WorldWideScience

Sample records for c-myb binds mll

  1. Conformational control of the binding of the transactivation domain of the MLL protein and c-Myb to the KIX domain of CREB.

    Directory of Open Access Journals (Sweden)

    Elif Nihal Korkmaz

    Full Text Available The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events.

  2. Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR

    Directory of Open Access Journals (Sweden)

    Finstad Samantha L

    2004-09-01

    Full Text Available Abstract Background Feline leukemia virus (FeLV induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. FeLV-945 is a viral variant identified as predominant in a cohort of naturally infected animals. FeLV-945 contains a unique sequence motif in the long terminal repeat (LTR comprised of a single copy of transcriptional enhancer followed by a 21-bp sequence triplicated in tandem. The LTR is precisely conserved among independent cases of multicentric lymphoma, myeloproliferative disease and anemia in animals from the cohort. The 21-bp triplication was previously shown to act as a transcriptional enhancer preferentially in hematopoietic cells and to confer a replicative advantage. The objective of the present study was to examine the molecular mechanism by which the 21-bp triplication exerts its influence and the selective advantage responsible for its precise conservation. Results Potential binding sites for the transcription factor, c-Myb, were identified across the repeat junctions of the 21-bp triplication. Such sites would not occur in the absence of the repeat; thus, a requirement for c-Myb binding to the repeat junctions of the triplication would exert a selective pressure to conserve its sequence precisely. Electrophoretic mobility shift assays demonstrated specific binding of c-Myb to the 21-bp triplication. Reporter gene assays showed that the triplication-containing LTR is responsive to c-Myb, and that responsiveness requires the presence of both c-Myb binding sites. Results further indicated that c-Myb in complex with the 21-bp triplication recruits the transcriptional co-activator, CBP, a regulator of normal hematopoiesis. FeLV-945 replication was shown to be positively regulated by CBP in a manner dependent on the presence of the 21-bp triplication. Conclusion Binding sites for c-Myb across the repeat junctions of the 21-bp triplication may account for its precise conservation in

  3. Multiple Copies of a Simple MYB-Binding Site Confers Trans-regulation by Specific Flavonoid-Related R2R3 MYBs in Diverse Species

    Directory of Open Access Journals (Sweden)

    Cyril Brendolise

    2017-10-01

    Full Text Available In apple, the MYB transcription factor MYB10 controls the accumulation of anthocyanins. MYB10 is able to auto-activate its expression by binding its own promoter at a specific motif, the R1 motif. In some apple accessions a natural mutation, termed R6, has more copies of this motif within the MYB10 promoter resulting in stronger auto-activation and elevated anthocyanins. Here we show that other anthocyanin-related MYBs selected from apple, pear, strawberry, petunia, kiwifruit and Arabidopsis are able to activate promoters containing the R6 motif. To examine the specificity of this motif, members of the R2R3 MYB family were screened against a promoter harboring the R6 mutation. Only MYBs from subgroups 5 and 6 activate expression by binding the R6 motif, with these MYBs sharing conserved residues in their R2R3 DNA binding domains. Insertion of the apple R6 motif into orthologous promoters of MYB10 in pear (PcMYB10 and Arabidopsis (AtMY75 elevated anthocyanin levels. Introduction of the R6 motif into the promoter region of an anthocyanin biosynthetic enzyme F3′5′H of kiwifruit imparts regulation by MYB10. This results in elevated levels of delphinidin in both tobacco and kiwifruit. Finally, an R6 motif inserted into the promoter the vitamin C biosynthesis gene GDP-L-Gal phosphorylase increases vitamin C content in a MYB10-dependent manner. This motif therefore provides a tool to re-engineer novel MYB-regulated responses in plants.

  4. DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

    International Nuclear Information System (INIS)

    Lutwyche, Jodi K.; Keough, Rebecca A.; Hunter, Julie; Coles, Leeanne S.; Gonda, Thomas J.

    2006-01-01

    Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor

  5. Interactions between the R2R3-MYB transcription factor, AtMYB61, and target DNA binding sites.

    Directory of Open Access Journals (Sweden)

    Michael B Prouse

    Full Text Available Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing. The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.

  6. HIPK1 interacts with c-Myb and modulates its activity through phosphorylation

    International Nuclear Information System (INIS)

    Matre, Vilborg; Nordgard, Oddmund; Alm-Kristiansen, Anne Hege; Ledsaak, Marit; Gabrielsen, Odd Stokke

    2009-01-01

    The transcription factor v-Myb is a potent inducer of myeloid leukaemias, and its cellular homologue c-Myb plays a crucial role in the regulation of haematopoiesis. In a yeast two-hybrid (Y2H) screening we identified the nuclear kinase HIPK1 as an interaction partner for human c-Myb. The interaction involves a C-terminal region of HIPK1, while a bipartite interaction surface was identified in c-Myb, including regions in its N-terminal DNA-binding domain as well as in its C-terminal region. HIPK1 and c-Myb co-localize in distinct nuclear foci upon co-transfection. c-Myb appears to be phosphorylated by HIPK1 in its negative regulatory domain as supported by both in vivo and in vitro data. A functional assay revealed that HIPK1 repressed the ability of c-Myb to activate a chromatin embedded target gene, mim-1, in haematopoetic cells. Our findings point to a novel link between an important kinase and a key regulator of haematopoiesis.

  7. Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex

    Science.gov (United States)

    Andrejka, Laura; Wen, Hong; Ashton, Jonathan; Grant, Megan; Iori, Kevin; Wang, Amy; Manak, J. Robert; Lipsick, Joseph S.

    2011-01-01

    Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics. PMID:21969598

  8. Role of c-Myb in chondrogenesis.

    Science.gov (United States)

    Oralová, V; Matalová, E; Janečková, E; Drobná Krejčí, E; Knopfová, L; Šnajdr, P; Tucker, A S; Veselá, I; Šmarda, J; Buchtová, M

    2015-07-01

    The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5-E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation. Copyright

  9. The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jing; Gurung, Buddha; Wan, Bingbing; Matkar, Smita; Veniaminova, Natalia A.; Wan, Ke; Merchant, Juanita L.; Hua, Xianxin; Lei, Ming (Michigan); (Michigan-Med); (UPENN-MED)

    2013-04-08

    Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

  10. Single molecule analysis of c-myb alternative splicing reveals novel classifiers for precursor B-ALL.

    Directory of Open Access Journals (Sweden)

    Ye E Zhou

    Full Text Available The c-Myb transcription factor, a key regulator of proliferation and differentiation in hematopoietic and other cell types, has an N-terminal DNA binding domain and a large C-terminal domain responsible for transcriptional activation, negative regulation and determining target gene specificity. Overexpression and rearrangement of the c-myb gene (MYB has been reported in some patients with leukemias and other types of cancers, implicating activated alleles of c-myb in the development of human tumors. Alternative RNA splicing can produce variants of c-myb with qualitatively distinct transcriptional activities that may be involved in transformation and leukemogenesis. Here, by performing a detailed, single molecule assay we found that c-myb alternative RNA splicing was elevated and much more complex in leukemia samples than in cell lines or CD34+ hematopoietic progenitor cells from normal donors. The results revealed that leukemia samples express more than 60 different c-myb splice variants, most of which have multiple alternative splicing events and were not detectable by conventional microarray or PCR approaches. For example, the single molecule assay detected 21 and 22 splice variants containing the 9B and 9S exons, respectively, most of which encoded unexpected variant forms of c-Myb protein. Furthermore, the detailed analysis identified some splice variants whose expression correlated with poor survival in a small cohort of precursor B-ALL samples. Our findings indicate that single molecule assays can reveal complexities in c-myb alternative splicing that have potential as novel biomarkers and could help explain the role of c-Myb variants in the development of human leukemia.

  11. GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter

    Czech Academy of Sciences Publication Activity Database

    Bartůněk, Petr; Králová, Jarmila; Blendiger, G.; Dvořák, Michal; Zenke, M.

    2003-01-01

    Roč. 22, č. 13 (2003), s. 1927-1935 ISSN 0950-9232 R&D Projects: GA ČR GV301/98/K042 Institutional research plan: CEZ:AV0Z5052915 Keywords : GATA-1 * c-myb * erythropoiesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.495, year: 2003

  12. c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min-Sun; Kim, Sun-Young; Arunachalam, Sankarganesh [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Hwang, Pyoung-Han [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Yi, Ho-Keun [Department of Biochemistry, School of Dentistry, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Nam, Sang-Yun [Department of Alternative Therapy, School of Alternative Medicine and Health Science, Jeonju University, Jeonju 561-712 (Korea, Republic of); Lee, Dae-Yeol, E-mail: leedy@chonbuk.ac.kr [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of)

    2009-07-17

    c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.

  13. c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

    International Nuclear Information System (INIS)

    Kim, Min-Sun; Kim, Sun-Young; Arunachalam, Sankarganesh; Hwang, Pyoung-Han; Yi, Ho-Keun; Nam, Sang-Yun; Lee, Dae-Yeol

    2009-01-01

    c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.

  14. Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

    Science.gov (United States)

    Grembecka, Jolanta; He, Shihan; Shi, Aibin; Purohit, Trupta; Muntean, Andrew G; Sorenson, Roderick J; Showalter, Hollis D; Murai, Marcelo J; Belcher, Amalia M; Hartley, Thomas; Hess, Jay L; Cierpicki, Tomasz

    2012-01-29

    Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

  15. Distinct regulation of c-myb gene expression by HoxA9, Meis1 and Pbx proteins in normal hematopoietic progenitors and transformed myeloid cells

    International Nuclear Information System (INIS)

    Dassé, E; Volpe, G; Walton, D S; Wilson, N; Del Pozzo, W; O'Neill, L P; Slany, R K; Frampton, J; Dumon, S

    2012-01-01

    The proto-oncogenic protein c-Myb is an essential regulator of hematopoiesis and is frequently deregulated in hematological diseases such as lymphoma and leukemia. To gain insight into the mechanisms underlying the aberrant expression of c-Myb in myeloid leukemia, we analyzed and compared c-myb gene transcriptional regulation using two cell lines modeling normal hematopoietic progenitor cells (HPCs) and transformed myelomonocytic blasts. We report that the transcription factors HoxA9, Meis1, Pbx1 and Pbx2 bind in vivo to the c-myb locus and maintain its expression through different mechanisms in HPCs and leukemic cells. Our analysis also points to a critical role for Pbx2 in deregulating c-myb expression in murine myeloid cells cotransformed by the cooperative activity of HoxA9 and Meis1. This effect is associated with an intronic positioning of epigenetic marks and RNA polymerase II binding in the orthologous region of a previously described alternative promoter for c-myb. Taken together, our results could provide a first hint to explain the abnormal expression of c-myb in leukemic cells

  16. The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation.

    Directory of Open Access Journals (Sweden)

    Ruiqin Zhong

    Full Text Available Wood is mainly composed of secondary walls, which constitute the most abundant stored carbon produced by vascular plants. Understanding the molecular mechanisms controlling secondary wall deposition during wood formation is not only an important issue in plant biology but also critical for providing molecular tools to custom-design wood composition suited for diverse end uses. Past molecular and genetic studies have revealed a transcriptional network encompassing a group of wood-associated NAC and MYB transcription factors that are involved in the regulation of the secondary wall biosynthetic program during wood formation in poplar trees. Here, we report the functional characterization of poplar orthologs of MYB46 and MYB83 that are known to be master switches of secondary wall biosynthesis in Arabidopsis. In addition to the two previously-described PtrMYB3 and PtrMYB20, two other MYBs, PtrMYB2 and PtrMYB21, were shown to be MYB46/MYB83 orthologs by complementation and overexpression studies in Arabidopsis. The functional roles of these PtrMYBs in regulating secondary wall biosynthesis were further demonstrated in transgenic poplar plants showing an ectopic deposition of secondary walls in PtrMYB overexpressors and a reduction of secondary wall thickening in their dominant repressors. Furthermore, PtrMYB2/3/20/21 together with two other tree MYBs, the Eucalyptus EgMYB2 and the pine PtMYB4, were shown to differentially bind to and activate the eight variants of the 7-bp SMRE consensus sequence, composed of ACC(A/TA(A/C(T/C. Together, our results indicate that the tree MYBs, PtrMYB2/3/20/21, EgMYB2 and PtMYB4, are master transcriptional switches that activate the SMRE sites in the promoters of target genes and thereby regulate secondary wall biosynthesis during wood formation.

  17. C-myb Regulates Autophagy for Pulp Vitality in Glucose Oxidative Stress.

    Science.gov (United States)

    Lee, Y H; Kim, H S; Kim, J S; Yu, M K; Cho, S D; Jeon, J G; Yi, H K

    2016-04-01

    Diabetes mellitus is closely related to oral-complicated diseases by oxidative stress. This study investigates whether cellular myeloblastosis (c-myb) could protect human dental pulp cells against glucose oxidative stress and regulate autophagy activity for pulp vitality. Diabetes mellitus was induced by streptozotocin in Sprague-Dawley rats, and their pulp tissue in teeth was analyzed in terms of pulp cavity and molecules by hematoxylin and eosin and immunohistochemistry staining. Human dental pulp cells were serially subcultured and treated with glucose oxidase in the presence of elevated glucose to generate glucose oxidative stress. The replication-deficient adenovirus c-myb and small interfering RNA c-myb were introduced for c-myb expression. The pulp tissue from the diabetic rats was structurally different from normal tissue in terms of narrow pulp capacity, reduced c-myb, and dentinogenesis molecules. Glucose oxidase treatment decreased c-myb and dentinogenesis molecules (bone morphogenetic protein 2 and 7, dentin matrix protein 1, and dentin sialophosphoprotein) in human dental pulp cells. However, overexpression of c-myb by adenovirus c-myb increased dentinogenesis, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B-light chain 3, and Beclin-1), and cell survival via p-AMPK/AKT signaling even with glucose oxidative stress. In contrast, the lack of c-myb decreased the above molecules and cell survival by downregulating p-AMPK/AKT signaling. The results indicate that diabetes leads to irreversible damage to dental pulp, which is related to downexpression of autophagy via the p-AMPK/AKT pathway by decline of c-myb. The findings of this study provide a new insight that c-myb could ameliorate autophagy activity and that it is applicable for monitoring complicated diseases of dental pulp. The involvement of c-myb in pulp pathology could serve a therapeutic target in oral-complicated diseases. © International & American Associations

  18. Initiation of MLL-rearranged AML is dependent on C/EBPα

    DEFF Research Database (Denmark)

    Ohlsson, Ewa; Hasemann, Marie Sigurd; Willer, Anton

    2014-01-01

    have compared gene expression profiles from human MLL-rearranged AML to normal progenitors and identified the myeloid tumor suppressor C/EBPα as a putative collaborator in MLL-rearranged AML. Interestingly, we find that deletion of Cebpa rendered murine hematopoietic progenitors completely resistant...

  19. Thrombin-induced, TNFR-dependent miR-181c downregulation promotes MLL1 and NF-κB target gene expression in human microglia.

    Science.gov (United States)

    Yin, Min; Chen, Zhiying; Ouyang, Yetong; Zhang, Huiyan; Wan, Zhigang; Wang, Han; Wu, Wei; Yin, Xiaoping

    2017-06-29

    Controlling thrombin-driven microglial activation may serve as a therapeutic target for intracerebral hemorrhage (ICH). Here, we investigated microRNA (miRNA)-based regulation of thrombin-driven microglial activation using an in vitro thrombin toxicity model applied to primary human microglia. A miRNA array identified 22 differential miRNA candidates. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) identified miR-181c as the most significantly downregulated miRNA. TargetScan analysis identified mixed lineage leukemia-1 (MLL1) as a putative gene target for miR-181c. qRT-PCR was applied to assess tumor necrosis factor-alpha (TNF-α), miR-181c, and MLL1 levels following thrombin or proteinase-activated receptor-4-specific activating peptide (PAR4AP) exposure. Anti-TNF-α antibodies and tumor necrosis factor receptor (TNFR) silencing were employed to test TNF-α/TNFR dependence. A dual-luciferase reporter system and miR-181c mimic transfection assessed whether mir-181c directly binds to and negatively regulates MLL1. Nuclear factor kappa-B (NF-κB)-dependent luciferase reporter assays and NF-κB target gene expression were assessed in wild-type (MLL1+) and MLL1-silenced cells. Thrombin or PAR4AP-induced miR-181c downregulation (p < 0.05) and MLL1 upregulation (p < 0.05) that were dependent upon TNF-α/TNFR. miR-181c decreased wild-type MLL1 3'-UTR luciferase reporter activity (p < 0.05), and a miR-181c mimic suppressed MLL1 expression (p < 0.05). Thrombin treatment increased, while miR-181c reduced, NF-κB activity and NF-κB target gene expression in both wild-type (MLL1+) and MLL1-silenced cells (p < 0.05). Thrombin-induced, TNF-α/TNFR-dependent miR-181c downregulation promotes MLL1 expression, increases NF-κB activity, and upregulates NF-κB target gene expression. As miR-181c opposes thrombin's stimulation of pro-inflammatory NF-κB activity, miR-181c mimic therapy may show promise in controlling thrombin

  20. Molecular characterization of the Jatropha curcas JcR1MYB1 gene encoding a putative R1-MYB transcription factor

    Directory of Open Access Journals (Sweden)

    Hui-Liang Li

    2014-09-01

    Full Text Available The cDNA encoding the R1-MYB transcription factor, designated as JcR1MYB1, was isolated from Jatropha curcas using rapid amplification of cDNA ends. JcR1MYB1 contains a 951 bp open reading frame that encodes 316 amino acids. The deduced JcR1MYB1 protein was predicted to possess the conserved, 56-amino acid-long DNA-binding domain, which consists of a single helix-turn-helix module and usually occurs in R1-MYBs. JcR1MYB1 is a member of the R1-MYB transcription factor subfamily. A subcellular localization study confirmed the nuclear localization of JcR1MYB1. Expression analysis showed that JcR1MYB1 transcripts accumulated in various examined tissues, with high expression levels in the root and low levels in the stem. JcR1MYB1 transcription was up-regulated by polyethylene glycol, NaCl, and cold treatments, as well as by abscisic acid, jasmonic acid, and ethylene treatment. Analysis of transgenic tobacco plants over-expressing JcR1MYB1 indicates an inportant function for this gene in salt stress.

  1. Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

    Directory of Open Access Journals (Sweden)

    O'Rourke John P

    2011-01-01

    Full Text Available Abstract Background The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. Methods We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. Results By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Conclusions Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer

  2. Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

    International Nuclear Information System (INIS)

    Quintana, Anita M; Liu, Fan; O'Rourke, John P; Ness, Scott A

    2011-01-01

    The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells

  3. Human Vav1 expression in hematopoietic and cancer cell lines is regulated by c-Myb and by CpG methylation.

    Directory of Open Access Journals (Sweden)

    Lena Ilan

    Full Text Available Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA. Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5' regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well.

  4. Dramatic repositioning of c-Myb to different promoters during the cell cycle observed by combining cell sorting with chromatin immunoprecipitation.

    Directory of Open Access Journals (Sweden)

    Anita M Quintana

    2011-02-01

    Full Text Available The c-Myb transcription factor is a critical regulator of proliferation and stem cell differentiation, and mutated alleles of c-Myb are oncogenic, but little is known about changes in c-Myb activity during the cell cycle. To map the association of c-Myb with specific target genes during the cell cycle, we developed a novel Fix-Sort-ChIP approach, in which asynchronously growing cells were fixed with formaldehyde, stained with Hoechst 33342 and separated into different cell cycle fractions by flow sorting, then processed for chromatin immunoprecipitation (ChIP assays. We found that c-Myb actively repositions, binding to some genes only in specific cell cycle phases. In addition, the specificity of c-Myb is dramatically different in small subpopulations of cells, for example cells in the G2/M phase of the cell cycle, than in the bulk population. The repositioning of c-Myb during the cell cycle is not due to changes in its expression and also occurs with ectopically expressed, epitope-tagged versions of c-Myb. The repositioning occurs in established cell lines, in primary human CD34+ hematopoietic progenitors and in primary human acute myeloid leukemia cells. The combination of fixation, sorting and ChIP analysis sheds new light on the dynamic nature of gene regulation during the cell cycle and provides a new type of tool for the analysis of gene regulation in small subsets of cells, such as cells in a specific phase of the cell cycle.

  5. The Arabidopsis Transcription Factor MYB112 Promotes Anthocyanin Formation during Salinity and under High Light Stress.

    Science.gov (United States)

    Lotkowska, Magda E; Tohge, Takayuki; Fernie, Alisdair R; Xue, Gang-Ping; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-11-01

    MYB transcription factors (TFs) are important regulators of flavonoid biosynthesis in plants. Here, we report MYB112 as a formerly unknown regulator of anthocyanin accumulation in Arabidopsis (Arabidopsis thaliana). Expression profiling after chemically induced overexpression of MYB112 identified 28 up- and 28 down-regulated genes 5 h after inducer treatment, including MYB7 and MYB32, which are both induced. In addition, upon extended induction, MYB112 also positively affects the expression of PRODUCTION OF ANTHOCYANIN PIGMENT1, a key TF of anthocyanin biosynthesis, but acts negatively toward MYB12 and MYB111, which both control flavonol biosynthesis. MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative polymerase chain reaction, we show that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters, revealing them as direct downstream target genes. We further show that MYB112 expression is up-regulated by salinity and high light stress, environmental parameters that both require the MYB112 TF for anthocyanin accumulation under these stresses. In contrast to several other MYB TFs affecting anthocyanin biosynthesis, MYB112 expression is not controlled by nitrogen limitation or an excess of carbon. Thus, MYB112 constitutes a regulator that promotes anthocyanin accumulation under abiotic stress conditions. © 2015 American Society of Plant Biologists. All Rights Reserved.

  6. Utilization of circular dichroism and electrospray ionization mass spectrometry to understand the formation and conversion of G-quadruplex DNA at the human c-myb proto-oncogene.

    Science.gov (United States)

    Fu, Hengqing; Yang, Pengfei; Hai, Jinhui; Li, Huihui

    2018-10-05

    G-quadruplex DNAs are involved in a number of key biological processes, including gene expression, transcription, and apoptosis. The c-myb oncogene contains a number of GGA repeats in its promoter which forms G-quadruplex, thus it could be used as a target in cancer therapeutics. Several in-vitro studies have used Circular Dichroism (CD) spectroscopy or electrospray ionization mass spectrometry (ESI-MS) to demonstrate formation and stability of G-quadruplex DNA structure in the promoter region of human c-myb oncogene. The factors affecting the c-myb G-quadruplex structures were investigated, such as cations (i.e. K + , NH 4 + and Na + ) and co-solutes (methanol and polyethylene glycol). The results indicated that the presence of cations and co-solutes could change the G-quadruplex structural population and promote its thermodynamic stabilization as indicated by CD melting curves. It indicated that the co-solutes preferentially stabilize the c-myb G-quadruplex structure containing both homo- and hetero-stacking. In addition, protopine was demonstrated as a binder of c-myb G-quadruplex as screened from a library of natural alkaloids using ESI-MS method. CD spectra showed that it could selectively stabilize the c-myb G-quadruplex structure compared to other six G-quadruplexes from tumor-related G-rich sequences and the duplex DNAs (both long and short-chain ones). The binding of protopine could induce the change in the G-quadruplex structural populations. Therefore, protopine with its high binding specificity could be considered as a precursor for the design of drugs to target and regulate c-myb oncogene transcription. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. DOF-binding sites additively contribute to guard cell-specificity of AtMYB60 promoter

    Directory of Open Access Journals (Sweden)

    Cominelli Eleonora

    2011-11-01

    Full Text Available Abstract Background We previously demonstrated that the Arabidopsis thaliana AtMYB60 protein is an R2R3MYB transcription factor required for stomatal opening. AtMYB60 is specifically expressed in guard cells and down-regulated at the transcriptional levels by the phytohormone ABA. Results To investigate the molecular mechanisms governing AtMYB60 expression, its promoter was dissected through deletion and mutagenesis analyses. By studying different versions of AtMYB60 promoter::GUS reporter fusions in transgenic plants we were able to demonstrate a modular organization for the AtMYB60 promoter. Particularly we defined: a minimal promoter sufficient to confer guard cell-specific activity to the reporter gene; the distinct roles of different DOF-binding sites organised in a cluster in the minimal promoter in determining guard cell-specific expression; the promoter regions responsible for the enhancement of activity in guard cells; a promoter region responsible for the negative transcriptional regulation by ABA. Moreover from the analysis of single and multiple mutants we could rule out the involvement of a group of DOF proteins, known as CDFs, already characterised for their involvement in flowering time, in the regulation of AtMYB60 expression. Conclusions These findings shed light on the regulation of gene expression in guard cells and provide new promoter modules as useful tools for manipulating gene expression in guard cells, both for physiological studies and future biotechnological applications.

  8. The Arabidopsis Transcription Factor MYB112 Promotes Anthocyanin Formation during Salinity and under High Light Stress1[OPEN

    Science.gov (United States)

    Lotkowska, Magda E.; Tohge, Takayuki; Fernie, Alisdair R.; Xue, Gang-Ping; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-01-01

    MYB transcription factors (TFs) are important regulators of flavonoid biosynthesis in plants. Here, we report MYB112 as a formerly unknown regulator of anthocyanin accumulation in Arabidopsis (Arabidopsis thaliana). Expression profiling after chemically induced overexpression of MYB112 identified 28 up- and 28 down-regulated genes 5 h after inducer treatment, including MYB7 and MYB32, which are both induced. In addition, upon extended induction, MYB112 also positively affects the expression of PRODUCTION OF ANTHOCYANIN PIGMENT1, a key TF of anthocyanin biosynthesis, but acts negatively toward MYB12 and MYB111, which both control flavonol biosynthesis. MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative polymerase chain reaction, we show that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters, revealing them as direct downstream target genes. We further show that MYB112 expression is up-regulated by salinity and high light stress, environmental parameters that both require the MYB112 TF for anthocyanin accumulation under these stresses. In contrast to several other MYB TFs affecting anthocyanin biosynthesis, MYB112 expression is not controlled by nitrogen limitation or an excess of carbon. Thus, MYB112 constitutes a regulator that promotes anthocyanin accumulation under abiotic stress conditions. PMID:26378103

  9. The A-myb transcription factor in neoplastic and normal B cells.

    Science.gov (United States)

    Golay, J; Facchinetti, V; Ying, G; Introna, M

    1997-07-01

    The myb family of transcription factors has been strongly implicated in the regulation of cell growth and differentiation in the haematopoietic system. The v-myb oncogene, carried by avian defective retroviruses, causes leukaemias in the chicken and transforms haematopoietic cells in vitro. Its normal cellular equivalent c-myb, has been shown to promote the proliferation and block the differentiation of haematopoietic cells in several experimental models and is required for fetal haematopoiesis. Two other members of the family have been cloned more recently, A-myb and B-myb, which show sequence homology with c-myb in several domains, of which the DNA binding domain as well as other regulatory domains. Both have been shown to be transcription factors. B-myb is also involved in the control of proliferation and differentiation, but, unlike c-myb, it is expressed in many cell types. The third member of the family, A-myb, shows the most restricted pattern of expression, suggesting a very specific role for this transcription factor. A-myb is expressed in a subpopulation of normal B lymphocytes activated in vivo and localised in the germinal center of peripheral lymphoid organs and is not detected at significant levels in all other mature or immature haematopoietic populations studied, including bone marrow cells, T lymphocytes, granulocytes, monocytes, either at rest or after in vitro activation. These studies indicate that A-myb plays a role during a narrow window of normal B cell differentiation. A-myb expression has also been studied in a wide range of neoplastic B cells, representing the whole spectrum of B cell differentiation. A-myb is strongly expressed in Burkitt's lymphomas (BL) and slg+ B-acute lymphoblastic leukaemias (B-ALL) and not in all other leukaemias/lymphomas tested, with the exception of a subset of CLL (about 25% of cases). It is intriguing that the A-myb genome has been localised relatively close to the c-myc gene on chromosome 8, suggesting that

  10. Analysis of the DNA-Binding Activities of the Arabidopsis R2R3-MYB Transcription Factor Family by One-Hybrid Experiments in Yeast.

    Directory of Open Access Journals (Sweden)

    Zsolt Kelemen

    Full Text Available The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs. Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.

  11. miR-150 Regulates Memory CD8 T Cell Differentiation via c-Myb

    Directory of Open Access Journals (Sweden)

    Zeyu Chen

    2017-09-01

    Full Text Available MicroRNAs play an important role in T cell responses. However, how microRNAs regulate CD8 T cell memory remains poorly defined. Here, we found that miR-150 negatively regulates CD8 T cell memory in vivo. Genetic deletion of miR-150 disrupted the balance between memory precursor and terminal effector CD8 T cells following acute viral infection. Moreover, miR-150-deficient memory CD8 T cells were more protective upon rechallenge. A key circuit whereby miR-150 repressed memory CD8 T cell development through the transcription factor c-Myb was identified. Without miR-150, c-Myb was upregulated and anti-apoptotic targets of c-Myb, such as Bcl-2 and Bcl-xL, were also increased, suggesting a miR-150-c-Myb survival circuit during memory CD8 T cell development. Indeed, overexpression of non-repressible c-Myb rescued the memory CD8 T cell defects caused by overexpression of miR-150. Overall, these results identify a key role for miR-150 in memory CD8 T cells through a c-Myb-controlled enhanced survival circuit.

  12. Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.

    Science.gov (United States)

    Liu, Xin; Yang, Lihua; Zhou, Xianyao; Zhou, Miaoping; Lu, Yan; Ma, Lingjian; Ma, Hongxiang; Zhang, Zengyan

    2013-05-01

    The disease take-all, caused by the fungus Gaeumannomyces graminis, is one of the most destructive root diseases of wheat worldwide. Breeding resistant cultivars is an effective way to protect wheat from take-all. However, little progress has been made in improving the disease resistance level in commercial wheat cultivars. MYB transcription factors play important roles in plant responses to environmental stresses. In this study, an R2R3-MYB gene in Thinopyrum intermedium, TiMYB2R-1, was cloned and characterized. The gene sequence includes two exons and an intron. The expression of TiMYB2R-1 was significantly induced following G. graminis infection. An in vitro DNA binding assay proved that TiMYB2R-1 protein could bind to the MYB-binding site cis-element ACI. Subcellular localization assays revealed that TiMYB2R-1 was localized in the nucleus. TiMYB2R-1 transgenic wheat plants were generated, characterized molecularly, and evaluated for take-all resistance. PCR and Southern blot analyses confirmed that TiMYB2R-1 was integrated into the genomes of three independent transgenic wheat lines by distinct patterns and the transgene was heritable. Reverse transcription-PCR and western blot analyses revealed that TiMYB2R-1 was highly expressed in the transgenic wheat lines. Based on disease response assessments for three successive generations, the significantly enhanced resistance to take-all was observed in the three TiMYB2R-1-overexpressing transgenic wheat lines. Furthermore, the transcript levels of at least six wheat defence-related genes were significantly elevated in the TiMYB2R-1 transgenic wheat lines. These results suggest that engineering and overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops.

  13. c-Myb and its target Bmi1 are required for p190BCR/ABL leukemogenesis in mouse and human cells.

    Science.gov (United States)

    Waldron, T; De Dominici, M; Soliera, A R; Audia, A; Iacobucci, I; Lonetti, A; Martinelli, G; Zhang, Y; Martinez, R; Hyslop, T; Bender, T P; Calabretta, B

    2012-04-01

    Expression of c-Myb is required for normal hematopoiesis and for proliferation of myeloid leukemia blasts and a subset of T-cell leukemia, but its role in B-cell leukemogenesis is unknown. We tested the role of c-Myb in p190(BCR/ABL)-dependent B-cell leukemia in mice transplanted with p190(BCR/ABL)-transduced marrow cells with a c-Myb allele (Myb(f/d)) and in double transgenic p190(BCR/ABL)/Myb(w/d) mice. In both models, loss of a c-Myb allele caused a less aggressive B-cell leukemia. In p190(BCR/ABL)-expressing human B-cell leukemia lines, knockdown of c-Myb expression suppressed proliferation and colony formation. Compared with c-Myb(w/f) cells, expression of Bmi1, a regulator of stem cell proliferation and maintenance, was decreased in pre-B cells from Myb(w/d) p190(BCR/ABL) transgenic mice. Ectopic expression of a mutant c-Myb or Bmi1 enhanced the proliferation and colony formation of Myb(w/d) p190(BCR/ABL) B-cells; by contrast, Bmi1 downregulation inhibited colony formation of p190(BCR/ABL)-expressing murine B cells and human B-cell leukemia lines. Moreover, c-Myb interacted with a segment of the human Bmi1 promoter and enhanced its activity. In blasts from 19 Ph(1) adult acute lymphoblastic leukemia patients, levels of c-Myb and Bmi1 showed a positive correlation. Together, these findings support the existence of a c-Myb-Bmi1 transcription-regulatory pathway required for p190(BCR/ABL) leukemogenesis.

  14. A role of c-Myb in ossification

    Czech Academy of Sciences Publication Activity Database

    Oralová, Veronika; Matalová, Eva; Knopfová, L.; Švandová, Eva; Šmarda, J.; Buchtová, Marcela

    2015-01-01

    Roč. 159, Suppl 1 (2015), S30-S31 ISSN 1213-8118. [Morphology 2015. International Congress of the Czech Anatomical Society /49./. Lojda Symposium on Histochemistry /52./. 06.09.2015-08.09.2015, Olomouc] R&D Projects: GA ČR GB14-37368G Institutional support: RVO:67985904 Keywords : c-Myb Subject RIV: EA - Cell Biology

  15. c-Myb Regulates the T-Bet-Dependent Differentiation Program in B Cells to Coordinate Antibody Responses

    Directory of Open Access Journals (Sweden)

    Dana Piovesan

    2017-04-01

    Full Text Available Summary: Humoral immune responses are tailored to the invading pathogen through regulation of key transcription factors and their networks. This is critical to establishing effective antibody-mediated responses, yet it is unknown how B cells integrate pathogen-induced signals to drive or suppress transcriptional programs specialized for each class of pathogen. Here, we detail the key role of the transcription factor c-Myb in regulating the T-bet-mediated anti-viral program. Deletion of c-Myb in mature B cells significantly increased serum IgG2c and CXCR3 expression by upregulating T-bet, normally suppressed during Th2-cell-mediated responses. Enhanced expression of T-bet resulted in aberrant plasma cell differentiation within the germinal center, mediated by CXCR3 expression. These findings identify a dual role for c-Myb in limiting inappropriate effector responses while coordinating plasma cell differentiation with germinal center egress. Identifying such intrinsic regulators of specialized antibody responses can assist in vaccine design and therapeutic intervention in B-cell-mediated immune disorders. : Piovesan et al. examine how B cells establish transcriptional programs that result in tailored responses to invading pathogens. The authors find that the transcription factor c-Myb represses the T-bet-mediated anti-viral program in B cells. c-Myb limits inappropriate effector responses while coordinating plasma cell differentiation with germinal center egress. Keywords: B cells, c-Myb, T-bet, immunoglobulin, CXCR3, plasma cell, germinal center

  16. Arabidopsis MYB68 in development and responses to environmental cues

    DEFF Research Database (Denmark)

    Feng, Caiping; Andreasson, E.; Maslak, A.

    2004-01-01

    The Arabidopsis MYB68 gene encodes a MYB family protein with N-terminal R2R3 DNA-binding domains. Analyses of MYB68 expression by RNA blot and a transposant gene-trap MYB68::GUS reporter indicated that MYB68 is expressed specifically in root pericycle cells. Root cultures of the myb68 mutant......, caused by the gene trap insertion in the first MYB68 exon, produced increased biomass and lignin levels compared to wild type. Under high temperature regimes, MYB68::GUS activity was elevated in roots, while vegetative growth of myb68 mutants was reduced compared to wild type. These data suggest that MYB...

  17. c-Myb is required for plasma cell migration to bone marrow after immunization or infection

    Science.gov (United States)

    O’Donnell, Kristy; Belz, Gabrielle T.; Nutt, Stephen L.

    2015-01-01

    Plasma cell migration is crucial to immunity, but little is known about the molecular regulators of their migratory programs. Here, we detail the critical role of the transcription factor c-Myb in determining plasma cell location. In the absence of c-Myb, no IgG+ antigen-specific plasma cells were detected in the bone marrow after immunization or virus infection. This was correlated with a dramatic reduction of plasma cells in peripheral blood, mislocalization in spleen, and an inability of c-Myb–deficient plasma cells to migrate along a CXCL12 gradient. Therefore, c-Myb plays an essential, novel role in establishing the long-lived plasma cell population in the BM via responsiveness to chemokine migration cues. PMID:26077717

  18. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes.

    Science.gov (United States)

    Xu, Haifeng; Wang, Nan; Liu, Jingxuan; Qu, Changzhi; Wang, Yicheng; Jiang, Shenghui; Lu, Ninglin; Wang, Deyun; Zhang, Zongying; Chen, Xuesen

    2017-05-01

    MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33. Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1-5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

  19. An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma

    Science.gov (United States)

    Drier, Yotam; Cotton, Matthew J.; Williamson, Kaylyn E.; Gillespie, Shawn M.; Ryan, Russell J.H.; Kluk, Michael J.; Carey, Christopher D.; Rodig, Scott J.; Sholl, Lynette M; Afrogheh, Amir H.; Faquin, William C.; Queimado, Lurdes; Qi, Jun; Wick, Michael J.; El-Naggar, Adel K.; Bradner, James E.; Moskaluk, Christopher A.; Aster, Jon C.; Knoechel, Birgit; Bernstein, Bradley E.

    2016-01-01

    Translocation events are frequent in cancer and may create chimeric fusions or ‘regulatory rearrangements’ that drive oncogene overexpression. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps reveal distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in the alternate ACC lineages. PMID:26829750

  20. Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

    International Nuclear Information System (INIS)

    Manzotti, G; Mariani, S A; Corradini, F; Bussolari, R; Cesi, V; Vergalli, J; Ferrari-Amorotti, G; Fragliasso, V; Soliera, A R; Cattelani, S; Raschellà, G; Holyoake, T L; Calabretta, B

    2012-01-01

    The c-Myb gene encodes the p75 c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p c-Mybex9b , which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of p c-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p c-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75 c-Myb , p c-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p c-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p c-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34 + cells, without affecting the levels of p75 c-Myb . Together, these studies indicate that expression of the low-abundance p c-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

  1. Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia

    International Nuclear Information System (INIS)

    Cerveira, Nuno; Snijder, Simone; Mariz, José M; Norton, Lucília; Mellink, Clemens H; Buijs, Arjan; Teixeira, Manuel R; Santos, Joana; Bizarro, Susana; Costa, Vera; Ribeiro, Franclim R; Lisboa, Susana; Correia, Cecília; Torres, Lurdes; Vieira, Joana

    2009-01-01

    A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature. Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient. When compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023) We found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2

  2. PhMYB4 fine-tunes the floral volatile signature of Petunia x hybrida through PhC4H.

    Science.gov (United States)

    Colquhoun, Thomas A; Kim, Joo Young; Wedde, Ashlyn E; Levin, Laura A; Schmitt, Kyle C; Schuurink, Robert C; Clark, David G

    2011-01-01

    In Petunia × hybrida cv 'Mitchell Diploid' (MD), floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis is controlled spatially, developmentally, and daily at molecular, metabolic, and biochemical levels. Multiple genes have been shown to encode proteins that either directly catalyse a biochemical reaction yielding FVBP compounds or are involved in metabolite flux prior to the formation of FVBP compounds. It was hypothesized that multiple transcription factors are involved in the precise regulation of all necessary genes, resulting in the specific volatile signature of MD flowers. After acquiring all available petunia transcript sequences with homology to Arabidopsis thaliana R2R3-MYB transcription factors, PhMYB4 (named for its close identity to AtMYB4) was identified, cloned, and characterized. PhMYB4 transcripts accumulate to relatively high levels in floral tissues at anthesis and throughout open flower stages, which coincides with the spatial and developmental distribution of FVBP production and emission. Upon RNAi suppression of PhMYB4 (ir-PhMYB4) both petunia cinnamate-4-hydroxylase (PhC4H1 and PhC4H2) gene transcript levels were significantly increased. In addition, ir-PhMYB4 plants emit higher levels of FVBP compounds derived from p-coumaric acid (isoeugenol and eugenol) compared with MD. Together, these results indicate that PhMYB4 functions in the repression of C4H transcription, indirectly controlling the balance of FVBP production in petunia floral tissue (i.e. fine-tunes).

  3. Transcription arrest by a G quadruplex forming-trinucleotide repeat sequence from the human c-myb gene.

    Science.gov (United States)

    Broxson, Christopher; Beckett, Joshua; Tornaletti, Silvia

    2011-05-17

    Non canonical DNA structures correspond to genomic regions particularly susceptible to genetic instability. The transcription process facilitates formation of these structures and plays a major role in generating the instability associated with these genomic sites. However, little is known about how non canonical structures are processed when encountered by an elongating RNA polymerase. Here we have studied the behavior of T7 RNA polymerase (T7RNAP) when encountering a G quadruplex forming-(GGA)(4) repeat located in the human c-myb proto-oncogene. To make direct correlations between formation of the structure and effects on transcription, we have taken advantage of the ability of the T7 polymerase to transcribe single-stranded substrates and of G4 DNA to form in single-stranded G-rich sequences in the presence of potassium ions. Under physiological KCl concentrations, we found that T7 RNAP transcription was arrested at two sites that mapped to the c-myb (GGA)(4) repeat sequence. The extent of arrest did not change with time, indicating that the c-myb repeat represented an absolute block and not a transient pause to T7 RNAP. Consistent with G4 DNA formation, arrest was not observed in the absence of KCl or in the presence of LiCl. Furthermore, mutations in the c-myb (GGA)(4) repeat, expected to prevent transition to G4, also eliminated the transcription block. We show T7 RNAP arrest at the c-myb repeat in double-stranded DNA under conditions mimicking the cellular concentration of biomolecules and potassium ions, suggesting that the G4 structure formed in the c-myb repeat may represent a transcription roadblock in vivo. Our results support a mechanism of transcription-coupled DNA repair initiated by arrest of transcription at G4 structures.

  4. Activation of anthocyanin biosynthesis by expression of the radish R2R3-MYB transcription factor gene RsMYB1.

    Science.gov (United States)

    Lim, Sun-Hyung; Song, Ji-Hye; Kim, Da-Hye; Kim, Jae Kwang; Lee, Jong-Yeol; Kim, Young-Mi; Ha, Sun-Hwa

    2016-03-01

    RsMYB1, a MYB TF of red radish origin, was characterized as a positive regulator to transcriptionally activate the anthocyanin biosynthetic machinery by itself in Arabidopsis and tobacco plants. Anthocyanins, providing the bright red-orange to blue-violet colors, are flavonoid-derived pigments with strong antioxidant activity that have benefits for human health. We isolated RsMYB1, which encodes an R2R3-MYB transcription factor (TF), from red radish plants (Raphanus sativus L.) that accumulate high levels of anthocyanins. RsMYB1 shows higher expression in red radish than in common white radish, in both leaves and roots, at different growth stages. Consistent with RsMYB1 function as an anthocyanin-promoting TF, red radishes showed higher expression of all six anthocyanin biosynthetic and two anthocyanin regulatory genes. Transient expression of RsMYB1 in tobacco showed that RsMYB1 is a positive regulator of anthocyanin production with better efficiency than the basic helix-loop-helix (bHLH) TF gene B-Peru. Also, the synergistic effect of RsMYB1 with B-Peru was larger than the effect of the MYB TF gene mPAP1D with B-peru. Arabidopsis plants stably expressing RsMYB1 produced red pigmentation throughout the plant, accompanied by up-regulation of the six structural and two regulatory genes for anthocyanin production. This broad transcriptional activation of anthocyanin biosynthetic machinery in Arabidopsis included up-regulation of TRANSPARENT TESTA8, which encodes a bHLH TF. These results suggest that overexpression of RsMYB1 promotes anthocyanin production by triggering the expression of endogenous bHLH genes as potential binding partners for RsMYB1. In addition, RsMYB1-overexpressing Arabidopsis plants had a higher antioxidant capacity than did non-transgenic control plants. Taken together, RsMYB1 is an actively positive regulator for anthocyanins biosynthesis in radish plants and it might be one of the best targets for anthocyanin production by single gene

  5. The Melanocyte Fate in Neural Crest is Triggered by Myb Proteins through Activation of c-kit

    Czech Academy of Sciences Publication Activity Database

    Karafiát, Vít; Dvořáková, Marta; Pajer, Petr; Čermák, Vladimír; Dvořák, Michal

    2007-01-01

    Roč. 64, č. 21 (2007), s. 2975-2984 ISSN 1420-682X R&D Projects: GA MŠk(CZ) LC06061; GA ČR GA204/06/1728 Institutional research plan: CEZ:AV0Z50520514 Keywords : c-myb proto-oncogene * v-mybAMV oncogene * neural crest * cell fate determination * melanocytes * c-kit signal Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.239, year: 2007

  6. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.

    Science.gov (United States)

    An, Xiu-Hong; Tian, Yi; Chen, Ke-Qin; Liu, Xiao-Juan; Liu, Dan-Dan; Xie, Xing-Bin; Cheng, Cun-Gang; Cong, Pei-Hua; Hao, Yu-Jin

    2015-04-01

    Anthocyanin and proanthocyanidin (PA) are important secondary metabolites and beneficial to human health. Their biosynthesis is induced by jasmonate (JA) treatment and regulated by MYB transcription factors (TFs). However, which and how MYB TFs regulate this process is largely unknown in apple. In this study, MdMYB9 and MdMYB11 which were induced by methyl jasmonate (MeJA) were functionally characterized. Overexpression of MdMYB9 or MdMYB11 promoted not only anthocyanin but also PA accumulation in apple calluses, and the accumulation was further enhanced by MeJA. Subsequently, yeast two-hybrid, pull-down and bimolecular fluorescence complementation assays showed that both MYB proteins interact with MdbHLH3. Moreover, Jasmonate ZIM-domain (MdJAZ) proteins interact with MdbHLH3. Furthermore, chromatin immunoprecipitation-quantitative PCR and yeast one-hybrid assays demonstrated that both MdMYB9 and MdMYB11 bind to the promoters of ANS, ANR and LAR, whereas MdbHLH3 is recruited to the promoters of MdMYB9 and MdMYB11 and regulates their transcription. In addition, transient expression assays indicated that overexpression of MdJAZ2 inhibits the recruitment of MdbHLH3 to the promoters of MdMYB9 and MdMYB11. Our findings provide new insight into the mechanism of how MeJA regulates anthocyanin and PA accumulation in apple. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. In utero exposure to benzene increases embryonic c-Myb and Pim-1 protein levels in CD-1 mice

    International Nuclear Information System (INIS)

    Wan, Joanne; Winn, Louise M.

    2008-01-01

    Benzene is a known human leukemogen, but its role as an in utero leukemogen remains controversial. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. We hypothesize that in utero exposure to benzene may cause leukemogenesis by affecting the embryonic c-Myb/Pim-1 signaling pathway and that this is mediated by oxidative stress. To investigate this hypothesis, pregnant CD-1 mice were treated with either 800 mg/kg of benzene or corn oil (i.p.) on days 10 and 11 of gestation and in some cases pretreated with 25 kU/kg of PEG-catalase. Phosphorylated and total embryonic c-Myb and Pim-1 protein levels were assessed using Western blotting and maternal and embryonic oxidative stress were assessed by measuring reduced to oxidized glutathione ratios. Our results show increased oxidative stress at 4 and 24 h after exposure, increased phosphorylated Pim-1 protein levels 4 h after benzene exposure, and increased Pim-1 levels at 24 and 48 h after benzene exposure. Embryonic c-Myb levels were elevated at 24 h after exposure. PEG-catalase pretreatment prevented benzene-mediated increases in embryonic c-Myb and Pim-1 protein levels, and benzene-induced oxidative stress. These results support a role for ROS in c-Myb and Pim-1 alterations after in utero benzene exposure

  8. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis

    Science.gov (United States)

    2012-01-01

    Background The MYB gene family comprises one of the richest groups of transcription factors in plants. Plant MYB proteins are characterized by a highly conserved MYB DNA-binding domain. MYB proteins are classified into four major groups namely, 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB based on the number and position of MYB repeats. MYB transcription factors are involved in plant development, secondary metabolism, hormone signal transduction, disease resistance and abiotic stress tolerance. A comparative analysis of MYB family genes in rice and Arabidopsis will help reveal the evolution and function of MYB genes in plants. Results A genome-wide analysis identified at least 155 and 197 MYB genes in rice and Arabidopsis, respectively. Gene structure analysis revealed that MYB family genes possess relatively more number of introns in the middle as compared with C- and N-terminal regions of the predicted genes. Intronless MYB-genes are highly conserved both in rice and Arabidopsis. MYB genes encoding R2R3 repeat MYB proteins retained conserved gene structure with three exons and two introns, whereas genes encoding R1R2R3 repeat containing proteins consist of six exons and five introns. The splicing pattern is similar among R1R2R3 MYB genes in Arabidopsis. In contrast, variation in splicing pattern was observed among R1R2R3 MYB members of rice. Consensus motif analysis of 1kb upstream region (5′ to translation initiation codon) of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. Real-time quantitative RT-PCR analysis showed that several members of MYBs are up-regulated by various abiotic stresses both in rice and Arabidopsis. Conclusion A comprehensive genome-wide analysis of chromosomal distribution, tandem repeats and phylogenetic relationship of MYB family genes in rice and Arabidopsis suggested their evolution via duplication. Genome-wide comparative analysis of MYB genes and their expression analysis

  9. Overexpression of a repressor MdMYB15L negatively regulates anthocyanin and cold tolerance in red-fleshed callus.

    Science.gov (United States)

    Xu, Haifeng; Yang, Guanxian; Zhang, Jing; Wang, Yicheng; Zhang, Tianliang; Wang, Nan; Jiang, Shenghui; Zhang, Zongying; Chen, Xuesen

    2018-04-14

    The cold-induced metabolic pathway and anthocyanin biosynthesis play important roles in plant growth. In this study, we identified a bHLH binding motif in the MdMYB15L protein using protein sequence analyses. Yeast two-hybrid and pull-down assays showed that MdMYB15L could interact with MdbHLH33. Overexpressing MdMYB15L in red-fleshed callus inhibited the expression of MdCBF2 and resulted in reduced cold tolerance but did not affect anthocyanin levels. Chip-PCR and EMSA analysis showed that MdMYB15L could bind the type II cis-acting element found in the MdCBF2 promoter. Overexpressing MdMYB15L in red-fleshed callus overexpressing MdbHLH33 also reduced cold tolerance and reduced MdbHLH33-induced anthocyanin biosynthesis. Knocking out the bHLH binding sequence of MdMYB15L (LBSMdMYB15L) prevented LBSMdMYB15L from interacting with MdbHLH33. Overexpressing LBSMdMYB15L in red-fleshed callus overexpressing MdbHLH33 also reduced cold tolerance and reduced MdbHLH33-induced anthocyanin biosynthesis. Together, these results suggested that an apple repressor MdMYB15L might play a key role in the cold signaling and anthocyanin metabolic pathways. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Effect of ischemic preconditioning on the expression of c-myb in the CA1 region of the gerbil hippocampus after ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Hui Young Lee

    2016-06-01

    Conclusion: Our results show that a lethal transient ischemia significantly decreased c-myb immunoreactivity in the SP of the CA1 region and that IPC well preserved c-myb immunoreactivity in the SP of the CA1 region. We suggest that the maintenance of c-myb might be related with IPC-mediated neuroprotection after a lethal ischemic insult.

  11. [MYB-like transcription factor SiMYB42 from foxtail millet (Setaria italica L.) enhances Arabidopsis tolerance to low-nitrogen stress].

    Science.gov (United States)

    Ding, Qing Qian; Wang, Xiao Ting; Hu, Li Qin; Qi, Xin; Ge, Lin Hao; Xu, Wei Ya; Xu, Zhao Shi; Zhou, Yong Bin; Jia, Guan Qing; Diao, Xian Min; Min, Dong Hong; Ma, You Zhi; Chen, Ming

    2018-04-20

    Myeloblastosis (MYB) transcription factors are one of the largest families of transcription factors in higher plants. They play an important role in plant development, defense response processes, and non-biological stresses, i.e., drought stress. Foxtail millet (Setaria italica L.), originated in China, is resistant to drought and low nutrition stresses and has been regarded as an ideal material for studying abiotic stress resistance in monocotyledon. In this study, we ran a transcription profile analysis of zheng 204 under low-nitrogen conditions and identified a MYB-like transcription factor SiMYB42, which was up-regulated under low-nitrogen stress. Phylogenetic tree analysis showed that SiMYB42 belongs to R2R3-MYB subfamily and has two MYB conserved domains. Expression pattern analysis showed that SiMYB42 was significantly up-regulated under various stress conditions, including low-nitrogen stress, high salt, drought and ABA conditions. The results of subcellular localization, quantitative real-time PCR and transcriptional activation analysis indicated that SiMYB42 protein localizes to the nucleus and cell membrane of plant cells, mainly expressed in the leaf or root of foxtail millet, and has transcription activation activity. Functional analysis showed that there was no significant difference between transgenic SiMYB42 Arabidopsis and wild-type (WT) Arabidopsis under normal conditions; however, under low-nitrogen condition, the root length, surface area and seedling fresh weight in transgenic SiMYB42 Arabidopsis, were significantly higher than their counterparts in WT. These results suggest that SiMYB42 transgenic plants exhibit higher tolerance to low-nitrogen stress. Expression levels of nitrate transporters genes NRT2.1, NRT2.4 and NRT2.5, which are the transcriptional targets of SiMYB42, were higher in transgenic SiMYB42 Arabidopsis plants than those in WT; the promoter regions of NRT2.1, NRT2.4 and NRT2.5 all have MYB binding sites. These results indicate

  12. McMYB12 Transcription Factors Co-regulate Proanthocyanidin and Anthocyanin Biosynthesis in Malus Crabapple.

    Science.gov (United States)

    Tian, Ji; Zhang, Jie; Han, Zhen-Yun; Song, Ting-Ting; Li, Jin-Yan; Wang, Ya-Ru; Yao, Yun-Cong

    2017-03-03

    The flavonoid compounds, proanthocyanidins (PAs), protect plants from biotic stresses, contribute to the taste of many fruits, and are beneficial to human health in the form of dietary antioxidants. In this study, we functionally characterized two Malus crabapple R2R3-MYB transcription factors, McMYB12a and McMYB12b, which co-regulate PAs and anthocyanin biosynthesis. McMYB12a was shown to be mainly responsible for upregulating the expression of anthocyanin biosynthetic genes by binding to their promoters, but to be only partially responsible for regulating PAs biosynthetic genes. In contrast, McMYB12b showed preferential binding to the promoters of PAs biosynthetic genes. Overexpression of McMYB12a and McMYB12b in tobacco (Nicotiana tabacum) altered the expression of flavonoid biosynthetic genes and promoted the accumulation of PAs and anthocyanins in tobacco petals. Conversely, transient silencing their expression in crabapple plants, using a conserved gene region, resulted in reduced PAs and anthocyanin production a green leaf phenotype. Meanwhile, transient overexpression of the two genes and silenced McMYB12s in apple (Malus domestica) fruit had a similar effect as overexpression in tobacco and silenced in crabapple. This study reveals a new mechanism for the coordinated regulation of PAs and anthocyanin accumulation in crabapple leaves, which depends on an auto-regulatory balance involving McMYB12a and McMYB12b expression.

  13. Dominant Repression by Arabidopsis Transcription Factor MYB44 Causes Oxidative Damage and Hypersensitivity to Abiotic Stress

    Directory of Open Access Journals (Sweden)

    Helene Persak

    2014-02-01

    Full Text Available In any living species, stress adaptation is closely linked with major changes of the gene expression profile. As a substrate protein of the rapidly stress-induced mitogen-activated protein kinase MPK3, Arabidopsis transcription factor MYB44 likely acts at the front line of stress-induced re-programming. We recently characterized MYB44 as phosphorylation-dependent positive regulator of salt stress signaling. Molecular events downstream of MYB44 are largely unknown. Although MYB44 binds to the MBSII element in vitro, it has no discernible effect on MBSII-driven reporter gene expression in plant co-transfection assays. This may suggest limited abundance of a synergistic co-regulator. MYB44 carries a putative transcriptional repression (Ethylene responsive element binding factor-associated Amphiphilic Repression, EAR motif. We employed a dominant repressor strategy to gain insights into MYB44-conferred stress resistance. Overexpression of a MYB44-REP fusion markedly compromised salt and drought stress tolerance—the opposite was seen in MYB44 overexpression lines. MYB44-mediated resistance likely results from induction of tolerance-enhancing, rather than from repression of tolerance-diminishing factors. Salt stress-induced accumulation of destructive reactive oxygen species is efficiently prevented in transgenic MYB44, but accelerated in MYB44-REP lines. Furthermore, heterologous overexpression of MYB44-REP caused tissue collapse in Nicotiana. A mechanistic model of MAPK-MYB-mediated enhancement in the antioxidative capacity and stress tolerance is proposed. Genetic engineering of MYB44 variants with higher trans-activating capacity may be a means to further raise stress resistance in crops.

  14. The role of MYB34, MYB51 and MYB122 in the regulation of camalexin biosynthesis in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Henning eFrerigmann

    2015-08-01

    Full Text Available The indolic phytoalexin camalexin is a crucial defence metabolite in the model plant Arabidopsis. Indolic phytoalexins and glucosinolates appear to have a common evolutionary origin and are interconnected on the biosynthetic level: a key intermediate in the biosynthesis of camalexin, indole-3-acetaldoxime (IAOx, is also required for the biosynthesis of indolic glucosinolates and is under tight control by the transcription factors MYB34, MYB51 and MYB122. The abundance of camalexin was strongly reduced in myb34/51 and myb51/122 double and in triple myb mutant, suggesting that these transcription factors are important in camalexin biosynthesis. Furthermore, expression of MYB51 and MYB122 was significantly increased by biotic and abiotic camalexin-inducing agents. Feeding of the triple myb34/51/122 mutant with IAOx or indole-3-acetonitrile largely restored camalexin biosynthesis. Conversely, tryptophan could not complement the low camalexin phenotype of this mutant, which supports a role for the three MYB factors in camalexin biosynthesis upstream of IAOx. Consistently expression of the camalexin biosynthesis genes CYP71B15/PAD3 and CYP71A13 was not negatively affected in the triple myb mutant and the MYBs could not activate pCYP71B15::uidA expression in trans-activation assays with cultured Arabidopsis cells. In conclusion, this study reveals the importance of MYB factors regulating the generation of IAOx as precursor of camalexin.

  15. Molecular and Epigenetic Mechanisms of MLL in Human Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Thomas A. Milne

    2012-09-01

    Full Text Available Epigenetics is often defined as the study of heritable changes in gene expression or chromosome stability that don’t alter the underlying DNA sequence. Epigenetic changes are established through multiple mechanisms that include DNA methylation, non-coding RNAs and the covalent modification of specific residues on histone proteins. It is becoming clear not only that aberrant epigenetic changes are common in many human diseases such as leukemia, but that these changes by their very nature are malleable, and thus are amenable to treatment. Epigenetic based therapies have so far focused on the use of histone deacetylase (HDAC inhibitors and DNA methyltransferase inhibitors, which tend to have more general and widespread effects on gene regulation in the cell. However, if a unique molecular pathway can be identified, diseases caused by epigenetic mechanisms are excellent candidates for the development of more targeted therapies that focus on specific gene targets, individual binding domains, or specific enzymatic activities. Designing effective targeted therapies depends on a clear understanding of the role of epigenetic mutations during disease progression. The Mixed Lineage Leukemia (MLL protein is an example of a developmentally important protein that controls the epigenetic activation of gene targets in part by methylating histone 3 on lysine 4. MLL is required for normal development, but is also mutated in a subset of aggressive human leukemias and thus provides a useful model for studying the link between epigenetic cell memory and human disease. The most common MLL mutations are chromosome translocations that fuse the MLL gene in frame with partner genes creating novel fusion proteins. In this review, we summarize recent work that argues MLL fusion proteins could function through a single molecular pathway, but we also highlight important data that suggests instead that multiple independent mechanisms underlie MLL mediated leukemogenesis.

  16. Molecular and Epigenetic Mechanisms of MLL in Human Leukemogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ballabio, Erica; Milne, Thomas A., E-mail: thomas.milne@imm.ox.ac.uk [MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital Headington, Oxford OX3 9DS (United Kingdom)

    2012-09-10

    Epigenetics is often defined as the study of heritable changes in gene expression or chromosome stability that don’t alter the underlying DNA sequence. Epigenetic changes are established through multiple mechanisms that include DNA methylation, non-coding RNAs and the covalent modification of specific residues on histone proteins. It is becoming clear not only that aberrant epigenetic changes are common in many human diseases such as leukemia, but that these changes by their very nature are malleable, and thus are amenable to treatment. Epigenetic based therapies have so far focused on the use of histone deacetylase (HDAC) inhibitors and DNA methyltransferase inhibitors, which tend to have more general and widespread effects on gene regulation in the cell. However, if a unique molecular pathway can be identified, diseases caused by epigenetic mechanisms are excellent candidates for the development of more targeted therapies that focus on specific gene targets, individual binding domains, or specific enzymatic activities. Designing effective targeted therapies depends on a clear understanding of the role of epigenetic mutations during disease progression. The Mixed Lineage Leukemia (MLL) protein is an example of a developmentally important protein that controls the epigenetic activation of gene targets in part by methylating histone 3 on lysine 4. MLL is required for normal development, but is also mutated in a subset of aggressive human leukemias and thus provides a useful model for studying the link between epigenetic cell memory and human disease. The most common MLL mutations are chromosome translocations that fuse the MLL gene in frame with partner genes creating novel fusion proteins. In this review, we summarize recent work that argues MLL fusion proteins could function through a single molecular pathway, but we also highlight important data that suggests instead that multiple independent mechanisms underlie MLL mediated leukemogenesis.

  17. Molecular and Epigenetic Mechanisms of MLL in Human Leukemogenesis

    International Nuclear Information System (INIS)

    Ballabio, Erica; Milne, Thomas A.

