2018-04-01

    Version 4 (v4) of the Extended Reconstructed Sea Surface Temperature (ERSST) dataset is compared with its precedent, the widely used version 3b (v3b). The essential upgrades applied to v4 lead to remarkable differences in the characteristics of the sea surface temperature (SST) anomaly (SSTa) in both the temporal and spatial domains. First, the largest discrepancy of the global mean SSTa values around the 1940s is due to ship-observation corrections made to reconcile observations from buckets and engine intake thermometers. Second, differences in global and regional mean SSTa values between v4 and v3b exhibit a downward trend (around -0.032°C per decade) before the 1940s, an upward trend (around 0.014°C per decade) during the period of 1950-2015, interdecadal oscillation with one peak around the 1980s, and two troughs during the 1960s and 2000s, respectively. This does not derive from treatments of the polar or the other data-void regions, since the difference of the SSTa does not share the common features. Third, the spatial pattern of the ENSO-related variability of v4 exhibits a wider but weaker cold tongue in the tropical region of the Pacific Ocean compared with that of v3b, which could be attributed to differences in gap-filling assumptions since the latter features satellite observations whereas the former features in situ ones. This intercomparison confirms that the structural uncertainty arising from underlying assumptions on the treatment of diverse SST observations even in the same SST product family is the main source of significant SST differences in the temporal domain. Why this uncertainty introduces artificial decadal oscillations remains unknown.

  7. Prognosis of Open Type 3-B Tibia Fractures By Ilizarov Technique: A Prospective Study

    OpenAIRE

    Janak Rathod, Taral Shah

    2012-01-01

    Background: Open fractures of the tibial shaft are both common and may be fraught with complications. Open fractures of the tibia, with associated vascular injuries, have historically had a very poor outcome. Malunion, delayed union, nonunion, and infection are all seen regularly after open tibia fractures. Objectives: Objective of the study is to explore advantage of primary or early ilizarov surgery in Open type 3- B fracture tibia. Methodology: A follow up study of 10 cases of open type 3-...

  8. Susceptibility of Hep3B cells in different phases of cell cycle to tBid.

    Science.gov (United States)

    Ma, Shi-Hong; Chen, George G; Ye, Caiguo; Leung, Billy C S; Ho, Rocky L K; Lai, Paul B S

    2011-01-01

    tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. On the unusual magnetic behaviour of CeRh3B2

    International Nuclear Information System (INIS)

    Galatanu, A; Yamamoto, E; Okubo, T; Yamada, M; Thamizhavel, A; Takeuchi, T; Sugiyama, K; Inada, Y; Onuki, Y

    2003-01-01

    CeRh 3 B 2 is a ferromagnet with an unexpectedly high Curie temperature and low saturation magnetic moments. The origin of its unusual magnetic behaviour remains unelucidated despite various investigations performed in the last two decades. Here we present magnetic results obtained on high-quality single crystals over a wide temperature range. Some new magnetic features are uncovered and possible approaches to explaining its magnetism are briefly discussed

  10. Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-06-01

    The protein methylome in mammalian mitochondria has been little studied until recently. Here, we describe that lysine-368 of human citrate synthase is methylated and that the modifying enzyme, localized in the mitochondrial matrix, is methyltransferase-like protein 12 (METTL12), a member of the family of 7β-strand methyltransferases. Lysine-368 is near the active site of citrate synthase, but removal of methylation has no effect on its activity. In mitochondria, it is possible that some or all of the enzymes of the citric acid cycle, including citrate synthase, are organized in metabolons to facilitate the channelling of substrates between participating enzymes. Thus, possible roles for the methylation of Lys-368 are in controlling substrate channelling itself, or in influencing protein-protein interactions in the metabolon. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  11. Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

    Energy Technology Data Exchange (ETDEWEB)

    Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina; Engel, Daniel A.; Derewenda, Zygmunt S.

    2017-08-15

    Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

  12. Assimilation of Feng-Yun-3B satellite microwave humidity sounder data over land

    Science.gov (United States)

    Chen, Keyi; Bormann, Niels; English, Stephen; Zhu, Jiang

    2018-03-01

    The ECMWF has been assimilating Feng-Yun-3B (FY-3B) satellite microwave humidity sounder (MWHS) data over ocean in an operational forecasting system since 24 September 2014. It is more difficult, however, to assimilate microwave observations over land and sea ice than over the open ocean due to higher uncertainties in land surface temperature, surface emissivity and less effective cloud screening. We compare approaches in which the emissivity is retrieved dynamically from MWHS channel 1 [150 GHz (vertical polarization)] with the use of an evolving emissivity atlas from 89 GHz observations from the MWHS onboard NOAA and EUMETSAT satellites. The assimilation of the additional data over land improves the fit of short-range forecasts to other observations, notably ATMS (Advanced Technology Microwave Sounder) humidity channels, and the forecast impacts are mainly neutral to slightly positive over the first five days. The forecast impacts are better in boreal summer and the Southern Hemisphere. These results suggest that the techniques tested allow for effective assimilation of MWHS/FY-3B data over land.

  13. Optical properties of Sr3B2O6:Dy3+/PMMA polymer nanocomposites

    Science.gov (United States)

    Khursheed, Sumara; Kumar, Vinay; Singh, Vivek K.; Sharma, Jitendra; Swart, H. C.

    2018-04-01

    The paper presents a facile way to synthesize luminescent polymer nanocomposite (PNC) films consisting of nanophosphors (NPs) of rare earth ions doped alkaline earth borates (Sr3B2O6:Dy3+) dispersed in a polymer (PMMA) matrix via a solution casting method and the results of their detailed structural and optical properties measurements. The PNC films were characterized using X-ray diffraction (XRD), Photoluminescence (PL), and differential scanning calorimetry (DSC). The crystallinity of the dispersed NPs did not suffer on account of being dispersed in the PMMA. The Rhombohedral structure and the formation of a single phase of Sr3B2O6:Dy3+ were confirmed by the XRD data of both the NP powders and the PNC films with an average particle size of 43 nm. Also, the observed PL emission and excitation spectra of the PNC films amply suggested that embedding of the nanophosphors in the PMMA matrix preserves their typical luminescence emission. The chromaticity coordinates (x = 0.37, y = 0.39) of the PNC films also validated the yellowish white emission of the nanophosphor. DSC scans on the PMMA only and the Sr3B2O6:Dy3+/PMMA films suggested an increase in the thermal stability of the PNC films as compared to pure PMMA although no significant change in the glass transition temperature was observed.

  14. Catecholamine-o-methyltransferase polymorphisms are associated with postoperative pain intensity.

    LENUS (Irish Health Repository)

    Lee, Peter J

    2011-02-01

    single nucleotide polymorphisms (SNPs) in the genes for catecholamine-O-methyltransferase (COMT), μ-opioid receptor and GTP cyclohydrolase (GCH1) have been linked to acute and chronic pain states. COMT polymorphisms are associated with experimental pain sensitivity and a chronic pain state. No such association has been identified perioperatively. We carried out a prospective observational clinical trial to examine associations between these parameters and the development of postoperative pain in patients undergoing third molar (M3) extraction.

  15. A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.; Feng, You; Clarke, Steven G.; Blobel, Günter; Stavropoulos, Pete

    2016-02-08

    Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

  16. Structure and possible mechanism of the CcbJ methyltransferase from Streptomyces caelestis

    Czech Academy of Sciences Publication Activity Database

    Bauer, J.; Ondrovičová, G.; Najmanová, Lucie; Pevala, V.; Kameník, Zdeněk; Koštan, J.; Janata, Jiří; Kutejová, Eva

    2014-01-01

    Roč. 70, APR 2014 (2014), s. 943-957 ISSN 0907-4449 R&D Projects: GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : CATECHOL-O-METHYLTRANSFERASE * SN2-LIKE TRANSITION-STATE * CRYSTAL-STRUCTURES Subject RIV: CE - Biochemistry Impact factor: 7.232, year: 2013

  17. Characterization of novel methyltransferases METTL22 and FAM86A.1

    OpenAIRE

    Ali, Qamar

    2012-01-01

    Proteins are subjected to various post-translational modifications (PTMs) that affect their activity, interaction and localization. Methylation is one such PTM that is well known to play a regulatory role in heterochromatin and euchromatin formation through methyl marks on histone tails, and it has recently been shown that regulation through methylation is also applicable to non-histone proteins. A recent characterization of protein methyltransferase (MTase) METTL21D led to the discovery of a...

  18. Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors

    Directory of Open Access Journals (Sweden)

    Orth Angela

    2009-04-01

    Full Text Available Abstract Background N-methyl-D-aspartate receptors (NMDARs are the most complex of ionotropic glutamate receptors (iGluRs. Subunits of this subfamily assemble into heteromers, which – depending on the subunit combination – may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity. Results Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg2+. Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants – possibly via disturbing the "proton shield" of NR1b splice variants. Conclusion While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors.

  19. Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nβ -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

    Science.gov (United States)

    Cázares-Flores, Paulo; Levac, Dylan; De Luca, Vincenzo

    2016-08-01

    Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ -methylajmaline, an unusual charged MIA found in R. serpentina. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  20. Identification of Aquifex aeolicus tRNA (m2(2G26) methyltransferase gene.

    Science.gov (United States)

    Takeda, Hiroshi; Hori, Hiroyuki; Endo, Yaeta

    2002-01-01

    The modifications of N2,N2-dimethylguanine (m2(2)G) are found in tRNAs and rRNAs from eukarya and archaea. In tRNAs, modification at position G26 is generated by tRNA (m2(2)G26) methyltransferase, which is encoded by the corresponding gene, trm1. This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the semi-conserved residue, G26, via the intermediate modified base, m2G26. Recent genome sequencing project has been reported that the putative trm1 is encoded in the genome of Aquifex aeolicus, a hyper-thermophilic eubacterium as only one exception among eubacteria. In order to confirm whether this bacterial trm1 gene product is a real tRNA (m2(2)G26) methyltransferase or not, we expressed this protein by wheat germ in vitro cell-free translation system. Our biochemical analysis clearly showed that this gene product possessed tRNA (m2(2)G26) methyltransferase activity.

  1. Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype

    International Nuclear Information System (INIS)

    Li Jiaxin; Waters, Stephen B.; Drobna, Zuzana; Devesa, Vicenta; Styblo, Miroslav; Thomas, David J.

    2005-01-01

    Inorganic arsenic is enzymatically methylated; hence, its ingestion results in exposure to the parent compound and various methylated arsenicals. Both experimental and epidemiological evidences suggest that some of the adverse health effects associated with chronic exposure to inorganic arsenic may be mediated by these methylated metabolites. If i As methylation is an activation process, then the phenotype for inorganic arsenic methylation may determine risk associated with exposure to this metalloid. We examined inorganic arsenic methylation phenotypes and arsenic (+3 oxidation state) methyltransferase genotypes in four species: three that methylate inorganic arsenic (human (Homo sapiens), rat (Rattus norwegicus), and mouse (Mus musculus)) and one that does not methylate inorganic arsenic (chimpanzee, Pan troglodytes). The predicted protein products from arsenic (+3 oxidation state) methyltransferase are similar in size for rat (369 amino acid residues), mouse (376 residues), and human (375 residues). By comparison, a 275-nucleotide deletion beginning at nucleotide 612 in the chimpanzee gene sequence causes a frameshift that leads to a nonsense mutation for a premature stop codon after amino acid 205. The null phenotype for inorganic arsenic methylation in the chimpanzee is likely due to the deletion in the gene for arsenic (+3 oxidation state) methyltransferase that yields an inactive truncated protein. This lineage-specific loss of function caused by the deletion event must have occurred in the Pan lineage after Homo-Pan divergence about 5 million years ago

  2. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia.

