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Sample records for mads-box protein positively

  1. SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

    PubMed

    Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

    2015-03-01

    To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development. PMID:25602439

  2. Ternary complex formation between MADS-box transcription factors and the histone fold protein NF-YB.

    PubMed

    Masiero, Simona; Imbriano, Carol; Ravasio, Federica; Favaro, Rebecca; Pelucchi, Nilla; Gorla, Mirella Sari; Mantovani, Roberto; Colombo, Lucia; Kater, Martin M

    2002-07-19

    MADS-box proteins are transcription factors present in different eukaryotic kingdoms. In contrast to plants, for mammalian and yeast MADS-box proteins ternary complex formation with unrelated transcription factors was reported. We show here the first identification of such ternary interaction in plants. A rice seed-specific NF-YB was identified as partner of OsMADS18 by two-hybrid screening. NF-YB contains a histone fold motif, HFM,(1) and is part of the trimeric CCAAT-binding NF-Y complex. OsMADS18, alone or in combination with a natural partner, interacts with OsNF-YB1 through the MADS and I regions. The mouse NF-YB also associates with OsMADS18 in vivo and in vitro as a NF-YB-NF-YC dimer. Other rice MADS-box proteins do not interact in these assays, indicating specificity for the interaction. OsNF-YB1 is capable of heterodimerizing with NF-YC, but not trimerizing with NF-YA, thus precluding CCAAT binding. Mutation of the variant Asp at position 99 of the HFM alpha2-helix into a conserved serine recovers the capacity to interact with NF-YA, but not with DNA. This is the first indication that members of the NF-YB family work through mechanisms independent of the CCAAT box. PMID:11971906

  3. DNA-binding specificity of Mcm1: operator mutations that alter DNA-bending and transcriptional activities by a MADS box protein.

    PubMed Central

    Acton, T B; Zhong, H; Vershon, A K

    1997-01-01

    The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins found in such diverse organisms as yeast, plants, flies, and humans. To explore the protein-DNA interactions of Mcm1 in vivo and in vitro, we have introduced an extensive series of base pair substitutions into an Mcm1 operator site and examined their effects on Mcm1-mediated transcriptional regulation and DNA-binding affinity. Our results show that Mcm1 uses a mechanism to contact the DNA that has some significant differences from the one used by the human serum response factor (SRF), a closely related MADS box protein in which the three-dimensional structure has been determined. One major difference is that 5-bromouracil-mediated photo-cross-linking experiments indicate that Mcm1 is in close proximity to functional groups in the major groove at the center of the recognition site whereas the SRF protein did not exhibit this characteristic. A more significant difference is that mutations at a position outside of the conserved CC(A/T)6GG site significantly reduce Mcm1-dependent DNA bending, while these substitutions have no effect on DNA bending by SRF. This result shows that the DNA bending by Mcm1 is sequence dependent and that the base-specific requirements for bending differ between Mcm1 and SRF. Interestingly, although these substitutions have a large effect on DNA bending and transcriptional activation by Mcm1, they have a relatively small effect on the DNA-binding affinity of the protein. This result suggests that the degree of DNA bending is important for transcriptional activation by Mcm1. PMID:9121436

  4. The study of two barley Type I-like MADS-box genes as potential targets of epigenetic regulation during seed development

    PubMed Central

    2012-01-01

    Background MADS-box genes constitute a large family of transcription factors functioning as key regulators of many processes during plant vegetative and reproductive development. Type II MADS-box genes have been intensively investigated and are mostly involved in vegetative and flowering development. A growing number of studies of Type I MADS-box genes in Arabidopsis, have assigned crucial roles for these genes in gamete and seed development and have demonstrated that a number of Type I MADS-box genes are epigenetically regulated by DNA methylation and histone modifications. However, reports on agronomically important cereals such as barley and wheat are scarce. Results Here we report the identification and characterization of two Type I-like MADS-box genes, from barley (Hordeum vulgare), a monocot cereal crop of high agronomic importance. Protein sequence and phylogenetic analysis showed that the putative proteins are related to Type I MADS-box proteins, and classified them in a distinct cereal clade. Significant differences in gene expression among seed developmental stages and between barley cultivars with varying seed size were revealed for both genes. One of these genes was shown to be induced by the seed development- and stress-related hormones ABA and JA whereas in situ hybridizations localized the other gene to specific endosperm sub-compartments. The genomic organization of the latter has high conservation with the cereal Type I-like MADS-box homologues and the chromosomal position of both genes is close to markers associated with seed quality traits. DNA methylation differences are present in the upstream and downstream regulatory regions of the barley Type I-like MADS-box genes in two different developmental stages and in response to ABA treatment which may be associated with gene expression differences. Conclusions Two barley MADS-box genes were studied that are related to Type I MADS-box genes. Differential expression in different seed developmental

  5. Genome-wide identification and analysis of the MADS-box gene family in sesame.

    PubMed

    Wei, Xin; Wang, Linhai; Yu, Jingyin; Zhang, Yanxin; Li, Donghua; Zhang, Xiurong

    2015-09-10

    MADS-box genes encode transcription factors that play crucial roles in plant growth and development. Sesame (Sesamum indicum L.) is an oil crop that contributes to the daily oil and protein requirements of almost half of the world's population; therefore, a genome-wide analysis of the MADS-box gene family is needed. Fifty-seven MADS-box genes were identified from 14 linkage groups of the sesame genome. Analysis of phylogenetic relationships with Arabidopsis thaliana, Utricularia gibba and Solanum lycopersicum MADS-box genes was performed. Sesame MADS-box genes were clustered into four groups: 28 MIKC(c)-type, 5 MIKC(⁎)-type, 14 Mα-type and 10 Mγ-type. Gene structure analysis revealed from 1 to 22 exons of sesame MADS-box genes. The number of exons in type II MADS-box genes greatly exceeded the number in type I genes. Motif distribution analysis of sesame MADS-box genes also indicated that type II MADS-box genes contained more motifs than type I genes. These results suggested that type II sesame MADS-box genes had more complex structures. By analyzing expression profiles of MADS-box genes in seven sesame transcriptomes, we determined that MIKC(C)-type MADS-box genes played significant roles in sesame flower and seed development. Although most MADS-box genes in the same clade showed similar expression features, some gene functions were diversified from the orthologous Arabidopsis genes. This research will contribute to uncovering the role of MADS-box genes in sesame development. PMID:25967387

  6. Banana Ovate Family Protein MaOFP1 and MADS-Box Protein MuMADS1 Antagonistically Regulated Banana Fruit Ripening

    PubMed Central

    Hu, Wei; Miao, Hongxia; Zhang, Jianbin; Jia, Caihong; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

    2015-01-01

    The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening. PMID:25886169

  7. Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening.

    PubMed

    Liu, Juhua; Zhang, Jing; Hu, Wei; Miao, Hongxia; Zhang, Jianbin; Jia, Caihong; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

    2015-01-01

    The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening. PMID:25886169

  8. The interaction of banana MADS-box protein MuMADS1 and ubiquitin-activating enzyme E-MuUBA in post-harvest banana fruit.

    PubMed

    Liu, Ju-Hua; Zhang, Jing; Jia, Cai-Hong; Zhang, Jian-Bin; Wang, Jia-Shui; Yang, Zi-Xian; Xu, Bi-Yu; Jin, Zhi-Qiang

    2013-01-01

    KEY MESSAGE : The interaction of MuMADS1 and MuUBA in banana was reported, which will help us to understand the mechanism of the MADS-box gene in regulating banana fruit development and ripening. The ubiquitin-activating enzyme E1 gene fragment MuUBA was obtained from banana (Musa acuminata L.AAA) fruit by the yeast two-hybrid method using the banana MADS-box gene MuMADS1 as bait and 2-day post-harvest banana fruit cDNA library as prey. MuMADS1 interacted with MuUBA. The interaction of MuMADS1 and MuUBA in vivo was further proved by bimolecular fluorescence complementation assay. Real-time quantitative PCR evaluation of MuMADS1 and MuUBA expression patterns in banana showed that they are highly expressed in the ovule 4 stage, but present in low levels in the stem, which suggests a simultaneously differential expression action exists for both MuMADS1 and MuUBA in different tissues and developmental fruits. MuMADS1 and MuUBA expression was highly stimulated by exogenous ethylene and suppressed by 1-methylcyclopropene. These results indicated that MuMADS1 and MuUBA were co-regulated by ethylene and might play an important role in post-harvest banana fruit ripening. PMID:23007689

  9. MADS1, a novel MADS-box protein, is involved in the response of Nicotiana benthamiana to bacterial harpin(Xoo).

    PubMed

    Zhang, Huajian; Teng, Wenjun; Liang, Jingang; Liu, Xinyu; Zhang, Haifeng; Zhang, Zhengguang; Zheng, Xiaobo

    2016-01-01

    MADS-box transcription factor genes are well known for their role in floral organ and seed development. In this study, a novel MADS-box-containing gene, designated NbMADS1, was isolated from leaves of Nicotiana benthamiana. The full-length cDNA was 666 bp and encoded a putative polypeptide of 221 aa with a mass of 24.3 kDa. To assess the role of NbMADS1 in the defence response to bacterial harpin(Xoo), an elicitor of the hypersensitive response, a loss-of-function experiment was performed in N. benthamiana plants using virus-induced gene silencing. Analyses of electrolyte leakage revealed more extensive cell death in the control plants than in NbMADS1-silenced plants. The NbMADS1-silenced plants showed impaired harpin(Xoo)-induced stomatal closure, decreased harpin(Xoo)-induced production of hydrogen peroxide (H2O2) and nitric oxide (NO) in guard cells, and reduced harpin(Xoo)-induced resistance to Phytophthora nicotianae. The compromised stomatal closure observed in the NbMADS1-silenced plants was inhibited by the application of H2O2 and sodium nitroprusside (an NO donor). Taken together, these results demonstrate that the NbMADS1-H2O2-NO pathway mediates multiple harpin(Xoo)-triggered responses, including stomatal closure, hypersensitive cell death, and defence-related gene expression, suggesting that NbMADS1 plays an important role in regulating the response to harpin(Xoo) in N. benthamiana plants. PMID:26466663

  10. Genome-wide analysis of the MADS-box gene family in Brassica rapa (Chinese cabbage).

    PubMed

    Duan, Weike; Song, Xiaoming; Liu, Tongkun; Huang, Zhinan; Ren, Jun; Hou, Xilin; Li, Ying

    2015-02-01

    The MADS-box gene family is an ancient and well-studied transcription factor family that functions in almost every developmental process in plants. There are a number of reports about the MADS-box family in different plant species, but systematic analysis of the MADS-box transcription factor family in Brassica rapa (Chinese cabbage) is still lacking. In this study, 160 MADS-box transcription factors were identified from the entire Chinese cabbage genome and compared with the MADS-box factors from 21 other representative plant species. A detailed list of MADS proteins from these 22 species was sorted. Phylogenetic analysis of the BrMADS genes, together with their Arabidopsis and rice counterparts, showed that the BrMADS genes were categorised into type I (Mα, Mβ, Mγ) and type II (MIKC(C), MIKC*) groups, and the MIKC(C) proteins were further divided into 13 subfamilies. The Chinese cabbage type II group has 95 members, which is twice as much as the Arabidopsis type II group, indicating that the Chinese cabbage type II genes have been retained more frequently than the type I genes. Finally, RNA-seq transcriptome data and quantitative real-time PCR analysis revealed that BrMADS genes are expressed in a tissue-specific manner similar to Arabidopsis. Interestingly, a number of BrMIKC genes showed responses to different abiotic stress treatments, suggesting a function for some of the genes in these processes as well. Taken together, the characterization of the B. rapa MADS-box family presented here, will certainly help in the selection of appropriate candidate genes and further facilitate functional studies in Chinese cabbage. PMID:25216934

  11. Isolation and characterisation of the carnation floral-specific MADS box gene, CMB2.

    PubMed

    Baudinette; Stevenson; Savin

    2000-06-29

    The cDNA clone KD81, was isolated from a carnation petal cDNA library based on its strong differential expression in petals compared with leaves. The deduced amino acid sequence of KD81 indicated high homology with members of the MADS box family of transcription factors. Identified within the deduced amino acid sequence are two conserved domains; an N-terminal, MADS box and a central, K box. The gene encoding KD81 was termed Carnation MADS Box gene 2 (CMB2). Expression of CMB2 is floral-specific and in petal, transcripts were persistent from the initial stages of development through flower opening. Transcripts were not detected in vegetative tissues. The CMB2 protein is most homologous to TDR6 from tomato, the product of the petal and stamen identity gene DEFICIENS (DEFA), and several DEFA homologues including SLM3, STDEF, PMADS1 and APETALA3. Southern blot analysis indicated that CMB2 is present as a single copy within the carnation genome. Characterisation of a genomic clone encoding CMB2, revealed the molecular structure of CMB2 to be consistent with that reported for other plant MADS box genes. Analysis of the CMB2 promoter sequence revealed the presence of two putative cis-acting elements known as serum response elements (SREs). These elements are proposed as the target for MADS box domain binding and may be involved in the regulation/autoregulation of gene expression. CMB2 represents the first reported isolation of a MADS box gene from carnation. PMID:10814815

  12. Bearded-Ear Encodes a MADS-box Transcription Factor Critical for Maize Floral Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We cloned bde by positional cloning and found that it encodes zag3, a MADS-box transcription factor in the conserved AGL6 clade. Mutants in the maize homolog of AGAMOUS, zag1, have a subset of bde floral defects. bde; zag1 double mutants have a severe ear phenotype, not observed in either single m...