    2012-01-01

    Epigenetics is often defined as the study of heritable changes in gene expression or chromosome stability that don’t alter the underlying DNA sequence. Epigenetic changes are established through multiple mechanisms that include DNA methylation, non-coding RNAs and the covalent modification of specific residues on histone proteins. It is becoming clear not only that aberrant epigenetic changes are common in many human diseases such as leukemia, but that these changes by their very nature are malleable, and thus are amenable to treatment. Epigenetic based therapies have so far focused on the use of histone deacetylase (HDAC) inhibitors and DNA methyltransferase inhibitors, which tend to have more general and widespread effects on gene regulation in the cell. However, if a unique molecular pathway can be identified, diseases caused by epigenetic mechanisms are excellent candidates for the development of more targeted therapies that focus on specific gene targets, individual binding domains, or specific enzymatic activities. Designing effective targeted therapies depends on a clear understanding of the role of epigenetic mutations during disease progression. The Mixed Lineage Leukemia (MLL) protein is an example of a developmentally important protein that controls the epigenetic activation of gene targets in part by methylating histone 3 on lysine 4. MLL is required for normal development, but is also mutated in a subset of aggressive human leukemias and thus provides a useful model for studying the link between epigenetic cell memory and human disease. The most common MLL mutations are chromosome translocations that fuse the MLL gene in frame with partner genes creating novel fusion proteins. In this review, we summarize recent work that argues MLL fusion proteins could function through a single molecular pathway, but we also highlight important data that suggests instead that multiple independent mechanisms underlie MLL mediated leukemogenesis

  18. Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

    Science.gov (United States)

    Reddy, Vaishnavi Amarr; Wang, Qian; Dhar, Niha; Kumar, Nadimuthu; Venkatesh, Prasanna Nori; Rajan, Chakravarthy; Panicker, Deepa; Sridhar, Vishweshwaran; Mao, Hui-Zhu; Sarojam, Rajani

    2017-09-01

    Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  19. McMYB12 Transcription Factors Co-regulate Proanthocyanidin and Anthocyanin Biosynthesis in Malus Crabapple

    OpenAIRE

    Tian, Ji; Zhang, Jie; Han, Zhen-yun; Song, Ting-ting; Li, Jin-yan; Wang, Ya-ru; Yao, Yun-cong

    2017-01-01

    The flavonoid compounds, proanthocyanidins (PAs), protect plants from biotic stresses, contribute to the taste of many fruits, and are beneficial to human health in the form of dietary antioxidants. In this study, we functionally characterized two Malus crabapple R2R3-MYB transcription factors, McMYB12a and McMYB12b, which co-regulate PAs and anthocyanin biosynthesis. McMYB12a was shown to be mainly responsible for upregulating the expression of anthocyanin biosynthetic genes by binding to th...

  20. Mll5 is required for normal spermatogenesis.

    Directory of Open Access Journals (Sweden)

    Damian B Yap

    Full Text Available Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis.Mll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated.Our data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects.

  1. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae.

    Science.gov (United States)

    Lin-Wang, Kui; Bolitho, Karen; Grafton, Karryn; Kortstee, Anne; Karunairetnam, Sakuntala; McGhie, Tony K; Espley, Richard V; Hellens, Roger P; Allan, Andrew C

    2010-03-21

    The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

  2. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

    Directory of Open Access Journals (Sweden)

    McGhie Tony K

    2010-03-01

    Full Text Available Abstract Background The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. Results We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry. Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. Conclusions This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

  3. The +37 kb Cebpa Enhancer Is Critical for Cebpa Myeloid Gene Expression and Contains Functional Sites that Bind SCL, GATA2, C/EBPα, PU.1, and Additional Ets Factors

    Science.gov (United States)

    Cooper, Stacy; Guo, Hong; Friedman, Alan D.

    2015-01-01

    The murine Cebpa gene contains an evolutionarily conserved 453 bp enhancer located at +37 kb that, together with its promoter, directs expression to myeloid progenitors and to long-term hematopoietic stem cells in transgenic mice. In human acute myeloid leukemia cases, the enhancer lacks point mutations but binds the RUNX1-ETO oncoprotein. The enhancer contains the H3K4me1 and H3K27Ac histone modifications, denoting an active enhancer, at progressively increasing levels as long-term hematopoietic stem cells transition to granulocyte-monocyte progenitors. We previously identified four enhancer sites that bind RUNX1 and demonstrated that their integrity is required for maximal enhancer activity in 32Dcl3 myeloid cells. The +37 kb Cebpa enhancer also contains C/EBP, Ets factor, Myb, GATA, and E-box consensus sites conserved in the human +42 kb CEBPA enhancer. Mutation of the two C/EBP, seven Ets, one Myb, two GATA, or two E-box sites reduces activity of an enhancer-promoter reporter in 32Dcl3 cells. In 293T gel shift assays, exogenous C/EBPα binds both C/EBP sites, c-Myb binds the Myb site, PU.1 binds the second Ets site, PU.1, Fli-1, ERG, and Ets1 bind the sixth Ets site, GATA2 binds both GATA sites, and SCL binds the second E-box. Endogenous hematopoietic RUNX1, PU.1, Fli-1, ERG, C/EBPα, GATA2, and SCL were previously shown to bind the enhancer, and we find that endogenous PU.1 binds the second Ets site in 32Dcl3 cells. Using CRISPR/Cas9, we developed 32Dcl3 lines in which the wild-type enhancer alleles are replaced with a variant mutant in the seven Ets sites. These lines have 20-fold reduced Cebpa mRNA when cultured in IL-3 or G-CSF, demonstrating a critical requirement for enhancer integrity for optimal Cebpa expression. In addition, these results indicate that the +37 kb Cebpa enhancer is the focus of multiple regulatory transcriptional pathways that impact its expression during normal hematopoiesis and potentially during myeloid transformation. PMID:25938608

  4. Transcriptional Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice

    Directory of Open Access Journals (Sweden)

    Shuchi eSmita

    2015-12-01

    Full Text Available MYB transcription factor (TF is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by top down and guide gene approaches. More than 50% of OsMYBs were strongly correlated under fifty experimental conditions with 51 hub genes via top down approach. Further, clusters were identified using Markov Clustering (MCL. To maximize the clustering performance, parameter evaluation of the MCL inflation score (I was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by guide gene approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought

  5. Mybs in mouse hair follicle development

    Czech Academy of Sciences Publication Activity Database

    Veselá, Barbora; Švandová, Eva; Šmarda, J.; Matalová, Eva

    2014-01-01

    Roč. 46, č. 5 (2014), s. 352-355 ISSN 0040-8166 R&D Projects: GA ČR GCP302/12/J059 Institutional support: RVO:67985904 Keywords : hair follicle * stem cells * c-Myb * B-Myb * development Subject RIV: EA - Cell Biology Impact factor: 1.252, year: 2014

  6. Transactivation mediated by B-Myb is dependent on TAF(II)250.

    Science.gov (United States)

    Bartusel, Thorsten; Klempnauer, Karl-Heinz

    2003-05-15

    B-Myb is a highly conserved member of the Myb family of transcription factors, which has been implicated in cell cycle regulation. B-Myb is expressed in most proliferating cells and its activity is highly regulated around the G1/S-phase border of the cell cycle. It is generally assumed that B-Myb regulates the expression of genes that are crucial for cell proliferation; however, the identity of these genes, the molecular mechanisms by which B-Myb stimulates their expression and the involvement of other proteins have not been sufficiently clarified. We have employed the hamster cell line ts13 as a tool to demonstrate a functional link between B-Myb and the coactivator TAF(II)250, a key component of the transcriptional machinery which itself is essential for cell proliferation. ts13 cells express a point-mutated version of TAF(II)250 whose intrinsic histone acetyl transferase activity is temperature sensitive. Transactivation of Myb-responsive reporter genes by B-Myb is temperature-dependent in ts13 cells but not in ts13 cells, which have been rescued by transfection with an expression vector for wild-type TAF(II)250. Furthermore, B-Myb and TAF(II)250 can be coprecipitated, suggesting that both proteins are present in a complex. The formation of this complex is dependent on the DNA-binding domain of B-Myb and not on its transactivation domain. Taken together, these observations provide the first evidence that the coactivator TAF(II)250 is involved in the activation of Myb responsive promoters by B-Myb. The finding that B-Myb transactivation is dependent on a key coactivator involved in cell cycle control is consistent with and strengthens the idea that B-Myb plays a crucial role as a transcription factor in proliferating cells.

  7. MLL-ENL cooperates with SCF to transform primary avian multipotent cells.

    Science.gov (United States)

    Schulte, Cathleen E; von Lindern, Marieke; Steinlein, Peter; Beug, Hartmut; Wiedemann, Leanne M

    2002-08-15

    The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.

  8. Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33, MYB65 and MYB101.

    Science.gov (United States)

    Xue, Tao; Liu, Zhenhua; Dai, Xuehuan; Xiang, Fengning

    2017-09-01

    Organ growth is a fundamental developmental process basing on cell proliferation and differentiation. The growth of the plant root is sustained by the activity of the root meristem, a process controlled in part by various transcription factors. Here, the miR159 has been identified as a post transcriptional repressor of root growth, on the basis that the mir159ab double mutant developed a larger meristem than did the wild type, and that it formed longer roots. In the mutant, the abundance of MYB33, MYB65 and MYB101 transcript was substantially increased. When MYB33, MYB65 and MYB101 were replaced by the miR159-resistant forms mMYB33, mMYB65 and mMYB101 respectively, the root meristem was similarly enlarged and the growth of the primary root enhanced. MYB65 activity promoted cell division in the root meristem by accelerating the cell cycle. The data suggest that miR159 acts as a key repressor of the primary root's growth, acting through its repression of MYB65 and consequent blocking of the cell cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. The Arabidopsis MYB96 transcription factor plays a role in seed dormancy.

    Science.gov (United States)

    Lee, Hong Gil; Lee, Kyounghee; Seo, Pil Joon

    2015-03-01

    Seed dormancy facilitates to endure environmental disadvantages by confining embryonic growth until the seeds encounter favorable environmental conditions for germination. Abscisic acid (ABA) and gibberellic acid (GA) play a pivotal role in the determination of the seed dormancy state. ABA establishes seed dormancy, while GA triggers seed germination. Here, we demonstrate that MYB96 contributes to the fine-tuning of seed dormancy regulation through the coordination of ABA and GA metabolism. The MYB96-deficient myb96-1 seeds germinated earlier than wild-type seeds, whereas delayed germination was observed in the activation-tagging myb96-1D seeds. The differences in germination rate disappeared after stratification or after-ripening. The MYB96 transcription factor positively regulates ABA biosynthesis genes 9-CIS-EPOXYCAROTENOID DIOXYGENASE 2 (NCED2), NCED5, NCED6, and NCED9, and also affects GA biosynthetic genes GA3ox1 and GA20ox1. Notably, MYB96 directly binds to the promoters of NCED2 and NCED6, primarily modulating ABA biosynthesis, which subsequently influences GA metabolism. In agreement with this, hyperdormancy of myb96-1D seeds was recovered by an ABA biosynthesis inhibitor fluridone, while hypodormancy of myb96-1 seeds was suppressed by a GA biosynthesis inhibitor paclobutrazol (PAC). Taken together, the metabolic balance of ABA and GA underlies MYB96 control of primary seed dormancy.

  10. TaMYB13-1, a R2R3 MYB transcription factor, regulates the fructan synthetic pathway and contributes to enhanced fructan accumulation in bread wheat

    Science.gov (United States)

    Kooiker, Maarten; Drenth, Janneke; Glassop, Donna; McIntyre, C. Lynne; Xue, Gang-Ping

    2013-01-01

    Fructans are the major component of temporary carbon reserve in the stem of temperate cereals, which is used for grain filling. Three families of fructosyltransferases are directly involved in fructan synthesis in the vacuole of Triticum aestivum. The regulatory network of the fructan synthetic pathway is largely unknown. Recently, a sucrose-upregulated wheat MYB transcription factor (TaMYB13-1) was shown to be capable of activating the promoter activities of sucrose:sucrose 1-fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT) in transient transactivation assays. This work investigated TaMYB13-1 target genes and their influence on fructan synthesis in transgenic wheat. TaMYB13-1 overexpression resulted in upregulation of all three families of fructosyltransferases including fructan:fructan 1-fructosyltransferase (1-FFT). A γ-vacuolar processing enzyme (γ-VPE1), potentially involved in processing the maturation of fructosyltransferases in the vacuole, was also upregulated by TaMYB13-1 overexpression. Multiple TaMYB13 DNA-binding motifs were identified in the Ta1-FFT1 and Taγ-VPE1 promoters and were bound strongly by TaMYB13-1. The expression profiles of these target genes and TaMYB13-1 were highly correlated in recombinant inbred lines and during stem development as well as the transgenic and non-transgenic wheat dataset, further supporting a direct regulation of these genes by TaMYB13-1. TaMYB13-1 overexpression in wheat led to enhanced fructan accumulation in the leaves and stems and also increased spike weight and grain weight per spike in transgenic plants under water-limited conditions. These data suggest that TaMYB13-1 plays an important role in coordinated upregulation of genes necessary for fructan synthesis and can be used as a molecular tool to improve the high fructan trait. PMID:23873993

  11. Two MYB-related transcription factors play opposite roles in sugar signaling in Arabidopsis.

    Science.gov (United States)

    Chen, Yi-Shih; Chao, Yi-Chi; Tseng, Tzu-Wei; Huang, Chun-Kai; Lo, Pei-Ching; Lu, Chung-An

    2017-02-01

    Sugar regulation of gene expression has profound effects at all stages of the plant life cycle. Although regulation at the transcriptional level is one of the most prominent mechanisms by which gene expression is regulated, only a few transcription factors have been identified and demonstrated to be involved in the regulation of sugar-regulated gene expression. OsMYBS1, an R1/2-type MYB transcription factor, has been demonstrated to be involved in sugar- and hormone-regulated α-amylase gene expression in rice. Arabidopsis contains two OsMYBS1 homologs. In the present study, we investigate MYBS1 and MYBS2 in sugar signaling in Arabidopsis. Our results indicate that MYBS1 and MYBS2 play opposite roles in regulating glucose and ABA signaling in Arabidopsis during seed germination and early seedling development. MYB proteins have been classified into four subfamilies: R2R3-MYB, R1/2-MYB, 3R-MYB, and 4R-MYB. An R1/2-type MYB transcription factor, OsMYBS1, has been demonstrated to be involved in sugar- and hormone-regulated α-amylase genes expression in rice. In this study, two genes homologous to OsMYBS1, MYBS1 and MYBS2, were investigated in Arabidopsis. Subcellular localization analysis showed that MYBS1 and MYBS2 were localized in the nucleus. Rice embryo transient expression assays indicated that both MYBS1 and MYBS2 could recognize the sugar response element, TA-box, in the promoter and induced promoter activity. mybs1 mutant exhibited hypersensitivity to glucose, whereas mybs2 seedlings were hyposensitive to it. MYBS1 and MYBS2 are involved in the control of glucose-responsive gene expression, as the mybs1 mutant displayed increased expression of a hexokinase gene (HXK1), chlorophyll a/b-binding protein gene (CAB1), ADP-glucose pyrophosphorylase gene (APL3), and chalcone synthase gene (CHS), whereas the mybs2 mutant exhibited decreased expression of these genes. mybs1 also showed an enhanced response to abscisic acid (ABA) in the seed germination and seedling

  12. Bone marrow mesenchymal stem cells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4 fusion gene

    Science.gov (United States)

    Catalina, Purificación; Rodríguez, René; Melen, Gustavo J.; Bueno, Clara; Arriero, Mar; García-Sánchez, Félix; Lassaletta, Alvaro; García-Sanz, Ramón

    2009-01-01

    MLL-AF4 fusion is a hallmark genetic abnormality in infant B-acute lymphoblastic leukemia (B-ALL) known to arise in utero. The cellular origin of leukemic fusion genes during human development is difficult to ascertain. The bone marrow (BM) microenvironment plays an important role in the pathogenesis of several hematological malignances. BM mesenchymal stem cells (BM-MSC) from 38 children diagnosed with cytogenetically different acute leukemias were screened for leukemic fusion genes. Fusion genes were absent in BM-MSCs of childhood leukemias carrying TEL-AML1, BCR-ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. However, MLL-AF4 was detected and expressed in BM-MSCs from all cases of MLL-AF4+ B-ALL. Unlike leukemic blasts, MLL-AF4+ BM-MSCs did not display monoclonal Ig gene rearrangements. Endogenous or ectopic expression of MLL-AF4 exerted no effect on MSC culture homeostasis. These findings suggest that MSCs may be in part tumor-related, highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4+ B-ALL. MLL-AF4 itself is not sufficient for MSC transformation and the expression of MLL-AF4 in MSCs is compatible with a mesenchymal phenotype, suggesting a differential impact in the hematopoietic system and mesenchyme. The absence of monoclonal rearrangements in MLL-AF4+ BM-MSCs precludes the possibility of cellular plasticity or de-differentiation of B-ALL blasts and suggests that MLL-AF4 might arise in a population of prehematopoietic precursors. PMID:19995953

  13. Low expression of Mda-7/IL-24 and high expression of C-myb in tumour tissues are predictors of poor prognosis for Burkitt lymphoma patients.

    Science.gov (United States)

    Ma, Ming; Zhao, Riyang; Yang, Xingxiao; Zhao, Lianmei; Liu, Lihua; Zhang, Cong; Wang, Xuexiao; Shan, Baoen

    2018-02-08

    Burkitt lymphoma is one of the most common types of haematopoietic malignancy in children and adolescents. Mda-7/IL-24 had been identified as a differentiation inducer of Burkitt lymphoma cells. Previous studies have revealed that knockdown of C-myb can also lead to the terminal differentiation of Burkitt lymphoma cells. The aim of the present study was to investigate the correlation between the expression of Mda-7/IL-24 and C-myb, as well as their prognostic significance, for Burkitt lymphoma patients. The tumour tissues were collected from 59 cases of Burkitt lymphoma patients and detected with Western blotting and immunohistochemistry. The results showed that the expression of Mda-7/IL-24 was lower, whereas the expression of C-myb was higher in Burkitt lymphoma tissues compared to specimens of normal lymph node tissues. Furthermore, C-myb expression was negatively correlated with Mda-7/IL-24 expression at the protein level in Burkitt lymphoma tissues and cell lines. Both the expression of Mda-7/IL-24 and C-myb in Burkitt lymphoma tissues was associated with some clinicopathological parameters, such as clinical stage, infiltration in the bone marrow, Ki67 and overall survival rates. These results indicated that low expression of Mda-7/IL-24 along with high expression of C-myb are predictors for poor prognosis of Burkitt lymphoma patients; this outcome suggests that Mda-7/IL-24 and C-myb might be potential targets for clinical treatment of Burkitt lymphoma. Mda-7/IL-24: melanoma differentiation associated gene7/interleukin 24; FCM: flow cytometry; Ecog: Eastern Cooperative Oncology Group; IPI: International lymphoma prognosis index.

  14. The MLL recombinome of acute leukemias in 2017.

    Science.gov (United States)

    Meyer, C; Burmeister, T; Gröger, D; Tsaur, G; Fechina, L; Renneville, A; Sutton, R; Venn, N C; Emerenciano, M; Pombo-de-Oliveira, M S; Barbieri Blunck, C; Almeida Lopes, B; Zuna, J; Trka, J; Ballerini, P; Lapillonne, H; De Braekeleer, M; Cazzaniga, G; Corral Abascal, L; van der Velden, V H J; Delabesse, E; Park, T S; Oh, S H; Silva, M L M; Lund-Aho, T; Juvonen, V; Moore, A S; Heidenreich, O; Vormoor, J; Zerkalenkova, E; Olshanskaya, Y; Bueno, C; Menendez, P; Teigler-Schlegel, A; Zur Stadt, U; Lentes, J; Göhring, G; Kustanovich, A; Aleinikova, O; Schäfer, B W; Kubetzko, S; Madsen, H O; Gruhn, B; Duarte, X; Gameiro, P; Lippert, E; Bidet, A; Cayuela, J M; Clappier, E; Alonso, C N; Zwaan, C M; van den Heuvel-Eibrink, M M; Izraeli, S; Trakhtenbrot, L; Archer, P; Hancock, J; Möricke, A; Alten, J; Schrappe, M; Stanulla, M; Strehl, S; Attarbaschi, A; Dworzak, M; Haas, O A; Panzer-Grümayer, R; Sedék, L; Szczepański, T; Caye, A; Suarez, L; Cavé, H; Marschalek, R

    2018-02-01

    Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

  15. Interaction of the transactivation domain of B-Myb with the TAZ2 domain of the coactivator p300: molecular features and properties of the complex.

    Directory of Open Access Journals (Sweden)

    Ojore Oka

    Full Text Available The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (K(d ~1.0-10 µM complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (~1200 Å(2. The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1, which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2.

  16. RFX2 is a candidate downstream amplifier of A-MYB regulation in mouse spermatogenesis

    Directory of Open Access Journals (Sweden)

    Kistler Malathi K

    2009-12-01

    Full Text Available Abstract Background Mammalian spermatogenesis involves formation of haploid cells from the male germline and then a complex morphological transformation to generate motile sperm. Focusing on meiotic prophase, some tissue-specific transcription factors are known (A-MYB or suspected (RFX2 to play important roles in modulating gene expression in pachytene spermatocytes. The current work was initiated to identify both downstream and upstream regulatory connections for Rfx2. Results Searches of pachytene up-regulated genes identified high affinity RFX binding sites (X boxes in promoter regions of several new genes: Adam5, Pdcl2, and Spag6. We confirmed a strong promoter-region X-box for Alf, a germ cell-specific variant of general transcription factor TFIIA. Using Alf as an example of a target gene, we showed that its promoter is stimulated by RFX2 in transfected cells and used ChIP analysis to show that the promoter is occupied by RFX2 in vivo. Turning to upstream regulation of the Rfx2 promoter, we identified a cluster of three binding sites (MBS for the MYB family of transcription factors. Because testis is one of the few sites of A-myb expression, and because spermatogenesis arrests in pachytene in A-myb knockout mice, the MBS cluster implicates Rfx2 as an A-myb target. Electrophoretic gel-shift, ChIP, and co-transfection assays all support a role for these MYB sites in Rfx2 expression. Further, Rfx2 expression was virtually eliminated in A-myb knockout testes. Immunohistology on testis sections showed that A-MYB expression is up-regulated only after pachytene spermatocytes have clearly moved away from the tubule wall, which correlates with onset of RFX2 expression, whereas B-MYB expression, by contrast, is prevalent only in earlier spermatocytes and spermatogonia. Conclusion With an expanding list of likely target genes, RFX2 is potentially an important transcriptional regulator in pachytene spermatocytes. Rfx2 itself is a good candidate to be

  17. AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling.

    Science.gov (United States)

    Shim, Jae Sung; Jung, Choonkyun; Lee, Sangjoon; Min, Kyunghun; Lee, Yin-Won; Choi, Yeonhee; Lee, Jong Seob; Song, Jong Tae; Kim, Ju-Kon; Choi, Yang Do

    2013-02-01

    The role of AtMYB44, an R2R3 MYB transcription factor, in signaling mediated by jasmonic acid (JA) and salicylic acid (SA) is examined. AtMYB44 is induced by JA through CORONATINE INSENSITIVE 1 (COI1). AtMYB44 over-expression down-regulated defense responses against the necrotrophic pathogen Alternaria brassicicola, but up-regulated WRKY70 and PR genes, leading to enhanced resistance to the biotrophic pathogen Pseudomonas syringae pv. tomato DC3000. The knockout mutant atmyb44 shows opposite effects. Induction of WRKY70 by SA is reduced in atmyb44 and npr1-1 mutants, and is totally abolished in atmyb44 npr1-1 double mutants, showing that WRKY70 is regulated independently through both NPR1 and AtMYB44. AtMYB44 over-expression does not change SA content, but AtMYB44 over-expression phenotypes, such as retarded growth, up-regulated PR1 and down-regulated PDF1.2 are reversed by SA depletion. The wrky70 mutation suppressed AtMYB44 over-expression phenotypes, including up-regulation of PR1 expression and down-regulation of PDF1.2 expression. β-estradiol-induced expression of AtMYB44 led to WRKY70 activation and thus PR1 activation. AtMYB44 binds to the WRKY70 promoter region, indicating that AtMYB44 acts as a transcriptional activator of WRKY70 by directly binding to a conserved sequence element in the WRKY70 promoter. These results demonstrate that AtMYB44 modulates antagonistic interaction by activating SA-mediated defenses and repressing JA-mediated defenses through direct control of WRKY70. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  18. The Expression of c-Myb Correlates with the Levels of Rhabdomyosarcoma-specific Marker Myogenin

    Czech Academy of Sciences Publication Activity Database

    Kašpar, Petr; Zíková, Martina; Bartůněk, Petr; Štěrba, J.; Strnad, Hynek; Křen, L.; Sedláček, Radislav

    2015-01-01

    Roč. 5, Oct 14 (2015) ISSN 2045-2322 R&D Projects: GA ČR GAP305/10/2133; GA ČR GAP301/12/1478; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : c-Myb * Rhabdomyosarcomas * C2C12 myoblast cell line * myogenin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.228, year: 2015

  19. Discovery of a Highly Potent, Cell-Permeable Macrocyclic Peptidomimetic (MM-589) Targeting the WD Repeat Domain 5 Protein (WDR5)–Mixed Lineage Leukemia (MLL) Protein–Protein Interaction

    Energy Technology Data Exchange (ETDEWEB)

    Karatas, Hacer; Li, Yangbing; Liu, Liu; Ji, Jiao; Lee, Shirley; Chen, Yong; Yang, Jiuling; Huang, Liyue; Bernard, Denzil; Xu, Jing; Townsend, Elizabeth C.; Cao, Fang; Ran, Xu; Li, Xiaoqin; Wen, Bo; Sun, Duxin; Stuckey, Jeanne A; Lei, Ming; Dou, Yali; Wang, Shaomeng (Michigan)

    2017-06-06

    We report herein the design, synthesis, and evaluation of macrocyclic peptidomimetics that bind to WD repeat domain 5 (WDR5) and block the WDR5–mixed lineage leukemia (MLL) protein–protein interaction. Compound 18 (MM-589) binds to WDR5 with an IC50 value of 0.90 nM (Ki value <1 nM) and inhibits the MLL H3K4 methyltransferase (HMT) activity with an IC50 value of 12.7 nM. Compound 18 potently and selectively inhibits cell growth in human leukemia cell lines harboring MLL translocations and is >40 times better than the previously reported compound MM-401. Cocrystal structures of 16 and 18 complexed with WDR5 provide structural basis for their high affinity binding to WDR5. Additionally, we have developed and optimized a new AlphaLISA-based MLL HMT functional assay to facilitate the functional evaluation of these designed compounds. Compound 18 represents the most potent inhibitor of the WDR5–MLL interaction reported to date, and further optimization of 18 may yield a new therapy for acute leukemia.

  20. Myb transcription factors and light regulate sporulation in the oomycete Phytophthora infestans.

    Science.gov (United States)

    Xiang, Qijun; Judelson, Howard S

    2014-01-01

    Life cycle progression in eukaryotic microbes is often influenced by environment. In the oomycete Phytophthora infestans, which causes late blight on potato and tomato, sporangia have been reported to form mostly at night. By growing P. infestans under different light regimes at constant temperature and humidity, we show that light contributes to the natural pattern of sporulation by delaying sporulation until the following dark period. However, illumination does not permanently block sporulation or strongly affect the total number of sporangia that ultimately form. Based on measurements of sporulation-induced genes such as those encoding protein kinase Pks1 and Myb transcription factors Myb2R1 and Myb2R3, it appears that most spore-associated transcripts start to rise four to eight hours before sporangia appear. Their mRNA levels oscillate with the light/dark cycle and increase with the amount of sporangia. An exception to this pattern of expression is Myb2R4, which is induced several hours before the other genes and declines after cultures start to sporulate. Transformants over-expressing Myb2R4 produce twice the number of sporangia and ten-fold higher levels of Myb2R1 mRNA than wild-type, and chromatin immunoprecipitation showed that Myb2R4 binds the Myb2R1 promoter in vivo. Myb2R4 thus appears to be an early regulator of sporulation. We attempted to silence eight Myb genes by DNA-directed RNAi, but succeeded only with Myb2R3, which resulted in suppressed sporulation. Ectopic expression studies of seven Myb genes revealed that over-expression frequently impaired vegetative growth, and in the case of Myb3R6 interfered with sporangia dormancy. We observed that the degree of silencing induced by a hairpin construct was correlated with its copy number, and ectopic expression was often unstable due to epigenetic silencing and transgene excision.

  1. Myb transcription factors and light regulate sporulation in the oomycete Phytophthora infestans.

    Directory of Open Access Journals (Sweden)

    Qijun Xiang

    Full Text Available Life cycle progression in eukaryotic microbes is often influenced by environment. In the oomycete Phytophthora infestans, which causes late blight on potato and tomato, sporangia have been reported to form mostly at night. By growing P. infestans under different light regimes at constant temperature and humidity, we show that light contributes to the natural pattern of sporulation by delaying sporulation until the following dark period. However, illumination does not permanently block sporulation or strongly affect the total number of sporangia that ultimately form. Based on measurements of sporulation-induced genes such as those encoding protein kinase Pks1 and Myb transcription factors Myb2R1 and Myb2R3, it appears that most spore-associated transcripts start to rise four to eight hours before sporangia appear. Their mRNA levels oscillate with the light/dark cycle and increase with the amount of sporangia. An exception to this pattern of expression is Myb2R4, which is induced several hours before the other genes and declines after cultures start to sporulate. Transformants over-expressing Myb2R4 produce twice the number of sporangia and ten-fold higher levels of Myb2R1 mRNA than wild-type, and chromatin immunoprecipitation showed that Myb2R4 binds the Myb2R1 promoter in vivo. Myb2R4 thus appears to be an early regulator of sporulation. We attempted to silence eight Myb genes by DNA-directed RNAi, but succeeded only with Myb2R3, which resulted in suppressed sporulation. Ectopic expression studies of seven Myb genes revealed that over-expression frequently impaired vegetative growth, and in the case of Myb3R6 interfered with sporangia dormancy. We observed that the degree of silencing induced by a hairpin construct was correlated with its copy number, and ectopic expression was often unstable due to epigenetic silencing and transgene excision.

  2. MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia

    OpenAIRE

    Mets, E; Van der Meulen, J; Van Peer, G; Boice, M; Mestdagh, P; Van de Walle, I; Lammens, T; Goossens, S; De Moerloose, B; Benoit, Y; Van Roy, N; Clappier, E; Poppe, B; Vandesompele, J; Wendel, H-G

    2014-01-01

    The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3′untranslated region–microRNA (miRNA) library screen and identified 33 p...

  3. Localization of c-MYB in differentiated cells during postnatal molar and alveolar bone development

    Czech Academy of Sciences Publication Activity Database

    Lungová, Vlasta; Buchtová, Marcela; Janečková, Eva; Tucker, A.S.; Knopfová, L.; Šmarda, J.; Matalová, Eva

    2012-01-01

    Roč. 120, č. 6 (2012), 495-504 ISSN 0909-8836 R&D Projects: GA ČR GCP302/12/J059 Institutional research plan: CEZ:AV0Z50450515 Keywords : c-myb * tooth * postnatal Subject RIV: FF - HEENT, Dentistry Impact factor: 1.420, year: 2012

  4. The MLL recombinome of acute leukemias in 2013

    Science.gov (United States)

    Meyer, C; Hofmann, J; Burmeister, T; Gröger, D; Park, T S; Emerenciano, M; Pombo de Oliveira, M; Renneville, A; Villarese, P; Macintyre, E; Cavé, H; Clappier, E; Mass-Malo, K; Zuna, J; Trka, J; De Braekeleer, E; De Braekeleer, M; Oh, S H; Tsaur, G; Fechina, L; van der Velden, V H J; van Dongen, J J M; Delabesse, E; Binato, R; Silva, M L M; Kustanovich, A; Aleinikova, O; Harris, M H; Lund-Aho, T; Juvonen, V; Heidenreich, O; Vormoor, J; Choi, W W L; Jarosova, M; Kolenova, A; Bueno, C; Menendez, P; Wehner, S; Eckert, C; Talmant, P; Tondeur, S; Lippert, E; Launay, E; Henry, C; Ballerini, P; Lapillone, H; Callanan, M B; Cayuela, J M; Herbaux, C; Cazzaniga, G; Kakadiya, P M; Bohlander, S; Ahlmann, M; Choi, J R; Gameiro, P; Lee, D S; Krauter, J; Cornillet-Lefebvre, P; Te Kronnie, G; Schäfer, B W; Kubetzko, S; Alonso, C N; zur Stadt, U; Sutton, R; Venn, N C; Izraeli, S; Trakhtenbrot, L; Madsen, H O; Archer, P; Hancock, J; Cerveira, N; Teixeira, M R; Lo Nigro, L; Möricke, A; Stanulla, M; Schrappe, M; Sedék, L; Szczepański, T; Zwaan, C M; Coenen, E A; van den Heuvel-Eibrink, M M; Strehl, S; Dworzak, M; Panzer-Grümayer, R; Dingermann, T; Klingebiel, T; Marschalek, R

    2013-01-01

    Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (∼90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements. PMID:23628958

  5. Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development

    KAUST Repository

    Park, Myoungryoul; Yun, Kilyoung; Mohanty, Bijayalaxmi; Herath, Venura; Xu, Fuyu; Wijaya, Edward; Bajic, Vladimir B.; Yun, Songjoong; De Los Reyes, Benildo G.

    2010-01-01

    acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co

  6. Situational Awareness: Regulation of the Myb Transcription Factor in Differentiation, the Cell Cycle and Oncogenesis

    Energy Technology Data Exchange (ETDEWEB)

    George, Olivia L.; Ness, Scott A., E-mail: sness@salud.unm.edu [Department of Internal Medicine, Section of Molecular Medicine, University of New Mexico Health Sciences Center, MSC07 4025-CRF 121, 1 University of New Mexico, Albuquerque, NM 87131 (United States)

    2014-10-02

    This review summarizes the mechanisms that control the activity of the c-Myb transcription factor in normal cells and tumors, and discusses how c-Myb plays a role in the regulation of the cell cycle. Oncogenic versions of c-Myb contribute to the development of leukemias and solid tumors such as adenoid cystic carcinoma, breast cancer and colon cancer. The activity and specificity of the c-Myb protein seems to be controlled through changes in protein-protein interactions, so understanding how it is regulated could lead to the development of novel therapeutic strategies.

  7. Myb proteins: angels and demons in normal and transformed cells.

    Science.gov (United States)

    Zhou, Ye; Ness, Scott A

    2011-01-01

    A key regulator of proliferation, differentiation and cell fate, the c-Myb transcription factor regulates the expression of hundreds of genes and is in turn regulated by numerous pathways and protein interactions. However, the most unique feature of c-Myb is that it can be converted into an oncogenic transforming protein through a few mutations that completely change its activity and specificity. The c-Myb protein is a myriad of interactions and activities rolled up in a protein that controls proliferation and differentiation in many different cell types. Here we discuss the background and recent progress that have led to a better understanding of this complex protein, and outline the questions that have yet to be answered.

  8. Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun; Kim, Eunhee; Cheong, Chaejoon; Cho, Myeon Haeng; Lee, Weontae

    2014-01-01

    Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2 1–64 ) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2 1–64 and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences

  9. Analysis list: Myb [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Myb Blood,Gonad + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Myb.1.t...sv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Myb.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-...u/mm9/target/Myb.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Myb.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Myb.Gonad.tsv http://dbarchive.biosciencedbc.jp.../kyushu-u/mm9/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Gonad.gml ...

  10. Characterization of the regulatory network of BoMYB2 in controlling anthocyanin biosynthesis in purple cauliflower.

    Science.gov (United States)

    Chiu, Li-Wei; Li, Li

    2012-10-01

    Purple cauliflower (Brassica oleracea L. var. botrytis) Graffiti represents a unique mutant in conferring ectopic anthocyanin biosynthesis, which is caused by the tissue-specific activation of BoMYB2, an ortholog of Arabidopsis PAP2 or MYB113. To gain a better understanding of the regulatory network of anthocyanin biosynthesis, we investigated the interaction among cauliflower MYB-bHLH-WD40 network proteins and examined the interplay of BoMYB2 with various bHLH transcription factors in planta. Yeast two-hybrid studies revealed that cauliflower BoMYBs along with the other regulators formed the MYB-bHLH-WD40 complexes and BobHLH1 acted as a bridge between BoMYB and BoWD40-1 proteins. Different BoMYBs exhibited different binding activity to BobHLH1. Examination of the BoMYB2 transgenic lines in Arabidopsis bHLH mutant backgrounds demonstrated that TT8, EGL3, and GL3 were all involved in the BoMYB2-mediated anthocyanin biosynthesis. Expression of BoMYB2 in Arabidopsis caused up-regulation of AtTT8 and AtEGL3 as well as a subset of anthocyanin structural genes encoding flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase. Taken together, our results show that MYB-bHLH-WD40 network transcription factors regulated the bHLH gene expression, which may represent a critical feature in the control of anthocyanin biosynthesis. BoMYB2 together with various BobHLHs specifically regulated the late anthocyanin biosynthetic pathway genes for anthocyanin biosynthesis. Our findings provide additional information for the complicated regulatory network of anthocyanin biosynthesis and the transcriptional regulation of transcription factors in vegetable crops.

  11. Identification and molecular characterization of MYB Transcription Factor Superfamily in C4 model plant foxtail millet (Setaria italica L.).

    Science.gov (United States)

    Muthamilarasan, Mehanathan; Khandelwal, Rohit; Yadav, Chandra Bhan; Bonthala, Venkata Suresh; Khan, Yusuf; Prasad, Manoj

    2014-01-01

    MYB proteins represent one of the largest transcription factor families in plants, playing important roles in diverse developmental and stress-responsive processes. Considering its significance, several genome-wide analyses have been conducted in almost all land plants except foxtail millet. Foxtail millet (Setaria italica L.) is a model crop for investigating systems biology of millets and bioenergy grasses. Further, the crop is also known for its potential abiotic stress-tolerance. In this context, a comprehensive genome-wide survey was conducted and 209 MYB protein-encoding genes were identified in foxtail millet. All 209 S. italica MYB (SiMYB) genes were physically mapped onto nine chromosomes of foxtail millet. Gene duplication study showed that segmental- and tandem-duplication have occurred in genome resulting in expansion of this gene family. The protein domain investigation classified SiMYB proteins into three classes according to number of MYB repeats present. The phylogenetic analysis categorized SiMYBs into ten groups (I-X). SiMYB-based comparative mapping revealed a maximum orthology between foxtail millet and sorghum, followed by maize, rice and Brachypodium. Heat map analysis showed tissue-specific expression pattern of predominant SiMYB genes. Expression profiling of candidate MYB genes against abiotic stresses and hormone treatments using qRT-PCR revealed specific and/or overlapping expression patterns of SiMYBs. Taken together, the present study provides a foundation for evolutionary and functional characterization of MYB TFs in foxtail millet to dissect their functions in response to environmental stimuli.

  12. Identification and molecular characterization of MYB Transcription Factor Superfamily in C4 model plant foxtail millet (Setaria italica L..

    Directory of Open Access Journals (Sweden)

    Mehanathan Muthamilarasan

    Full Text Available MYB proteins represent one of the largest transcription factor families in plants, playing important roles in diverse developmental and stress-responsive processes. Considering its significance, several genome-wide analyses have been conducted in almost all land plants except foxtail millet. Foxtail millet (Setaria italica L. is a model crop for investigating systems biology of millets and bioenergy grasses. Further, the crop is also known for its potential abiotic stress-tolerance. In this context, a comprehensive genome-wide survey was conducted and 209 MYB protein-encoding genes were identified in foxtail millet. All 209 S. italica MYB (SiMYB genes were physically mapped onto nine chromosomes of foxtail millet. Gene duplication study showed that segmental- and tandem-duplication have occurred in genome resulting in expansion of this gene family. The protein domain investigation classified SiMYB proteins into three classes according to number of MYB repeats present. The phylogenetic analysis categorized SiMYBs into ten groups (I-X. SiMYB-based comparative mapping revealed a maximum orthology between foxtail millet and sorghum, followed by maize, rice and Brachypodium. Heat map analysis showed tissue-specific expression pattern of predominant SiMYB genes. Expression profiling of candidate MYB genes against abiotic stresses and hormone treatments using qRT-PCR revealed specific and/or overlapping expression patterns of SiMYBs. Taken together, the present study provides a foundation for evolutionary and functional characterization of MYB TFs in foxtail millet to dissect their functions in response to environmental stimuli.

  13. Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10.

    Science.gov (United States)

    Feng, Shouqian; Wang, Yanling; Yang, Song; Xu, Yuting; Chen, Xuesen

    2010-06-01

    Skin color is an important factor in pear breeding programs. The degree of red coloration is determined by the content and composition of anthocyanins. In plants, many MYB transcriptional factors are involved in regulating anthocyanin biosynthesis. In this study, a R2R3-MYB transcription factor gene, PyMYB10, was isolated from Asian pear (Pyrus pyrifolia) cv. 'Aoguan'. Sequence analysis suggested that the PyMYB10 gene was an ortholog of MdMYB10 gene, which regulates anthocyanin biosynthesis in red fleshed apple (Malus x domestica) cv. 'Red Field'. PyMYB10 was identified at the genomic level and had three exons, with its upstream sequence containing core sequences of cis-acting regulatory elements involved in light responsiveness. Fruit bagging showed that light could induce expression of PyMYB10 and anthocyanin biosynthesis. Quantitative real-time PCR revealed that PyMYB10 was predominantly expressed in pear skins, buds, and young leaves, and the level of transcription in buds was higher than in skin and young leaves. In ripening fruits, the transcription of PyMYB10 in the skin was positively correlated with genes in the anthocyanin pathway and with anthocyanin biosynthesis. In addition, the transcription of PyMYB10 and genes of anthocyanin biosynthesis were more abundant in red-skinned pear cultivars compared to blushed cultivars. Transgenic Arabidopsis plants overexpressing PyMYB10 exhibited ectopic pigmentation in immature seeds. The study suggested that PyMYB10 plays a role in regulating anthocyanin biosynthesis and the overexpression of PyMYB10 was sufficient to induce anthocyanin accumulation.

  14. Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Ji-Hye [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Won Kyung [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Eunhee; Cheong, Chaejoon [Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang, Chungbuk 363-883 (Korea, Republic of); Cho, Myeon Haeng [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2014-09-26

    Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.

  15. The Onion (Allium cepa L.) R2R3-MYB Gene MYB1 Regulates Anthocyanin Biosynthesis

    Science.gov (United States)

    Schwinn, Kathy E.; Ngo, Hanh; Kenel, Fernand; Brummell, David A.; Albert, Nick W.; McCallum, John A.; Pither-Joyce, Meeghan; Crowhurst, Ross N.; Eady, Colin; Davies, Kevin M.

    2016-01-01

    Bulb color is an important consumer trait for onion (Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow), and chalcones (bright yellow). Flavonoid regulation is poorly characterized in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressed and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic (Allium sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum majus of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species. PMID:28018399

  16. The onion (Allium cepa L. R2R3-MYB gene MYB1 regulates anthocyanin biosynthesis

    Directory of Open Access Journals (Sweden)

    Kathy Schwinn

    2016-12-01

    Full Text Available Bulb colour is an important consumer trait for onion (Allium cepa L., Allioideae, Asparagales. The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red, flavonols (pale yellow and chalcones (bright yellow. Flavonoid regulation is poorly characterised in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs that commonly activate anthocyanin (SG6, MYB1 or flavonol (SG7, MYB29 production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5. MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressd and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic (A. sativum L. plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum majus of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species.

  17. The Onion (Allium cepa L.) R2R3-MYB Gene MYB1 Regulates Anthocyanin Biosynthesis.

    Science.gov (United States)

    Schwinn, Kathy E; Ngo, Hanh; Kenel, Fernand; Brummell, David A; Albert, Nick W; McCallum, John A; Pither-Joyce, Meeghan; Crowhurst, Ross N; Eady, Colin; Davies, Kevin M

    2016-01-01

    Bulb color is an important consumer trait for onion ( Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow), and chalcones (bright yellow). Flavonoid regulation is poorly characterized in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressed and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic ( Allium sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum maju s of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species.

  18. An R2R3-MYB transcription factor, OjMYB1, functions in anthocyanin biosynthesis in Oenanthe javanica.

    Science.gov (United States)

    Feng, Kai; Xu, Zhi-Sheng; Que, Feng; Liu, Jie-Xia; Wang, Feng; Xiong, Ai-Sheng

    2018-02-01

    This study showed that an R2R3-MYB transcription factor, OjMYB1, is involved in anthocyanin biosynthesis and accumulation in Oenanthe javanica. Anthocyanins can be used as safe natural food colorants, obtained from many plants. R2R3-MYB transcription factors (TFs) play important roles in anthocyanins biosynthesis during plant development. Oenanthe javanica is a popular vegetable with high nutritional values and numerous medical functions. O. javanica has purple petioles that are mainly due to anthocyanins accumulation. In the present study, the gene encoding an R2R3-MYB TF, OjMYB1, was isolated from purple O. javanica. Sequencing results showed that OjMYB1 contained a 912-bp open reading frame encoding 303 amino acids. Sequence alignments revealed that OjMYB1 contained bHLH-interaction motif ([DE]Lx2[RK]x3Lx6Lx3R) and ANDV motif ([A/G]NDV). Phylogenetic analysis indicated that the OjMYB1 classified into the anthocyanins biosynthesis clade. Subcellular localization assay showed that OjMYB1 was a nuclear protein in vivo. The heterologous expression of OjMYB1 in Arabidopsis could enhance the anthocyanins content and up-regulate the expression levels of the structural genes-related anthocyanins biosynthesis. Yeast two-hybrid assay indicated that OjMYB1 could interact with AtTT8 and AtEGL3 proteins. Enzymatic analysis revealed that overexpression of OjMYB1 gene up-regulated the enzyme activity of 3-O-glycosyltransferase encoded by AtUGT78D2 in transgenic Arabidopsis. Our results provided a comprehensive understanding of the structure and function of OjMYB1 TF in O. javanica.

  19. Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development

    KAUST Repository

    Park, Myoungryoul

    2010-09-28

    The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Cold-dependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development. © 2010 Blackwell Publishing Ltd.

  20. Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development.

    Science.gov (United States)

    Park, Myoung-Ryoul; Yun, Kil-Young; Mohanty, Bijayalaxmi; Herath, Venura; Xu, Fuyu; Wijaya, Edward; Bajic, Vladimir B; Yun, Song-Joong; De Los Reyes, Benildo G

    2010-12-01

    The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Cold-dependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development. © 2010 Blackwell Publishing Ltd.

  1. A MYB transcription factor, DcMYB6, is involved in regulating anthocyanin biosynthesis in purple carrot taproots.

    Science.gov (United States)

    Xu, Zhi-Sheng; Feng, Kai; Que, Feng; Wang, Feng; Xiong, Ai-Sheng

    2017-03-27

    Carrots are widely grown and enjoyed around the world. Purple carrots accumulate rich anthocyanins in the taproots, while orange, yellow, and red carrots accumulate rich carotenoids in the taproots. Our previous studies indicated that variation in the activity of regulatory genes may be responsible for variations in anthocyanin production among various carrot cultivars. In this study, an R2R3-type MYB gene, designated as DcMYB6, was isolated from a purple carrot cultivar. In a phylogenetic analysis, DcMYB6 was grouped into an anthocyanin biosynthesis-related MYB clade. Sequence analyses revealed that DcMYB6 contained the conserved bHLH-interaction motif and two atypical motifs of anthocyanin regulators. The expression pattern of DcMYB6 was correlated with anthocyanin production. DcMYB6 transcripts were detected at high levels in three purple carrot cultivars but at much lower levels in six non-purple carrot cultivars. Overexpression of DcMYB6 in Arabidopsis led to enhanced anthocyanin accumulation in both vegetative and reproductive tissues and upregulated transcript levels of all seven tested anthocyanin-related structural genes. Together, these results show that DcMYB6 is involved in regulating anthocyanin biosynthesis in purple carrots. Our results provide new insights into the regulation of anthocyanin synthesis in purple carrot cultivars.

  2. MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Mets, E; Van der Meulen, J; Van Peer, G; Boice, M; Mestdagh, P; Van de Walle, I; Lammens, T; Goossens, S; De Moerloose, B; Benoit, Y; Van Roy, N; Clappier, E; Poppe, B; Vandesompele, J; Wendel, H-G; Taghon, T; Rondou, P; Soulier, J; Van Vlierberghe, P; Speleman, F

    2015-04-01

    The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3'untranslated region-microRNA (miRNA) library screen and identified 33 putative MYB-targeting miRNAs. Subsequently, transcriptome data from two independent T-ALL cohorts and different subsets of normal T-cells were used to select miRNAs with relevance in the context of normal and malignant T-cell transformation. Hereby, miR-193b-3p was identified as a novel bona fide tumor-suppressor miRNA that targets MYB during malignant T-cell transformation thereby offering an entry point for efficient MYB targeting-oriented therapies for human T-ALL.

  3. Identification of transcription factors ZmMYB111and ZmMYB148 involved in phenylpropanoid metabolism

    Directory of Open Access Journals (Sweden)

    Junjie eZhang

    2016-02-01

    Full Text Available Maize is the leading crop worldwide in terms of both planting area and total yields, but environmental stresses cause significant losses in productivity. Phenylpropanoid compounds play an important role in plant stress resistance; however, the mechanism of their synthesis is not fully understood, especially in regard to the expression and regulation of key genes. Phenylalanine ammonia-lyase (PAL is the first key enzyme involved in phenylpropanoid metabolism, and it has a significant effect on the synthesis of important phenylpropanoid compounds. According to the results of sequence alignments and functional prediction, we selected two conserved R2R3-MYB transcription factors as candidate genes for the regulation of phenylpropanoid metabolism. The two candidate R2R3-MYB genes, which we named ZmMYB111and ZmMYB148, were cloned, and then their structural characteristics and phylogenetic placement were predicted and analyzed. In addition, a series of evaluations were performed, including expression profiles, subcellular localization, transcription activation, protein-DNA interaction, and transient expression in maize endosperm. Our results indicated that both ZmMYB111 and ZmMYB148 are indeed R2R3-MYB transcription factors and that they may play a regulatory role in PAL gene expression.

  4. The MYB182 protein down-regulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes.

    Science.gov (United States)

    Yoshida, Kazuko; Ma, Dawei; Constabel, C Peter

    2015-03-01

    Trees in the genus Populus (poplar) contain phenolic secondary metabolites including the proanthocyanidins (PAs), which help to adapt these widespread trees to diverse environments. The transcriptional activation of PA biosynthesis in response to herbivory and ultraviolet light stress has been documented in poplar leaves, and a regulator of this process, the R2R3-MYB transcription factor MYB134, has been identified. MYB134-overexpressing transgenic plants show a strong high-PA phenotype. Analysis of these transgenic plants suggested the involvement of additional MYB transcription factors, including repressor-like MYB factors. Here, MYB182, a subgroup 4 MYB factor, was found to act as a negative regulator of the flavonoid pathway. Overexpression of MYB182 in hairy root culture and whole poplar plants led to reduced PA and anthocyanin levels as well as a reduction in the expression of key flavonoid genes. Similarly, a reduced accumulation of transcripts of a MYB PA activator and a basic helix-loop-helix cofactor was observed in MYB182-overexpressing hairy roots. Transient promoter activation assays in poplar cell culture demonstrated that MYB182 can disrupt transcriptional activation by MYB134 and that the basic helix-loop-helix-binding motif of MYB182 was essential for repression. Microarray analysis of transgenic plants demonstrated that down-regulated targets of MYB182 also include shikimate pathway genes. This work shows that MYB182 plays an important role in the fine-tuning of MYB134-mediated flavonoid metabolism. © 2015 American Society of Plant Biologists. All Rights Reserved.

  5. An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development.

    Science.gov (United States)

    Vimolmangkang, Sornkanok; Han, Yuepeng; Wei, Guochao; Korban, Schuyler S

    2013-11-07

    Red coloration of fruit is an important trait in apple, and it is mainly attributed to the accumulation of anthocyanins, a class of plant flavonoid metabolites. Anthocyanin biosynthesis is genetically determined by structural and regulatory genes. Plant tissue pigmentation patterns are mainly controlled by expression profiles of regulatory genes. Among these regulatory genes are MYB transcription factors (TFs), wherein the class of two-repeats (R2R3) is deemed the largest, and these are associated with the anthocyanin biosynthesis pathway. Although three MdMYB genes, almost identical in nucleotide sequences, have been identified in apple, it is likely that there are other R2R3 MYB TFs that are present in the apple genome that are also involved in the regulation of coloration of red color pigmentation of the skin of apple fruits. In this study, a novel R2R3 MYB gene has been isolated and characterized in apple. This MYB gene is closely related to the Arabidopsis thaliana AtMYB3, and has been designated as MdMYB3. This TF belongs to the subgroup 4 R2R3 family of plant MYB transcription factors. This apple MdMYB3 gene is mapped onto linkage group 15 of the integrated apple genetic map. Transcripts of MdMYB3 are detected in all analyzed tissues including leaves, flowers, and fruits. However, transcripts of MdMYB3 are higher in excocarp of red-skinned apple cultivars than that in yellowish-green skinned apple cultivars. When this gene is ectopically expressed in Nicotiana tabacum cv. Petite Havana SR1, flowers of transgenic tobacco lines carrying MdMYB3 have exhibited increased pigmentation and accumulate higher levels of anthocyanins and flavonols than wild-type flowers. Overexpression of MdMYB3 has resulted in transcriptional activation of several flavonoid pathway genes, including CHS, CHI, UFGT, and FLS. Moreover, peduncles of flowers and styles of pistils of transgenic plants overexpressing MdMYB3 are longer than those of wild-type plants, thus suggesting that this

  6. C-myb Plays an Essential Role in the Protective Function of IGF-1 on Cytotoxicity Induced by Aβ25-35 via the PI3K/Akt Pathway.

    Science.gov (United States)

    Zhang, Jingyu; Shu, Yongwei; Qu, Yang; Zhang, Lina; Chu, Tingting; Zheng, Yonghui; Zhao, Hong

    2017-12-01

    There have been numerous reports about neurodegenerative diseases, including Alzheimer's disease. Nevertheless, the molecules responsible for the neurodegeneration in Alzheimer's disease are basically unknown. Recent findings indicate that the cellular myeloblastosis (c-myb) regulates neural progenitor cell proliferation. In the current study, the function of insulin-like growth factor-1 (IGF-1) against cell toxicity in SH-SY5Y cells induced by β-amyloid 25-35 (Aβ 25-35 ) and its molecular mechanism were investigated. It was found that p25 protein production was raised by Aβ 25-35 (25 μM), similar to the increased expression of μ-calpain. The results also showed that Aβ 25-35 reduced c-myb, elevated tau hyper-phosphorylation, and induced death of SH-SY5Y cells. Loss of cell viability and apoptosis of SH-SY5Y cells induced by Aβ 25-35 were attenuated by IGF-1 pretreatment in a dose-dependent manner. In addition, IGF-1 blocked μ-calpain expression, which was induced by Aβ 25-35 and reduced p25 formation and tau hyper-phosphorylation. Moreover, the expression of c-myb in SH-SY5Y cells was increased by combining IGF-1 with Aβ 25-35 or IGF-1 alone. The neuroprotective function of IGF-1 was attenuated in the SH-SY5Y cells, which were transfected with a c-myb small interfering RNA. Furthermore, LY294002, a specific PI3K inhibitor, reduced c-myb expression and abolished IGF-1's protective function in SH-SY5Y cell apoptosis induced by Aβ 25-35 . The facts above indicate that c-myb has a role in IGF-1-mediated protection from Aβ 25-35 -induced cytotoxicity via the PI3K/Akt pathway.

  7. Genome-Wide Identification, Evolution and Functional Divergence of MYB Transcription Factors in Chinese White Pear (Pyrus bretschneideri).

    Science.gov (United States)

    Li, Xiaolong; Xue, Cheng; Li, Jiaming; Qiao, Xin; Li, Leiting; Yu, Li'ang; Huang, Yuhua; Wu, Jun

    2016-04-01

    The MYB superfamily is large and functionally diverse in plants. To date, MYB family genes have not yet been identified in Chinese white pear (Pyrus bretschneideri), and their functions remain unclear. In this study, we identified 231 genes as candidate MYB genes and divided them into four subfamilies. The R2R3-MYB (PbrMYB) family shared an R2R3 domain with 104 amino acid residues, including five conserved tryptophan residues. The Pbr MYB family was divided into 37 functional subgroups including 33 subgroups which contained both MYB genes of Rosaceae plants and AtMYB genes, and four subgroups which included only Rosaceae MYB genes or AtMYB genes. PbrMYB genes with similar functions clustered into the same subgroup, indicating functional conservation. We also found that whole-genome duplication (WGD) and dispersed duplications played critical roles in the expansion of the MYB family. The 87 Pbr MYB duplicated gene pairs dated back to the two WGD events. Purifying selection was the primary force driving Pbr MYB gene evolution. The 15 gene pairs presented 1-7 codon sites under positive selection. A total of 147 expressed genes were identified from RNA-sequencing data of fruit, and six Pbr MYB members in subgroup C1 were identified as important candidate genes in the regulation of lignin synthesis by quantitative real-time PCR analysis. Further correlation analysis revealed that six PbrMYBs were significantly correlated with five structural gene families (F5H, HCT, CCR, POD and C3'H) in the lignin pathway. The phylogenetic, evolution and expression analyses of the MYB gene family in Chinese white pear establish a solid foundation for future comprehensive functional analysis of Pbr MYB genes. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. The MLL recombinome of acute leukemias in 2013

    DEFF Research Database (Denmark)

    Meyer, C; Hofmann, Julian; Burmeister, T

    2013-01-01

    patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79......Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia...... patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All...

  9. Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited

    Directory of Open Access Journals (Sweden)

    Sun Jian-Sheng

    2006-01-01

    Full Text Available Abstract Background Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL, considers the spatial proximity of loci in interphase nuclei (static "contact first" model. The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model. Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4 and a less frequent partner gene (ENL, should elucidate the MLL translocation mechanism. Methods Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. Results In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value loci would indicate a greater probability of the occurrence of t(11;19(q23;p13.3 compared to t(4;11(q21;q23. However this is in contradiction to the epidemiology of 11q23 translocation. Conclusion The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.

  10. 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.

  11. Phenylpropanoids accumulation in eggplant fruit: characterization of biosynthetic genes and regulation by a MYB transcription factor

    Directory of Open Access Journals (Sweden)

    Teresa eDocimo

    2016-01-01

    Full Text Available Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena fruits. Chlorogenic acid (CGA accounts for 70 to 90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena.Higher contents of CGA, Delphinidin 3-rutinoside and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group 6 MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties.In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation.Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9 resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of

  12. Ectopic Overexpression of a Novel R2R3-MYB, NtMYB2 from Chinese Narcissus Represses Anthocyanin Biosynthesis in Tobacco

    Directory of Open Access Journals (Sweden)

    Muhammad Anwar

    2018-03-01

    Full Text Available R2R3 MYB transcription factors play key functions in the regulation of secondary metabolites. In the present study, a R2R3 MYB transcriptional factor NtMYB2 was identified from Chinese narcissus (Narcissus tazetta L. var. Chinensis Roem and functionally characterized. NtMYB2 belongs to subgroup 4 of the R2R3 MYB transcription factor family that are related to repressor MYBs involved in the regulation of anthocyanin and flavonoids. Transient expression confirmed that NtMYB2 strongly reduced the red pigmentation induced by MYB- anthocyanin activators in agro-infiltrated tobacco leaves. Ectopic expression of NtMYB2 in tobacco significantly reduced the pigmentation and altered the floral phenotypes in transgenic tobacco flowers. Gene expression analysis suggested that NtMYB2 repressed the transcript levels of structural genes involved in anthocyanin biosynthesis pathway, especially the UFGT gene. NtMYB2 gene is expressed in all examined narcissus tissues; the levels of transcription in petals and corona is higher than other tissues and the transcription level at the bud stage was highest. These results show that NtMYB2 is involved in the regulation of anthocyanin biosynthesis pathway and may act as a repressor by down regulating the transcripts of key enzyme genes in Chinese narcissus.

  13. Ectopic Overexpression of a Novel R2R3-MYB, NtMYB2 from Chinese Narcissus Represses Anthocyanin Biosynthesis in Tobacco.

    Science.gov (United States)

    Anwar, Muhammad; Wang, Guiqing; Wu, Jiacheng; Waheed, Saquib; Allan, Andrew C; Zeng, Lihui

    2018-03-28

    R2R3 MYB transcription factors play key functions in the regulation of secondary metabolites. In the present study, a R2R3 MYB transcriptional factor NtMYB2 was identified from Chinese narcissus ( Narcissus tazetta L. var. Chinensis Roem) and functionally characterized. NtMYB2 belongs to subgroup 4 of the R2R3 MYB transcription factor family that are related to repressor MYBs involved in the regulation of anthocyanin and flavonoids. Transient expression confirmed that NtMYB2 strongly reduced the red pigmentation induced by MYB- anthocyanin activators in agro-infiltrated tobacco leaves. Ectopic expression of NtMYB2 in tobacco significantly reduced the pigmentation and altered the floral phenotypes in transgenic tobacco flowers. Gene expression analysis suggested that NtMYB2 repressed the transcript levels of structural genes involved in anthocyanin biosynthesis pathway, especially the UFGT gene. NtMYB2 gene is expressed in all examined narcissus tissues; the levels of transcription in petals and corona is higher than other tissues and the transcription level at the bud stage was highest. These results show that NtMYB2 is involved in the regulation of anthocyanin biosynthesis pathway and may act as a repressor by down regulating the transcripts of key enzyme genes in Chinese narcissus.