    Science.gov (United States)

    Cao, Fang; Townsend, Elizabeth C; Karatas, Hacer; Xu, Jing; Li, Li; Lee, Shirley; Liu, Liu; Chen, Yong; Ouillette, Peter; Zhu, Jidong; Hess, Jay L; Atadja, Peter; Lei, Ming; Qin, Zhaohui S; Malek, Sami; Wang, Shaomeng; Dou, Yali

    2014-01-23

    Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Structural insights into mechanisms of the small RNA methyltransferase HEN1

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ying; Ji, Lijuan; Huang, Qichen; Vassylyev, Dmitry G.; Chen, Xuemei; Ma, Jin-Biao; (UAB); (UCR)

    2010-02-22

    RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of {approx}20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-L-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 {angstrom} crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-L-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg{sup 2+}-dependent 2'-O-methylation mechanism.

  4. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    International Nuclear Information System (INIS)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Lamballeire, Xavier de; Brisbare, Nadege; Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno; Gould, Ernest; Forrester, Naomi; Bolognesi, Martino

    2006-01-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup

  5. Methylated nucleosides in tRNA and tRNA methyltransferases

    Directory of Open Access Journals (Sweden)

    Hiroyuki eHori

    2014-05-01

    Full Text Available To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s. Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon–anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed.

  6. A Reverse Genetics Approach for the Design of Methyltransferase-Defective Live Attenuated Avian Metapneumovirus Vaccines.

    Science.gov (United States)

    Zhang, Yu; Sun, Jing; Wei, Yongwei; Li, Jianrong

    2016-01-01

    Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. aMPV belongs to the family Paramyxoviridae which includes many important human pathogens such as human respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (PIV3). The family also includes highly lethal emerging pathogens such as Nipah virus and Hendra virus, as well as agriculturally important viruses such as Newcastle disease virus (NDV). For many of these viruses, there is no effective vaccine. Here, we describe a reverse genetics approach to develop live attenuated aMPV vaccines by inhibiting the viral mRNA cap methyltransferase. The viral mRNA cap methyltransferase is an excellent target for the attenuation of paramyxoviruses because it plays essential roles in mRNA stability, efficient viral protein translation and innate immunity. We have described in detail the materials and methods used to generate recombinant aMPVs that lack viral mRNA cap methyltransferase activity. We have also provided methods to evaluate the genetic stability, pathogenesis, and immunogenicity of live aMPV vaccine candidates in turkeys.

  7. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    Energy Technology Data Exchange (ETDEWEB)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy); Lamballeire, Xavier de; Brisbare, Nadege [Unité des Virus Emergents, Faculté de Médecine, 27 Boulevard Jean Moulin, 13005 Marseille (France); Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS ESIL, Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France); Gould, Ernest; Forrester, Naomi [CEH Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom); Bolognesi, Martino, E-mail: martino.bolognesi@unimi.it [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy)

    2006-08-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.

  8. Preparation of novel pyrrolo [2,3-b]pyridine derivatives via a new concise synthetic approach

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Na; Jia, Haiyong; You, Xing; Jiang, Du; Lu, Kui; Yu, Peng [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Sino-French Joint Lab of Food Nutrition/Safety and Medicinal Chemistry, Tianjin University of Science and Technology, Tianjin (China)

    2015-04-15

    The pyrrolo[2,3-b]pyridine core structure, a bioisostere of quinolones, is found in several molecules that possess important biological activity. We describe here a new, concise, three-step synthesis of pyrrolo[2,3-b]pyridines starting from L-alanine. A series of 4,7-dihydro-4-oxo-1H-pyrrolo[2,3-b]pyridine-5-carboxylic acid derivatives, which have not been previously reported, were synthesized using this approach.

  9. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

    International Nuclear Information System (INIS)

    Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

    2013-01-01

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation

  10. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun, E-mail: yizc@buaa.edu.cn

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation.

  11. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells.

    Science.gov (United States)

    Suriguga; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. © 2013.

  12. TRMM-3B43 Bias Correction over the High Elevations of the Contiguous United States

    Science.gov (United States)

    Hashemi, H.; Nordin, K. M.; Lakshmi, V.; Knight, R. J.

    2016-12-01

    Precipitation can be quantified using a rain gauge network, or a remotely sensed precipitation product. Ultimately, the choice of dataset depends on the particular application, the catchment size, climate and the time period of study. In a region with a long record and a dense rain gauge network, the elevation-modified ground-based precipitation product, PRISM, has been found to work well. However, in poorly gauged regions the use of remotely sensed precipitation products is an absolute necessity. The Tropical Rainfall Measuring Mission (TRMM) has provided valuable precipitation datasets for hydrometeorological studies over the past two decades (1998-2015). One concern regarding the usage of TRMM data is the accuracy of the precipitation estimates, when compared to those obtained using PRISM. The reason for this concern is that TRMM and PRISM do not always agree and, typically, TRMM underestimates PRISM over the mountainous regions of the United States. In this study, we develop a correction function to improve the accuracy of the TRMM monthly product (TRMM-3B43) by estimating and removing the bias in the satellite data using the ground-based precipitation product, PRISM. We observe a strong relationship between the bias and land surface elevation; TRMM-3B43 tends to underestimate the PRISM product at altitudes greater than 1500 m above mean sea level (m.amsl) in the contiguous United States. A relationship is developed between TRMM-PRISM bias and elevation. The correction function is used to adjust the TRMM monthly precipitation using PRISM and elevation data. The model is calibrated using 25% of the available time period and the remaining 75% of the time period is used for validation. The corrected TRMM-3B43 product is verified for the high elevations over the contiguous United States and two local regions in the mountainous areas of the western United States. The results show a significant improvement in the accuracy of the TRMM product in the high elevations of

  13. Genome-wide Association Study Implicates PARD3B-based AIDS Restriction

    Science.gov (United States)

    Nelson, George W.; Lautenberger, James A.; Chinn, Leslie; McIntosh, Carl; Johnson, Randall C.; Sezgin, Efe; Kessing, Bailey; Malasky, Michael; Hendrickson, Sher L.; Pontius, Joan; Tang, Minzhong; An, Ping; Winkler, Cheryl A.; Limou, Sophie; Le Clerc, Sigrid; Delaneau, Olivier; Zagury, Jean-François; Schuitemaker, Hanneke; van Manen, Daniëlle; Bream, Jay H.; Gomperts, Edward D.; Buchbinder, Susan; Goedert, James J.; Kirk, Gregory D.; O'Brien, Stephen J.

    2011-01-01

    Background. Host genetic variation influences human immunodeficiency virus (HIV) infection and progression to AIDS. Here we used clinically well-characterized subjects from 5 pretreatment HIV/AIDS cohorts for a genome-wide association study to identify gene associations with rate of AIDS progression. Methods.  European American HIV seroconverters (n = 755) were interrogated for single-nucleotide polymorphisms (SNPs) (n = 700,022) associated with progression to AIDS 1987 (Cox proportional hazards regression analysis, co-dominant model). Results.  Association with slower progression was observed for SNPs in the gene PARD3B. One of these, rs11884476, reached genome-wide significance (relative hazard = 0.3; P =3. 370 × 10−9) after statistical correction for 700,022 SNPs and contributes 4.52% of the overall variance in AIDS progression in this study. Nine of the top-ranked SNPs define a PARD3B haplotype that also displays significant association with progression to AIDS (hazard ratio, 0.3; P = 3.220 × 10−8). One of these SNPs, rs10185378, is a predicted exonic splicing enhancer; significant alteration in the expression profile of PARD3B splicing transcripts was observed in B cell lines with alternate rs10185378 genotypes. This SNP was typed in European cohorts of rapid progressors and was found to be protective for AIDS 1993 definition (odds ratio, 0.43, P = .025). Conclusions. These observations suggest a potential unsuspected pathway of host genetic influence on the dynamics of AIDS progression. PMID:21502085

  14. LC3B is indispensable for selective autophagy of p62 but not basal autophagy

    International Nuclear Information System (INIS)

    Maruyama, Yoko; Sou, Yu-Shin; Kageyama, Shun; Takahashi, Takao; Ueno, Takashi; Tanaka, Keiji; Komatsu, Masaaki; Ichimura, Yoshinobu

    2014-01-01

    Highlights: • Knockdown of LC3 or GABARAP families did not affect the basal autophagy. • LC3B has a higher affinity for the autophagy-specific substrate, p62, than GABARAPs. • siRNA-mediated knockdown of LC3B, but not that of GABARAPs, resulted in significant accumulation of p62. - Abstract: Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs

  15. Analysis of diversity and linkage disequilibrium along chromosome 3B of bread wheat (Triticum aestivum L.).

    Science.gov (United States)

    Horvath, Aniko; Didier, Audrey; Koenig, Jean; Exbrayat, Florence; Charmet, Gilles; Balfourier, François

    2009-11-01

    A highly polymorphic core collection of bread wheat and a more narrow-based breeding material, gathered from pedigrees of seven modern cultivars, was analysed in order to compare genetic diversity indices and linkage disequilibrium (LD) patterns along the chromosome 3B with microsatellite (SSR) and Diversity Arrays Technology markers. Five ancestral gene pools could be identified within the core collection, indicating a strong geographical structure (Northwest Europe, Southeast Europe, CIMMYT-ICARDA group, Asia, Nepal). The breeding material showed a temporal structure, corresponding to different periods of breeding programmes [old varieties (from old landraces to 1919), semi-modern varieties (1920-1959), modern varieties (1960-2006)]. Basic statistics showed a higher genetic diversity in the core collection than in the breeding material, indicating a stronger selection pressure in this latter material. More generally, the chromosome 3B had a lower diversity than the whole B-genome. LD was weak in all studied materials. Amongst geographical groups, the CIMMYT-ICARDA pool presented the longest ranged LD in contrast to Asian accessions. In the breeding material, LD increased from old cultivars to modern varieties. Genitors of seven modern cultivars were found to be different; most marker pairs in significant LD were observed amongst genitors of Alexandre and Koreli varieties, indicating an important inbreeding effect. At low genetic distances (0-5 cM), the breeding material had higher LD than the core collection, but globally the two materials had similar values in all classes. Marker pairs in significant LD are generally observed around the centromere in both arms and at distal position on the short arm of the chromosome 3B.

  16. Birefringence Bragg Binary (3B) grating, quasi-Bragg grating and immersion gratings

    Science.gov (United States)

    Ebizuka, Noboru; Morita, Shin-ya; Yamagata, Yutaka; Sasaki, Minoru; Bianco, Andorea; Tanabe, Ayano; Hashimoto, Nobuyuki; Hirahara, Yasuhiro; Aoki, Wako

    2014-07-01

    A volume phase holographic (VPH) grating achieves high angular dispersion and very high diffraction efficiency for the first diffraction order and for S or P polarization. However the VPH grating could not achieve high diffraction efficiency for non-polarized light at a large diffraction angle because properties of diffraction efficiencies for S and P polarizations are different. Furthermore diffraction efficiency of the VPH grating extinguishes toward a higher diffraction order. A birefringence binary Bragg (3B) grating is a thick transmission grating with optically anisotropic material such as lithium niobate or liquid crystal. The 3B grating achieves diffraction efficiency up to 100% for non-polarized light by tuning of refractive indices for S and P polarizations, even in higher diffraction orders. We fabricated 3B grating with liquid crystal and evaluated the performance of the liquid crystal grating. A quasi-Bragg (QB) grating, which consists long rectangle mirrors aligned in parallel precisely such as a window shade, also achieves high diffraction efficiency toward higher orders. We fabricated QB grating by laminating of silica glass substrates and glued by pressure fusion of gold films. A quasi-Bragg immersion (QBI) grating has smooth mirror hypotenuse and reflector array inside the hypotenuse, instead of step-like grooves of a conventional immersion grating. An incident beam of the QBI grating reflects obliquely at a reflector, then reflects vertically at the mirror surface and reflects again at the same reflector. We are going to fabricate QBI gratings by laminating of mirror plates as similar to fabrication of the QB grating. We will also fabricate silicon and germanium immersion gratings with conventional step-like grooves by means of the latest diamond machining methods. We introduce characteristics and performance of these gratings.