  13. SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

    PubMed Central

    Immink, Richard GH; Tonaco, Isabella AN; de Folter, Stefan; Shchennikova, Anna; van Dijk, Aalt DJ; Busscher-Lange, Jacqueline; Borst, Jan W; Angenent, Gerco C

    2009-01-01

    Background Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study. Results In total, 106 multimeric complexes were identified; in more than half of these at least one SEP protein was present. Besides the known complexes involved in determining floral organ identity, various complexes consisting of combinations of proteins known to play a role in floral organ identity specification, and flowering time determination were discovered. The capacity to form this latter type of complex suggests that homeotic factors play essential roles in down-regulation of the MADS box genes involved in floral timing in the flower via negative auto-regulatory loops. Furthermore, various novel complexes were identified that may be important for the direct regulation of the floral transition process. A subsequent detailed analysis of the APETALA3, PISTILLATA, and SEP3 proteins in living plant cells suggests the formation of a multimeric complex in vivo. Conclusions Overall, these results provide strong indications that higher-order complex formation is a general and essential molecular mechanism for plant MADS box protein functioning and attribute a pivotal role to the SEP3 'glue' protein in mediating multimerization. PMID:19243611

  14. Conserved homeodomain proteins interact with MADS box protein Mcm1 to restrict ECB-dependent transcription to the M/G1 phase of the cell cycle

    PubMed Central

    Pramila, Tata; Miles, Shawna; GuhaThakurta, Debraj; Jemiolo, Dave; Breeden, Linda L.

    2002-01-01

    Two homeodomain proteins, Yox1 and Yhp1, act as repressors at early cell cycle boxes (ECBs) to restrict their activity to the M/G1 phase of the cell cycle in budding yeast. These proteins bind to Mcm1 and to a typical homeodomain binding site. The expression of Yox1 is periodic and directly correlated with its binding to, and repression of, ECB activity. The absence of Yox1 and Yhp1 or the constitutive expression of Yox1 leads to the loss of cell-cycle regulation of ECB activity. Therefore, the cell-cycle-regulated expression of these repressors defines the interval of ECB-dependent transcription. Twenty-eight genes, including MCM2-7, CDC6, SWI4, CLN3, and a number of genes required during late M phase have been identified that are coordinately regulated by this pathway. PMID:12464633

  15. Phytoplasma effector SAP54 hijacks plant reproduction by degrading MADS-box proteins and promotes insect colonization in a RAD23-dependent manner.

    PubMed

    MacLean, Allyson M; Orlovskis, Zigmunds; Kowitwanich, Krissana; Zdziarska, Anna M; Angenent, Gerco C; Immink, Richard G H; Hogenhout, Saskia A

    2014-04-01

    Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of the MADS-domain transcription factor (MTF) family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23) family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants). PMID:24714165

  16. Phytoplasma Effector SAP54 Hijacks Plant Reproduction by Degrading MADS-box Proteins and Promotes Insect Colonization in a RAD23-Dependent Manner

    PubMed Central

    MacLean, Allyson M.; Orlovskis, Zigmunds; Kowitwanich, Krissana; Zdziarska, Anna M.; Angenent, Gerco C.; Immink, Richard G. H.; Hogenhout, Saskia A.

    2014-01-01

    Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of the MADS-domain transcription factor (MTF) family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23) family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants). PMID:24714165

  17. Transcriptome-wide analysis of the MADS-box gene family in the orchid Erycina pusilla.

    PubMed

    Lin, Choun-Sea; Hsu, Chen-Tran; Liao, De-Chih; Chang, Wan-Jung; Chou, Ming-Lun; Huang, Yao-Ting; Chen, Jeremy J W; Ko, Swee-Suak; Chan, Ming-Tsair; Shih, Ming-Che

    2016-01-01

    Orchids exhibit a range of unique flower shapes and are a valuable ornamental crop. MADS-box transcription factors are key regulatory components in flower initiation and development. Changing the flower shape and flowering time can increase the value of the orchid in the ornamental horticulture industry. In this study, 28 MADS-box genes were identified from the transcriptome database of the model orchid Erycina pusilla. The full-length genomic sequences of these MADS-box genes were obtained from BAC clones. Of these, 27 were MIKC-type EpMADS (two truncated forms) and one was a type I EpMADS. Eleven EpMADS genes contained introns longer than 10 kb. Phylogenetic analysis classified the 24 MIKC(c) genes into nine subfamilies. Three specific protein motifs, AG, FUL and SVP, were identified and used to classify three subfamilies. The expression profile of each EpMADS gene correlated with its putative function. The phylogenetic analysis was highly correlated with the protein domain identification and gene expression results. Spatial expression of EpMADS6, EpMADS12 and EpMADS15 was strongly detected in the inflorescence meristem, floral bud and seed via in situ hybridization. The subcellular localization of the 28 EpMADS proteins was also investigated. Although EpMADS27 lacks a complete MADS-box domain, EpMADS27-YFP was localized in the nucleus. This characterization of the orchid MADS-box family genes provides useful information for both orchid breeding and studies of flowering and evolution. PMID:25917508

  18. Toward the Analysis of the Petunia MADS Box Gene Family by Reverse and Forward Transposon Insertion Mutagenesis Approaches: B, C, and D Floral Organ Identity Functions Require SEPALLATA-Like MADS Box Genes in Petunia

    PubMed Central

    Vandenbussche, Michiel; Zethof, Jan; Souer, Erik; Koes, Ronald; Tornielli, Giovanni B.; Pezzotti, Mario; Ferrario, Silvia; Angenent, Gerco C.; Gerats, Tom

    2003-01-01

    We have initiated a systematic functional analysis of the MADS box, intervening region, K domain, C domain-type MADS box gene family in petunia. The starting point for this has been a reverse-genetics approach, aiming to select for transposon insertions into any MADS box gene. We have developed and applied a family signature insertion screening protocol that is highly suited for this purpose, resulting in the isolation of 32 insertion mutants in 20 different MADS box genes. In addition, we identified three more MADS box gene insertion mutants using a candidate-gene approach. The defined insertion lines provide a sound foundation for a systematic functional analysis of the MADS box gene family in petunia. Here, we focus on the analysis of Floral Binding Protein2 (FBP2) and FBP5 genes that encode the E-function, which in Arabidopsis has been shown to be required for B and C floral organ identity functions. fbp2 mutants display sepaloid petals and ectopic inflorescences originating from the third floral whorl, whereas fbp5 mutants appear as wild type. In fbp2 fbp5 double mutants, reversion of floral organs to leaf-like organs is increased further. Strikingly, ovules are replaced by leaf-like structures in the carpel, indicating that in addition to the B- and C-functions, the D-function, which specifies ovule development, requires E-function activity. Finally, we compare our data with results obtained using cosuppression approaches and conclude that the latter might be less suited for assigning functions to individual members of the MADS box gene family. PMID:14576291

  19. MADS-Box Transcription Factor SsMADS Is Involved in Regulating Growth and Virulence in Sclerotinia sclerotiorum

    PubMed Central

    Qu, Xiaoyan; Yu, Baodong; Liu, Jinliang; Zhang, Xianghui; Li, Guihua; Zhang, Dongjing; Li, Le; Wang, Xueliang; Wang, Lu; Chen, Jingyuan; Mu, Wenhui; Pan, Hongyu; Zhang, Yanhua

    2014-01-01

    MADS-box proteins, a well-conserved family of transcription factors in eukaryotic organisms, specifically regulate a wide range of cellular functions, including primary metabolism, cell cycle, and cell identity. However, little is known about roles of the MADS-box protein family in the fungal pathogen Sclerotinia sclerotiorum. In this research, the S. sclerotiorum MADS-box gene SsMADS was cloned; it encodes a protein that is highly similar to Mcm1 orthologs from Saccharomyces cerevisiae and other fungi, and includes a highly conserved DNA-binding domain. MADS is a member of the MADS box protein SRF (serum response factor) lineage. SsMADS function was investigated using RNA interference. Silenced strains were obtained using genetic transformation of the RNA interference vectors pS1-SsMADS and pSD-SsMADS. SsMADS expression levels in silenced strains were analyzed using RT-PCR. The results showed that SsMADS mRNA expression in these silenced strains was reduced to different degrees, and growth rate in these silenced strains was significantly decreased. Infecting tomato leaflets with silenced strains indicated that SsMADS was required for leaf pathogenesis in a susceptible host. Our results suggest that the MADS-box transcription factor SsMADS is involved in S. sclerotiorum growth and virulence. PMID:24815067

  20. Diverse roles for MADS box genes in Arabidopsis development.

    PubMed Central

    Rounsley, S D; Ditta, G S; Yanofsky, M F

    1995-01-01

    Members of the MADS box gene family play important roles in flower development from the early step of determining the identity of floral meristems to specifying the identity of floral organ primordia later in flower development. We describe here the isolation and characterization of six additional members of this family, increasing the number of reported Arabidopsis MADS box genes to 17. All 11 members reported prior to this study are expressed in flowers, and the majority of them are floral specific. RNA expression analyses of the six genes reported here indicate that two genes, AGL11 and AGL13 (AGL for AGAMOUS-like), are preferentially expressed in ovules, but each has a distinct expression pattern. AGL15 is preferentially expressed in embryos, with its onset at or before the octant stage early in embryo development. AGL12, AGL14, and AGL17 are all preferentially expressed in root tissues and therefore represent the only characterized MADS box genes expressed in roots. Phylogenetic analyses showed that the two genes expressed in ovules are closely related to previously isolated MADS box genes, whereas the four genes showing nonfloral expression are more distantly related. Data from this and previous studies indicate that in addition to their proven role in flower development, MADS box genes are likely to play roles in many other aspects of plant development. PMID:7549482

  1. Characterization and expression analysis of six MADS-box genes in maize (Zea mays L.).

    PubMed

    Zhang, Zhongbao; Li, Huiyong; Zhang, Dengfeng; Liu, Yinghui; Fu, Jing; Shi, Yunsu; Song, Yanchun; Wang, Tianyu; Li, Yu

    2012-05-15

    MADS-box genes encode a family of transcription factors, which control diverse developmental processes in flowering plants, with organs ranging from roots, flowers and fruits. In this study, six maize cDNAs encoding MADS-box proteins were isolated. BLASTX searches and phylogenetic analysis indicated that the six MADS-box genes belonging to the AGL2-like clade. qRT-PCR analysis revealed that these genes had differential expression patterns in different organs in maize. The results of yeast one-hybrid system indicated that the protein ZMM3-1, ZMM3-2, ZMM6, ZMM7-L, ZMM8-L and ZMM14-L had transcriptional activation activity. Subcellular localization of ZMM7-L demonstrated that the fluorescence of ZMM7-L-GFP was mainly detected in the nuclei of onion epidermal cells. qRT-PCR analysis for expression pattern of ZMM7-L showed that the gene was up-regulated by abiotic stresses and down-regulated by exogenous ABA. The germination rates of over-expression transgenic lines were lower than that of the wild type on medium with 150 mM NaCl, 350 mM mannitol. These results indicated that ZMM7-L might be a negative transcription factor responsive to abiotic stresses. PMID:22440334

  2. Phylogenetic analysis and molecular evolution of the dormancy associated MADS-box genes from peach

    PubMed Central

    Jiménez, Sergio; Lawton-Rauh, Amy L; Reighard, Gregory L; Abbott, Albert G; Bielenberg, Douglas G

    2009-01-01

    Background Dormancy associated MADS-box (DAM) genes are candidates for the regulation of growth cessation and terminal bud formation in peach. These genes are not expressed in the peach mutant evergrowing, which fails to cease growth and enter dormancy under dormancy-inducing conditions. We analyzed the phylogenetic relationships among and the rates and patterns of molecular evolution within DAM genes in the phylogenetic context of the MADS-box gene family. Results The peach DAM genes grouped with the SVP/StMADS11 lineage of type II MIKCC MADS-box genes. Phylogenetic analyses suggest that the peach SVP/StMADS11-like gene family, which contains significantly more members than annual model plants, expanded through serial tandem gene duplication. We found evidence of strong purifying selection acting to constrain functional divergence among the peach DAM genes and only a single codon, located in the C-terminal region, under significant positive selection. Conclusion Because all DAM genes are expressed in peach and are subjected to strong purifying selection we suggest that the duplicated genes have been maintained by subfunctionalization and/or neofunctionalization. In addition, this pattern of selection suggests that the DAM genes are important for peach growth and development. PMID:19558704