  14. Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited

    International Nuclear Information System (INIS)

    Gué, Michaël; Sun, Jian-Sheng; Boudier, Thomas

    2006-01-01

    Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static 'contact first' model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic 'breakage first' model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism. Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static 'contact first' model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation. The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static 'contact first' model of translocation. The recently proposed dynamic 'breakage first' model offers an attractive alternative explanation

  15. Over-expression of TaMYB33 encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis.

    Science.gov (United States)

    Qin, Yuxiang; Wang, Mengcheng; Tian, Yanchen; He, Wenxing; Han, Lu; Xia, Guangmin

    2012-06-01

    Salt and drought stresses often adversely affect plant growth and productivity, MYB transcription factors have been shown to participate in the response to these stresses. Here we identified a new R2R3-type MYB transcription factor gene TaMYB33 from wheat (Triticum aestivum). TaMYB33 was induced by NaCl, PEG and ABA treatments, and its promoter sequence contains putative ABRE, MYB and other abiotic stress related cis-elements. Ectopic over-expression of TaMYB33 in Arabidopsis thaliana remarkably enhanced its tolerance to drought and NaCl stresses, but not to LiCl and KCl treatments. The expressions of AtP5CS and AtZAT12 which mirror the activities of proline and ascorbate peroxidase synthesis respectively were induced in TaMYB33 over-expression lines, indicating TaMYB33 promotes the ability for osmotic pressure balance-reconstruction and reactive oxidative species (ROS) scavenging. The up-regulation of AtAAO3 along with down-regulation of AtABF3, AtABI1 in TaMYB33 over-expression lines indicated that ABA synthesis was elevated while its signaling was restricted. These results suggest that TaMYB33 enhances salt and drought tolerance partially through superior ability for osmotic balance reconstruction and ROS detoxification.

  16. Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

    Science.gov (United States)

    Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

    2018-01-15

    The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

  17. The MLL recombinome of acute leukemias in 2017

    NARCIS (Netherlands)

    C. Meyer; T. Burmeister; D. Gröger (D.); G. Tsaur; L. Fechina; A. Renneville; R. Sutton; N. Venn; M. Emerenciano (M.); Pombo-De-Oliveira, M.S. (M. S.); Barbieri Blunck, C. (C.); Almeida Lopes, B. (B.); J. Zuna; J. Trka (Jan); Ballerini, P. (P.); Lapillonne, H. (H.); E. de Braekeleer; G. Cazzaniga (Gianni); Corral Abascal, L. (L.); V.H.J. van der Velden (Vincent); E. Delabesse; Park, T.S. (T. S.); S.H. Oh (S.); M.L.M. Silva (M. L M); T. Lund-Aho (T.); V. Juvonen (V.); A.S. Moore (A.); O. Heidenreich; Vormoor, J. (J.); Zerkalenkova, E. (E.); Olshanskaya, Y. (Y.); Bueno, C. (C.); P. Menéndez (Pablo); A. Teigler-Schlegel; U. zur Stadt; Lentes, J. (J.); G. Göhring (Gudrun); Kustanovich, A. (A.); O. Aleinikova (O.); Schäfer, B.W. (B. W.); S. Kubetzko (S.); H.O. Madsen; Gruhn, B. (B.); Duarte, X. (X.); P. Gameiro; E. Lippert (Eric); Bidet, A. (A.); J.-M. Cayuela (Jean-Michel); E. Clappier; C.N. Alonso (Cristina); C.M. Zwaan (Christian Michel); M.M. van den Heuvel-Eibrink (Marry); S. Izraeli (Shai); L. Trakhtenbrot; P. Archer (P.); J. Hancock; A. Möricke; Alten, J. (J.); M. Schrappe (Martin); M. Stanulla (Martin); S. Strehl; A. Attarbaschi (Andishe); M.N. Dworzak (Michael); Haas, O.A. (O. A.); R. Panzer-Grümayer (Renate); L. Sedek (Lukasz); Szczepa, T. (T.); A. Caye (Aurélie); Suarez, L. (L.); H. Cavé (Helene); R. Marschalek (Rolf)

    2018-01-01

    textabstractChromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner

  18. MLL-ENL cooperates with SCF to transform primary avian multipotent cells

    NARCIS (Netherlands)

    Schulte, Cathleen E.; von Lindern, Marieke; Steinlein, Peter; Beug, Hartmut; Wiedemann, Leanne M.

    2002-01-01

    The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic

  19. MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation

    Science.gov (United States)

    Sebastian, Soji; Sreenivas, Prethish; Sambasivan, Ramkumar; Cheedipudi, Sirisha; Kandalla, Prashanth; Pavlath, Grace K.; Dhawan, Jyotsna

    2009-01-01

    Most cells in adult tissues are nondividing. In skeletal muscle, differentiated myofibers have exited the cell cycle permanently, whereas satellite stem cells withdraw transiently, returning to active proliferation to repair damaged myofibers. We have examined the epigenetic mechanisms operating in conditional quiescence by analyzing the function of a predicted chromatin regulator mixed lineage leukemia 5 (MLL5) in a culture model of reversible arrest. MLL5 is induced in quiescent myoblasts and regulates both the cell cycle and differentiation via a hierarchy of chromatin and transcriptional regulators. Knocking down MLL5 delays entry of quiescent myoblasts into S phase, but hastens S-phase completion. Cyclin A2 (CycA) mRNA is no longer restricted to S phase, but is induced throughout G0/G1, with activation of the cell cycle regulated element (CCRE) in the CycA promoter. Overexpressed MLL5 physically associates with the CCRE and impairs its activity. MLL5 also regulates CycA indirectly: Cux, an activator of CycA promoter and S phase is induced in RNAi cells, and Brm/Brg1, CCRE-binding repressors that promote differentiation are repressed. In knockdown cells, H3K4 methylation at the CCRE is reduced, reflecting quantitative global changes in methylation. MLL5 appears to lack intrinsic histone methyl transferase activity, but regulates expression of histone-modifying enzymes LSD1 and SET7/9, suggesting an indirect mechanism. Finally, expression of muscle regulators Pax7, Myf5, and myogenin is impaired in MLL5 knockdown cells, which are profoundly differentiation defective. Collectively, our results suggest that MLL5 plays an integral role in novel chromatin regulatory mechanisms that suppress inappropriate expression of S-phase-promoting genes and maintain expression of determination genes in quiescent cells. PMID:19264965

  20. The ABA receptor PYL8 promotes lateral root growth by enhancing MYB77-dependent transcription of auxin-responsive genes.

    Science.gov (United States)

    Zhao, Yang; Xing, Lu; Wang, Xingang; Hou, Yueh-Ju; Gao, Jinghui; Wang, Pengcheng; Duan, Cheng-Guo; Zhu, Xiaohong; Zhu, Jian-Kang

    2014-06-03

    The phytohormone abscisic acid (ABA) regulates plant growth, development, and abiotic stress responses. ABA signaling is mediated by a group of receptors known as the PYR1/PYL/RCAR family, which includes the pyrabactin resistance 1-like protein PYL8. Under stress conditions, ABA signaling activates SnRK2 protein kinases to inhibit lateral root growth after emergence from the primary root. However, even in the case of persistent stress, lateral root growth eventually recovers from inhibition. We showed that PYL8 is required for the recovery of lateral root growth, following inhibition by ABA. PYL8 directly interacted with the transcription factors MYB77, MYB44, and MYB73. The interaction of PYL8 and MYB77 increased the binding of MYB77 to its target MBSI motif in the promoters of multiple auxin-responsive genes. Compared to wild-type seedlings, the lateral root growth of pyl8 mutant seedlings and myb77 mutant seedlings was more sensitive to inhibition by ABA. The recovery of lateral root growth was delayed in pyl8 mutant seedlings in the presence of ABA, and the defect was rescued by exposing pyl8 mutant seedlings to the auxin IAA (3-indoleacetic acid). Thus, PYL8 promotes lateral root growth independently of the core ABA-SnRK2 signaling pathway by enhancing the activities of MYB77 and its paralogs, MYB44 and MYB73, to augment auxin signaling. Copyright © 2014, American Association for the Advancement of Science.

  1. CsMYB5a and CsMYB5e from Camellia sinensis differentially regulate anthocyanin and proanthocyanidin biosynthesis.

    Science.gov (United States)

    Jiang, Xiaolan; Huang, Keyi; Zheng, Guangshun; Hou, Hua; Wang, Peiqiang; Jiang, Han; Zhao, Xuecheng; Li, Mingzhuo; Zhang, Shuxiang; Liu, Yajun; Gao, Liping; Zhao, Lei; Xia, Tao

    2018-05-01

    Tea is one of the most widely consumed nonalcoholic beverages worldwide. Polyphenols are nutritional compounds present in the leaves of tea plants. Although numerous genes are functionally characterized to encode enzymes that catalyze the formation of diverse polyphenolic metabolites, transcriptional regulation of those different pathways such as late steps of the proanthcoyanidin (PA) pathway remains unclear. In this study, using different tea transcriptome databases, we screened at least 140 R2R3-MYB transcription factors (TFs) and grouped them according to the basic function domains of the R2R3 MYB TF superfamily. Among 140 R2R3 TFs, CsMYB5a and CsMYB5e were chosen for analysis because they may be involved in PA biosynthesis regulation. CsMYB5a-overexpressing tobacco plants exhibited downregulated anthocyanin accumulation but a high polymeric PA content in the flowers. Overexpression of CsMYB5e in tobacco plants did not change the anthocyanin content but increased the dimethylaminocinnamaldehyde-stained PA content. RNA-seq and qRT-PCR analyses revealed that genes related to PA and anthocyanin biosynthesis pathways were markedly upregulated in both CsMYB5a- and CsMYB5e-overexpressing flowers. Three UGTs and four GSTs were identified as involved in PA and anthocyanin glycosylation and transportation in transgenic plants. These results provide new insights into the regulation of PA and anthocyanin biosynthesis in Camellia sinensis. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    International Nuclear Information System (INIS)

    Do, To Uyen; Ho, Bay; Shih, Shyh-Jen; Vaughan, Andrew

    2012-01-01

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient

  3. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    Energy Technology Data Exchange (ETDEWEB)

    Do, To Uyen [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Ho, Bay; Shih, Shyh-Jen [Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Vaughan, Andrew, E-mail: Andrew.vaughan@ucdmc.ucdavis.edu [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States)

    2012-12-15

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

  4. Overexpression of PtrMYB119, a R2R3-MYB transcription factor from Populus trichocarpa, promotes anthocyanin production in hybrid poplar.

    Science.gov (United States)

    Cho, Jin-Seong; Nguyen, Van Phap; Jeon, Hyung-Woo; Kim, Min-Ha; Eom, Seok Hyun; Lim, You Jin; Kim, Won-Chan; Park, Eung-Jun; Choi, Young-Im; Ko, Jae-Heung

    2016-09-01

    Anthocyanins are a group of colorful and bioactive natural pigments with important physiological and ecological functions in plants. We found an MYB transcription factor (PtrMYB119) from Populus trichocarpa that positively regulates anthocyanin production when expressed under the control of the CaMV 35S promoter in transgenic Arabidopsis Amino acid sequence analysis revealed that PtrMYB119 is highly homologous to Arabidopsis PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), a well-known transcriptional activator of anthocyanin biosynthesis. Independently produced transgenic poplars overexpressing PtrMYB119 or PtrMYB120 (a paralogous gene to PtrMYB119) (i.e., 35S::PtrMYB119 and 35S::PtrMYB120, respectively) showed elevated accumulation of anthocyanins in the whole plants, including leaf, stem and even root tissues. Using a reverse-phase high-performance liquid chromatography, we confirmed that the majority of the accumulated anthocyanin in our transgenic poplar is cyanidin-3-O-glucoside. Gene expression analyses revealed that most of the genes involved in the anthocyanin biosynthetic pathway were highly upregulated in 35S::PtrMYB119 poplars compared with the nontransformed control poplar. Among these genes, expression of PtrCHS1 (Chalcone Synthase1) and PtrANS2 (Anthocyanin Synthase2), which catalyze the initial and last steps of anthocyanin biosynthesis, respectively, was upregulated by up to 350-fold. Subsequent transient activation assays confirmed that PtrMYB119 activated the transcription of both PtrCHS1 and PtrANS2 Interestingly, expression of MYB182, a repressor of both anthocyanin and proanthocyanidin (PA) biosynthesis, was largely suppressed in 35S::PtrMYB119 poplars, while expression of MYB134, an activator of PA biosynthesis, was not changed significantly. More interestingly, high-level accumulation of anthocyanins in 35S::PtrMYB119 poplars did not have an adverse effect on plant growth. Taken together, our results demonstrate that PtrMYB119 and PtrMYB120

  5. 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.

  6. An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean.

    Directory of Open Access Journals (Sweden)

    Shanshan Chu

    2017-05-01

    Full Text Available Isoflavones comprise a group of secondary metabolites produced almost exclusively by plants in the legume family, including soybean [Glycine max (L. Merr.]. They play vital roles in plant defense and have many beneficial effects on human health. Isoflavone content is a complex quantitative trait controlled by multiple genes, and the genetic mechanisms underlying isoflavone biosynthesis remain largely unknown. Via a genome-wide association study (GWAS, we identified 28 single nucleotide polymorphisms (SNPs that are significantly associated with isoflavone concentrations in soybean. One of these 28 SNPs was located in the 5'-untranslated region (5'-UTR of an R2R3-type MYB transcription factor, GmMYB29, and this gene was thus selected as a candidate gene for further analyses. A subcellular localization study confirmed that GmMYB29 was located in the nucleus. Transient reporter gene assays demonstrated that GmMYB29 activated the IFS2 (isoflavone synthase 2 and CHS8 (chalcone synthase 8 gene promoters. Overexpression and RNAi-mediated silencing of GmMYB29 in soybean hairy roots resulted in increased and decreased isoflavone content, respectively. Moreover, a candidate-gene association analysis revealed that 11 natural GmMYB29 polymorphisms were significantly associated with isoflavone contents, and regulation of GmMYB29 expression could partially contribute to the observed phenotypic variation. Taken together, these results provide important genetic insights into the molecular mechanisms underlying isoflavone biosynthesis in soybean.

  7. Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa).

    Science.gov (United States)

    Park, Jong-Sug; Kim, Jung-Bong; Cho, Kang-Jin; Cheon, Choong-Ill; Sung, Mi-Kyung; Choung, Myoung-Gun; Roh, Kyung-Hee

    2008-06-01

    The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches.

  8. Expression and characterization of c-Myb in prenatal odontogenesis

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Buchtová, Marcela; Tucker, A. S.; Bender, T. P.; Janečková, Eva; Lungová, V.; Balková, Simona; Šmarda, J.

    2011-01-01

    Roč. 53, č. 6 (2011), s. 793-803 ISSN 0012-1592 R&D Projects: GA AV ČR KJB500450802; GA ČR GAP304/11/1418; GA ČR(CZ) GP304/08/P289; GA ČR GC524/08/J032 Institutional research plan: CEZ:AV0Z50450515 Keywords : morphogenesis * mouse * Myb Subject RIV: FF - HEENT, Dentistry Impact factor: 2.210, year: 2011

  9. Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4.

    Science.gov (United States)

    Herz, Hans-Martin; Mohan, Man; Garruss, Alexander S; Liang, Kaiwei; Takahashi, Yoh-Hei; Mickey, Kristen; Voets, Olaf; Verrijzer, C Peter; Shilatifard, Ali

    2012-12-01

    Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers.

  10. Genomic Survey and Expression Profiling of the MYB Gene Family in Watermelon

    Directory of Open Access Journals (Sweden)

    Qing XU

    2018-01-01

    Full Text Available Myeloblastosis (MYB proteins constitute one of the largest transcription factor (TF families in plants. They are functionally diverse in regulating plant development, metabolism, and multiple stress responses. However, the function of watermelon MYB proteins remains elusive to date. Here, a genome-wide identification of watermelon MYB TFs was performed by bioinformatics analysis. A total of 162 MYB genes were identified from watermelon (ClaMYB. A comprehensive overview of the ClaMYB genes was undertaken, including the gene structures, chromosomal distribution, gene duplication, conserved protein motif, and phylogenetic relationship. According to the analyses, the watermelon MYB genes were categorized into three groups (R1R2R3-MYB, R2R3-MYB, and MYB-related. Amino acid alignments for all MYB motifs of ClaMYBs demonstrated high conservation. Investigation of their chromosomal localization revealed that these ClaMYB genes distributed across the 11 watermelon chromosomes. Gene duplication analyses showed that tandem duplication events contributed predominantly to the expansion of the MYB gene family in the watermelon genome. Phylogenetic comparison of the ClaMYB proteins with Arabidopsis MYB proteins revealed that watermelon MYB proteins underwent a more diverse evolution after divergence from Arabidopsis. Some watermelon MYBs were found to cluster into the functional clades of Arabidopsis MYB proteins. Expression analysis under different stress conditions identified a group of watermelon MYB proteins implicated in the plant stress responses. The comprehensive investigation of watermelon MYB genes in this study provides a useful reference for future cloning and functional analysis of watermelon MYB proteins. Keywords: watermelon, MYB transcription factor, abiotic stress, phylogenetic analysis

  11. The incidence and distribution characteristics of MLL rearrangements in Chinese acute myeloid leukemia patients by multiplex nested RT-PCR.

    Science.gov (United States)

    Yang, Hua; Cao, Tingting; Gao, Li; Wang, Lili; Zhu, Chengying; Xu, Yuanyuan; Jing, Yu; Zhu, Haiyan; Lv, Na; Yu, Li

    2017-07-20

    Occurrence of MLL (Mixed Lineage Leukemia) gene rearrangements indicates poor prognosis in acute myeloid leukemia (AML) patients. This is the first study to report the positive rate and distribution characteristics of MLL rearrangements in AML patients in north China. We used multiplex nested real time PCR (RT-PCR) to screen for incidence of 11 MLL rearrangements in 433 AML patients. Eleven MLL rearrangements included (MLL-PTD, MLL-AF9, MLL-ELL, MLL-AF10, MLL-AF17, MLL-AF6, MLL-ENL, MLL-AF1Q, MLL-CBP, MLL-AF1P, MLL-AFX1). There were 68 AML patients with MLL rearrangements, and the positive rate was 15.7%. MLL-PTD (4.84%) was detected in 21 patients, MLL-AF9 in 15, (3.46%), MLL-ELL in 10 (2.31%), MLL-AF10 in 8 (1.85%), MLL-AF1Q in 2 (0.46%), 3 cases each of MLL-AF17, MLL-AF6, MLL-ENL (0.69% each), a and single case each of MLL-CBP, MLL-AF1P, and MLL-AFX1 (0.23% each). The highest rate of MLL rearrangements was found in 24 patients with M5 subtype AML, occurring in 24 cases (35.3%). MLL rearrangements occurred in 21 patients with M2 subtype AML (30.9%), and in 10 patients with M4 subtype AML (14.7%). Screening fusion genes by multiplex nested RT-PCR is a convenient, fast, economical, and accurate method for diagnosis and predicting prognosis of AML.

  12. Molecular characterization of BrMYB28 and BrMYB29 paralogous transcription factors involved in the regulation of aliphatic glucosinolate profiles in Brassica rapa ssp. pekinensis.

    Science.gov (United States)

    Baskar, Venkidasamy; Park, Se Won

    2015-07-01

    Glucosinolates (GSL) are one of the major secondary metabolites of the Brassicaceae family. In the present study, we aim at characterizing the multiple paralogs of aliphatic GSL regulators, such as BrMYB28 and BrMYB29 genes in Brassica rapa ssp. pekinensis, by quantitative real-time PCR (qRT-PCR) analysis in different tissues and at various developmental stages. An overlapping gene expression pattern between the BrMYBs as well as their downstream genes (DSGs) was found at different developmental stages. Among the BrMYB28 and BrMYB29 paralogous genes, the BrMYB28.3 and BrMYB29.1 genes were dominantly expressed in most of the developmental stages, compared to the other paralogs of the BrMYB genes. Furthermore, the differential expression pattern of the BrMYBs was observed under various stress treatments. Interestingly, BrMYB28.2 showed the least expression in most developmental stages, while its expression was remarkably high in different stress conditions. More specifically, the BrMYB28.2, BrMYB28.3, and BrMYB29.1 genes were highly responsive to various abiotic and biotic stresses, further indicating their possible role in stress tolerance. Moreover, the in silico cis motif analysis in the upstream regulatory regions of BrMYBs showed the presence of various putative stress-specific motifs, which further indicated their responsiveness to biotic and abiotic stresses. These observations suggest that the dominantly expressed BrMYBs, both in different developmental stages and under various stress treatments (BrMYB28.3 and BrMYB29.1), may be potential candidate genes for altering the GSL level through genetic modification studies in B. rapa ssp. pekinensis. Copyright © 2015. Published by Elsevier SAS.

  13. Frequent alteration of MLL3 frameshift mutations in microsatellite deficient colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Watanabe

    Full Text Available MLL3 is a histone 3-lysine 4 methyltransferase with tumor-suppressor properties that belongs to a family of chromatin regulator genes potentially altered in neoplasia. Mutations in MLL3 were found in a whole genome analysis of colorectal cancer but have not been confirmed by a separate study.We analyzed mutations of coding region and promoter methylation in MLL3 using 126 cases of colorectal cancer. We found two isoforms of MLL3 and DNA sequencing revealed frameshift and other mutations affecting both isoforms of MLL3 in colorectal cancer cells and 19 of 134 (14% primary colorectal samples analyzed. Moreover, frameshift mutations were more common in cases with microsatellite instability (31% both in CRC cell lines and primary tumors. The largest isoform of MLL3 is transcribed from a CpG island-associated promoter that has highly homology with a pseudo-gene on chromosome 22 (psiTPTE22. Using an assay which measured both loci simultaneously we found prominent age related methylation in normal colon (from 21% in individuals less than 25 years old to 56% in individuals older than 70, R = 0.88, p<0.001 and frequent hypermethylation (83% in both CRC cell lines and primary tumors. We next studied the two loci separately and found that age and cancer related methylation was solely a property of the pseudogene CpG island and that the MLL3 loci was unmethylated.We found that frameshift mutations of MLL3 in both CRC cells and primary tumor that were more common in cases with microsatellite instability. Moreover, we have shown CpG island-associated promoter of MLL3 gene has no DNA methylation in CRC cells but also primary tumor and normal colon, and this region has a highly homologous of pseudo gene (psiTPTE22 that was age relate DNA methylation.

  14. Functional Characterization of Tea (Camellia sinensis MYB4a Transcription Factor Using an Integrative Approach

    Directory of Open Access Journals (Sweden)

    Mingzhuo Li

    2017-06-01

    Full Text Available Green tea (Camellia sinensis, Cs abundantly produces a diverse array of phenylpropanoid compounds benefiting human health. To date, the regulation of the phenylpropanoid biosynthesis in tea remains to be investigated. Here, we report a cDNA isolated from leaf tissues, which encodes a R2R3-MYB transcription factor. Amino acid sequence alignment and phylogenetic analysis indicate that it is a member of the MYB4-subgroup and named as CsMYB4a. Transcriptional and metabolic analyses show that the expression profile of CsMYB4a is negatively correlated to the accumulation of six flavan-3-ols and other phenolic acids. GFP fusion analysis shows CsMYB4a’s localization in the nucleus. Promoters of five tea phenylpropanoid pathway genes are isolated and characterized to contain four types of AC-elements, which are targets of MYB4 members. Interaction of CsMYB4a and five promoters shows that CsMYB4a decreases all five promoters’ activity. To further characterize its function, CsMYB4a is overexpressed in tobacco plants. The resulting transgenic plants show dwarf, shrinking and yellowish leaf, and early senescence phenotypes. A further genome-wide transcriptomic analysis reveals that the expression levels of 20 tobacco genes involved in the shikimate and the phenylpropanoid pathways are significantly downregulated in transgenic tobacco plants. UPLC-MS and HPLC based metabolic profiling reveals significant reduction of total lignin content, rutin, chlorogenic acid, and phenylalanine in CsMYB4a transgenic tobacco plants. Promoter sequence analysis of the 20 tobacco genes characterizes four types of AC-elements. Further CsMYB4a-AC element and CsMYB4a-promoter interaction analyses indicate that the negative regulation of CsMYB4a on the shikimate and phenylpropanoid pathways in tobacco is via reducing promoter activity. Taken together, all data indicate that CsMYB4a negatively regulates the phenylpropanoid and shikimate pathways.Highlight: A tea (Camellia

  15. Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL

    Energy Technology Data Exchange (ETDEWEB)

    Khan,J.; Dunn, B.; Tong, L.

    2005-01-01

    Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.

  16. Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes

    Science.gov (United States)

    Matus, José Tomás; Aquea, Felipe; Arce-Johnson, Patricio

    2008-01-01

    Background The MYB superfamily constitutes the most abundant group of transcription factors described in plants. Members control processes such as epidermal cell differentiation, stomatal aperture, flavonoid synthesis, cold and drought tolerance and pathogen resistance. No genome-wide characterization of this family has been conducted in a woody species such as grapevine. In addition, previous analysis of the recently released grape genome sequence suggested expansion events of several gene families involved in wine quality. Results We describe and classify 108 members of the grape R2R3 MYB gene subfamily in terms of their genomic gene structures and similarity to their putative Arabidopsis thaliana orthologues. Seven gene models were derived and analyzed in terms of gene expression and their DNA binding domain structures. Despite low overall sequence homology in the C-terminus of all proteins, even in those with similar functions across Arabidopsis and Vitis, highly conserved motif sequences and exon lengths were found. The grape epidermal cell fate clade is expanded when compared with the Arabidopsis and rice MYB subfamilies. Two anthocyanin MYBA related clusters were identified in chromosomes 2 and 14, one of which includes the previously described grape colour locus. Tannin related loci were also detected with eight candidate homologues in chromosomes 4, 9 and 11. Conclusion This genome wide transcription factor analysis in Vitis suggests that clade-specific grape R2R3 MYB genes are expanded while other MYB genes could be well conserved compared to Arabidopsis. MYB gene abundance, homology and orientation within particular loci also suggests that expanded MYB clades conferring quality attributes of grapes and wines, such as colour and astringency, could possess redundant, overlapping and cooperative functions. PMID:18647406

  17. Functional diversification of grapevine MYB5a and MYB5b in the control of flavonoid biosynthesis in a petunia anthocyanin regulatory mutant.

    Science.gov (United States)

    Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Guzzo, Flavia; Zamboni, Anita; Avesani, Linda; Tornielli, Giovanni Battista

    2014-03-01

    Flavonoids play a key role in grapevine physiology and also contribute substantially to the quality of berries and wines. VvMYB5a and VvMYB5b are R2R3-MYB transcription factors previously proposed to control the spatiotemporal expression of flavonoid structural genes during berry development. We investigated the functions of these two proteins in detail by heterologous expression in a petunia an2 mutant, which has negligible anthocyanin levels in the petals because it lacks the MYB protein PhAN2. We also expressed VvMYBA1, the grapevine ortholog of petunia PhAN2, in the same genetic background. The anthocyanin profiles induced by expressing these transgenes in the petals revealed that VvMYBA1 is the functional ortholog of PhAN2 and that, unlike VvMYB5a, VvMYB5b can partially complement the an2 mutation. Transcriptomic analysis of petals by microarray hybridization and quantitative PCR confirmed that VvMYB5b up-regulates a subset of anthocyanin structural genes, whereas VvMYB5a has a more limited impact on the expression of genes related to anthocyanin biosynthesis. Furthermore, we identified additional specific and common targets of these two regulators, related to vacuolar acidification and membrane remodeling. Taken together, these data provide insight into the role of VvMYB5a and VvMYB5b in flavonoid biosynthesis and provide evidence for additional regulatory roles in distinct pathways.

  18. MYB52 Negatively Regulates Pectin Demethylesterification in Seed Coat Mucilage.

    Science.gov (United States)

    Shi, Dachuan; Ren, Angyan; Tang, Xianfeng; Qi, Guang; Xu, Zongchang; Chai, Guohua; Hu, Ruibo; Zhou, Gongke; Kong, Yingzhen

    2018-04-01

    Pectin, which is a major component of the plant primary cell walls, is synthesized and methyl-esterified in the Golgi apparatus and then demethylesterified by pectin methylesterases (PMEs) located in the cell wall. The degree of methylesterification affects the functional properties of pectin, and thereby influences plant growth, development and defense. However, little is known about the mechanisms that regulate pectin demethylesterification. Here, we show that in Arabidopsis ( Arabidopsis thaliana ) seed coat mucilage, the absence of the MYB52 transcription factor is correlated with an increase in PME activity and a decrease in the degree of pectin methylesterification. Decreased methylesterification in the myb52 mutant is also correlated with an increase in the calcium content of the seed mucilage. Chromatin immunoprecipitation analysis and molecular genetic studies suggest that MYB52 transcriptionally activates PECTIN METHYLESTERASE INHIBITOR6 ( PMEI6 ), PMEI14 , and SUBTILISIN-LIKE SER PROTEASE1.7 ( SBT1.7 ) by binding to their promoters. PMEI6 and SBT1.7 have previously been shown to be involved in seed coat mucilage demethylesterification. Our characterization of two PMEI14 mutants suggests that PMEI14 has a role in seed coat mucilage demethylesterification, although its activity may be confined to the seed coat in contrast to PMEI6, which functions in the whole seed. Our demonstration that MYB52 negatively regulates pectin demethylesterification in seed coat mucilage, and the identification of components of the molecular network involved, provides new insight into the regulatory mechanism controlling pectin demethylesterification and increases our understanding of the transcriptional regulation network involved in seed coat mucilage formation. © 2018 American Society of Plant Biologists. All Rights Reserved.

  19. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.

    Science.gov (United States)

    Aharoni, A; De Vos, C H; Wein, M; Sun, Z; Greco, R; Kroon, A; Mol, J N; O'Connell, A P

    2001-11-01

    Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co-ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3-rutinoside (an anthocyanin) and of quercetin-glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were adversely affected by FaMYB1 overexpression. Two-hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C-terminus of FaMYB1 contains the motif pdLNL(D)/(E)Lxi(G)/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.

  20. c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix-dependent breast cancer cell invasion and metastasis

    Czech Academy of Sciences Publication Activity Database

    Knopfová, L.; Beneš, P.; Pekarčíková, L.; Hermanová, M.; Masařík, M.; Pernicová, Zuzana; Souček, Karel; Šmarda, J.

    2012-01-01

    Roč. 11, MAR 23 (2012), ID 15 ISSN 1476-4598 R&D Projects: GA MZd NS9600 Grant - others:GA AV ČR(CZ) IAA501630901 Institutional support: RVO:68081707 Keywords : c-Myb * Metastasis * Breast cancer Subject RIV: BO - Biophysics Impact factor: 5.134, year: 2012

  1. Estudio molecular del gen MLL en 30 pacientes con leucemias agudas Molecular study of MLL gen in 30 patients with acute leukemias

    Directory of Open Access Journals (Sweden)

    Raquel Levón Herrera

    2000-04-01

    Full Text Available Los reordenamientos del gen MLL en la banda cromosómica 11q23 son frecuentes en leucemias agudas (LA en niños y en las LA secundarias desarrolladas después de la terapia con inhibidores de la enzima topoisomerasa II. En menor medida también se aprecia en adultos con LA. La presencia de estos reordenamientos se considera un indicador de mal pronóstico asociado con resultados clínicos desfavorables, por ello es muy importante realizar su determinación en las LA. En este trabajo mostramos los resultados preliminares de la introducción del estudio del gen MLL en nuestro país mediante la técnica de Southern. Analizamos ADN de 30 pacientes con LA, incluidos niños y adultos, que en el momento del estudio se encontraban al debut o en recaída. El estudio molecular se realizó con la sonda FA4, que es un inserto genómico del gen MLL. Sólo uno de los 30 pacientes mostró bandas de reordenamiento con 2 enzimas de restricción diferentes, el resto mostró el gen MLL en configuración germinal. Es interesante destacar que el paciente con el reordenamiento era un niño con leucemia mieloblástica aguda subtipo M5b, lo cual concuerda con la literatura, donde se describe que estos reordenamientos están estrechamente correlacionados con los subtipos mielomonocítico (M4 y monocítico (M5 de leucemia mieloide aguda (LMARearrangements of MLL gen in llq23 chromosomal band are frequents in childhood type of acute leukemia (AL and in secondary AL, developed after therapy with II topoisomerase enzyme. To a lesser extent also is seen in adults with AL. Presence of theses rearrangements is considered to be a worse prognosis indicator, associated with unfavourable clinical results, that is why it is very important to carry our its assessment in AL. In this paper authors present preliminary results from introduction of study on MLL gen in our country through Southern technique. DNA from 30 patients was analized, including children and adults, that at the

  2. The MLL recombinome of acute leukemias in 2017

    DEFF Research Database (Denmark)

    Meyer, C; Burmeister, T; Gröger, D

    2018-01-01

    Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs)...... of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.Leukemia advance online publication, 8 August 2017; doi:10.1038/leu.2017.213....

  3. MLL duplication in a pediatric patient with B-cell lymphoblastic lymphoma.

    Science.gov (United States)

    Mater, David Van; Goodman, Barbara K; Wang, Endi; Gaca, Ana M; Wechsler, Daniel S

    2012-04-01

    Lymphoblastic lymphoma is the second most common type of non-Hodgkin lymphoma seen in children. Approximately, 90% of lymphoblastic lymphomas arise from T cells, with the remaining 10% being B-cell-lineage derived. Although T-cell lymphoblastic lymphoma most frequently occurs in the anterior mediastinum (thymus), B-cell lymphoblastic lymphoma (B-LBL) predominates in extranodal sites such as skin and bone. Here, we describe a pediatric B-LBL patient who presented with extensive abdominal involvement and whose lymphoma cells displayed segmental duplication of the mixed lineage leukemia (MLL) gene. MLL duplication/amplification has been described primarily in acute myeloid leukemia and myelodysplastic syndrome with no published reports of discrete MLL duplication/amplification events in B-LBL. The MLL gene duplication noted in this case may represent a novel mechanism for tumorigenesis in B-LBL.

  4. Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha

    International Nuclear Information System (INIS)

    Valtieri, M.; Venturelli, D.; Care, A.; Fossati, C.; Pelosi, E.; Labbaye, C.; Mattia, G.; Gewirtz, A.M.; Calabretta, B.; Peschle, C.

    1991-01-01

    These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited. Using purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase

  5. WRKY2/34–VQ20 Modules in Arabidopsis thaliana Negatively Regulate Expression of a Trio of Related MYB Transcription Factors During Pollen Development

    Directory of Open Access Journals (Sweden)

    Rihua Lei

    2018-03-01

    Full Text Available Male gametogenesis in plants is tightly controlled and involves the complex and precise regulation of transcriptional reprogramming. Interactions between WRKY proteins and VQ motif-containing proteins are required to control these complicated transcriptional networks. However, our understanding of the mechanisms by which these complexes affect downstream gene expression is quite limited. In this study, we found that WRKY2 and WKRY34 repress MYB97, MYB101, and MYB120 expression during male gametogenesis. MYB expression was up-regulated in the wrky2-1 wrky34-1 vq20-1 triple mutant during male gametogenesis. The expression levels of six potential targets of the three MYBs increased the most in the wrky2-1 wrky34-1 vq20-1 triple mutant, followed by the wrky2-1 wrky34-1 double mutant, compared with in wild-type. Yeast one-hybrid and dual luciferase reporter assays indicated that WRKY2 and WRKY34 recognized the MYB97 promoter by binding to its W-boxes. MYB97 overexpression caused defects in pollen germination and pollen tube length, which impacted male fertility. Thus, WRKY2/34–VQ20 complexes appear to negatively regulate the expression of certain MYBs during plant male gametogenesis.

  6. An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds.

    Science.gov (United States)

    Sutoh, Keita; Washio, Kenji; Imai, Ryozo; Wada, Masamitsu; Nakai, Tomonori; Yamauchi, Daisuke

    2015-01-01

    The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213 N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1.

  7. VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in Chili pepper leaves

    Directory of Open Access Journals (Sweden)

    zhen ezhang

    2015-07-01

    Full Text Available The purple coloration of pepper leaves arises from the accumulation of anthocyanin. Three regulatory and 12 structural genes have been characterized for their involvement in the anthocyanin biosynthesis. Examination of the abundance of these genes in leaves showed that the majority of them differed between anthocyanin pigmented line Z1 and non-pigmented line A3. Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation. Moreover, the expression of multiple genes was altered in the silenced leaves. The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern. Most structural genes including CHS, CHI, F3H, F3’5’H, DFR, ANS, UFGT, ANP and GST were repressed in CaMYB-silenced foliage with the exception of PAL, C4H and 4CL. These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes. Besides CaMYB silenced leaves rendered more sporulation of Phytophthora capsici Leonian indicating that CaMYB might be involved in the defense response to pathogens.

  8. Transcrition factor c-Myb is involved in the regulation of the epithelial-mesenchymal transition in the avian neural crest

    Czech Academy of Sciences Publication Activity Database

    Karafiát, Vít; Dvořáková, Marta; Krejčí, E.; Králová, Jarmila; Pajer, Petr; Šnajdr, P.; Mandíková, Sonja; Bartůněk, Petr; Grim, M.; Dvořák, Michal

    2005-01-01

    Roč. 62, č. 21 (2005), s. 2516-2525 ISSN 1420-682X R&D Projects: GA ČR GA304/03/0463; GA AV ČR IAA5052309 Institutional research plan: CEZ:AV0Z50520514 Keywords : c-myb gene * epithelial-mesenchymal transition * neural crest Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.582, year: 2005

  9. Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes

    Directory of Open Access Journals (Sweden)

    Arce-Johnson Patricio

    2008-07-01

    Full Text Available Abstract Background The MYB superfamily constitutes the most abundant group of transcription factors described in plants. Members control processes such as epidermal cell differentiation, stomatal aperture, flavonoid synthesis, cold and drought tolerance and pathogen resistance. No genome-wide characterization of this family has been conducted in a woody species such as grapevine. In addition, previous analysis of the recently released grape genome sequence suggested expansion events of several gene families involved in wine quality. Results We describe and classify 108 members of the grape R2R3 MYB gene subfamily in terms of their genomic gene structures and similarity to their putative Arabidopsis thaliana orthologues. Seven gene models were derived and analyzed in terms of gene expression and their DNA binding domain structures. Despite low overall sequence homology in the C-terminus of all proteins, even in those with similar functions across Arabidopsis and Vitis, highly conserved motif sequences and exon lengths were found. The grape epidermal cell fate clade is expanded when compared with the Arabidopsis and rice MYB subfamilies. Two anthocyanin MYBA related clusters were identified in chromosomes 2 and 14, one of which includes the previously described grape colour locus. Tannin related loci were also detected with eight candidate homologues in chromosomes 4, 9 and 11. Conclusion This genome wide transcription factor analysis in Vitis suggests that clade-specific grape R2R3 MYB genes are expanded while other MYB genes could be well conserved compared to Arabidopsis. MYB gene abundance, homology and orientation within particular loci also suggests that expanded MYB clades conferring quality attributes of grapes and wines, such as colour and astringency, could possess redundant, overlapping and cooperative functions.

  10. Striking a balance: regulation of transposable elements by Zfp281 and Mll2 in mouse embryonic stem cells.

    Science.gov (United States)

    Dai, Qian; Shen, Yang; Wang, Yan; Wang, Xin; Francisco, Joel Celio; Luo, Zhuojuan; Lin, Chengqi

    2017-12-01

    Transposable elements (TEs) compose about 40% of the murine genome. Retrotransposition of active TEs such as LINE-1 (L1) tremendously impacts genetic diversification and genome stability. Therefore, transcription and transposition activities of retrotransposons are tightly controlled. Here, we show that the Krüppel-like zinc finger protein Zfp281 directly binds and suppresses a subset of retrotransposons, including the active young L1 repeat elements, in mouse embryonic stem (ES) cells. In addition, we find that Zfp281-regulated L1s are highly enriched for 5-hydroxymethylcytosine (5hmC) and H3K4me3. The COMPASS-like H3K4 methyltransferase Mll2 is the major H3K4me3 methylase at the Zfp281-regulated L1s and required for their proper expression. Our studies also reveal that Zfp281 functions partially through recruiting the L1 regulators DNA hydroxymethylase Tet1 and Sin3A, and restricting Mll2 at these active L1s, leading to their balanced expression. In summary, our data indicate an instrumental role of Zfp281 in suppressing the young active L1s in mouse ES cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries.

    Science.gov (United States)

    Deluc, Laurent; Bogs, Jochen; Walker, Amanda R; Ferrier, Thilia; Decendit, Alain; Merillon, Jean-Michel; Robinson, Simon P; Barrieu, François

    2008-08-01

    Among the dramatic changes occurring during grape berry (Vitis vinifera) development, those affecting the flavonoid pathway have provoked a number of investigations in the last 10 years. In addition to producing several compounds involved in the protection of the berry and the dissemination of the seeds, final products of this pathway also play a critical role in berry and wine quality. In this article, we describe the cloning and functional characterization of VvMYB5b, a cDNA isolated from a grape berry (V. vinifera 'Cabernet Sauvignon') library. VvMYB5b encodes a protein belonging to the R2R3-MYB family of transcription factors and displays significant similarity with VvMYB5a, another MYB factor recently shown to regulate flavonoid synthesis in grapevine. The ability of VvMYB5a and VvMYB5b to activate the grapevine promoters of several structural genes of the flavonoid pathway was confirmed by transient expression of the corresponding cDNAs in grape cells. Overexpression of VvMYB5b in tobacco (Nicotiana tabacum) leads to an up-regulation of genes encoding enzymes of the flavonoid pathway and results in the accumulation of anthocyanin- and proanthocyanidin-derived compounds. The ability of VvMYB5b to regulate particularly the anthocyanin and the proanthocyanidin pathways is discussed in relation to other recently characterized MYB transcription factors in grapevine. Taken together, data presented in this article give insight into the transcriptional mechanisms associated with the regulation of the flavonoid pathway throughout grape berry development.

  12. MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study.

    Science.gov (United States)

    Makrythanasis, P; van Bon, B W; Steehouwer, M; Rodríguez-Santiago, B; Simpson, M; Dias, P; Anderlid, B M; Arts, P; Bhat, M; Augello, B; Biamino, E; Bongers, E M H F; Del Campo, M; Cordeiro, I; Cueto-González, A M; Cuscó, I; Deshpande, C; Frysira, E; Izatt, L; Flores, R; Galán, E; Gener, B; Gilissen, C; Granneman, S M; Hoyer, J; Yntema, H G; Kets, C M; Koolen, D A; Marcelis, C l; Medeira, A; Micale, L; Mohammed, S; de Munnik, S A; Nordgren, A; Psoni, S; Reardon, W; Revencu, N; Roscioli, T; Ruiterkamp-Versteeg, M; Santos, H G; Schoumans, J; Schuurs-Hoeijmakers, J H M; Silengo, M C; Toledo, L; Vendrell, T; van der Burgt, I; van Lier, B; Zweier, C; Reymond, A; Trembath, R C; Perez-Jurado, L; Dupont, J; de Vries, B B A; Brunner, H G; Veltman, J A; Merla, G; Antonarakis, S E; Hoischen, A

    2013-12-01

    Recently, pathogenic variants in the MLL2 gene were identified as the most common cause of Kabuki (Niikawa-Kuroki) syndrome (MIM#147920). To further elucidate the genotype-phenotype correlation, we studied a large cohort of 86 clinically defined patients with Kabuki syndrome (KS) for mutations in MLL2. All patients were assessed using a standardized phenotype list and all were scored using a newly developed clinical score list for KS (MLL2-Kabuki score 0-10). Sequencing of the full coding region and intron-exon boundaries of MLL2 identified a total of 45 likely pathogenic mutations (52%): 31 nonsense, 10 missense and four splice-site mutations, 34 of which were novel. In five additional patients, novel, i.e. non-dbSNP132 variants of clinically unknown relevance, were identified. Patients with likely pathogenic nonsense or missense MLL2 mutations were usually more severely affected (median 'MLL2-Kabuki score' of 6) as compared to the patients without MLL2 mutations (median 'MLL2-Kabuki score' of 5), a significant difference (p < 0.0014). Several typical facial features such as large dysplastic ears, arched eyebrows with sparse lateral third, blue sclerae, a flat nasal tip with a broad nasal root, and a thin upper and a full lower lip were observed more often in mutation positive patients. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. A R2R3-MYB Gene LfMYB113 is Responsible for Autumn Leaf Coloration in Formosan sweet gum (Liquidambar formosana Hance).

    Science.gov (United States)

    Wen, Chi-Hsiang; Chu, Fang-Hua

    2017-03-01

    The regulation of autumn leaf coloration in deciduous trees has long been an enigma. Due to the fact that different coloration phenotypes may be considered when planting, more understanding of the regulation mechanism is needed. In this study, a R2R3-MYB transcription factor gene LfMYB113 was identified from a subtropical deciduous tree species Formosan sweet gum (Liquidambar formosana Hance). The expression patterns of LfMYB113 in four selected phenotypes were different and were positively correlated with leaf anthocyanin content. In a 35S::LfMYB113 transgenic Nicotiana tabacum plant, both the early and late genes in the anthocyanin biosynthetic pathway were shown to be up-regulated. It was also shown that LfMYB113 can activate the promoter sequence of LfDFR1 and LfDFR2. Transient overexpression of LfMYB113 in Nicotiana benthamiana showed strong anthocyanin accumulation and pre-senescence; the latter was confirmed by up-regulation of senescence-associated genes. In addition, the activation of proLfSGR::YFP by LfMYB113 in transient experiments indicated that LfMYB113 may have a role in regulation of Chl degradation. To our knowledge, this is the first time a R2R3-MYB transcription factor has been functionally identified as one of the key regulators of autumn leaf coloration and autumn leaf senescence. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. The Phenylpropanoid Pathway Is Controlled at Different Branches by a Set of R2R3-MYB C2 Repressors in Grapevine1

    Science.gov (United States)

    Cavallini, Erika; Matus, José Tomás; Finezzo, Laura; Zenoni, Sara; Loyola, Rodrigo; Guzzo, Flavia; Schlechter, Rudolf; Ageorges, Agnès; Arce-Johnson, Patricio

    2015-01-01

    Because of the vast range of functions that phenylpropanoids possess, their synthesis requires precise spatiotemporal coordination throughout plant development and in response to the environment. The accumulation of these secondary metabolites is transcriptionally controlled by positive and negative regulators from the MYB and basic helix-loop-helix protein families. We characterized four grapevine (Vitis vinifera) R2R3-MYB proteins from the C2 repressor motif clade, all of which harbor the ethylene response factor-associated amphiphilic repression domain but differ in the presence of an additional TLLLFR repression motif found in the strong flavonoid repressor Arabidopsis (Arabidopsis thaliana) AtMYBL2. Constitutive expression of VvMYB4a and VvMYB4b in petunia (Petunia hybrida) repressed general phenylpropanoid biosynthetic genes and selectively reduced the amount of small-weight phenolic compounds. Conversely, transgenic petunia lines expressing VvMYBC2-L1 and VvMYBC2-L3 showed a severe reduction in petal anthocyanins and seed proanthocyanidins together with a higher pH of crude petal extracts. The distinct function of these regulators was further confirmed by transient expression in tobacco (Nicotiana benthamiana) leaves and grapevine plantlets. Finally, VvMYBC2-L3 was ectopically expressed in grapevine hairy roots, showing a reduction in proanthocyanidin content together with the down-regulation of structural and regulatory genes of the flavonoid pathway as revealed by a transcriptomic analysis. The physiological role of these repressors was inferred by combining the results of the functional analyses and their expression patterns in grapevine during development and in response to ultraviolet B radiation. Our results indicate that VvMYB4a and VvMYB4b may play a key role in negatively regulating the synthesis of small-weight phenolic compounds, whereas VvMYBC2-L1 and VvMYBC2-L3 may additionally fine tune flavonoid levels, balancing the inductive effects of

  15. Conifer R2R3-MYB transcription factors: sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca

    Directory of Open Access Journals (Sweden)

    Grima-Pettenati Jacqueline

    2007-03-01

    Full Text Available Abstract Background Several members of the R2R3-MYB family of transcription factors act as regulators of lignin and phenylpropanoid metabolism during wood formation in angiosperm and gymnosperm plants. The angiosperm Arabidopsis has over one hundred R2R3-MYBs genes; however, only a few members of this family have been discovered in gymnosperms. Results We isolated and characterised full-length cDNAs encoding R2R3-MYB genes from the gymnosperms white spruce, Picea glauca (13 sequences, and loblolly pine, Pinus taeda L. (five sequences. Sequence similarities and phylogenetic analyses placed the spruce and pine sequences in diverse subgroups of the large R2R3-MYB family, although several of the sequences clustered closely together. We searched the highly variable C-terminal region of diverse plant MYBs for conserved amino acid sequences and identified 20 motifs in the spruce MYBs, nine of which have not previously been reported and three of which are specific to conifers. The number and length of the introns in spruce MYB genes varied significantly, but their positions were well conserved relative to angiosperm MYB genes. Quantitative RTPCR of MYB genes transcript abundance in root and stem tissues revealed diverse expression patterns; three MYB genes were preferentially expressed in secondary xylem, whereas others were preferentially expressed in phloem or were ubiquitous. The MYB genes expressed in xylem, and three others, were up-regulated in the compression wood of leaning trees within 76 hours of induction. Conclusion Our survey of 18 conifer R2R3-MYB genes clearly showed a gene family structure similar to that of Arabidopsis. Three of the sequences are likely to play a role in lignin metabolism and/or wood formation in gymnosperm trees, including a close homolog of the loblolly pine PtMYB4, shown to regulate lignin biosynthesis in transgenic tobacco.

  16. HOXA9 is required for survival in human MLL-rearranged acute leukemias

    NARCIS (Netherlands)

    J. Faber (Joerg); A.V. Krivtsov (Andrei); M.C. Stubbs (Matthew); R. Wright (Renee); T.N. Davis (Tina); M.M. van den Heuvel-Eibrink (Marry); C.M. Zwaan (Christian Michel); A.L. Kung (Andrew); S.A. Armstrong (Scott)

    2009-01-01

    textabstractLeukemias that harbor translocations involving the mixed lineage leukemia gene (MLL) possess unique biologic characteristics and often have an unfavorable prognosis. Gene expression analyses demonstrate a distinct profile for MLL-rearranged leukemias with consistent high-level expression

  17. MYT3, a Myb-like transcription factor, affects fungal development and pathogenicity of Fusarium graminearum.

    Directory of Open Access Journals (Sweden)

    Yongsoo Kim

    Full Text Available We previously characterized members of the Myb protein family, MYT1 and MYT2, in Fusarium graminearum. MYT1 and MYT2 are involved in female fertility and perithecium size, respectively. To expand knowledge of Myb proteins in F. graminearum, in this study, we characterized the functions of the MYT3 gene, which encodes a putative Myb-like transcription factor containing two Myb DNA-binding domains and is conserved in the subphylum Pezizomycotina of Ascomycota. MYT3 proteins were localized in nuclei during most developmental stages, suggesting the role of MYT3 as a transcriptional regulator. Deletion of MYT3 resulted in impairment of conidiation, germination, and vegetative growth compared to the wild type, whereas complementation of MYT3 restored the wild-type phenotype. Additionally, the Δmyt3 strain grew poorly on nitrogen-limited media; however, the mutant grew robustly on minimal media supplemented with ammonium. Moreover, expression level of nitrate reductase gene in the Δmyt3 strain was decreased in comparison to the wild type and complemented strain. On flowering wheat heads, the Δmyt3 strain exhibited reduced pathogenicity, which corresponded with significant reductions in trichothecene production and transcript levels of trichothecene biosynthetic genes. When the mutant was selfed, mated as a female, or mated as a male for sexual development, perithecia were not observed on the cultures, indicating that the Δmyt3 strain lost both male and female fertility. Taken together, these results demonstrate that MYT3 is required for pathogenesis and sexual development in F. graminearum, and will provide a robust foundation to establish the regulatory networks for all Myb-like proteins in F. graminearum.

  18. Ectopic Expression of the Coleus R2R3 MYB-Type Proanthocyanidin Regulator Gene SsMYB3 Alters the Flower Color in Transgenic Tobacco.

    Directory of Open Access Journals (Sweden)

    Qinlong Zhu

    Full Text Available Proanthocyanidins (PAs play an important role in plant disease defense and have beneficial effects on human health. We isolated and characterized a novel R2R3 MYB-type PA-regulator SsMYB3 from a well-known ornamental plant, coleus (Solenostemon scutellarioides, to study the molecular regulation of PAs and to engineer PAs biosynthesis. The expression level of SsMYB3 was correlated with condensed tannins contents in various coleus tissues and was induced by wounding and light. A complementation test in the Arabidopsis tt2 mutant showed that SsMYB3 could restore the PA-deficient seed coat phenotype and activated expression of the PA-specific gene ANR and two related genes, DFR and ANS. In yeast two-hybrid assays, SsMYB3 interacted with the Arabidopsis AtTT8 and AtTTG1 to reform the ternary transcriptional complex, and also interacted with two tobacco bHLH proteins (NtAn1a and NtJAF13-1 and a WD40 protein, NtAn11-1. Ectopic overexpression of SsMYB3 in transgenic tobacco led to almost-white flowers by greatly reducing anthocyanin levels and enhancing accumulation of condensed tannins. This overexpression of SsMYB3 upregulated the key PA genes (NtLAR and NtANR and late anthocyanin structural genes (NtDFR and NtANS, but downregulated the expression of the final anthocyanin gene NtUFGT. The formative SsMYB3-complex represses anthocyanin accumulation by directly suppressing the expression of the final anthocyanin structural gene NtUFGT, through competitive inhibition or destabilization of the endogenous NtAn2-complex formation. These results suggested that SsMYB3 may form a transcription activation complex to regulate PA biosynthesis in the Arabidopsis tt2 mutant and transgenic tobacco. Our findings suggest that SsMYB3 is involved in the regulation of PA biosynthesis in coleus and has the potential as a molecular tool for manipulating biosynthesis of PAs in fruits and other crops using metabolic engineering.

  19. Coordinated transcriptional regulation of two key genes in the lignin branch pathway--CAD and CCR--is mediated through MYB- binding sites.

    Science.gov (United States)

    Rahantamalala, Anjanirina; Rech, Philippe; Martinez, Yves; Chaubet-Gigot, Nicole; Grima-Pettenati, Jacqueline; Pacquit, Valérie

    2010-06-28

    Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the final steps in the biosynthesis of monolignols, the monomeric units of the phenolic lignin polymers which confer rigidity, imperviousness and resistance to biodegradation to cell walls. We have previously shown that the Eucalyptus gunnii CCR and CAD2 promoters direct similar expression patterns in vascular tissues suggesting that monolignol production is controlled, at least in part, by the coordinated transcriptional regulation of these two genes. Although consensus motifs for MYB transcription factors occur in most gene promoters of the whole phenylpropanoid pathway, functional evidence for their contribution to promoter activity has only been demonstrated for a few of them. Here, in the lignin-specific branch, we studied the functional role of MYB elements as well as other cis-elements identified in the regulatory regions of EgCAD2 and EgCCR promoters, in the transcriptional activity of these gene promoters. By using promoter deletion analysis and in vivo footprinting, we identified an 80 bp regulatory region in the Eucalyptus gunnii EgCAD2 promoter that contains two MYB elements, each arranged in a distinct module with newly identified cis-elements. A directed mutagenesis approach was used to introduce block mutations in all putative cis-elements of the EgCAD2 promoter and in those of the 50 bp regulatory region previously delineated in the EgCCR promoter. We showed that the conserved MYB elements in EgCAD2 and EgCCR promoters are crucial both for the formation of DNA-protein complexes in EMSA experiments and for the transcriptional activation of EgCAD2 and EgCCR promoters in vascular tissues in planta. In addition, a new regulatory cis-element that modulates the balance between two DNA-protein complexes in vitro was found to be important for EgCAD2 expression in the cambial zone. Our assignment of functional roles to the identified cis-elements clearly demonstrates the

  20. Sensitivity of MLL-rearranged AML cells to all-trans retinoic acid is associated with the level of H3K4me2 in the RARα promoter region

    International Nuclear Information System (INIS)

    Sakamoto, K; Imamura, T; Yano, M; Yoshida, H; Fujiki, A; Hirashima, Y; Hosoi, H

    2014-01-01

    All-trans retinoic acid (ATRA) is well established as differentiation therapy for acute promyelocytic leukemia (APL) in which the PML–RARα (promyelocytic leukemia-retinoic acid receptor α) fusion protein causes blockade of the retinoic acid (RA) pathway; however, in types of acute myeloid leukemia (AML) other than APL, the mechanism of RA pathway inactivation is not fully understood. This study revealed the potential mechanism of high ATRA sensitivity of mixed-lineage leukemia (MLL)-AF9-positive AML compared with MLL-AF4/5q31-positive AML. Treatment with ATRA induced significant myeloid differentiation accompanied by upregulation of RARα, C/EBPα, C/EBPε and PU.1 in MLL-AF9-positive but not in MLL-AF4/5q31-positive cells. Combining ATRA with cytarabine had a synergistic antileukemic effect in MLL-AF9-positive cells in vitro. The level of dimethyl histone H3 lysine 4 (H3K4me2) in the RARα gene-promoter region, PU.1 upstream regulatory region (URE) and RUNX1+24/+25 intronic enhancer was higher in MLL-AF9-positive cells than in MLL-AF4-positive cells, and inhibiting lysine-specific demethylase 1, which acts as a histone demethylase inhibitor, reactivated ATRA sensitivity in MLL-AF4-positive cells. These findings suggest that the level of H3K4me2 in the RARα gene-promoter region, PU.1 URE and RUNX1 intronic enhancer is determined by the MLL-fusion partner. Our findings provide insight into the mechanisms of ATRA sensitivity in AML and novel treatment strategies for ATRA-resistant AML

  1. Nucleoporin Nup98 associates with Trx/MLL and NSL histone-modifying complexes and regulates Hox gene expression.

    Science.gov (United States)

    Pascual-Garcia, Pau; Jeong, Jieun; Capelson, Maya

    2014-10-23

    The nuclear pore complex is a transport channel embedded in the nuclear envelope and made up of 30 different components termed nucleoporins (Nups). In addition to their classical role in transport, a subset of Nups has a conserved role in the regulation of transcription via direct binding to chromatin. The molecular details of this function remain obscure, and it is unknown how metazoan Nups are recruited to their chromatin locations or what transcription steps they regulate. Here, we demonstrate genome-wide and physical association between Nup98 and histone-modifying complexes MBD-R2/NSL [corrected] and Trx/MLL. Importantly, we identify a requirement for MBD-R2 in recruitment of Nup98 to many of its genomic target sites. Consistent with its interaction with the Trx/MLL complex, Nup98 is shown to be necessary for Hox gene expression in developing fly tissues. These findings introduce roles of Nup98 in epigenetic regulation that may underlie the basis of oncogenicity of Nup98 fusions in leukemia.

  2. Genome-wide analysis of the MYB gene family in physic nut (Jatropha curcas L.).

    Science.gov (United States)

    Zhou, Changpin; Chen, Yanbo; Wu, Zhenying; Lu, Wenjia; Han, Jinli; Wu, Pingzhi; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2015-11-01

    The MYB proteins comprise one of the largest transcription factor families in plants, and play key roles in regulatory networks controlling development, metabolism, and stress responses. A total of 125 MYB genes (JcMYB) have been identified in the physic nut (Jatropha curcas L.) genome, including 120 2R-type MYB, 4 3R-MYB, and 1 4R-MYB genes. Based on exon-intron arrangement of MYBs from both lower (Physcomitrella patens) and higher (physic nut, Arabidopsis, and rice) plants, we can classify plant MYB genes into ten groups (MI-X), except for MIX genes which are nonexistent in higher plants. We also observed that MVIII genes may be one of the most ancient MYB types which consist of both R2R3- and 3R-MYB genes. Most MYB genes (76.8% in physic nut) belong to the MI group which can be divided into 34 subgroups. The JcMYB genes were nonrandomly distributed on its 11 linkage groups (LGs). The expansion of MYB genes across several subgroups was observed and resulted from genome triplication of ancient dicotyledons and from both ancient and recent tandem duplication events in the physic nut genome. The expression patterns of several MYB duplicates in the physic nut showed differences in four tissues (root, stem, leaf, and seed), and 34 MYB genes responded to at least one abiotic stressor (drought, salinity, phosphate starvation, and nitrogen starvation) in leaves and/or roots based on the data analysis of digital gene expression tags. Overexpression of the JcMYB001 gene in Arabidopsis increased its sensitivity to drought and salinity stresses. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Molecular Characterization of MYB28 Involved in Aliphatic Glucosinolate Biosynthesis in Chinese Kale (Brassica oleracea var. alboglabra Bailey

    Directory of Open Access Journals (Sweden)

    Ling Yin

    2017-06-01

    Full Text Available Glucosinolates are Brassicaceae-specific secondary metabolites that act as crop protectants, flavor precursors, and cancer-prevention agents, which shows strong evidences of anticarcinogentic, antioxidant, and antimicrobial activities. MYB28, the R2R3-MYB28 transcription factor, directly activates genes involved in aliphatic glucosinolate biosynthesis. In this study, the MYB28 homology (BoaMYB28 was identified in Chinese kale (Brassica oleracea var. alboglabra Bailey. Analysis of the nucleotide sequence indicated that the cDNA of BoaMYB28 was 1257 bp with an ORF of 1020 bp. The deduced BoaMYB28 protein was a polypeptide of 339 amino acid with a putative molecular mass of 38 kDa and a pI of 6.87. Sequence homology and phylogenetic analysis showed that BoaMYB28 was most closely related to MYB28 homologs from the Brassicaceae family. The expression levels of BoaMYB28 varies across the tissues and developmental stages. BoaMYB28 transcript levels were higher in leaves and stems compared with those in cotyledons, flowers, and siliques. BoaMYB28 was expressed across all developmental leaf stages, with higher transcript accumulation in mature and inflorescence leaves. Over-expression and RNAi studies showed that BoaMYB28 retains the basic MYB28 gene function as a major transcriptional regulator of aliphatic glucosinolate pathway. The results indicated that over-expression and RNAi lines showed no visible difference on plant morphology. The contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes increased in over-expression lines and decreased in RNAi lines. In over-expression lines, aliphatic glucosinolate contents were 1.5- to 3-fold higher than those in the wild-type, while expression levels of aliphatic glucosinolate biosynthesis genes were 1.5- to 4-fold higher than those in the wild-type. In contrast, the contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate

  4. Molecular Characterization of MYB28 Involved in Aliphatic Glucosinolate Biosynthesis in Chinese Kale (Brassica oleracea var. alboglabra Bailey).