  17. LC3B is indispensable for selective autophagy of p62 but not basal autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yoko [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Department of Pediatrics, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Sou, Yu-Shin; Kageyama, Shun [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Takahashi, Takao [Department of Pediatrics, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Ueno, Takashi [Division of Proteomics and Biomolecular Science, Center for Biomedical Research Resources, Juntendo University Graduate School of Medicine, Tokyo 113-8421 (Japan); Tanaka, Keiji [Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Komatsu, Masaaki, E-mail: komatsu-ms@igakuken.or.jp [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Department of Biochemistry, School of Medicine, Niigata University, Niigata 951-8510 (Japan); Ichimura, Yoshinobu, E-mail: ichimura-ys@igakuken.or.jp [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan)

    2014-03-28

    Highlights: • Knockdown of LC3 or GABARAP families did not affect the basal autophagy. • LC3B has a higher affinity for the autophagy-specific substrate, p62, than GABARAPs. • siRNA-mediated knockdown of LC3B, but not that of GABARAPs, resulted in significant accumulation of p62. - Abstract: Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.

  18. INFRARED AND KINEMATIC PROPERTIES OF THE SUBSTELLAR OBJECT G 196-3 B

    International Nuclear Information System (INIS)

    Zapatero Osorio, M. R.; Caballero, J. A.; Rebolo, R.; Bihain, G.; Bejar, V. J. S.; Alvarez, C.

    2010-01-01

    We report unusual near- and mid-infrared photometric properties of G 196-3 B, the young substellar companion at 16'' from the active M2.5-type star G 196-3 A, using data taken with the IRAC and MIPS instruments onboard Spitzer. G 196-3 B shows markedly redder colors at all wavelengths from 1.6 up to 24 μm than expected for its spectral type, which is determined at L3 from optical and near-infrared spectra. We discuss various physical scenarios to account for its reddish nature and conclude that a low-gravity atmosphere with enshrouded upper atmospheric layers and/or a warm dusty disk/envelope provides the most likely explanations, the two of them consistent with an age in the interval 20-300 Myr. We also present new and accurate separate proper motion measurements for G 196-3 A and B confirming that both objects are gravitationally linked and share the same motion within a few mas yr -1 . After integration of the combined spectrophotometric spectral energy distributions, we obtain the result that the difference in the bolometric magnitudes of G 196-3 A and B is 6.15 ± 0.10 mag. Kinematic consideration of the Galactic space motions of the system for distances in the interval 15-30 pc suggests that the pair is a likely member of the Local Association and that it lies near the past positions of young star clusters like α Persei less than 85 Myr ago, where the binary might have originated. At these young ages, the mass of G 196-3 B would be in the range 12-25 M Jup , close to the frontier between planets and brown dwarfs.

  19. Magnetic and crystallographic properties of HoCo3B2

    International Nuclear Information System (INIS)

    Caspi, E.N.; Dubman, M.; Ettedgui, H.; Shaked, H.; Short, S.; Jorgensen, J.D.

    2005-01-01

    AC-susceptibility measurements for HoCo 3 B 2 show paramagnetism below room temperature and a small, and large anomalies at 150 and 10K, respectively. Neutron powder diffraction measurements show a ferromagnetic order below 10K, where the ferromagnetic Ho and Co sublattices are ferromagnetically coupled. The magnetic axis direction is perpendicular to the c axis. The observed magnitudes of the magnetic moments at 4.5K are 5.08(4) and 0.11(2) μ B for Ho and Co, respectively. No long range magnetic order was detected above 10K

  20. LC3B globular structures correlate with survival in esophageal adenocarcinoma.

    LENUS (Irish Health Repository)

    El-Mashed, Shereen

    2015-01-01

    Esophageal adenocarcinoma has the fastest growing incidence of any solid tumor in the Western world. Prognosis remains poor with overall five-year survival rates under 25 %. Only a limited number of patients benefit from chemotherapy and there are no biomarkers that can predict outcome. Previous studies have indicated that induction of autophagy can influence various aspects of tumor cell biology, including chemosensitivity. The objective of this study was to assess whether expression of the autophagy marker (LC3B) correlated with patient outcome.

  1. DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphoma-derived B-cell line: potential involvement of direct inhibition of cAMP-responsive element (CRE)-binding protein/CRE modulator/activation transcription factor binding.

    Science.gov (United States)

    Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

    2010-06-18

    Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

  2. The evolution of CHROMOMETHYLASES and gene body DNA methylation in plants

    OpenAIRE

    Leebens-Mack, Jim; Griffin, Patrick; Rohr, Nicholas; Niederhuth, Chad; Ji, Lexiang; Bewick, Adam; Schmitz, Robert

    2017-01-01

    Background The evolution of gene body methylation (gbM), its origins, and its functional consequences are poorly understood. By pairing the largest collection of transcriptomes (>1000) and methylomes (77) across Viridiplantae, we provide novel insights into the evolution of gbM and its relationship to CHROMOMETHYLASE (CMT) proteins. Results CMTs are evolutionary conserved DNA methyltransferases in Viridiplantae. Duplication events gave rise to what are now referred to as CMT1, 2 and 3. Indepe...

  3. Functional characterization of two CITED3 homologs (gcCITED3a and gcCITED3b in the hypoxia-tolerant grass carp, Ctenopharyngodon idellus

    Directory of Open Access Journals (Sweden)

    Yu Richard MK

    2009-11-01

    Full Text Available Abstract Background CITED proteins belong to a family of non-DNA-binding transcriptional co-regulators that are characterized by a conserved ED-rich domain at the C-terminus. This family of genes is involved in the regulation of a variety of transcriptional responses through interactions with the CBP/p300 integrators and various transcription factors. In fish, very little is known about the expression and functions of CITEDs. Results We have characterized two closely related but distinct CITED3 genes, gcCited3a and gcCited3b, from the hypoxia-tolerant grass carp. The deduced gcCITED3a and gcCITED3b proteins share 72% amino acid identity, and are highly similar to the CITED3 proteins of both chicken and Xenopus. Northern blot analysis indicates that the mRNA expression of gcCited3a and gcCited3b is strongly induced by hypoxia in the kidney and liver, respectively. Luciferase reporter assays demonstrated that both gene promoters are activated by gcHIF-1. Further, ChIP assays comparing normal and hypoxic conditions reveal differential in vivo binding of gcHIF-1 to both gene promoters in kidney and liver tissues. HRE-luciferase reporter assays demonstrated that both gcCITED3a and gcCITED3b proteins inhibit gcHIF-1 transcriptional activity, and GST pull-down assays confirmed that both proteins bind specifically to the CH1 domain of the grass carp p300 protein. Conclusion The grass carp gcCITED3a and gcCITED3b genes are differentially expressed and regulated in different fish organs in response to hypoxic stress. This is the first report demonstrating in vivo regulation of two closely-related CITED3 isogenes by HIF-1, as well as CITED3 regulation of HIF-1 transcriptional activity in fish. Overall, our findings suggest that unique molecular mechanisms operate through these two gcCITED3 isoforms that likely play an important regulatory role in the hypoxic response in the grass carp.

  4. Regulated production and anti-HIV type 1 activities of cytidine deaminases APOBEC3B, 3F, and 3G.

    Science.gov (United States)

    Rose, Kristine M; Marin, Mariana; Kozak, Susan L; Kabat, David

    2005-07-01

    APOBEC3G and 3F (A3G and A3F) cytidine deaminases incorporate into retroviral cores where they lethally hypermutate nascent DNA reverse transcripts. As substantiated here, the viral infectivity factor (Vif) encoded by human immunodeficiency virus type-1 (HIV-1) binds A3G and A3F and induces their degradation, thereby precluding their incorporation into viral progeny. Previous evidence suggested that A3G is expressed in H9 and other nonpermissive cells that contain this antiviral defense but not in several permissive cells, and that overexpression of A3G or A3F makes permissive cells nonpermissive. Using a broader panel of cell lines, we confirmed a correlation between A3G and cellular abilities to inactivate HIV-1(Deltavif). However, there was a quantitative discrepancy because several cells with weak antiviral activities had similar amounts of wild-type A3G mRNA and protein compared to H9 cells. Antiviral activity of H9 cells was also attenuated in some conditions. These quantitative discrepancies could not be explained by the presence of A3F or other A3G paralogs in some of the cell lines. Thus, A3A, A3B, and A3C had weak but significant anti-HIV-1 activities and did not dominantly interfere with A3G or A3F antiviral functions. Control of A3G synthesis by the protein kinase C/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway was also similar in permissive and nonpermissive cells. A3G in highly permissive cells is degraded by Vif, suggesting that it is not in a sequestered site, and is specifically incorporated in low amounts into HIV-1(Deltavif). Although A3G and/or A3F inactivate HIV-1(Deltavif) and are neutralized by Vif, the antiviral properties of cell lines are also influenced by other cellular and viral factors.

  5. HSD3B and gene-gene interactions in a pathway-based analysis of genetic susceptibility to bladder cancer.

    Directory of Open Access Journals (Sweden)

    Angeline S Andrew

    Full Text Available Bladder cancer is the 4(th most common cancer among men in the U.S. We analyzed variant genotypes hypothesized to modify major biological processes involved in bladder carcinogenesis, including hormone regulation, apoptosis, DNA repair, immune surveillance, metabolism, proliferation, and telomere maintenance. Logistic regression was used to assess the relationship between genetic variation affecting these processes and susceptibility in 563 genotyped urothelial cell carcinoma cases and 863 controls enrolled in a case-control study of incident bladder cancer conducted in New Hampshire, U.S. We evaluated gene-gene interactions using Multifactor Dimensionality Reduction (MDR and Statistical Epistasis Network analysis. The 3'UTR flanking variant form of the hormone regulation gene HSD3B2 was associated with increased bladder cancer risk in the New Hampshire population (adjusted OR 1.85 95%CI 1.31-2.62. This finding was successfully replicated in the Texas Bladder Cancer Study with 957 controls, 497 cases (adjusted OR 3.66 95%CI 1.06-12.63. The effect of this prevalent SNP was stronger among males (OR 2.13 95%CI 1.40-3.25 than females (OR 1.56 95%CI 0.83-2.95, (SNP-gender interaction P = 0.048. We also identified a SNP-SNP interaction between T-cell activation related genes GATA3 and CD81 (interaction P = 0.0003. The fact that bladder cancer incidence is 3-4 times higher in males suggests the involvement of hormone levels. This biologic process-based analysis suggests candidate susceptibility markers and supports the theory that disrupted hormone regulation plays a role in bladder carcinogenesis.

  6. Magnetic properties and magnetocaloric effect of HoCo3B2 compound

    Directory of Open Access Journals (Sweden)

    X. Q. Zheng

    2018-05-01

    Full Text Available A sample of HoCo3B2 compound was synthesized, and the magnetic and MCE properties were investigated. Compound shows a change corresponding to R-R (R = rare earth sublattice magnetic order transition and the transition temperature is determined to be 11.8 K (TC. The characteristic of Arrott plots with positive slope around TC was observed, indicating a second-order phase transition. Based on isothermal magnetization data, together with Maxwell’s relationship, the magnetic entropy change (-ΔSM was calculated. The maximum -ΔSM reaches 7.8, 12.7 and 14.4 J/kg K for field range of 0-2 T, 0-5 T and 0-7 T, respectively. Accordingly, the value of RC (refrigerant capacity is 99, 289 and 432 J/kg for above field ranges. The large MCE of HoCo3B2 compound indicates its potential application for magnetic refrigeration in low temperature range.

  7. Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec.

    Science.gov (United States)

    Lister, James A; Lane, Brandon M; Nguyen, Anhthu; Lunney, Katherine

    2011-11-01

    The MiT family comprises four genes in mammals: Mitf, Tfe3, Tfeb, and Tfec, which encode transcription factors of the basic-helix-loop-helix/leucine zipper class. Mitf is well-known for its essential role in the development of melanocytes, however the functions of the other members of this family, and of interactions between them, are less well understood. We have now characterized the complete set of MiT genes from zebrafish, which totals six instead of four. The zebrafish genome contain two mitf (mitfa and mitfb), two tfe3 (tfe3a and tfe3b), and single tfeb and tfec genes; this distribution is shared with other teleosts. We present here the sequence and embryonic expression patterns for the zebrafish tfe3b, tfeb, and tfec genes, and identify a new isoform of tfe3a. These findings will assist in elucidating the roles of the MiT gene family over the course of vertebrate evolution. Copyright © 2011 Wiley-Liss, Inc.