  3. Comparative phylogenetic analysis and transcriptional profiling of MADS-box gene family identified DAM and FLC-like genes in apple (Malusx domestica)

    PubMed Central

    Kumar, Gulshan; Arya, Preeti; Gupta, Khushboo; Randhawa, Vinay; Acharya, Vishal; Singh, Anil Kumar

    2016-01-01

    The MADS-box transcription factors play essential roles in various processes of plant growth and development. In the present study, phylogenetic analysis of 142 apple MADS-box proteins with that of other dicotyledonous species identified six putative Dormancy-Associated MADS-box (DAM) and four putative Flowering Locus C-like (FLC-like) proteins. In order to study the expression of apple MADS-box genes, RNA-seq analysis of 3 apical and 5 spur bud stages during dormancy, 6 flower stages and 7 fruit development stages was performed. The dramatic reduction in expression of two MdDAMs, MdMADS063 and MdMADS125 and two MdFLC-like genes, MdMADS135 and MdMADS136 during dormancy release suggests their role as flowering-repressors in apple. Apple orthologs of Arabidopsis genes, FLOWERING LOCUS T, FRIGIDA, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 and LEAFY exhibit similar expression patterns as reported in Arabidopsis, suggesting functional conservation in floral signal integration and meristem determination pathways. Gene ontology enrichment analysis of predicted targets of DAM revealed their involvement in regulation of reproductive processes and meristematic activities, indicating functional conservation of SVP orthologs (DAM) in apple. This study provides valuable insights into the functions of MADS-box proteins during apple phenology, which may help in devising strategies to improve important traits in apple. PMID:26856238

  4. New MADS-box gene in fern: cloning and expression analysis of DfMADS1 from Dryopteris fragrans.

    PubMed

    Huang, Qingyang; Li, Wenhua; Fan, Ruifeng; Chang, Ying

    2014-01-01

    MADS genes encode a family of transcription factors, some of which control the identities of floral organs in flowering plants. Most of the MADS-box genes in fern have been cloned and analyzed in model plants, such as Ceratopteris richardii and Ceratopteris pteridoides. In this study, a new MADS-box gene, DfMADS1(GU385475), was cloned from Dryopteris fragrans (L.) Schott to better understand the role of MADS genes in the evolution of floral organs. The full-length DfMADS1 cDNA was 973 bp in length with a 75 bp 5'-UTR and a 169 bp 3'-UTR. The DfMADS1 protein was predicted to contain a typical MIKC-type domain structure consisting of a MADS domain, a short I region, a K domain, and a C-terminal region. The DfMADS1 protein showed high homology with MADS box proteins from other ferns. Phylogenetic analysis revealed that DfMADS1 belongs to the CRM1-like subfamily. RT-PCR analysis indicated that DfMADS1 is expressed in both the gametophytes and the sporophytes of D. fragrans. PMID:24466046

  5. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor

    PubMed Central

    Yao, Jia-Long; Dong, Yi-Hu; Morris, Bret A. M.

    2001-01-01

    Fruit development in higher plants normally requires pollination and fertilization to stimulate cell division of specific floral tissues. In some cases, parthenocarpic fruit development proceeds without either pollination or fertilization. Parthenocarpic fruit without seed has higher commercial value than seeded fruit. Several apple (Malus domestica) mutants (Rae Ime, Spencer Seedless and Wellington Bloomless) are known to produce only apetalous flowers that readily go on to develop into parthenocarpic fruit. Through genetics, a single recessive gene has been identified to control this trait in apple. Flower phenotypes of these apple mutants are strikingly similar to those of the Arabidopsis mutant pistillata (pi), which produces flowers where petals are transformed to sepals and stamens to carpels. In this study, we have cloned the apple PI homolog (MdPI) that shows 64% amino acid sequence identity and closely conserved intron positions and mRNA expression patterns to the Arabidopsis PI. We have identified that in the apetalous mutants MdPI has been mutated by a retrotransposon insertion in intron 4 in the case of Rae Ime and in intron 6 in the case of Spencer Seedless and Wellington Bloomless. The insertion apparently abolishes the normal expression of the MdPI gene. We conclude that the loss of function mutation in the MdPI MADS-box transcription factor confers parthenocarpic fruit development in these apple varieties and demonstrates another function for the MADS- box gene family. The knowledge generated here could be used to produce parthenocarpic fruit cultivars through genetic engineering. PMID:11158635

  6. Cloning, Characterization, Regulation, and Function of Dormancy-Associated MADS-Box Genes from Leafy Spurge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DORMANCY-ASSOCIATED MADS-BOX (DAM) genes are SHORT VEGETATIVE PHASE–Like MADS box transcription factors linked to endodormancy induction. We have cloned and characterized several cDNA and genomic clones of DAM genes from the model perennial weed leafy spurge (Euphorbia esula). We present evidence fo...

  7. MADS-box genes in maize: Frequent targets of selection during domestication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MADS-box genes encode transcription factors that are key regulators of plant inflorescence and flower development. We examined DNA sequence variation in 32 maize MADS-box genes and 32 random loci from the maize genome and investigated their involvement in maize domestication and improvement. Using n...

  8. Expression of paralogous SEP-, FUL-, AG- and STK-like MADS-box genes in wild-type and peloric Phalaenopsis flowers

    PubMed Central

    Acri-Nunes-Miranda, Roberta; Mondragón-Palomino, Mariana

    2014-01-01

    The diverse flowers of Orchidaceae are the result of several major morphological transitions, among them the most studied is the differentiation of the inner median tepal into the labellum, a perianth organ key in pollinator attraction. Type A peloria lacking stamens and with ectopic labella in place of inner lateral tepals are useful for testing models on the genes specifying these organs by comparing their patterns of expression between wild-type and peloric flowers. Previous studies focused on DEFICIENS- and GLOBOSA-like MADS-box genes because of their conserved role in perianth and stamen development. The “orchid code” model summarizes this work and shows in Orchidaceae there are four paralogous lineages of DEFICIENS/AP3-like genes differentially expressed in each floral whorl. Experimental tests of this model showed the conserved, higher expression of genes from two specific DEF-like gene lineages is associated with labellum development. The present study tests whether eight MADS-box candidate SEP-, FUL-, AG-, and STK-like genes have been specifically duplicated in the Orchidaceae and are also differentially expressed in association with the distinct flower organs of Phalaenopsis hyb. “Athens.” The gene trees indicate orchid-specific duplications. In a way analogous to what is observed in labellum-specific DEF-like genes, a two-fold increase in the expression of SEP3-like gene PhaMADS7 was measured in the labellum-like inner lateral tepals of peloric flowers. The overlap between SEP3-like and DEF-like genes suggests both are associated with labellum specification and similar positional cues determine their domains of expression. In contrast, the uniform messenger levels of FUL-like genes suggest they are involved in the development of all organs and their expression in the ovary suggests cell differentiation starts before pollination. As previously reported AG-like and STK-like genes are exclusively expressed in gynostemium and ovary, however no

  9. MADS goes genomic in conifers: towards determining the ancestral set of MADS-box genes in seed plants

    PubMed Central

    Gramzow, Lydia; Weilandt, Lisa; Theißen, Günter

    2014-01-01

    Background and Aims MADS-box genes comprise a gene family coding for transcription factors. This gene family expanded greatly during land plant evolution such that the number of MADS-box genes ranges from one or two in green algae to around 100 in angiosperms. Given the crucial functions of MADS-box genes for nearly all aspects of plant development, the expansion of this gene family probably contributed to the increasing complexity of plants. However, the expansion of MADS-box genes during one important step of land plant evolution, namely the origin of seed plants, remains poorly understood due to the previous lack of whole-genome data for gymnosperms. Methods The newly available genome sequences of Picea abies, Picea glauca and Pinus taeda were used to identify the complete set of MADS-box genes in these conifers. In addition, MADS-box genes were identified in the growing number of transcriptomes available for gymnosperms. With these datasets, phylogenies were constructed to determine the ancestral set of MADS-box genes of seed plants and to infer the ancestral functions of these genes. Key Results Type I MADS-box genes are under-represented in gymnosperms and only a minimum of two Type I MADS-box genes have been present in the most recent common ancestor (MRCA) of seed plants. In contrast, a large number of Type II MADS-box genes were found in gymnosperms. The MRCA of extant seed plants probably possessed at least 11–14 Type II MADS-box genes. In gymnosperms two duplications of Type II MADS-box genes were found, such that the MRCA of extant gymnosperms had at least 14–16 Type II MADS-box genes. Conclusions The implied ancestral set of MADS-box genes for seed plants shows simplicity for Type I MADS-box genes and remarkable complexity for Type II MADS-box genes in terms of phylogeny and putative functions. The analysis of transcriptome data reveals that gymnosperm MADS-box genes are expressed in a great variety of tissues, indicating diverse roles of MADS-box

  10. Functional Characterization of OsMADS18, a Member of the AP1/SQUA Subfamily of MADS Box Genes1[w

    PubMed Central

    Fornara, Fabio; Pařenicová, Lucie; Falasca, Giuseppina; Pelucchi, Nilla; Masiero, Simona; Ciannamea, Stefano; Lopez-Dee, Zenaida; Altamura, Maria Maddalena; Colombo, Lucia; Kater, Martin M.

    2004-01-01

    MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function. PMID:15299121

  11. MADS-box genes in plant ontogeny and phylogeny: Haeckel's 'biogenetic law' revisited.

    PubMed

    Theissen, G; Saedler, H

    1995-10-01

    Data currently accumulating with impressive speed indicate that the molecular evolution of MADS-box genes was a decisive aspect of the morphological evolution of plants. Studies on MADS-box genes in diverse plant species thus help us to understand the emergence of morphological novelties, such as the flower, in evolution. This furthers our understanding of the relationship between ontogeny and phylogeny, which has been a controversial issue since Ernst Haeckel published his 'biogenetic law' more than a century ago. PMID:8664551

  12. Unique and redundant functional domains of APETALA1 and CAULIFLOWER, two recently duplicated Arabidopsis thaliana floral MADS-box genes.

    PubMed

    Alvarez-Buylla, Elena R; García-Ponce, Berenice; Garay-Arroyo, Adriana

    2006-01-01

    APETALA1 (AP1) and CAULIFLOWER (CAL) are closely related MADS box genes that are partially redundant during Arabidopsis thaliana floral meristem determination. AP1 is able to fully substitute for CAL functions, but not vice versa, and AP1 has unique sepal and petal identity specification functions. In this study, the unique and redundant functions of these two genes has been mapped to the four protein domains that characterize type-II MADS-domain proteins by expressing all 15 chimeric combinations of AP1 and CAL cDNA regions under control of the AP1 promoter in ap1-1 loss-of-function plants. The "in vivo" function of these chimeric genes was analysed in Arabidopsis plants by expressing the chimeras. Rescue of flower meristem and sepal/petal identities was scored in single and multiple insert homozygous transgenic lines. Using these chimeric lines, it was found that distinct residues of the AP1 K domain not shared by the same CAL domain are necessary and sufficient for complete recovery of floral meristem identity, in the context of the CAL protein sequence, while both AP1 COOH and K domains are indispensable for complete rescue of sepal identity. By contrast, either one of these two AP1 domains is necessary and sufficient for complete petal identity recovery. It was also found that there were positive and negative synergies among protein domains and their combinations, and that multiple-insert lines showed relatively better rescue than equivalent single-insert lines. Finally, several lines had flowers with extra sepals and petals suggesting that chimeric proteins yield abnormal transcriptional complexes that may alter the expression or regulation of genes that control floral organ number under normal conditions. PMID:16893974

  13. Overexpression of a novel MADS-box gene SlFYFL delays senescence, fruit ripening and abscission in tomato

    NASA Astrophysics Data System (ADS)

    Xie, Qiaoli; Hu, Zongli; Zhu, Zhiguo; Dong, Tingting; Zhao, Zhiping; Cui, Baolu; Chen, Guoping

    2014-03-01

    MADS-domain proteins are important transcription factors involved in many biological processes of plants. In our study, a tomato MADS-box gene, SlFYFL, was isolated. SlFYFL is expressed in all tissues of tomato and significantly higher in mature leave, fruit of different stages, AZ (abscission zone) and sepal. Delayed leaf senescence and fruit ripening, increased storability and longer sepals were observed in 35S:FYFL tomato. The accumulation of carotenoid was reduced, and ethylene content, ethylene biosynthetic and responsive genes were down-regulated in 35S:FYFL fruits. Abscission zone (AZ) did not form normally and abscission zone development related genes were declined in AZs of 35S:FYFL plants. Yeast two-hybrid assay revealed that SlFYFL protein could interact with SlMADS-RIN, SlMADS1 and SlJOINTLESS, respectively. These results suggest that overexpression of SlFYFL regulate fruit ripening and development of AZ via interactions with the ripening and abscission zone-related MADS box proteins.