    Science.gov (United States)

    Yin, Ling; Chen, Hancai; Cao, Bihao; Lei, Jianjun; Chen, Guoju

    2017-01-01

    Glucosinolates are Brassicaceae-specific secondary metabolites that act as crop protectants, flavor precursors, and cancer-prevention agents, which shows strong evidences of anticarcinogentic, antioxidant, and antimicrobial activities. MYB28 , the R2R3-MYB28 transcription factor, directly activates genes involved in aliphatic glucosinolate biosynthesis. In this study, the MYB28 homology ( BoaMYB28 ) was identified in Chinese kale ( Brassica oleracea var. alboglabra Bailey). Analysis of the nucleotide sequence indicated that the cDNA of BoaMYB28 was 1257 bp with an ORF of 1020 bp. The deduced BoaMYB28 protein was a polypeptide of 339 amino acid with a putative molecular mass of 38 kDa and a pI of 6.87. Sequence homology and phylogenetic analysis showed that BoaMYB28 was most closely related to MYB28 homologs from the Brassicaceae family. The expression levels of BoaMYB28 varies across the tissues and developmental stages. BoaMYB28 transcript levels were higher in leaves and stems compared with those in cotyledons, flowers, and siliques. BoaMYB28 was expressed across all developmental leaf stages, with higher transcript accumulation in mature and inflorescence leaves. Over-expression and RNAi studies showed that BoaMYB28 retains the basic MYB28 gene function as a major transcriptional regulator of aliphatic glucosinolate pathway. The results indicated that over-expression and RNAi lines showed no visible difference on plant morphology. The contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes increased in over-expression lines and decreased in RNAi lines. In over-expression lines, aliphatic glucosinolate contents were 1.5- to 3-fold higher than those in the wild-type, while expression levels of aliphatic glucosinolate biosynthesis genes were 1.5- to 4-fold higher than those in the wild-type. In contrast, the contents of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthesis genes in

  5. Functional Characterization of Cotton GaMYB62L, a Novel R2R3 TF in Transgenic Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Hamama Islam Butt

    Full Text Available Drought stress can trigger the production of ABA in plants, in response to adverse conditions, which induces the transcript of stress-related marker genes. The R2R3 MYB TFs are implicated in regulation of various plants developmental, metabolic and multiple environmental stress responses. Here, a R2R3-MYB cloned gene, GaMYB62L, was transformed in Arabidopsis and was functionally characterized. The GaMYB62L protein contains two SANT domains with a conserved R2R3 imperfect repeats. The GaMYB62L cDNA is 1,017 bp with a CDS of 879, encodes a 292-residue polypeptide with MW of 38.78 kD and a pI value of 8.91. Overexpressed GaMYB62L transgenic Arabidopsis have increased proline and chlorophyll content, superior seed germination rate under salt and osmotic stress, less water loss rate with reduced stomatal apertures, high drought avoidance as compared to WT on water deprivation and also significant plant survival rates at low temperature. In addition, overexpressed GaMYB62L lines were more sensitive to ABA mediated germination and root elongation assay. Moreover, ABA induced GaMYB62L overexpression, enhanced the expression of ABA stress related marker genes like RD22, COR15A, ADH1, and RD29A. Together, overexpression of GaMYB62L suggested having developed better drought, salt and cold tolerance in transgenic Arabidopsis and thus presented it as a prospective candidate gene to achieve better abiotic stress tolerance in cotton crop.

  6. Functional Characterization of a Novel R2R3-MYB Transcription Factor Modulating the Flavonoid Biosynthetic Pathway from Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2017-07-01

    Full Text Available Epimedium species have been widely used both as traditional Chinese medicinal plants and ornamental perennials. Both flavonols, acting as the major bioactive components (BCs and anthocyanins, predominantly contributing to the color diversity of Epimedium flowers belong to different classes of flavonoids. It is well-acknowledged that flavonoid biosynthetic pathway is predominantly regulated by R2R3-MYB transcription factor (TF as well as bHLH TF and WD40 protein at the transcriptional level. MYB TFs specifically regulating anthocyanin or flavonol biosynthetic pathway have been already isolated and functionally characterized from Epimedium sagittatum, but a R2R3-MYB TF involved in regulating both these two pathways has not been functionally characterized to date in Epimedium plants. In this study, we report the functional characterization of EsMYB9, a R2R3-MYB TF previously isolated from E. sagittatum. The previous study indicated that EsMYB9 belongs to a small subfamily of R2R3-MYB TFs containing grape VvMYB5a and VvMYB5b TFs, which regulate flavonoid biosynthetic pathway. The present studies show that overexpression of EsMYB9 in tobacco leads to increased transcript levels of flavonoid pathway genes and increased contents of anthocyanins and flavonols. Yeast two-hybrid assay indicates that the C-terminal region of EsMYB9 contributes to the autoactivation activity, and EsMYB9 interacts with EsTT8 or AtTT8 bHLH regulator. Transient reporter assay shows that EsMYB9 slightly activates the expression of EsCHS (chalcone synthase promoter in transiently transformed leaves of Nicotiana benthamiana, but the addition of AtTT8 or EsTT8 bHLH regulator strongly enhances the transcriptional activation of EsMYB9 against five promoters of the flavonoid pathway genes except EsFLS (flavonol synthase. In addition, co-transformation of EsMYB9 and EsTT8 in transiently transfected tobacco leaves strongly induces the expressions of flavonoid biosynthetic genes. The

  7. Mouse Incisor Stem Cell Niche and Myb Transcription Factors

    Czech Academy of Sciences Publication Activity Database

    Švandová, Eva; Veselá, Barbora; Šmarda, J.; Hampl, A.; Radlanski, R.J.; Matalová, Eva

    2015-01-01

    Roč. 44, č. 5 (2015), s. 338-344 ISSN 0340-2096 R&D Projects: GA ČR GAP304/11/1418; GA ČR GCP302/12/J059 Institutional support: RVO:67985904 Keywords : c-Myb * stem cell niches Subject RIV: EA - Cell Biology Impact factor: 0.615, year: 2015

  8. Characterization of a New Pink-Fruited Tomato Mutant Results in the Identification of a Null Allele of the SlMYB12 Transcription Factor.

    Science.gov (United States)

    Fernandez-Moreno, Josefina-Patricia; Tzfadia, Oren; Forment, Javier; Presa, Silvia; Rogachev, Ilana; Meir, Sagit; Orzaez, Diego; Aharoni, Aspah; Granell, Antonio

    2016-07-01

    The identification and characterization of new tomato (Solanum lycopersicum) mutants affected in fruit pigmentation and nutritional content can provide valuable insights into the underlying biology, as well as a source of new alleles for breeding programs. To date, all characterized pink-pigmented tomato fruit mutants appear to result from low SlMYB12 transcript levels in the fruit skin. Two new mutant lines displaying a pink fruit phenotype (pf1 and pf2) were characterized in this study. In the pf mutants, SlMYB12 transcripts accumulated to wild-type levels but exhibited the same truncation, which resulted in the absence of the essential MYB activation domain coding region. Allelism and complementation tests revealed that both pf mutants were allelic to the y locus and showed the same recessive null allele in homozygosis: Δy A set of molecular and metabolic effects, reminiscent of those observed in the Arabidopsis (Arabidopsis thaliana) myb11 myb12 myb111 triple mutant, were found in the tomato Δy mutants. To our knowledge, these have not been described previously, and our data support the idea of their being null mutants, in contrast to previously described transcriptional hypomorphic pink fruit lines. We detected a reduction in the expression of several flavonol glycosides and some associated glycosyl transferases. Transcriptome analysis further revealed that the effects of the pf mutations extended beyond the flavonoid pathway into the interface between primary and secondary metabolism. Finally, screening for Myb-binding sites in the candidate gene promoter sequences revealed that 141 of the 152 co-down-regulated genes may be direct targets of SlMYB12 regulation. © 2016 American Society of Plant Biologists. All Rights Reserved.

  9. Morus alba Leaf Lectin (MLL) Sensitizes MCF-7 Cells to Anoikis by Inhibiting Fibronectin Mediated Integrin-FAK Signaling through Ras and Activation of P38 MAPK

    Science.gov (United States)

    Saranya, Jayaram; Shilpa, Ganesan; Raghu, Kozhiparambil G.; Priya, Sulochana

    2017-01-01

    Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK. PMID:28223935

  10. McMYB10 regulates coloration via activating McF3'H and later structural genes in ever-red leaf crabapple.

    Science.gov (United States)

    Tian, Ji; Peng, Zhen; Zhang, Jie; Song, Tingting; Wan, Huihua; Zhang, Meiling; Yao, Yuncong

    2015-09-01

    The ever-red leaf trait, which is important for breeding ornamental and higher anthocyanin plants, rarely appears in Malus families, but little is known about the regulation of anthocyanin biosynthesis involved in the red leaves. In our study, HPLC analysis showed that the anthocyanin concentration in ever-red leaves, especially cyanidin, was significantly higher than that in evergreen leaves. The transcript level of McMYB10 was significantly correlated with anthocyanin synthesis between the 'Royalty' and evergreen leaf 'Flame' cultivars during leaf development. We also found the ever-red leaf colour cultivar 'Royalty' contained the known R6 : McMYB10 sequence, but was not in the evergreen leaf colour cultivar 'Flame', which have been reported in apple fruit. The distinction in promoter region maybe is the main reason why higher expression level of McMYB10 in red foliage crabapple cultivar. Furthermore, McMYB10 promoted anthocyanin biosynthesis in crabapple leaves and callus at low temperatures and during long-day treatments. Both heterologous expression in tobacco (Nicotiana tabacum) and Arabidopsis pap1 mutant, and homologous expression in crabapple and apple suggested that McMYB10 could promote anthocyanins synthesis and enhanced anthocyanin accumulation in plants. Interestingly, electrophoretic mobility shift assays, coupled with yeast one-hybrid analysis, revealed that McMYB10 positively regulates McF3'H via directly binding to AACCTAAC and TATCCAACC motifs in the promoter. To sum up, our results demonstrated that McMYB10 plays an important role in ever-red leaf coloration, by positively regulating McF3'H in crabapple. Therefore, our work provides new perspectives for ornamental fruit tree breeding. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  11. dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid.

    Science.gov (United States)

    Lau, Su-Ee; Schwarzacher, Trude; Othman, Rofina Yasmin; Harikrishna, Jennifer Ann

    2015-08-11

    The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape. Flower bud development in the Dendrobium hybrid was characterised into seven stages and the time of meiosis was determined as between stages 3 to 5 when the bud is approximately half of the mature size. Scanning electron microscopy characterisation of adaxial epidermal cells of the flower perianth, showed that the petals and sepals each are divided into two distinct domains based on cell shape and size, while the labellum comprises seven domains. Thirty-two partial cDNA fragments representing R2R3-MYB gene sequences were isolated from D. hybrida. Phylogenetic analysis revealed that nine of the translated sequences were clustered with MYB sequences that are known to be involved in cell shape development and from these, DhMYB1 was selected for full length cDNA cloning and functional study. Direct application of a 430 bp dsRNA from the 3' region of DhMYB1 to emerging orchid flower buds reduced expression of DhMYB1 RNA compared with untreated control. Scanning electron microscopy of adaxial epidermal cells within domain one of the labellum of flowers treated with DhMYB1 dsRNA showed flattened epidermal cells whilst those of control flowers were conical. DhMYB1 is expressed throughout flower bud development and is involved in the development of the conical cell shape of the epidermal cells of the Dendrobium hybrida flower labellum. The direct application of dsRNA changed the phenotype of

  12. Identification of CD34+ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia

    Science.gov (United States)

    Aoki, Yuki; Watanabe, Takashi; Saito, Yoriko; Kuroki, Yoko; Hijikata, Atsushi; Takagi, Masatoshi; Tomizawa, Daisuke; Eguchi, Mariko; Eguchi-Ishimae, Minenori; Kaneko, Akiko; Ono, Rintaro; Sato, Kaori; Suzuki, Nahoko; Fujiki, Saera; Koh, Katsuyoshi; Ishii, Eiichi; Shultz, Leonard D.; Ohara, Osamu; Mizutani, Shuki

    2015-01-01

    Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34+CD38+CD19+ and CD34−CD19+ cells initiated leukemia, and in MLL-AF9 patients, CD34−CD19+ cells were LICs. In MLL-ENL patients, either CD34+ or CD34− cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34+CD38−CD19−CD33− cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34+CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4+ single positive (SP), CD8+ SP, and CD4+CD8+ double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34+CD38+ and CD34− LICs but not in CD34+CD38−CD19−CD33− HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia. PMID:25538041

  13. A MYB transcription factor, DcMYB6, is involved in regulating anthocyanin biosynthesis in purple carrot taproots

    OpenAIRE

    Xu, Zhi-Sheng; Feng, Kai; Que, Feng; Wang, Feng; Xiong, Ai-Sheng

    2017-01-01

    Carrots are widely grown and enjoyed around the world. Purple carrots accumulate rich anthocyanins in the taproots, while orange, yellow, and red carrots accumulate rich carotenoids in the taproots. Our previous studies indicated that variation in the activity of regulatory genes may be responsible for variations in anthocyanin production among various carrot cultivars. In this study, an R2R3-type MYB gene, designated as DcMYB6, was isolated from a purple carrot cultivar. In a phylogenetic an...

  14. Differential effects of v-Jun and c-Jun proteins on v-myb-transformed monoblasts

    Czech Academy of Sciences Publication Activity Database

    Ševčíková, S.; Souček, Karel; Kubala, Lukáš; Bryja, Vítězslav; Šmarda, J.

    2002-01-01

    Roč. 59, č. 10 (2002), s. 1690-1705 ISSN 1420-682X R&D Projects: GA ČR GA301/01/0040 Institutional research plan: CEZ:AV0Z5004920 Keywords : v-myb * Jun * differentiation Subject RIV: BO - Biophysics Impact factor: 5.259, year: 2002

  15. QsMYB1 expression is modulated in response to heat and drought stresses and during plant recovery in Quercus suber.

    Science.gov (United States)

    Almeida, Tânia; Pinto, Glória; Correia, Barbara; Santos, Conceição; Gonçalves, Sónia

    2013-12-01

    Cork oak is an economically important forest species showing a great tolerance to high temperatures and shortage of water. However, the mechanisms underlying this plasticity are still poorly understood. Among the stress regulators, transcription factors (TFs) are especially important since they can control a wide range of stress-inducible genes, which make them powerful targets for genetic engineering of stress tolerance. Here we evaluated the influence of increasing temperatures (up to 55 °C) or drought (18% field capacity, FC) on the expression profile of an R2R3-MYB transcription factor of cork oak, the QsMYB1. QsMYB1 was previously identified as being preferentially expressed in cork tissues and as having an associated alternative splicing mechanism, which results in two different transcripts (QsMYB1.1 and QsMYB1.2). Expression analysis by reverse transcription quantitative PCR (RT-qPCR) revealed that increasing temperatures led to a gradual down-regulation of QsMYB1 transcripts with more effect on QsMYB1.1 abundance. On the other hand, under drought condition, expression of QsMYB1 variants, mainly the QsMYB1.2, was transiently up-regulated shortly after the stress imposition. Recovery from each stress has also resulted in a differential response by both QsMYB1 transcripts. Several physiological and biochemical parameters (plant water status, chlorophyll fluorescence, lipid peroxidation and proline content) were determined in order to monitor the plant performance under stress and recovery. In conclusion, this report provides the first evidence that QsMYB1 TF may have a putative function in the regulatory network of cork oak response to heat and drought stresses and during plant recovery. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  16. TGIF1 is a negative regulator of MLL-rearranged acute myeloid leukemia

    DEFF Research Database (Denmark)

    Willer, Anton; Jakobsen, Janus Schou; Ohlsson, E

    2015-01-01

    orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general......Members of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing transcription factors are important downstream effectors of oncogenic fusion proteins involving the mixed lineage leukemia (MLL) gene. A well-characterized member of this protein family is MEIS1, which...... influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML.Leukemia...

  17. MLL/WDR5 Complex Regulates Kif2A Localization to Ensure Chromosome Congression and Proper Spindle Assembly during Mitosis.

    Science.gov (United States)

    Ali, Aamir; Veeranki, Sailaja Naga; Chinchole, Akash; Tyagi, Shweta

    2017-06-19

    Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. A Myb transcription factor of Phytophthora sojae, regulated by MAP kinase PsSAK1, is required for zoospore development.

    Directory of Open Access Journals (Sweden)

    Meng Zhang

    Full Text Available PsSAK1, a mitogen-activated protein (MAP kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b direct germination of sporangia, and c afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae.

  19. Genome-Wide Classification and Evolutionary and Expression Analyses of Citrus MYB Transcription Factor Families in Sweet Orange

    Science.gov (United States)

    Hou, Xiao-Jin; Li, Si-Bei; Liu, Sheng-Rui; Hu, Chun-Gen; Zhang, Jin-Zhi

    2014-01-01

    MYB family genes are widely distributed in plants and comprise one of the largest transcription factors involved in various developmental processes and defense responses of plants. To date, few MYB genes and little expression profiling have been reported for citrus. Here, we describe and classify 177 members of the sweet orange MYB gene (CsMYB) family in terms of their genomic gene structures and similarity to their putative Arabidopsis orthologs. According to these analyses, these CsMYBs were categorized into four groups (4R-MYB, 3R-MYB, 2R-MYB and 1R-MYB). Gene structure analysis revealed that 1R-MYB genes possess relatively more introns as compared with 2R-MYB genes. Investigation of their chromosomal localizations revealed that these CsMYBs are distributed across nine chromosomes. Sweet orange includes a relatively small number of MYB genes compared with the 198 members in Arabidopsis, presumably due to a paralog reduction related to repetitive sequence insertion into promoter and non-coding transcribed region of the genes. Comparative studies of CsMYBs and Arabidopsis showed that CsMYBs had fewer gene duplication events. Expression analysis revealed that the MYB gene family has a wide expression profile in sweet orange development and plays important roles in development and stress responses. In addition, 337 new putative microsatellites with flanking sequences sufficient for primer design were also identified from the 177 CsMYBs. These results provide a useful reference for the selection of candidate MYB genes for cloning and further functional analysis forcitrus. PMID:25375352

  20. The C-terminal region (640-967) of Arabidopsis CPL1 interacts with the abiotic stress- and ABA-responsive transcription factors

    International Nuclear Information System (INIS)

    Bang, Woo Young; Kim, Se Won; Jeong, In Sil; Koiwa, Hisashi; Bahk, Jeong Dong

    2008-01-01

    Proteins in CPL1 family are unique to plants and contain a phosphatase catalytic domain and double-stranded RNA (dsRNA)-binding motifs (DRMs) in a single peptide. Though DRMs are important for the function of Arabidopsis CPL1 in vivo, the role of CPL1 DRM has been obscure. We have isolated two transcription factors, ANAC019 (At1g52890) and AtMYB3 (At1g22640), which specifically interact with the C-terminal region (640-967) of AtCPL1 containing two DRMs. Detailed interaction analysis indicated that AtMYB3 specifically interacted with the first DRM but not with the second DRM in CPL1 C-terminal fragment. GFP-fusion analysis indicated that AtMYB3 localized in nuclei-like CPL1, and its expression is induced by abiotic stress and ABA treatment. These results suggest that AtMYB3 function in abiotic stress signaling in concert with CPL1

  1. Multiple R2R3-MYB transcription factors involved in the regulation of anthocyanin accumulation in peach flower

    Directory of Open Access Journals (Sweden)

    Hui Zhou

    2016-10-01

    Full Text Available Anthocyanin accumulation is responsible for flower coloration in peach. Here, we report the identification and functional characterization of eight flavonoid-related R2R3-MYB transcription factors, designated PpMYB10.2, PpMYB9, PpMYBPA1, Peace, PpMYB17, PpMYB18, PpMYB19 and PpMYB20, respectively, in peach flower transcriptome. PpMYB10.2 and PpMYB9 are able to activate transcription of anthocyanin biosynthetic genes, whilst PpMYBPA1 and Peace have a strong activation on the promoters of proanthocyanin (PA biosynthetic genes. PpMYB17-20 show a strong repressive effect on transcription of flavonoid pathway genes such as DFR. These results indicate that anthocyanin accumulation in peach flower is coordinately regulated by a set of R2R3-MYB genes. In addition, PpMYB9 and PpMYB10.2 are closely related but separated into two groups, designated MYB9 and MYB10, respectively. PpMYB9 shows a strong activation on the PpUGT78A2 promoter, but with no effect on the promoter of PpUGT78B (commonly called PpUFGT in previous studies. In contrast, PpMYB10.2 is able to activate the PpUFGT promoter, but not for the PpUGT78A2 promoter. Unlike the MYB10 gene that is universally present in plants, the MYB9 gene is lost in most dicot species. Therefore, the PpMYB9 gene represents a novel group of anthocyanin-related MYB activators, which may have diverged in function from the MYB10 genes. Our study will aid in understanding the complex mechanism regulating floral pigmentation in peach and functional divergence of the R2R3-MYB gene family in plants.

  2. Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control

    DEFF Research Database (Denmark)

    Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K

    2015-01-01

    an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme...... that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein...

  3. Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers.

    Science.gov (United States)

    Nakatsuka, Takashi; Yamada, Eri; Saito, Misa; Fujita, Kohei; Nishihara, Masahiro

    2013-12-01

    Single-repeat MYB transcription factors, GtMYB1R1 and GtMYB1R9 , were isolated from gentian. Overexpression of these genes reduced anthocyanin accumulation in tobacco flowers, demonstrating their applicability to modification of flower color. RNA interference (RNAi) has recently been used to successfully modify flower color intensity in several plant species. In most floricultural plants, this technique requires prior isolation of target flavonoid biosynthetic genes from the same or closely related species. To overcome this limitation, we developed a simple and efficient method for reducing floral anthocyanin accumulation based on genetic engineering using novel transcription factor genes isolated from Japanese gentians. We identified two single-repeat MYB genes--GtMYB1R and GtMYB1R9--predominantly expressed in gentian petals. Transgenic tobacco plants expressing these genes were produced, and their flowers were analyzed for flavonoid components and expression of flavonoid biosynthetic genes. Transgenic tobacco plants expressing GtMYB1R1 or GtMYB1R9 exhibited significant reductions in floral anthocyanin accumulation, resulting in white-flowered phenotypes. Expression levels of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) genes were preferentially suppressed in these transgenic tobacco flowers. A yeast two-hybrid assay demonstrated that both GtMYB1R1 and GtMYB1R9 proteins interacted with the GtbHLH1 protein, previously identified as an anthocyanin biosynthesis regulator in gentian flowers. In addition, a transient expression assay indicated that activation of the gentian GtDFR promoter by the GtMYB3-GtbHLH1 complex was partly canceled by addition of GtMYB1R1 or GtMYB1R9. These results suggest that GtMYB1R1 and GtMYB1R9 act as antagonistic transcription factors of anthocyanin biosynthesis in gentian flowers. These genes should consequently be useful for manipulating anthocyanin accumulation via genetic engineering in

  4. A novel spliced fusion of MLL with CT45A2 in a pediatric biphenotypic acute leukemia

    International Nuclear Information System (INIS)

    Cerveira, Nuno; Marschalek, Rolf; Teixeira, Manuel R; Meyer, Claus; Santos, Joana; Torres, Lurdes; Lisboa, Susana; Pinheiro, Manuela; Bizarro, Susana; Correia, Cecília; Norton, Lucília

    2010-01-01

    Abnormalities of 11q23 involving the MLL gene are found in approximately 10% of human leukemias. To date, nearly 100 different chromosome bands have been described in rearrangements involving 11q23 and 64 fusion genes have been cloned and characterized at the molecular level. In this work we present the identification of a novel MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia. Cytogenetics, fluorescence in situ hybridization (FISH), molecular studies (RT-PCR and LDI-PCR), and bioinformatic sequence analysis were used to characterize the CT45A2 gene as novel MLL fusion partner in pediatric acute leukemia. Fluorescence in situ hybridization of bone marrow G-banded metaphases demonstrated a cryptic insertion of 11q23 in Xq26.3 involving the MLL gene. Breakpoint fusion analysis revealed that a DNA fragment of 653 kb from 11q23, containing MLL exons 1-9 in addition to 16 other 11q23 genes, was inserted into the upstream region of the CT45A2 gene located at Xq26.3. In addition, a deletion at Xq26.3 encompassing the 3' region of the DDX26B gene (exons 9-16) and the entire CT45A1 gene was identified. RNA analysis revealed the presence of a novel MLL-CT45A2 fusion transcript in which the first 9 exons of the MLL gene were fused in-frame to exon 2 of the CT45A2 gene, resulting in a spliced MLL fusion transcript with an intact open reading frame. The resulting chimeric transcript predicts a fusion protein where the N-terminus of MLL is fused to the entire open reading frame of CT45A2. Finally, we demonstrate that all breakpoint regions are rich in long repetitive motifs, namely LINE/L1 and SINE/Alu sequences, but all breakpoints were exclusively identified outside these repetitive DNA sequences. We have identified CT45A2 as a novel spliced MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia, as a result of a cryptic insertion of 11q23 in Xq26.3. Since CT45A2 is the first Cancer/Testis antigen family gene

  5. MLL-AF4 fusion transcripts in Acute Lymphocytic Leukemia patientsin Children hospital of Tabriz

    Directory of Open Access Journals (Sweden)

    Amir Monfaredan

    2014-08-01

    Full Text Available Background: MLL-AF4 positive Leukemias comprise about 50-70% of acute lymphoid leukemias in children and about 5% of adolescents and adults Despite recent advances in the treatment of hematologic malignancies of children with ALL in particular, but it seems that poor results are obtained from treating this type of malignancy. Perhaps it is due to the lack of enough knowledge about the expression pattern of the fusion gene induced by chromosomal translocations. This study aims to consider several aspects of the common chromosomal disorder, t (4 11: due to lack of accurate statistical results for this type of translocation in our country, acceptable results are provided Sprevalence of isoforms of recombinant genes involved in MLL-AF4 are explained. Materials and methods: Of 36 patients with ALL between 4 months -11 years of age, peripheral blood sampling was done and total RNA extracted and cDNA was made. Then cDNA was amplified in two steps with the PCR and Nested PCR reactions. After electrophoresis the products were compared and analyzed in comparison with the internal control. Results: The results showed that MLL-AF4 recombinant gene expression in the age between 4 to 12 months range is maximum in the second stage by Nested PCR. Also the highest frequency of fusion isoforms of the gene involved in the same age range is e11-e4 isoform with the frequency of 0.13. Conclusion: It seems that investigation of translocation and chromosomal abnormalities using molecular techniques is one of the most accurate and suitable methods for identifying chromosomal characteristics in patients with acute leukemia, particularly ALL.

  6. A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

    Directory of Open Access Journals (Sweden)

    Granier Thierry

    2011-08-01

    Full Text Available Abstract Background Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR. In grapevine (Vitis vinifera L., several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. Results In this work, we describe the effects of a single amino acid substitution (R69L located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. Conclusions Taken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s. In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment

  7. Downregulation of B-myb promotes senescence via the ROS-mediated p53/p21 pathway, in vascular endothelial cells.

    Science.gov (United States)

    Zhou, Zhihui; Yin, Yanlin; Chang, Qun; Sun, Guanqun; Lin, Jiahui; Dai, Yalei

    2017-04-01

    To reveal whether B-myb is involved in preventing senescence of vascular endothelial cells, and if so, to identify possible mechanisms for it. C57/BL6 male mice and primary human aortic endothelial cells (HAECs) were used. Bleomycin was applied to induce stress-related premature senescence. B-myb knockdown was achieved using an siRNA technique and cell senescence was assessed using the senescence-associated β-galactosidase (SA-β-gal) assay. Intracellular reactive oxygen species (ROS) production was analysed using an ROS assay kit and cell proliferation was evaluated using KFluor488 EdU kit. Capillary tube network formation was determined by Matrigel assay. Expressions of mRNA and protein levels were detected by real-time PCR and western blotting. B-myb expression significantly decreased, while p53 and p21 expressions increased in the aortas of aged mice. This expression pattern was also found in replicative senescent HAECs and senescent HAECs induced by bleomycin. B-myb knockdown resulted in upregulation of p22 phox , ROS accumulation and cell senescence of HAECs. Downregulation of B-myb significantly inhibited cell proliferation and capillary tube network formation and activated the p53/p21 signalling pathway. Blocking ROS production or inhibiting p53 activation remarkably attenuated SA-β-gal activity and delayed cell senescence induced by B-myb-silencing. Downregulation of B-myb induced senescence by upregulation of p22 phox and activation of the ROS/p53/p21 pathway, in our vascular endothelial cells, suggesting that B-myb may be a novel candidate for regulating cell senescence to protect against endothelial senescence-related cardiovascular diseases. © 2016 John Wiley & Sons Ltd.

  8. The Jasmonate-ZIM-Domain Proteins Interact with the WD-Repeat/bHLH/MYB Complexes to Regulate Jasmonate-Mediated Anthocyanin Accumulation and Trichome Initiation in Arabidopsis thaliana[C][W

    Science.gov (United States)

    Qi, Tiancong; Song, Susheng; Ren, Qingcuo; Wu, Dewei; Huang, Huang; Chen, Yan; Fan, Meng; Peng, Wen; Ren, Chunmei; Xie, Daoxin

    2011-01-01

    Jasmonates (JAs) mediate plant responses to insect attack, wounding, pathogen infection, stress, and UV damage and regulate plant fertility, anthocyanin accumulation, trichome formation, and many other plant developmental processes. Arabidopsis thaliana Jasmonate ZIM-domain (JAZ) proteins, substrates of the CORONATINE INSENSITIVE1 (COI1)–based SCFCOI1 complex, negatively regulate these plant responses. Little is known about the molecular mechanism for JA regulation of anthocyanin accumulation and trichome initiation. In this study, we revealed that JAZ proteins interact with bHLH (Transparent Testa8, Glabra3 [GL3], and Enhancer of Glabra3 [EGL3]) and R2R3 MYB transcription factors (MYB75 and Glabra1), essential components of WD-repeat/bHLH/MYB transcriptional complexes, to repress JA-regulated anthocyanin accumulation and trichome initiation. Genetic and physiological evidence showed that JA regulates WD-repeat/bHLH/MYB complex-mediated anthocyanin accumulation and trichome initiation in a COI1-dependent manner. Overexpression of the MYB transcription factor MYB75 and bHLH factors (GL3 and EGL3) restored anthocyanin accumulation and trichome initiation in the coi1 mutant, respectively. We speculate that the JA-induced degradation of JAZ proteins abolishes the interactions of JAZ proteins with bHLH and MYB factors, allowing the transcriptional function of WD-repeat/bHLH/MYB complexes, which subsequently activate respective downstream signal cascades to modulate anthocyanin accumulation and trichome initiation. PMID:21551388

  9. Functional characterization of TRICHOMELESS2, a new single-repeat R3 MYB transcription factor in the regulation of trichome patterning in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Gan Lijun

    2011-12-01

    Full Text Available Abstract Background Single-repeat R3 MYB transcription factors (single-repeat MYBs play important roles in controlling trichome patterning in Arabidopsis. It was proposed that single-repeat MYBs negatively regulate trichome formation by competing with GLABRA1 (GL1 for binding GLABRA3/ENHANCER OF GLABRA3 (GL3/EGL3, thus inhibiting the formation of activator complex TTG1(TRANSPARENT TESTA GLABRA1-GL3/EGL3-GL1 that is required for the activation of GLABRA2 (GL2, whose product is a positive regulator of trichome formation. Previously we identified a novel single-repeat MYB transcription factor, TRICHOMELESS1 (TCL1, which negatively regulates trichome formation on the inflorescence stems and pedicels by directly suppressing the expression of GL1. Results We analyzed here the role of TRICHOMELESS2 (TCL2, a previously-uncharacterized single-repeat MYB transcription factor in trichome patterning in Arabidopsis. We showed that TCL2 is closely related to TCL1, and like TCL1 and other single-repeat MYBs, TCL2 interacts with GL3. Overexpression of TCL2 conferred glabrous phenotype while knockdown of TCL2 via RNAi induced ectopic trichome formation on the inflorescence stems and pedicels, a phenotype that was previously observed in tcl1 mutants. These results suggested that TCL2 may have overlapping function with TCL1 in controlling trichome formation on inflorescences. On the other hand, although the transcription of TCL2, like TCL1, is not controlled by the activator complex formed by GL1 and GL3, and TCL2 and TCL1 proteins are more than 80% identical at the amino acid level, the expression of TCL2 under the control of TCL1 promoter only partially recovered the mutant phenotype of tcl1, implying that TCL2 and TCL1 are not fully functional equivalent. Conclusions TCL2 function redundantly with TCL1 in controlling trichome formation on inflorescences, but they are not fully functional equivalent. Transcription of TCL2 is not controlled by activator complex

  10. AtMyb7, a subgroup 4 R2R3 Myb, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5

    KAUST Repository

    Kim, Junhyeok; Hyun, Wooyoung; Nguyen, Hoai Nguyen; Jeong, Chanyoung; Xiong, Liming; Hong, Sukwhan; Lee, Hojoung

    2014-01-01

    Various Myb proteins have been shown to play crucial roles in plants, including primary and secondary metabolism, determination of cell fate and identity, regulation of development and involvement in responses to biotic and abiotic stresses. The 126 R2R3 Myb proteins (with two Myb repeats) have been found in Arabidopsis; however, the functions of most of these proteins remain to be fully elucidated. In the present study, we characterized the function of AtMyb7 using molecular biological and genetic analyses. We used qRT-PCR to determine the levels of stress-response gene transcripts in wild-type and atmyb7 plants. We showed that ArabidopsisAtMyb7 plays a critical role in seed germination. Under abscisic acid (ABA) and high-salt stress conditions, atmyb7 plants showed a lower germination rate than did wild-type plants. Furthermore, AtMyb7 promoter:GUS seeds exhibited different expression patterns in response to variations in the seed imbibition period. AtMyb7 negatively controls the expression of the gene encoding bZIP transcription factor, ABI5, which is a key transcription factor in ABA signalling and serves as a crucial regulator of germination inhibition in Arabidopsis. © 2014 John Wiley & Sons Ltd.

  11. AtMyb7, a subgroup 4 R2R3 Myb, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5

    KAUST Repository

    Kim, Junhyeok

    2014-08-27

    Various Myb proteins have been shown to play crucial roles in plants, including primary and secondary metabolism, determination of cell fate and identity, regulation of development and involvement in responses to biotic and abiotic stresses. The 126 R2R3 Myb proteins (with two Myb repeats) have been found in Arabidopsis; however, the functions of most of these proteins remain to be fully elucidated. In the present study, we characterized the function of AtMyb7 using molecular biological and genetic analyses. We used qRT-PCR to determine the levels of stress-response gene transcripts in wild-type and atmyb7 plants. We showed that ArabidopsisAtMyb7 plays a critical role in seed germination. Under abscisic acid (ABA) and high-salt stress conditions, atmyb7 plants showed a lower germination rate than did wild-type plants. Furthermore, AtMyb7 promoter:GUS seeds exhibited different expression patterns in response to variations in the seed imbibition period. AtMyb7 negatively controls the expression of the gene encoding bZIP transcription factor, ABI5, which is a key transcription factor in ABA signalling and serves as a crucial regulator of germination inhibition in Arabidopsis. © 2014 John Wiley & Sons Ltd.

  12. Identification and expression analyses of MYB and WRKY transcription factor genes in Papaver somniferum L.

    Science.gov (United States)

    Kakeshpour, Tayebeh; Nayebi, Shadi; Rashidi Monfared, Sajad; Moieni, Ahmad; Karimzadeh, Ghasem

    2015-10-01

    Papaver somniferum L. is an herbaceous, annual and diploid plant that is important from pharmacological and strategic point of view. The cDNA clones of two putative MYB and WRKY genes were isolated (GeneBank accession numbers KP411870 and KP203854, respectively) from this plant, via the nested-PCR method, and characterized. The MYB transcription factor (TF) comprises 342 amino acids, and exhibits the structural features of the R2R3MYB protein family. The WRKY TF, a 326 amino acid-long polypeptide, falls structurally into the group II of WRKY protein family. Quantitative real-time PCR (qRT-PCR) analyses indicate the presence of these TFs in all organs of P. somniferum L. and Papaver bracteatum L. Highest expression levels of these two TFs were observed in the leaf tissues of P. somniferum L. while in P. bracteatum L. the espression levels were highest in the root tissues. Promoter analysis of the 10 co-expressed gene clustered involved in noscapine biosynthesis pathway in P. somniferum L. suggested that not only these 10 genes are co-expressed, but also share common regulatory motifs and TFs including MYB and WRKY TFs, and that may explain their common regulation.

  13. Neuronal Kmt2a/Mll1 histone methyltransferase is essential for prefrontal synaptic plasticity and working memory.

    Science.gov (United States)

    Jakovcevski, Mira; Ruan, Hongyu; Shen, Erica Y; Dincer, Aslihan; Javidfar, Behnam; Ma, Qi; Peter, Cyril J; Cheung, Iris; Mitchell, Amanda C; Jiang, Yan; Lin, Cong L; Pothula, Venu; Stewart, A Francis; Ernst, Patricia; Yao, Wei-Dong; Akbarian, Schahram

    2015-04-01

    Neuronal histone H3-lysine 4 methylation landscapes are defined by sharp peaks at gene promoters and other cis-regulatory sequences, but molecular and cellular phenotypes after neuron-specific deletion of H3K4 methyl-regulators remain largely unexplored. We report that neuronal ablation of the H3K4-specific methyltransferase, Kmt2a/Mixed-lineage leukemia 1 (Mll1), in mouse postnatal forebrain and adult prefrontal cortex (PFC) is associated with increased anxiety and robust cognitive deficits without locomotor dysfunction. In contrast, only mild behavioral phenotypes were observed after ablation of the Mll1 ortholog Kmt2b/Mll2 in PFC. Impaired working memory after Kmt2a/Mll1 ablation in PFC neurons was associated with loss of training-induced transient waves of Arc immediate early gene expression critical for synaptic plasticity. Medial prefrontal layer V pyramidal neurons, a major output relay of the cortex, demonstrated severely impaired synaptic facilitation and temporal summation, two forms of short-term plasticity essential for working memory. Chromatin immunoprecipitation followed by deep sequencing in Mll1-deficient cortical neurons revealed downregulated expression and loss of the transcriptional mark, trimethyl-H3K4, at <50 loci, including the homeodomain transcription factor Meis2. Small RNA-mediated Meis2 knockdown in PFC was associated with working memory defects similar to those elicited by Mll1 deletion. Therefore, mature prefrontal neurons critically depend on maintenance of Mll1-regulated H3K4 methylation at a subset of genes with an essential role in cognition and emotion. Copyright © 2015 the authors 0270-6474/15/355097-12$15.00/0.

  14. ARID5B polymorphism confers an increased risk to acquire specific MLL rearrangements in early childhood leukemia

    Science.gov (United States)

    2014-01-01

    Background Acute leukemia in early age (EAL) is characterized by acquired genetic alterations such as MLL rearrangements (MLL-r). The aim of this case-controlled study was to investigate whether single nucleotide polymorphisms (SNPs) of IKZF1, ARID5B, and CEBPE could be related to the onset of EAL cases (diagnosis). Methods The SNPs (IKZF1 rs11978267, ARID5B rs10821936 and rs10994982, CEBPE rs2239633) were genotyped in 265 cases [169 acute lymphoblastic leukemia (ALL) and 96 acute myeloid leukaemia (AML)] and 505 controls by Taqman allelic discrimination assay. Logistic regression was used to evaluate the association between SNPs of cases and controls, adjusted on skin color and/or age. The risk was determined by calculating odds ratios (ORs) with 95% confidence interval (CI). Results Children with the IKZF1 SNP had an increased risk of developing MLL-germline ALL in white children. The heterozygous/mutant genotype in ARID5B rs10994982 significantly increased the risk for MLL-germline leukemia in white and non-white children (OR 2.60, 95% CI: 1.09-6.18 and OR 3.55, 95% CI: 1.57-8.68, respectively). The heterozygous genotype in ARID5B rs10821936 increased the risk for MLL-r leukemia in both white and non-white (OR 2.06, 95% CI: 1.12-3.79 and OR 2.36, 95% CI: 1.09-5.10, respectively). Furthermore, ARID5B rs10821936 conferred increased risk for MLL-MLLT3 positive cases (OR 7.10, 95% CI:1.54-32.68). Our data do not show evidence that CEBPE rs2239633 confers increased genetic susceptibility to EAL. Conclusions IKZF1 and CEBPE variants seem to play a minor role in genetic susceptibility to EAL, while ARID5B rs10821936 increased the risk of MLL-MLLT3. This result shows that genetic susceptibility could be associated with the differences regarding MLL breakpoints and partner genes. PMID:24564228

  15. Genome-wide identification, functional prediction, and evolutionary analysis of the R2R3-MYB superfamily in Brassica napus.

    Science.gov (United States)

    Hajiebrahimi, Ali; Owji, Hajar; Hemmati, Shiva

    2017-10-01

    R2R3-MYB transcription factors (TFs) have been shown to play important roles in plants, including in development and in various stress conditions. Phylogenetic analysis showed the presence of 249 R2R3-MYB TFs in Brassica napus, called BnaR2R3-MYB TFs, clustered into 38 clades. BnaR2R3-MYB TFs were distributed on 19 chromosomes of B. napus. Sixteen gene clusters were identified. BnaR2R3-MYB TFs were characterized by motif prediction, gene structure analysis, and gene ontology. Evolutionary analysis revealed that BnaR2R3-MYB TFs are mainly formed as a result of whole-genome duplication. Orthologs and paralogs of BnaR2R3-MYB TFs were identified in B. napus, B. rapa, B. oleracea, and Arabidopsis thaliana using synteny-based methods. Purifying selection was pervasive within R2R3-MYB TFs. K n /K s values lower than 0.3 indicated that BnaR2R3-MYB TFs are being functionally converged. The role of gene conversion in the formation of BnaR2R3-MYB TFs was significant. Cis-regulatory elements in the upstream regions of BnaR2R3-MYB genes, miRNA targeting BnaR2R3MYB TFs, and post translational modifications were identified. Digital expression data revealed that BnaR2R3-MYB genes were highly expressed in the roots and under high salinity treatment after 24 h. BnaMYB21, BnaMYB141, and BnaMYB148 have been suggested for improving salt-tolerant B. napus. BnaR2R3-MYB genes were mostly up regulated on the 14th day post inoculation with Leptosphaeria biglobosa and L. maculan. BnaMYB150 is a candidate for increased tolerance to Leptospheria in B. napus.

  16. Novel bioresources for studies of Brassica oleracea: identification of a kale MYB transcription factor responsible for glucosinolate production.

    Science.gov (United States)

    Araki, Ryoichi; Hasumi, Akiko; Nishizawa, Osamu Ishizaki; Sasaki, Katsunori; Kuwahara, Ayuko; Sawada, Yuji; Totoki, Yasushi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Li, Yimeng; Saito, Kazuki; Ogawa, Toshiya; Hirai, Masami Yokota

    2013-10-01

    Plants belonging to the Brassicaceae family exhibit species-specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health-promoting properties. Among them, glucoraphanin (aliphatic 4-methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full-length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild-type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  17. MYB Transcription Factors in Chinese Pear (Pyrus bretschneideri Rehd.: Genome-Wide Identification, Classification and Expression Profiling during Fruit Development

    Directory of Open Access Journals (Sweden)

    Yun Peng eCao

    2016-04-01

    Full Text Available The MYB family is one of the largest families of transcription factors in plants. Although some MYBs have been reported to play roles in secondary metabolism, no comprehensive study of the MYB family in Chinese pear (Pyrus bretschneideri Rehd. has been reported. In the present study, we performed genome-wide analysis of MYB genes in Chinese pear, designated as PbMYBs, including analyses of their phylogenic relationships, structures, chromosomal locations, promoter regions, GO annotations and collinearity. A total of 129 PbMYB genes were identified in the pear genome and were divided into 31 subgroups based on phylogenetic analysis. These PbMYBs were unevenly distributed among 16 chromosomes (total of 17 chromosomes. The occurrence of gene duplication events indicated that whole-genome duplication and segmental duplication likely played key roles in expansion of the PbMYB gene family. Ka/Ks analysis suggested that the duplicated PbMYBs mainly experienced purifying selection with restrictive functional divergence after the duplication events. Interspecies microsynteny analysis revealed maximum orthology between pear and peach, followed by plum and strawberry. Subsequently, the expression patterns of 20 PbMYB genes that may be involved in lignin biosynthesis according to their phylogenetic relationships were examined throughout fruit development. Among the twenty genes examined, PbMYB25 and PbMYB52 exhibited expression patterns consistent with the typical variations in the lignin content previously reported. Moreover, sub-cellular localization analysis revealed that two proteins PbMYB25 and PbMYB52 were localized to the nucleus. All together, PbMYB25 and PbMYB52 were inferred to be candidate genes involved in the regulation of lignin biosynthesis during the development of pear fruit. This study provides useful information for further functional analysis of the MYB gene family in pear.

  18. Molecular characterization of Quercus suber MYB1, a transcription factor up-regulated in cork tissues.

    Science.gov (United States)

    Almeida, Tânia; Menéndez, Esther; Capote, Tiago; Ribeiro, Teresa; Santos, Conceição; Gonçalves, Sónia

    2013-01-15

    The molecular processes associated with cork development in Quercus suber L. are poorly understood. A previous molecular approach identified a list of genes potentially important for cork formation and differentiation, providing a new basis for further molecular studies. This report is the first molecular characterization of one of these candidate genes, QsMYB1, coding for an R2R3-MYB transcription factor. The R2R3-MYB gene sub-family has been described as being involved in the phenylpropanoid and lignin pathways, both involved in cork biosynthesis. The results showed that the expression of QsMYB1 is putatively mediated by an alternative splicing (AS) mechanism that originates two different transcripts (QsMYB1.1 and QsMYB1.2), differing only in the 5'-untranslated region, due to retention of the first intron in one of the variants. Moreover, within the retained intron, a simple sequence repeat (SSR) was identified. The upstream regulatory region of QsMYB1 was extended by a genome walking approach, which allowed the identification of the putative gene promoter region. The relative expression pattern of QsMYB1 transcripts determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that both transcripts were up-regulated in cork tissues; the detected expression was several times higher in newly formed cork harvested from trees producing virgin, second or reproduction cork when compared with wood. Moreover, the expression analysis of QsMYB1 in several Q. suber organs showed very low expression in young branches and roots, whereas in leaves, immature acorns or male flowers, no expression was detected. These preliminary results suggest that QsMYB1 may be related to secondary growth and, in particular, with the cork biosynthesis process with a possible alternative splicing mechanism associated with its regulatory function. Copyright © 2012 Elsevier GmbH. All rights reserved.

  19. A novel RT-qPCR assay for quantification of the MLL-MLLT3 fusion transcript in acute myeloid leukaemia

    DEFF Research Database (Denmark)

    Abildgaard, Lotte; Ommen, Hans Beier; Lausen, Birgitte Frederiksen

    2013-01-01

    OBJECTIVES: Patients with acute myeloid leukaemia (AML) of the monocytic lineage often lack molecular markers for minimal residual disease (MRD) monitoring. The MLL-MLLT3 fusion transcript found in patients with AML harbouring t(9;11) is amenable to RT-qPCR quantification but because...... of the heterogeneity of translocation break points, the MLL-MLLT3 fusion gene is a challenging target. We hypothesised that MRD monitoring using MLL-MLLT3 as a RT-qPCR marker is feasible in the majority of patients with t(9;11)-positive AML. METHODS: Using a locked nucleic acid probe, we developed a sensitive RT......-qPCR assay for quantification of the most common break point region of the MLL-MLLT3 fusion gene. Five paediatric patients with t(9;11)-positive AML were monitored using the MLL-MLLT3 assay. RESULTS: A total of 43 bone marrow (BM) and 52 Peripheral blood (PB) samples were collected from diagnosis until...

  20. Comparative transcriptome analysis of oil palm flowers reveals an EAR-motif-containing R2R3-MYB that modulates phenylpropene biosynthesis.

    Science.gov (United States)

    Li, Ran; Reddy, Vaishnavi Amarr; Jin, Jingjing; Rajan, Chakaravarthy; Wang, Qian; Yue, Genhua; Lim, Chin Huat; Chua, Nam-Hai; Ye, Jian; Sarojam, Rajani

    2017-11-23

    Oil palm is the most productive oil crop and the efficiency of pollination has a direct impact on the yield of oil. Pollination by wind can occur but maximal pollination is mediated by the weevil E. kamerunicus. These weevils complete their life cycle by feeding on male flowers. Attraction of weevils to oil palm flowers is due to the emission of methylchavicol by both male and female flowers. In search for male flowers, the weevils visit female flowers by accident due to methylchavicol fragrance and deposit pollen. Given the importance of methylchavicol emission on pollination, we performed comparative transcriptome analysis of oil palm flowers and leaves to identify candidate genes involved in methylchavicol production in flowers. RNA sequencing (RNA-Seq) of male open flowers, female open flowers and leaves was performed using Illumina HiSeq 2000 platform. Analysis of the transcriptome data revealed that the transcripts of methylchavicol biosynthesis genes were strongly up-regulated whereas transcripts encoding genes involved in lignin production such as, caffeic acid O-methyltransferase (COMT) and Ferulate-5-hydroxylase (F5H) were found to be suppressed in oil palm flowers. Among the transcripts encoding transcription factors, an EAR-motif-containing R2R3-MYB transcription factor (EgMYB4) was found to be enriched in oil palm flowers. We determined that EgMYB4 can suppress the expression of a monolignol pathway gene, EgCOMT, in vivo by binding to the AC elements present in the promoter region. EgMYB4 was further functionally characterized in sweet basil which also produces phenylpropenes like oil palm. Transgenic sweet basil plants showed significant reduction in lignin content but produced more phenylpropenes. Our results suggest that EgMYB4 possibly restrains lignin biosynthesis in oil palm flowers thus allowing enhanced carbon flux into the phenylpropene pathway. This study augments our understanding of the diverse roles that EAR-motif-containing MYBs play to

  1. Mutations in Mll2, an H3K4 Methyltransferase, Result in Insulin Resistance and Impaired Glucose Tolerance in Mice

    Science.gov (United States)

    Schröter, David; Matthews, Helen C.; Bogani, Debora; Moir, Lee; Long, Anna; Church, Christopher; Hugill, Alison; Anstee, Quentin M.; Goldin, Rob; Thursz, Mark; Hollfelder, Florian; Cox, Roger D.

    2013-01-01

    We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level. PMID:23826075

  2. Mutations in Mll2, an H3K4 methyltransferase, result in insulin resistance and impaired glucose tolerance in mice.

    Directory of Open Access Journals (Sweden)

    Michelle Goldsworthy

    Full Text Available We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7 gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.

  3. The small ubiquitin-like modifier E3 ligase MdSIZ1 promotes anthocyanin accumulation by sumoylating MdMYB1 under low-temperature conditions in apple.

    Science.gov (United States)

    Zhou, Li-Jie; Li, Yuan-Yuan; Zhang, Rui-Fen; Zhang, Chun-Ling; Xie, Xing-Bin; Zhao, Cheng; Hao, Yu-Jin

    2017-10-01

    MdMYB1 acts as a crucial component of the MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis in red-skinned apples (Malus domestica), but little is known about its post-translational regulation. Here, a small ubiquitin-like modifier E3 ligase MdSIZ1 was screened out as an MdMYB1-interacting protein with a yeast two-hybridization approach. The interaction between MdSIZ1 and MdMYB1 was further verified with pull-down and CoIP assays. Furthermore, it was found that MdSIZ1 directly sumoylated MdMYB1 proteins in vivo and in vitro, especially under moderately low temperature (17 °C) conditions, and that this sumoylation was required for MdMYB1 protein stability. Moreover, the transcription level of MdSIZ1 gene was remarkably induced by low temperature and phosphorus deficiency, and MdSIZ1 overexpression exerted a large positive influence on anthocyanin accumulation and red fruit coloration, suggesting its important role in the regulation of anthocyanin biosynthesis under stress conditions. Our findings reveal an important role for a small ubiquitin-like modifier modification of MYB transcription factors in regulation of anthocyanin biosynthesis in plants. © 2017 John Wiley & Sons Ltd.

  4. The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells.

    Science.gov (United States)

    Zhan, Ming; Riordon, Daniel R; Yan, Bin; Tarasova, Yelena S; Bruweleit, Sarah; Tarasov, Kirill V; Li, Ronald A; Wersto, Robert P; Boheler, Kenneth R

    2012-01-01

    Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.

  5. Overexpression of SbMyb60 in sorghum bicolor impacts both primary and secondary metabolism

    Science.gov (United States)

    Few transcription factors have been identified in C4 grasses that either positively or negatively regulate monolignol biosynthesis. Previously, overexpression of SbMyb60 in sorghum (Sorghum bicolor (L.) Moench) was shown to induce monolignol synthesis, which led to elevated lignin deposition and al...

  6. The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system.

    Science.gov (United States)

    Kim, Cha Young; Ahn, Young Ock; Kim, Sun Ha; Kim, Yun-Hee; Lee, Haeng-Soon; Catanach, Andrew S; Jacobs, Jeanne M E; Conner, Anthony J; Kwak, Sang-Soo

    2010-07-01

    MYB transcription factors play important roles in transcriptional regulation of many secondary metabolites including anthocyanins. We cloned the R2R3-MYB type IbMYB1 complementary DNAs from the purple-fleshed sweet potato (Ipomoea batatas L. cv Sinzami) and investigated the expression patterns of IbMYB1 gene with IbMYB1a and IbMYB1b splice variants in leaf and root tissues of various sweet potato cultivars by reverse transcription-polymerase chain reaction. The transcripts of IbMYB1 were predominantly expressed in the purple-fleshed storage roots and they were also detectable in the leaf tissues accumulating anthocyanin pigments. In addition, transcript levels of IbMYB1 gene were up-regulated by treatment with methyl jasmonate or salicylic acid in leaf and root tissues of cv. White Star. To set up the intragenic vector system in sweet potato, we first evaluated the utilization of the IbMYB1 gene as a visible selectable marker. The IbMYB1a was transiently expressed in tobacco leaves under the control of a constitutive cauliflower mosaic virus 35S promoter, a root-specific and sucrose-inducible sporamin promoter, and an oxidative stress-inducible sweet potato anionic peroxidase2 promoter. We also showed that overexpression of IbMYB1a induced massive anthocyanin pigmentation in tobacco leaves and up-regulated the transcript levels of the structural genes in anthocyanin biosynthetic pathway. Furthermore, high-performance liquid chromatography analysis revealed that the expression of IbMYB1a led to production of cyanidin as a major core molecule of anthocyanidins in tobacco leaves. These results suggest that the IbMYB1 gene can be applicable to a visible marker for sweet potato transformation with intragenic vectors, as well as the production of anthocyanin as important nutritive value in other plant species.

  7. An Evolutionary Conserved Epigenetic Mark of Polycomb Response Elements Implemented by Trx/MLL/COMPASS.

    Science.gov (United States)

    Rickels, Ryan; Hu, Deqing; Collings, Clayton K; Woodfin, Ashley R; Piunti, Andrea; Mohan, Man; Herz, Hans-Martin; Kvon, Evgeny; Shilatifard, Ali

    2016-07-21

    Polycomb response elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs in mammals remains debated. Accumulating evidence supports a model in which CpG islands recruit Polycomb group (PcG) complexes; however, which subset of CGIs is selected to serve as PREs is unclear. Trithorax (Trx) positively regulates gene expression in Drosophila and co-occupies PREs to antagonize Polycomb-dependent silencing. Here we demonstrate that Trx-dependent H3K4 dimethylation (H3K4me2) marks Drosophila PREs and maintains the developmental expression pattern of nearby genes. Similarly, the mammalian Trx homolog, MLL1, deposits H3K4me2 at CpG-dense regions that could serve as PREs. In the absence of MLL1 and H3K4me2, H3K27me3 levels, a mark of Polycomb repressive complex 2 (PRC2), increase at these loci. By inhibiting PRC2-dependent H3K27me3 in the absence of MLL1, we can rescue expression of these loci, demonstrating a functional balance between MLL1 and PRC2 activities at these sites. Thus, our study provides rules for identifying cell-type-specific functional mammalian PREs within the human genome. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Validation and Structural Characterization of the LEDGF/p75-MLL Interface as a New Target for the Treatment of MLL-Dependent Leukemia

    Czech Academy of Sciences Publication Activity Database

    Čermáková, K.; Těšina, Petr; Demeulemeester, J.; El Ashkar, S.; Méreau, H.; Schwaller, J.; Řezáčová, Pavlína; Veverka, Václav; De Rijck, J.

    2014-01-01

    Roč. 74, č. 18 (2014), s. 5139-5151 ISSN 0008-5472 R&D Projects: GA MŠk(CZ) LK11205; GA MŠk(CZ) 7E08066; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : MLL * leukemia * LEDGF/p75 Subject RIV: CE - Biochemistry Impact factor: 9.329, year: 2014

  9. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

    Science.gov (United States)

    Gonzalez, Antonio; Zhao, Mingzhe; Leavitt, John M; Lloyd, Alan M

    2008-03-01

    In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

  10. Genome-Wide Identification of R2R3-MYB Genes and Expression Analyses During Abiotic Stress in Gossypium raimondii

    Science.gov (United States)

    He, Qiuling; Jones, Don C.; Li, Wei; Xie, Fuliang; Ma, Jun; Sun, Runrun; Wang, Qinglian; Zhu, Shuijin; Zhang, Baohong

    2016-01-01

    The R2R3-MYB is one of the largest families of transcription factors, which have been implicated in multiple biological processes. There is great diversity in the number of R2R3-MYB genes in different plants. However, there is no report on genome-wide characterization of this gene family in cotton. In the present study, a total of 205 putative R2R3-MYB genes were identified in cotton D genome (Gossypium raimondii), that are much larger than that found in other cash crops with fully sequenced genomes. These GrMYBs were classified into 13 groups with the R2R3-MYB genes from Arabidopsis and rice. The amino acid motifs and phylogenetic tree were predicted and analyzed. The sequences of GrMYBs were distributed across 13 chromosomes at various densities. The results showed that the expansion of the G. Raimondii R2R3-MYB family was mainly attributable to whole genome duplication and segmental duplication. Moreover, the expression pattern of 52 selected GrMYBs and 46 GaMYBs were tested in roots and leaves under different abiotic stress conditions. The results revealed that the MYB genes in cotton were differentially expressed under salt and drought stress treatment. Our results will be useful for determining the precise role of the MYB genes during stress responses with crop improvement. PMID:27009386

  11. Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals.

    Science.gov (United States)

    Li, Chonghui; Qiu, Jian; Ding, Ling; Huang, Mingzhong; Huang, Surong; Yang, Guangsui; Yin, Junmei

    2017-03-01

    Dendrobium hybrids orchid are popular throughout the world. They have various floral color and pigmentation patterns that are mainly caused by anthocyanins. It is well established that anthocyanin biosynthesis is regulated by the interplay between MYB and bHLH transcription factors (TF) in most plants. In this study, we identified one R2R3-MYB gene, DhMYB2, and one bHLH gene, DhbHLH1, from a Dendrobium hybrid. Their expression profiles were related to anthocyanin pigmentation in Dendrobium petals. Transient over-expression of these two TF genes showed that both DhMYB2 and DhbHLH1 resulted in anthocyanin production in white petals. The interaction between the two TFs was observed in vitro. In different Dendrobium hybrids petals with various pigmentations, DhMYB2 and DhbHLH1 were co-expressed with DhDFR and DhANS, which are regarded as potential regulatory targets of the two TFs. In flowers with distinct purple lips but white or yellow petals/sepals, the expression of DhbHLH1 was only related to anthocyanin accumulation in the lips. Taken together, DhMYB2 interacted with DhbHLH1 to regulate anthocyanin production in Dendrobium hybrid petals. DhbHLH1 was also responsible for the distinct anthocyanin pigmentation in lip tissues. The functional characterization of DhMYB2 and DhbHLH1 will improve understanding of anthocyanin biosynthesis modulation in Dendrobium orchids. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

    Science.gov (United States)

    Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

    2017-01-17

    FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Functional Characterization of NAC and MYB Transcription Factors Involved in Regulation of Biomass Production in Switchgrass (Panicum virgatum.

    Directory of Open Access Journals (Sweden)

    Ruiqin Zhong

    Full Text Available Switchgrass is a promising biofuel feedstock due to its high biomass production and low agronomic input requirements. Because the bulk of switchgrass biomass used for biofuel production is lignocellulosic secondary walls, studies on secondary wall biosynthesis and its transcriptional regulation are imperative for designing strategies for genetic improvement of biomass production in switchgrass. Here, we report the identification and functional characterization of a group of switchgrass transcription factors, including several NACs (PvSWNs and a MYB (PvMYB46A, for their involvement in regulating secondary wall biosynthesis. PvSWNs and PvMYB46A were found to be highly expressed in stems and their expression was closely associated with sclerenchyma cells. Overexpression of PvSWNs and PvMYB46A in Arabidopsis was shown to result in activation of the biosynthetic genes for cellulose, xylan and lignin and ectopic deposition of secondary walls in normally parenchymatous cells. Transactivation and complementation studies demonstrated that PvSWNs were able to activate the SNBE-driven GUS reporter gene and effectively rescue the secondary wall defects in the Arabidopsis snd1 nst1 double mutant, indicating that they are functional orthologs of Arabidopsis SWNs. Furthermore, we showed that PvMYB46A could activate the SMRE-driven GUS reporter gene and complement the Arabidopsis myb46 myb83 double mutant, suggesting that it is a functional ortholog of Arabidopsis MYB46/MYB83. Together, these results indicate that PvSWNs and PvMYB46A are transcriptional switches involved in regulating secondary wall biosynthesis, which provides molecular tools for genetic manipulation of biomass production in switchgrass.

  14. Identification and expression pattern analysis of BoMYB51 involved in indolic glucosinolate biosynthesis from broccoli (Brassica oleracea var. italica).