  8. Optimization and characterization of biosurfactant production from marine Vibrio sp. strain 3B-2

    Science.gov (United States)

    Hu, Xiaoke; Wang, Caixia; Wang, Peng

    2015-01-01

    A biosurfactant-producing bacterium, designated 3B-2, was isolated from marine sediment and identified as Vibrio sp. by 16S rRNA gene sequencing. The culture medium composition was optimized to increase the capability of 3B-2 for producing biosurfactant. The produced biosurfactant was characterized in terms of protein concentration, surface tension, and oil-displacement efficiency. The optimal medium for biosurfactant production contained: 0.5% lactose, 1.1% yeast extract, 2% sodium chloride, and 0.1% disodium hydrogen phosphate. Under optimal conditions (28°C), the surface tension of crude biosurfactant could be reduced to 41 from 71.5 mN/m (water), while its protein concentration was increased to up to 6.5 g/L and the oil displacement efficiency was improved dramatically at 6.5 cm. Two glycoprotein fractions with the molecular masses of 22 and 40 kDa were purified from the biosurfactant, which held great potential for applications in microbial enhanced oil recovery and bioremediation. PMID:26441908

  9. Magnetic properties and magnetocaloric effect of HoCo3B2 compound

    Science.gov (United States)

    Zheng, X. Q.; Xu, J. W.; Zhang, H.; Zhang, J. Y.; Wang, S. G.; Zhang, Y.; Xu, Z. Y.; Wang, L. C.; Shen, B. G.

    2018-05-01

    A sample of HoCo3B2 compound was synthesized, and the magnetic and MCE properties were investigated. Compound shows a change corresponding to R-R (R = rare earth) sublattice magnetic order transition and the transition temperature is determined to be 11.8 K (TC). The characteristic of Arrott plots with positive slope around TC was observed, indicating a second-order phase transition. Based on isothermal magnetization data, together with Maxwell's relationship, the magnetic entropy change (-ΔSM) was calculated. The maximum -ΔSM reaches 7.8, 12.7 and 14.4 J/kg K for field range of 0-2 T, 0-5 T and 0-7 T, respectively. Accordingly, the value of RC (refrigerant capacity) is 99, 289 and 432 J/kg for above field ranges. The large MCE of HoCo3B2 compound indicates its potential application for magnetic refrigeration in low temperature range.

  10. CALIFA, a Dedicated Calorimeter for the R{sup 3}B/FAIR

    Energy Technology Data Exchange (ETDEWEB)

    Cortina-Gil, D., E-mail: d.cortina@usc.es [Universidad de Santiago de Compostela, E-15782 Santiago de Compostela (Spain); Alvarez-Pol, H. [Universidad de Santiago de Compostela, E-15782 Santiago de Compostela (Spain); Aumann, T. [Technische Universität Darmstadt, D-64289 Darmstadt (Germany); Helmholtzzentrum für Schwerionenforschung, D-64291 Darmstadt (Germany); Avdeichikov, V. [University of Lund, SE 221 00 Lund (Sweden); Bendel, M. [Technische Universität München, D-85748 Garching (Germany); Benlliure, J. [Universidad de Santiago de Compostela, E-15782 Santiago de Compostela (Spain); Bertini, D. [Helmholtzzentrum für Schwerionenforschung, D-64291 Darmstadt (Germany); Bezbakh, A. [Joint Institute for Nuclear Research, RU-141980 Dubna (Russian Federation); Bloch, T. [Technische Universität Darmstadt, D-64289 Darmstadt (Germany); Böhmer, M. [Technische Universität München, D-85748 Garching (Germany); Borge, M.J.G.; Briz, J.A. [Instituto Estructura de la Materia, CSIC, E-28006 Madrid (Spain); Cabanelas, P. [Universidad de Santiago de Compostela, E-15782 Santiago de Compostela (Spain); Casarejos, E. [Universidad de Vigo, E-36310 Vigo (Spain); Carmona Gallardo, M. [Instituto Estructura de la Materia, CSIC, E-28006 Madrid (Spain); Cederkäll, J. [University of Lund, SE 221 00 Lund (Sweden); Chulkov, L. [Nuclear Reseach Center, Kurchatov Institute, RU-123182 Moscow (Russian Federation); Dierigl, M. [Technische Universität München, D-85748 Garching (Germany); Di Julio, D. [University of Lund, SE 221 00 Lund (Sweden); Durán, I. [Universidad de Santiago de Compostela, E-15782 Santiago de Compostela (Spain); and others

    2014-06-15

    The R{sup 3}B experiment (Reactions with Relativistic Radioactive Beams) at FAIR (Facility for Antiproton and Ion Research) is a versatile setup dedicated to the study of reactions induced by high-energy radioactive beams. It will provide kinematically complete measurements with high efficiency, acceptance and resolution, making possible a broad physics program with rare-isotopes. CALIFA (CALorimeter for In-Flight detection of gamma-rays and high energy charged pArticles), is a complex detector based on scintillation crystals, that will surround the target of the R{sup 3}B experiment. CALIFA will act as a total absorption gamma-calorimeter and spectrometer, as well as identifier of charged particles from target residues. This versatility is its most challenging requirement, demanding a huge dynamic range, to cover from low energy gamma-rays up to 300 MeV protons. This fact, along with the high-energy of the beams determine the conceptual design of the detector, presented in this paper, together with the technical solutions proposed for its construction.

  11. The superfamily of C3b/C4b-binding proteins

    DEFF Research Database (Denmark)

    Kristensen, Torsten; D'Eustachio, P; Ogata, R T

    1987-01-01

    The determination of primary structures by amino acid and nucleotide sequencing for the C3b-and/or C4b-binding proteins H, C4BP, CR1, B, and C2 has revealed the presence of a common structural element. This element is approximately 60 amino acids long and is repeated in a tandem fashion, commencing...... at the amino-terminal end of each molecule. Two other complement components, C1r and C1s, have two of these repeating units in the carboxy-terminal region of their noncatalytic A chains. Three noncomplement proteins, beta 2-glycoprotein I (beta 2I), the interleukin 2 receptor (IL 2 receptor), and the b chain...... of factor XIII, have 4, 2 and 10 of these repeating units, respectively. These proteins obviously belong to the above family, although there is no evidence that they interact with C3b and/or C4b. Human haptoglobin and rat leukocyte common antigen also contain two and three repeating units, respectively...

  12. Analysis of the big halo event observed by Pamir emulsion chamber -P3B-90-

    International Nuclear Information System (INIS)

    Yamashita, Seibun

    1986-01-01

    There is observed a big air family (named as 'P3B-90') with a large halo by Pamir carbon chamber, called P3B, in USSR-Japan joint experiment at Pamir plateau (4370 m above sea level). Total observed energy of this halo is estimated to be 19000 TeV. It penetrates through both the upper and the lower chamber without decreasing its size. Such strong penetrative power is due to the existence of many hadrons in the halo region which are produced through the atmospheric successive interactions. The energy ratio of hadronic part to electromagnetic part is 1/3 and it is nearly equal to the case of 'Andromeda' observed in Chacaltaya experiment. On the other hand, 298 shower spots (>1 TeV) with total observed energy 1163 TeV are found within about 30 cm of radius from the center of the family. Among the observed shower spots, 45 spots are identified as hadronic ones, whose total observed energy is 345 TeV and 253 spots are electromagnetic ones with total observed energy 818 TeV. There exists a large difference in the power index of the energy spectrum between the electromagnetic and the hadronic components. That is, the hadronic component has a harder spectrum than the electromagnetic component. It shows that there exist a lot of hadrons in high energy region among the observed shower spots. This seems to be a general tendency of the super-high energy families. (author)

  13. Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

    Science.gov (United States)

    Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

    2010-01-01

    N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

  14. Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

    Science.gov (United States)

    Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

    2007-02-23

    N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

  15. Association of variation in Fc gamma receptor 3B gene copy number with rheumatoid arthritis in Caucasian samples

    NARCIS (Netherlands)

    McKinney, Cushla; Fanciulli, Manuela; Merriman, Marilyn E.; Phipps-Green, Amanda; Alizadeh, Behrooz Z.; Koeleman, Bobby P. C.; Dalbeth, Nicola; Gow, Peter J.; Harrison, Andrew A.; Highton, John; Jones, Peter B.; Stamp, Lisa K.; Steer, Sophia; Barrera, Pilar; Coenen, Marieke J. H.; Franke, Barbara; van Riel, Piet L. C. M.; Vyse, Tim J.; Aitman, Tim J.; Radstake, Timothy R. D. J.; Merriman, Tony R.

    2010-01-01

    Objective There is increasing evidence that variation in gene copy number (CN) influences clinical phenotype. The low-affinity Fc gamma receptor 3B (FCGR3B) located in the FCGR gene cluster is a CN polymorphic gene involved in the recruitment to sites of inflammation and activation of

  16. Association of variation in Fcgamma receptor 3B gene copy number with rheumatoid arthritis in Caucasian samples.

    NARCIS (Netherlands)

    McKinney, C.; Fanciulli, M.; Merriman, M.E.; Phipps-Green, A.; Alizadeh, B.Z.; Koeleman, B.P.; Dalbeth, N.; Gow, P.J.; Harrison, A.A.; Highton, J.; Jones, P.B.; Stamp, L.K.; Steer, S.; Barrera, P.; Coenen, M.J.H.; Franke, B.; Riel, P.L.C.M. van; Vyse, T.J.; Aitman, T.J.; Radstake, T.R.D.J.; Merriman, T.R.

    2010-01-01

    OBJECTIVE: There is increasing evidence that variation in gene copy number (CN) influences clinical phenotype. The low-affinity Fcgamma receptor 3B (FCGR3B) located in the FCGR gene cluster is a CN polymorphic gene involved in the recruitment to sites of inflammation and activation of

  17. Presence of a consensus DNA motif at nearby DNA sequence of the mutation susceptible CG nucleotides.

    Science.gov (United States)

    Chowdhury, Kaushik; Kumar, Suresh; Sharma, Tanu; Sharma, Ankit; Bhagat, Meenakshi; Kamai, Asangla; Ford, Bridget M; Asthana, Shailendra; Mandal, Chandi C

    2018-01-10

    Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA "T/AGC/GAGGA/TG" sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing

  18. DNA Methylation Modulates Nociceptive Sensitization after Incision.

    Directory of Open Access Journals (Sweden)

    Yuan Sun

    Full Text Available DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT inhibitor 5-Aza-2'-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2'-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.

  19. Long-Term Outcomes of Total Exudative Retinal Detachments in Stage 3B Coats Disease.

    Science.gov (United States)

    Li, Albert S; Capone, Antonio; Trese, Michael T; Sears, Jonathan E; Kychenthal, Andres; De la Huerta, Irina; Ferrone, Philip J

    2018-06-01

    To evaluate the long-term outcomes of treatment of total exudative retinal detachments (ERDs) secondary to Coats disease (stage 3B) and the role of vitrectomy. Retrospective, observational case series. A total of 16 eyes in 16 patients undergoing treatment for total ERDs secondary to Coats disease with at least 5 years of follow-up. We reviewed the records of patients with stage 3B Coats disease. The interventions, including the timing of vitrectomy if used, and clinical course were recorded. The primary outcome measures were visual acuity at the most recent appointment, whether there was progression to neovascular glaucoma (NVG) or phthisis bulbi, and need for enucleation. All patients received ablative treatment (photocoagulation or cryotherapy), with 8 having scleral buckling (SB) and 6 having external drainage of subretinal fluid (XD). Of the 12 patients who had pars plana vitrectomy (PPV), 8 had early PPV (EV) in the first year after presenting, and 4 of 8 in the expectant management group had late PPV (late vitrectomy) at a mean of 4.3 years post-presentation for treatment of significant traction retinal detachment (TRD). The other 4 patients of 8 in the expectant management group did not require vitrectomy. Mean follow-up overall was 9 1/2 years. At the date of last follow-up, 50% had no light perception or light perception vision, which was consistent across the subgroups that underwent EV (4/8), late vitrectomy (2/4), or no PPV (2/4). A total of 4 of 16 patients had progression to NVG or phthisis, 1 of whom required enucleation. In this retrospective series of patients with Stage 3B Coats disease, ablative therapy with a combination of PPV, XD, or SB was effective in preventing progression to NVG or phthisis in the majority of patients, thus preserving the globe. Half of the patients (4/8) in this series who did not undergo PPV in the early vitrectomy group developed late-onset TRD, suggesting a possible role for early prophylactic vitrectomy with possible

  20. Molecular cloning and characterization of juvenile hormone acid methyltransferase in the honey bee, Apis mellifera, and its differential expression during caste differentiation.