  14. Members of the tomato FRUITFULL MADS-box family regulate style abscission and fruit ripening

    PubMed Central

    Wang, Shufen; Lu, Gang; Hou, Zheng; Luo, Zhidan; Wang, Taotao; Li, Hanxia; Zhang, Junhong; Ye, Zhibiao

    2014-01-01

    The tomato (Solanum lycopersicum) protein MADS-RIN plays important roles in fruit ripening. In this study, the functions of two homologous tomato proteins, FUL1 and FUL2, which contain conserved MIKC domains that typify plant MADS-box proteins, and which interact with MADS-RIN, were analysed. Transgenic functional analysis showed that FUL1 and FUL2 function redundantly in fruit ripening regulation, but exhibit distinct roles in the regulation of cellular differentiation and expansion. Over-expression of FUL2 in tomato resulted in a pointed tip at the blossom end of the fruit, together with a thinner pericarp, reduced stem diameter, and smaller leaves, but no obvious phenotypes resulted from FUL1 over-expression. Dual suppression of FUL1 and FUL2 substantially inhibited fruit ripening by blocking ethylene biosynthesis and decreasing carotenoid accumulation. In addition, the levels of transcript corresponding to ACC SYNTHASE2 (ACS2), which plays a key role in ethylene biosynthesis, were significantly decreased in the FUL1/FUL2 knock-down tomato fruits. Overall, our results suggest that FUL proteins can regulate tomato fruit ripening through fine-tuning ethylene biosynthesis and the expression of ripening-related genes. PMID:24723399

  15. MADS-box transcription factor AGL21 regulates lateral root development and responds to multiple external and physiological signals.

    PubMed

    Yu, Lin-Hui; Miao, Zi-Qing; Qi, Guo-Feng; Wu, Jie; Cai, Xiao-Teng; Mao, Jie-Li; Xiang, Cheng-Bin

    2014-11-01

    Plant root system morphology is dramatically influenced by various environmental cues. The adaptation of root system architecture to environmental constraints, which mostly depends on the formation and growth of lateral roots, is an important agronomic trait. Lateral root development is regulated by the external signals coordinating closely with intrinsic signaling pathways. MADS-box transcription factors are known key regulators of the transition to flowering and flower development. However, their functions in root development are still poorly understood. Here we report that AGL21, an AGL17-clade MADS-box gene, plays a crucial role in lateral root development. AGL21 was highly expressed in root, particularly in the root central cylinder and lateral root primordia. AGL21 overexpression plants produced more and longer lateral roots while agl21 mutants showed impaired lateral root development, especially under nitrogen-deficient conditions. AGL21 was induced by many plant hormones and environmental stresses, suggesting a function of this gene in root system plasticity in response to various signals. Furthermore, AGL21 was found positively regulating auxin accumulation in lateral root primordia and lateral roots by enhancing local auxin biosynthesis, thus stimulating lateral root initiation and growth. We propose that AGL21 may be involved in various environmental and physiological signals-mediated lateral root development and growth. PMID:25122697

  16. MADS-Box Transcription Factor AGL21 Regulates Lateral Root Development and Responds to Multiple External and Physiological Signals

    PubMed Central

    Yu, Lin-Hui; Miao, Zi-Qing; Qi, Guo-Feng; Wu, Jie; Cai, Xiao-Teng; Mao, Jie-Li; Xiang, Cheng-Bin

    2014-01-01

    Plant root system morphology is dramatically influenced by various environmental cues. The adaptation of root system architecture to environmental constraints, which mostly depends on the formation and growth of lateral roots, is an important agronomic trait. Lateral root development is regulated by the external signals coordinating closely with intrinsic signaling pathways. MADS-box transcription factors are known key regulators of the transition to flowering and flower development. However, their functions in root development are still poorly understood. Here we report that AGL21, an AGL17-clade MADS-box gene, plays a crucial role in lateral root development. AGL21 was highly expressed in root, particularly in the root central cylinder and lateral root primordia. AGL21 overexpression plants produced more and longer lateral roots while agl21 mutants showed impaired lateral root development, especially under nitrogen-deficient conditions. AGL21 was induced by many plant hormones and environmental stresses, suggesting a function of this gene in root system plasticity in response to various signals. Furthermore, AGL21 was found positively regulating auxin accumulation in lateral root primordia and lateral roots by enhancing local auxin biosynthesis, thus stimulating lateral root initiation and growth. We propose that AGL21 may be involved in various environmental and physiological signals-mediated lateral root development and growth. PMID:25122697

  17. A MADS-box gene of Populus deltoides expressed during flower development and in vegetative organs.

    PubMed

    Zhang, Bingyu; Su, Xiaohua; Zhou, Xiangming

    2008-06-01

    A MADS-box gene (PdPI) was isolated from a cDNA library constructed from male flower buds of Populus deltoides Bartr. ex Marsh. According to an analysis of genomic DNA structure and putative protein structure, and a phylogenetic study, PdPI is an ortholog of the Arabidopsis PI gene. Relative-quantitative real-time polymerase chain reaction analysis showed that PdPI has a broader expression pattern than PI in Arabidopsis. PdPI was strongly expressed in floral buds and roots and weakly expressed in immature xylem, leaves and apical buds of the male P. deltoides tree. In male inflorescences, PdPI expression was abundant in the perianth and anther, and weak in the peduncle and mature pollen. The large differences in PdPI expression at various phases of male floral bud development were closely related to the development of flower organs (perianth and stamen) and pollen. PdPI was also expressed in female inflorescences. Our results suggest that PdPI has multiple functions in the development of P. deltoides. PMID:18381273

  18. Cloning and characterization of a PI-like MADS-box gene in Phalaenopsis orchid.

    PubMed

    Guo, Bin; Hexige, Saiyin; Zhang, Tian; Pittman, Jon K; Chen, Donghong; Ming, Feng

    2007-11-30

    The highly evolved flowers of orchids have colorful sepals and fused columns that offer an opportunity to discover new genes involved in floral development in monocotyledon species. In this investigation, we cloned and characterized the homologous PISTALLATA-like (PI-like) gene PhPI15 (Phalaenopsis PI STILLATA # 15), from the Phalaenopsis hybrid cultivar. The protein sequence encoded by PhPI15 contains a typical PI-motif. Its sequence also formed a subclade with other monocot PI-type genes in phylogenetic analysis. Southern analysis showed that PhPI15 was present in the Phalaenopsis orchid genome as a single copy. Furthermore, it was expressed in all the whorls of the Phalaenopsis flower, while no expression was detected in vegetative organs. The flowers of transgenic tobacco plants ectopically expressing PhPI15 showed male-sterile phenotypes. Thus, as a Class-B MADS-box gene, PhPI15 specifies floral organ identity in orchids. PMID:18047777

  19. Cloning, characterization, regulation, and function of dormancy-associated MADS-BOX genes from leafy spurge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DORMANCY-ASSOCIATED MADS-BOX (DAM) genes are transcription factors that have been linked to endodormancy induction. The evergrowing mutation in peach, which renders it incapable of entering endodormancy, resulted from a deletion in a series of DAM genes (Bielenberg et al. 2008). Likewise, DAM genes ...

  20. Cloning, Characterization, Regulation, and Function of DORMANCY-ASSOCIATED MADS-BOX Genes from Leafy Spurge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DORMANCY-ASSOCIATED MADS-BOX (DAM) genes are transcription factors that have been linked to endodormancy induction. The evergrowing mutation in peach, which renders it incapable of entering endodormancy, resulted from a deletion in a series of DAM genes (Bielenberg et al. 2008). Likewise, DAM genes ...

  1. Analysis of the Petunia TM6 MADS box gene reveals functional divergence within the DEF/AP3 lineage.

    PubMed

    Rijpkema, Anneke S; Royaert, Stefan; Zethof, Jan; van der Weerden, Gerard; Gerats, Tom; Vandenbussche, Michiel

    2006-08-01

    Antirrhinum majus DEFICIENS (DEF) and Arabidopsis thaliana APETALA3 (AP3) MADS box proteins are required to specify petal and stamen identity. Sampling of DEF/AP3 homologs revealed two types of DEF/AP3 proteins, euAP3 and TOMATO MADS BOX GENE6 (TM6), within core eudicots, and we show functional divergence in Petunia hybrida euAP3 and TM6 proteins. Petunia DEF (also known as GREEN PETALS [GP]) is expressed mainly in whorls 2 and 3, and its expression pattern remains unchanged in a blind (bl) mutant background, in which the cadastral C-repression function in the perianth is impaired. Petunia TM6 functions as a B-class organ identity protein only in the determination of stamen identity. Atypically, Petunia TM6 is regulated like a C-class rather than a B-class gene, is expressed mainly in whorls 3 and 4, and is repressed by BL in the perianth, thereby preventing involvement in petal development. A promoter comparison between DEF and TM6 indicates an important change in regulatory elements during or after the duplication that resulted in euAP3- and TM6-type genes. Surprisingly, although TM6 normally is not involved in petal development, 35S-driven TM6 expression can restore petal development in a def (gp) mutant background. Finally, we isolated both euAP3 and TM6 genes from seven solanaceous species, suggesting that a dual euAP3/TM6 B-function system might be the rule in the Solanaceae. PMID:16844905

  2. Organization of the MADS box from human SRF revealed by tyrosine perturbation.

    PubMed

    Profantová, Barbora; Coïc, Yves-Marie; Profant, Václav; Štěpánek, Josef; Kopecký, Vladimír; Turpin, Pierre-Yves; Alpert, Bernard; Zentz, Christian

    2015-02-01

    MADS box family transcription factors are involved in signal transduction and development control through DNA specific sequence recognition. The DNA binding domain of these proteins contains a conservative 55-60 amino acid sequence which defines the membership of this large family. Here we present a thorough study of the MADS segment of serum response factor (MADS(SRF)). Fluorescence, UV-absorption, and Raman spectroscopy studies were performed in order to disclose its behavior and basic functional properties in an aqueous environment. The secondary structure of MADS(SRF) estimated by analysis of Raman spectra and supported by CD has revealed only the C-terminal part as homologous with those of free core-SRF, while the N-terminal part has lost the stable α-helical structure found in both the free core-SRF and its specific complex with DNA. The three tyrosine residues of the MADS(SRF) were used as spectroscopic inner probes. The effect of environmental conditions, especially pH variations and addition of variously charged quenchers, on their spectra was examined. Two-component fluorescence quenching was revealed using factor analysis and corresponding Stern-Volmer constants determined. Factor analysis of absorbance and fluorescence pH titration led to determination of three dissociation constants pKa1 = 6.4 ± 0.2, pKa2 = 7.3 ± 0.2, and pKa3 = 9.6 ± 0.6. Critical comparison of all experiments identified the deprotonation of His193 hydrogen bonded to Tyr195 as a candidate for pKa1 (and that of Tyr158 as a candidate for pKa2). Within MADS(SRF), His193 is a key intermediary between the N-terminal primary DNA binding element and the hydrophobic C-terminal protein dimerization element. PMID:25558766

  3. An Atlas of Type I MADS Box Gene Expression during Female Gametophyte and Seed Development in Arabidopsis[W

    PubMed Central

    Bemer, Marian; Heijmans, Klaas; Airoldi, Chiara; Davies, Brendan; Angenent, Gerco C.

    2010-01-01

    Members of the plant type I MADS domain subfamily have been reported to be involved in reproductive development in Arabidopsis (Arabidopsis thaliana). However, from the 61 type I genes in the Arabidopsis genome, only PHERES1, AGAMOUS-LIKE80 (AGL80), DIANA, AGL62, and AGL23 have been functionally characterized, which revealed important roles for these genes during female gametophyte and early seed development. The functions of the other genes are still unknown, despite the fact that the available single T-DNA insertion mutants have been largely investigated. The lack of mutant phenotypes is likely due to a considerable number of recent intrachromosomal duplications in the type I subfamily, resulting in nonfunctional genes in addition to a high level of redundancy. To enable a breakthrough in type I MADS box gene characterization, a framework needs to be established that allows the prediction of the functionality and redundancy of the type I genes. Here, we present a complete atlas of their expression patterns during female gametophyte and seed development in Arabidopsis, deduced from reporter lines containing translational fusions of the genes to green fluorescent protein and β-glucuronidase. All the expressed genes were revealed to be active in the female gametophyte or developing seed, indicating that the entire type I subfamily is involved in reproductive development in Arabidopsis. Interestingly, expression was predominantly observed in the central cell, antipodal cells, and chalazal endosperm. The combination of our expression results with phylogenetic and protein interaction data allows a better identification of putative redundantly acting genes and provides a useful tool for the functional characterization of the type I MADS box genes in Arabidopsis. PMID:20631316

  4. An atlas of type I MADS box gene expression during female gametophyte and seed development in Arabidopsis.

    PubMed

    Bemer, Marian; Heijmans, Klaas; Airoldi, Chiara; Davies, Brendan; Angenent, Gerco C

    2010-09-01

    Members of the plant type I MADS domain subfamily have been reported to be involved in reproductive development in Arabidopsis (Arabidopsis thaliana). However, from the 61 type I genes in the Arabidopsis genome, only PHERES1, AGAMOUS-LIKE80 (AGL80), DIANA, AGL62, and AGL23 have been functionally characterized, which revealed important roles for these genes during female gametophyte and early seed development. The functions of the other genes are still unknown, despite the fact that the available single T-DNA insertion mutants have been largely investigated. The lack of mutant phenotypes is likely due to a considerable number of recent intrachromosomal duplications in the type I subfamily, resulting in nonfunctional genes in addition to a high level of redundancy. To enable a breakthrough in type I MADS box gene characterization, a framework needs to be established that allows the prediction of the functionality and redundancy of the type I genes. Here, we present a complete atlas of their expression patterns during female gametophyte and seed development in Arabidopsis, deduced from reporter lines containing translational fusions of the genes to green fluorescent protein and beta-glucuronidase. All the expressed genes were revealed to be active in the female gametophyte or developing seed, indicating that the entire type I subfamily is involved in reproductive development in Arabidopsis. Interestingly, expression was predominantly observed in the central cell, antipodal cells, and chalazal endosperm. The combination of our expression results with phylogenetic and protein interaction data allows a better identification of putative redundantly acting genes and provides a useful tool for the functional characterization of the type I MADS box genes in Arabidopsis. PMID:20631316

  5. Evolutionary and expression analysis of a MADS-box gene superfamily involved in ovule development of seeded and seedless grapevines.