    Science.gov (United States)

    Yu, Qingyue; Hao, Guodong; Zhou, Jianxin; Wang, Jingying; Evivie, Ejiroghene Ruona; Li, Jing

    2018-06-22

    Glucosinolates are a class of amino acid-derived specialized metabolites characteristic of the Brassicales order. Trp derived indolic glucosinolates are essential for the effective plant defense responses to a wide range of pathogens and herbivores. In Arabidopsis, MYB51 is the key transcription factor positively regulates indolic glucosinolate production by activating certain biosynthetic genes. In this study, we report the isolation and identification of a MYB51 from broccoli designated as BoMYB51. Overexpression of BoMYB51 in Arabidopsis increased indolic glucosinolate production by upregulating biosynthetic genes and resulted in enhanced flagellin22 (Flg22) induced callose deposition. The spatial expression pattern and responsive expression of BoMYB51 to several hormones and stress treatments were investigated by expressing the β-glucuronidase (GUS) reporter gene driven by BoMYB51 promotor in Arabidopsis and quantitative real-time PCR analysis in broccoli. Our study provides information on molecular characteristics of BoMYB51 and possible physiological process BoMYB51 may involve. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages

    Directory of Open Access Journals (Sweden)

    Julian Buchrieser

    2017-02-01

    Full Text Available Tissue-resident macrophages, such as microglia, Kupffer cells, and Langerhans cells, derive from Myb-independent yolk sac (YS progenitors generated before the emergence of hematopoietic stem cells (HSCs. Myb-independent YS-derived resident macrophages self-renew locally, independently of circulating monocytes and HSCs. In contrast, adult blood monocytes, as well as infiltrating, gut, and dermal macrophages, derive from Myb-dependent HSCs. These findings are derived from the mouse, using gene knockouts and lineage tracing, but their applicability to human development has not been formally demonstrated. Here, we use human induced pluripotent stem cells (iPSCs as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy, we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent, RUNX1-, and SPI1 (PU.1-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages, such as alveolar and kidney macrophages, microglia, Kupffer cells, and Langerhans cells.

  16. Failure to launch: the self-regulating Md-MYB10 R6 gene from apple is active in flowers but not leaves of Petunia.

    Science.gov (United States)

    Boase, Murray R; Brendolise, Cyril; Wang, Lei; Ngo, Hahn; Espley, Richard V; Hellens, Roger P; Schwinn, Kathy E; Davies, Kevin M; Albert, Nick W

    2015-10-01

    The Md - MYB10 R6 gene from apple is capable of self-regulating in heterologous host species and enhancing anthocyanin pigmentation, but the activity of MYB10 is dependent on endogenous protein partners. Coloured foliage due to anthocyanin pigments (bronze/red/black) is an attractive trait that is often lacking in many bedding, ornamental and horticultural plants. Apples (Malus × domestica) containing an allelic variant of the anthocyanin regulator, Md-MYB10 R6 , are highly pigmented throughout the plant, due to autoregulation by MYB10 upon its own promoter. We investigated whether Md-MYB10 R6 from apple is capable of functioning within the heterologous host Petunia hybrida to generate plants with novel pigmentation patterns. The Md-MYB10 R6 transgene (MYB10-R6 pro :MYB10:MYB10 term ) activated anthocyanin synthesis when transiently expressed in Antirrhinum rosea (dorsea) petals and petunia leaf discs. Stable transgenic petunias containing Md-MYB10 R6 lacked foliar pigmentation but had coloured flowers, complementing the an2 phenotype of 'Mitchell' petunia. The absence of foliar pigmentation was due to the failure of the Md-MYB10 R6 gene to self-activate in vegetative tissues, suggesting that additional protein partners are required for Md-MYB10 to activate target genes in this heterologous system. In petunia flowers, where endogenous components including MYB-bHLH-WDR (MBW) proteins were present, expression of the Md-MYB10 R6 promoter was initiated, allowing auto-regulation to occur and activating anthocyanin production. Md-MYB10 is capable of operating within the petunia MBW gene regulation network that controls the expression of the anthocyanin biosynthesis genes, AN1 (bHLH) and MYBx (R3-MYB repressor) in petals.

  17. The crucial role of PpMYB10.1 in anthocyanin accumulation in peach and relationships between its allelic type and skin color phenotype.

    Science.gov (United States)

    Tuan, Pham Anh; Bai, Songling; Yaegaki, Hideaki; Tamura, Takayuki; Hihara, Seisuke; Moriguchi, Takaya; Oda, Kenji

    2015-11-18

    Red coloration of fruit skin is one of the most important traits in peach (Prunus persica), and it is mainly due to the accumulation of anthocyanins. Three MYB10 genes, PpMYB10.1, PpMYB10.2, and PpMYB10.3, have been reported as important regulators of red coloration and anthocyanin biosynthesis in peach fruit. In this study, contribution of PpMYB10.1/2/3 to anthocyanin accumulation in the fruit skin was investigated in the Japanese peach cultivars, white-skinned 'Mochizuki' and red-skinned 'Akatsuki'. We then investigated the relationships between allelic type of PpMYB10.1 and skin color phenotype in 23 Japanese peach cultivars for future establishment of DNA-marker. During the fruit development of 'Mochizuki' and 'Akatsuki', anthocyanin accumulation was observed only in the skin of red 'Akatsuki' fruit in the late ripening stages concomitant with high mRNA levels of the last step gene leading to anthocyanin accumulation, UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT). This was also correlated with the expression level of PpMYB10.1. Unlike PpMYB10.1, expression levels of PpMYB10.2/3 were low in the skin of both 'Mochizuki' and 'Akatsuki' throughout fruit development. Moreover, only PpMYB10.1 revealed expression levels associated with total anthocyanin accumulation in the leaves and flowers of 'Mochizuki' and 'Akatsuki'. Introduction of PpMYB10.1 into tobacco increased the expression of tobacco UFGT, resulting in higher anthocyanin accumulation and deeper red transgenic tobacco flowers; however, overexpression of PpMYB10.2/3 did not alter anthocyanin content and color of transgenic tobacco flowers when compared with wild-type flowers. Dual-luciferase assay showed that the co-infiltration of PpMYB10.1 with PpbHLH3 significantly increased the activity of PpUFGT promoter. We also found close relationships of two PpMYB10.1 allelic types, MYB10.1-1/MYB10.1-2, with the intensity of red skin coloration. We showed that PpMYB10.1 is a major regulator of anthocyanin

  18. Expression of the sweetpotato R2R3-type IbMYB1a gene induces anthocyanin accumulation in Arabidopsis.

    Science.gov (United States)

    Chu, Hyosub; Jeong, Jae Cheol; Kim, Wook-Jin; Chung, Dong Min; Jeon, Hyo Kon; Ahn, Young Ock; Kim, Sun Ha; Lee, Haeng-Soon; Kwak, Sang-Soo; Kim, Cha Young

    2013-06-01

    R2R3-type MYB transcription factors (TFs) play important roles in transcriptional regulation of anthocyanins. The R2R3-type IbMYB1 is known to be a key regulator of anthocyanin biosynthesis in the storage roots of sweetpotato. We previously showed that transient expression of IbMYB1a led to anthocyanin pigmentation in tobacco leaves. In this article, we generated transgenic Arabidopsis plants expressing the IbMYB1a gene under the control of CaMV 35S promoter, and the sweetpotato SPO and SWPA2 promoters. Overexpression of IbMYBa in transgenic Arabidopsis produced strong anthocyanin pigmentation in seedlings and generated a deep purple color in leaves, stems and seeds. Reverse transcription-polymerase chain reaction analysis showed that IbMYB1a expression induced upregulation of several structural genes in the anthocyanin biosynthetic pathway, including 4CL, CHI, F3'H, DFR, AGT, AAT and GST. Furthermore, overexpression of IbMYB1a led to enhanced expression of the AtTT8 (bHLH) and PAP1/AtMYB75 genes. high-performance liquid chromatography analysis revealed that IbMYB1a expression led to the production of cyanidin as a major core molecule of anthocyanidins in Arabidopsis, as occurs in the purple leaves of sweetpotato (cv. Sinzami). This result shows that the IbMYB1a TF is sufficient to induce anthocyanin accumulation in seedlings, leaves, stems and seeds of Arabidopsis plants. Copyright © Physiologia Plantarum 2012.

  19. Genome-wide organization and expression profiling of the R2R3-MYB transcription factor family in pineapple (Ananas comosus).

    Science.gov (United States)

    Liu, Chaoyang; Xie, Tao; Chen, Chenjie; Luan, Aiping; Long, Jianmei; Li, Chuhao; Ding, Yaqi; He, Yehua

    2017-07-01

    The MYB proteins comprise one of the largest families of plant transcription factors, which are involved in various plant physiological and biochemical processes. Pineapple (Ananas comosus) is one of three most important tropical fruits worldwide. The completion of pineapple genome sequencing provides a great opportunity to investigate the organization and evolutionary traits of pineapple MYB genes at the genome-wide level. In the present study, a total of 94 pineapple R2R3-MYB genes were identified and further phylogenetically classified into 26 subfamilies, as supported by the conserved gene structures and motif composition. Collinearity analysis indicated that the segmental duplication events played a crucial role in the expansion of pineapple MYB gene family. Further comparative phylogenetic analysis suggested that there have been functional divergences of MYB gene family during plant evolution. RNA-seq data from different tissues and developmental stages revealed distinct temporal and spatial expression profiles of the AcMYB genes. Further quantitative expression analysis showed the specific expression patterns of the selected putative stress-related AcMYB genes in response to distinct abiotic stress and hormonal treatments. The comprehensive expression analysis of the pineapple MYB genes, especially the tissue-preferential and stress-responsive genes, could provide valuable clues for further function characterization. In this work, we systematically identified AcMYB genes by analyzing the pineapple genome sequence using a set of bioinformatics approaches. Our findings provide a global insight into the organization, phylogeny and expression patterns of the pineapple R2R3-MYB genes, and hence contribute to the greater understanding of their biological roles in pineapple.

  20. Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis.

    Science.gov (United States)

    Trentin, Luca; Bresolin, Silvia; Giarin, Emanuela; Bardini, Michela; Serafin, Valentina; Accordi, Benedetta; Fais, Franco; Tenca, Claudya; De Lorenzo, Paola; Valsecchi, Maria Grazia; Cazzaniga, Giovanni; Kronnie, Geertruy Te; Basso, Giuseppe

    2016-10-04

    To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

  1. The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation in Medicago truncatula.

    Science.gov (United States)

    Jun, Ji Hyung; Liu, Chenggang; Xiao, Xirong; Dixon, Richard A

    2015-10-01

    Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes. © 2015 American Society of Plant Biologists. All rights reserved.

  2. Identification of Arabidopsis MYB56 as a novel substrate for CRL3(BPM) E3 ligases.

    Science.gov (United States)

    Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

    2015-02-01

    Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  3. The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation in Medicago truncatula[OPEN

    Science.gov (United States)

    2015-01-01

    Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes. PMID:26410301

  4. The FOUR LIPS and MYB88 transcription factor genes are widely expressed in Arabidopsis thaliana during development.

    Science.gov (United States)

    Lei, Qin; Lee, EunKyoung; Keerthisinghe, Sandra; Lai, Lien; Li, Meng; Lucas, Jessica R; Wen, Xiaohong; Ren, Xiaolin; Sack, Fred D

    2015-09-01

    The FOUR LIPS (FLP) and MYB88 transcription factors, which are closely related in structure and function, control the development of stomata, as well as entry into megasporogenesis in Arabidopsis thaliana. However, other locations where these transcription factors are expressed are poorly described. Documenting additional locations where these genes are expressed might define new functions for these genes. Expression patterns were examined throughout vegetative and reproductive development. The expression from two transcriptional-reporter fusions were visualized with either β-glucuronidase (GUS) or green fluorescence protein (GFP). Both flp and myb88 genes were expressed in many, previously unreported locations, consistent with the possibility of additional functions for FLP and MYB88. Moreover, expression domains especially of FLP display sharp cutoffs or boundaries. In addition to stomatal and reproductive development, FLP and MYB88, which are R2R3 MYB transcription factor genes, are expressed in many locations in cells, tissues, and organs. © 2015 Botanical Society of America.

  5. Hoxa9 and Hoxa10 induce CML myeloid blast crisis development through activation of Myb expression.

    Science.gov (United States)

    Negi, Vijay; Vishwakarma, Bandana A; Chu, Su; Oakley, Kevin; Han, Yufen; Bhatia, Ravi; Du, Yang

    2017-11-17

    Mechanisms underlying the progression of Chronic Myeloid Leukemia (CML) from chronic phase to myeloid blast crisis are poorly understood. Our previous studies have suggested that overexpression of SETBP1 can drive this progression by conferring unlimited self-renewal capability to granulocyte macrophage progenitors (GMPs). Here we show that overexpression of Hoxa9 or Hoxa10 , both transcriptional targets of Setbp1 , is also sufficient to induce self-renewal of primary myeloid progenitors, causing their immortalization in culture. More importantly, both are able to cooperate with BCR/ABL to consistently induce transformation of mouse GMPs and development of aggressive leukemias resembling CML myeloid blast crisis, suggesting that either gene can drive CML progression by promoting the self-renewal of GMPs. We further identify Myb as a common critical target for Hoxa9 and Hoxa10 in inducing self-renewal of myeloid progenitors as Myb knockdown significantly reduced colony-forming potential of myeloid progenitors immortalized by the expression of either gene. Interestingly, Myb is also capable of immortalizing primary myeloid progenitors in culture and cooperating with BCR/ABL to induce leukemic transformation of mouse GMPs. Significantly increased levels of MYB transcript also were detected in all human CML blast crisis samples examined over chronic phase samples, further suggesting the possibility that MYB overexpression may play a prevalent role in driving human CML myeloid blast crisis development. In summary, our results identify overexpression of HOXA9 , HOXA10 , and MYB as critical drivers of CML progression, and suggest MYB as a key therapeutic target for inhibiting the self-renewal of leukemia-initiating cells in CML myeloid blast crisis patients.

  6. A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    Full Text Available Herba epimedii (Epimedium, a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1 from Epimedium sagittatum (Sieb. Et Zucc. Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR and anthocyanidin synthase (ANS. In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

  7. A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

    Science.gov (United States)

    Huang, Wenjun; Sun, Wei; Lv, Haiyan; Luo, Ming; Zeng, Shaohua; Pattanaik, Sitakanta; Yuan, Ling; Wang, Ying

    2013-01-01

    Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

  8. A DNA probe combination for improved detection of MLL/11q23 breakpoints by double-color interphase-FISH in acute leukemias.

    NARCIS (Netherlands)

    Bergh, A. von; Emanuel, B.; Zelderen-Bhola, S. van; Smetsers, A.F.C.M.; Soest, R. van; Stul, M.; Vranckx, H.; Schuuring, E.; Hagemeijer, A.; Kluin, P.

    2000-01-01

    Reciprocal translocations involving the MLL gene on chromosome band 11q23 have been observed in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In AML, identification of MLL breakpoints is an important prognostic factor. Breakpoints are clustered in an 8 kb DNA fragment

  9. A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates.

    Directory of Open Access Journals (Sweden)

    Ida Elken Sønderby

    Full Text Available BACKGROUND: Glucosinolates are natural metabolites in the order Brassicales that defend plants against both herbivores and pathogens and can attract specialized insects. Knowledge about the genes controlling glucosinolate regulation is limited. Here, we identify three R2R3 MYB transcription factors regulating aliphatic glucosinolate biosynthesis in Arabidopsis by combining several systems biology tools. METHODOLOGY/PRINCIPAL FINDINGS: MYB28 was identified as a candidate regulator of aliphatic glucosinolates based on its co-localization within a genomic region controlling variation both in aliphatic glucosinolate content (metabolite QTL and in transcript level for genes involved in the biosynthesis of aliphatic glucosinolates (expression QTL, as well as its co-expression with genes in aliphatic glucosinolate biosynthesis. A phylogenetic analysis with the R2R3 motif of MYB28 showed that it and two homologues, MYB29 and MYB76, were members of an Arabidopsis-specific clade that included three characterized regulators of indole glucosinolates. Over-expression of the individual MYB genes showed that they all had the capacity to increase the production of aliphatic glucosinolates in leaves and seeds and induce gene expression of aliphatic biosynthetic genes within leaves. Analysis of leaves and seeds of single knockout mutants showed that mutants of MYB29 and MYB76 have reductions in only short-chained aliphatic glucosinolates whereas a mutant in MYB28 has reductions in both short- and long-chained aliphatic glucosinolates. Furthermore, analysis of a double knockout in MYB28 and MYB29 identified an emergent property of the system since the absence of aliphatic glucosinolates in these plants could not be predicted by the chemotype of the single knockouts. CONCLUSIONS/SIGNIFICANCE: It seems that these cruciferous-specific MYB regulatory genes have evolved both overlapping and specific regulatory capacities. This provides a unique system within which to

  10. A spontaneous dominant-negative mutation within a 35S::AtMYB90 transgene inhibits flower pigment production in tobacco.

    Science.gov (United States)

    Velten, Jeff; Cakir, Cahid; Cazzonelli, Christopher I

    2010-03-29

    In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation. A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production. Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors.

  11. Transient spontaneous remission in congenital MLL-AF10 rearranged acute myeloid leukemia presenting with cardiorespiratory failure and meconium ileus.

    Science.gov (United States)

    Gyárfás, Tobias; Wintgens, Juergen; Biskup, Wolfgang; Oschlies, Ilske; Klapper, Wolfram; Siebert, Reiner; Bens, Susanne; Haferlach, Claudia; Meisel, Roland; Kuhlen, Michaela; Borkhardt, Arndt

    2016-12-01

    Neonatal leukemia is a rare disease with an estimated prevalence of about one to five in a million neonates. The majority being acute myeloid leukemia (AML), neonatal leukemia can present with a variety of symptoms including hyperleucocytosis, cytopenia, hepatosplenomegaly, and skin infiltrates. Chromosomal rearrangements including mixed lineage leukemia (MLL) translocations are common in neonatal AML. A female neonate born at 34 weeks gestation presented with cardiorespiratory failure, hepatosplenomegaly, pancytopenia, and coagulopathy. She required intensive care treatment including mechanical ventilation, high-dose catecholamine therapy, and multiple transfusions. Small intestinal biopsy obtained during laparotomy for meconium ileus revealed an infiltrate by an undifferentiated monoblastic, MLL-rearranged leukemia. No other manifestations of leukemia could be detected. After spontaneous clinical remission, lasting 5 months without any specific treatment, the patient presented with leukemia cutis and full-blown monoblastic leukemia. MLL-AF10-rearranged AML could be re-diagnosed and successfully treated with chemotherapy and hematopoietic stem cell transplantation. Our patient exhibited a unique manifestation of neonatal MLL-AF10 rearranged AML with cardiorespiratory failure and intestinal infiltration. It highlights the importance of leukemia in the differential diagnosis of neonatal distress, congenital hematological abnormalities, and skin lesions.

  12. LcMYB1 is a key determinant of differential anthocyanin accumulation among genotypes, tissues, developmental phases and ABA and light stimuli in Litchi chinensis.

    Directory of Open Access Journals (Sweden)

    Biao Lai

    Full Text Available The red coloration of litchi fruit depends on the accumulation of anthocyanins. The anthocyanins level in litchi fruit varies widely among cultivars, developmental stages and environmental stimuli. Previous studies on various plant species demonstrate that anthocyanin biosynthesis is controlled at the transcriptional level. Here, we describe a litchi R2R3-MYB transcription factor gene, LcMYB1, which demonstrates a similar sequence as other known anthocyanin regulators. The transcription levels of the LcMYB1 and anthocyanin biosynthetic genes were investigated in samples with different anthocyanin levels. The expression of LcMYB1 was strongly associated with tissue anthocyanin content. LcMYB1 transcripts were only detected in anthocyanin-accumulating tissues and were positively correlated with anthocyanin accumulation in the pericarps of 12 genotypes. ABA and sunlight exposure promoted, whereas CPPU and bagging inhibited the expression of LcMYB1 and anthocyanin accumulation in the pericarp. Cis-elements associated with light responsiveness and abscisic acid responsiveness were identified in the promoter region of LcMYB1. Among the 6 structural genes tested, only LcUFGT was highly correlated with LcMYB1. These results suggest that LcMYB1 controls anthocyanin biosynthesis in litchi and LcUFGT might be the structural gene that is targeted and regulated by LcMYB1. Furthermore, the overexpression of LcMYB1 induced anthocyanin accumulation in all tissues in tobacco, confirming the function of LcMYB1 in the regulation of anthocyanin biosynthesis. The upregulation of NtAn1b in response to LcMYB1 overexpression seems to be essential for anthocyanin accumulation in the leaf and pedicel. In the reproductive tissues of transgenic tobacco, however, increased anthocyanin accumulation is independent of tobacco's endogenous MYB and bHLH transcriptional factors, but associated with the upregulation of specific structural genes.

  13. LcMYB1 is a key determinant of differential anthocyanin accumulation among genotypes, tissues, developmental phases and ABA and light stimuli in Litchi chinensis.

    Science.gov (United States)

    Lai, Biao; Li, Xiao-Jing; Hu, Bing; Qin, Yong-Hua; Huang, Xu-Ming; Wang, Hui-Cong; Hu, Gui-Bing

    2014-01-01

    The red coloration of litchi fruit depends on the accumulation of anthocyanins. The anthocyanins level in litchi fruit varies widely among cultivars, developmental stages and environmental stimuli. Previous studies on various plant species demonstrate that anthocyanin biosynthesis is controlled at the transcriptional level. Here, we describe a litchi R2R3-MYB transcription factor gene, LcMYB1, which demonstrates a similar sequence as other known anthocyanin regulators. The transcription levels of the LcMYB1 and anthocyanin biosynthetic genes were investigated in samples with different anthocyanin levels. The expression of LcMYB1 was strongly associated with tissue anthocyanin content. LcMYB1 transcripts were only detected in anthocyanin-accumulating tissues and were positively correlated with anthocyanin accumulation in the pericarps of 12 genotypes. ABA and sunlight exposure promoted, whereas CPPU and bagging inhibited the expression of LcMYB1 and anthocyanin accumulation in the pericarp. Cis-elements associated with light responsiveness and abscisic acid responsiveness were identified in the promoter region of LcMYB1. Among the 6 structural genes tested, only LcUFGT was highly correlated with LcMYB1. These results suggest that LcMYB1 controls anthocyanin biosynthesis in litchi and LcUFGT might be the structural gene that is targeted and regulated by LcMYB1. Furthermore, the overexpression of LcMYB1 induced anthocyanin accumulation in all tissues in tobacco, confirming the function of LcMYB1 in the regulation of anthocyanin biosynthesis. The upregulation of NtAn1b in response to LcMYB1 overexpression seems to be essential for anthocyanin accumulation in the leaf and pedicel. In the reproductive tissues of transgenic tobacco, however, increased anthocyanin accumulation is independent of tobacco's endogenous MYB and bHLH transcriptional factors, but associated with the upregulation of specific structural genes.

  14. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae (on linr)

    OpenAIRE

    Wang, Kui-Lin; Bolitho, Karen; Grafton, Karryn; Kortstee, A.J.; Karunairetnam, Sakuntala; McGhie, T.K.; Espley, R.V.; Hellens, R.P.; Allan, A.C.

    2010-01-01

    Background - The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all th...

  15. Identification of Arabidopsis MYB56 as a novel substrate for CRL3BPM E3 ligases.

    Science.gov (United States)

    Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

    2014-10-24

    Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies pointed out that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling the flowering time point in plants. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

  16. Favorable outcome in non-infant children with MLL-AF4-positive acute lymphoblastic leukemia: a report from the Tokyo Children's Cancer Study Group.

    Science.gov (United States)

    Tomizawa, Daisuke; Kato, Motohiro; Takahashi, Hiroyuki; Fujimura, Junya; Inukai, Takeshi; Fukushima, Takashi; Kiyokawa, Nobutaka; Koh, Katsuyoshi; Manabe, Atsushi; Ohara, Akira

    2015-11-01

    Unlike acute lymphoblastic leukemia (ALL) in infants, MLL gene rearrangement (MLL-r) is rare in ALL children (≥1 year old). The outcome and optimal treatment options for MLL-r ALL remain controversial. Among the 1827 children enrolled in the Tokyo Children's Cancer Study Group ALL studies L95-14, L99-15, L99-1502, L04-16, and L07-1602 (1995-2009), 25 MLL-r ALL patients (1.3 %) were identified. Their median age and leukocyte count at diagnosis was 2 years old (range 1-15 years) and 27,690/μL (range 1800-1,113,000/μL), respectively. All but one patient achieved complete remission (CR) after induction therapy, and 19 underwent allogeneic hematopoietic stem cell transplantation (HSCT) in first CR according to the protocol. The 5-year event-free survival (EFS) and overall survival (OS) rate were 60.0 % [standard error (SE), 9.7 %] and 64.0 % (SE 9.6 %), respectively. Notably, 9/12 cases with MLL-AF4-positive ALL are alive in continuous CR with a 75.0 % (SE 12.5 %) EFS rate. The causes of treatment failure were as follows: one induction failure, five relapses, and five transplant-related deaths. With intensive chemotherapy and allogeneic HSCT, favorable outcome of children (≥1 year old) with MLL-AF4-positive ALL was observed. However, considering the risk of acute and late toxicities associated with HSCT, its indication should be restricted.

  17. Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra) fruit by a R2R3 MYB transcription factor.

    Science.gov (United States)

    Niu, Shan-Shan; Xu, Chang-Jie; Zhang, Wang-Shu; Zhang, Bo; Li, Xian; Lin-Wang, Kui; Ferguson, Ian B; Allan, Andrew C; Chen, Kun-Song

    2010-03-01

    Chinese bayberry (Myrica rubra) is a fruit crop with cultivars producing fruit ranging from white (Shuijing, SJ) to red (Dongkui, DK) and dark red-purple (Biqi, BQ), as a result of different levels of anthocyanin accumulation. Genes encoding the anthocyanin biosynthesis enzymes chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDPglucose: flavonoid 3-O-glucosyltransferase (UFGT), as well as MrMYB1, a R2R3 MYB transcription factor homologous to known activators of anthocyanin biosynthesis, were isolated from ripe fruit of BQ. Differences in mRNA abundance of MrF3H, MrF3'H, MrDFR1, MrANS and MrUFGT were highly correlated with differential accumulation of anthocyanins between cultivars, suggesting coordinated regulation by transcription factors. The transcript level of MrMYB1 was strongly associated with the anthocyanin content in ripe fruit of the three cultivars, as well as different anthocyanin containing tissues of BQ fruit. Fruit bagging strongly inhibited anthocyanin accumulation in fruit as well as the expression of all anthocyanin biosynthetic genes and MrMYB1. Overexpression of MrMYB1 stimulated both anthocyanin accumulation and activated an Arabidopsis-DFR promoter in tobacco (Nicotiana tabacum). MrMYB1d, an allele with a 1 bp deletion at nucleotide 30 of coding sequence, was observed in SJ and DK fruit, suggesting that a nonsense mutation of the MYB1 protein may be responsible for no or low expression of MYB1 in the white and red fruit. These results show that coordinated expression of multiple biosynthetic genes is involved in anthocyanin accumulation in Chinese bayberry fruit, and this is regulated by MrMYB1.

  18. DOT1L inhibitor EPZ-5676 displays synergistic antiproliferative activity in combination with standard of care drugs and hypomethylating agents in MLL-rearranged leukemia cells.

    Science.gov (United States)

    Klaus, Christine R; Iwanowicz, Dorothy; Johnston, Danielle; Campbell, Carly A; Smith, Jesse J; Moyer, Mikel P; Copeland, Robert A; Olhava, Edward J; Scott, Margaret Porter; Pollock, Roy M; Daigle, Scott R; Raimondi, Alejandra

    2014-09-01

    EPZ-5676 [(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol], a small-molecule inhibitor of the protein methyltransferase DOT1L, is currently under clinical investigation for acute leukemias bearing MLL-rearrangements (MLL-r). In this study, we evaluated EPZ-5676 in combination with standard of care (SOC) agents for acute leukemias as well as other chromatin-modifying drugs in cellular assays with three human acute leukemia cell lines: MOLM-13 (MLL-AF9), MV4-11 (MLL-AF4), and SKM-1 (non-MLL-r). Studies were performed to evaluate the antiproliferative effects of EPZ-5676 combinations in a cotreatment model in which the second agent was added simultaneously with EPZ-5676 at the beginning of the assay, or in a pretreatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. EPZ-5676 was found to act synergistically with the acute myeloid leukemia (AML) SOC agents cytarabine or daunorubicin in MOLM-13 and MV4-11 MLL-r cell lines. EPZ-5676 is selective for MLL-r cell lines as demonstrated by its lack of effect either alone or in combination in the nonrearranged SKM-1 cell line. In MLL-r cells, the combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the chemotherapeutic agents, suggesting that EPZ-5676 sets up a durable, altered chromatin state that enhances the chemotherapeutic effects. Our evaluation of EPZ-5676 in conjunction with other chromatin-modifying drugs also revealed a consistent combination benefit, including synergy with DNA hypomethylating agents. These results indicate that EPZ-5676 is highly efficacious as a single agent and synergistically acts with other chemotherapeutics, including AML SOC drugs and DNA hypomethylating agents in MLL-r cells. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Combinatorial analysis of lupulin gland transcription factors from R2R3Myb, bHLH and WDR families indicates a complex regulation of chs_H1 genes essential for prenylflavonoid biosynthesis in hop (Humulus Lupulus L.

    Directory of Open Access Journals (Sweden)

    Matoušek Jaroslav

    2012-02-01

    Full Text Available Abstract Background Lupulin glands of hop produce a specific metabolome including hop bitter acids valuable for the brewing process and prenylflavonoids with promising health-beneficial activities. The detailed analysis of the transcription factor (TF-mediated regulation of the oligofamily of one of the key enzymes, i.e., chalcone synthase CHS_H1 that efficiently catalyzes the production of naringenin chalcone, a direct precursor of prenylflavonoids in hop, constitutes an important part of the dissection of the biosynthetic pathways leading to the accumulation of these compounds. Results Homologues of flavonoid-regulating TFs HlMyb2 (M2, HlbHLH2 (B2 and HlWDR1 (W1 from hop were cloned using a lupulin gland-specific cDNA library from the hop variety Osvald's 72. Using a "combinatorial" transient GUS expression system it was shown that these unique lupulin-gland-associated TFs significantly activated the promoter (P of chs_H1 in ternary combinations of B2, W1 and either M2 or the previously characterized HlMyb3 (M3. The promoter activation was strongly dependent on the Myb-P binding box TCCTACC having a core sequence CCWACC positioned on its 5' end region and it seems that the complexity of the promoter plays an important role. M2B2W1-mediated activation significantly exceeded the strength of expression of native chs_H1 gene driven by the 35S promoter of CaMV, while M3B2W1 resulted in 30% of the 35S:chs_H1 expression level, as quantified by real-time PCR. Another newly cloned hop TF, HlMyb7, containing a transcriptional repressor-like motif pdLNLD/ELxiG/S (PDLNLELRIS, was identified as an efficient inhibitor of chs_H1-activating TFs. Comparative analyses of hop and A. thaliana TFs revealed a complex activation of Pchs_H1 and Pchs4 in combinatorial or independent manners. Conclusions This study on the sequences and functions of various lupulin gland-specific transcription factors provides insight into the complex character of the regulation of the

  20. A dominant negative mutant of an Arabidopsis R2R3 Myb (AtMyb90) blocks flower pigment production in tobacco

    Science.gov (United States)

    A spontaneous mutation converted a hyper-pigmented (anthocyanins), CaMV-35S-pro::AtMYB90 containing, transgenic tobacco line into one displaying wild-type pigmentation in all tissues except for flower petals, which, counter-intuitively, showed anthocyanin levels dramatically below wild-type in the p...

  1. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  2. ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells.

    Science.gov (United States)

    Garrison, Brian S; Rybak, Adrian P; Beerman, Isabel; Heesters, Balthasar; Mercier, Francois E; Scadden, David T; Bryder, David; Baron, Roland; Rossi, Derrick J

    2017-08-03

    The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521 / Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521 -deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.

  3. Alcohol binding in the C1 (C1A + C1B) domain of protein kinase C epsilon

    Science.gov (United States)

    Pany, Satyabrata; Das, Joydip

    2015-01-01

    Background Alcohol regulates the expression and function of protein kinase C epsilon (PKCε). In a previous study we identified an alcohol binding site in the C1B, one of the twin C1 subdomains of PKCε. Methods In this study, we investigated alcohol binding in the entire C1 domain (combined C1A and C1B) of PKCε. Fluorescent phorbol ester, SAPD and fluorescent diacylglycerol (DAG) analog, dansyl-DAG were used to study the effect of ethanol, butanol, and octanol on the ligand binding using fluorescence resonance energy transfer (FRET). To identify alcohol binding site(s), PKCεC1 was photolabeled with 3-azibutanol and 3-azioctanol, and analyzed by mass spectrometry. The effects of alcohols and the azialcohols on PKCε were studied in NG108-15 cells. Results In the presence of alcohol, SAPD and dansyl-DAG showed different extent of FRET, indicating differential effects of alcohol on the C1A and C1B subdomains. Effects of alcohols and azialcohols on PKCε in NG108-15 cells were comparable. Azialcohols labeled Tyr-176 of C1A and Tyr-250 of C1B. Inspection of the model structure of PKCεC1 reveals that these residues are 40 Å apart from each other indicating that these residues form two different alcohol binding sites. Conclusions The present results provide evidence for the presence of multiple alcohol-binding sites on PKCε and underscore the importance of targeting this PKC isoform in developing alcohol antagonists. PMID:26210390

  4. Purification of tracer for somatomedin C radioimmunoassay by hydrophobic interaction chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Baxter, R.C.; Brown, A.S.

    1982-03-01

    A tracer for use in the somatomedin C radiommunoassay by hydrophobic interaction chromatography was purified. Material showing greatest immunoreactivity binds to Octyl Sepharose CL-4B (Pharmacia) in a buffer mixture consisting of 130 mL of acetonitrile and 870 mL of 0.1 mol/L NH/sub 4/HCO/sub 3/, pH 7.8, but is eluted by increasing the acetonitrile content to 180 mL/L. As compared with tracer purified by binding to specific antiserum in liquid phase, precipitating the complex with second antibody, and then dissociating by gel chromatography at acid pH, this tracer shows equal immunoreactivity against specific somatomedin C antiserum. Either preparation allows excellent discrimination between extracts of normal, acromegalic, and hypopituitary plasma samples; thus either is suitable for use in the somatomedin C radioimmunoassay. Tracer purification by hydrophobic interaction chromatography is rapid and inexpensive. It may be useful in preparing highly immunoreactive tracers for other peptide radioimmunoassays.

  5. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation.

    Science.gov (United States)

    Koues, Olivia I; Mehta, Ninad T; Truax, Agnieszka D; Dudley, R Kyle; Brooks, Jeanne K; Greer, Susanna F

    2010-02-04

    Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription. Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS) complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  6. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation

    Directory of Open Access Journals (Sweden)

    Koues Olivia I

    2010-02-01

    Full Text Available Abstract Background Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC-II and class II transactivator (CIITA promoters, implicating Sug1 in events required to initiate mammalian transcription. Results Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL/complex of proteins associated with Set I (COMPASS complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Conclusion Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  7. The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

    Science.gov (United States)

    Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

    2017-12-05

    Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Genome-wide identification and characterization of R2R3MYB family in Rosaceae.

    Science.gov (United States)

    González, Máximo; Carrasco, Basilio; Salazar, Erika

    2016-09-01

    Transcription factors R2R3MYB family have been associated with the control of secondary metabolites, development of structures, cold tolerance and response to biotic and abiotic stress, among others. In recent years, genomes of Rosaceae botanical family are available. Although this information has been used to study the karyotype evolution of these species from an ancestral genome, there are no studies that treat the evolution and diversity of gene families present in these species or in the botanical family. Here we present the first comparative study of the R2R3MYB subfamily of transcription factors in three species of Rosaceae family (Malus domestica, Prunus persica and Fragaria vesca). We described 186, 98 and 86 non-redundant gene models for apple, peach and strawberry, respectively. In this research, we analyzed the intron-exon structure and genomic distribution of R2R3MYB families mentioned above. The phylogenetic comparisons revealed putative functions of some R2R3MYB transcription factors. This analysis found 44 functional subgroups, seven of which were unique for Rosaceae. In addition, our results showed a highly collinearity among some genes revealing the existence of conserved gene models between the three species studied. Although some gene models in these species have been validated under several approaches, more research in the Rosaceae family is necessary to determine gene expression patterns in specific tissues and development stages to facilitate understanding of the regulatory and biochemical mechanism in this botanical family.

  9. Overexpression of the IbMYB1 gene in an orange-fleshed sweet potato cultivar produces a dual-pigmented transgenic sweet potato with improved antioxidant activity.

    Science.gov (United States)

    Park, Sung-Chul; Kim, Yun-Hee; Kim, Sun Ha; Jeong, Yu Jeong; Kim, Cha Young; Lee, Joon Seol; Bae, Ji-Yeong; Ahn, Mi-Jeong; Jeong, Jae Cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

    2015-04-01

    The R2R3-type protein IbMYB1 is a key regulator of anthocyanin biosynthesis in the storage roots of sweet potato [Ipomoea batatas (L.) Lam]. Previously, we demonstrated that IbMYB1 expression stimulated anthocyanin pigmentation in tobacco leaves and Arabidopsis. Here, we generated dual-pigmented transgenic sweet potato plants that accumulated high levels of both anthocyanins and carotenoids in a single sweet potato storage root. An orange-fleshed cultivar with high carotenoid levels was transformed with the IbMYB1 gene under the control of either the storage root-specific sporamin 1 (SPO1) promoter or the oxidative stress-inducible peroxidase anionic 2 (SWPA2) promoter. The SPO1-MYB transgenic lines exhibited higher anthocyanin levels in storage roots than empty vector control (EV) or SWPA2-MYB plants, but carotenoid content was unchanged. SWPA2-MYB transgenic lines exhibited higher levels of both anthocyanin and carotenoids than EV plants. Analysis of hydrolyzed anthocyanin extracts indicated that cyanidin and peonidin predominated in both overexpression lines. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that IbMYB1 expression in both IbMYB1 transgenic lines strongly induced the upregulation of several genes in the anthocyanin biosynthetic pathway, whereas the expression of carotenoid biosynthetic pathway genes varied between transgenic lines. Increased anthocyanin levels in transgenic plants also promoted the elevation of proanthocyanidin and total phenolic levels in fresh storage roots. Consequently, all IbMYB1 transgenic plants displayed much higher antioxidant activities than EV plants. In field cultivations, storage root yields varied between the transgenic lines. Taken together, our results indicate that overexpression of IbMYB1 is a highly promising strategy for the generation of transgenic plants with enhanced antioxidant capacity. © 2014 Scandinavian Plant Physiology Society.

  10. Ion Binding Energies Determining Functional Transport of ClC Proteins

    Science.gov (United States)

    Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

    2014-06-01

    The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

  11. Two LcbHLH transcription factors interacting with LcMYB1 in regulating late structural genes of anthocyanin biosynthesis in Nicotiana and Litchi chinensis during anthocyanin accumulation

    Directory of Open Access Journals (Sweden)

    Biao eLai

    2016-02-01

    Full Text Available Anthocyanin biosynthesis requires the MYB-bHLH-WD40 protein complex to activate the late biosynthetic genes. LcMYB1 was thought to act as key regulator in anthocyanin biosynthesis of litchi. However, basic helix-loop-helix proteins (bHLHs as partners have not been identified yet. The present study describes the functional characterization of three litchi bHLH candidate anthocyanin regulators, LcbHLH1, LcbHLH2 and LcbHLH3. Although these three litchi bHLHs phylogenetically clustered with bHLH proteins involved in anthcoyanin biosynthesis in other plant, only LcbHLH1 and LcbHLH3 were found to localize in the nucleus and physically interact with LcMYB1. The transcription levels of all these bHLHs were not coordinated with anthocyanin accumulation in different tissues and during development. However, when co-infiltrated with LcMYB1, both LcbHLH1 and LcbHLH3 enhanced anthocyanin accumulation in tobacco leaves with LcbHLH3 being the best inducer. Significant accumulation of anthocyanins in leaves transformed with the combination of LcMYB1 and LcbHLH3 were noticed, And this was associated with the up-regulation of two tobacco endogenous bHLH regulators, NtAn1a and NtAn1b, and late structural genes, like NtDFR and NtANS. Significant activity of the ANS promoter was observed in transient expression assays either with LcMYB1-LcbHLH1 or LcMYB1-LcbHLH3, while only minute activity was detected after transformation with only LcMYB1. In contrast, no activity was measured after induction with the combination of LcbHLH2 and LcMYB1. Higher DFR expression was also oberseved in paralleling with higher anthocyanins in co-transformed lines. LcbHLH1 and LcbHLH3 are essential partner of LcMYB1 in regulating the anthocyanin production in tobacco and probably also in litchi. The LcMYB1-LcbHLH complex enhanced anthocyanin accumulation may associate with activating the transcription of DFR and ANS.

  12. The Jasmonate-ZIM-domain proteins interact with the WD-Repeat/bHLH/MYB complexes to regulate Jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana.

    Science.gov (United States)

    Qi, Tiancong; Song, Susheng; Ren, Qingcuo; Wu, Dewei; Huang, Huang; Chen, Yan; Fan, Meng; Peng, Wen; Ren, Chunmei; Xie, Daoxin

    2011-05-01

    Jasmonates (JAs) mediate plant responses to insect attack, wounding, pathogen infection, stress, and UV damage and regulate plant fertility, anthocyanin accumulation, trichome formation, and many other plant developmental processes. Arabidopsis thaliana Jasmonate ZIM-domain (JAZ) proteins, substrates of the CORONATINE INSENSITIVE1 (COI1)-based SCF(COI1) complex, negatively regulate these plant responses. Little is known about the molecular mechanism for JA regulation of anthocyanin accumulation and trichome initiation. In this study, we revealed that JAZ proteins interact with bHLH (Transparent Testa8, Glabra3 [GL3], and Enhancer of Glabra3 [EGL3]) and R2R3 MYB transcription factors (MYB75 and Glabra1), essential components of WD-repeat/bHLH/MYB transcriptional complexes, to repress JA-regulated anthocyanin accumulation and trichome initiation. Genetic and physiological evidence showed that JA regulates WD-repeat/bHLH/MYB complex-mediated anthocyanin accumulation and trichome initiation in a COI1-dependent manner. Overexpression of the MYB transcription factor MYB75 and bHLH factors (GL3 and EGL3) restored anthocyanin accumulation and trichome initiation in the coi1 mutant, respectively. We speculate that the JA-induced degradation of JAZ proteins abolishes the interactions of JAZ proteins with bHLH and MYB factors, allowing the transcriptional function of WD-repeat/bHLH/MYB complexes, which subsequently activate respective downstream signal cascades to modulate anthocyanin accumulation and trichome initiation.

  13. Enhancers of Polycomb EPC1 and EPC2 sustain the oncogenic potential of MLL leukemia stem cells

    Science.gov (United States)

    Huang, Xu; Spencer, Gary J; Lynch, James T; Ciceri, Filippo; Somerville, Tim D D; Somervaille, Tim C P

    2013-01-01

    Through a targeted knockdown (KD) screen of chromatin regulatory genes we identified the EP400 complex components EPC1 and EPC2 as critical oncogenic co-factors in acute myeloid leukemia (AML). EPC1 and EPC2 were required for the clonogenic potential of human AML cells of multiple molecular subtypes. Focusing on MLL-mutated AML as an exemplar, Epc1 or Epc2 KD induced apoptosis of murine MLL-AF9 AML cells and abolished leukemia stem cell potential. By contrast, normal hematopoietic stem and progenitor cells (HSPC) were spared. Similar selectivity was observed for human primary AML cells versus normal CD34+ HSPC. In keeping with these distinct functional consequences, Epc1 or Epc2 KD induced divergent transcriptional consequences in murine MLL-AF9 granulocyte-macrophage progenitor-like (GMP) cells versus normal GMP, with a signature of increased MYC activity in leukemic but not normal cells. This was caused by accumulation of MYC protein and was also observed following KD of other EP400 complex genes. Pharmacological inhibition of MYC:MAX dimerization, or concomitant MYC KD, reduced apoptosis following EPC1 KD, linking the accumulation of MYC to cell death. Therefore EPC1 and EPC2 are components of a complex which directly or indirectly serves to prevent MYC accumulation and AML cell apoptosis, thus sustaining oncogenic potential. PMID:24166297

  14. Characterization of the dextran-binding domain in the glucan-binding protein C of Streptococcus mutans.

    Science.gov (United States)

    Takashima, Y; Fujita, K; Ardin, A C; Nagayama, K; Nomura, R; Nakano, K; Matsumoto-Nakano, M

    2015-10-01

    Streptococcus mutans produces multiple glucan-binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell-surface-associated protein involved in dextran-induced aggregation. The purpose of the present study was to characterize the dextran-binding domain of GbpC using bioinformatics analysis and molecular techniques. Bioinformatics analysis specified five possible regions containing molecular binding sites termed GB1 through GB5. Next, truncated recombinant GbpC (rGbpC) encoding each region was produced using a protein expression vector and five deletion mutant strains were generated, termed CDGB1 through CDGB5 respectively. The dextran-binding rates of truncated rGbpC that included the GB1, GB3, GB4 and GB5 regions in the upstream sequences were higher than that of the construct containing GB2 in the downstream region. In addition, the rates of dextran-binding for strains CDGB4 and CD1, which was entire gbpC deletion mutant, were significantly lower than for the other strains, while those of all other deletion mutants were quite similar to that of the parental strain MT8148. Biofilm structures formed by CDGB4 and CD1 were not as pronounced as that of MT8148, while those formed by other strains had greater density as compared to that of CD1. Our results suggest that the dextran-binding domain may be located in the GB4 region in the interior of the gbpC gene. Bioinformatics analysis is useful for determination of functional domains in many bacterial species. © 2015 The Society for Applied Microbiology.

  15. Metabolic engineering of apple by overexpression of the MdMyb10 gene

    Directory of Open Access Journals (Sweden)

    Khaled A.L. Rihani

    2017-06-01

    In the present study, the flavonoid pathway was successfully modified in apple by overexpressing the MdMyb10 transcription factor to validate the hypothesis of increased effect on plant disease resistance.

  16. MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny.

    NARCIS (Netherlands)

    Horton, S.J.; Jaques, J.; Woolthuis, C.; Dijk, J. van; Mesuraca, M.; Huls, G.A.; Morrone, G.; Vellenga, E.; Schuringa, J.J.

    2013-01-01

    The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of

  17. Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor.

    Science.gov (United States)

    Cheng, Yu-Jie; Kim, Myoung-Duck; Deng, Xi-Ping; Kwak, Sang-Soo; Chen, Wei

    2013-12-01

    IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes.

  18. LEDGF/p75 as a target for treatment of MLL-dependent leukemia and HIV infection

    Czech Academy of Sciences Publication Activity Database

    Čermáková, K.; Těšina, Petr; Demeulemeester, J.; El Ashkar, S.; Méreau, H.; Schwaller, J.; Christ, F.; Debyser, Z.; Řezáčová, Pavlína; Veverka, Václav; De Rijck, J.

    2015-01-01

    Roč. 22, č. 1 (2015), s. 30 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 Keywords : LEDGF/p75 * leukemia * MLL Subject RIV: CE - Biochemistry

  19. Morel-Lavallee Lesion (MLL) Mimicking A Soft Tissue Neoplasm.

    Science.gov (United States)

    Kumar, Sandeep; Hasan, Roumina; Kadavigere, Rajagopal; Maddukuri, Satish Babu; Puppala, Radha

    2015-04-01

    Morel-lavallee lesion (MLL) represents post traumatic subcutaneous cyst generally overlying bony prominences like greater trochanter, lower back, knee and scapula. A 51-year-old man presented with a swelling in left thigh since six years which was insidious in onset, gradually progressive in size and not associated with pain, fever or discharge. There was no history of trauma or any associated constitutional symptoms. Since there was no history of trauma recalled by the patient the clinical dilemma was between soft tissue sarcoma and cold abscess. We report a case of slow growing painless mass lesion of thigh, diagnosed on Magnetic Resonance Imaging (MRI) as morel lavallee lesion and describe its salient imaging features with treatment options.

  20. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae (on linr)

    NARCIS (Netherlands)

    Wang, Kui-Lin; Bolitho, Karen; Grafton, Karryn; Kortstee, A.J.; Karunairetnam, Sakuntala; McGhie, T.K.; Espley, R.V.; Hellens, R.P.; Allan, A.C.

    2010-01-01

    Background - The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the

  1. The oncoprotein v-Myb activates transcription of Gremlin 2 during in vitro differentiation of the chicken neural crest to melanoblasts

    Czech Academy of Sciences Publication Activity Database

    Starostová, Michaela; Čermák, Vladimír; Dvořáková, Marta; Karafiát, Vít; Kosla, Jan; Dvořák, Michal

    2014-01-01

    Roč. 540, č. 1 (2014), s. 122-129 ISSN 0378-1119 R&D Projects: GA AV ČR KAN200520801 Institutional support: RVO:68378050 Keywords : Melanocyte development * Tamoxifen-inducible v-Myb * v-Myb-dependent genes * PRDC * KRT19 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.138, year: 2014

  2. Upregulation of miR-150* and miR-630 induces apoptosis in pancreatic cancer cells by targeting IGF-1R.

    Directory of Open Access Journals (Sweden)

    Lulu Farhana

    Full Text Available MicroRNAs have been implicated in many critical cellular processes including apoptosis. We have previously found that apoptosis in pancreatic cancer cells was induced by adamantyl retinoid-related (ARR molecule 3-Cl-AHPC. Here we report that 3-Cl-AHPC-dependent apoptosis involves regulating a number of microRNAs including miR-150* and miR-630. 3-Cl-AHPC stimulated miR-150* expression and caused decreased expression of c-Myb and IGF-1R in the pancreatic cancer cells. 3-Cl-AHPC-mediated reduction of c-Myb resulted in diminished binding of c-Myb with IGF-1R and Bcl-2 promoters, thereby causing repression of their transcription and protein expression. Over-expression of miR-150* also resulted in diminished levels of c-Myb and Bcl-2 proteins. Furthermore, the addition of the miRNA inhibitor 2'-O-methylated miR-150 blocked 3-Cl-AHPC-mediated increase in miR-150* levels and abrogated loss of c-Myb protein. Knockdown of c-Myb in PANC-1 cells resulted in enhanced apoptosis both in the presence or absence of 3-Cl-AHPC confirming the anti-apoptotic property of c-Myb. Overexpression of miR-630 also induced apoptosis in the pancreatic cancer cells and inhibited target protein IGF-1R mRNA and protein expression. Together these results implicate key roles for miR-150* and miR-630 and their targeting of IGF-1R to promote apoptosis in pancreatic cancer cells.

  3. Los factores de transcripción tipo Myb, una familia de reguladores de la diferenciación celular conservada en los organismos eucariontes

    Directory of Open Access Journals (Sweden)

    Jenny Arratia

    2013-01-01

    Full Text Available La familia de proteínas Myb, ubicua en los eucariontes, se caracteriza por la presencia de un dominio de unión al ADN característico denominado dominio Myb. Éste consiste en una secuencia de aminoácidos conservados (50-53 aminoácidos que puede estar repetida entre dos (dominio mínimo de unión al ADN y hasta cuatro veces en la misma proteína. En las plantas, la familia Myb es muy numerosa, mientras que en los animales sólo se encuentran tres miembros, y en otros eucariontes se ha identificado al menos a un miembro de esta familia. Las proteínas Myb participan como activadores o represores transcripcionales en la regulación de procesos celulares fundamentales en el metabolismo o la diferenciación celular. La actividad de las proteínas Myb se regula a través de diversas modificaciones post-traduccionales dentro de las que destacan el estado redox, la fosforilación y la ubiquitinación.

  4. Identification of a R2R3-MYB gene regulating anthocyanin biosynthesis and relationships between its variation and flower color difference in lotus (Nelumbo Adans.

    Directory of Open Access Journals (Sweden)

    Shan-Shan Sun

    2016-09-01

    Full Text Available The lotus (Nelumbonaceae: Nelumbo Adans. is a highly desired ornamental plant, comprising only two extant species, the sacred lotus (N. nucifera Gaerten. with red flowers and the American lotus (N. lutea Willd. with yellow flowers. Flower color is the most obvious difference of two species. To better understand the mechanism of flower color differentiation, the content of anthocyanins and the expression levels of four key structural genes (e.g., DFR, ANS, UFGT and GST were analyzed in two species. Our results revealed that anthocyanins were detected in red flowers, not yellow flowers. Expression analysis showed that no transcripts of GST gene and low expression level of three UFGT genes were detected in yellow flowers. In addition, three regulatory genes (NnMYB5, NnbHLH1 and NnTTG1 were isolated from red flowers and showed a high similarity to corresponding regulatory genes of other species. Sequence analysis of MYB5, bHLH1 and TTG1 in two species revealed striking differences in coding region and promoter region of MYB5 gene. Population analysis identified three MYB5 variants in Nelumbo: a functional allele existed in red flowers and two inactive forms existed in yellow flowers. This result revealed that there was an association between allelic variation in MYB5 gene and flower color difference. Yeast two-hybrid experiments showed that NnMYB5 interacts with NnbHLH1, NlbHLH1 and NnTTG1, and NnTTG1 also interacts with NnbHLH1 and NlbHLH1. The over-expression of NnMYB5 led to anthocyanin accumulation in immature seeds and flower stalks and up-regulation of expression of TT19 in Arabidopsis. Therefore, NnMYB5 is a transcription activator of anthocyanin synthesis. This study helps to elucidate the function of NnMYB5 and will contribute to clarify the mechanism of flower coloration and genetic engineering of flower color in lotus.

  5. Frequency and clinical impact of ETV6/RUNX1, AF4‑MLL, and BCR ...

    African Journals Online (AJOL)

    Background: Variations in disease presentation and outcome of leukemia treatment has been associated with the presence of certain mutant genes. Three major translocations (ETV6‑RUNX1, BCR‑ABL, and AF4‑MLL) in acute lymphoblastic leukemia (ALL) have been shown to affect treatment outcome. This study is aimed ...

  6. Three R2R3 MYB transcription factor genes from Capsicum annuum ...

    African Journals Online (AJOL)

    Jane

    2011-08-08

    Aug 8, 2011 ... between plants and microbes, and in male fertility of some species .... (Gerbera hybrid, CAD87010), AmROSEA1 (Antirrhinum majus,ABB83826), ..... MYB26 results in male sterility due to non-dehiscent anthers. Plant J.

  7. Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

    Directory of Open Access Journals (Sweden)

    Alvaro Muñoz-López

    2016-10-01

    Full Text Available Induced pluripotent stem cells (iPSCs are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

  8. Modulation of flavonoid metabolites in Arabidopsis thaliana through overexpression of the MYB75 transcription factor: role of kaempferol-3,7-dirhamnoside in resistance to the specialist insect herbivore Pieris brassicae.

    Science.gov (United States)

    Onkokesung, Nawaporn; Reichelt, Michael; van Doorn, Arjen; Schuurink, Robert C; van Loon, Joop J A; Dicke, Marcel

    2014-05-01

    Anthocyanins and flavonols are secondary metabolites that can function in plant defence against herbivores. In Arabidopsis thaliana, anthocyanin and flavonol biosynthesis are regulated by MYB transcription factors. Overexpression of MYB75 (oxMYB75) in Arabidopsis results in increasing anthocyanin and flavonol levels which enhances plant resistance to generalist caterpillars. However, how these metabolites affect specialist herbivores has remained unknown. Performance of a specialist aphid (Brevicoryne brassicae) was unaffected after feeding on oxMYB75 plants, whereas a specialist caterpillar (Pieris brassicae) gained significantly higher body mass when feeding on this plant. An increase in anthocyanin and total flavonol glycoside levels correlated negatively with the body mass of caterpillars fed on oxMYB75 plants. However, a significant reduction of kaempferol-3,7-dirhamnoside (KRR) corresponded to an increased susceptibility of oxMYB75 plants to caterpillar feeding. Pieris brassicae caterpillars also grew less on an artificial diet containing KRR or on oxMYB75 plants that were exogenously treated with KRR, supporting KRR's function in direct defence against this specialist caterpillar. The results show that enhancing the activity of the anthocyanin pathway in oxMYB75 plants results in re-channelling of quercetin/kaempferol metabolites which has a negative effect on the accumulation of KRR, a novel defensive metabolite against a specialist caterpillar.

  9. Reflection-Based Python-C++ Bindings

    International Nuclear Information System (INIS)

    Generowicz, Jacek; Lavrijsen, Wim T.L.P.; Marino, Massimo; Mato, Pere

    2004-01-01

    Python is a flexible, powerful, high-level language with excellent interactive and introspective capabilities and a very clean syntax. As such, it can be a very effective tool for driving physics analysis. Python is designed to be extensible in low-level C-like languages, and its use as a scientific steering language has become quite widespread. To this end, existing and custom-written C or C++ libraries are bound to the Python environment as so-called extension modules. A number of tools for easing the process of creating such bindings exist, such as SWIG and Boost. Python. Yet, the process still requires a considerable amount of effort and expertise. The C++ language has few built-in introspective capabilities, but tools such as LCGDict and CINT add this by providing so-called dictionaries: libraries that contain information about the names, entry points, argument types, etc. of other libraries. The reflection information from these dictionaries can be used for the creation of bindings and so the process can be fully automated, as dictionaries are already provided for many end-user libraries for other purposes, such as object persistency. PyLCGDict is a Python extension module that uses LCG dictionaries, as PyROOT uses CINT reflection information, to allow /cwPython users to access C++ libraries with essentially no preparation on the users' behalf. In addition, and in a similar way, PyROOT gives ROOT users access to Python libraries

  10. Heterologous expression of AtMYB12 in kale (Brassica oleracea var. acephala) leads to high flavonol accumulation.

    Science.gov (United States)

    Lännenpää, Mika

    2014-08-01

    Overexpression of Arabidopsis AtMYB12 transcription factor greatly increases the total phenolic and flavonol content in transgenic kale leaves. Flavonoids are a diverse group of plant secondary metabolites exhibiting a number of health-promoting effects. There has been a growing interest to develop biotechnological methods for the enhanced production of flavonoids in crop plants. AtMYB12 is an Arabidopsis transcription factor which specifically activates flavonol synthesis and its overexpression has led to increased flavonol accumulation in several transgenic plants. In the present study, AtMYB12 was overexpressed in a commercial cultivar of kale and the transgenic plants were tested both in in vitro and in semi-field conditions in cages under natural light. Using this method, a severalfold increase in both total phenolics content and flavonol accumulation was achieved. This study provides a reliable and efficient transformation protocol for kale and suggests the potential of this flavonol-enriched vegetable for the production of kaempferol.

  11. Frequency and clinical impact of ETV6/RUNX1, AF4‑MLL, and BCR ...

    African Journals Online (AJOL)

    2015-05-12

    May 12, 2015 ... features of prognostic importance, which include total white blood cell count (P = 0.416) and FAB subtype (P = 0.576). Conclusion: Presence of fusion ... Frequency and clinical impact of ETV6/RUNX1, AF4‑MLL, and BCR/ABL fusion ... define distinct clinic‑pathological subgroups and have been used in risk ...

  12. Modulation of flavonoid metabolites in Arabidopsis thaliana through overexpression of the MYB75 transcription factor: role of kaempferol-3,7-dirhamnoside in resistance to the specialist insect herbivore Pieris brassicae

    NARCIS (Netherlands)

    Onkokesung, N.; Reichelt, M.; Doorn, van A.; Schuurink, R.C.; Loon, van J.J.A.; Dicke, M.

    2014-01-01

    Anthocyanins and flavonols are secondary metabolites that can function in plant defence against herbivores. In Arabidopsis thaliana, anthocyanin and flavonol biosynthesis are regulated by MYB transcription factors. Overexpression of MYB75 (oxMYB75) in Arabidopsis results in increasing anthocyanin

  13. Improvement of Haramay Fiber by Pre-treatment of Retting Process withPhosphoric Acid

    International Nuclear Information System (INIS)

    Kuntari-Sasas; Neni-Rustini Eriawati

    2000-01-01

    Haramay as bast fiber contains of cellulose fiber as the main part, mixedwith hemi cellulose, pectin, and lignin as binding material for cellulosefiber to keep it together in the bundle form. For textile material, this bastfiber has to be freed from its binding material, called as retting process,before subjecting to scouring, dyeing and finishing process in textileindustry. In the retting process the dissolve of binding material can be doneeither by using enzyme in bio technology or extraction with strong alkalinecondition in common technology. Using sodium hydroxide for dissolving thebinding material can be carried out easily with good dissolving ability, butcan render the strength retention of the cellulose fiber. Pre-treatment ofthe bast fiber with phosphoric acid (H 3 PO 4 ), is expected to hydrolyze someof the binding materials that can not be dissolved in alkaline condition,including natural pigment that colored the fiber with creamy white. In thisstudy, the pre-treatment process before retting with phosphoric acid wascarried out in various condition, such as concentration of phosphoric acid (5ml/l- 25 ml/l), time and temperature of pre-treatment (1-3 hours at 50 o C or12-24 hours at room temperature), followed by neutralization in dilutealkaline. The retting process was carried out by means of scouring in variousconcentration of sodium hydroxide (NaOH 38 o Be, 10 ml/l-30 m/l), and then wascontinued with bleaching process in hydrogen peroxide solution. Aftercarrying out those experiment, the bast fiber that called haramay wassubjected to testing for weight reduction, strength retention and degree ofwhiteness. From the testing results it is concluded that pre-treatment withphosphoric acid can increase the weight reduction, strength retention ortenacity and degree of whiteness of haramay fiber compared to the oneswithout pre-treatment with phosphoric acid. The best result was obtained bypre-treatment with 5 ml/l H 3 PO 4 at 50 o C for 2 hours, continued by

  14. Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Vesa Kirjavainen

    Full Text Available Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS O-antigen (O-ag and outer core (OC do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp, an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

  15. MdCOP1 Ubiquitin E3 Ligases Interact with MdMYB1 to Regulate Light-Induced Anthocyanin Biosynthesis and Red Fruit Coloration in Apple1[W][OA

    Science.gov (United States)

    Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

    2012-01-01

    MdMYB1 is a crucial regulator of light-induced anthocyanin biosynthesis and fruit coloration in apple (Malus domestica). In this study, it was found that MdMYB1 protein accumulated in the light but degraded via a ubiquitin-dependent pathway in the dark. Subsequently, the MdCOP1-1 and MdCOP1-2 genes were isolated from apple fruit peel and were functionally characterized in the Arabidopsis (Arabidopsis thaliana) cop1-4 mutant. Yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that MdMYB1 interacts with the MdCOP1 proteins. Furthermore, in vitro and in vivo experiments indicated that MdCOP1s are necessary for the ubiquitination and degradation of MdMYB1 protein in the dark and are therefore involved in the light-controlled stability of the MdMYB1 protein. Finally, a viral vector-based transformation approach demonstrated that MdCOP1s negatively regulate the peel coloration of apple fruits by modulating the degradation of the MdMYB1 protein. Our findings provide new insight into the mechanism by which light controls anthocyanin accumulation and red fruit coloration in apple and even other plant species. PMID:22855936

  16. Trichostatin a suppresses transformation by the v-myb oncogene in BM2 cells

    Czech Academy of Sciences Publication Activity Database

    Nemajerová, A.; Šmarda, J.; Jurdic, P.; Kubala, Lukáš; Souček, Karel; Šmardová, J.