    Directory of Open Access Journals (Sweden)

    Wenfeng Li

    Full Text Available Juvenile hormone acid methyltransferase (JHAMT is an enzyme involved in one of the final steps of juvenile hormone biosynthesis in insects. It transfers a methyl group from S-adenosyl-L-methionine (SAM to the carboxyl group of either farnesoic acid (FA or JH acid (JHA. Several genes coding for JHAMT have been cloned and characterized from insects from different orders, and they have been shown to play critical roles in metamorphosis and reproduction. However, the significance of JHAMT in Hymenopteran insects is unknown. We used RACE amplification method to clone JHAMT cDNA from the honey bee, Apis mellifera (AmJHAMT. The full length cDNA of AmJHAMT that we cloned is 1253bp long and encodes a 278-aa protein that shares 32-36% identity with known JHAMTs. A SAM-binding motif, conserved in the SAM-dependent methyltransferase (SAM-MT superfamily, is present in AmJHAMT. Its secondary structure also contains a typical SAM-MT fold. Most of the active sites bound with SAM and substrates (JHA or FA are conserved in AmJHAMT as in other JHAMT orthologs. Phylogenetic analysis clustered AmJHAMT with the other orthologs from Hymenoptera to form a major clade in the phylogenetic tree. Purified recombinant AmJHAMT protein expressed in E. coli was used to produce polyclonal antibodies and to verify the identity of AmJHAMT by immunoblotting and mass spectrometry. Quantitative RT-PCR and immunoblotting analyses revealed that queen larvae contained significantly higher levels of AmJHAMT mRNA and protein than worker larvae during the periods of caste development. The temporal profiles of both AmJHAMT mRNA and protein in queens and workers showed a similar pattern as the JH biosynthesis. These results suggest that the gene that we cloned codes for a functional JHAMT that catalyzes the final reactions of JH biosynthesis in honey bees. In addition, AmJHAMT may play an important role in honey bee caste differentiation.

  1. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    Science.gov (United States)

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

    2015-01-01

    Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

  2. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Karminska, K. H.; Purta, E.; Hansen, L .H.

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase...

  3. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

    Science.gov (United States)

    Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

    2016-04-04

    Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  4. The 29.5 kb APOBEC3B Deletion Polymorphism Is Not Associated with Clinical Outcome of Breast Cancer.

    Science.gov (United States)

    Liu, Jingjing; Sieuwerts, Anieta M; Look, Maxime P; van der Vlugt-Daane, Michelle; Meijer-van Gelder, Marion E; Foekens, John A; Hollestelle, Antoinette; Martens, John W M

    2016-01-01

    Increased APOBEC3B mRNA levels are associated with a hypermutator phenotype and poor prognosis in ER-positive breast cancer patients. In addition, a 29.5 kb deletion polymorphism of APOBEC3B, resulting in an APOBEC3A-B hybrid transcript, has been associated with an increased breast cancer risk and the hypermutator phenotype. Here we evaluated whether the APOBEC3B deletion polymorphism also associates with clinical outcome of breast cancer. Copy number analysis was performed by quantitative PCR (qPCR) in primary tumors of 1,756 Dutch breast cancer patients. The APOBEC3B deletion was found in 187 patients of whom 16 carried a two-copy deletion and 171 carried a one-copy deletion. The prognostic value of the APOBEC3B deletion for the natural course of the disease was evaluated among 1,076 lymph-node negative (LNN) patients who did not receive adjuvant systemic treatment. No association was found between APOBEC3B copy number values and the length of metastasis-free survival (MFS; hazard ratio (HR) = 1.00, 95% confidence interval (CI) = 0.90-1.11, P = 0.96). Subgroup analysis by ER status also did not reveal an association between APOBEC3B copy number values and the length of MFS. The predictive value of the APOBEC3B deletion was assessed among 329 ER-positive breast cancer patients who received tamoxifen as the first-line therapy for recurrent disease and 226 breast cancer patients who received first-line chemotherapy for recurrent disease. No association between APOBEC3B copy number values and the overall response rate (ORR) to either tamoxifen (odds ratio (OR) = 0.88, 95% CI = 0.69-1.13, P = 0.31) or chemotherapy (OR = 0.97, 95% CI = 0.71-1.33, P = 0.87) was found. Thus, in contrast to APOBEC3B mRNA levels, the APOBEC3B deletion polymorphism has neither a prognostic nor a predictive value for breast cancer patients. Although a correlation exists between APOBEC3B copy number and mRNA expression, it is relatively weak. This suggests that other mechanisms exist that may

  5. Na+/H+ and Na+/NH4+ exchange activities of zebrafish NHE3b expressed in Xenopus oocytes

    Science.gov (United States)

    Ito, Yusuke; Kato, Akira; Hirata, Taku; Hirose, Shigehisa

    2014-01-01

    Zebrafish Na+/H+ exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na+ is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na+ absorption but not leakage. However, zNHE3b-mediated Na+ absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na+-free media resulted in significant decrease in intracellular pH (pHi) and intracellular Na+ activity (aNai). aNai increased significantly when the cytoplasm was acidified by media containing CO2-HCO3− or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na+/H+ exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH3/NH4+ resulted in significant decreases in pHi and aNai and significant increase in intracellular NH4+ activity, indicating that zNHE3b mediates the Na+/NH4+ exchange. In low-Na+ (0.5 mM) media, zNHE3b oocytes maintained aNai of 1.3 mM, and Na+-influx was observed when pHi was decreased by media containing CO2-HCO3− or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na+ absorption from ion-poor fresh water by its Na+/H+ and Na+/NH4+ exchange activities. PMID:24401990

  6. Dynamic Wind-Tunnel Testing of a Sub-Scale Iced S-3B Viking

    Science.gov (United States)

    Lee, Sam; Barnhart, Billy; Ratvasky, Thomas P.

    2012-01-01

    The effect of ice accretion on a 1/12-scale complete aircraft model of S-3B Viking was studied in a rotary-balance wind tunnel. Two types of ice accretions were considered: ice protection system failure shape and runback shapes that form downstream of the thermal ice protection system. The results showed that the ice shapes altered the stall characteristics of the aircraft. The ice shapes also reduced the control surface effectiveness, but mostly near the stall angle of attack. There were some discrepancies with the data with the flaps deflected that were attributed to the low Reynolds number of the test. Rotational and forced-oscillation studies showed that the effects of ice were mostly in the longitudinal forces, and the effects on the lateral forces were relatively minor.

  7. The time development of hadronic showers and the T3B experiment

    International Nuclear Information System (INIS)

    Soldner, Christian

    2013-01-01

    The compact linear collider (CLIC) is a future linear e + e - collider operated at a center of mass energy of up to 3 TeV and with a collision rate of particle bunches of up to 2 GHz. This poses challenging requirements on the detector system. The accumulation of background events, such as γγ→hadrons resulting from Beamstrahlung, must be minimized through a precise time stamping capability in all subdetector systems. In the event reconstruction, the energy depositions within the calorimeters will be used to assign events precisely to a small set of consecutive bunch crossings. The finite time evolution of hadronic showers, on the other hand, requires an extended integration time to achieve a satisfactory energy resolution in the calorimeter. The energy resolution is also deteriorated by the leakage of shower particles. Tungsten is foreseen as dense absorber material, but the time evolution of hadron showers within such a calorimeter is not sufficiently explored yet. In the context of this thesis, the T3B experiment (short for Tungsten Timing Test Beam) was designed and constructed. It is optimized to measure the time development and the contribution of delayed energy depositions within hadronic cascades. The T3B experiment consists of 15 scintillator cells assembled in a strip. The scintillation light generated within the cells is detected by novel silicon photomultiplier whose signal is read out with fast oscilloscopes providing a sampling rate of 1.25 GHz. This strip was positioned behind two different calorimeter prototypes of the CALICE collaboration which use a tungsten and steel (for comparison) absorber structure. T3B was part of the CALICE test beam campaign 2010/2011 carried out at the PS and SPS at CERN and acquired data on hadronic showers in an energy range of 2-300 GeV. A test beam optimized data acquisition software was developed from scratch. With the development and application of a novel waveform decomposition algorithm, the time of arrival of

  8. Magnetic ordering and spin-reorientation transitions in TbCo3B2

    International Nuclear Information System (INIS)

    Dubman, Moshe; Caspi, El'ad N.; Ettedgui, Hanania; Keller, Lukas; Melamud, Mordechai; Shaked, Hagai

    2005-01-01

    The magnetic structure of the compound TbCo 3 B 2 has been studied in the temperature range 1.5 K≤T≤300 K by means of neutron powder diffraction, magnetization, magnetic ac susceptibility, and heat capacity measurements. The compound is of hexagonal symmetry and is paramagnetic at 300 K, undergoes a magnetic Co-Co ordering transition at ∼170 K, and a second magnetic Tb-Tb ordering transition at ∼30 K. The latter induces a spin-reorientation transition, in which the magnetic axis rotates from the c axis toward the basal plane. Below this transition a symmetry decrease (γ magnetostriction) sets in, leading to an orthorhombic distortion of the crystal lattice. The crystal and magnetic structures and interactions and their evolution with temperature are discussed using a microscopic physical model

  9. The time development of hadronic showers and the T3B experiment

    Energy Technology Data Exchange (ETDEWEB)

    Soldner, Christian

    2013-06-06

    The compact linear collider (CLIC) is a future linear e{sup +}e{sup -} collider operated at a center of mass energy of up to 3 TeV and with a collision rate of particle bunches of up to 2 GHz. This poses challenging requirements on the detector system. The accumulation of background events, such as {gamma}{gamma}{yields}hadrons resulting from Beamstrahlung, must be minimized through a precise time stamping capability in all subdetector systems. In the event reconstruction, the energy depositions within the calorimeters will be used to assign events precisely to a small set of consecutive bunch crossings. The finite time evolution of hadronic showers, on the other hand, requires an extended integration time to achieve a satisfactory energy resolution in the calorimeter. The energy resolution is also deteriorated by the leakage of shower particles. Tungsten is foreseen as dense absorber material, but the time evolution of hadron showers within such a calorimeter is not sufficiently explored yet. In the context of this thesis, the T3B experiment (short for Tungsten Timing Test Beam) was designed and constructed. It is optimized to measure the time development and the contribution of delayed energy depositions within hadronic cascades. The T3B experiment consists of 15 scintillator cells assembled in a strip. The scintillation light generated within the cells is detected by novel silicon photomultiplier whose signal is read out with fast oscilloscopes providing a sampling rate of 1.25 GHz. This strip was positioned behind two different calorimeter prototypes of the CALICE collaboration which use a tungsten and steel (for comparison) absorber structure. T3B was part of the CALICE test beam campaign 2010/2011 carried out at the PS and SPS at CERN and acquired data on hadronic showers in an energy range of 2-300 GeV. A test beam optimized data acquisition software was developed from scratch. With the development and application of a novel waveform decomposition algorithm

  10. STUDY ON THE RETRIEVAL OF SNOW DEPTH FROM FY3B/MWRI IN THE ATCTIC

    Directory of Open Access Journals (Sweden)

    L. Li

    2016-06-01

    Full Text Available temperatures. Given the high albedo and low thermal conductivity, snow is regarded as one of the key reasons for the amplification of the warming in polar regions. The distributions of sea ice and snow depth are essential to the whole thermal conduction in the Arctic. This study focused on the retrieval of snow depth on sea ice from brightness temperatures of the MicroWave Radiometer Imager (MWRI onboard the FengYun (FY-3B satellite during the period from December 1, 2010 to April 30, 2011. After cross calibrated to the Advanced Microwave Scanning Radiometer–EOS (AMSR-E Level 2A data, the MWRI brightness temperatures were applied to calculate the sea ice concentrations based on the Arctic Radiation and Turbulence Interaction Study Sea Ice (ASI algorithm. According to the proportional relationship between the snow depth and the surface scattering in 18.7 and 36.5 GHz, the snow depths were derived. In order to eliminate the influence of uncertainties in grain sizes of snow as well as sporadic weather effects, the seven-day averaged snow depths were calculated. Then the results were compared with the snow depths from the AMSR-E Level 3 Sea Ice products. The bias of differences between the MWRI and the AMSR-E Level 3 products are ranged between −1.09 and −0.32 cm,while the standard deviations and the correlation coefficients are ranged from 2.47 to 2.88 cm and from 0.78 to 0.90 for different months. As a result, it could be summarized that FY3B/MWRI showed a promising prospect in retrieving snow depth on sea ice.