    PubMed

    Wang, Li; Yin, Xiangjing; Cheng, Chenxia; Wang, Hao; Guo, Rongrong; Xu, Xiaozhao; Zhao, Jiao; Zheng, Yi; Wang, Xiping

    2015-06-01

    MADS-box transcription factors are involved in many aspects of plant growth and development, such as floral organ determination, fruit ripening, and embryonic development. Yet not much is known about grape (Vitis vinifera) MADS-box genes in a relatively comprehensive genomic and functional way during ovule development. Accordingly, we identified 54 grape MADS-box genes, aiming to enhance our understanding of grape MADS-box genes from both evolutionary and functional perspectives. Synteny analysis indicated that both segmental and tandem duplication events contributed to the expansion of the grape MADS-box family. Furthermore, synteny analysis between the grape and Arabidopsis genomes suggested that several grape MADS-box genes arose before divergence of the two species. Phylogenetic analysis and comparisons of exon-intron structures provided further insight into the evolutionary relationships between the genes, as well as their putative functions. Based on phylogenetic tree analysis, grape MADS-box genes were divided into type I and type II subgroups. Tissue-specific expression analysis suggested roles in both vegetative and reproductive tissue development. Expression analysis of the MADS-box genes following gibberellic acid (GA3) treatment revealed their response to GA3 treatment and that seedlessness caused by GA3 treatment underwent a different mechanism from that of normal ovule abortion. Expression profiling of MADS-box genes from six cultivars suggests their function in ovule development and may represent potential ovule identity genes involved in parthenocarpy. The results presented provide a few candidate genes involved in ovule development for future study, which may be useful in seedlessness-related molecular breeding programs. PMID:25429734

  6. Involvement of a banana MADS-box transcription factor gene in ethylene-induced fruit ripening.

    PubMed

    Liu, Juhua; Xu, Biyu; Hu, Lifang; Li, Meiying; Su, Wei; Wu, Jing; Yang, Jinghao; Jin, Zhiqiang

    2009-01-01

    To investigate the regulation of MADS-box genes in banana (Musa acuminata L. AAA group cv. Brazilian) fruit development and postharvest ripening, we isolated from banana fruit a MADS-box gene designated MuMADS1. Amino acid alignment indicated MuMADS1 belongs to the AGAMOUS subfamily, and phylogenetic analysis indicates that this gene is most similar to class D MADS-box genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that MuMADS1 is expressed in the stamen and pistil of male and female flowers and in the rhizome, the vegetative reproductive organ of the banana plant. In preharvest banana fruit, MuMADS1 is likely expressed throughout banana fruit development. In postharvest banana ripening, MuMADS1 is associated with ethylene biosynthesis. Expression patterns of MuMADS1 during postharvest ripening as determined by real-time RT-PCR suggest that differential expression of MuMADS1 may not only be induced by ethylene biosynthesis associated with postharvest banana ripening, but also may be induced by exogenous ethylene. PMID:18820933

  7. A Novel Sucrose-Regulatory MADS-Box Transcription Factor GmNMHC5 Promotes Root Development and Nodulation in Soybean (Glycine max [L.] Merr.).

    PubMed

    Liu, Wei; Han, Xiangdong; Zhan, Ge; Zhao, Zhenfang; Feng, Yongjun; Wu, Cunxiang

    2015-01-01

    The MADS-box protein family includes many transcription factors that have a conserved DNA-binding MADS-box domain. The proteins in this family were originally recognized to play prominent roles in floral development. Recent findings, especially with regard to the regulatory roles of the AGL17 subfamily in root development, have greatly broadened their known functions. In this study, a gene from soybean (Glycine max [L.] Merr.), GmNMHC5, was cloned from the Zigongdongdou cultivar and identified as a member of the AGL17 subfamily. Real-time fluorescence quantitative PCR analysis showed that GmNMHC5 was expressed at much higher levels in roots and nodules than in other organs. The activation of expression was first examined in leaves and roots, followed by shoot apexes. GmNMHC5 expression levels rose sharply when the plants were treated under short-day conditions (SD) and started to pod, whereas low levels were maintained in non-podding plants under long-day conditions (LD). Furthermore, overexpression of GmNMHC5 in transgenic soybean significantly promoted lateral root development and nodule building. Moreover, GmNMHC5 is upregulated by exogenous sucrose. These results indicate that GmNMHC5 can sense the sucrose signal and plays significant roles in lateral root development and nodule building. PMID:26404246

  8. A Novel Sucrose-Regulatory MADS-Box Transcription Factor GmNMHC5 Promotes Root Development and Nodulation in Soybean (Glycine max [L.] Merr.)

    PubMed Central

    Liu, Wei; Han, Xiangdong; Zhan, Ge; Zhao, Zhenfang; Feng, Yongjun; Wu, Cunxiang

    2015-01-01

    The MADS-box protein family includes many transcription factors that have a conserved DNA-binding MADS-box domain. The proteins in this family were originally recognized to play prominent roles in floral development. Recent findings, especially with regard to the regulatory roles of the AGL17 subfamily in root development, have greatly broadened their known functions. In this study, a gene from soybean (Glycine max [L.] Merr.), GmNMHC5, was cloned from the Zigongdongdou cultivar and identified as a member of the AGL17 subfamily. Real-time fluorescence quantitative PCR analysis showed that GmNMHC5 was expressed at much higher levels in roots and nodules than in other organs. The activation of expression was first examined in leaves and roots, followed by shoot apexes. GmNMHC5 expression levels rose sharply when the plants were treated under short-day conditions (SD) and started to pod, whereas low levels were maintained in non-podding plants under long-day conditions (LD). Furthermore, overexpression of GmNMHC5 in transgenic soybean significantly promoted lateral root development and nodule building. Moreover, GmNMHC5 is upregulated by exogenous sucrose. These results indicate that GmNMHC5 can sense the sucrose signal and plays significant roles in lateral root development and nodule building. PMID:26404246

  9. Divergence of recently duplicated M{gamma}-type MADS-box genes in Petunia.

    PubMed

    Bemer, Marian; Gordon, Jonathan; Weterings, Koen; Angenent, Gerco C

    2010-02-01

    The MADS-box transcription factor family has expanded considerably in plants via gene and genome duplications and can be subdivided into type I and MIKC-type genes. The two gene classes show a different evolutionary history. Whereas the MIKC-type genes originated during ancient genome duplications, as well as during more recent events, the type I loci appear to experience high turnover with many recent duplications. This different mode of origin also suggests a different fate for the type I duplicates, which are thought to have a higher chance to become silenced or lost from the genome. To get more insight into the evolution of the type I MADS-box genes, we isolated nine type I genes from Petunia, which belong to the Mgamma subclass, and investigated the divergence of their coding and regulatory regions. The isolated genes could be subdivided into two categories: two genes were highly similar to Arabidopsis Mgamma-type genes, whereas the other seven genes showed less similarity to Arabidopsis genes and originated more recently. Two of the recently duplicated genes were found to contain deleterious mutations in their coding regions, and expression analysis revealed that a third paralog was silenced by mutations in its regulatory region. However, in addition to the three genes that were subjected to nonfunctionalization, we also found evidence for neofunctionalization of one of the Petunia Mgamma-type genes. Our study shows a rapid divergence of recently duplicated Mgamma-type MADS-box genes and suggests that redundancy among type I paralogs may be less common than expected. PMID:19933156

  10. Flower development: the evolutionary history and functions of the AGL6 subfamily MADS-box genes.

    PubMed

    Dreni, Ludovico; Zhang, Dabing

    2016-04-01

    AGL6 is an ancient subfamily of MADS-box genes found in both gymnosperms and angiosperms. Its functions remained elusive despite the fact that the MADS-box genes and the ABC model have been studied for >20 years. Nevertheless, recent discoveries in petunia, rice, and maize support its involvement in the 'E' function of floral development, very similar to the closely related AGL2 (SEPALLATA) subfamily which has been well characterized. The known functions of AGL6 span from ancient conserved roles to new functions acquired in specific plant families. The AGL6 genes are involved in floral meristem regulation, in floral organs, and ovule (integument) and seed development, and have possible roles in both male and female germline and gametophyte development. In grasses, they are also important for the development of the first whorl of the flower, whereas in Arabidopsis they may play additional roles before floral meristem formation. This review covers these recent insights and some other aspects that are not yet fully elucidated, which deserve more studies in the future. PMID:26956504

  11. Phenotypic alterations of petal and sepal by ectopic expression of a rice MADS box gene in tobacco.

    PubMed

    Kang, H G; Noh, Y S; Chung, Y Y; Costa, M A; An, K; An, G

    1995-10-01

    Floral organ development is controlled by a group of regulatory factors containing the MADS domain. In this study, we have isolated and characterized a cDNA clone from rice, OsMADS3, which encodes a MADS-domain containing protein. The OsMADS3 amino acid sequence shows over 60% identity to AG of Arabidopsis, PLE of Antirrhinum majus, and AG/PLE homologues of petunia, tobacco, tomato, Brassica napus, and maize. Homology in the MADS box region is most conserved. RNA blot analysis indicated that the rice MADS gene was preferentially expressed in reproductive organs, especially in stamen and carpel. In situ localization studies showed that the transcript was present primarily in stamen and carpel. The function of the rice OsMADS3 was elucidated by ectopic expression of the gene under the control of the CaMV 35S promoter in a heterologous tobacco plant system. Transgenic plants exhibited an altered morphology and coloration of the perianth organs. Sepals were pale green and elongated. Limbs of the corolla were split into sections which in some plants became antheroid structures attached to tubes that resembled filaments. The phenotypes mimic the results of ectopic expression of dicot AG gene or AG homologues. These results indicate that the OsMADS3 gene is possibly an AG homologue and that the AG genes appear to be structurally and functionally conserved between dicot and monocot. PMID:7579155

  12. Male and female flowers of the dioecious plant sorrel show different patterns of MADS box gene expression.

    PubMed Central

    Ainsworth, C; Crossley, S; Buchanan-Wollaston, V; Thangavelu, M; Parker, J

    1995-01-01

    Male and female flowers of the dioecious plant sorrel (Rumex acetosa) each produce three whorls of developed floral organs: two similar whorls of three perianth segments and either six stamens (in the male) or a gynoecium consisting of a fertile carpel and two sterile carpels (in the female). In the developing male flower, there is no significant proliferation of cells in the center of the flower, in the position normally occupied by the carpels of a hermaphrodite plant. In the female flower, small stamen primordia are formed. To determine whether the organ differences are associated with differences in the expression of organ identity genes, cDNA clones representing the putative homologs of B and C function MADS box genes were isolated and used in an in situ hybridization analysis. The expression of RAD1 and RAD2 (two different DEFICIENS homologs) in males and females was confined to the stamen whorl; the lack of expression in the second, inner perianth whorl correlated with the sepaloid nature of the inner whorl of perianth segments. Expression of RAP1 (a PLENA homolog) occurred in the carpel and stamen whorls in very young flower primordia from both males and females. However, as soon as the inappropriate set of organs ceased to develop, RAP1 expression became undetectable in those organs. The absence of expression of RAP1 may be the cause of the arrest in organ development or may be a consequence. PMID:7580253

  13. Coordinating expression of FLOWERING LOCUS T by DORMANCY ASSOCIATED MADS-BOX-like genes in leafy spurge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leafy spurge is a noxious perennial weed that produces underground adventitious buds, which are crucial for generating new vegetative shoots following periods of freezing temperatures or exposure to various control measures. DORMANCY ASSOCIATED MADS-BOX (DAM) genes have been proposed to play a direc...