    2003-01-01

    Roč. 12, č. 2 (2003), s. 225-235 ISSN 1525-8165 Institutional research plan: CEZ:AV0Z5004920 Keywords : v-myb * trichostatin A * differentation Subject RIV: BO - Biophysics Impact factor: 1.800, year: 2003

  17. Los factores de transcripción tipo Myb, una familia de reguladores de la diferenciación celular conservada en los organismos eucariontes

    OpenAIRE

    Arratia, Jenny; Aguirre, Jesús

    2013-01-01

    La familia de proteínas Myb, ubicua en los eucariontes, se caracteriza por la presencia de un dominio de unión al ADN característico denominado dominio Myb. Éste consiste en una secuencia de aminoácidos conservados (50-53 aminoácidos) que puede estar repetida entre dos (dominio mínimo de unión al ADN) y hasta cuatro veces en la misma proteína. En las plantas, la familia Myb es muy numerosa, mientras que en los animales sólo se encuentran tres miembros, y en otros eucariontes se ha identificad...

  18. C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

    Science.gov (United States)

    Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

    2015-09-11

    In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Conformational Ensembles of an Intrinsically Disordered Protein pKID with and without a KIX Domain in Explicit Solvent Investigated by All-Atom Multicanonical Molecular Dynamics

    Directory of Open Access Journals (Sweden)

    Haruki Nakamura

    2012-02-01

    Full Text Available The phosphorylated kinase-inducible activation domain (pKID adopts a helix–loop–helix structure upon binding to its partner KIX, although it is unstructured in the unbound state. The N-terminal and C-terminal regions of pKID, which adopt helices in the complex, are called, respectively, αA and αB. We performed all-atom multicanonical molecular dynamics simulations of pKID with and without KIX in explicit solvents to generate conformational ensembles. Although the unbound pKID was disordered overall, αA and αB exhibited a nascent helix propensity; the propensity of αA was stronger than that of αB, which agrees with experimental results. In the bound state, the free-energy landscape of αB involved two low free-energy fractions: native-like and non-native fractions. This result suggests that αB folds according to the induced-fit mechanism. The αB-helix direction was well aligned as in the NMR complex structure, although the αA helix exhibited high flexibility. These results also agree quantitatively with experimental observations. We have detected that the αB helix can bind to another site of KIX, to which another protein MLL also binds with the adopting helix. Consequently, MLL can facilitate pKID binding to the pKID-binding site by blocking the MLL-binding site. This also supports experimentally obtained results.

  20. Purification and characterization of cGMP binding protein-phosphodiesterase from rat lung

    International Nuclear Information System (INIS)

    Francis, S.H.; Walseth, T.F.; Corbin, J.D.

    1986-01-01

    The cGMP binding protein-phosphodiesterase (cG-BPP) with a phosphodiesterase specific activity of 7 μM/min/mg has been purified from rat lung by sequential chromatography on DEAE-cellulose, Blue-Sepharose, zinc chelate affinity adsorbent and HPLC-DEAE. Migration of the major band on SDS-PAGE corresponds to a MW of ∼93,000. Both cGMP phosphodiesterase activity and cGMP binding from the HPLC-DEAE profile correlate with this band. Since the authors previous work has determined the native MW to be ∼177,000, this suggests a dimeric structure comprised of two 93,000 MW subunits for the rat lung cG-BPP. At low cGMP concentrations, cGMP binding is stimulated ∼20-fold by histone and ∼5-fold by 3-isobutyl-1-methylxanthine(IBMX). The purified protein has one component of cGMP dissociation with a rate constant of 0.045/min. Photolysis of the purified protein in the presence of 32 P-cGMP labels the 93,000 MW band and this labeling is increased by IBMX, indicating that the 93,000 MW band is a subunit of the cGMP-BPP. This implies that the enzyme preparation is nearly homogeneous, a conclusion also supported by a minimum [ 3 H]-cGMP binding stoichiometry of 0.5 mol per 93,000 subunit. An additional protein band with a MW of ∼90,000 also occurs in these preparations which exhibits behavior similar to the 93,000 MW protein. N 2 -Hexyl-cGMP inhibits phosphodiesterase activity by competing with cGMP for hydrolysis at the catalytic site but not at the binding site. N 2 -Hexyl cGMP actually increases cGMP binding. This provides the first evidence that cGMP binding is increased by compounds hydrolyzed at the catalytic site. This interaction between the binding and phosphodiesterase sites could be important in the regulation of the functions of these sites in vivo

  1. Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning.

    Science.gov (United States)

    Albert, Nick W; Lewis, David H; Zhang, Huaibi; Schwinn, Kathy E; Jameson, Paula E; Davies, Kevin M

    2011-03-01

    We present an investigation of anthocyanin regulation over the entire petunia plant, determining the mechanisms governing complex floral pigmentation patterning and environmentally induced vegetative anthocyanin synthesis. DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) encode members of the R2R3-MYB transcription factor family that regulate anthocyanin synthesis in petunia, and control anthocyanin production in vegetative tissues and contribute to floral pigmentation. In addition to these two MYB factors, the basic helix-loop-helix (bHLH) factor ANTHOCYANIN1 (AN1) and WD-repeat protein AN11, are also essential for vegetative pigmentation. The induction of anthocyanins in vegetative tissues by high light was tightly correlated to the induction of transcripts for PHZ and AN1. Interestingly, transcripts for PhMYB27, a putative R2R3-MYB active repressor, were highly expressed during non-inductive shade conditions and repressed during high light. The competitive inhibitor PhMYBx (R3-MYB) was expressed under high light, which may provide feedback repression. In floral tissues DPL regulates vein-associated anthocyanin pigmentation in the flower tube, while PHZ determines light-induced anthocyanin accumulation on exposed petal surfaces (bud-blush). A model is presented suggesting how complex floral and vegetative pigmentation patterns are derived in petunia in terms of MYB, bHLH and WDR co-regulators. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  2. Mycobacterium tuberculosis cAMP Receptor Protein (Rv3676) Differs from the Escherichia coli Paradigm in Its cAMP Binding and DNA Binding Properties and Transcription Activation Properties*

    Science.gov (United States)

    Stapleton, Melanie; Haq, Ihtshamul; Hunt, Debbie M.; Arnvig, Kristine B.; Artymiuk, Peter J.; Buxton, Roger S.; Green, Jeffrey

    2010-01-01

    The pathogen Mycobacterium tuberculosis produces a burst of cAMP upon infection of macrophages. Bacterial cyclic AMP receptor proteins (CRP) are transcription factors that respond to cAMP by binding at target promoters when cAMP concentrations increase. Rv3676 (CRPMt) is a CRP family protein that regulates expression of genes (rpfA and whiB1) that are potentially involved in M. tuberculosis persistence and/or emergence from the dormant state. Here, the CRPMt homodimer is shown to bind two molecules of cAMP (one per protomer) at noninteracting sites. Furthermore, cAMP binding by CRPMt was relatively weak, entropy driven, and resulted in a relatively small enhancement in DNA binding. Tandem CRPMt-binding sites (CRP1 at −58.5 and CRP2 at −37.5) were identified at the whiB1 promoter (PwhiB1). In vitro transcription reactions showed that CRP1 is an activating site and that CRP2, which was only occupied in the presence of cAMP or at high CRPMt concentrations in the absence of cAMP, is a repressing site. Binding of CRPMt to CRP1 was not essential for open complex formation but was required for transcription activation. Thus, these data suggest that binding of CRPMt to the PwhiB1 CRP1 site activates transcription at a step after open complex formation. In contrast, high cAMP concentrations allowed occupation of both CRP1 and CRP2 sites, resulting in inhibition of open complex formation. Thus, M. tuberculosis CRP has evolved several distinct characteristics, compared with the Escherichia coli CRP paradigm, to allow it to regulate gene expression against a background of high concentrations of cAMP. PMID:20028978

  3. The Eucalyptus grandis R2R3-MYB transcription factor family: evidence for woody growth-related evolution and function.

    Science.gov (United States)

    Soler, Marçal; Camargo, Eduardo Leal Oliveira; Carocha, Victor; Cassan-Wang, Hua; San Clemente, Hélène; Savelli, Bruno; Hefer, Charles A; Paiva, Jorge A Pinto; Myburg, Alexander A; Grima-Pettenati, Jacqueline

    2015-06-01

    The R2R3-MYB family, one of the largest transcription factor families in higher plants, controls a wide variety of plant-specific processes including, notably, phenylpropanoid metabolism and secondary cell wall formation. We performed a genome-wide analysis of this superfamily in Eucalyptus, one of the most planted hardwood trees world-wide. A total of 141 predicted R2R3-MYB sequences identified in the Eucalyptus grandis genome sequence were subjected to comparative phylogenetic analyses with Arabidopsis thaliana, Oryza sativa, Populus trichocarpa and Vitis vinifera. We analysed features such as gene structure, conserved motifs and genome location. Transcript abundance patterns were assessed by RNAseq and validated by high-throughput quantitative PCR. We found some R2R3-MYB subgroups with expanded membership in E. grandis, V. vinifera and P. trichocarpa, and others preferentially found in woody species, suggesting diversification of specific functions in woody plants. By contrast, subgroups containing key genes regulating lignin biosynthesis and secondary cell wall formation are more conserved across all of the species analysed. In Eucalyptus, R2R3-MYB tandem gene duplications seem to disproportionately affect woody-preferential and woody-expanded subgroups. Interestingly, some of the genes belonging to woody-preferential subgroups show higher expression in the cambial region, suggesting a putative role in the regulation of secondary growth. © 2014 The Authors New Phytologist © 2014 New Phytologist Trust.

  4. Binding properties of Clostridium botulinum type C progenitor toxin to mucins.

    Science.gov (United States)

    Nakamura, Toshio; Takada, Noriko; Tonozuka, Takashi; Sakano, Yoshiyuki; Oguma, Keiji; Nishikawa, Atsushi

    2007-04-01

    It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327-333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl alpha2-3 lactose and sialyl alpha2-6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.

  5. Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

    Directory of Open Access Journals (Sweden)

    Birgit Piater

    Full Text Available The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF. Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX. CLN64 and a previously described single-stranded DNA (ssDNA aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.

  6. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    Science.gov (United States)

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-09

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  7. C5a binding to human polymorphonuclear leukocyte plasma membrane (PMNLM) receptors

    International Nuclear Information System (INIS)

    Conway, R.G.; Mollison, K.W.; Carter, G.W.; Lane, B.

    1986-01-01

    Previous investigations of the C5a receptor have been performed using intact human PMNL. To circumvent some of the potential problems with such whole cell assays (e.g. internalization or metabolism of radioligand) the authors have developed a PMNLM binding assay. Human PMNLM were prepared by nitrogen cavitation and Percoll gradient centrifugation. Specific binding of [ 125 I]C5a to PMNLM was: high affinity, K/sub D/ = 0.6 nM; saturable, B/sub max/ = 8.7 pmol/mg protein; and reversible. Kinetic measurements agree with the K/sub D/ value obtained by Scatchard analysis. Furthermore, the binding activity of C5a correlates with biological activity as measured by myeloperoxidase release from human PMNL. Human serum C5a and recombinant C5a bind with similar affinities when measured by competition or direct binding and label the same number of sites in human PMNLM. The nonhydrolyzable GTP analog, GppNHp, induces a low affinity state of the C5a receptor (4-6 fold shift in K/sub D/) with little effect on B/sub max/. In summary, the criteria have been satisfied for identification of a biologically relevant C5a binding site in human PMNLM. Regulation of the C5a receptor and its membrane transduction mechanism(s) appears to involve guanyl nucleotides, as has been found for other chemoattractant receptors

  8. Factor VII and protein C are phosphatidic acid-binding proteins.

    Science.gov (United States)

    Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H

    2013-08-20

    Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

  9. Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

    Science.gov (United States)

    Neupane, Durga P; Avalos, Dante; Fullam, Stephanie; Roychowdhury, Hridindu; Yukl, Erik T

    2017-10-20

    Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Binding of complement proteins C1q and C4bp to serum amyloid P component (SAP) in solid contra liquid phase

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Nielsen, EH; Andersen, Ove

    1996-01-01

    Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy....... Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')2 anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high...... affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate...

  11. Staphylococcus aureus manganese transport protein C (MntC is an extracellular matrix- and plasminogen-binding protein.

    Directory of Open Access Journals (Sweden)

    Natália Salazar

    Full Text Available Infections caused by Staphylococcus aureus--particularly nosocomial infections--represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM. Manganese transport protein C (MntC, a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA. The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

  12. The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

    Science.gov (United States)

    Rohrbeck, Astrid; Höltje, Markus; Adolf, Andrej; Oms, Elisabeth; Hagemann, Sandra; Ahnert-Hilger, Gudrun; Just, Ingo

    2017-10-27

    The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to β1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Ion-Regulated Allosteric Binding of Fullerenes (C-60 and C-70) by Tetrathiafulvalene-Calix[4]pyrroles

    DEFF Research Database (Denmark)

    Davis, C. M.; Lim, J. M.; Larsen, K. R.

    2014-01-01

    of the C4P in a ball-and-socket binding mode. The interactions between the TTF-C4P receptors and the fullerene guests are highly influenced by both the nature of halide anions and their counter tetraalkylammonium cations. Three halides (F-, Cl-, and Br-) were studied. All three potentiate the binding...

  14. Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers

    International Nuclear Information System (INIS)

    Pan, B.S.; Solaro, R.J.

    1987-01-01

    In order to obtain information with regard to behavior of the Ca 2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca 2+ -binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca 2+ -binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca 2+ -Mg 2+ and Ca 2+ -specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45 Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca 2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca 2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca 2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45 Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca 2+ -binding sites whose off-rate constant for Ca 2+ is significantly lower than the Ca 2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC

  15. Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor l-arginine

    International Nuclear Information System (INIS)

    Garnett, James A.; Baumberg, Simon; Stockley, Peter G.; Phillips, Simon E. V.

    2007-01-01

    The crystal structure of the C-terminal domain hexameric core of AhrC, with bound corepressor (l-arginine), has been solved at 1.95 Å resolution. Binding of l-arginine results in a rotation between the two trimers of the hexamer, leading to the activation of the DNA-binding state. The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolic sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and l-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor l-arginine. Here, the crystal structure refined to 1.95 Å is presented

  16. Overexpression of MYB115, AAD2, or AAD3 in Arabidopsis thaliana seeds yields contrasting omega-7 contents

    Science.gov (United States)

    To, Alexandra; Barthole, Guillaume; Lepiniec, Loïc

    2018-01-01

    Omega-7 monoenoic fatty acids (ω-7 FAs) are increasingly exploited both for their positive effects on health and for their industrial potential. Some plant species produce fruits or seeds with high amounts of ω-7 FAs. However, the low yields and poor agronomic properties of these plants preclude their commercial use. As an alternative, the metabolic engineering of oilseed crops for sustainable ω-7 FA production has been proposed. Two palmitoyl-ACP desaturases (PADs) catalyzing ω-7 FA biosynthesis were recently identified and characterized in Arabidopsis thaliana, together with MYB115 and MYB118, two transcription factors that positively control the expression of the corresponding PAD genes. In the present research, we examine the biotechnological potential of these new actors of ω-7 metabolism for the metabolic engineering of plant-based production of ω-7 FAs. We placed the PAD and MYB115 coding sequences under the control of a promoter strongly induced in seeds and evaluated these different constructs in A. thaliana. Seeds were obtained that exhibit ω-7 FA contents ranging from 10 to >50% of the total FAs, and these major compositional changes have no detrimental effect on seed germination. PMID:29381741

  17. CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis.

    Science.gov (United States)

    Zhu, Hui-Fen; Fitzsimmons, Karen; Khandelwal, Abha; Kranz, Robert G

    2009-07-01

    Single-repeat R3 MYB transcription factors like CPC (CAPRICE) are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S::CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3-MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs (e.g. root hairs, trichomes), but can influence anthocyanin pigment synthesis.

  18. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein

    NARCIS (Netherlands)

    Hateboer, G.; Timmers, H.T.M.; Rustgi, A.K.; Billaud, Marc; Veer, L.J. Van 't; Bernards, R.A.

    1993-01-01

    Using a protein binding assay, we show that the amino-teminal 204 amino acids of the c-Myc protein interact di y with a key component of the basal p tdon factor TFID, the TATA box-binding protein (TBP). Essentialy the same region of the c-Myc protein alo binds the product of the retinoblatoma

  19. Systematic chemical and molecular profiling of MLL-rearranged infant acute lymphoblastic leukemia reveals efficacy of romidepsin

    Science.gov (United States)

    Cruickshank, M N; Ford, J; Cheung, L C; Heng, J; Singh, S; Wells, J; Failes, T W; Arndt, G M; Smithers, N; Prinjha, R K; Anderson, D; Carter, K W; Gout, A M; Lassmann, T; O'Reilly, J; Cole, C H; Kotecha, R S; Kees, U R

    2017-01-01

    To address the poor prognosis of mixed lineage leukemia (MLL)-rearranged infant acute lymphoblastic leukemia (iALL), we generated a panel of cell lines from primary patient samples and investigated cytotoxic responses to contemporary and novel Food and Drug Administration-approved chemotherapeutics. To characterize representation of primary disease within cell lines, molecular features were compared using RNA-sequencing and cytogenetics. High-throughput screening revealed variable efficacy of currently used drugs, however identified consistent efficacy of three novel drug classes: proteasome inhibitors, histone deacetylase inhibitors and cyclin-dependent kinase inhibitors. Gene expression of drug targets was highly reproducible comparing iALL cell lines to matched primary specimens. Histone deacetylase inhibitors, including romidepsin (ROM), enhanced the activity of a key component of iALL therapy, cytarabine (ARAC) in vitro and combined administration of ROM and ARAC to xenografted mice further reduced leukemia burden. Molecular studies showed that ROM reduces expression of cytidine deaminase, an enzyme involved in ARAC deactivation, and enhances the DNA damage–response to ARAC. In conclusion, we present a valuable resource for drug discovery, including the first systematic analysis of transcriptome reproducibility in vitro, and have identified ROM as a promising therapeutic for MLL-rearranged iALL. PMID:27443263

  20. Empirical tight-binding parameters for solid C60

    International Nuclear Information System (INIS)

    Tit, N.; Kumar, V.

    1993-01-01

    We present a tight-binding model for the electronic structure of C 60 using four (1s and 3p) orbitals per carbon atom. The model has been developed by fitting the tight-binding parameters to the ab-initio pseudopotential calculation of Troullier and Martins (Phys. Rev. B46, 1754 (1992)) in the face-centered cubic (Fm3-bar) phase. Following this, calculations of the energy bands and the density of electronic states have been carried out as a function of the lattice constant. Good agreement has been obtained with the observed lattice-constant dependence of T c using McMillan's formula. Furthermore, calculations of the electronic structure are presented in the simple cubic (Pa3-bar) phase. (author). 43 refs, 3 figs, 1 tab

  1. Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

    Science.gov (United States)

    Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

    2014-03-14

    Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

  2. p53 induces differentiation but not apoptosis of v-Myb-transformed monoblasts

    Czech Academy of Sciences Publication Activity Database

    Navrátilová, J.; Horváth, Viktor; Kozubík, Alois; Lojek, Antonín; Šmarda, J.

    2006-01-01

    Roč. 18, č. 1 (2006), S38-S38 ISSN 1107-3756. [The 11th World Congress on Advances in Oncology and 9th International Symposium on Molecular Medicine . 12.10.2006-14.10.2006, Hersonissos] R&D Projects: GA ČR(CZ) GA301/06/0036 Institutional research plan: CEZ:AV0Z50040507 Keywords : p53 * v-Myb * BM2 Subject RIV: BO - Biophysics

  3. Activity of cAMP-dependent protein kinases and cAMP-binding proteins of rat kidney cytosol during dehydration

    International Nuclear Information System (INIS)

    Zelenina, M.N.; Solenov, E.I.; Ivanova, L.N.

    1985-01-01

    The activity of cAMP-dependent protein kinases, the binding of cAMP, and the spectrum of cAMP-binding proteins in the cytosol of the renal papilla was studied in intact rats and in rats after 24 h on a water-deprived diet. It was found that the activation of protein kinases by 10 -6 M cAMP is significantly higher in the experimental animals than in the intact animals. In chromatography on DEAE-cellulose, the positions of the peaks of specific reception of cAMP corresponded to the peaks of the regulatory subunits of cAMP-dependent protein kinases of types I and II. In this case, in intact animals more than 80% of the binding activity was detected in peaks II, whereas in rats subjected to water deprivation, more than 60% of the binding was observed in peak I. The general regulatory activity of the cytosol was unchanged in the experimental animals in comparison with intact animals. It is suggested that during dehydration there is an induction of the synthesis of the regulatory subunit of type I cAMP-dependent protein kinase in the renal papilla

  4. Retinoic acid enhances differentiation of v-myb-transformed monoblasts induced by okadaic acid

    Czech Academy of Sciences Publication Activity Database

    Beneš, P.; Macečková, V.; Zatloukalová, Jiřina; Kovářová, L.; Šmardová, J.; Šmarda, J.

    2007-01-01

    Roč. 31, č. 10 (2007), s. 1421-1431 ISSN 0145-2126 Grant - others:GA ČR(CZ) GP301/03/D022; GA ČR(CZ) GA301/06/0036 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : v-myb * monoblast * macrophage Subject RIV: BO - Biophysics Impact factor: 2.561, year: 2007

  5. Covalent bindings in proteins following UV-C irradiation

    International Nuclear Information System (INIS)

    Diezel, W.; Meffert, H.; Soennichsen, N.; Reinicke, C.

    1980-01-01

    Following a UV-C irradiation of catalase cross-linked catalase subunits could be detected by sodium dodecylsulfate gel electrophoresis. The subunits of aldolase were not cross-linked. The origin of covalent bindings in the catalase molecule is suggested to be effected by a free radical chain reaction induced by the heme component of catalase after UV-C irradiation. (author)

  6. Mannan-binding lectin activates C3 and the

    DEFF Research Database (Denmark)

    Selander, B.; Martensson, U.; Weintraub, A.

    2006-01-01

    Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL) or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera...... and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen-specific oligosaccharides derived from Salmonella typhimurium, S...

  7. shRNA screening identifies JMJD1C as being required for leukemia maintenance

    DEFF Research Database (Denmark)

    Sroczynska, Patrycja; Cruickshank, V Adam; Bukowski, John-Paul

    2014-01-01

    Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid and lymphoid leukemia (AML and ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic...... candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro, as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines......, with the strongest effect observed in the MLL-rearranged ALL cell line, SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia....

  8. Clostridium botulinum C2 toxin. Identification of the binding site for chloroquine and related compounds and influence of the binding site on properties of the C2II channel.

    Science.gov (United States)

    Neumeyer, Tobias; Schiffler, Bettina; Maier, Elke; Lang, Alexander E; Aktories, Klaus; Benz, Roland

    2008-02-15

    Clostridium botulinum C2 toxin belongs to the family of binary AB type toxins that are structurally organized into distinct enzyme (A, C2I) and binding (B, C2II) components. The proteolytically activated 60-kDa C2II binding component is essential for C2I transport into target cells. It oligomerizes into heptamers and forms channels in lipid bilayer membranes. The C2II channel is cation-selective and can be blocked by chloroquine and related compounds. Residues 303-330 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which has been implicated in the formation of two amphipathic beta-strands involved in membrane insertion and channel formation. In the present study, C2II mutants created by substitution of different negatively charged amino acids by alanine-scanning mutagenesis were analyzed in artificial lipid bilayer membranes. The results suggested that most of the C2II mutants formed SDS-resistant oligomers (heptamers) similar to wild type. The mutated negatively charged amino acids did not influence channel properties with the exception of Glu(399) and Asp(426), which are probably localized in the vestibule near the channel entrance. These mutants show a dramatic decrease in their affinity for binding of chloroquine and its analogues. Similarly, F428A, which represents the Phi-clamp in anthrax protective antigen, was mutated in C2II in several other amino acids. The C2II mutants F428A, F428D, F428Y, and F428W not only showed altered chloroquine binding but also had drastically changed single channel properties. The results suggest that amino acids Glu(399), Asp(426), and Phe(428) have a major impact on the function of C2II as a binding protein for C2I delivery into target cells.

  9. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    Science.gov (United States)

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

    Science.gov (United States)

    Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

    2014-01-21

    Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. Pathogenetic, Clinical, and Prognostic Features of Adult t(4;11(q21;q23/MLL-AF4 Positive B-Cell Acute Lymphoblastic Leukemia

    Directory of Open Access Journals (Sweden)

    F. Marchesi

    2011-01-01

    Full Text Available Translocation t(4;11(q21;q23 leading to formation of MLL-AF4 fusion gene is found in about 10% of newly diagnosed B-cell acute lymphoblastic leukemia (ALL in adult patients. Patients expressing this chromosomal aberration present typical biological, immunophenotypic, and clinical features. This form of leukemia is universally recognized as high-risk leukemia and treatment intensification with allogeneic hematopoietic stem cell transplantation (HSCT in first complete remission (CR could be a valid option to improve prognosis, but data obtained from the literature are controversial. In this review, we briefly describe pathogenetic, clinical, and prognostic characteristics of adult t(4;11(q21;q23/MLL-AF4 positive ALL and provide a review of the clinical outcome reported by the most important cooperative groups worldwide.

  12. An .I.ex vivo ./I.model to study v-Myb-induced leukemogenicity

    Czech Academy of Sciences Publication Activity Database

    Dvořáková, Marta; Králová, Jarmila; Karafiát, Vít; Bartůněk, Petr; Dvořák, Michal

    2001-01-01

    Roč. 27, č. 2 (2001), s. 437-445 ISSN 1079-9796 R&D Projects: GA ČR GV301/98/K042; GA ČR GA204/00/0554; GA AV ČR IPP2052002 Grant - others:HHMI(US) 75195-540401 Institutional research plan: CEZ:AV0Z5052915 Keywords : v-Myb oncoprotein * PEST domain * leucine zipper Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.703, year: 2001

  13. Different sensitivities to competitive inhibition of benzodiazepine receptor binding of {sup 11}C-iomazenil and {sup 11}C-flumazenil in rhesus monkey brain

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Osamu; Hosoi, Rie; Kobayashi, Kaoru [Osaka Univ., Suita (Japan). Medical School; Itoh, Takashi; Gee, A.; Suzuki, Kazutoshi

    2001-04-01

    The in vivo binding kinetics of {sup 11}C-iomazenil were compared with those of {sup 11}C-flumazenil binding in rhesus monkey brain. The monkey was anesthetized with ketamine and intravenously injected with either {sup 11}C-iomazenil or {sup 11}C-flumazenil in combination with the coadministration of different doses of non-radioactive flumazenil (0, 5 and 20 {mu}g/kg). The regional distribution of {sup 11}C-iomazenil in the brain was similar to that of {sup 11}C-flumazenil, but the sensitivity of {sup 11}C-iomazenil binding to competitive inhibition by non-radioactive flumazenil was much less than that of {sup 11}C-flumazenil binding. A significant reduction in {sup 11}C-flumazenil binding in the cerebral cortex was observed with 20 {mu}g/kg of flumazenil, whereas a relatively smaller inhibition of {sup 11}C-iomazenil binding in the same region was observed with the same dose of flumazenil. These results suggest that {sup 11}C-flumazenil may be a superior radiotracer for estimating benzodiazepine receptor occupancy in the intact brain. (author)

  14. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Nuemket, Nipawan [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu [Creative Research Institution ' Sousei,' Hokkaido University, Sapporo 001-0021 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tsukamoto, Kentaro; Tsuji, Takao [Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192 (Japan); Nakamura, Keiji; Kozaki, Shunji [Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531 (Japan); Yao, Min [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao, E-mail: tanaka@castor.sci.hokudai.ac.jp [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan)

    2011-07-29

    Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

  15. A dominant-negative mutation within AtMYB90 blocks flower pigment production in transgenic tobacco.

    Science.gov (United States)

    During de novo shoot induction in cultured transgenic tobacco callus a spontaneous mutation within the coding region of a AtMYB90 transgene produced a plant line in which the original transgene-induced over-pigmented phenotype (dark red/purple from anthocyanin overproduction in most tissues) was los...

  16. Interaction between cardiac myosin-binding protein C and formin Fhod3.

    Science.gov (United States)

    Matsuyama, Sho; Kage, Yohko; Fujimoto, Noriko; Ushijima, Tomoki; Tsuruda, Toshihiro; Kitamura, Kazuo; Shiose, Akira; Asada, Yujiro; Sumimoto, Hideki; Takeya, Ryu

    2018-05-08

    Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C-binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C-binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C-null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C-null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C-null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C-mediated regulation of cardiac function via direct interaction.

  17. 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...

  18. LcMYB1 Is a Key Determinant of Differential Anthocyanin Accumulation among Genotypes, Tissues, Developmental Phases and ABA and Light Stimuli in Litchi chinensis

    OpenAIRE

    Lai, Biao; Li, Xiao-Jing; Hu, Bing; Qin, Yong-Hua; Huang, Xu-Ming; Wang, Hui-Cong; Hu, Gui-Bing

    2014-01-01

    The red coloration of litchi fruit depends on the accumulation of anthocyanins. The anthocyanins level in litchi fruit varies widely among cultivars, developmental stages and environmental stimuli. Previous studies on various plant species demonstrate that anthocyanin biosynthesis is controlled at the transcriptional level. Here, we describe a litchi R2R3-MYB transcription factor gene, LcMYB1, which demonstrates a similar sequence as other known anthocyanin regulators. The transcription level...

  19. GWA Mapping of Anthocyanin Accumulation Reveals Balancing Selection of MYB90 in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Johanna A Bac-Molenaar

    Full Text Available Induction of anthocyanin accumulation by osmotic stress was assessed in 360 accessions of Arabidopsis thaliana. A wide range of natural variation, with phenotypes ranging from green to completely red/purple rosettes, was observed. A genome wide association (GWA mapping approach revealed that sequence diversity in a small 15 kb region on chromosome 1 explained 40% of the variation observed. Sequence and expression analyses of alleles of the candidate gene MYB90 identified a causal polymorphism at amino acid (AA position 210 of this transcription factor of the anthocyanin biosynthesis pathway. This amino acid discriminates the two most frequent alleles of MYB90. Both alleles are present in a substantial part of the population, suggesting balancing selection between these two alleles. Analysis of the geographical origin of the studied accessions suggests that the macro climate is not the driving force behind positive or negative selection for anthocyanin accumulation. An important role for local climatic conditions is, therefore, suggested. This study emphasizes that GWA mapping is a powerful approach to identify alleles that are under balancing selection pressure in nature.

  20. Bacillus subtilis SepF binds to the C-terminus of FtsZ.

    Science.gov (United States)

    Król, Ewa; van Kessel, Sebastiaan P; van Bezouwen, Laura S; Kumar, Neeraj; Boekema, Egbert J; Scheffers, Dirk-Jan

    2012-01-01

    Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.

  1. Bacillus subtilis SepF binds to the C-terminus of FtsZ.

    Directory of Open Access Journals (Sweden)

    Ewa Król

    Full Text Available Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.

  2. Systematic in-vitro evaluation of the NCI/NIH Developmental Therapeutics Program Approved Oncology Drug Set for the identification of a candidate drug repertoire for MLL-rearranged leukemia

    Directory of Open Access Journals (Sweden)

    Hoeksema KA

    2011-09-01

    measurable cytotoxicity against leukemia cells, and among these, 12 were effective against all five MLL-rearranged cell lines (IC50 [half maximal inhibitory concentration] < 1 µM. These 12 agents include cladribine, dactinomycin, daunorubicin, docetaxel, etoposide, gemcitabine, mitomycin C, mitoxantrone, teniposide, topotecan, triethylenemelamine, and vinblastine. We show that the Approved Oncology Drug Set II contains a number of agents with potent antileukemic activity in the tested cell lines. As approved drugs, these agents have been used in clinical settings for many years for other malignancies, thus their toxicity profile, pharmacokinetics, and other properties are readily available. Further evaluation of their use in future clinical trials for pediatric leukemia with MLL abnormalities should be considered.Keywords: drug screening, therapeutic repertoire

  3. Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

    Science.gov (United States)

    Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

    2013-02-15

    PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

  4. Distribution and binding of [14C]acrylamide to macromolecules in SENCAR and BALB/c mice following oral and topical administration

    International Nuclear Information System (INIS)

    Carlson, G.P.; Weaver, P.M.

    1985-01-01

    To determine if differences in acrylamide distribution or its binding to DNA could be responsible for the reported higher incidence of skin papillomas observed after oral administration compared to topical application, [ 14 C]acrylamide was administered by topical application and oral intubation to male SENCAR and BALB/mice. Portions of lung, liver, stomach, testes, and skin were removed, and 14 C was measured at 15 min, 30 min, 1, 6, 12, 24, and 48 hr. Binding to DNA, RNA, and protein was measured at 6 and 48 hr. Following oral administration, few strain differences in distribution or binding were noted. After topical application, SENCAR mice generally showed higher tissue concentrations than did the BALB/c mice at the early time periods but not at the later ones. Comparing the two routes, comparable concentrations were observed in all tissues except the skin where the amount of [ 14 C]acrylamide after topical application was approximately 100 times that observed after oral administration. At 48 hr, binding to DNA was sevenfold higher after topical than after oral administration. The effect of route on papilloma formation cannot be explained, therefore, on the basis of either a difference in distribution or binding to DNA in the target organ. The binding of acrylamide to DNA in skin was similar in both SENCAR and BALB/c mice indicating that the much greater susceptibility of the SENCAR mice to tumorigenesis cannot be explained simply on the basis of distribution or macromolecular binding

  5. Thermodynamic and structural properties of the specific binding between Ag⁺ ion and C:C mismatched base pair in duplex DNA to form C-Ag-C metal-mediated base pair.

    Science.gov (United States)

    Torigoe, Hidetaka; Okamoto, Itaru; Dairaku, Takenori; Tanaka, Yoshiyuki; Ono, Akira; Kozasa, Tetsuo

    2012-11-01

    Metal ion-nucleic acid interactions have attracted considerable interest for their involvement in structure formation and catalytic activity of nucleic acids. Although interactions between metal ion and mismatched base pair duplex are important to understand mechanism of gene mutations related to heavy metal ions, they have not been well-characterized. We recently found that the Ag(+) ion stabilized a C:C mismatched base pair duplex DNA. A C-Ag-C metal-mediated base pair was supposed to be formed by the binding between the Ag(+) ion and the C:C mismatched base pair to stabilize the duplex. Here, we examined specificity, thermodynamics and structure of possible C-Ag-C metal-mediated base pair. UV melting indicated that only the duplex with the C:C mismatched base pair, and not of the duplexes with the perfectly matched and other mismatched base pairs, was specifically stabilized on adding the Ag(+) ion. Isothermal titration calorimetry demonstrated that the Ag(+) ion specifically bound with the C:C base pair at 1:1 molar ratio with a binding constant of 10(6) M(-1), which was significantly larger than those for nonspecific metal ion-DNA interactions. Electrospray ionization mass spectrometry also supported the specific 1:1 binding between the Ag(+) ion and the C:C base pair. Circular dichroism spectroscopy and NMR revealed that the Ag(+) ion may bind with the N3 positions of the C:C base pair without distorting the higher-order structure of the duplex. We conclude that the specific formation of C-Ag-C base pair with large binding affinity would provide a binding mode of metal ion-DNA interactions, similar to that of the previously reported T-Hg-T base pair. The C-Ag-C base pair may be useful not only for understanding of molecular mechanism of gene mutations related to heavy metal ions but also for wide variety of potential applications of metal-mediated base pairs in various fields, such as material, life and environmental sciences. Copyright © 2012 Elsevier

  6. 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...

  7. CMYB1 Encoding a MYB Transcriptional Activator Is Involved in Abiotic Stress and Circadian Rhythm in Rice

    Directory of Open Access Journals (Sweden)

    Min Duan

    2014-01-01

    Full Text Available Through analysis of cold-induced transcriptome, a novel gene encoding a putative MYB transcription factor was isolated and designated Cold induced MYB 1 (CMYB1. Tissue-specific gene expression analysis revealed that CMYB1 was highly expressed in rice stems and nodes. qRT-PCR assay indicated that CMYB1 was dramatically induced by cold stress (>100-folds and induced by exogenous ABA and osmotic stress. Interestingly, CMYB1 showed rhythmic expression profile in rice leaves at different developmental stages. Subcellular localization assay suggested that CMYB1-GFP (green fluorescent protein fusion protein was localized in the nuclei. Moreover, CMYB1 exhibited the transcriptional activation activity when transiently expressed in rice protoplast cells. Taken together, CMYB1 probably functions as a transcriptional activator in mediating stress and rhythm responsive gene expression in rice.

  8. Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

    Science.gov (United States)

    Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

    2002-12-24

    KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

  9. Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in sorghum bicolor

    Science.gov (United States)

    The phenylpropanoid biosynthesis pathway that generates lignin subunits represents a significant target to alter the abundance and composition of lignin. The major regulators of phenylpropanoid metabolism are myb transcription factors, which have been shown to modulate secondary cell wall compositi...

  10. A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional Chinese medicinal plant, Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2016-07-01

    Full Text Available Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from E. sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase and EsFLS (flavonol synthase, but not the promoters of EsDFRs (dihydroflavonol 4-reductase and EsANS (anthocyanidin synthase in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase, NtCHI (chalcone isomerase, NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived bioactive components in E

  11. Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C.

    Science.gov (United States)

    Provenzani, Riccardo; Tarvainen, Ilari; Brandoli, Giulia; Lempinen, Antti; Artes, Sanna; Turku, Ainoleena; Jäntti, Maria Helena; Talman, Virpi; Yli-Kauhaluoma, Jari; Tuominen, Raimo K; Boije Af Gennäs, Gustav

    2018-01-01

    Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain-targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

  12. Binding kinetics of 11C-N-methyl piperidyl benzilate (11C-NMPB) in a rhesus monkey brain using the cerebellum as a reference region

    International Nuclear Information System (INIS)

    Itoh, Takashi; Tanaka, Masayasu; Suzuki, Kasutoshi; Kobayashi, Kaoru; Inoue, Osamu

    2005-01-01

    The binding kinetics of' 11 C-N-methyl piperidyl benzilate ( 11 C-NMPB) in rhesus monkey brain were studied using animal positron emission tomography (PET) (SHR2000). This study is intended to assess the validity of the method using the cerebellum as a reference region, and to evaluate the effects of anesthesia on 11 C -NMPB binding. Two monkeys, anesthetized with ketamine, received intravenous 11 C-NMPB alone (370-760 MBq, 11 C-NMPB accumulated densely in the striatum and cerebral cortex with time. In contrast, the tracer accumulation significantly decreased with increased doses of nonradioactive NMPB. In the cerebellum, on the other hand, the accumulation of 11 C-NMPB remained low and the tracer was slowly eliminated from the brain following the injection. 11 C-NMPB binding in the cerebellum was barely affected by the increased dose of nonradioactive NMPB. We thus concluded that the specific 11 C-NMPB binding was negligible in the cerebellum, and performed simplified evaluation of 11 C-NMPB binding in each brain region by a graphical method using the cerebellum as a reference region. PET was conducted 26 times, in total both in ketamine-anesthetized and awake monkeys (n=3 each). Measurements of 11 C-NMPB binding showed good run-to-run reproducibility within individual animals. When 11 C-NMPB binding was compared between ketamine-treated and awake animals, a significant increase in 11 C-NMPB binding was observed in the striatum but not in other brain regions of ketamine-treated animals. (author)

  13. Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T-cell therapy.

    Science.gov (United States)

    Gardner, Rebecca; Wu, David; Cherian, Sindhu; Fang, Min; Hanafi, Laïla-Aïcha; Finney, Olivia; Smithers, Hannah; Jensen, Michael C; Riddell, Stanley R; Maloney, David G; Turtle, Cameron J

    2016-05-19

    Administration of lymphodepletion chemotherapy followed by CD19-specific chimeric antigen receptor (CAR)-modified T cells is a remarkably effective approach to treating patients with relapsed and refractory CD19(+) B-cell malignancies. We treated 7 patients with B-cell acute lymphoblastic leukemia (B-ALL) harboring rearrangement of the mixed lineage leukemia (MLL) gene with CD19 CAR-T cells. All patients achieved complete remission (CR) in the bone marrow by flow cytometry after CD19 CAR-T-cell therapy; however, within 1 month of CAR-T-cell infusion, 2 of the patients developed acute myeloid leukemia (AML) that was clonally related to their B-ALL, a novel mechanism of CD19-negative immune escape. These reports have implications for the management of patients with relapsed and refractory MLL-B-ALL who receive CD19 CAR-T-cell therapy. © 2016 by The American Society of Hematology.

  14. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  15. Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

    OpenAIRE

    Shishmarev, Dmitry; Wang, Yao; Mason, Claire E.; Su, Xun-Cheng; Oakley, Aaron J.; Graham, Bim; Huber, Thomas; Dixon, Nicholas E.; Otting, Gottfried

    2013-01-01

    Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron den...

  16. GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis.

    Science.gov (United States)

    Dietrich, Philipp A; Yang, Chen; Leung, Halina H L; Lynch, Jennifer R; Gonzales, Estrella; Liu, Bing; Haber, Michelle; Norris, Murray D; Wang, Jianlong; Wang, Jenny Yingzi

    2014-11-20

    β-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML. © 2014 by The American Society of Hematology.

  17. Metabolic and molecular analyses of white mutant Vaccinium berries show down-regulation of MYBPA1-type R2R3 MYB regulatory factor.

    Science.gov (United States)

    Primetta, Anja K; Karppinen, Katja; Riihinen, Kaisu R; Jaakola, Laura

    2015-09-01

    MYBPA1-type R2R3 MYB transcription factor shows down-regulation in white mutant berries of Vaccinium uliginosum deficient in anthocyanins but not proanthocyanidins suggesting a role in the regulation of anthocyanin biosynthesis. Berries of the genus Vaccinium are among the best natural sources of flavonoids. In this study, the expression of structural and regulatory flavonoid biosynthetic genes and the accumulation of flavonoids in white mutant and blue-colored wild-type bog bilberry (V. uliginosum) fruits were measured at different stages of berry development. In contrast to high contents of anthocyanins in ripe blue-colored berries, only traces were detected by HPLC-ESI-MS in ripe white mutant berries. However, similar profile and high levels of flavonol glycosides and proanthocyanidins were quantified in both ripe white and ripe wild-type berries. Analysis with qRT-PCR showed strong down-regulation of structural genes chalcone synthase (VuCHS), dihydroflavonol 4-reductase (VuDFR) and anthocyanidin synthase (VuANS) as well as MYBPA1-type transcription factor VuMYBPA1 in white berries during ripening compared to wild-type berries. The profiles of transcript accumulation of chalcone isomerase (VuCHI), anthocyanidin reductase (VuANR), leucoanthocyanidin reductase (VuLAR) and flavonoid 3'5' hydroxylase (VuF3'5'H) were more similar between the white and the wild-type berries during fruit development, while expression of UDP-glucose: flavonoid 3-O-glucosyltransferase (VuUFGT) showed similar trend but fourfold lower level in white mutant. VuMYBPA1, the R2R3 MYB family member, is a homologue of VmMYB2 of V. myrtillus and VcMYBPA1 of V. corymbosum and belongs to MYBPA1-type MYB family which members are shown in some species to be related with proanthocyanidin biosynthesis in fruits. Our results combined with earlier data of the role of VmMYB2 in white mutant berries of V. myrtillus suggest that the regulation of anthocyanin biosynthesis in Vaccinium species could differ

  18. Different patterns of gene expression in rice varieties undergoing a ...

    African Journals Online (AJOL)

    Some genes specifically up-regulated in infected 9804-Rxo1 were defenserelated, including the genes encoding pathogenesis-related protein, terpene synthase family, transcription factors (TFs) AP2 domain containing protein, myb-like deoxyribonucleic acid (DNA)- binding domain containing protein, and C2H2-type ...

  19. Significant decreases in frontal and temporal [11C]-raclopride binding after THC challenge.

    Science.gov (United States)

    Stokes, Paul R A; Egerton, Alice; Watson, Ben; Reid, Alistair; Breen, Gerome; Lingford-Hughes, Anne; Nutt, David J; Mehta, Mitul A

    2010-10-01

    Delta9-tetrahydrocannabinol (THC) increases prefrontal cortical dopamine release in animals, but this is yet to be examined in humans. In man, striatal dopamine release can be indexed using [11C]-raclopride positron emission tomography (PET), and recent reports suggest that cortical [11C]-raclopride binding may also be sensitive to dopaminergic challenges. Using an existing dataset we examined whether THC alters [11C]-raclopride binding potential (BP(ND)) in cortical regions. Thirteen healthy volunteers underwent two [11C]-raclopride PET scans following either oral 10 mg THC or placebo. Significant areas of decreased cortical [11C]-raclopride BP(ND) were identified using whole brain voxel-wise analysis and quantified using a region of interest (ROI) ratio analysis. Effect of blood flow on binding was estimated using a simplified reference tissue model analysis. Results were compared to [11C]-raclopride test-retest reliability in the ROIs identified using a separate cohort of volunteers. Voxel-wise analysis identified three significant clusters of decreased [11C]-raclopride BP(ND) after THC in the right middle frontal gyrus, left superior frontal gyrus and left superior temporal gyrus. Decreases in [11C]-raclopride BPND following THC were greater than test-retest variability in these ROIs. R1, an estimate of blood flow, significantly decreased in the left superior frontal gyrus in the THC condition but was unchanged in the other ROIs. Decreased frontal binding significantly correlated to catechol-o-methyl transferase (COMT) val108 status. We have demonstrated for the first time significant decreases in bilateral frontopolar cortical and left superior temporal gyrus [11C]-raclopride binding after THC. The interpretation of these findings in relation to prefrontal dopamine release is discussed. Copyright 2010 Elsevier Inc. All rights reserved.

  20. Plasma Triglyceride Levels May Be Modulated by Gene Expression of IQCJ, NXPH1, PHF17 and MYB in Humans

    Directory of Open Access Journals (Sweden)

    Bastien Vallée Marcotte

    2017-01-01

    Full Text Available A genome-wide association study (GWAS by our group identified loci associated with the plasma triglyceride (TG response to ω-3 fatty acid (FA supplementation in IQCJ, NXPH1, PHF17 and MYB. Our aim is to investigate potential mechanisms underlying the associations between single nucleotide polymorphisms (SNPs in the four genes and TG levels following ω-3 FA supplementation. 208 subjects received 3 g/day of ω-3 FA (1.9–2.2 g of EPA and 1.1 g of docosahexaenoic acid (DHA for six weeks. Plasma TG were measured before and after the intervention. 67 SNPs were selected to increase the density of markers near GWAS hits. Genome-wide expression and methylation analyses were conducted on respectively 30 and 35 participants’ blood sample together with in silico analyses. Two SNPs of IQCJ showed different affinities to splice sites depending on alleles. Expression levels were influenced by genotype for one SNP in NXPH1 and one in MYB. Associations between 12 tagged SNPs of IQCJ, 26 of NXPH1, seven of PHF17 and four of MYB and gene-specific CpG site methylation levels were found. The response of plasma TG to ω-3 FA supplementation may be modulated by the effect of DNA methylation on expression levels of genes revealed by GWAS.

  1. Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

    The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

  2. Potential of [11C]DASB for measuring endogenous serotonin with PET: binding studies

    International Nuclear Information System (INIS)

    Lundquist, Pinelopi; Wilking, Helena; Hoeglund, A. Urban; Sandell, Johan; Bergstroem, Mats; Hartvig, Per; Langstroem, Bengt

    2005-01-01

    The serotonin transporter radioligand [ 11 C]-3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile, or [ 11 C]DASB, was examined in order to assess its potential for measuring fluctuations in endogenous serotonin concentrations with positron emission tomography. Binding characteristics of [ 11 C]DASB and the propensity for serotonin to displace the tracer were explored in rat brain homogenates. Experiments showed that serotonin displaced [ 11 C]DASB in vitro. Ex vivo experiments performed after tranylcypromine injection (3 or 15 mg/kg) showed a dose-dependent trend in radioactivity uptake and suggested that serotonin may compete with [ 11 C]DASB for transporter binding

  3. Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong (Harvard-Med); (UAH); (Maastricht); (Chinese Aca. Sci.)

    2010-11-03

    Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

  4. Eculizumab treatment: stochastic occurrence of C3 binding to individual PNH erythrocytes

    Directory of Open Access Journals (Sweden)

    Michela Sica

    2017-06-01

    Full Text Available Abstract Background C5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH. However, C3-bound PNH red blood cells (RBCs, arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+ and without (C3− C3 binding. Methods To investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway. Results When RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes PNH RBCs. Conclusions This study indicates that the distinct (C3+ and C3− PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.

  5. Incipient localization and tight-binding superconductivity: Tsub(c) calculation

    International Nuclear Information System (INIS)

    Kolley, E.; Kolley, W.

    1984-01-01

    Localization effects on the superconducting transition temperature Tsub(c) are examined in strongly disordered three-dimensional systems. A tight-binding formulation of strong-coupling superconductivity is combined, after configuration averaging, with the self-consistent treatment of Anderson localization developed by Vollhardt and Woelfle. The Coulomb interaction becomes retarded via the joint local local density of states, giving rise to an enhancement of the pseudopotential. Numerical Tsub(c) results as a function of disorder are compared with another theoretical work and experimental values for some high-Tsub(c) materials. (orig.)

  6. The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain.

    Science.gov (United States)

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J; Sivakumaran, Andrew; Yoon, Je-Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, B Göran; Wilce, Jacqueline A; Díaz-Moreno, Irene

    2014-01-01

    T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.

  7. Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I

    International Nuclear Information System (INIS)

    Clemmons, D.R.; Elgin, R.G.; Han, V.K.; Casella, S.J.; D'Ercole, A.J.; Van Wyk, J.J.

    1986-01-01

    We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in 125 I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in 125 I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt 125 I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that 125 I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. 125 I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds 125 I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding

  8. The binding of cytochrome c to neuroglobin: A docking and surface plasmon resonance study

    DEFF Research Database (Denmark)

    Bønding, Signe Helbo; Henty, K.; Dingley, A.J.

    2008-01-01

    is associated with a small unfavourable enthalpy change (1.9 kcal mol-1) and a moderately large, favourable entropy change (14.8 cal mol-1 deg-1). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions....... one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved....... surface plasmon resonance studies provide a value of 45 μM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation...

  9. Reduced parahippocampal and lateral temporal GABAA-[11C]flumazenil binding in major depression: preliminary results

    International Nuclear Information System (INIS)

    Klumpers, Ursula M.H.; Veltman, Dick J.; Drent, Madeleine L.; Boellaard, Ronald; Lammertsma, Adriaan A.; Comans, Emile F.I.; Meynen, Gerben; Hoogendijk, Witte J.G.

    2010-01-01

    Major depressive disorder (MDD) has been related to both a dysfunctional γ-amino butyric acid (GABA) system and to hyperactivity of the hypothalamic-pituitary-adrenal axis (HPA). Although GABA has been suggested to inhibit HPA axis activity, their relationship has never been studied at the level of the central GABA A -benzodiazepine receptor in depressed patients or in relation to antidepressant treatment. Eleven depressed outpatients were compared, before and after treatment with citalopram, with nine age-matched healthy controls. The subjects were scanned using the positron emission tomography (PET) tracer [ 11 C]flumazenil ([ 11 C]FMZ). Parametric voxel-by-voxel Logan plots were compared with methods based on regions of interest (ROI), to provide volume of distribution (V T ) and binding potential (BP ND ) values. Plasma GABA levels were determined and a dexamethasone-corticotropin releasing hormone (DEX-CRH) test was performed. In MDD, parametric voxel-by-voxel Logan plots showed bilateral reduced [ 11 C]FMZ binding in the parahippocampal gyrus and right lateral superior temporal gyrus (p uncorrected ≤0.001). In the temporal area, [ 11 C]FMZ binding showed a strong inverse correlation with HPA axis activity. Plasma GABA did not discriminate MDD from controls, but correlated inversely with [ 11 C]FMZ binding in the right insula. Following treatment with citalopram, voxel-based analysis revealed reduced binding in the right lateral temporal gyrus and dorsolateral prefrontal cortex. The bilateral reduction in limbic parahippocampal and right temporal [ 11 C]FMZ binding found in MDD indicates decreased GABA A -benzodiazepine receptor complex affinity and/or number. The inverse relationship between GABA A binding in the temporal lobe and HPA axis activity, suggests that HPA axis hyperactivity is partly due to reduced GABA-ergic inhibition. (orig.)

  10. Cassava C-repeat binding factor 1 gene responds to low temperature and enhances cold tolerance when overexpressed in Arabidopsis and cassava.

    Science.gov (United States)

    An, Dong; Ma, Qiuxiang; Wang, Hongxia; Yang, Jun; Zhou, Wenzhi; Zhang, Peng

    2017-05-01

    Cassava MeCBF1 is a typical CBF transcription factor mediating cold responses but its low expression in apical buds along with a retarded response cause inefficient upregulation of downstream cold-related genes, rendering cassava chilling-sensitive. Low temperature is a major abiotic stress factor affecting survival, productivity and geographic distribution of important crops worldwide. The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB) are important regulators of abiotic stress response in plants. In this study, MeCBF1, a CBF-like gene, was identified in the tropical root crop cassava (Manihot esculenta Crantz). The MeCBF1 encodes a protein that shares strong homology with DREB1As/CBFs from Arabidopsis as well as other species. The MeCBF1 was localized to the nucleus and is mainly expressed in stem and mature leaves, but not in apical buds or stem cambium. MeCBF1 expression was not only highly responsive to cold, but also significantly induced by salt, PEG and ABA treatment. Several stress-associated cis-elements were found in its promoter region, e.g., ABRE-related, MYC recognition sites, and MYB responsive element. Compared with AtCBF1, the MeCBF1 expression induced by cold in cassava was retarded and upregulated only after 4 h, which was also confirmed by its promoter activity. Overexpression of MeCBF1 in transgenic Arabidopsis and cassava plants conferred enhanced crytolerance. The CBF regulon was smaller and not entirely co-regulated with MeCBF1 expression in overexpressed cassava. The retarded MeCBF1 expression in response to cold and attenuated CBF-regulon might lead cassava to chilling sensitivity.

  11. Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

    Science.gov (United States)

    Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

    2017-01-01

    As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin ( Ts -CRT), a Ca 2+ -binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts -CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts -CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts -CRT (r Ts -CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts -CRT on the surface of newborn larvae (NBL) of T. spiralis with anti- Ts -CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis -expressed Ts -CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

  12. [11C]Harmine Binding to Brain Monoamine Oxidase A: Test-Retest Properties and Noninvasive Quantification.

    Science.gov (United States)

    Zanderigo, Francesca; D'Agostino, Alexandra E; Joshi, Nandita; Schain, Martin; Kumar, Dileep; Parsey, Ramin V; DeLorenzo, Christine; Mann, J John

    2018-02-08

    Inhibition of the isoform A of monoamine oxidase (MAO-A), a mitochondrial enzyme catalyzing deamination of monoamine neurotransmitters, is useful in treatment of depression and anxiety disorders. [ 11 C]harmine, a MAO-A PET radioligand, has been used to study mood disorders and antidepressant treatment. However, [ 11 C]harmine binding test-retest characteristics have to date only been partially investigated. Furthermore, since MAO-A is ubiquitously expressed, no reference region is available, thus requiring arterial blood sampling during PET scanning. Here, we investigate [ 11 C]harmine binding measurements test-retest properties; assess effects of using a minimally invasive input function estimation on binding quantification and repeatability; and explore binding potentials estimation using a reference region-free approach. Quantification of [ 11 C]harmine distribution volume (V T ) via kinetic models and graphical analyses was compared based on absolute test-retest percent difference (TRPD), intraclass correlation coefficient (ICC), and identifiability. The optimal procedure was also used with a simultaneously estimated input function in place of the measured curve. Lastly, an approach for binding potentials quantification in absence of a reference region was evaluated. [ 11 C]harmine V T estimates quantified using arterial blood and kinetic modeling showed average absolute TRPD values of 7.7 to 15.6 %, and ICC values between 0.56 and 0.86, across brain regions. Using simultaneous estimation (SIME) of input function resulted in V T estimates close to those obtained using arterial input function (r = 0.951, slope = 1.073, intercept = - 1.037), with numerically but not statistically higher test-retest difference (range 16.6 to 22.0 %), but with overall poor ICC values, between 0.30 and 0.57. Prospective studies using [ 11 C]harmine are possible given its test-retest repeatability when binding is quantified using arterial blood. Results with SIME of

  13. In vitro covalent binding of 3-[14C]methylindole metabolites in goat tissues

    International Nuclear Information System (INIS)

    Bray, T.M.; Carlson, J.R.; Nocerini, M.R.

    1984-01-01

    Covalent binding of 3-[ 14 C]methylindole (3[ 14 C]MI) in crude microsomal preparations of goat lung, liver, and kidney was measured to determine if a reactive intermediate was formed during the in vitro metabolism of 3-methylindole (3MI). The bound radioactivity was highest in lung compared to liver and kidney. The amount of bound radioactivity per nanomole of cytochrome P-450 was approximately 10 times higher in the lung compared to the liver. No detectable bound radioactivity was found when 3-[ 3 H]methyloxindole was used as the substrate. Cofactor requirements and the effects of inhibitors indicate that a mixed function oxidase (MFO) system is involved in formation of a reactive intermediate. Inhibitors and conjugating agents that are known to reduce the severity of 3MI-induced lung injury such as piperonyl butoxide (MFO inhibitor) and glutathione (conjugating agent) significantly decreased the in vitro binding of 3[ 14 C]MI. The results indicate that a reactive intermediate is produced during the metabolism of 3MI by the MFO system. The organ specificity in binding suggests that covalent binding by lung microsomes may be related to the mechanism of 3MI-induced lung injury

  14. FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

    Science.gov (United States)

    Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

    2014-04-01

    Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

  15. Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

    International Nuclear Information System (INIS)

    Ahluwalia, Unnati; Deep, Shashank

    2012-01-01

    Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

  16. Python bindings for C++ using PyROOT/cppyy: the experience from PyCool in COOL

    CERN Multimedia

    CERN. Geneva

    2016-01-01

    The COOL software is used by the ATLAS and LHCb experiments to handle the time variation and versioning of their conditions data, using a variety of different relational database technologies. While the COOL core libraries are written in C++ and are integrated in the experiment C++ frameworks, a package offering Python bindings of the COOL C++ APIs, PyCool, is also provided and has been an essential component of the ATLAS conditions data management toolkit for over 10 years. Almost since the beginning, the implementation of PyCool has been based on ROOT to generate Python bindings for C++, initially using Reflex and PyROOT in ROOT5 and more recently using clang and cppyy in ROOT6. This presentation will describe the PyCool experience with using ROOT to generate Python bindings for C++, throughout the many evolutions of the underlying technology.

  17. Binding proteins for the regulatory subunit (RII-B) of brain cAMP-dependent protein kinase II: isolation and initial characterization of cDNA clones

    International Nuclear Information System (INIS)

    Bregman, D.B.; Hu, E.; Rubin, C.S.

    1987-01-01

    In mammalian brain several proteins bind RII-B with high affinity. An example is P75, which co-purifies with RII-B and also complexes Ca 2+ -calmodulin. Thus, RII-B binding proteins (RBPs) might play a role in integrating the Ca 2+ and cAMP signalling pathways in the CNS. In order to study the structure and function of these polypeptides they have isolated cloned cDNAs for RBPs by screening brain λgt11 expression libraries using a functional assay: the binding of 32 P-labeled RII to fusion proteins produced by recombinants expressing RII binding domains. Inserts from rat brain recombinant clones λ7B and λ10B both hybridize to a brain mRNA of 7000 nucleotides. Northern gel analyses indicate that the putative RBP mRNA is also expressed in lung, but not in several other tissues. The λ7B insert was subcloned into the expression plasmid pINIA. A 50 kDa high affinity RII-B binding polypeptide accumulated in E. coli transformed with pINIA-7B. Two RBP cDNAs (λ77, λ100A) have been retrieved from a bovine λgt 11 library using a monoclonal antibody directed against P75 and the binding assay respectively. On Southern blots the insert from λ100A hybridizes to the cDNA insert from clones λ77, suggesting that λ 77 cDNA might contain sequences coding for both an RII binding domain and a P75 epitope. The bovine λ100A insert also hybridizes with the rat λ7B clone indicating that an RII binding domain is conserved in the two species

  18. Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

    International Nuclear Information System (INIS)

    Mueller, H.; Fehr, J.

    1986-01-01

    The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

  19. Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Based on our recent finding that cardiac myosin binding protein C (cMyBP-C phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302, DAD (Asp273-Ala282-Asp302, SAS (Ser273-Ala282-Ser302, and t/t (cMyBP-C null genotypes, and the results were compared to transgenic mice expressing wide-type (WT cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi, and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc, and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

  20. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides

    OpenAIRE

    Yoga, Yano M. K.; Traore, Daouda A. K.; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R.; Barker, Andrew; Leedman, Peter J.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

    2012-01-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA...

  1. Species differences in [11C]clorgyline binding in brain

    International Nuclear Information System (INIS)

    Fowler, Joanna S.; Ding, Yu-Shin; Logan, Jean; MacGregor, Robert R.; Shea, Colleen; Garza, Victor; Gimi, Raomond; Volkow, Nora D.; Wang, Gene-Jack; Schlyer, David; Ferrieri, Richard; Gatley, S. John; Alexoff, David; Carter, Pauline; King, Payton; Pappas, Naomi; Arnett, Carroll D.

    2001-01-01

    [ 11 C]Clorgyline selectively binds to MAO A in the human brain. This contrasts with a recent report that [ 11 C]clorgyline (in contrast to other labeled MAO A inhibitors) is not retained in the rhesus monkey brain . To explore this difference, we compared [ 11 C]clorgyline in the baboon brain before and after clorgyline pretreatment and we also synthesized deuterium substituted [ 11 C]clorgyline (and its nor-precursor) for comparison. [ 11 C]Clorgyline was not retained in the baboon brain nor was it influenced by clorgyline pretreatment or by deuterium substitution, contrasting to results in humans. This suggests a species difference in the susceptibility of MAO A to inhibition by clorgyline and represents an unusual example of where the behavior of a radiotracer in the baboon brain does not predict its behavior in the human brain

  2. Ultraviolet B (UVB) induction of the c-fos promoter is mediated by phospho-cAMP response element binding protein (CREB) binding to CRE and c-fos activator protein 1 site (FAP1) cis elements.