  11. Structure of receptive fields in a computational model of area 3b of primary sensory cortex.

    Science.gov (United States)

    Detorakis, Georgios Is; Rougier, Nicolas P

    2014-01-01

    In a previous work, we introduced a computational model of area 3b which is built upon the neural field theory and receives input from a simplified model of the index distal finger pad populated by a random set of touch receptors (Merkell cells). This model has been shown to be able to self-organize following the random stimulation of the finger pad model and to cope, to some extent, with cortical or skin lesions. The main hypothesis of the model is that learning of skin representations occurs at the thalamo-cortical level while cortico-cortical connections serve a stereotyped competition mechanism that shapes the receptive fields. To further assess this hypothesis and the validity of the model, we reproduced in this article the exact experimental protocol of DiCarlo et al. that has been used to examine the structure of receptive fields in area 3b of the primary somatosensory cortex. Using the same analysis toolset, the model yields consistent results, having most of the receptive fields to contain a single region of excitation and one to several regions of inhibition. We further proceeded our study using a dynamic competition that deeply influences the formation of the receptive fields. We hypothesized this dynamic competition to correspond to some form of somatosensory attention that may help to precisely shape the receptive fields. To test this hypothesis, we designed a protocol where an arbitrary region of interest is delineated on the index distal finger pad and we either (1) instructed explicitly the model to attend to this region (simulating an attentional signal) (2) preferentially trained the model on this region or (3) combined the two aforementioned protocols simultaneously. Results tend to confirm that dynamic competition leads to shrunken receptive fields and its joint interaction with intensive training promotes a massive receptive fields migration and shrinkage.

  12. Structure of Receptive Fields in a Computational Model of Area 3b of Primary Sensory Cortex

    Directory of Open Access Journals (Sweden)

    Georgios eDetorakis

    2014-07-01

    Full Text Available In a previous work, we introduced a computational model of area 3b which is built upon the neural field theory and receives input from a simplified model of the index distal finger pad populated by a random set of touch receptors(Merkell cells. This model has been shown to be able to self-organize following the random stimulation of the finger pad model and to cope, to some extent, with cortical or skin lesions. The main hypothesis of the model is that learning of skin representations occurs at the thalamo-cortical level while cortico-cortical connections serve a stereotyped competition mechanism that shapes the receptive fields. To further assess this hypothesis and the validity of the model, we reproduced in this article the exact experimental protocol of DiCarlo et al. that has been used to examine the structure of receptive fields in area 3b of the primary somatosensory cortex. Using the same analysis toolset, the model yields consistent results, having most of the receptive fields to contain a single region of excitation and one to severalregions of inhibition. We further proceeded our study using a dynamic competition that deeply influences the formation of the receptive fields. We hypothesized this dynamic competition to correspond to some form of somatosensory attention that may help to precisely shape the receptive fields. To test this hypothesis, we designed a protocol where an arbitrary region of interest is delineated on the index distal finger pad and we either (1 instructed explicitly the model to attend to this region (simulating an attentional signal (2 preferentially trained the model on this region or (3combined the two aforementioned protocols simultaneously. Results tend to confirm that dynamic competition leads to shrunken receptive fields and its joint interaction with intensive training promotes a massive receptive fields migration and shrinkage.

  13. Development of a graphic interface for the Ramona-3B code

    International Nuclear Information System (INIS)

    Maldonado D, D.; Santos O, M.A.

    2003-01-01

    In this work a graphic interface that interprets the data of the Ramona-3B code is presented. The Ramona-3B code it is a computer program, that it uses text files as input and its generate output also of this type. The quantity of generated information is so big that always it is necessary to process this information with graphic tools to be able to analyze the results of the simulations of nuclear centrals with boiling water reactors. When having a modern tool that it translates text in graphics in an automatic way and that it is of great versatility, one can obtain a graphic interface that facilitates the interpretation of how a BWR nuclear plant behaves. To achieve this tool the key it has been a program that it reads chains of previously indicated characters that keeps the data in a file for later to manipulate them in the creation of the graphic interface. It is used a software of easy access that resists the processing of a great one quantity of data and that later its have been able to graph. Another important function of this interface it is allowing the modification of the input file for Ramona using graphic unfolding and helps in it lines without necessarily to go to the file with input data. For the design of graphic interface it was decided first to show the more representative variables of a BWR type nuclear plant. It is used Mat lab as platform on several options, as PHP, Lab view or C ' . The obtained graphs allow monitoring the plant and to have the control of selected variables. For the graphic interface only is necessary to indicate it the variable to simulate for to be able to interpret graphically the behavior of the BWR type nuclear plant. This tool is of great utility for the teaching of students that they are interested in this type of nuclear topics. (Author)

  14. An easy-to-perform photometric assay for methyltransferase activity measurements.

    Science.gov (United States)

    Schäberle, Till F; Siba, Christian; Höver, Thomas; König, Gabriele M

    2013-01-01

    Methyltransferases (MTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a suitable substrate. Such methylations are important modifications in secondary metabolisms, especially on natural products produced by polyketide synthases and nonribosomal peptide synthetases, many of which are of special interest due to their prominent pharmacological activities (e.g., lovastatin, cyclosporin). To gain basic biochemical knowledge on the methylation process, it is of immense relevance to simplify methods concerning experimental problems caused by a large variety in substrates. Here, we present a photometric method to analyze MT activity by measuring SAM consumption in a coupled enzyme assay. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Methyl transfer in glucosinolate biosynthesis mediated by indole glucosinolate O-Methyltransferase 5

    DEFF Research Database (Denmark)

    Pfalz, Marina; Mukhaimar, Maisara; Perreau, François

    2016-01-01

    in position 1 (1-IG modification) or 4 (4-IG modification). Products of the 4-IG modification pathway mediate plant-enemy interactions and are particularly important for Arabidopsis innate immunity. While CYP81Fs encoding cytochrome P450 monooxygenases and IGMTs encoding indole glucosinolate O...... with moderate similarity to previously characterized IGMTs, encodes the methyltransferase that is responsible for the conversion of 1OHI3M to 1MOI3M. Disruption of IGMT5 function increases resistance against the root-knot nematode Meloidogyne javanica and suggests a potential role for the 1-IG modification...

  16. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  17. Reasons of carcinogenesis indicate a big-bang inside: a hypothesis for the aberration of DNA methylation.

    Science.gov (United States)

    Roy, A; Roy Chattopadhyay, N

    2013-07-01

    Cancer involves various sets of altered gene functions which embrace all the three basic mechanisms of regulation of gene expression. However, no common mechanism is inferred till date for this versatile disease and thus no full proof remedy can be offered. Here we show that the basic mechanisms are interlinked and indicate towards one of those mechanisms as being the superior one; the methylation of cytosines in specific DNA sequences, for the initiation and maintenance of carcinogenesis. The analyses of the previous reports and the nucleotide sequences of the DNA methyltransferases strongly support the assumption that the mutation(s) in the DNA-binding site(s) of DNA-methyltransferases acts as a master regulator; though it continues the cycle from mutation to repair to methylation. We anticipate that our hypothesis will start a line of study for the proposal of a treatment regime for cancers by introducing wild type methyltransferases in the diseased cells and/or germ cells, and/or by targeting ligands to the altered binding domain(s) where a mutation in the concerned enzyme(s) is seen. Copyright © 2013. Published by Elsevier Ltd.

  18. Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

    Science.gov (United States)

    Bate-Eya, Laurel T; Gierman, Hinco J; Ebus, Marli E; Koster, Jan; Caron, Huib N; Versteeg, Rogier; Dolman, M Emmy M; Molenaar, Jan J

    2017-04-01

    Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. let-7 Modulates Chromatin Configuration and Target Gene Repression through Regulation of the ARID3B Complex

    Directory of Open Access Journals (Sweden)

    Tsai-Tsen Liao

    2016-01-01

    Full Text Available Let-7 is crucial for both stem cell differentiation and tumor suppression. Here, we demonstrate a chromatin-dependent mechanism of let-7 in regulating target gene expression in cancer cells. Let-7 directly represses the expression of AT-rich interacting domain 3B (ARID3B, ARID3A, and importin-9. In the absence of let-7, importin-9 facilitates the nuclear import of ARID3A, which then forms a complex with ARID3B. The nuclear ARID3B complex recruits histone demethylase 4C to reduce histone 3 lysine 9 trimethylation and promotes the transcription of stemness factors. Functionally, expression of ARID3B is critical for the tumor initiation in let-7-depleted cancer cells. An inverse association between let-7 and ARID3A/ARID3B and prognostic significance is demonstrated in head and neck cancer patients. These results highlight a chromatin-dependent mechanism where let-7 regulates cancer stemness through ARID3B.

  20. BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

    Science.gov (United States)

    Rodríguez, Andrea E; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

    2016-01-01

    Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.

  1. EphrinB1/EphB3b Coordinate Bidirectional Epithelial-Mesenchymal Interactions Controlling Liver Morphogenesis and Laterality

    DEFF Research Database (Denmark)

    Cayuso, Jordi; Dzementsei, Aliaksandr; Fischer, Johanna C

    2016-01-01

    Positioning organs in the body often requires the movement of multiple tissues, yet the molecular and cellular mechanisms coordinating such movements are largely unknown. Here, we show that bidirectional signaling between EphrinB1 and EphB3b coordinates the movements of the hepatic endoderm...... and adjacent lateral plate mesoderm (LPM), resulting in asymmetric positioning of the zebrafish liver. EphrinB1 in hepatoblasts regulates directional migration and mediates interactions with the LPM, where EphB3b controls polarity and movement of the LPM. EphB3b in the LPM concomitantly repels hepatoblasts...

  2. Association between TPMT*3C and decreased thiopurine S-methyltransferase activity in patients with neuromyelitis optica spectrum disorders in China.

    Science.gov (United States)

    Gong, Xiaoqing; Mei, Shenghui; Li, Xindi; Li, Xingang; Zhou, Heng; Liu, Yonghong; Zhou, Anna; Yang, Li; Zhao, Zhigang; Zhang, Xinghu

    2018-06-01

    Thiopurines are effective drugs in treating neuromyelitis optica spectrum disorders and other diseases. Thiopurines' toxicity is mainly imputed to thiopurine S-methyltransferase activity. In Chinese population, the most common and important variation of thiopurine S-methyltransferase is TPMT*3C (rs1142345). This study aims to reveal the association between thiopurine S-methyltransferase activity and genetic polymorphisms of thiopurine S-methyltransferase in patients with neuromyelitis optica spectrum disorders in China. A liquid chromatography tandem mass/mass method was used to evaluate the thiopurine S-methyltransferase activity by using 6-mercapthioprine as the substrate in human erythrocyte haemolysate via 1 h incubation at 37 °C to form its methylated product 6-methylmercaptopurine. The amount of 6-methylmercaptopurine was adjusted by haematocrit and normalized to 8 × 10 8 erythrocytes. The selected polymorphisms of thiopurine S-methyltransferase were identified using MassARRAY system (Sequenom) and multiple SNaPshot technique. In 69 patients with neuromyelitis optica spectrum disorders, thiopurine S-methyltransferase activity was 80.29-154.53 (127.51 ± 16.83) pmol/h/8 × 10 8 erythrocytes. TPMT*3C (rs1142345) was associated with lower thiopurine S-methyltransferase activity (BETA = -25.37, P = 0.011). Other selected variants were not associated with thiopurine S-methyltransferase activity. TPMT*3C affects TPMT activity in Chinese patients with neuromyelitis optica spectrum disorders. Further studies are warranted to confirm the results. TPRs = thiopurines; NMOSD = neuromyelitis optica spectrum disorders; TPMT = thiopurine S-methyltransferase; LC-MS/MS = liquid chromatography tandem mass/mass; 6-MMP = 6-methylmercaptopurine; IS = internal standard; SNP = single nucleotide polymorphism; MAF = minor allele frequency; HWE = Hardy-Weinberg equilibrium; BETA = regression coefficients; UTR-3 = untranslated region 3.