  14. The regulation of MADS-box gene expression during ripening of banana and their regulatory interation with ethylene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MADS-box genes (MaMADS1-6), potential components of the developmental control of ripening have been cloned from Grand Nain banana cultivar. Similarity of these genes to tomato LeRIN is very low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns...

  15. Changes in ethylene signaling and MADS box gene expression are associated with banana finger drop.

    PubMed

    Hubert, O; Piral, G; Galas, C; Baurens, F-C; Mbéguié-A-Mbéguié, D

    2014-06-01

    Banana finger drop was examined in ripening banana harvested at immature (iMG), early (eMG) and late mature green (lMG) stages, with contrasting ripening rates and ethylene sensitivities. Concomitantly, 11 ethylene signal transduction components (ESTC) and 6 MADS box gene expressions were comparatively studied in median (control zone, CZ) and pedicel rupture (drop zone DZ) areas in peel tissue. iMG fruit did not ripen or develop finger drop while eMG and lMG fruits displayed a similar finger drop pattern. Several ESTC and MADS box gene mRNAs were differentially induced in DZ and CZ and sequentially in eMG and lMG fruits. MaESR2, 3 and MaEIL1, MaMADS2 and MaMADS5 had a higher mRNA level in eMG and acted earlier, whereas MaERS1, MaCTR1, MaEIL3/AB266319, MaEIL4/AB266320 and MaEIL5/AB266321, MaMADS4 and to a lesser extent MaMADS2 and 5 acted later in lMG. In this fruit, MaERS1 and 3, MaCTR1, MaEIL3, 4 and MaEIL5/AB266321, and MaMADS4 were enhanced by finger drop, suggesting their specific involvement in this process. MaEIL1, MaMADS1 and 3, induced at comparable levels in DZ and CZ, are probably related to the overall fruit ripening process. These findings led us to consider that developmental cues are the predominant finger drop regulation factor. PMID:24767119

  16. Fruit Ripening Regulation of α-Mannosidase Expression by the MADS Box Transcription Factor RIPENING INHIBITOR and Ethylene

    PubMed Central

    Irfan, Mohammad; Ghosh, Sumit; Meli, Vijaykumar S.; Kumar, Anil; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2016-01-01

    α-Mannosidase (α-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression. PMID:26834776

  17. Fruit Ripening Regulation of α-Mannosidase Expression by the MADS Box Transcription Factor RIPENING INHIBITOR and Ethylene.

    PubMed

    Irfan, Mohammad; Ghosh, Sumit; Meli, Vijaykumar S; Kumar, Anil; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2016-01-01

    α-Mannosidase (α-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression. PMID:26834776

  18. A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de-vernalization responses.

    PubMed

    Périlleux, Claire; Pieltain, Alexandra; Jacquemin, Guillaume; Bouché, Frédéric; Detry, Nathalie; D'Aloia, Maria; Thiry, Laura; Aljochim, Pierre; Delansnay, Martin; Mathieu, Anne-Sophie; Lutts, Stanley; Tocquin, Pierre

    2013-08-01

    Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT-PCR and named FLC-LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over-expression of CiFL1 in Arabidopsis caused late flowering and prevented up-regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post-vernalization temperature was favorable to flowering and when it de-vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re-activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold-induced down-regulation of a MADS box floral repressor and its re-activation by high temperature thus appear to be conserved features of the vernalization and de-vernalization responses in distant species. PMID:23581257

  19. Perspectives on MADS-box expression during orchid flower evolution and development.

    PubMed

    Mondragón-Palomino, Mariana

    2013-01-01

    The diverse morphology of orchid flowers and their complex, often deceptive strategies to become pollinated have fascinated researchers for a long time. However, it was not until the 20th century that the ontogeny of orchid flowers, the genetic basis of their morphology and the complex phylogeny of Orchidaceae were investigated. In parallel, the improvement of techniques for in vitro seed germination and tissue culture, together with studies on biochemistry, physiology, and cytology supported the progress of what is now a highly productive industry of orchid breeding and propagation. In the present century both basic research in orchid flower evo-devo and the interest for generating novel horticultural varieties have driven the characterization of many members of the MADS-box family encoding key regulators of flower development. This perspective summarizes the picture emerging from these studies and discusses the advantages and limitations of the comparative strategy employed so far. I address the growing role of natural and horticultural mutants in these studies and the emergence of several model species in orchid evo-devo and genomics. In this context, I make a plea for an increasingly integrative approach. PMID:24065980

  20. Perspectives on MADS-box expression during orchid flower evolution and development

    PubMed Central

    Mondragón-Palomino, Mariana

    2013-01-01

    The diverse morphology of orchid flowers and their complex, often deceptive strategies to become pollinated have fascinated researchers for a long time. However, it was not until the 20th century that the ontogeny of orchid flowers, the genetic basis of their morphology and the complex phylogeny of Orchidaceae were investigated. In parallel, the improvement of techniques for in vitro seed germination and tissue culture, together with studies on biochemistry, physiology, and cytology supported the progress of what is now a highly productive industry of orchid breeding and propagation. In the present century both basic research in orchid flower evo-devo and the interest for generating novel horticultural varieties have driven the characterization of many members of the MADS-box family encoding key regulators of flower development. This perspective summarizes the picture emerging from these studies and discusses the advantages and limitations of the comparative strategy employed so far. I address the growing role of natural and horticultural mutants in these studies and the emergence of several model species in orchid evo-devo and genomics. In this context, I make a plea for an increasingly integrative approach. PMID:24065980

  1. The role of MADS-box transcription factors in secondary metabolism and sexual development in the maize pathogen Fusarium verticillioides.

    PubMed

    Ortiz, Carlos S; Shim, Won-Bo

    2013-11-01

    MADS-box transcription factors (TFs) regulate functionally diverse gene targets in eukaryotes. In select ascomycetes, MADS-box TFs have been shown to play a role in virulence, and vegetative and sexual development. Here, we characterized Fusarium verticillioides MADS-box TFs, Mads1 and Mads2, in terms of their roles in secondary metabolism and sexual mating. Sequence analyses showed that MADS1 and MADS2 encode TFs with a SRF-type dimerization domain and a MEF2-type dimerization domain, respectively. The MADS1 and MADS2 knockout mutants (Fmt1 and Fmt2 strains, respectively) exhibited decreased vegetative growth and FB1 production when compared to the wild-type. Fmt1 showed reduced expression of 14 polyketide synthase (PKS) genes present in the organism, whereas Fmt2 did not display a change in PKS gene expression. Significantly, the deletion of MADS1 and MADS2 in the MAT1-2 genotype (Fmt4 and Fmt5 strains, respectively) led to strains that failed to produce perithecia and ascospores when crossed with the MAT1-1 wild-type strain. Notably, deletion of either gene did not have an effect on the ability of the fungus to colonize maize stalk or kernels. FB1 production and PKS expression data suggest that Mads1 is a broad regulator of secondary metabolism in F. verticillioides, and may target regulons upstream of Mads2 to influence FB1 production. In addition, MADS-box TFs in F. verticillioides play a critical role in the perithecia development. PMID:23985144

  2. Characterization and Expression Analysis of PtAGL24, a SHORT VEGETATIVE PHASE/AGAMOUS-LIKE 24 (SVP/AGL24)-Type MADS-Box Gene from Trifoliate Orange (Poncirus trifoliata L. Raf.)

    PubMed Central

    Sun, Lei-Ming; Zhang, Jin-Zhi; Hu, Chun-Gen

    2016-01-01

    The transition from vegetative to reproductive growth in perennial woody plants does not occur until after several years of repeated seasonal changes and alternative growth. To better understand the molecular basis of flowering regulation in citrus, a MADS-box gene was isolated from trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.). Sequence alignment and phylogenetic analysis showed that the MADS-box gene is more closely related to the homologs of the AGAMOUS-LIKE 24 (AGL24) lineage than to any of the other MADS-box lineages known from Arabidopsis; it is named PtAGL24. Expression analysis indicated that PtAGL24 was widely expressed in the most organs of trifoliate orange, with the higher expression in mature flowers discovered by real-time PCR. Ectopic expression of PtAGL24 in wild-type Arabidopsis promoted early flowering and caused morphological changes in class I transgenic Arabidopsis. Yeast two-hybrid assay revealed that PtAGL24 interacted with Arabidopsis AtAGL24 and other partners of AtAGL24, suggesting that the abnormal morphology of PtAGL24 overexpression in transgenic Arabidopsis was likely due to the inappropriate interactions between exogenous and endogenous proteins. Also, PtAGL24 interacted with SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (PtSOC1) and APETALA1 (PtAP1) of citrus. These results suggest that PtAGL24 may play an important role in the process of floral transition but may have diverse functions in citrus development. PMID:27375669

  3. Heat stress yields a unique MADS box transcription factor in determining seed size and thermal sensitivity.

    PubMed

    Chen, Chen; Begcy, Kevin; Liu, Kan; Folsom, Jing J; Wang, Zhen; Zhang, Chi; Walia, Harkamal

    2016-05-01

    Early seed development events are highly sensitive to increased temperature. This high sensitivity to a short-duration temperature spike reduces seed viability and seed size at maturity. The molecular basis of heat stress sensitivity during early seed development is not known. We selected rice (Oryza sativa), a highly heat-sensitive species, to explore this phenomenon. Here, we elucidate the molecular pathways that contribute to the heat sensitivity of a critical developmental window during which the endosperm transitions from syncytium to the cellularization stage in young seeds. A transcriptomic comparison of seeds exposed to moderate (35°C) and severe (39°C) heat stress with control (28°C) seeds identified a set of putative imprinted genes, which were down-regulated under severe heat stress. Several type I MADS box genes specifically expressed during the syncytial stage were differentially regulated under moderate and severe heat stress. The suppression and overaccumulation of these genes are associated with precocious and delayed cellularization under moderate and severe stress, respectively. We show that modulating the expression of OsMADS87, one of the heat-sensitive, imprinted genes associated with syncytial stage endosperm, regulates rice seed size. Transgenic seeds deficient in OsMADS87 exhibit accelerated endosperm cellularization. These seeds also have lower sensitivity to a moderate heat stress in terms of seed size reduction compared with seeds from wild-type plants and plants overexpressing OsMADS87 Our findings suggest that OsMADS87 and several other genes identified in this study could be potential targets for improving the thermal resilience of rice during reproductive development. PMID:26936896

  4. Homoeologous copy-specific expression patterns of MADS-box genes for floral formation in allopolyploid wheat.

    PubMed

    Tanaka, Miku; Tanaka, Hiroko; Shitsukawa, Naoki; Kitagawa, Satoshi; Takumi, Shigeo; Murai, Koji

    2016-01-01

    The consensus model for floral organ formation in higher plants, the so-called ABCDE model, proposes that floral whorl-specific combinations of class A, B, C, D, and E genes specify floral organ identity. Class A, B, C, D and E genes encode MADS-box transcription factors; the single exception being the class A gene APETALA2. Bread wheat (Triticum aestivum) is a hexaploid species with a genome constitution AABBDD; the hexaploid originated from a cross between tetraploid T. turgidum (AABB) and diploid Aegilops tauschii (DD). Tetraploid wheat is thought to have originated from a cross between the diploid species T. urartu (AA) and Ae. speltoides (BB). Consequently, the hexaploid wheat genome contains triplicated homoeologous copies (homoeologs) of each gene derived from the different ancestral diploid species. In this study, we examined the expression patterns of homoeologs of class B, C and D MADS-box genes during floral development. For the class B gene wheat PISTILLATA2 (WPI2), the homoeologs from the A and D genomes were expressed, while expression of the B genome homoeolog was suppressed. For the class C gene wheat AGAMOUS1 (WAG1), the homoeologs on the A and B genomes were expressed, while expression of the D genome homoeolog was suppressed. For the class D gene wheat SEEDSTICK (WSTK), the B genome homoeolog was preferentially expressed. These differential patterns of homoeolog expression were consistently observed among different hexaploid wheat varieties and synthetic hexaploid wheat lines developed by artificial crosses between tetraploid wheat and Ae. tauschii. These results suggest that homoeolog-specific regulation of the floral MADS-box genes occurs in allopolyploid wheat. PMID:26616759

  5. The MADS-box transcription factor FgMcm1 regulates cell identity and fungal development in Fusarium graminearum.

    PubMed

    Yang, Cui; Liu, Huiquan; Li, Guotian; Liu, Meigang; Yun, Yingzi; Wang, Chenfang; Ma, Zhonghua; Xu, Jin-Rong