    Science.gov (United States)

    Gonzales, Melissa; Bowden, G Tim

    2002-06-26

    The ultraviolet B (UVB) portion (280-320 nm) of the ultraviolet spectrum has been shown to contribute to the development of non-melanoma skin cancer in humans. Research in the human keratinocyte cell line, HaCaT, revealed that UVB irradiation caused the upregulation of the transcription factor activator protein-1 (AP-1). The AP-1 complex formed in UVB-irradiated HaCaT cells is specifically composed of c-fos and Jun D. c-Fos expression was induced in a manner that correlated with the UVB-induced activation of AP-1. To investigate how c-fos expression is regulated by UVB irradiation, the role of each of four cis elements within the c-fos promoter was evaluated. Clustered point mutations at the sis inducible element (SIE), serum response element (SRE), c-fos AP-1 site (FAP1), or cyclic AMP response elements (CRE) significantly inhibited UVB induction of the c-fos promoter. This indicated that all four cis elements are required for maximum promoter activity. The CRE and FAP1 elements were the two most active cis elements that mediate the UVB transactivation of c-fos. Homodimers of phosphorylated cAMP response element binding protein (CREB) were induced by UVB irradiation to bind to each of these elements. Therefore, CREB may function as an important regulatory protein in the UVB-induced expression of c-fos.

  3. A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

    Science.gov (United States)

    Cholko, Timothy; Chen, Wei; Tang, Zhiye; Chang, Chia-en A.

    2018-05-01

    Abnormal activity of cyclin-dependent kinase 8 (CDK8) along with its partner protein cyclin C (CycC) is a common feature of many diseases including colorectal cancer. Using molecular dynamics (MD) simulations, this study determined the dynamics of the CDK8-CycC system and we obtained detailed breakdowns of binding energy contributions for four type-I and five type-II CDK8 inhibitors. We revealed system motions and conformational changes that will affect ligand binding, confirmed the essentialness of CycC for inclusion in future computational studies, and provide guidance in development of CDK8 binders. We employed unbiased all-atom MD simulations for 500 ns on twelve CDK8-CycC systems, including apoproteins and protein-ligand complexes, then performed principal component analysis (PCA) and measured the RMSF of key regions to identify protein dynamics. Binding pocket volume analysis identified conformational changes that accompany ligand binding. Next, H-bond analysis, residue-wise interaction calculations, and MM/PBSA were performed to characterize protein-ligand interactions and find the binding energy. We discovered that CycC is vital for maintaining a proper conformation of CDK8 to facilitate ligand binding and that the system exhibits motion that should be carefully considered in future computational work. Surprisingly, we found that motion of the activation loop did not affect ligand binding. Type-I and type-II ligand binding is driven by van der Waals interactions, but electrostatic energy and entropic penalties affect type-II binding as well. Binding of both ligand types affects protein flexibility. Based on this we provide suggestions for development of tighter-binding CDK8 inhibitors and offer insight that can aid future computational studies.

  4. Serotonin 2A receptor agonist binding in the human brain with [11C]Cimbi-36

    DEFF Research Database (Denmark)

    Ettrup, Anders; Svarer, Claus; McMahon, Brenda

    2016-01-01

    INTRODUCTION: [(11)C]Cimbi-36 is a recently developed serotonin 2A (5-HT2A) receptor agonist positron emission tomography (PET) radioligand that has been successfully applied for human neuroimaging. Here, we investigate the test-retest variability of cerebral [(11)C]Cimbi-36 PET and compare [(11)C...... test-retest variability in [(11)C]Cimbi-36 binding measures, and another eight were scanned after a bolus plus constant infusion with [(18)F]altanserin. Regional differences in the brain distribution of [(11)C]Cimbi-36 and [(18)F]altanserin were assessed with a correlation of regional binding measures...... and with voxel-based analysis. RESULTS: Test-retest variability of [(11)C]Cimbi-36 non-displaceable binding potential (BPND) was consistently correlation between regional...

  5. Novel insights into regulation of asparagine synthetase in conifers

    Directory of Open Access Journals (Sweden)

    Javier eCanales

    2012-05-01

    Full Text Available Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4. In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait., PpAS1 and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1 was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed.

  6. Study on the Effect of cellulolytic strain MYB3 for Corn Stover Fermentation

    Science.gov (United States)

    Yan, Han; Bai, Bing; Cheng, Xiao-Xiao; Li, Guang-Chun; Huang, Shi-Chen; Piao, Chun-Xiang

    2018-03-01

    The effects of corn stover fermentation with the Bacillus megaterium MYB3 was studied in this paper. The results showed that the decomposition rates of cellulose and hemicellulose were 49.6%, 43.46% after 20 days respectively, after fermentation, pH was changed to 5.68, and adjusted to corn stover initial pH 3 to achieve the purpose of sterilization. The decomposition rate was significantly increased by adding corn flour. After adjusting fermentation composes with the ratio of the corn stove to corn flour was 15 : 1, the decomposition rate of cellulose would be 52.37% for 10 days.

  7. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  8. X-ray diffraction study of the binding of the antisickling agent 12C79 to human hemoglobin

    International Nuclear Information System (INIS)

    Wireko, R.C.; Abraham, D.J.

    1991-01-01

    The hemoglobin binding site of the antisickling agent 12C79 has been determined by x-ray crystallography. 12C79 is recognized as one of the first molecules to reach clinical trials that was designed, de novo, from x-ray-determined atomic coordinates of a protein. Several previous attempts to verify the proposed Hb binding sites via crystallographic studies have failed. Using revised experimental procedures, the authors obtained 12C79-deoxhemoglobin crystals grown after reaction with oxyhemoglobin and cyanoborohydride reduction to stabilize the Schiff base linkage. The difference electron-density Fourier maps show that two 12C79 molecules bind covalently to both symmetry-related N-terminal amino groups of the hemoglobin α chains. This is in contrast to the original design that proposed the binding of one drug molecule that spans the molecular dyad to interact with both N-terminal α-amino groups

  9. c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

    Science.gov (United States)

    Lively, T N; Ferguson, H A; Galasinski, S K; Seto, A G; Goodrich, J A

    2001-07-06

    c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

  10. Microtubule Binding and Disruption and Induction of Premature Senescence by Disorazole C1S⃞

    Science.gov (United States)

    Tierno, Marni Brisson; Kitchens, Carolyn A.; Petrik, Bethany; Graham, Thomas H.; Wipf, Peter; Xu, Fengfeng L.; Saunders, William S.; Raccor, Brianne S.; Balachandran, Raghavan; Day, Billy W.; Stout, Jane R.; Walczak, Claire E.; Ducruet, Alexander P.; Reese, Celeste E.; Lazo, John S.

    2009-01-01

    Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A1, was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G2/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C1 inhibited purified bovine tubulin polymerization, with an IC50 of 11.8 ± 0.4 μM, and inhibited [3H]vinblastine binding noncompetitively, with a Ki of 4.5 ± 0.6 μM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C1, with a Ki of 10.6 ± 1.5 μM, indicating that disorazole C1 bound tubulin uniquely among known antimitotic agents. Disorazole C1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence. PMID:19066338

  11. NTL8 Regulates Trichome Formation in Arabidopsis by Directly Activating R3 MYB Genes TRY and TCL1.

    Science.gov (United States)

    Tian, Hainan; Wang, Xianling; Guo, Hongyan; Cheng, Yuxin; Hou, Chunjiang; Chen, Jin-Gui; Wang, Shucai

    2017-08-01

    The NAM, ATAF1/2, and CUC (NAC) are plant-specific transcription factors that regulate multiple aspects of plant growth and development and plant response to environmental stimuli. We report here the identification of NTM1-LIKE8 (NTL8), a membrane-associated NAC transcription factor, as a novel regulator of trichome formation in Arabidopsis ( Arabidopsis thaliana ). From an activation-tagged Arabidopsis population, we identified a dominant, gain-of-function mutant with glabrous inflorescence stem. By using plasmid rescue and RT-PCR analyses, we found that NTL8 was tagged; thus, the mutant was named ntl8-1 Dominant ( ntl8-1D ). Recapitulation experiment further confirmed that the phenotype observed in the ntl8-1D mutant was caused by elevated expression of NTL8 Quantitative RT-PCR results showed that the expression level of the single-repeat R3 MYB genes TRIPTYCHON ( TRY ) and TRICHOMELESS1 ( TCL1 ) was elevated in the ntl8-1D mutant. Genetic analyses demonstrated that NTL8 acts upstream of TRY and TCL1 in the regulation of trichome formation. When recruited to the promoter region of the reporter gene Gal4:GUS by a fused GAL4 DNA-binding domain, NTL8 activated the expression of the reporter gene. Chromatin immunoprecipitation results indicated that TRY and TCL1 are direct targets of NTL8. However, NTL8 did not interact with SQUAMOSA PROMOTER BINDING PROTEIN LIKE9, another transcription factor that regulates the expression of TRY and TCL1 , in yeast and plant cells. Taken together, our results suggest that NTL8 negatively regulates trichome formation in Arabidopsis by directly activating the expression of TRY and TCL1 . © 2017 American Society of Plant Biologists. All Rights Reserved.

  12. Surface complement C3 fragments and cellular binding of microparticles in patients with SLE

    DEFF Research Database (Denmark)

    Winberg, Line Kjær; Nielsen, Claus Henrik; Jacobsen, Søren

    2017-01-01

    Objectives: To examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing the ability of MPs to bind to circulating phagocytes and erythrocytes. These fea......Objectives: To examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing the ability of MPs to bind to circulating phagocytes and erythrocytes...

  13. Evolution of AF6-RAS association and its implications in mixed-lineage leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Matthew J.; Ottoni, Elizabeth; Ishiyama, Noboru; Goudreault, Marilyn; Haman, André; Meyer, Claus; Tucholska, Monika; Gasmi-Seabrook, Genevieve; Menezes, Serena; Laister, Rob C.; Minden, Mark D.; Marschalek, Rolf; Gingras, Anne-Claude; Hoang, Trang; Ikura, Mitsuhiko

    2017-10-23

    Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.

  14. The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

    Directory of Open Access Journals (Sweden)

    Caileen M Brison

    Full Text Available The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

  15. Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity.

    Science.gov (United States)

    Le Bihan, Yann-Vaï; Matot, Béatrice; Pietrement, Olivier; Giraud-Panis, Marie-Josèphe; Gasparini, Sylvaine; Le Cam, Eric; Gilson, Eric; Sclavi, Bianca; Miron, Simona; Le Du, Marie-Hélène

    2013-03-01

    Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.

  16. Transcriptomic analysis of grape (Vitis vinifera L. leaves after exposure to ultraviolet C irradiation.

    Directory of Open Access Journals (Sweden)

    Huifen Xi

    Full Text Available BACKGROUND: Only a small amount of solar ultraviolet C (UV-C radiation reaches the Earth's surface. This is because of the filtering effects of the stratospheric ozone layer. Artificial UV-C irradiation is used on leaves and fruits to stimulate different biological processes in plants. Grapes are a major fruit crop and are grown in many parts of the world. Research has shown that UV-C irradiation induces the biosynthesis of phenols in grape leaves. However, few studies have analyzed the overall changes in gene expression in grape leaves exposed to UV-C. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, transcriptional responses were investigated in grape (Vitis vinifera L. leaves before and after exposure to UV-C irradiation (6 W·m-2 for 10 min using an Affymetrix Vitis vinifera (Grape Genome Array (15,700 transcripts. A total of 5274 differentially expressed probe sets were defined, including 3564 (67.58% probe sets that appeared at both 6 and 12 h after exposure to UV-C irradiation but not before exposure. A total of 468 (8.87% probe sets and 1242 (23.55% probe sets were specifically expressed at these times. The probe sets were associated with a large number of important traits and biological pathways, including cell rescue (i.e., antioxidant enzymes, protein fate (i.e., HSPs, primary and secondary metabolism, and transcription factors. Interestingly, some of the genes involved in secondary metabolism, such as stilbene synthase, responded intensely to irradiation. Some of the MYB and WRKY family transcription factors, such as VvMYBPA1, VvMYB14, VvMYB4, WRKY57-like, and WRKY 65, were also strongly up-regulated (about 100 to 200 fold. CONCLUSIONS: UV-C irridiation has an important role in some biology processes, especially cell rescue, protein fate, secondary metabolism, and regulation of transcription.These results opened up ways of exploring the molecular mechanisms underlying the effects of UV-C irradiation on grape leaves and have

  17. Transcriptomic analysis of grape (Vitis vinifera L.) leaves after exposure to ultraviolet C irradiation.

    Science.gov (United States)

    Xi, Huifen; Ma, Ling; Liu, Guotian; Wang, Nian; Wang, Junfang; Wang, Lina; Dai, Zhanwu; Li, Shaohua; Wang, Lijun

    2014-01-01

    Only a small amount of solar ultraviolet C (UV-C) radiation reaches the Earth's surface. This is because of the filtering effects of the stratospheric ozone layer. Artificial UV-C irradiation is used on leaves and fruits to stimulate different biological processes in plants. Grapes are a major fruit crop and are grown in many parts of the world. Research has shown that UV-C irradiation induces the biosynthesis of phenols in grape leaves. However, few studies have analyzed the overall changes in gene expression in grape leaves exposed to UV-C. In the present study, transcriptional responses were investigated in grape (Vitis vinifera L.) leaves before and after exposure to UV-C irradiation (6 W·m-2 for 10 min) using an Affymetrix Vitis vinifera (Grape) Genome Array (15,700 transcripts). A total of 5274 differentially expressed probe sets were defined, including 3564 (67.58%) probe sets that appeared at both 6 and 12 h after exposure to UV-C irradiation but not before exposure. A total of 468 (8.87%) probe sets and 1242 (23.55%) probe sets were specifically expressed at these times. The probe sets were associated with a large number of important traits and biological pathways, including cell rescue (i.e., antioxidant enzymes), protein fate (i.e., HSPs), primary and secondary metabolism, and transcription factors. Interestingly, some of the genes involved in secondary metabolism, such as stilbene synthase, responded intensely to irradiation. Some of the MYB and WRKY family transcription factors, such as VvMYBPA1, VvMYB14, VvMYB4, WRKY57-like, and WRKY 65, were also strongly up-regulated (about 100 to 200 fold). UV-C irridiation has an important role in some biology processes, especially cell rescue, protein fate, secondary metabolism, and regulation of transcription.These results opened up ways of exploring the molecular mechanisms underlying the effects of UV-C irradiation on grape leaves and have great implications for further studies.

  18. Species differences in [{sup 11}C]clorgyline binding in brain

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, Joanna S. E-mail: fowler@bnl.gov; Ding, Yu-Shin; Logan, Jean; MacGregor, Robert R.; Shea, Colleen; Garza, Victor; Gimi, Raomond; Volkow, Nora D.; Wang, Gene-Jack; Schlyer, David; Ferrieri, Richard; Gatley, S. John; Alexoff, David; Carter, Pauline; King, Payton; Pappas, Naomi; Arnett, Carroll D

    2001-10-01

    [{sup 11}C]Clorgyline selectively binds to MAO A in the human brain. This contrasts with a recent report that [{sup 11}C]clorgyline (in contrast to other labeled MAO A inhibitors) is not retained in the rhesus monkey brain . To explore this difference, we compared [{sup 11}C]clorgyline in the baboon brain before and after clorgyline pretreatment and we also synthesized deuterium substituted [{sup 11}C]clorgyline (and its nor-precursor) for comparison. [{sup 11}C]Clorgyline was not retained in the baboon brain nor was it influenced by clorgyline pretreatment or by deuterium substitution, contrasting to results in humans. This suggests a species difference in the susceptibility of MAO A to inhibition by clorgyline and represents an unusual example of where the behavior of a radiotracer in the baboon brain does not predict its behavior in the human brain.

  19. Characterization of guinea pig myocardial leukotriene C4 binding sites. Regulation by cations and sulfhydryl-directed reagents

    International Nuclear Information System (INIS)

    Hogaboom, G.K.; Mong, S.; Stadel, J.M.; Crooke, S.T.

    1985-01-01

    Using [ 3 H]leukotriene C4 (LTC4) and radioligand-binding techniques, specific leukotriene C4 binding sites have been identified in membranes derived from guinea pig ventricular myocardium. High performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 2% of membrane-bound [ 3 H]LTC4 was converted at 20 degrees to [ 3 H]leukotriene D4 or [ 3 H]leukotriene E4. The specific binding of 4 nM [ 3 H]LTC4, in the presence of 80 mM L-serine-borate, reached a stable steady state within 15 min at 20 degrees (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded a dissociation constant (Kd) of 27.5 +/- 6.0 nM and a maximum number of binding sites (Bmax) of 19.9 +/- 5.2 pmol/mg of membrane protein. Competition binding studies of [ 3 H]LTC4 with synthetic leukotriene C4, leukotriene D4, and leukotriene E4 and the putative peptidoleukotriene antagonists FPL 55712, SKF 88046, and 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid revealed an order of potency of leukotriene C4 much greater than 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid greater than SKF 88046 greater than LTE4 greater than LTD4 greater than FPL 55712. The specific [ 3 H]LTC4 binding was stimulated by the divalent cations Ca2+, Mg2+, and Mn2+ and to a lesser degree by the monovalent cations Na+, K+, Li+, and NH4+. CaCl2 (3 mM) and NaCl (150 mM) stimulated the LTC4 binding by increasing the Bmax to 42.6 +/- 5.9 and 35.0 +/- 2.0 pmol/mg, respectively, but had minimal effects on Kd

  20. Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival.

    Science.gov (United States)

    Caine, Jennifer A; Lin, Yi-Pin; Kessler, Julie R; Sato, Hiromi; Leong, John M; Coburn, Jenifer

    2017-12-01

    Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced. © 2017 John Wiley & Sons Ltd.

  1. Reduced parahippocampal and lateral temporal GABA{sub A}-[{sup 11}C]flumazenil binding in major depression: preliminary results

    Energy Technology Data Exchange (ETDEWEB)

    Klumpers, Ursula M.H. [VU University Medical Center, Department of Psychiatry, Amsterdam (Netherlands); GGZ inGeest, partner of VUmc, Department of Psychiatry, Amsterdam (Netherlands); VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam (Netherlands); Veltman, Dick J. [VU University Medical Center, Department of Psychiatry, Amsterdam (Netherlands); VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam (Netherlands); Drent, Madeleine L. [VU University Medical Center, Department of Endocrinology, Amsterdam (Netherlands); Boellaard, Ronald; Lammertsma, Adriaan A. [VU University Medical Center, Department of Nuclear Medicine and PET Research, Amsterdam (Netherlands); VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam (Netherlands); Comans, Emile F.I. [VU University Medical Center, Department of Nuclear Medicine and PET Research, Amsterdam (Netherlands); Meynen, Gerben [VU University Medical Center, Department of Psychiatry, Amsterdam (Netherlands); Hoogendijk, Witte J.G. [VU University Medical Center, Department of Psychiatry, Amsterdam (Netherlands); VU University Medical Center, Center for Neurogenomics and Cognitive Research, Amsterdam (Netherlands); VU University Medical Center, Neuroscience Campus Amsterdam, Amsterdam (Netherlands)

    2010-03-15

    Major depressive disorder (MDD) has been related to both a dysfunctional {gamma}-amino butyric acid (GABA) system and to hyperactivity of the hypothalamic-pituitary-adrenal axis (HPA). Although GABA has been suggested to inhibit HPA axis activity, their relationship has never been studied at the level of the central GABA{sub A}-benzodiazepine receptor in depressed patients or in relation to antidepressant treatment. Eleven depressed outpatients were compared, before and after treatment with citalopram, with nine age-matched healthy controls. The subjects were scanned using the positron emission tomography (PET) tracer [{sup 11}C]flumazenil ([{sup 11}C]FMZ). Parametric voxel-by-voxel Logan plots were compared with methods based on regions of interest (ROI), to provide volume of distribution (V{sub T}) and binding potential (BP{sub ND}) values. Plasma GABA levels were determined and a dexamethasone-corticotropin releasing hormone (DEX-CRH) test was performed. In MDD, parametric voxel-by-voxel Logan plots showed bilateral reduced [{sup 11}C]FMZ binding in the parahippocampal gyrus and right lateral superior temporal gyrus (p uncorrected {<=}0.001). In the temporal area, [{sup 11}C]FMZ binding showed a strong inverse correlation with HPA axis activity. Plasma GABA did not discriminate MDD from controls, but correlated inversely with [{sup 11}C]FMZ binding in the right insula. Following treatment with citalopram, voxel-based analysis revealed reduced binding in the right lateral temporal gyrus and dorsolateral prefrontal cortex. The bilateral reduction in limbic parahippocampal and right temporal [{sup 11}C]FMZ binding found in MDD indicates decreased GABA{sub A}-benzodiazepine receptor complex affinity and/or number. The inverse relationship between GABA{sub A} binding in the temporal lobe and HPA axis activity, suggests that HPA axis hyperactivity is partly due to reduced GABA-ergic inhibition. (orig.)

  2. CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

    Energy Technology Data Exchange (ETDEWEB)

    Villarreal, Seth A.; Pattanayek, Rekha; Williams, Dewight R.; Mori, Tetsuya; Qin, Ximing; Johnson, Carl H.; Egli, Martin; Stewart, Phoebe L. [Case Western; (Vanderbilt); (Vanderbilt-MED)

    2014-10-02

    The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB–KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein–protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.

  3. A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale (Brassica Oleracea var. acephala f. tricolor).

    Science.gov (United States)

    Zhang, Bin; Hu, Zongli; Zhang, Yanjie; Li, Yali; Zhou, Shuang; Chen, Guoping

    2012-02-01

    The purple kale (Brassica Oleracea var. acephala f. tricolor) is a mutation in kales, giving the mutant phenotype of brilliant purple color in the interior. Total anthocyanin analysis showed that the amount of anthocyanins in the purple kale was up to 1.73 mg g(-1) while no anthocyanin was detected in the white kale. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the purple kale, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the purple cultivar "Red Dove" and the white cultivar "White Dove". The result showed that nearly all the anthocyanin biosynthetic genes showed higher expression levels in the purple cultivar than in the white cultivar, especially for DFR and ANS, they were barely detected in the white cultivar. Interestingly, the fact that a R2R3 MYB transcription factor named BoPAP1 was extremely up-regulated in the purple kale and induced by low temperature attracted our attention. Further sequence analysis showed that BoPAP1 shared high similarity with AtPAP1 and BoMYB1. In addition, the anthocyanin accumulation in the purple kale is strongly induced by the low temperature stress. The total anthocyanin contents in the purple kale under low temperature were about 50-fold higher than the plants grown in the greenhouse. The expression of anthocyanin biosynthetic genes C4H, F3H, DFR, ANS and UFGT were all enhanced under the low temperature. These evidences strongly suggest that BoPAP1 may play an important role in activating the anthocyanin structural genes for the abundant anthocyanin accumulation in the purple kale.

  4. Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

    Science.gov (United States)

    Pavan Kumar, Y; Saha, Puja; Saha, Dhurjhoti; Bessi, Irene; Schwalbe, Harald; Chowdhury, Shantanu; Dash, Jyotirmayee

    2016-03-02

    The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Regional brain [(11)C]carfentanil binding following tobacco smoking.

    Science.gov (United States)

    Domino, Edward F; Hirasawa-Fujita, Mika; Ni, Lisong; Guthrie, Sally K; Zubieta, Jon Kar

    2015-06-03

    To determine if overnight tobacco abstinent carriers of the AG or GG (*G) vs. the AA variant of the human mu opioid receptor (OPRM1) A118G polymorphism (rs1799971) differ in [(11)C]carfentanil binding after tobacco smoking. Twenty healthy American male smokers who abstained from tobacco overnight were genotyped and completed positron emission tomography (PET) scans with the mu opioid receptor agonist, [(11)C]carfentanil. They smoked deniconized (denic) and average nicotine (avnic) cigarettes during the PET scans. Smoking avnic cigarette decreased the binding potential (BPND) of [(11)C]carfentanil in the right medial prefrontal cortex (mPfc; 6, 56, 18), left anterior medial prefrontal cortex (amPfc; -2, 46, 44), right ventral striatum (vStr; 16, 3, -10), left insula (Ins; -42, 10, -12), right hippocampus (Hippo; 18, -6, -14) and left cerebellum (Cbl; -10, -88, -34), and increased the BPND in left amygdala (Amy; -20, 0, -22), left putamen (Put; -22, 10, -6) and left nucleus accumbens (NAcc; -10, 12, -8). In the AA allele carriers, avnic cigarette smoking significantly changed the BPND compared to after denic smoking in most brain areas listed above. However in the *G carriers the significant BPND changes were confirmed in only amPfc and vStr. Free mu opioid receptor availability was significantly less in the *G than the AA carriers in the Amy and NAcc. The present study demonstrates that BPND changes induced by avnic smoking in OPRM1 *G carriers were blunted compared to the AA carriers. Also *G smokers had less free mu opioid receptor availability in Amy and NAcc. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Radioligand binding analysis of α 2 adrenoceptors with [11C]yohimbine in brain in vivo: Extended Inhibition Plot correction for plasma protein binding

    DEFF Research Database (Denmark)

    Phan, Jenny-Ann; Landau, Anne M.; Jakobsen, Steen

    2017-01-01

    We describe a novel method of kinetic analysis of radioligand binding to neuroreceptors in brain in vivo, here applied to noradrenaline receptors in rat brain. The method uses positron emission tomography (PET) of [11C]yohimbine binding in brain to quantify the density and affinity of α 2...... Inhibition Plot introduced here yielded an estimate of the volume of distribution of non-displaceable ligand in brain tissue that increased with the increase of the free fraction of the radioligand in plasma. The resulting binding potentials of the radioligand declined by 50-60% in the presence of unlabeled...

  7. Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport.

    Science.gov (United States)

    Rehan, Medhat; Furnholm, Teal; Finethy, Ryan H; Chu, Feixia; El-Fadly, Gomaah; Tisa, Louis S

    2014-09-01

    Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller "leaf-like" structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics' analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu(2+) stress. After 5 days of Cu(2+) stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu(2+)-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu(2+)-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.

  8. MYB transcription factor gene involved in sex determination in Asparagus officinalis.

    Science.gov (United States)

    Murase, Kohji; Shigenobu, Shuji; Fujii, Sota; Ueda, Kazuki; Murata, Takanori; Sakamoto, Ai; Wada, Yuko; Yamaguchi, Katsushi; Osakabe, Yuriko; Osakabe, Keishi; Kanno, Akira; Ozaki, Yukio; Takayama, Seiji

    2017-01-01

    Dioecy is a plant mating system in which individuals of a species are either male or female. Although many flowering plants evolved independently from hermaphroditism to dioecy, the molecular mechanism underlying this transition remains largely unknown. Sex determination in the dioecious plant Asparagus officinalis is controlled by X and Y chromosomes; the male and female karyotypes are XY and XX, respectively. Transcriptome analysis of A. officinalis buds showed that a MYB-like gene, Male Specific Expression 1 (MSE1), is specifically expressed in males. MSE1 exhibits tight linkage with the Y chromosome, specific expression in early anther development and loss of function on the X chromosome. Knockout of the MSE1 orthologue in Arabidopsis induces male sterility. Thus, MSE1 acts in sex determination in A. officinalis. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  9. Serotonin 2A receptor agonist binding in the human brain with [C]Cimbi-36

    DEFF Research Database (Denmark)

    Ettrup, A.; da Cunha-Bang, S.; McMahon, Barry P.

    2014-01-01

    [C]Cimbi-36 was recently developed as a selective serotonin 2A (5-HT) receptor agonist radioligand for positron emission tomography (PET) brain imaging. Such an agonist PET radioligand may provide a novel, and more functional, measure of the serotonergic system and agonist binding is more likely ....... Thus, we here describe [C]Cimbi-36 as the first agonist PET radioligand to successfully image and quantify 5-HT receptors in the human brain.Journal of Cerebral Blood Flow & Metabolism advance online publication, 30 April 2014; doi:10.1038/jcbfm.2014.68....... than antagonist binding to reflect 5-HT levels in vivo. Here, we show data from a first-in-human clinical trial with [C]Cimbi-36. In 29 healthy volunteers, we found high brain uptake and distribution according to 5-HT receptors with [C]Cimbi-36 PET. The two-tissue compartment model using arterial input...

  10. Improved outcome with hematopoietic stem cell transplantation in a poor prognostic subgroup of infants with mixed-lineage-leukemia (MLL)-rearranged acute lymphoblastic leukemia: results from the Interfant-99 Study

    DEFF Research Database (Denmark)

    Mann, Georg; Attarbaschi, Andishe; Schrappe, Martin

    2010-01-01

    received chemotherapy only (P = .03). However, the advantage was restricted to a subgroup with 2 additional unfavorable prognostic features: age less than 6 months and either poor response to steroids at day 8 or leukocytes more than or equal to 300 g/L. Ninety-seven of 297 MLL(+) patients (33%) had...

  11. Novel thrombopoietin mimetic peptides bind c-Mpl receptor: Synthesis, biological evaluation and molecular modeling.

    Science.gov (United States)

    Liu, Yaquan; Tian, Fang; Zhi, Dejuan; Wang, Haiqing; Zhao, Chunyan; Li, Hongyu

    2017-02-01

    Thrombopoietin (TPO) acts in promoting the proliferation of hematopoietic stem cells and by initiating specific maturation events in megakaryocytes. Now, TPO-mimetic peptides with amino acid sequences unrelated to TPO are of considerable pharmaceutical interest. In the present paper, four new TPO mimetic peptides that bind and activate c-Mpl receptor have been identified, synthesized and tested by Dual-Luciferase reporter gene assay for biological activities. The molecular modeling research was also approached to understand key molecular mechanisms and structural features responsible for peptide binding with c-Mpl receptor. The results presented that three of four mimetic peptides showed significant activities. In addition, the molecular modeling approaches proved hydrophobic interactions were the driven positive forces for binding behavior between peptides and c-Mpl receptor. TPO peptide residues in P7, P13 and P7' positions were identified by the analysis of hydrogen bonds and energy decompositions as the key ones for benefiting better biological activities. Our data suggested the synthesized peptides have considerable potential for the future development of stable and highly active TPO mimetic peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. A new BODIPY/nanoparticle/Ni affinity system for binding of cytochrome c

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Kursunlu, Ahmed Nuri [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Arslan, Gulsin [Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey)

    2015-09-15

    Highlights: • BODIPY was synthesized, and then attached to magnetic nanoparticles. • Ni(II) ions were chelated on prepared material. • The binding of cytochrome c to obtained material was studied. - Abstract: In this study, 3,5-{Bis[4,4-difluoro, 8-(2,6-diethyl, 1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene)]}benzoylchloride (BODIPY) was synthesized for the improving of a new immobilized metal affinity supporting material. Firstly, the synthesized BODIPY was immobilized on iron oxide superparamagnetic nanoparticles (SPIONs) and then, Ni(II) ions were chelated with the active terminals of BODIPY on nanoparticles surfaces to prepare an immobilized metal affinity (IMA) adsorbent for protein adsorption. The amount of BODIPY coated on SPIONs was about 29.7 μM at 10 mg nanoparticles. 738 μmol of Ni(II) ions were loaded to 10 mg of the SPIONs/BODIPY. The binding amount of cytochrome c was found to be 170 μg to the SPIONs/BODIPY/Ni at pH 7.4. The binding amount of the molecules on SPIONs was analyzed by using UV–vis, fluorescence and atomic absorption spectroscopy. The characterization of the prepared surfaces was performed by FT-IR, SEM and TEM.

  13. Influence of alcoholism and cholesterol on TSPO binding in brain: PET [11C]PBR28 studies in humans and rodents.

    Science.gov (United States)

    Kim, Sung Won; Wiers, Corinde E; Tyler, Ryan; Shokri-Kojori, Ehsan; Jang, Yeon Joo; Zehra, Amna; Freeman, Clara; Ramirez, Veronica; Lindgren, Elsa; Miller, Gregg; Cabrera, Elizabeth A; Stodden, Tyler; Guo, Min; Demiral, Şükrü B; Diazgranados, Nancy; Park, Luke; Liow, Jeih-San; Pike, Victor; Morse, Cheryl; Vendruscolo, Leandro F; Innis, Robert B; Koob, George F; Tomasi, Dardo; Wang, Gene-Jack; Volkow, Nora D

    2018-05-03

    Neuroinflammation appears to contribute to neurotoxicity observed with heavy alcohol consumption. To assess whether chronic alcohol results in neuroinflammation we used PET and [ 11 C]PBR28, a ligand that binds to the 18-kDa translocator protein (TSPO), to compare participants with an alcohol use disorder (AUD: n = 19) with healthy controls (HC: n = 17), and alcohol-dependent (n = 9) with -nondependent rats (n = 10). Because TSPO is implicated in cholesterol's transport for steroidogenesis, we investigated whether plasma cholesterol levels influenced [ 11 C]PBR28 binding. [ 11 C]PBR28 binding did not differ between AUD and HC. However, when separating by TSPO genotype rs6971, we showed that medium-affinity binders AUD participants showed lower [ 11 C]PBR28 binding than HC in regions of interest (whole brain, gray and white matter, hippocampus, and thalamus), but no group differences were observed in high-affinity binders. Cholesterol levels inversely correlated with brain [ 11 C]PBR28 binding in combined groups, due to a correlation in AUD participants. In rodents, we observed no differences in brain [ 11 C]PBR28 uptake between alcohol-dependent and -nondependent rats. These findings, which are consistent with two previous [ 11 C]PBR28 PET studies, may indicate lower activation of microglia in AUD, whereas failure to observe alcohol effects in the rodent model indicate that species differences do not explain the discrepancy with prior rodent autoradiographic studies reporting increases in TSPO binding with chronic alcohol. However, reduced binding in AUD participants could also reflect competition from endogenous TSPO ligands such as cholesterol; and since the rs6971 polymorphism affects the cholesterol-binding domain of TSPO this could explain why differences were observed only in medium-affinity binders.

  14. Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent.

    Science.gov (United States)

    Kim, Eun-Mi; Joung, Min-Hee; Lee, Chang-Moon; Jeong, Hwan-Jeong; Lim, Seok Tae; Sohn, Myung-Hee; Kim, Dong Wook

    2010-07-15

    The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the 'click-to-chelate' protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12, [(99m)Tc(CO)(3)]13, and [(99m)Tc(CO)(3)]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)(3)]12, [Re(CO)(3)]13, and [Re(CO)(3)]14, were shown to have high binding affinities (0.13 microM, 0.06 microM, and 0.16 microM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12-14) to c-Met receptor positive U87MG cells. 2010 Elsevier Ltd. All rights reserved.

  15. Quantification of (R)-[11C]PK11195 binding in rheumatoid arthritis

    International Nuclear Information System (INIS)

    Kropholler, M.A.; Boellaard, R.; Kloet, R.W.; Lammertsma, A.A.; Elzinga, E.H.; Voskuyl, A.E.; Laken, C.J. van der; Dijkmans, B.A.C.; Maruyama, K.

    2009-01-01

    Rheumatoid arthritis (RA) involves migration of macrophages into inflamed areas. (R)-[ 11 C]PK11195 binds to peripheral benzodiazepine receptors, expressed on macrophages, and may be used to quantify inflammation using positron emission tomography (PET). This study evaluated methods for the quantification of (R)-[ 11 C]PK11195 binding in the knee joints of RA patients. Data from six patients with RA were analysed. Dynamic PET scans were acquired in 3-D mode following (R)-[ 11 C]PK11195 injection. During scanning arterial radioactivity concentrations were measured to determine the plasma (R)-[ 11 C]PK11195 concentrations. Data were analysed using irreversible and reversible one-tissue and two-tissue compartment models and input functions with various types of metabolite correction. Model preferences according to the Akaike information criterion (AIC) and correlations between measures were evaluated. Correlations between distribution volume (V d ) and standardized uptake values (SUV) were evaluated. AIC indicated optimal performance for a one-tissue reversible compartment model including blood volume. High correlations were observed between V d obtained using different input functions (R 2 =0.80-1.00) and between V d obtained with one- and two-tissue reversible compartment models (R 2 =0.75-0.94). A high correlation was observed between optimal V d and SUV after injection (R 2 =0.73). (R)-[ 11 C]PK11195 kinetics in the knee were best described by a reversible single-tissue compartment model including blood volume. Applying metabolite corrections did not increase sensitivity. Due to the high correlation with V d , SUV is a practical alternative for clinical use. (orig.)

  16. C to U RNA editing mediated by APOBEC1 requires RNA-binding protein RBM47.

    Science.gov (United States)

    Fossat, Nicolas; Tourle, Karin; Radziewic, Tania; Barratt, Kristen; Liebhold, Doreen; Studdert, Joshua B; Power, Melinda; Jones, Vanessa; Loebel, David A F; Tam, Patrick P L

    2014-08-01

    Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA-binding protein A1CF. We identify and characterise here a novel RNA-binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1-mediated editing in vitro. Editing is further impaired in Rbm47-deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing. © 2014 The Authors.

  17. Preliminary X-ray crystallographic study of the receptor-binding domain of the D/C mosaic neurotoxin from Clostridium botulinum

    International Nuclear Information System (INIS)

    Nuemket, Nipawan; Tanaka, Yoshikazu; Tsukamoto, Kentaro; Tsuji, Takao; Nakamura, Keiji; Kozaki, Shunji; Yao, Min; Tanaka, Isao

    2010-01-01

    To determine the binding mechanism of BoNT/OFD05 and its ganglioside receptors on neuronal cells, recombinant BoNT/OFD05 receptor-binding domain has been expressed, purified and crystallized. Botulinum toxin (BoNT) from Clostridium botulinum OFD05, isolated from bovine botulism, is a D/C mosaic-type BoNT. BoNTs possess binding, translocation and catalytic domains. The BoNT/OFD05 binding domain exhibits significant sequence identity to BoNT/C, which requires a single ganglioside as a binding receptor on neuronal cells, while BoNT/A and BoNT/B require two receptors for specific binding. To determine the binding mechanism of BoNT/OFD05 and its ganglioside receptors on neuronal cells, recombinant BoNT/OFD05 receptor-binding domain has been expressed, purified and crystallized. Native and SeMet-derivative crystals showed X-ray diffraction to 2.8 and 3.1 Å resolution, respectively. The crystals belonged to space group P2 1 2 1 2 1

  18. Tunable GLUT-Hexose Binding and Transport via Modulation of Hexose C-3 Hydrogen-Bonding Capabilities.

    Science.gov (United States)

    Kumar Kondapi, Venkata Pavan; Soueidan, Olivier-Mohamad; Cheeseman, Christopher I; West, Frederick G

    2017-06-12

    The importance of the hydrogen bonding interactions in the GLUT-hexose binding process (GLUT=hexose transporter) has been demonstrated by studying the binding of structurally modified d-fructose analogues to GLUTs, and in one case its transport into cells. The presence of a hydrogen bond donor at the C-3 position of 2,5-anhydro-d-mannitol derivatives is essential for effective binding to GLUT5 and transport into tumor cells. Surprisingly, installation of a group that can function only as a hydrogen bond acceptor at C-3 resulted in selective recognition by GLUT1 rather than GLUT5. A fluorescently labelled analogue clearly showed GLUT-mediated transport and low efflux properties of the probe. This study reveals that a single positional modification of a 2,5-anhydro-d-mannitol derivative is sufficient to switch its binding preference from GLUT5 to GLUT1, and uncovers general scaffolds that are suitable for the potential selective delivery of molecular payloads into tumor cells via GLUT transport machinery. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

    Energy Technology Data Exchange (ETDEWEB)

    Chinchilla, Diana, E-mail: Diana_Chinchilla@yahoo.com; Kilheeney, Heather, E-mail: raindropszoo@yahoo.com; Vitello, Lidia B., E-mail: lvitello@niu.edu; Erman, James E., E-mail: jerman@niu.edu

    2014-01-03

    Highlights: •Cytochrome c peroxidase (CcP) binds acrylonitrile in a pH-independent fashion. •The spectrum of the CcP/acrylonitrile complex is that of a 6c–ls ferric heme. •The acrylonitrile/CcP complex has a K{sub D} value of 1.1 ± 0.2 M. •CcP compound I oxidizes acrylonitrile with a maximum turnover rate of 0.61 min{sup −1}. -- Abstract: Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M{sup −1} s{sup −1} and 0.34 ± 0.15 s{sup −1}, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min{sup −1} at pH 6.0.

  20. Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.

    Science.gov (United States)

    Twerdochlib, Adriana L; Chubatsu, Leda S; Souza, Emanuel M; Pedrosa, Fábio O; Steffens, M Berenice R; Yates, M Geoffrey; Rigo, Liu U

    2003-07-01

    NtrC is a bacterial enhancer-binding protein (EBP) that activates transcription by the sigma54 RNA polymerase holoenzyme. NtrC has a three domain structure typical of EBP family. In Herbaspirillum seropedicae, an endophytic diazotroph, NtrC regulates several operons involved in nitrogen assimilation, including glnAntrBC. In order to over-express and purify the NtrC protein, DNA fragments containing the complete structural gene for the whole protein, and for the N-terminal+Central and Central+C-terminal domains were cloned into expression vectors. The NtrC and NtrC(N-terminal+Central) proteins were over-expressed as His-tag fusion proteins upon IPTG addition, solubilized using N-lauryl-sarcosyl and purified by metal affinity chromatography. The over-expressed His-tag-NtrC(Central+C-terminal) fusion protein was partially soluble and was also purified by affinity chromatography. DNA band-shift assays showed that the NtrC protein and the Central+C-terminal domains bound specifically to the H. seropedicae glnA promoter region. The C-terminal domain is presumably necessary for DNA-protein interaction and DNA-binding does not require a phosphorylated protein.

  1. Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum

    International Nuclear Information System (INIS)

    Leberer, E.; Charuk, J.H.M.; MacLennan, D.H.; Green, N.M.

    1989-01-01

    Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1,308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH 2 -terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca 2+ . The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45 C 2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca 2+ with the membrane. The authors propose that this lumenal Ca 2+ binding glycoprotein of the sarcoplasmic reticulum be designated sarcalumenin

  2. Docking Studies of Binding of Ethambutol to the C-Terminal Domain of the Arabinosyltransferase from Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Guillermo Salgado-Moran

    2013-01-01

    Full Text Available The binding of ethambutol to the C-terminal domain of the arabinosyltransferase from Mycobacterium tuberculosis was studied. The analysis was performed using an in silico approach in order to find out, by docking calculations and energy descriptors, the conformer of Ethambutol that forms the most stable complex with the C-terminal domain of arabinosyltransferase. The complex shows that location of the Ethambutol coincides with the cocrystallization ligand position and that amino acid residues ASH1051, ASN740, ASP1052, and ARG1055 should be critical in the binding of Ethambutol to C-terminal domain EmbC.

  3. Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus C binding and replication.

    Science.gov (United States)

    Bochkov, Yury A; Watters, Kelly; Ashraf, Shamaila; Griggs, Theodor F; Devries, Mark K; Jackson, Daniel J; Palmenberg, Ann C; Gern, James E

    2015-04-28

    Members of rhinovirus C (RV-C) species are more likely to cause wheezing illnesses and asthma exacerbations compared with other rhinoviruses. The cellular receptor for these viruses was heretofore unknown. We report here that expression of human cadherin-related family member 3 (CDHR3) enables the cells normally unsusceptible to RV-C infection to support both virus binding and replication. A coding single nucleotide polymorphism (rs6967330, C529Y) was previously linked to greater cell-surface expression of CDHR3 protein, and an increased risk of wheezing illnesses and hospitalizations for childhood asthma. Compared with wild-type CDHR3, cells transfected with the CDHR3-Y529 variant had about 10-fold increases in RV-C binding and progeny yields. We developed a transduced HeLa cell line (HeLa-E8) stably expressing CDHR3-Y529 that supports RV-C propagation in vitro. Modeling of CDHR3 structure identified potential binding sites that could impact the virus surface in regions that are highly conserved among all RV-C types. Our findings identify that the asthma susceptibility gene product CDHR3 mediates RV-C entry into host cells, and suggest that rs6967330 mutation could be a risk factor for RV-C wheezing illnesses.

  4. SPM analysis of parametric (R)-[11C]PK11195 binding images: plasma input versus reference tissue parametric methods.

    Science.gov (United States)

    Schuitemaker, Alie; van Berckel, Bart N M; Kropholler, Marc A; Veltman, Dick J; Scheltens, Philip; Jonker, Cees; Lammertsma, Adriaan A; Boellaard, Ronald

    2007-05-01

    (R)-[11C]PK11195 has been used for quantifying cerebral microglial activation in vivo. In previous studies, both plasma input and reference tissue methods have been used, usually in combination with a region of interest (ROI) approach. Definition of ROIs, however, can be labourious and prone to interobserver variation. In addition, results are only obtained for predefined areas and (unexpected) signals in undefined areas may be missed. On the other hand, standard pharmacokinetic models are too sensitive to noise to calculate (R)-[11C]PK11195 binding on a voxel-by-voxel basis. Linearised versions of both plasma input and reference tissue models have been described, and these are more suitable for parametric imaging. The purpose of this study was to compare the performance of these plasma input and reference tissue parametric methods on the outcome of statistical parametric mapping (SPM) analysis of (R)-[11C]PK11195 binding. Dynamic (R)-[11C]PK11195 PET scans with arterial blood sampling were performed in 7 younger and 11 elderly healthy subjects. Parametric images of volume of distribution (Vd) and binding potential (BP) were generated using linearised versions of plasma input (Logan) and reference tissue (Reference Parametric Mapping) models. Images were compared at the group level using SPM with a two-sample t-test per voxel, both with and without proportional scaling. Parametric BP images without scaling provided the most sensitive framework for determining differences in (R)-[11C]PK11195 binding between younger and elderly subjects. Vd images could only demonstrate differences in (R)-[11C]PK11195 binding when analysed with proportional scaling due to intersubject variation in K1/k2 (blood-brain barrier transport and non-specific binding).

  5. Recoveries of rat lymph FA after administration of specific structured C-13-TAG

    DEFF Research Database (Denmark)

    Vistisen, Bodil; Mu, Huiling; Høy, Carl-Erik

    2003-01-01

    of intragastrically administered L*L*L*, M*M*M*, ML*M, and ML*L* (where * = C-13-labeled FA) in rats. Lymph lipids were separated into lipid classes and analyzed by GC combustion isotope ratio MS. The recoveries of lymph TAG 18:2n-6 8 h after administration of L*L*L*, ML*M, and ML*L* were 38.6, 48.4, and 49...... rapid recovery of exogenous long-chain FA following administration of specific structured TAG compared with long-chain TAG was probably due to fast hydrolysis. The lymphatic recovery of 8:0 was 2.2% 24 h after administration of M*M*M*. This minor lymphatic recovery of exogenous 8:0 was probably due...... to low stimulation of chylomicron formation. These results demonstrate tendencies toward faster lymphatic recovery of long-chain FA after administration of specific structured TAG compared with long-chain TAG....

  6. Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode.

    Science.gov (United States)

    Nieto, Lidia; Tharun, Inga M; Balk, Mark; Wienk, Hans; Boelens, Rolf; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

    2015-11-20

    The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the "two-pronged plug two-hole socket" model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

  7. C-terminal low-complexity sequence repeats of Mycobacterium smegmatis Ku modulate DNA binding.

    Science.gov (United States)

    Kushwaha, Ambuj K; Grove, Anne

    2013-01-24

    Ku protein is an integral component of the NHEJ (non-homologous end-joining) pathway of DSB (double-strand break) repair. Both eukaryotic and prokaryotic Ku homologues have been characterized and shown to bind DNA ends. A unique feature of Mycobacterium smegmatis Ku is its basic C-terminal tail that contains several lysine-rich low-complexity PAKKA repeats that are absent from homologues encoded by obligate parasitic mycobacteria. Such PAKKA repeats are also characteristic of mycobacterial Hlp (histone-like protein) for which they have been shown to confer the ability to appose DNA ends. Unexpectedly, removal of the lysine-rich extension enhances DNA-binding affinity, but an interaction between DNA and the PAKKA repeats is indicated by the observation that only full-length Ku forms multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer. The C-terminal extension promotes DNA end-joining by T4 DNA ligase, suggesting that the PAKKA repeats also contribute to efficient end-joining. We suggest that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins.

  8. Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.

    Science.gov (United States)

    Dey, Sanjay; Biswas, Maitree; Sen, Udayaditya; Dasgupta, Jhimli

    2015-04-03

    Bacterial enhancer-binding proteins (bEBPs) oligomerize through AAA(+) domains and use ATP hydrolysis-driven energy to isomerize the RNA polymerase-σ(54) complex during transcriptional initiation. Here, we describe the first structure of the central AAA(+) domain of the flagellar regulatory protein FlrC (FlrC(C)), a bEBP that controls flagellar synthesis in Vibrio cholerae. Our results showed that FlrC(C) forms heptamer both in nucleotide (Nt)-free and -bound states without ATP-dependent subunit remodeling. Unlike the bEBPs such as NtrC1 or PspF, a novel cis-mediated "all or none" ATP binding occurs in the heptameric FlrC(C), because constriction at the ATPase site, caused by loop L3 and helix α7, restricts the proximity of the trans-protomer required for Nt binding. A unique "closed to open" movement of Walker A, assisted by trans-acting "Glu switch" Glu-286, facilitates ATP binding and hydrolysis. Fluorescence quenching and ATPase assays on FlrC(C) and mutants revealed that although Arg-349 of sensor II, positioned by trans-acting Glu-286 and Tyr-290, acts as a key residue to bind and hydrolyze ATP, Arg-319 of α7 anchors ribose and controls the rate of ATP hydrolysis by retarding the expulsion of ADP. Heptameric state of FlrC(C) is restored in solution even with the transition state mimicking ADP·AlF3. Structural results and pulldown assays indicated that L3 renders an in-built geometry to L1 and L2 causing σ(54)-FlrC(C) interaction independent of Nt binding. Collectively, our results underscore a novel mechanism of ATP binding and σ(54) interaction that strives to understand the transcriptional mechanism of the bEBPs, which probably interact directly with the RNA polymerase-σ(54) complex without DNA looping. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Analysis of Tb3+- and melittin-binding with the C-terminal domain of centrin in Euplotes octocarinatus

    International Nuclear Information System (INIS)

    Zhao Yaqin; Diao Xiuling; Yan Jun; Feng Yanan; Wang Zhijun; Liang Aihua; Yang Binsheng

    2012-01-01

    Centrin is a low molecular mass (20 KDa) protein that belongs to the EF-hand superfamily. In this work, the interaction between the Tb 3+ -saturated C-terminal domain of Euplotes octocarinatus centrin (Tb 2 -C-EoCen) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was investigated using difference UV–vis spectra and the fluorescence spectra methods. In 100 mM N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (Hepes) at pH 7.4, with the addition of Tb 2 -C-EoCen, four new peaks were observed at 265 nm, 278 nm, 317 nm and 360 nm by absorptivity compared with blank solution of TNS. At the same time, the reaction could be measured by fluorescence spectra. The fluorescence emission of TNS was shifted from 480 nm to 445 nm in the presence of Tb 2 -C-EoCen. Meanwhile, its fluorescence intensity was increased markedly. The 1:1 stoichiometric ratio of C-EoCen to TNS was confirmed by fluorescence titration curves. The conditional binding constants of TNS with C-EoCen and Tb 2 -C-EoCen were calculated to be log K (C-EoCen-TNS) =5.32±0.04 M −1 and log K (Tb2-C-EoCen-TNS) =5.58±0.12 M −1 , respectively. In addition, the protein of Tb 2 -C-EoCen binding with melittin was also studied. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of Tb 2 -C-EoCen to melittin was confirmed. And the conditional binding constant of C-EoCen with melittin was calculated to be log Ka′=6.79±0.17 M −1 . - Highlights: ► Tb 3+ induced conformational changes of protein C-EoCen from closed state to open state. ► Conformational changes resulted in the exposure of hydrophobic surfaces on C-EoCen. ► Tb 2 -C-EoCen may bind with target peptide melittin.

  10. Characterization of the hormone-binding domain of the chicken c-erbA/thyroid hormone receptor protein

    DEFF Research Database (Denmark)

    Muñoz, A; Zenke, M; Gehring, U

    1988-01-01

    mutations present in the carboxy-terminal half of P75gag-v-erbA co-operate in abolishing hormone binding, and that the ligand-binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand-binding domains of P75......To identify and characterize the hormone-binding domain of the thyroid hormone receptor, we analyzed the ligand-binding capacities of proteins representing chimeras between the normal receptor and P75gag-v-erbA, the retrovirus-encoded form deficient in binding ligand. Our results show that several......gag-v-erbA and that renders it biologically inactive fails to affect hormone binding by the c-erbA protein. These results suggest that the mutation changed the ability of P75gag-v-erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone...

  11. Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

    Science.gov (United States)

    Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe

    2015-01-01

    Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles. PMID:26580551

  12. Radioligand binding analysis of α 2 adrenoceptors with [11C]yohimbine in brain in vivo: Extended Inhibition Plot correction for plasma protein binding.

    Science.gov (United States)

    Phan, Jenny-Ann; Landau, Anne M; Jakobsen, Steen; Wong, Dean F; Gjedde, Albert

    2017-11-22

    We describe a novel method of kinetic analysis of radioligand binding to neuroreceptors in brain in vivo, here applied to noradrenaline receptors in rat brain. The method uses positron emission tomography (PET) of [ 11 C]yohimbine binding in brain to quantify the density and affinity of α 2 adrenoceptors under condition of changing radioligand binding to plasma proteins. We obtained dynamic PET recordings from brain of Spraque Dawley rats at baseline, followed by pharmacological challenge with unlabeled yohimbine (0.3 mg/kg). The challenge with unlabeled ligand failed to diminish radioligand accumulation in brain tissue, due to the blocking of radioligand binding to plasma proteins that elevated the free fractions of the radioligand in plasma. We devised a method that graphically resolved the masking of unlabeled ligand binding by the increase of radioligand free fractions in plasma. The Extended Inhibition Plot introduced here yielded an estimate of the volume of distribution of non-displaceable ligand in brain tissue that increased with the increase of the free fraction of the radioligand in plasma. The resulting binding potentials of the radioligand declined by 50-60% in the presence of unlabeled ligand. The kinetic unmasking of inhibited binding reflected in the increase of the reference volume of distribution yielded estimates of receptor saturation consistent with the binding of unlabeled ligand.

  13. Small molecule inhibition of hepatitis C virus E2 binding to CD81

    International Nuclear Information System (INIS)

    Van Compernolle, Scott E.; Wiznycia, Alexander V.; Rush, Jeremy R.; Dhanasekaran, Muthu; Baures, Paul W.; Todd, Scott C.

    2003-01-01

    The hepatitis C virus (HCV) is a causal agent of chronic liver infection, cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. CD81 is a receptor for HCV envelope glycoprotein E2. Although the binding of HCV-E2 with CD81 is well documented the role of this interaction in the viral life cycle remains unclear. Host specificity and mutagenesis studies suggest that the helix D region of CD81 mediates binding to HCV-E2. Structural analysis of CD81 has enabled the synthesis of small molecules designed to mimic the space and hydrophobic features of the solvent-exposed face on helix D. Utilizing a novel bis-imidazole scaffold a series of over 100 compounds has been synthesized. Seven related, imidazole-based compounds were identified that inhibit binding of HCV-E2 to CD81. The inhibitory compounds have no short-term effect on cellular expression of CD81 or other tetraspanins, do not disrupt CD81 associations with other cell surface proteins, and bind reversibly to HCV-E2. These results provide an important proof of concept that CD81-based mimics can disrupt binding of HCV-E2 to CD81

  14. C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

    Science.gov (United States)

    Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

    2016-10-01

    The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

  15. Conformational control of the binding of diatomic gases to cytochrome c'.

    Science.gov (United States)

    Manole, Andreea; Kekilli, Demet; Svistunenko, Dimitri A; Wilson, Michael T; Dobbin, Paul S; Hough, Michael A

    2015-06-01

    The cytochromes c' (CYTcp) are found in denitrifying, methanotrophic and photosynthetic bacteria. These proteins are able to form stable adducts with CO and NO but not with O2. The binding of NO to CYTcp currently provides the best structural model for the NO activation mechanism of soluble guanylate cyclase. Ligand binding in CYTcps has been shown to be highly dependent on residues in both the proximal and distal heme pockets. Group 1 CYTcps typically have a phenylalanine residue positioned close to the distal face of heme, while for group 2, this residue is typically leucine. We have structurally, spectroscopically and kinetically characterised the CYTcp from Shewanella frigidimarina (SFCP), a protein that has a distal phenylalanine residue and a lysine in the proximal pocket in place of the more common arginine. Each monomer of the SFCP dimer folds as a 4-alpha-helical bundle in a similar manner to CYTcps previously characterised. SFCP exhibits biphasic binding kinetics for both NO and CO as a result of the high level of steric hindrance from the aromatic side chain of residue Phe 16. The binding of distal ligands is thus controlled by the conformation of the phenylalanine ring. Only a proximal 5-coordinate NO adduct, confirmed by structural data, is observed with no detectable hexacoordinate distal NO adduct.

  16. Stable MCC binding to the APC/C is required for a functional spindle assembly checkpoint

    DEFF Research Database (Denmark)

    Hein, Jamin B; Nilsson, Jakob

    2014-01-01

    stably to the APC/C. Whether MCC formation per se is sufficient for a functional SAC or MCC association with the APC/C is required remains unclear. Here, we analyze the role of two conserved motifs in Cdc20, IR and C-Box, in binding of the MCC to the APC/C. Mutants in both motifs assemble the MCC....../C is critical for a functional SAC....

  17. Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

    Science.gov (United States)

    Nakamura, Toshio; Tonozuka, Takashi; Ide, Azusa; Yuzawa, Takayuki; Oguma, Keiji; Nishikawa, Atsushi

    2008-02-22

    Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.

  18. Steroid hormones affect binding of the sigma ligand 11C-SA4503 in tumour cells and tumour-bearing rats

    International Nuclear Information System (INIS)

    Rybczynska, Anna A.; Elsinga, Philip H.; Sijbesma, Jurgen W.; Jong, Johan R. de; Vries, Erik F. de; Dierckx, Rudi A.; Waarde, Aren van; Ishiwata, Kiichi

    2009-01-01

    Sigma receptors are implicated in memory and cognitive functions, drug addiction, depression and schizophrenia. In addition, sigma receptors are strongly overexpressed in many tumours. Although the natural ligands are still unknown, steroid hormones are potential candidates. Here, we examined changes in binding of the sigma-1 agonist 11 C-SA4503 in C6 glioma cells and in living rats after modification of endogenous steroid levels. 11 C-SA4503 binding was assessed in C6 monolayers by gamma counting and in anaesthetized rats by microPET scanning. C6 cells were either repeatedly washed and incubated in steroid-free medium or exposed to five kinds of exogenous steroids (1 h or 5 min before tracer addition, respectively). Tumour-bearing male rats were repeatedly treated with pentobarbital (a condition known to result in reduction of endogenous steroid levels) or injected with progesterone. Binding of 11 C-SA4503 to C6 cells was increased (∝50%) upon removal and decreased (∝60%) upon addition of steroid hormones (rank order of potency: progesterone > allopregnanolone = testosterone = androstanolone > dehydroepiandrosterone-3-sulphate, IC 50 progesterone 33 nM). Intraperitoneally administered progesterone reduced tumour uptake and tumour-to-muscle contrast (36%). Repeated treatment of animals with pentobarbital increased the PET standardized uptake value of 11 C-SA4503 in tumour (16%) and brain (27%), whereas the kinetics of blood pool radioactivity was unaffected. The binding of 11 C-SA4503 is sensitive to steroid competition. Since not only increases but also decreases of steroid levels affect ligand binding, a considerable fraction of the sigma-1 receptor population in cultured tumour cells or tumour-bearing animals is normally occupied by endogenous steroids. (orig.)

  19. C-terminal region of MAP7 domain containing protein 3 (MAP7D3 promotes microtubule polymerization by binding at the C-terminal tail of tubulin.

    Directory of Open Access Journals (Sweden)

    Saroj Yadav

    Full Text Available MAP7 domain containing protein 3 (MAP7D3, a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7 family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD with the C-terminal tail (CT of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0 ± 0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics.

  20. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    International Nuclear Information System (INIS)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-01-01

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

  1. Novel interactions of ankyrins-G at the costameres: The muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

    International Nuclear Information System (INIS)

    Maiweilidan, Yimingjiang; Klauza, Izabela; Kordeli, Ekaterini

    2011-01-01

    Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein-protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein-protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres

  2. Patterns of binding of aluminum-containing adjuvants to Haemophilus influenzae type b and meningococcal group C conjugate vaccines and components

    Science.gov (United States)

    Otto, Robert B.D.; Burkin, Karena; Amir, Saba Erum; Crane, Dennis T.; Bolgiano, Barbara

    2015-01-01

    The basis of Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroup C (MenC) glycoconjugates binding to aluminum-containing adjuvants was studied. By measuring the amount of polysaccharide and protein in the non-adsorbed supernatant, the adjuvant, aluminum phosphate, AlPO4, was found to be less efficient than aluminum hydroxide, Al(OH)3 at binding to the conjugates, at concentrations relevant to licensed vaccine formulations and when equimolar. At neutral pH, binding of TT conjugates to AlPO4 was facilitated through the carrier protein, with only weak binding of AlPO4 to CRM197 being observed. There was slightly higher binding of either adjuvant to tetanus toxoid conjugates, than to CRM197 conjugates. This was verified in AlPO4 formulations containing DTwP–Hib, where the adsorption of TT-conjugated Hib was higher than CRM197-conjugated Hib. At neutral pH, the anionic Hib and MenC polysaccharides did not appreciably bind to AlPO4, but did bind to Al(OH)3, due to electrostatic interactions. Phosphate ions reduced the binding of the conjugates to the adjuvants. These patterns of adjuvant adsorption can form the basis for future formulation studies with individual and combination vaccines containing saccharide-protein conjugates. PMID:26194164

  3. Inhibition of [11C]mirtazapine binding by alpha2-adrenoceptor antagonists studied by positron emission tomography in living porcine brain

    DEFF Research Database (Denmark)

    Smith, Donald F; Dyve, Suzan; Minuzzi, Luciano

    2006-01-01

    Inhibition of [11C]mirtazapine binding by alpha2-adrenoceptor antagonists studied by positron emission tomography in living porcine brain......Inhibition of [11C]mirtazapine binding by alpha2-adrenoceptor antagonists studied by positron emission tomography in living porcine brain...