  3. Regulation of DNA Methylation Patterns by CK2-Mediated Phosphorylation of Dnmt3a

    Directory of Open Access Journals (Sweden)

    Rachel Deplus

    2014-08-01

    Full Text Available DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.

  4. Modeling DNA

    Science.gov (United States)

    Robertson, Carol

    2016-01-01

    Deoxyribonucleic acid (DNA) is life's most amazing molecule. It carries the genetic instructions that almost every organism needs to develop and reproduce. In the human genome alone, there are some three billion DNA base pairs. The most difficult part of teaching DNA structure, however, may be getting students to visualize something as small as a…

  5. Thiopurines inhibit bovine viral diarrhea virus production in a thiopurine methyltransferase-dependent manner.

    Science.gov (United States)

    Hoover, Spencer; Striker, Rob

    2008-04-01

    The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine (AZA) has in vitro activity against bovine viral diarrhea virus (BVDV). While the mechanism of inhibition is unknown, AZA and related thiopurine nucleoside analogues have been used as immunosuppressants for decades and both AZA metabolites and cellular genes involved in AZA metabolism have been extensively characterized. Here, we show that only certain riboside metabolites have antiviral activity and identify the most potent known antiviral AZA metabolite as 6-methylmercaptopurine riboside (6MMPr). The antiviral activity of 6MMPr is antagonized by adenosine, and is specific to BVDV and not to the related yellow fever virus. An essential step in the conversion of AZA to 6MMPr is the addition of a methyl group onto the sulfur atom attached to position six of the purine ring. Intracellularly, the methyl group is added by thiopurine methyltransferase (TPMT), an S-adenosyl methionine-dependent methyltransferase. Either chemically bypassing or inhibiting TPMT modulates antiviral activity of AZA metabolites. TPMT exists in several variants with varying levels of activity and since 6MMPr is a potent antiviral, the antiviral activity of AZA may be modulated by host genetics.

  6. Crystallization and preliminary X-ray crystallographic studies of O-methyltransferase from Anabaena PCC 7120

    International Nuclear Information System (INIS)

    Li, Guoming; Tang, Zhenting; Meng, Geng; Dai, Kesheng; Zhao, Jindong; Zheng, Xiaofeng

    2009-01-01

    The O-methyltransferase (OMT) from the Anabaena PCC 7120 has been overexpressed in a soluble form in E. coli, purified and crystallized. The crystals belonged to space group C222 1 and diffracted to 2.4 Å resolution. O-Methyltransferase (OMT) is a ubiquitous enzyme that exists in bacteria, plants and humans and catalyzes a methyl-transfer reaction using S-adenosyl-l-methionine as a methyl donor and a wide range of phenolics as acceptors. To investigate the structure and function of OMTs, omt from Anabaena PCC 7120 was cloned into expression vector pET21a and expressed in a soluble form in Escherichia coli strain BL21 (DE3). The recombinant OMT protein was purified to homogeneity using a two-step strategy. Crystals of OMT that diffracted to a resolution of 2.4 Å were obtained using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222 1 , with unit-cell parameters a = 131.620, b = 227.994, c = 150.777 Å, α = β = γ = 90°. There are eight molecules per asymmetric unit

  7. Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C; Rife, Jason P; Ban, Nenad

    2012-03-23

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

  8. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis*

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C.; Rife, Jason P.; Ban, Nenad

    2012-01-01

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3′-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. PMID:22308031

  9. Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard

    International Nuclear Information System (INIS)

    LeDuc, Danika L.; AbdelSamie, Manal; Montes-Bayon, Maria; Wu, Carol P.; Reisinger, Sarah J.; Terry, Norman

    2006-01-01

    A major goal of our selenium (Se) phytoremediation research is to use genetic engineering to develop fast-growing plants with an increased ability to tolerate, accumulate, and volatilize Se. To this end we incorporated a gene (encoding selenocysteine methyltransferase, SMT) from the Se hyperaccumulator, Astragalus bisulcatus, into Indian mustard (LeDuc, D.L., Tarun, A.S., Montes-Bayon, M., Meija, J., Malit, M.F., Wu, C.P., AbdelSamie, M., Chiang, C.-Y., Tagmount, A., deSouza, M., Neuhierl, B., Boeck, A., Caruso, J., Terry, N., 2004. Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation Plant Physiol. 135, 377-383.). The resulting transgenic plants successfully enhanced Se phytoremediation in that the plants tolerated and accumulated Se from selenite significantly better than wild type. However, the advantage conferred by the SMT enzyme was much less when Se was supplied as selenate. In order to enhance the phytoremediation of selenate, we developed double transgenic plants that overexpressed the gene encoding ATP sulfurylase (APS) in addition to SMT, i.e., APS x SMT. The results showed that there was a substantial improvement in Se accumulation from selenate (4 to 9 times increase) in transgenic plants overexpressing both APS and SMT. - Simultaneous overexpression of APS and SMT genes in Indian mustard greatly increases ability to accumulate selenate

  10. The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

    Science.gov (United States)

    Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

    2012-01-01

    Summary Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified. PMID:22554051

  11. Structure and Mechanism of the Rebeccamycin Sugar 4'-O-Methyltransferase RebM

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shanteri; McCoy, Jason G.; Zhang, Changsheng; Bingman, Craig A.; Phillips, Jr., George N.; Thorson, Jon S. (UW)

    2008-12-12

    The 2.65-{angstrom} crystal structure of the rebeccamycin 4'-O-methyltransferase RebM in complex with S-adenosyl-l-homocysteine revealed RebM to adopt a typical S-adenosylmethionine-binding fold of small molecule O-methyltransferases (O-MTases) and display a weak dimerization domain unique to MTases. Using this structure as a basis, the RebM substrate binding model implicated a predominance of nonspecific hydrophobic interactions consistent with the reported ability of RebM to methylate a wide range of indolocarbazole surrogates. This model also illuminated the three putative RebM catalytic residues (His{sup 140/141} and Asp{sup 166}) subsequently found to be highly conserved among sequence-related natural product O-MTases from GC-rich bacteria. Interrogation of these residues via site-directed mutagenesis in RebM demonstrated His{sup 140} and Asp{sup 166} to be most important for catalysis. This study reveals RebM to be a member of the general acid/base-dependent O-MTases and, as the first crystal structure for a sugar O-MTase, may also present a template toward the future engineering of natural product MTases for combinatorial applications.

  12. The Histone Methyltransferase Activity of MLL1 Is Dispensable for Hematopoiesis and Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Bibhu P. Mishra

    2014-05-01

    Full Text Available Despite correlations between histone methyltransferase (HMT activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4 methyltransferase critical for maintaining hematopoietic stem cells (HSCs. Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16 acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

  13. Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

    Science.gov (United States)

    Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

    2013-01-01

    Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

  14. Improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine n-methyltransferase

    International Nuclear Information System (INIS)

    Bowsher, R.R.; Henry, D.P.

    1986-01-01

    Radioenzymatic assays have been developed for catecholamines using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for norepinephrine (NE) and require minimal manipulative effort but until now have been less sensitive than the more complex procedures using COMT. The authors report an improved purification scheme for bovine PNMT which has permitted development of an NE assay with dramatically improved sensitivity (0.5 pg), specificity and reproducibility (C.V. < 5%). PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sepharcryl S-200 and Phenyl-Boronate Agarose. Recovery of PNMT through the purification scheme was 50%, while blank recovery was <.001%. NE can be directly quantified in 25 ul of human plasma and an 80 tube assay can be completed within 4 h. The capillary to venous plasma NE gradient was examined in 8 normotensive male subjects. Capillary plasma (NE (211.2 +/- 61.3 pg/ml)) was lower than venous plasma NE (366.6 +/- 92.5 pg/ml) in all subjects (p < 0.005). This difference suggests that capillary (NE) may be a unique indicator of sympathetic nervous system activity in vivo. In conclusion, purification of PNMT has facilitated development of an improved radioenzymatic for NE with significantly improved sensitivity

  15. An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

    Science.gov (United States)

    Kalb, Daniel; Heinekamp, Thorsten; Schieferdecker, Sebastian; Nett, Markus; Brakhage, Axel A; Hoffmeister, Dirk

    2016-10-04

    S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. De novo DNA methylation during monkey pre-implantation embryogenesis.

    Science.gov (United States)

    Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

    2017-04-01

    Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis.

  17. 188Re DD-3B6/22 Fab' for use in therapy of ovarian cancer: labelling and animal studies

    International Nuclear Information System (INIS)

    Schmidt, Peter F.; Smith, Suzanne V.; Bundesen, Peter G.

    1998-01-01

    A fast and high yielding method of 188 Re radiolabelling DD-3B6/22 Fab' is described. An inert atmosphere [N 2 (g)] and ascorbic acid was essential for preparation and storage of therapeutic levels (≤2 GBq/mg) for up to 24 h. Immunoreactivity was greater than 75%. Pharmacokinetic studies in nu/nu mice demonstrated localisation of 188 Re DD-3B6/22 Fab' was equivalent and correlated well with the behaviour observed for 99m Tc DD-3B6/22 Fab' used to image ovarian cancer. Excellent stability at the target site in vivo supports the potential use of 188 Re DD-3B6/22 Fab' in the therapy of ovarian cancer

  18. TiO2/beads as a photocatalyst for the degradation of X3B azo dye

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The feasibility of photocatalytic degradation of X3B azo dye by TiO2/beads photocatalyst was studied. The effects of parameters such as the amount of TiO2/beads, airflow, as well as the concentrations of H2O2, Fe3+, Mg2+ and Na+ on the photocatalytic degradation of X3B azo dye were also studied. The results showed that 25 mg/dm3 X3B azo dye can be photocatalytically degraded completely by 30 min illumination with a 375W medium pressure mercury lamp. Adding a small amount of H2O2 or Fe3+, the efficiencies of photocatalytic degradation of X3B azo dye were increased rapidly. The mechanisms of the reaction and the role of the additives were also investigated. After 120 hours TiO2/beads showed no significant loss of the photocatalytic activity.