    2015-08-01

    In eukaryotic cells, MADS-box genes are known to play major regulatory roles in various biological processes by combinatorial interactions with other transcription factors. In this study, we functionally characterized the FgMCM1 MADS-box gene in Fusarium graminearum, the causal agent of wheat and barley head blight. Deletion of FgMCM1 resulted in the loss of perithecium production and phialide formation. The Fgmcm1 mutant was significantly reduced in virulence, deoxynivalenol biosynthesis and conidiation. In yeast two-hybrid assays, FgMcm1 interacted with Mat1-1-1 and Fst12, two transcription factors important for sexual reproduction. Whereas Fgmcm1 mutants were unstable and produced stunted subcultures, Fgmcm1 mat1-1-1 but not Fgmcm1 fst12 double mutants were stable. Furthermore, spontaneous suppressor mutations occurred frequently in stunted subcultures to recover growth rate. Ribonucleic acid sequencing analysis indicated that a number of sexual reproduction-related genes were upregulated in stunted subcultures compared with the Fgmcm1 mutant, which was downregulated in the expression of genes involved in pathogenesis, secondary metabolism and conidiation. We also showed that culture instability was not observed in the Fvmcm1 mutants of the heterothallic Fusarium verticillioides. Overall, our data indicate that FgMcm1 plays a critical role in the regulation of cell identity, sexual and asexual reproduction, secondary metabolism and pathogenesis in F. graminearum. PMID:25627073

  6. MADS-Box Transcription Factor VdMcm1 Regulates Conidiation, Microsclerotia Formation, Pathogenicity, and Secondary Metabolism of Verticillium dahliae

    PubMed Central

    Xiong, Dianguang; Wang, Yonglin; Tian, Longyan; Tian, Chengming

    2016-01-01

    Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species resulting in devastating yield losses worldwide. Due to its ability to colonize plant xylem and form microsclerotia, V. dahliae is highly persistent and difficult to control. In this study, we show that the MADS-box transcription factor VdMcm1 is a key regulator of conidiation, microsclerotia formation, virulence, and secondary metabolism of V. dahliae. In addition, our findings suggest that VdMcm1 is involved in cell wall integrity. Finally, comparative RNA-Seq analysis reveals 823 significantly downregulated genes in the VdMcm1 deletion mutant, with diverse biological functions in transcriptional regulation, plant infection, cell adhesion, secondary metabolism, transmembrane transport activity, and cell secretion. When taken together, these data suggest that VdMcm1 performs pleiotropic functions in V. dahliae. PMID:27536281

  7. MADS-Box Transcription Factor VdMcm1 Regulates Conidiation, Microsclerotia Formation, Pathogenicity, and Secondary Metabolism of Verticillium dahliae.

    PubMed

    Xiong, Dianguang; Wang, Yonglin; Tian, Longyan; Tian, Chengming

    2016-01-01

    Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species resulting in devastating yield losses worldwide. Due to its ability to colonize plant xylem and form microsclerotia, V. dahliae is highly persistent and difficult to control. In this study, we show that the MADS-box transcription factor VdMcm1 is a key regulator of conidiation, microsclerotia formation, virulence, and secondary metabolism of V. dahliae. In addition, our findings suggest that VdMcm1 is involved in cell wall integrity. Finally, comparative RNA-Seq analysis reveals 823 significantly downregulated genes in the VdMcm1 deletion mutant, with diverse biological functions in transcriptional regulation, plant infection, cell adhesion, secondary metabolism, transmembrane transport activity, and cell secretion. When taken together, these data suggest that VdMcm1 performs pleiotropic functions in V. dahliae. PMID:27536281

  8. GRCD1, an AGL2-like MADS Box Gene, Participates in the C Function during Stamen Development in Gerbera hybrida

    PubMed Central

    Kotilainen, Mika; Elomaa, Paula; Uimari, Anne; Albert, Victor A.; Yu, Deyue; Teeri, Teemu H.

    2000-01-01

    Despite the differences in flower form, the underlying mechanism in determining the identity of floral organs is largely conserved among different angiosperms, but the details of how the functions of A, B, and C are specified varies greatly among plant species. Here, we report functional analysis of a Gerbera MADS box gene, GRCD1, which is orthologous to AGL2-like MADS box genes. Members of this group of genes are being reported in various species in growing numbers, but their functions remained largely unsettled. GRCD1 expression is detected in all four whorls, but the strongest signal is seen in the developing stamen and carpel. Downregulating GRCD1 expression by antisense transformation revealed that lack of GRCD1 caused homeotic changes in one whorl only: sterile staminodes, which normally develop in whorl 3 of marginal female florets, were changed into petals. This indicates that the GRCD1 gene product is active in determining stamen identity. Transgenic downregulation of GRCD1 causes a homeotic change similar to that in the downregulation of the Gerbera C function genes GAGA1 and GAGA2, but one that is limited to whorl 3. Downregulation of GRCD1 expression does not reduce expression of GAGA1 or GAGA2, or vice versa; and in yeast two-hybrid analysis, GRCD1 is able to interact with GAGA1 and GAGA2. We propose that a heterodimer between the GRCD1 and GAGA1/2 gene products is needed to fulfill the C function in whorl 3 in Gerbera. PMID:11041884

  9. Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways.

    PubMed

    Zhao, Ying; Liang, Haiying; Li, Lan; Tang, Sha; Han, Xiao; Wang, Congpeng; Xia, Xinli; Yin, Weilun

    2015-01-01

    Metasequoia glyptostroboides is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as 5-to-7 years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE) tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD) and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia. PMID:26157452

  10. Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear (Pyrus pyrifolia white pear group) flower bud

    PubMed Central

    Niu, Qingfeng; Li, Jianzhao; Cai, Danying; Qian, Minjie; Jia, Huimin; Bai, Songling; Hussain, Sayed; Liu, Guoqin; Teng, Yuanwen; Zheng, Xiaoyan

    2016-01-01

    Bud dormancy in perennial plants is indispensable to survival over winter and to regrowth and development in the following year. However, the molecular pathways of endo-dormancy induction, maintenance, and release are still unclear, especially in fruit crops. To identify genes with roles in regulating endo-dormancy, 30 MIKCC-type MADS-box genes were identified in the pear genome and characterized. The 30 genes were analysed to determine their phylogenetic relationships with homologous genes, genome locations, gene structure, tissue-specific transcript profiles, and transcriptional patterns during flower bud dormancy in ‘Suli’ pear (Pyrus pyrifolia white pear group). The roles in regulating bud dormancy varied among the MIKC gene family members. Yeast one-hybrid and transient assays showed that PpCBF enhanced PpDAM1 and PpDAM3 transcriptional activity during the induction of dormancy, probably by binding to the C-repeat/DRE binding site, while DAM proteins inhibited the transcriptional activity of PpFT2 during dormancy release. In the small RNA-seq analysis, 185 conserved, 24 less-conserved, and 32 pear-specific miRNAs with distinct expression patterns during bud dormancy were identified. Joint analyses of miRNAs and MIKC genes together with degradome data showed that miR6390 targeted PpDAM transcripts and degraded them to release PpFT2. Our data show that cross-talk among PpCBF, PpDAM, PpFT2, and miR6390 played important roles in regulating endo-dormancy. A model for the molecular mechanism of dormancy transition is proposed: short-term chilling in autumn activates the accumulation of CBF, which directly promotes DAM expression; DAM subsequently inhibits FT expression to induce endo-dormancy, and miR6390 degrades DAM genes to release endo-dormancy. PMID:26466664

  11. Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways

    PubMed Central

    Zhao, Ying; Liang, Haiying; Li, Lan; Tang, Sha; Han, Xiao; Wang, Congpeng; Xia, Xinli; Yin, Weilun

    2015-01-01

    Metasequoia glyptostroboides is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as 5-to-7 years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE) tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD) and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia. PMID:26157452

  12. The regulation of MADS-box gene expression during ripening of banana and their regulatory interaction with ethylene.

    PubMed

    Elitzur, Tomer; Vrebalov, Julia; Giovannoni, James J; Goldschmidt, Eliezer E; Friedman, Haya

    2010-03-01

    Six MaMADS-box genes have been cloned from the banana fruit cultivar Grand Nain. The similarity of these genes to tomato LeRIN is low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns, specifically in fruit and the induction during ripening and in response to ethylene and 1-MCP, suggest that some of these genes may participate in ripening. MaMADS1, 2, and 3, are highly expressed in fruit only, while the others are expressed in fruit as well as in other organs. Moreover, the suites of MaMADS-box genes and their temporal expression differ in peel and pulp during ripening. In the pulp, the increase in MaMADS2, 3, 4, and 5 expression preceded an increase in ethylene production, but coincides with the CO(2) peak. However, MaMADS1 expression in pulp coincided with ethylene production, but a massive increase in its expression occurred late during ripening, together with a second wave in the expression of MaMADS2, 3, and 4. In the peel, on the other hand, an increase in expression of MaMADS1, 3, and to a lesser degree also of MaMADS4 and 2 coincided with an increase in ethylene production. Except MaMADS3, which was induced by ethylene in pulp and peel, only MaMADS4, and 5 in pulp and MaMADS1 in peel were induced by ethylene. 1-MCP applied at the onset of the increase in ethylene production, increased the levels of MaMADS4 and MaMADS1 in pulp, while it decreased MaMADS1, 3, 4, and 5 in peel, suggesting that MaMADS4 and MaMADS1 are negatively controlled by ethylene at the onset of ethylene production only in pulp. Only MaMADS2 is neither induced by ethylene nor by 1-MCP, and it is expressed mainly in pulp. Our results suggest that two independent ripening programs are employed in pulp and peel which involve the activation of mainly MaMADS2, 4, and 5 and later on also MaMADS1 in pulp, and mainly MaMADS1, and 3 in peel. Hence, our results are consistent with MaMADS2, a SEP3 homologue, acting in the pulp upstream of the

  13. Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS box genes in angiosperms.

    PubMed Central

    Kramer, Elena M; Jaramillo, M Alejandra; Di Stilio, Verónica S

    2004-01-01

    Members of the AGAMOUS (AG) subfamily of MIKC-type MADS-box genes appear to control the development of reproductive organs in both gymnosperms and angiosperms. To understand the evolution of this subfamily in the flowering plants, we have identified 26 new AG-like genes from 15 diverse angiosperm species. Phylogenetic analyses of these genes within a large data set of AG-like sequences show that ancient gene duplications were critical in shaping the evolution of the subfamily. Before the radiation of extant angiosperms, one event produced the ovule-specific D lineage and the well-characterized C lineage, whose members typically promote stamen and carpel identity as well as floral meristem determinacy. Subsequent duplications in the C lineage resulted in independent instances of paralog subfunctionalization and maintained functional redundancy. Most notably, the functional homologs AG from Arabidopsis and PLENA (PLE) from Antirrhinum are shown to be representatives of separate paralogous lineages rather than simple genetic orthologs. The multiple subfunctionalization events that have occurred in this subfamily highlight the potential for gene duplication to lead to dissociation among genetic modules, thereby allowing an increase in morphological diversity. PMID:15020484

  14. MOSAIC FLORAL ORGANS1, an AGL6-Like MADS Box Gene, Regulates Floral Organ Identity and Meristem Fate in Rice[W

    PubMed Central

    Ohmori, Shinnosuke; Kimizu, Mayumi; Sugita, Maiko; Miyao, Akio; Hirochika, Hirohiko; Uchida, Eiji; Nagato, Yasuo; Yoshida, Hitoshi

    2009-01-01

    Floral organ identity and meristem determinacy in plants are controlled by combinations of activities mediated by MADS box genes. AGAMOUS-LIKE6 (AGL6)-like genes are MADS box genes expressed in floral tissues, but their biological functions are mostly unknown. Here, we describe an AGL6-like gene in rice (Oryza sativa), MOSAIC FLORAL ORGANS1 (MFO1/MADS6), that regulates floral organ identity and floral meristem determinacy. In the flower of mfo1 mutants, the identities of palea and lodicule are disturbed, and mosaic organs were observed. Furthermore, the determinacy of the floral meristem was lost, and extra carpels or spikelets developed in mfo1 florets. The expression patterns of floral MADS box genes were disturbed in the mutant florets. Suppression of another rice AGL6-like gene, MADS17, caused no morphological abnormalities in the wild-type background, but it enhanced the phenotype in the mfo1 background, indicating that MADS17 has a minor but redundant function with that of MFO1. Whereas single mutants in either MFO1 or the SEPALLATA-like gene LHS1 showed moderate phenotypes, the mfo1 lhs1 double mutant showed a severe phenotype, including the loss of spikelet meristem determinacy. We propose that rice AGL6-like genes help to control floral organ identity and the establishment and determinacy of the floral meristem redundantly with LHS1. PMID:19820190