  4. The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

    Directory of Open Access Journals (Sweden)

    Deng W-M

    2009-02-01

    Full Text Available Abstract Background Dystroglycan (Dg is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. Results We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. Conclusion Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

  5. Identification of a 48 kDa tubulin or tubulin-like C6/36 mosquito cells protein that binds dengue virus 2 using mass spectrometry

    International Nuclear Information System (INIS)

    Chee, H.-Y.; AbuBakar, Sazaly

    2004-01-01

    Binding of dengue virus 2 (DENV-2) to C6/36 mosquito cells protein was investigated. A 48 kDa DENV-2-binding C6/36 cells protein (D2BP) was detected in a virus overlay protein-binding assay. The binding occurred only to the C6/36 cells cytosolic protein fraction and it was inhibited by free D2BP. D2BP was shown to bind to DENV-2 E in the far-Western-binding studies and using mass spectrometry (MS) and MS/MS, peptide masses of the D2BP that matched to β-tubulin and α-tubulin chains were identified. These findings suggest that DENV-2 through DENV-2 E binds directly to a 48 kDa tubulin or tubulin-like protein of C6/36 mosquito cells

  6. A BPTTF-based self-assembled electron-donating triangle capable of C60 binding.

    Science.gov (United States)

    Goeb, Sébastien; Bivaud, Sébastien; Dron, Paul Ionut; Balandier, Jean-Yves; Chas, Marcos; Sallé, Marc

    2012-03-25

    A kinetically stable self-assembled redox-active triangle is isolated. The resulting electron-donating cavity, which incorporates three BPTTF units, exhibits a remarkable binding ability for electron-deficient C(60), supported by a favorable combination of structural and electronic features.

  7. Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

    Science.gov (United States)

    Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

    2015-02-01

    Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. CACTA-superfamily transposable element is inserted in MYB transcription factor gene of soybean line producing variegated seeds.

    Science.gov (United States)

    Yan, Fan; Di, Shaokang; Takahashi, Ryoji

    2015-08-01

    The R gene of soybean, presumably encoding a MYB transcription factor, controls seed coat color. The gene consists of multiple alleles, R (black), r-m (black spots and (or) concentric streaks on brown seed), and r (brown seed). This study was conducted to determine the structure of the MYB transcription factor gene in a near-isogenic line (NIL) having r-m allele. PCR amplification of a fragment of the candidate gene Glyma.09G235100 generated a fragment of about 1 kb in the soybean cultivar Clark, whereas a fragment of about 14 kb in addition to fragments of 1 and 1.4 kb were produced in L72-2040, a Clark 63 NIL with the r-m allele. Clark 63 is a NIL of Clark with the rxp and Rps1 alleles. A DNA fragment of 13 060 bp was inserted in the intron of Glyma.09G235100 in L72-2040. The fragment had the CACTA motif at both ends, imperfect terminal inverted repeats (TIR), inverse repetition of short sequence motifs close to the 5' and 3' ends, and a duplication of three nucleotides at the site of integration, indicating that it belongs to a CACTA-superfamily transposable element. We designated the element as Tgm11. Overall nucleotide sequence, motifs of TIR, and subterminal repeats were similar to those of Tgm1 and Tgs1, suggesting that these elements comprise a family.

  9. A single-repeat R3-MYB transcription factor MYBC1 negatively regulates freezing tolerance in Arabidopsis

    International Nuclear Information System (INIS)

    Zhai, Hong; Bai, Xi; Zhu, Yanming; Li, Yong; Cai, Hua; Ji, Wei; Ji, Zuojun; Liu, Xiaofei; Liu, Xin; Li, Jing

    2010-01-01

    We had previously identified the MYBC1 gene, which encodes a single-repeat R3-MYB protein, as a putative osmotic responding gene; however, no R3-MYB transcription factor has been reported to regulate osmotic stress tolerance. Thus, we sought to elucidate the function of MYBC1 in response to osmotic stresses. Real-time RT-PCR analysis indicated that MYBC1 expression responded to cold, dehydration, salinity and exogenous ABA at the transcript level. mybc1 mutants exhibited an increased tolerance to freezing stress, whereas 35S::MYBC1 transgenic plants exhibited decreased cold tolerance. Transcript levels of some cold-responsive genes, including CBF/DREB genes, KIN1, ADC1, ADC2 and ZAT12, though, were not altered in the mybc1 mutants or the 35S::MYBC1 transgenic plants in response to cold stress, as compared to the wild type. Microarray analysis results that are publically available were investigated and found transcript level of MYBC1 was not altered by overexpression of CBF1, CBF2, and CBF3, suggesting that MYBC1 is not down regulated by these CBF family members. Together, these results suggested that MYBC1is capable of negatively regulating the freezing tolerance of Arabidopsis in the CBF-independent pathway. In transgenic Arabidopsis carrying an MYBC1 promoter driven β-glucuronidase (GUS) construct, GUS activity was observed in all tissues and was relatively stronger in the vascular tissues. Fused MYBC1 and GFP protein revealed that MYBC1 was localized exclusively in the nuclear compartment.

  10. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

  11. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    International Nuclear Information System (INIS)

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

    2002-01-01

    The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that

  12. Patterns of binding of aluminum-containing adjuvants to Haemophilus influenzae type b and meningococcal group C conjugate vaccines and components.

    Science.gov (United States)

    Otto, Robert B D; Burkin, Karena; Amir, Saba Erum; Crane, Dennis T; Bolgiano, Barbara

    2015-09-01

    The basis of Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroup C (MenC) glycoconjugates binding to aluminum-containing adjuvants was studied. By measuring the amount of polysaccharide and protein in the non-adsorbed supernatant, the adjuvant, aluminum phosphate, AlPO4, was found to be less efficient than aluminum hydroxide, Al(OH)3 at binding to the conjugates, at concentrations relevant to licensed vaccine formulations and when equimolar. At neutral pH, binding of TT conjugates to AlPO4 was facilitated through the carrier protein, with only weak binding of AlPO4 to CRM197 being observed. There was slightly higher binding of either adjuvant to tetanus toxoid conjugates, than to CRM197 conjugates. This was verified in AlPO4 formulations containing DTwP-Hib, where the adsorption of TT-conjugated Hib was higher than CRM197-conjugated Hib. At neutral pH, the anionic Hib and MenC polysaccharides did not appreciably bind to AlPO4, but did bind to Al(OH)3, due to electrostatic interactions. Phosphate ions reduced the binding of the conjugates to the adjuvants. These patterns of adjuvant adsorption can form the basis for future formulation studies with individual and combination vaccines containing saccharide-protein conjugates. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  13. 5-HT(1A) receptor binding in euthymic bipolar patients using positron emission tomography with [carbonyl-(11)C]WAY-100635.

    Science.gov (United States)

    Sargent, Peter A; Rabiner, Eugenii A; Bhagwagar, Zubin; Clark, Luke; Cowen, Philip; Goodwin, Guy M; Grasby, Paul M

    2010-06-01

    This study was undertaken to examine whether brain 5-HT(1A) receptor binding is reduced in euthymic bipolar patients. Eight medicated euthymic bipolar patients and 8 healthy volunteers underwent positron emission tomography scanning using the selective 5-HT(1A) receptor radioligand [carbonyl-(11)C]WAY-100635. No significant difference in global postsynaptic parametric binding potential (BP(ND)) was found between euthymic bipolar patients (mean + or - SD, 4.24 + or - 0.76) and healthy volunteers (mean + or - SD, 4.34 + or - 0.86). Ninety five percent Confidence Intervals for the difference in group mean global postsynaptic BP(ND) were -0.77 to 0.97. Analysis of regional BP(ND) did not reveal regional differences between patients and healthy controls. The number of subjects studied was limited and all subjects were on medication. In contrast to previous findings of reduced 5-HT(1A) receptor binding in untreated unipolar and bipolar depressed patients [Sargent, P.A., Kjaer, K.H., Bench, C.J., Rabiner, E.A., Messa, C., Meyer, J., Gunn, R.N., Grasby, P.M., Cowen, P.J., 2000. Brain serotonin1A receptor binding measured by positron emission tomography with [(11)C]WAY-100635: effects of depression and antidepressant treatment. Arch. Gen. Psychiatry 57, 174-180]; [Drevets, W.C., Frank, E., Price, J.C., Kupfer, D.J., Holt, D., Greer, P.J., Huang, Y., Gautier, C., Mathis, C., 1999. PET imaging of serotonin1A receptor binding in depression. Biol. Psychiatry 46, 1375-1387] and in recovered unipolar depressed patients [Bhagwagar, Z., Rabiner, E.A., Sargent, P.A., Grasby, P.M., Cowen, P.J., 2004. Persistent reduction in brain serotonin1A receptor binding in recovered depressed men measured by positron emission tomography with [(11)C]WAY-100635. Mol. Psychiatry 9, 386-92], this study found no difference in 5-HT(1A) receptor BP(ND) between medicated euthymic bipolar patients and healthy controls. Normal 5-HT(1A) receptor BP(ND) in these patients may be a result of drug treatment or

  14. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

    Science.gov (United States)

    Csermely, P; Szamel, M; Resch, K; Somogyi, J

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

  15. A simple two-step purification procedure for the iC3b binding collectin conglutinin

    DEFF Research Database (Denmark)

    Krogh-Meibom, Thomas; Ingvartsen, Klaus Lønne; Tornoe, Ida

    2010-01-01

    Bovine conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step proced...

  16. The C-terminal domain of the Arabidopsis AtMBD7 protein confers strong chromatin binding activity

    International Nuclear Information System (INIS)

    Zemach, Assaf; Paul, Laju K.; Stambolsky, Perry; Efroni, Idan; Rotter, Varda; Grafi, Gideon

    2009-01-01

    The Arabidopsis MBD7 (AtMBD7) - a naturally occurring poly MBD protein - was previously found to be functional in binding methylated-CpG dinucleotides in vitro and localized to highly methylated chromocenters in vivo. Furthermore, AtMBD7 has significantly lower mobility within the nucleus conferred by cooperative activity of its three MBD motifs. Here we show that besides the MBD motifs, AtMBD7 possesses a strong chromatin binding domain located at its C-terminus designated sticky-C (StkC). Mutational analysis showed that a glutamic acid residue near the C-terminus is essential though not sufficient for the StkC function. Further analysis demonstrated that this motif can render nuclear proteins highly immobile both in plant and animal cells, without affecting their native subnuclear localization. Thus, the C-terminal, StkC motif plays an important role in fastening AtMBD7 to its chromosomal, CpG-methylated sites. It may be possible to utilize this motif for fastening nuclear proteins to their chromosomal sites both in plant and animal cells for research and gene therapy applications.

  17. Association of cardiac myosin binding protein-C with the ryanodine receptor channel: putative retrograde regulation?

    Science.gov (United States)

    Stanczyk, Paulina J; Seidel, Monika; White, Judith; Viero, Cedric; George, Christopher H; Zissimopoulos, Spyros; Lai, F Anthony

    2018-06-21

    The cardiac muscle ryanodine receptor-Ca 2+ release channel (RyR2) constitutes the sarcoplasmic reticulum (SR) Ca 2+ efflux mechanism that initiates myocyte contraction, while cardiac myosin binding protein-C (cMyBP-C) mediates regulation of acto-myosin cross-bridge cycling. In this report, we provide the first evidence for the presence of direct interaction between these two proteins, forming a RyR2:cMyBP-C complex. The C-terminus of cMyBP-C binds with the RyR2 N-terminus in mammalian cells and is not mediated by a fibronectin-like domain. Notably, we detected complex formation between both recombinant cMyBP-C and RyR2, as well as with the native proteins in cardiac tissue. Cellular Ca 2+ dynamics in HEK293 cells is altered upon co-expression of cMyBP-C and RyR2, with lowered frequency of RyR2-mediated spontaneous Ca 2+ oscillations, suggesting cMyBP-C exerts a potential inhibitory effect on RyR2-dependent Ca 2+ release. Discovery of a functional RyR2 association with cMyBP-C provides direct evidence for a putative mechanistic link between cytosolic soluble cMyBP-C and SR-mediated Ca 2+ release, via RyR2. Importantly, this interaction may have clinical relevance to the observed cMyBP-C and RyR2 dysfunction in cardiac pathologies, such as hypertrophic cardiomyopathy. © 2018. Published by The Company of Biologists Ltd.

  18. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  19. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    International Nuclear Information System (INIS)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-01-01

    Research highlights: → A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. → HCV-3a NS5A increases mature SREBP-1c protein level. → HCV-3a NS5A activates SREBP-1c transcription. → Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. → Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  20. Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate. [Spinacia oleracea

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Genhai; Jensen, R.G. (Univ. of Arizona, Tucson (USA))

    1990-05-01

    The properties of the tight and specific binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO{sub 2} and Mg{sup 2+}, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of ({sup 14}C)CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported. The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg{sup 2+}. Addition of ({sup 14}C)CABP and EDTA stopped binding of Mg{sup 2+} and allowed tight binding of the radiolabel only to sites which were CO{sub 2}/Mg{sup 2+}-activated at that moment. The rate of CO{sub 2} fixation was proportional to the CO{sub 2}/Mg{sup 2+}-activated sites. During light-dependent CO{sub 2} fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg{sup 2+} and CO{sub 2}. Lysis of chloroplasts in media with ({sup 14}C)CABP plus EDTA estimated those carbamylated sites having Mg{sup 2+}. The loss of Rubisco activation during illumination was partially due to the lack of Mg{sup 2+} to stabilize the carbamylated sites.

  1. Regional increases in [11C]flumazenil binding after epilepsy surgery

    International Nuclear Information System (INIS)

    Savic, I.; Blomqvist, G.; Halldin, C.; Litton, J.E.; Gulyas, B.

    1998-01-01

    Introduction - Animal experiments suggest that epileptic seizures alter the expression of mRNA for neuro-receptors. PET measurements with [ 11 C]flumazenil show that patients with partial seizures have a reduced density of benzodiazepine (BZ) receptors in the epileptogenic regions (ER) and some of the target areas for seizure activity, the so called projection areas. Recent data suggest that the degree of BZ receptor reduction in ER is correlated to seizure frequency. We therefore hypothesized that seizure activity can alter the BZ receptor binding, and that some of these changes could normalize when the seizures disappeared. Methods - In 4 patients whose seizures were generated by mesial temporal lobe structures, BZ receptor density was measured with [ 11 C]flumazenil PET before, and 1 year after the epilepsy surgery and cessation of seizures. By use of a computerized anatomical brain atlas the same regions were analyzed in both PET scans, and the results related to data from 7 healthy controls. Results - Presurgical PET scans showed reductions in BZ receptor density in the epileptogenic regions and some of its primary projection areas. Other cortical regions had normal values. Postsurgically, the calculated BZ receptor density normalized (29±17% increase) in several of the affected projection areas, whereas the values in other cortical regions remained unaltered. Conclusion - Regional reductions in BZ receptor density may be dynamic and related to seizures. The present preliminary observations encourage further studies on seizure-related changes in regional receptor binding in humans. (au)

  2. Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I.

    Science.gov (United States)

    Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B; Erman, James E

    2014-01-03

    Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32±0.16 M(-1) s(-1) and 0.34±0.15 s(-1), respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1±0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a "peroxygenase"-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min(-1) at pH 6.0. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Interaction between the C-terminal domains of measles virus nucleoprotein and phosphoprotein: a tight complex implying one binding site.

    Science.gov (United States)

    Blocquel, David; Habchi, Johnny; Costanzo, Stéphanie; Doizy, Anthony; Oglesbee, Michael; Longhi, Sonia

    2012-10-01

    The intrinsically disordered C-terminal domain (N(TAIL) ) of the measles virus (MeV) nucleoprotein undergoes α-helical folding upon binding to the C-terminal X domain (XD) of the phosphoprotein. The N(TAIL) region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489-506. In the previous studies published in this journal, we obtained experimental evidence supporting a K(D) for the N(TAIL) -XD binding reaction in the nM range and also showed that an additional N(TAIL) region (Box3, aa 517-525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (K(D) in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re-evaluate the role of Box3 in N(TAIL) -XD binding. Since our previous studies relied on N(TAIL) -truncated forms possessing an irrelevant Flag sequence appended at their C-terminus, we, herein, generated an N(TAIL) devoid of Box3 and any additional C-terminal residues, as well as a form encompassing only residues 482-525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these N(TAIL) forms. Results effectively argue for the presence of a single XD-binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high-affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point. Copyright © 2012 The Protein Society.

  4. Quantification of [11C]yohimbine binding to α2 adrenoceptors in rat brain in vivo

    DEFF Research Database (Denmark)

    Phan, Jenny-Ann; Landau, Anne M.; Wong, Dean F.

    2015-01-01

    We quantified the binding potentials (BPND) of [11C]yohimbine binding in rat brain to alpha-2 adrenoceptors to evaluate [11C]yohimbine as an in vivo marker of noradrenergic neurotransmission and to examine its sensitivity to the level of noradrenaline. Dual [11C]yohimbine dynamic positron emissio...... is sensitive to a challenge known to increase the extracellular level of noradrenaline, which can benefit future PET investigations of pathologic conditions related to disrupted noradrenergic neurotransmission....... challenge with amphetamine induced a significant decline of [11C]yohimbine BPND of ∼38% in all volumes of interest. The BPND was greatest in the thalamus and striatum, followed in descending order by, frontal cortex, pons, and cerebellum. The experimental data demonstrate that [11C]yohimbine binding...

  5. Mapping a candidate gene (MdMYB10 for red flesh and foliage colour in apple

    Directory of Open Access Journals (Sweden)

    Allan Andrew C

    2007-07-01

    Full Text Available Abstract Background Integrating plant genomics and classical breeding is a challenge for both plant breeders and molecular biologists. Marker-assisted selection (MAS is a tool that can be used to accelerate the development of novel apple varieties such as cultivars that have fruit with anthocyanin through to the core. In addition, determining the inheritance of novel alleles, such as the one responsible for red flesh, adds to our understanding of allelic variation. Our goal was to map candidate anthocyanin biosynthetic and regulatory genes in a population segregating for the red flesh phenotypes. Results We have identified the Rni locus, a major genetic determinant of the red foliage and red colour in the core of apple fruit. In a population segregating for the red flesh and foliage phenotype we have determined the inheritance of the Rni locus and DNA polymorphisms of candidate anthocyanin biosynthetic and regulatory genes. Simple Sequence Repeats (SSRs and Single Nucleotide Polymorphisms (SNPs in the candidate genes were also located on an apple genetic map. We have shown that the MdMYB10 gene co-segregates with the Rni locus and is on Linkage Group (LG 09 of the apple genome. Conclusion We have performed candidate gene mapping in a fruit tree crop and have provided genetic evidence that red colouration in the fruit core as well as red foliage are both controlled by a single locus named Rni. We have shown that the transcription factor MdMYB10 may be the gene underlying Rni as there were no recombinants between the marker for this gene and the red phenotype in a population of 516 individuals. Associating markers derived from candidate genes with a desirable phenotypic trait has demonstrated the application of genomic tools in a breeding programme of a horticultural crop species.

  6. Effect of NAD on binding and liberation of 14C-GABA in administration of the convulsion producing drug

    International Nuclear Information System (INIS)

    Fomenko, A.I.; Stepanenko, S.P.; Parkhomets, P.K.; Donchenko, G.V.

    1993-01-01

    Administration of corazole into animals led to a decrease in content of NAD and gamma-aminobutyric acid (GABA) in brain. Under these conditions, binding of 14 C-GABA was increased and its liberation was inhibited in the synaptosomes of the brain cortex. Additional administration of incotinamide, accompanied by considerable increase in content of NAD and GABA, caused a decrease in accumulation of exogenous GABA in the synaptosomes and removed the effects produced by the convulsant agent. Kinetics of 14 C-GABA binding in the presence of NAD demonstrated that the more effective inhibition of the binding occurred in the animals treated with the convulsant drug. NAD appears to affect the GABA-ergic transmission at the postsynaptic level

  7. Steroid hormones affect binding of the sigma ligand {sup 11}C-SA4503 in tumour cells and tumour-bearing rats

    Energy Technology Data Exchange (ETDEWEB)

    Rybczynska, Anna A.; Elsinga, Philip H.; Sijbesma, Jurgen W.; Jong, Johan R. de; Vries, Erik F. de; Dierckx, Rudi A.; Waarde, Aren van [University of Groningen, Nuclear Medicine and Molecular Imaging, University of Groningen Medical Center, Groningen (Netherlands); Ishiwata, Kiichi [Tokyo Metropolitan Institute of Gerontology, Positron Medical Center, Tokyo (Japan)

    2009-07-15

    Sigma receptors are implicated in memory and cognitive functions, drug addiction, depression and schizophrenia. In addition, sigma receptors are strongly overexpressed in many tumours. Although the natural ligands are still unknown, steroid hormones are potential candidates. Here, we examined changes in binding of the sigma-1 agonist {sup 11}C-SA4503 in C6 glioma cells and in living rats after modification of endogenous steroid levels. {sup 11}C-SA4503 binding was assessed in C6 monolayers by gamma counting and in anaesthetized rats by microPET scanning. C6 cells were either repeatedly washed and incubated in steroid-free medium or exposed to five kinds of exogenous steroids (1 h or 5 min before tracer addition, respectively). Tumour-bearing male rats were repeatedly treated with pentobarbital (a condition known to result in reduction of endogenous steroid levels) or injected with progesterone. Binding of {sup 11}C-SA4503 to C6 cells was increased ({proportional_to}50%) upon removal and decreased ({proportional_to}60%) upon addition of steroid hormones (rank order of potency: progesterone > allopregnanolone = testosterone = androstanolone > dehydroepiandrosterone-3-sulphate, IC{sub 50} progesterone 33 nM). Intraperitoneally administered progesterone reduced tumour uptake and tumour-to-muscle contrast (36%). Repeated treatment of animals with pentobarbital increased the PET standardized uptake value of {sup 11}C-SA4503 in tumour (16%) and brain (27%), whereas the kinetics of blood pool radioactivity was unaffected. The binding of {sup 11}C-SA4503 is sensitive to steroid competition. Since not only increases but also decreases of steroid levels affect ligand binding, a considerable fraction of the sigma-1 receptor population in cultured tumour cells or tumour-bearing animals is normally occupied by endogenous steroids. (orig.)

  8. (/sup 14/C)-L-valine binding to membranes of the frontal cortex in hepatic encephalopathy

    Energy Technology Data Exchange (ETDEWEB)

    Kienzl, E.; Riederer, P.; Jellinger, K. (Krankenhaus der Stadt Wien-Lainz (Austria). Ludwig Boltzmann Inst. fuer Neurobiologie); Kleinberger, G. (Vienna Univ. (Austria). 1. Medizinische Klinik)

    1982-01-01

    Serotonin (5-HT) receptors are modulated by L-valine (VAL). For further characterization of this effect a binding assay of (/sup 14/C)-L-VAL has been developed. A brief description of the experimental conditions is given. Moreover, measurement of VAL-binding has been applied to human brain tissue either from controls or hepatic failure. A marked increase of VAL-binding sites with no change in affinity was noted in hepatic coma, while in patients treated with parenteral nutrition plus VAL no such change could be measured. It is concluded that the beneficial therapeutic effects of VAL in hepatic encephalopathy are, at least in part, due to its modulating action on postsynaptic receptor membranes.

  9. Bridging Binding Modes of Phosphine-Stabilized Nitrous Oxide to Zn(C6F5)2

    NARCIS (Netherlands)

    Neu, Rebecca C.; Otten, Edwin; Stephan, Douglas W.

    2009-01-01

    Reaction of [tBu3PN2O(B(C6H4F)3)] with 1, 1.5, or 2 equivalents of Zn(C6F5)2 affords the species [{tBu3PN2OZn(C6F5)2}2], [{tBu3PN2OZn(C6F5)2}2Zn(C6F5)2], and [tBu3PN2O{Zn(C6F5)2}2] displaying unique binding modes of Zn to the phosphine-stabilized N2O fragment.

  10. The Impact of XmnI-HBG2, BCL11A and HBS1L-MYB Single Nucleotide Polymorphisms on Hb F Variation of Hematologically Normal Iranian Individuals.

    Science.gov (United States)

    Keyhani, Elaheh; Jafari Vesiehsari, Mahjoobeh; Talebi Kakroodi, Setareh; Darabi, Elham; Zamani, Fahimeh; Karimlou, Masoud; Kamali, Koorosh; Neishabury, Maryam

    2016-06-01

    The impact of Hb F on severity of sickle cell disease and β-thalassemia (β-thal) is well documented. The XmnI-HBG2, BCL11A and HBS1L-MYB single nucleotide polymorphisms (SNPs) have been introduced as the most important factors causing variation in fetal hemoglobin (Hb F) levels in different population studies. However, the extent of their effect could be population-specific. In this study, multivariate linear regression analysis was used to evaluate the association of Hb F with age, sex, and eight SNPs, including XmnI-HBG2, four BCL11A, two HBS1L-MYB SNPs and the polymorphic palindromic 5' hypersensitive 4-locus control region (5'HS4-LCR). One hundred and twenty-two hematologically normal individuals, from a previous study cohort, constituted our study population. In multivariate regression analyses, no association of Hb F was observed with age or sex of the individuals and SNPs in this study. We conducted a univariate regression analysis to further investigate the results, which among all the factors only detected XmnI-HBG2 and 5'HS4 SNPs as significant modifiers of Hb F. The significance of these two factors disappeared in a bivariate analysis. These results suggest that either XmnI-HBG2 or 5'HS4-LCR have a stronger contribution in Hb F variations of the Iranian population than BCL11A and HBS1L-MYB SNPs. Furthermore, the effect of low population size and technical limitations on obtained results could not be ruled out.

  11. Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release

    Energy Technology Data Exchange (ETDEWEB)

    Voleti, Rashmi; Tomchick, Diana R.; Südhof, Thomas C.; Rizo, Josep

    2017-09-18

    Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.

  12. C-terminal β9-strand of the cyclic nucleotide-binding homology domain stabilizes activated states of Kv11.1 channels.

    Directory of Open Access Journals (Sweden)

    Chai Ann Ng

    Full Text Available Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K(+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels.

  13. Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

    Science.gov (United States)

    Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

    2012-01-01

    Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

  14. Kinetic analysis of [11C]vorozole binding in the human brain with positron emission tomography.

    Science.gov (United States)

    Logan, Jean; Kim, Sung Won; Pareto, Deborah; Telang, Frank; Wang, Gene-Jack; Fowler, Joanna S; Biegon, Anat

    2014-01-01

    Using positron emission tomography, we investigated the kinetics of [11C]vorozole ([11C]VOR), a radiotracer for the enzyme aromatase that catalyzes the last step in estrogen biosynthesis. Six subjects were scanned under baseline conditions followed by retest 2 weeks later. The retest was followed by a blocking study with 2.5 mg of the aromatase inhibitor letrozole. The binding potential (BP(A)ND) was estimated from a Lassen plot using the total tissue distribution volume (VT) for baseline and blocked. for the thalamus was found to be 15 times higher than that for the cerebellum. From the letrozole studies, we found that [11C]VOR exhibits a slow binding compartment (small k4) that has a nonspecific and a blockable component. Because of the sensitivity of VT to variations in k4, a common value was used for the four highest binding regions. We also considered the tissue uptake to plasma ratio for 60 to 90 minutes as an outcome measure. Using the ratio method, the difference between the highest and lowest was 2.4 compared to 3.5 for the VT. The ratio method underestimates the high regions but is less variable and may be more suitable for patient studies. Because of its kinetics and distribution, this tracer is not a candidate for a bolus infusion or reference tissue methods.

  15. Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states.

    Science.gov (United States)

    Mazhab-Jafari, Mohammad T; Das, Rahul; Fotheringham, Steven A; SilDas, Soumita; Chowdhury, Somenath; Melacini, Giuseppe

    2007-11-21

    cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a

  16. Arabidopsis CDS blastp result: AK064765 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064765 002-120-H12 At4g11400.1 ARID/BRIGHT DNA-binding domain-containing protein ...otein ELD/OSA1 [Homo sapiens] GI:18568414; contains Pfam profiles PF01388: ARID/BRIGHT DNA binding domain, PF01448: ELM2 domain, PF00249: Myb-like DNA-binding domain 3e-45 ...

  17. Arabidopsis CDS blastp result: AK059905 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059905 006-208-H04 At4g11400.1 ARID/BRIGHT DNA-binding domain-containing protein ...otein ELD/OSA1 [Homo sapiens] GI:18568414; contains Pfam profiles PF01388: ARID/BRIGHT DNA binding domain, PF01448: ELM2 domain, PF00249: Myb-like DNA-binding domain 4e-29 ...

  18. Comparison of the binding of the irreversible monoamine oxidase tracers, [11C]clorgyline and [11C]l-deprenyl in brain and peripheral organs in humans

    International Nuclear Information System (INIS)

    Fowler, Joanna S.; Logan, Jean; Wang, Gene-Jack; Volkow, Nora D.; Telang, Frank; Ding Yushin; Shea, Colleen; Garza, Victor; Xu Youwen; Li Zizhong; Alexoff, David; Vaska, Paul; Ferrieri, Richard; Schlyer, David; Zhu Wei; John Gatley, S.

    2004-01-01

    The monoamine oxidase A and B (MAO A and B) radiotracers [ 11 C]clorgyline (CLG) and [ 11 C]L-deprenyl (DEP) and their deuterium labeled counterparts (CLG-D and DEP-D) were compared to determine whether their distribution and kinetics in humans are consistent with their physical, chemical and pharmacological properties and the reported ratios of MAO A:MAO B in post-mortem human tissues. Irreversible binding was consistently higher for DEP in brain, heart, kidneys and spleen but not lung where CLG >DEP and not in thyroid where there is no DEP binding. The generally higher DEP binding is consistent with its higher enzyme affinity and larger free fraction in plasma while differences in regional distribution for CLG and DEP in brain, heart, thyroid and lungs are consistent with different relative ratios of MAO A and B in humans

  19. Chorion gene activation and repression is dependent on BmC/EBP expression and binding to cognate cis-elements.

    Science.gov (United States)

    Papantonis, Argyris; Sourmeli, Sissy; Lecanidou, Rena

    2008-05-09

    From the different cis-elements clustered on silkmoth chorion gene promoters, C/EBP binding sites predominate. Their sequence composition and dispersal vary amongst promoters of diverse developmental specificity. Occupancy of these sites by BmC/EBP was examined through Southwestern and ChIP assays modified to suit ovarian follicular cells. For the genes studied, binding of BmC/EBP coincided with the respective stages of transcriptional activation. However, the factor was reloaded on promoter sequences long after individual gene repression. Furthermore, suppression of BmC/EBP transcription in developing follicles resulted in de-regulation of chorion gene expression. A biphasic function of BmC/EBP, according to which it may act as both an activator and a repressor during silkmoth choriogenesis, is considered under the light of the presented data.

  20. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  1. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  2. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

    OpenAIRE

    Kadamur, Ganesh; Ross, Elliott M.

    2016-01-01

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in...

  3. Identification of TTAGGG-binding proteins in Neurospora crassa, a fungus with vertebrate-like telomere repeats.

    Science.gov (United States)

    Casas-Vila, Núria; Scheibe, Marion; Freiwald, Anja; Kappei, Dennis; Butter, Falk

    2015-11-17

    To date, telomere research in fungi has mainly focused on Saccharomyces cerevisiae and Schizosaccharomyces pombe, despite the fact that both yeasts have degenerated telomeric repeats in contrast to the canonical TTAGGG motif found in vertebrates and also several other fungi. Using label-free quantitative proteomics, we here investigate the telosome of Neurospora crassa, a fungus with canonical telomeric repeats. We show that at least six of the candidates detected in our screen are direct TTAGGG-repeat binding proteins. While three of the direct interactors (NCU03416 [ncTbf1], NCU01991 [ncTbf2] and NCU02182 [ncTay1]) feature the known myb/homeobox DNA interaction domain also found in the vertebrate telomeric factors, we additionally show that a zinc-finger protein (NCU07846) and two proteins without any annotated DNA-binding domain (NCU02644 and NCU05718) are also direct double-strand TTAGGG binders. We further find two single-strand binders (NCU02404 [ncGbp2] and NCU07735 [ncTcg1]). By quantitative label-free interactomics we identify TTAGGG-binding proteins in Neurospora crassa, suggesting candidates for telomeric factors that are supported by phylogenomic comparison with yeast species. Intriguingly, homologs in yeast species with degenerated telomeric repeats are also TTAGGG-binding proteins, e.g. in S. cerevisiae Tbf1 recognizes the TTAGGG motif found in its subtelomeres. However, there is also a subset of proteins that is not conserved. While a rudimentary core TTAGGG-recognition machinery may be conserved across yeast species, our data suggests Neurospora as an emerging model organism with unique features.

  4. Test-retest reproducibility of [{sup 11}C]PBR28 binding to TSPO in healthy control subjects

    Energy Technology Data Exchange (ETDEWEB)

    Collste, K.; Forsberg, A.; Varrone, A.; Amini, N.; Halldin, C.; Farde, L.; Cervenka, S. [Karolinska Institutet, Department of Clinical Neuroscience, Centre for Psychiatry Research, Stockholm (Sweden); Aeinehband, S. [Karolinska Institutet, Department of Clinical Neuroscience, Neuroimmunology Unit, Stockholm (Sweden); Yakushev, I. [Karolinska Institutet, Department of Clinical Neuroscience, Centre for Psychiatry Research, Stockholm (Sweden); Technische Universitaet Muenchen, Department of Nuclear Medicine and TUM Neuroimaging Center (TUM-NIC), Munich (Germany)

    2016-01-15

    The PET radioligand [{sup 11}C]PBR28 binds to the translocator protein (TSPO), a marker of brain immune activation. We examined the reproducibility of [{sup 11}C]PBR28 binding in healthy subjects with quantification on a regional and voxel-by-voxel basis. In addition, we performed a preliminary analysis of diurnal changes in TSPO availability. Twelve subjects were examined using a high-resolution research tomograph and [{sup 11}C]PBR28, six in the morning and afternoon of the same day, and six in the morning on two separate days. Regional volumes of distribution (V{sub T}) were derived using a region-of-interest based two-tissue compartmental analysis (2TCM), as well as a parametric approach. Metabolite-corrected arterial plasma was used as input function. For the whole sample, the mean absolute variability in V{sub T} in the grey matter (GM) was 18.3 ± 12.7 %. Intraclass correlation coefficients in GM regions ranged from 0.90 to 0.94. Reducing the time of analysis from 91 to 63 min yielded a variability of 16.9 ± 14.9 %. There was a strong correlation between the parametric and 2TCM-derived GM values (r = 0.99). A significant increase in GM V{sub T} was observed between the morning and afternoon examinations when using secondary methods of quantification (p = 0.028). In the subjects examined at the same time of the day, the absolute variability was 15.9 ± 12.2 % for the 91-min 2TCM data. V{sub T} of [{sup 11}C]PBR28 binding showed medium reproducibility and high reliability in GM regions. Our findings support the use of parametric approaches for determining [{sup 11}C]PBR28 V{sub T} values, and indicate that the acquisition time could be shortened. Diurnal changes in TSPO binding in the brain may be a potential confounder in clinical studies and should be investigated further. (orig.)

  5. The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

    Directory of Open Access Journals (Sweden)

    Clara L Oeste

    2011-01-01

    Full Text Available Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184 in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14-PGJ(2 (15d-PGJ(2 and Δ(12-PGJ(2 selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO, a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2. Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

  6. DNA clasping by mycobacterial HU: the C-terminal region of HupB mediates increased specificity of DNA binding.

    Directory of Open Access Journals (Sweden)

    Sandeep Kumar

    Full Text Available BACKGROUND: HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb has only one subunit of HU coded by ORF Rv2986c (hupB gene. One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB(Mtb bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function. METHODOLOGY/PRINCIPAL FINDINGS: With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB(Mtb and another which expresses only the N terminal region (first 95 amino acid of hupB (HupB(MtbN. Gel retardation assays revealed that HupB(MtbN is almost like E. coli HU (heat stable nucleoid protein in terms of its DNA binding, with a binding constant (K(d for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HUalphaalpha and HUalphabeta forms. However CTR (C-terminal Region of HupB(Mtb imparts greater specificity in DNA binding. HupB(Mtb protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton's reaction, were compared. It was also observed that both the proteins have DNA binding preference for A:T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb. CONCLUSIONS/SIGNIFICANCE: These data thus point that HupB(Mtb may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.

  7. Insulin-like growth factor binding protein-2: contributions of the C-terminal domain to insulin-like growth factor-1 binding.

    Science.gov (United States)

    Kibbey, Megan M; Jameson, Mark J; Eaton, Erin M; Rosenzweig, Steven A

    2006-03-01

    Signaling by the insulin-like growth factor (IGF)-1 receptor (IGF-1R) has been implicated in the promotion and aggressiveness of breast, prostate, colorectal, and lung cancers. The IGF binding proteins (IGFBPs) represent a class of natural IGF antagonists that bind to and sequester IGF-1/2 from the IGF-1R, making them attractive candidates as therapeutics for cancer prevention and control. Recombinant human IGFBP-2 significantly attenuated IGF-1-stimulated MCF-7 cell proliferation with coaddition of 20 or 100 nM IGFBP-2 (50 or 80% inhibition, respectively). We previously identified IGF-1 contact sites both upstream and downstream of the CWCV motif (residues 247-250) in human IGFBP-2 (J Biol Chem 276:2880-2889, 2001). To further test their contributions to IGFBP-2 function, the single tryptophan in human IGFBP-2, Trp-248, was selectively cleaved with 2-(2'nitrophenylsulfenyl)-3-methyl-3 bromoindolenine (BNPS-skatole) and the BNPS-skatole products IGFBP-2(1-248) and IGFBP-2(249-289) as well as IGFBP-2(1-190) were expressed as glutathione S-transferase-fusion proteins and purified. Based on competition binding analysis, deletion of residues 249 to 289 caused an approximately 20-fold decrease in IGF-1 binding affinity (IGFBP-2 EC50 = 0.35 nM and IGFBP-2(1-248) = 7 nM). Removal of the remainder of the C-terminal domain had no further effect on affinity (IGFBP-2(1-190) EC50 = 9.2 nM). In kinetic assays, IGFBP-2(1-248) and IGFBP-2(1-190) exhibited more rapid association and dissociation rates than full-length IGFBP-2. These results confirm that regions upstream and downstream of the CWCV motif participate in IGF-1 binding. They further support the development of full-length IGFBP-2 as a cancer therapeutic.

  8. Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

    Science.gov (United States)

    Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

    2004-08-01

    Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

  9. C-peptide prevents SMAD3 binding to alpha promoters to inhibit collagen type IV synthesis.

    Science.gov (United States)

    Li, Yanning; Zhong, Yan; Gong, Wenjian; Gao, Xuehan; Qi, Huanli; Liu, Kun; Qi, Jinsheng

    2018-07-01

    Activation of transforming growth factor β1 (TGFB1)/SMAD3 signaling may lead to additional synthesis of collagen type IV (COL4), which is a major contributor to extracellular matrix (ECM) accumulation in diabetic nephropathy (DN). C-peptide can attenuate fibrosis to have unique beneficial effects in DN. However, whether and how C-peptide affects TGFB1/SMAD3-activated COL4 synthesis is unclear. In this study, pathological changes, expression of COL4 a1-a5 chains ( Col4a1-a5 ), COL4 distribution and protein and TGFB1 and SMAD3 protein were first assessed in a rat model of diabetes. Then, rat mesangial cells were treated with high glucose (HG) and/or C-peptide to investigate the underlying mechanism. Col4a1-a5 expression, COL4 protein and secretion, TGFB1 protein, SMAD3 nuclear translocation and binding of SMAD3 to its cognate sites in the promoters of Col4a1a2 , Col4a3a4 and Col4a5 were measured. It was found that C-peptide attenuated glomerular pathological changes and suppressed renal Col4a1 -a5 mRNA expression, COL4 protein content and TGFB1 protein content. C-peptide had a dose-dependent effect to inhibit Col4a1-a5 mRNA expression, COL4 protein content and secretion, in HG-stimulated mesangial cells. In addition, the HG-induced increase in TGFB1 protein content was significantly reduced by C-peptide. Although not apparently affecting SMAD3 nuclear translocation, C-peptide prevented SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters in HG-stimulated mesangial cells. In conclusion, C-peptide could prevent SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters, to inhibit COL4 generation. These results may provide a mechanism for the alleviation of fibrosis in DN by C-peptide. © 2018 Society for Endocrinology.

  10. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  11. Two distinct binding modes define the interaction of Brox with the C-terminal tails of CHMP5 and CHMP4B.

    Science.gov (United States)

    Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Sette, Paola; Rudd, Victoria; Chuenchor, Watchalee; Bello, Nana F; Bouamr, Fadila; Xiao, Tsan Sam

    2012-05-09

    Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic α helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an α helix, the CHMP5 C-terminal tail adopts a tandem β-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a β-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4 involved in RNA silencing.

    Directory of Open Access Journals (Sweden)

    Katia Marrocco

    Full Text Available Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome is a master ubiquitin protein ligase (E3 that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA. This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2 of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.

  13. Iron hexacyanide/cytochrome-C - intramolecular electron transfer and binding constants - (pulse radiolytic study). Progress report

    International Nuclear Information System (INIS)

    Ilan, Y.; Shafferman, A.

    Internal oxidation and reduction rates of horse cytochrome-c in the complexes, CII.Fe/sup III/(CN) -3 6 and CIII.Fe/sup II/(CN) -4 6 , are 4.6.10 4 s -1 and 3.3.10 2 s -1 , respectively. The binding sites of the iron hexacyanide ions on either CII or CIII are kinetically almost indistinguishable; binding constants range from 0.87.10 3 to 2.10 3 M -1 . The present pulse radiolytic kinetic data are compared with that from N.M.R, T-jump and equilibrium dialysis studies

  14. Cyclic adenosine 3',5'-monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter.

    Science.gov (United States)

    Clem, Brian F; Hudson, Elizabeth A; Clark, Barbara J

    2005-03-01

    Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5'-TGACGTCA-3') within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.

  15. A and MdMYB1 allele-specific markers controlling apple (Malus x domestica Borkh.) skin color and suitability for marker-assisted selection.

    Science.gov (United States)

    Zhang, X J; Wang, L X; Chen, X X; Liu, Y L; Meng, R; Wang, Y J; Zhao, Z Y

    2014-10-31

    Pre-selection for fruit skin color at the seedling stage would be highly advantageous, with marker-assisted selection offering a potential method for apple pre-selection. A and MdMYB1 alleles are allele-specific DNA markers that are potentially associated with apple skin color, and co-segregate with the Rf and Rni loci, respectively. Here, we assessed the potential application of these 2 alleles for marker-assisted breeding across 30 diverse cultivars and 2 apple seedling progenies. The red skin color phenotype was usually associated with the MdMYB1-1 allele and A(1) allele, respectively, while the 2 molecular markers provided approximately 91% predictability in the 'Fuji' x 'Cripps Pink' and 'Fuji' x 'Gala' progenies. The results obtained from the 30 cultivars and 2 progenies were consistent for the 2 molecular markers. Hence, the results supported that Rf and Rni could be located in a gene cluster, or even correspond to alleles of the same gene. Our results are consistent with the hypothesis that red/yellow dimorphism is controlled by a monogenic system, with the presence of the red anthocyanin pigmentation being dominant. In addition, our results supported that the practical utilization of the 2 function markers to efficiently and accurately select red-skinned apple cultivars in apple scion breeding programs.

  16. Assembly of human C-terminal binding protein (CtBP) into tetramers.

    Science.gov (United States)

    Bellesis, Andrew G; Jecrois, Anne M; Hayes, Janelle A; Schiffer, Celia A; Royer, William E

    2018-06-08

    C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD + - and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer. © 2018 Bellesis et al.

  17. Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

    OpenAIRE

    Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

    1981-01-01

    We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

  18. Effects of acute nicotine on hemodynamics and binding of [11C]raclopride to dopamine D2,3 receptors in pig brain.

    Science.gov (United States)

    Cumming, Paul; Rosa-Neto, Pedro; Watanabe, Hideaki; Smith, Donald; Bender, Dirk; Clarke, Paul B S; Gjedde, Albert

    2003-07-01

    Positive reinforcing properties of nicotine and the psychostimulants have been attributed to elevated dopamine release in the basal ganglia. It is well known that the specific binding of [(11)C]raclopride to dopamine D(2,3) receptors in living striatum is reduced by cocaine and amphetamines, revealing increased competition between endogenous dopamine and [(11)C]raclopride for dopamine D(2,3) receptors. However, the sensitivity of [(11)C]raclopride binding to nicotine-induced dopamine release is less well documented. In order to provide the basis for mapping effects of nicotine, we first optimized reference tissue methods for quantifying [(11)C]raclopride binding sites in striatum of living pigs (n = 16). In the same animals, the rate of cerebral blood flow (CBF) was mapped using [(15)O]water. Neither a low dose of nicotine (50 mu kg(-1), iv) nor a high dose of nicotine (500 microg kg(-1), iv) altered CBF in the pig brain, an important condition for calculating the binding of radioligands when using a reference tissue to estimate the free ligand concentration. The methods of Logan and of Lammertsma were compared using the cerebellum or the occipital cortex as reference tissues for calculating the binding potential (pB) of [(11)C]raclolpride in brain. Irrespective of the method used, the mean undrugged baseline pB in striatum (ca. 2.0) was significantly asymmetric, with highest binding in the left caudate and right putamen. Test-retest estimates of pB were stable. Subtraction of Logan pB maps revealed that the low dose of nicotine reduced the pB of [(11)C]raclopride by 10% in a cluster of voxels in the left anteroventral striatum, but this effect did not persist after correction for multiple comparisons. The high dose of nicotine (n = 9) acutely reduced pB by 10% bilaterally in the ventral striatum; 3 h after the high nicotine dose, the reductions had shifted dorsally and caudally into the caudate and putamen. Evidently, nicotine challenge enhances the competition

  19. [11C]Doxepin binding to histamine H1 receptors in living human brain in association with attentive waking and circadian rhythm

    Directory of Open Access Journals (Sweden)

    Kazuhiko eYanai

    2012-06-01

    Full Text Available Molecular imaging in neuroscience is a new research field that enables visualization of the impact of molecular events on brain structure and function in humans. While magnetic resonance-based imaging techniques can provide complex information at the level of system, positron emission tomography (PET enables determination of the distribution and density of receptor and enzyme in the human brain. Previous studies using [11C]raclopride revealed that the release of neuronal dopamine was increased in human brain by psychostimulants or reward stimuli. Following on from these previous studies, we examined whether the levels of neuronal release of histamine might change [11C]doxepin binding to the H1 receptors under the influence of physiological stimuli. The purpose of the present study was to evaluate the test-retest reliability of quantitative measurement of [11C]doxepin binding between morning and afternoon and between resting and attentive waking conditions in healthy human subjects. There was a trend for a decrease in [11C]doxepin binding during attentive calculation tasks compared with that in resting conditions, but the difference (approximately 10% was not significant. In contrast, the binding potential of [11C]doxepin in the anterior cingulate gyrus was significantly higher in the morning than that in the afternoon (approximately 19%, suggesting that higher histamine release in the morning would decrease the [11C]doxepin binding in the afternoon. This study suggests that non-invasive measurement of neuronal histamine release is feasible in humans by PET ligand-activation study, although the development of a tracer with better signal-to-noise properties is needed.

  20. CRIS-a novel cAMP-binding protein controlling spermiogenesis and the development of flagellar bending.

    Directory of Open Access Journals (Sweden)

    Anke Miriam Krähling

    Full Text Available The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD. Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+-regulated proteins that--in mature sperm--are involved in flagellar bending.

  1. Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize.

    Science.gov (United States)

    Johnson, Eric T; Berhow, Mark A; Dowd, Patrick F

    2007-04-18

    Hi II maize (Zea mays) plants were engineered to express maize p1 cDNA, a Myb transcription factor, controlled by a putative silk specific promoter, for secondary metabolite production and corn earworm resistance. Transgene expression did not enhance silk color, but about half of the transformed plant silks displayed browning when cut, which indicated the presence of p1-produced secondary metabolites. Levels of maysin, a secondary metabolite with insect toxicity, were highest in newly emerged browning silks. The insect resistance of transgenic silks was also highest at emergence, regardless of maysin levels, which suggests that other unidentified p1-induced molecules likely contributed to larval mortality. Mean survivor weights of corn earworm larvae fed mature browning transgenic silks were significantly lower than weights of those fed mature nonbrowning transgenic silks. Some transgenic pericarps browned with drying and contained similar molecules found in pericarps expressing a dominant p1 allele, suggesting that the promoter may not be silk-specific.

  2. Regional increases in [{sup 11}C]flumazenil binding after epilepsy surgery

    Energy Technology Data Exchange (ETDEWEB)

    Savic, I. [Huddinge Hospital, Dept. of Neurology (Sweden); Blomqvist, G. [Karolinska Hospital, Dept. of Clinical Neurophysiology, Stockholm (Sweden); Halldin, C. [Karolinska Hospital, Dept. of Psychiatry, Stockholm (Sweden); Litton, J.E. [Karolinska Hospital, Dept. of Psychology, Stockholm (Sweden); Gulyas, B. [Karolinska Institute, Div. of Human Brain Research (Sweden)

    1998-05-01

    Introduction - Animal experiments suggest that epileptic seizures alter the expression of mRNA for neuro-receptors. PET measurements with [{sup 11}C]flumazenil show that patients with partial seizures have a reduced density of benzodiazepine (BZ) receptors in the epileptogenic regions (ER) and some of the target areas for seizure activity, the so called projection areas. Recent data suggest that the degree of BZ receptor reduction in ER is correlated to seizure frequency. We therefore hypothesized that seizure activity can alter the BZ receptor binding, and that some of these changes could normalize when the seizures disappeared. Methods - In 4 patients whose seizures were generated by mesial temporal lobe structures, BZ receptor density was measured with [{sup 11}C]flumazenil PET before, and 1 year after the epilepsy surgery and cessation of seizures. By use of a computerized anatomical brain atlas the same regions were analyzed in both PET scans, and the results related to data from 7 healthy controls. Results - Presurgical PET scans showed reductions in BZ receptor density in the epileptogenic regions and some of its primary projection areas. Other cortical regions had normal values. Postsurgically, the calculated BZ receptor density normalized (29{+-}17% increase) in several of the affected projection areas, whereas the values in other cortical regions remained unaltered. Conclusion - Regional reductions in BZ receptor density may be dynamic and related to seizures. The present preliminary observations encourage further studies on seizure-related changes in regional receptor binding in humans. (au) 41 refs.

  3. Determination of the extraction efficiency for {sup 233}U source α-recoil ions from the MLL buffer-gas stopping cell

    Energy Technology Data Exchange (ETDEWEB)

    Wense, Lars v.d.; Seiferle, Benedict; Thirolf, Peter G. [Ludwig-Maximilians-Universitaet Muenchen, Garching (Germany); Laatiaoui, Mustapha [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, Darmstadt (Germany); Helmholtz Institut Mainz, Mainz (Germany)

    2015-03-01

    Following the α decay of {sup 233}U, {sup 229}Th recoil ions are shown to be extracted in a significant amount from the MLL buffer-gas stopping cell. The produced recoil ions and subsequent daughter nuclei are mass purified with the help of a customized quadrupole mass spectrometer. The combined extraction and mass purification efficiency for {sup 229}Th{sup 3+} is determined via MCP-based measurements and via the direct detection of the {sup 229}Th α decay. A large value of (10±2)% for the combined extraction and mass purification efficiency of {sup 229}Th{sup 3+} is obtained at a mass resolution of about 1u/e. In addition to {sup 229}Th, also other α-recoil ions of the {sup 233,} {sup 232}U decay chains are addressed. (orig.)

  4. A Wheat R2R3-type MYB Transcription Factor TaODORANT1 Positively Regulates Drought and Salt Stress Responses in Transgenic Tobacco Plants

    Directory of Open Access Journals (Sweden)

    Qiuhui Wei

    2017-08-01

    Full Text Available MYB transcription factors play important roles in plant responses to biotic and abiotic stress. In this study, TaODORANT1, a R2R3-MYB gene, was cloned from wheat (Triticum aestivum L.. TaODORANT1 was localized in the nucleus and functioned as a transcriptional activator. TaODORANT1 was up-regulated in wheat under PEG6000, NaCl, ABA, and H2O2 treatments. TaODORANT1-overexpressing transgenic tobacco plants exhibited higher relative water content and lower water loss rate under drought stress, as well as lower Na+ accumulation in leaves under salt stress. The transgenic plants showed higher CAT activity but lower ion leakage, H2O2 and malondialdehyde contents under drought and salt stresses. Besides, the transgenic plants also exhibited higher SOD activity under drought stress. Our results also revealed that TaODORANT1 overexpression up-regulated the expression of several ROS- and stress-related genes in response to both drought and salt stresses, thus enhancing transgenic tobacco plants tolerance. Our studies demonstrate that TaODORANT1 positively regulates plant tolerance to drought and salt stresses.

  5. [14C]mechlorethamine binding to proteins of the human keratinocyte

    International Nuclear Information System (INIS)

    Deaton, M.A.; Jones, G.P.; Bowman, P.D.

    1990-01-01

    Much mustard agent research has focused on mustard/DNA interactions. Mustard also interacts with proteins, however, and to reach the DNA any agent must first pass through the cytoplasm. We hypothesized that the cell's proteins would covalently bind mustard, and thereby limit its access to the DNA. Keratinocyte proteins were radiolabeled with [ 14 C]mechlorethamine and separated by electrophoresis. The banding patterns that resulted were made visible on x-ray films, then compared with control patterns. A correspondence of almost one-to-one was observed, which supports the hypothesis that many cellular proteins are susceptible to mustard alkylation. It follows that some mustard symptoms probably result from effects on existing proteins

  6. Hepatitis C Virus Resistance to Carbohydrate-Binding Agents.

    Directory of Open Access Journals (Sweden)

    Laure Izquierdo

    Full Text Available Carbohydrate binding agents (CBAs, including natural lectins, are more and more considered as broad-spectrum antivirals. These molecules are able to directly inhibit many viruses such as Human Immunodeficiency Virus (HIV, Hepatitis C Virus (HCV, Dengue Virus, Ebola Virus or Severe Acute Respiratory Syndrome Coronavirus through binding to envelope protein N-glycans. In the case of HIV, it has been shown that CBAs select for mutant viruses with N-glycosylation site deletions which are more sensitive to neutralizing antibodies. In this study we aimed at evaluating the HCV resistance to CBAs in vitro. HCV was cultivated in the presence of increasing Galanthus nivalis agglutinin (GNA, Cyanovirin-N, Concanavalin-A or Griffithsin concentrations, during more than eight weeks. At the end of lectin exposure, the genome of the isolated strains was sequenced and several potential resistance mutations in the E1E2 envelope glycoproteins were identified. The effect of these mutations on viral fitness as well as on sensitivity to inhibition by lectins, soluble CD81 or the 3/11 neutralizing antibody was assessed. Surprisingly, none of these mutations, alone or in combination, conferred resistance to CBAs. In contrast, we observed that some mutants were more sensitive to 3/11 or CD81-LEL inhibition. Additionally, several mutations were identified in the Core and the non-structural proteins. Thus, our results suggest that in contrast to HIV, HCV resistance to CBAs is not directly conferred by mutations in the envelope protein genes but could occur through an indirect mechanism involving mutations in other viral proteins. Further investigations are needed to completely elucidate the underlying mechanisms.

  7. Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.

    Science.gov (United States)

    Kuroda, Taruho S; Fukuda, Mitsunori

    2005-01-01

    Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

  8. The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

    Science.gov (United States)

    Smelter, Dan F; de Lange, Willem J; Cai, Wenxuan; Ge, Ying; Ralphe, J Carter

    2018-06-01

    Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of

  9. An efficiently cleaved HIV-1 clade C Env selectively binds to neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Saikat Boliar

    Full Text Available An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.

  10. Irreversible binding of 14C-labelled trichloroethylene to mice liver constituents in vivo and in vitro

    International Nuclear Information System (INIS)

    Uehleke, H.; Poplawski-Tabarelli, S.

    1977-01-01

    1) 14 C-labelled trichloroethylene was injected i.p. into male mice (10 μmole/g of b.w.). The radioactivity irreversibly bound to hepatic protein reached highest levels after 6 h : 2 nmole/mg in cytosol protein, 4.4 nmole/mg in mitochondrial protein, and 7.6 nmole/mg in microsomal protein. 2) The commercial trichloroethylene contained radioactive impurities binding to proteins without metabolic activation. Purification by various extraction removed 60-70% of those materials. In aerobic incubates of mice hepatic microsomes and NADPH the covalent binding rate of the purified trichloroethylene was 1.4 nmole/mg protein in 60 min. The activity of rat liver microsomes was approximately 40% less. Covalent binding increased 2-fold with microsomes of mice pretreated with phenobarbital. (orig.) [de

  11. A C-terminal PDZ domain-binding sequence is required for striatal distribution of the dopamine transporter

    DEFF Research Database (Denmark)

    Rickhag, Karl Mattias; Hansen, Freja Herborg; Sørensen, Gunnar

    2013-01-01

    believed to bind synaptic scaffolding proteins, but its functional significance is uncertain. Here we demonstrate that two different dopamine transporter knock-in mice with disrupted PDZ-binding motifs (dopamine transporter-AAA and dopamine transporter+Ala) are characterized by dramatic loss of dopamine......The dopamine transporter mediates reuptake of dopamine from the synaptic cleft. The cellular mechanisms controlling dopamine transporter levels in striatal nerve terminals remain poorly understood. The dopamine transporters contain a C-terminal PDZ (PSD-95/Discs-large/ZO-1) domain-binding sequence...... transporter expression in the striatum, causing hyperlocomotion and attenuated response to amphetamine. In cultured dopaminergic neurons and striatal slices from dopamine transporter-AAA mice, we find markedly reduced dopamine transporter surface levels and evidence for enhanced constitutive internalization...

  12. The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

    International Nuclear Information System (INIS)

    Sun Jian; Yu Jisheng; Yu Zhiwu; Zha Xiao; Wu Yuqing

    2012-01-01

    Graphial abstract: The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1. Highlights: ► Several driving forces contribute to the interaction between heparin and peptides. ► C-terminal of HPV L1 is a potential candidate for the attachment to host cells. ► The C-terminal peptides of HPV-16 and -18 L1 have different heparin-binding. ► The different heparin-binding provides an explanation for the distinct prevalences. - Abstract: The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.

  13. Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Harndahl, Mikkel; Stryhn, Anette

    2014-01-01

    MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide...... library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices...... representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide...

  14. The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors.

    Directory of Open Access Journals (Sweden)

    Philip D Townsend

    Full Text Available The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition.

  15. RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

    Science.gov (United States)

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

    2013-01-01

    T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

  16. Direct binding and activation of protein kinase C isoforms by steroid hormones.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2008-10-01

    The non-genomic action of steroid hormones regulates a wide variety of cellular responses including regulation of ion transport, cell proliferation, migration, death and differentiation. In order to achieve such plethora of effects steroid hormones utilize nearly all known signal transduction pathways. One of the key signalling molecules regulating the non-genomic action of steroid hormones is protein kinase C (PKC). It is thought that rapid action of steroids hormones results from the activation of plasma membrane receptors; however, their molecular identity remains elusive. In recent years, an increasing number of studies have pointed at the selective binding and activation of specific PKC isoforms by steroid hormones. This has led to the hypothesis that PKC could act as a receptor as well as a transducer of the non-genomic effects of these hormones. In this review we summarize the current knowledge of the direct binding and activation of PKC by steroid hormones.

  17. Use of 13NMR to delineate the mode of association or binding of 13C-labeled pollutants with humic materials

    International Nuclear Information System (INIS)

    Bortiatynski, J.M.; Minard, R.D.; Hatcher, P.G.

    1993-01-01

    13 C NMR has recently been shown to be a powerful technique for the examination of the covalent binding of pollutants to humic materials when the latter are enriched with 13 C. Enhanced signals are observed for the carbons that are highly enriched with 13 C while the remaining signals due to naturally abundant 13 C form unlabeled pollutant carbons or humic substances are at the baseline noise level. If covalent bonding and/or non covalent associations take place at or near the site of the 13 C label(s), the nature of the bonding or association can be discerned and the adsorption coefficients can be calculated. In this paper, the authors present the results of such binding studies which demonstrate the great potential of this technique

  18. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jin, Aishun [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Immunology, College of Basic Medical Sciences, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081 (China); Kishi, Hiroyuki, E-mail: immkishi@med.u-toyama.ac.jp [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Muraguchi, Atsushi [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

  19. Evaluation of in vivo selective binding of [11C]doxepin to histamine H1 receptors in five animal species

    International Nuclear Information System (INIS)

    Ishiwata, Kiichi; Kawamura, Kazunori; Wang Weifang; Tsukada, Hideo; Harada, Norihiro; Mochizuki, Hideki; Kimura, Yuichi; Ishii, Kenji; Iwata, Ren; Yanai, Kazuhiko

    2004-01-01

    The specific binding of [ 11 C]doxepin, which has been used as a radioligand for mapping histamine H 1 receptors in human brain by positron emission tomography, was evaluated in five animal species. In mice the [ 11 C]doxepin uptake was reduced by treatment with cold doxepin and two H 1 receptor antagonists, but not with H 2 /H 3 antagonists. The specific binding evaluated with treatment with (+)-chlorpheniramine (H 1 antagonist) was in the range of 10-30% in mouse, rat, rabbit, and monkey, but was not detected in guinea pig

  20. PH4 of petunia is an R2R3-MYB protein that activates vacuolar acidification through interactions with Basic-Helix-Loop transcription factors of the anthocyanin pathway.

    NARCIS (Netherlands)

    Quattrocchio, F.M.; Verweij, C.W.; Kroon, A.R.; Spelt, C.E.; Mol, J.N.M.; Koes, R.E.

    2006-01-01

    The Petunia hybrids genes ANTHOCYANIN1 (AN1) and AN2 encode transcription factors with a basic-helix-loop-helix (BHLH) and a MYB domain, respectively, that are required for anthocyanin synthesis and acidification of the vacuole in petal cells. Mutation of PH4 results in a bluer flower color,