  19. The admissibility criterion under article 35(3)(b) ECHR: a "significant disadvantage" to human rights protection? / Nikos Vogiatzis

    Index Scriptorium Estoniae

    Vogiatzis, Nikos

    2016-01-01

    Vastuvõetavuse kriteeriumist Inimõiguste ja põhivabaduste kaitse konventsiooni artikli 35(3)(b) alusel, artiklist tulenevast piirangust õigusemõistmisele rahvusvahelisel tasandil ja individuaalkaebuse esitamise piirangutest Euroopa Inimõiguste Kohtusse

  20. DNMT3B -579 G>T Promoter Polymorphism and the Risk of Gastric Cancer in the West of Iran.

    Science.gov (United States)

    Ahmadi, Kulsom; Soleimani, Azam; Irani, Shiva; Kiani, Aliasghar; Ghanadi, Kourosh; Noormohamadi, Zahra; Sakinejad, Foroozan

    2018-06-01

    Many studies have suggested that modulation of DNMT3B function caused by single nucleotide polymorphisms of the DNMT3B promoter region may underlie the susceptibility to various cancers such as tumors of the digestive system. The aim of this study was to investigate the effect of -579 G>T polymorphism in the promoter of the DNMT3B gene on risk of gastric cancer in a population from West Iran. We conducted a case-control study in 100 gastric cancer patients and 112 cancer-free controls to assess the correlation between DNMT3B -579 G>T (rs1569686) polymorphism and the risk of gastric cancer. Detection of genotypes of DNMT3B G39179T polymorphism was analyzed by PCR-RFLP. There was no significant difference in the distribution of DNMT3B -579 G>T genotypes between the cases and controls. However, in the stratified analysis by clinicopathological characteristic types, we found that statistically, the risk susceptibility to gastric cancer was significantly associated with tumor grade II and GT/TT genotype of patients, compared to patients having GG genotype, (OR = 5.4737, 95% CI = 1.4746. 20.3184, P = 0.01). Our study suggested that the -579 T allele may increase the relative risk for the progression of clinicopathological characteristic of tumor grade of gastric cancer patients.

  1. Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

    International Nuclear Information System (INIS)

    Mizuno, Kouichi; Matsuzaki, Masahiro; Kanazawa, Shiho; Tokiwano, Tetsuo; Yoshizawa, Yuko; Kato, Misako

    2014-01-01

    Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl- 14 C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with

  2. Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

    Energy Technology Data Exchange (ETDEWEB)

    Mizuno, Kouichi, E-mail: koumno@akita-pu.ac.jp [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Matsuzaki, Masahiro [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kanazawa, Shiho [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan); Tokiwano, Tetsuo; Yoshizawa, Yuko [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kato, Misako [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan)

    2014-10-03

    Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-{sup 14}C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or

  3. Folic Acid Supplementation Delays Atherosclerotic Lesion Development by Modulating MCP1 and VEGF DNA Methylation Levels In Vivo and In Vitro

    Science.gov (United States)

    Cui, Shanshan; Li, Wen; Lv, Xin; Wang, Pengyan; Gao, Yuxia; Huang, Guowei

    2017-01-01

    The pathogenesis of atherosclerosis has been partly acknowledged to result from aberrant epigenetic mechanisms. Accordingly, low folate levels are considered to be a contributing factor to promoting vascular disease because of deregulation of DNA methylation. We hypothesized that increasing the levels of folic acid may act via an epigenetic gene silencing mechanism to ameliorate atherosclerosis. Here, we investigated the atheroprotective effects of folic acid and the resultant methylation status in high-fat diet-fed ApoE knockout mice and in oxidized low-density lipoprotein-treated human umbilical vein endothelial cells. We analyzed atherosclerotic lesion histology, folate concentration, homocysteine concentration, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and DNA methyltransferase activity, as well as monocyte chemotactic protein-1 (MCP1) and vascular endothelial growth factor (VEGF) expression and promoter methylation. Folic acid reduced atherosclerotic lesion size in ApoE knockout mice. The underlying folic acid protective mechanism appears to operate through regulating the normal homocysteine state, upregulating the SAM: SAH ratio, elevating DNA methyltransferase activity and expression, altering MCP1 and VEGF promoter methylation, and inhibiting MCP1 and VEGF expression. We conclude that folic acid supplementation effectively prevented atherosclerosis by modifying DNA methylation through the methionine cycle, improving DNA methyltransferase activity and expression, and thus changing the expression of atherosclerosis-related genes. PMID:28475147

  4. Neutron-capture reactions with the R{sup 3}B-CaveC setup

    Energy Technology Data Exchange (ETDEWEB)

    Heine, Marcel [IKP, TU Darmstadt (Germany)

    2014-07-01

    Recent research has shown that the (n,γ) transition-rates on light nuclei can have an influence on the neutron-balance during the r-process. Especially neutron rich carbon isotopes play an important role in r-process nucleo synthesis network calculations which include light nuclei, since these nuclei are aligned along major flow-paths. In particular {sup 18}C is of interest, because it can be interpreted as a waiting point. The {sup 17}C(n,γ){sup 18}C rate could so far only be estimated theoretically and has an uncertainty of a factor of ten [1]. At the R{sup 3}B-CaveC setup at GSI we have measured the (n,γ) time reversed reaction, i.e. {sup 18}C(γ,n){sup 17}C for the above mentioned nucleus, via the Coulomb-breakup of {sup 18}C beam. The kinematically complete measurement allows extracting energy dependent neutron-capture cross section with respect to the excitation energy by using the invariant-mass method. Experimental results are presented in comparison to theoretical calculations.

  5. Preparation of Fly ash Based Adsorbents for Removal Active Red X-3B from Dying Wastewater

    Directory of Open Access Journals (Sweden)

    Li Jinping

    2016-01-01

    Full Text Available Fly ash with a large number of active sites can occur with the adsorbent chemical and physical adsorption, and therefore have a strong adsorption capacity. The original fly ash and raw fly ash compared to the physical and chemical properties to a significant change. On the fly ash in industrial water treatment application were outlined. The purpose is to focus on the modification methods of fly ash and comparison of raw fly ash and fly ash in the effect of dyeing wastewater. Single factor test method; select the appropriate modifier to study the dosage, pH, stirring time on the modification of adsorption properties of fly ash before and after. The results showed that the modified fly ash was better than the adsorption. Greatly improves on active red X-3B dye wastewater removal capacity, pH = 5, 6, dosage is 5g / L, the mixing time is 30min, COD removal rate reached 73.07%. This modified material can be used as adsorbent for pre-treating dying wastewater.

  6. Correction of TRMM 3B42V7 Based on Linear Regression Models over China

    Directory of Open Access Journals (Sweden)

    Shaohua Liu

    2016-01-01

    Full Text Available High temporal-spatial precipitation is necessary for hydrological simulation and water resource management, and remotely sensed precipitation products (RSPPs play a key role in supporting high temporal-spatial precipitation, especially in sparse gauge regions. TRMM 3B42V7 data (TRMM precipitation is an essential RSPP outperforming other RSPPs. Yet the utilization of TRMM precipitation is still limited by the inaccuracy and low spatial resolution at regional scale. In this paper, linear regression models (LRMs have been constructed to correct and downscale the TRMM precipitation based on the gauge precipitation at 2257 stations over China from 1998 to 2013. Then, the corrected TRMM precipitation was validated by gauge precipitation at 839 out of 2257 stations in 2014 at station and grid scales. The results show that both monthly and annual LRMs have obviously improved the accuracy of corrected TRMM precipitation with acceptable error, and monthly LRM performs slightly better than annual LRM in Mideastern China. Although the performance of corrected TRMM precipitation from the LRMs has been increased in Northwest China and Tibetan plateau, the error of corrected TRMM precipitation is still significant due to the large deviation between TRMM precipitation and low-density gauge precipitation.

  7. Molecular basis of substrate promiscuity for the SAM-dependent O-methyltransferase NcsB1, involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin.

    Science.gov (United States)

    Cooke, Heather A; Guenther, Elizabeth L; Luo, Yinggang; Shen, Ben; Bruner, Steven D

    2009-10-13

    The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.

  8. Cloning, expression, purification and initial crystallographic studies of UbiG: a methyltransferase involved in ubiquinone biosynthesis in Escherichia coli

    International Nuclear Information System (INIS)

    Costa, M.A.F.; Magalhaes, R.D.; Nagem, R.A.P.; Ferreira-Junior, J.R.; Barros, M.H.

    2012-01-01

    Full text: Ubiquinone is a molecule that functions as an electron carrier in the respiratory chain in living organisms. Some clinical phenotypes, including, encephalomyopathy, has been associated with ubiquinone deficiency, raising the interest in the biosynthetic pathway of this molecule. This pathway was proposed mainly from the results of the genetic analysis of mutants of E. coli. UbiG is a methyltransferase involved in ubiquinone biosynthesis in E. coli. In this work we have cloned, expressed, purified and made initial crystallographic assessments of UbiG for later determination of its three-dimensional structure. The gene encoding UbiG was amplified from E. coli genomic DNA by polymerase chain reaction. The 753 bases pairs amplicon was inserted into the expression plasmid pMCSG7 by ligation independent cloning system and transformed into BL21(DE3) E. coli strain. The expression of UbiG, verified by SDS polyacrylamide gel, showed a protein of approximately 29kDa after IPTG induction. The recombinant UbiG, in the soluble fraction of the cellular lysate, was purified by affinity chromatography and the molecular weight of recombinant UbiG of approximately 29 kDa was confirmed by mass spectrometry. After removal of His-tag by TEV protease, another affinity chromatography was performed and UbiG, without His-tag, was observed in flow-through fraction. In Size-Exclusion Chromatography (SEC), the recombinant UbiG showed a unique peak with correct molecular weight of a monomer. Analysis of CD indicated that recombinant UbiG has 31,80% of alpha helix at 20 deg C and DLS showed that 70.9% of the sample is still monomeric in solution even five days after purification. Initial crystallization studies were performed with Crystal Screen 1 and Crystal Screen 2 from Hampton Research. Needle-shaped microcrystals of UbiG were obtained using a precipitant solution consisting of 0,1M lithium sulfate, 0,1M Tris pH 7,5 and 30% w/v polyethylene glycol 4,000. (author)

  9. Cloning, expression, purification and initial crystallographic studies of UbiG: a methyltransferase involved in ubiquinone biosynthesis in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Costa, M.A.F.; Magalhaes, R.D.; Nagem, R.A.P. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil); Ferreira-Junior, J.R.; Barros, M.H. [Universidade de Sao Paulo (USP), SP (Brazil)

    2012-07-01

    Full text: Ubiquinone is a molecule that functions as an electron carrier in the respiratory chain in living organisms. Some clinical phenotypes, including, encephalomyopathy, has been associated with ubiquinone deficiency, raising the interest in the biosynthetic pathway of this molecule. This pathway was proposed mainly from the results of the genetic analysis of mutants of E. coli. UbiG is a methyltransferase involved in ubiquinone biosynthesis in E. coli. In this work we have cloned, expressed, purified and made initial crystallographic assessments of UbiG for later determination of its three-dimensional structure. The gene encoding UbiG was amplified from E. coli genomic DNA by polymerase chain reaction. The 753 bases pairs amplicon was inserted into the expression plasmid pMCSG7 by ligation independent cloning system and transformed into BL21(DE3) E. coli strain. The expression of UbiG, verified by SDS polyacrylamide gel, showed a protein of approximately 29kDa after IPTG induction. The recombinant UbiG, in the soluble fraction of the cellular lysate, was purified by affinity chromatography and the molecular weight of recombinant UbiG of approximately 29 kDa was confirmed by mass spectrometry. After removal of His-tag by TEV protease, another affinity chromatography was performed and UbiG, without His-tag, was observed in flow-through fraction. In Size-Exclusion Chromatography (SEC), the recombinant UbiG showed a unique peak with correct molecular weight of a monomer. Analysis of CD indicated that recombinant UbiG has 31,80% of alpha helix at 20 deg C and DLS showed that 70.9% of the sample is still monomeric in solution even five days after purification. Initial crystallization studies were performed with Crystal Screen 1 and Crystal Screen 2 from Hampton Research. Needle-shaped microcrystals of UbiG were obtained using a precipitant solution consisting of 0,1M lithium sulfate, 0,1M Tris pH 7,5 and 30% w/v polyethylene glycol 4,000. (author)

  10. A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids1[OPEN

    Science.gov (United States)

    Levac, Dylan; Cázares, Paulo; Yu, Fang

    2016-01-01

    Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

  11. Impaired Homocysteine Transmethylation and Protein-Methyltransferase Activity Reduce Expression of Selenoprotein P: Implications for Obesity and Metabolic Syndrome

    Science.gov (United States)

    Obesity causes Metabolic Syndrome and Type-II Diabetes, disrupting hepatic function, methionine (Met)/homocysteine (Hcy) transmethylation and methyltransferase (PRMT) activities. Selenoprotein P (SEPP1), exported from the liver, is the predominate form of plasma selenium (Se) and the physiological S...