  15. Heat stress yields a unique MADS box transcription factor in determining seed size and thermal sensitivity1[OPEN

    PubMed Central

    Begcy, Kevin; Liu, Kan; Wang, Zhen; Zhang, Chi

    2016-01-01

    Early seed development events are highly sensitive to increased temperature. This high sensitivity to a short-duration temperature spike reduces seed viability and seed size at maturity. The molecular basis of heat stress sensitivity during early seed development is not known. We selected rice (Oryza sativa), a highly heat-sensitive species, to explore this phenomenon. Here, we elucidate the molecular pathways that contribute to the heat sensitivity of a critical developmental window during which the endosperm transitions from syncytium to the cellularization stage in young seeds. A transcriptomic comparison of seeds exposed to moderate (35°C) and severe (39°C) heat stress with control (28°C) seeds identified a set of putative imprinted genes, which were down-regulated under severe heat stress. Several type I MADS box genes specifically expressed during the syncytial stage were differentially regulated under moderate and severe heat stress. The suppression and overaccumulation of these genes are associated with precocious and delayed cellularization under moderate and severe stress, respectively. We show that modulating the expression of OsMADS87, one of the heat-sensitive, imprinted genes associated with syncytial stage endosperm, regulates rice seed size. Transgenic seeds deficient in OsMADS87 exhibit accelerated endosperm cellularization. These seeds also have lower sensitivity to a moderate heat stress in terms of seed size reduction compared with seeds from wild-type plants and plants overexpressing OsMADS87. Our findings suggest that OsMADS87 and several other genes identified in this study could be potential targets for improving the thermal resilience of rice during reproductive development. PMID:26936896

  16. ODDSOC2 is a MADS box floral repressor that is down-regulated by vernalization in temperate cereals.

    PubMed

    Greenup, Aaron G; Sasani, Shahryar; Oliver, Sandra N; Talbot, Mark J; Dennis, Elizabeth S; Hemming, Megan N; Trevaskis, Ben

    2010-07-01

    In temperate cereals, such as wheat (Triticum aestivum) and barley (Hordeum vulgare), the transition to reproductive development can be accelerated by prolonged exposure to cold (vernalization). We examined the role of the grass-specific MADS box gene ODDSOC2 (OS2) in the vernalization response in cereals. The barley OS2 gene (HvOS2) is expressed in leaves and shoot apices but is repressed by vernalization. Vernalization represses OS2 independently of VERNALIZATION1 (VRN1) in a VRN1 deletion mutant of einkorn wheat (Triticum monococcum), but VRN1 is required to maintain down-regulation of OS2 in vernalized plants. Furthermore, barleys that carry active alleles of the VRN1 gene (HvVRN1) have reduced expression of HvOS2, suggesting that HvVRN1 down-regulates HvOS2 during development. Overexpression of HvOS2 delayed flowering and reduced spike, stem, and leaf length in transgenic barley plants. Plants overexpressing HvOS2 showed reduced expression of barley homologs of the Arabidopsis (Arabidopsis thaliana) gene FLOWERING PROMOTING FACTOR1 (FPF1) and increased expression of RNase-S-like genes. FPF1 promotes floral development and enhances cell elongation, so down-regulation of FPF1-like genes might explain the phenotypes of HvOS2 overexpression lines. We present an extended model of the genetic pathways controlling vernalization-induced flowering in cereals, which describes the regulatory relationships between VRN1, OS2, and FPF1-like genes. Overall, these findings highlight differences and similarities between the vernalization responses of temperate cereals and the model plant Arabidopsis. PMID:20431086

  17. Identification and Characterization of Three Orchid MADS-Box Genes of the AP1/AGL9 Subfamily during Floral Transition1

    PubMed Central

    Yu, Hao; Goh, Chong Jin

    2000-01-01

    Gene expressions associated with in vitro floral transition in an orchid hybrid (Dendrobium grex Madame Thong-In) were investigated by differential display. One clone, orchid transitional growth related gene 7 (otg7), encoding a new MADS-box gene, was identified to be specifically expressed in the transitional shoot apical meristem (TSAM). Using this clone as a probe, three orchid MADS-box genes, DOMADS1, DOMADS2, and DOMADS3, were subsequently isolated from the TSAM cDNA library. Phylogenetic analyses show that DOMADS1 and DOMADS2 are new members of the AGL2 subfamily and SQUA subfamily, respectively. DOMADS3 contains the signature amino acids as with the members in the independent OSMADS1 subfamily separated from the AGL2 subfamily. All three of the DOMADS genes were expressed in the TSAM during floral transition and later in mature flowers. DOMADS1 RNA was uniformly expressed in both of the inflorescence meristem and the floral primordium and later localized in all of the floral organs. DOMADS2 showed a novel expression pattern that has not been previously characterized for any other MADS-box genes. DOMADS2 transcript was expressed early in the 6-week-old vegetative shoot apical meristem in which the obvious morphological change to floral development had yet to occur. It was expressed throughout the process of floral transition and later in the columns of mature flowers. The onset of DOMADS3 transcription was in the early TSAM at the stage before the differentiation of the first flower primordium. Later, DOMADS3 transcript was only detectable in the pedicel tissues. Our results suggest that the DOMADS genes play important roles in the process of floral transition. PMID:10938351

  18. Gain of function mutation in tobacco MADS box promoter switch on the expression of flowering class B genes converting sepals to petals.

    PubMed

    Mahajan, Monika; Yadav, Sudesh Kumar

    2014-02-01

    One mutant transgenic line displaying homeotic conversion of sepals to petals with other phenotypic aberrations was selected and characterized at molecular level. The increased transcript level of gene encoding anthocyanidin synthase and petal specific class B genes, GLOBOSA and DEFECIENS in sepals of mutant line may be responsible for its homeotic conversion to petaloid organs. While characterizing this mutant line for locus identification, T-DNA was found to be inserted in 3' untranslated region of promoter of class B MADS box gene, GLOBOSA. Here, CaMV 35S promoter of T-DNA might be deriving the expression of class B genes. PMID:24362510

  19. Floral homeotic genes were recruited from homologous MADS-box genes preexisting in the common ancestor of ferns and seed plants

    PubMed Central

    Münster, Thomas; Pahnke, Jens; Di Rosa, Alexandra; Kim, Jan T.; Martin, William; Saedler, Heinz; Theissen, Günter

    1997-01-01

    Flowers sensu lato are short, specialized axes bearing closely aggregated sporophylls. They are typical for seed plants (spermatophytes) and are prominent in flowering plants sensu stricto (angiosperms), where they often comprise an attractive perianth. There is evidence that spermatophytes evolved from gymnosperm-like plants with a fern-like mode of reproduction called progymnosperms. It seems plausible, therefore, that the stamens/carpels and pollen sacs/nucelli of spermatophytes are homologous to fern sporophylls and sporangia, respectively. However, the exact mode and molecular basis of early seed and flower evolution is not yet known. Comparing flower developmental control genes to their homologs from lower plants that do not flower may help to clarify the issue. We have isolated and characterized MADS-box genes expressed in gametophytes and sporophytes of the fern Ceratopteris. The data indicate that at least two different MADS-box genes homologous to floral homeotic genes existed in the last common ancestor of contemporary vascular plants, some descendants of which underwent multiple duplications and diversifications and were recruited into novel developmental networks during the evolution of floral organs. PMID:9122209

  20. Ectopic expression of two MADS box genes from orchid (Oncidium Gower Ramsey) and lily (Lilium longiflorum) alters flower transition and formation in Eustoma grandiflorum.

    PubMed

    Thiruvengadam, Muthu; Yang, Chang-Hsien

    2009-10-01

    Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and alpha-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks' duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation. PMID:19639326

  1. A MADS-box gene NtSVP regulates pedicel elongation by directly suppressing a KNAT1-like KNOX gene NtBPL in tobacco (Nicotiana tabacum L.)

    PubMed Central

    Wang, Di; Chen, Xiaobo; Zhang, Zenglin; Liu, Danmei; Song, Gaoyuan; Kong, Xingchen; Geng, Shuaifeng; Yang, Jiayue; Wang, Bingnan; Wu, Liang; Li, Aili; Mao, Long

    2015-01-01

    Optimal inflorescence architecture is important for plant reproductive success by affecting the ultimate number of flowers that set fruits and for plant competitiveness when interacting with biotic or abiotic conditions. The pedicel is one of the key contributors to inflorescence architecture diversity. To date, knowledge about the molecular mechanisms of pedicel development is derived from Arabidopsis. Not much is known regarding other plants. Here, an SVP family MADS-box gene, NtSVP, in tobacco (Nicotiana tabacum) that is required for pedicel elongation was identified. It is shown that knockdown of NtSVP by RNA interference (RNAi) caused elongated pedicels, while overexpression resulted in compact inflorescences with much shortened pedicels. Moreover, an Arabidopsis BREVIPEDECELLUS/KNAT1 homologue NtBP-Like (NtBPL) was significantly up-regulated in NtSVP-RNAi plants. Disruption of NtBPL decreased pedicel lengths and shortened cortex cells. Consistent with the presence of a CArG-box at the NtBPL promoter, the direct binding of NtSVP to the NtBPL promoter was demonstrated by yeast one-hybrid assay, electrophoretic mobility shift assay, and dual-luciferase assay, in which NtSVP may act as a repressor of NtBPL. Microarray analysis showed that down-regulation of NtBPL resulted in differential expression of genes associated with a number of hormone biogenesis and signalling genes such as those for auxin and gibberellin. These findings together suggest the function of a MADS-box transcription factor in plant pedicel development, probably via negative regulation of a BP-like class I KNOX gene. The present work thus postulates the conservation and divergence of the molecular regulatory pathways underlying the development of plant inflorescence architecture. PMID:26175352

  2. A MADS-box gene NtSVP regulates pedicel elongation by directly suppressing a KNAT1-like KNOX gene NtBPL in tobacco (Nicotiana tabacum L.).

    PubMed

    Wang, Di; Chen, Xiaobo; Zhang, Zenglin; Liu, Danmei; Song, Gaoyuan; Kong, Xingchen; Geng, Shuaifeng; Yang, Jiayue; Wang, Bingnan; Wu, Liang; Li, Aili; Mao, Long

    2015-10-01

    Optimal inflorescence architecture is important for plant reproductive success by affecting the ultimate number of flowers that set fruits and for plant competitiveness when interacting with biotic or abiotic conditions. The pedicel is one of the key contributors to inflorescence architecture diversity. To date, knowledge about the molecular mechanisms of pedicel development is derived from Arabidopsis. Not much is known regarding other plants. Here, an SVP family MADS-box gene, NtSVP, in tobacco (Nicotiana tabacum) that is required for pedicel elongation was identified. It is shown that knockdown of NtSVP by RNA interference (RNAi) caused elongated pedicels, while overexpression resulted in compact inflorescences with much shortened pedicels. Moreover, an Arabidopsis BREVIPEDECELLUS/KNAT1 homologue NtBP-Like (NtBPL) was significantly up-regulated in NtSVP-RNAi plants. Disruption of NtBPL decreased pedicel lengths and shortened cortex cells. Consistent with the presence of a CArG-box at the NtBPL promoter, the direct binding of NtSVP to the NtBPL promoter was demonstrated by yeast one-hybrid assay, electrophoretic mobility shift assay, and dual-luciferase assay, in which NtSVP may act as a repressor of NtBPL. Microarray analysis showed that down-regulation of NtBPL resulted in differential expression of genes associated with a number of hormone biogenesis and signalling genes such as those for auxin and gibberellin. These findings together suggest the function of a MADS-box transcription factor in plant pedicel development, probably via negative regulation of a BP-like class I KNOX gene. The present work thus postulates the conservation and divergence of the molecular regulatory pathways underlying the development of plant inflorescence architecture. PMID:26175352

  3. TOMATO AGAMOUS1 and ARLEQUIN/TOMATO AGAMOUS-LIKE1 MADS-box genes have redundant and divergent functions required for tomato reproductive development.

    PubMed

    Gimenez, Estela; Castañeda, Laura; Pineda, Benito; Pan, Irvin L; Moreno, Vicente; Angosto, Trinidad; Lozano, Rafael

    2016-07-01

    Within the tomato MADS-box gene family, TOMATO AGAMOUS1 (TAG1) and ARLEQUIN/TOMATO AGAMOUS LIKE1 (hereafter referred to as TAGL1) are, respectively, members of the euAG and PLE lineages of the AGAMOUS clade. They perform crucial functions specifying stamen and carpel development in the flower and controlling late fruit development. To gain insight into the roles of TAG1 and TAGL1 genes and to better understand their functional redundancy and diversification, we characterized single and double RNAi silencing lines of these genes and analyzed expression profiles of regulatory genes involved in reproductive development. Double RNAi lines did show cell abnormalities in stamens and carpels and produced extremely small fruit-like organs displaying some sepaloid features. Expression analyses indicated that TAG1 and TAGL1 act together to repress fourth whorl sepal development, most likely through the MACROCALYX gene. Results also proved that TAG1 and TAGL1 have diversified their functions in fruit development: while TAG1 controls placenta and seed formation, TAGL1 participates in cuticle development and lignin biosynthesis inhibition. It is noteworthy that both TAG1 and double RNAi plants lacked seed development due to abnormalities in pollen formation. This seedless phenotype was not associated with changes in the expression of B-class stamen identity genes Tomato MADS-box 6 and Tomato PISTILLATA observed in silencing lines, suggesting that other regulatory factors should participate in pollen formation. Taken together, results here reported support the idea that both redundant and divergent functions of TAG1 and TAGL1 genes are needed to control tomato reproductive development. PMID:27125648

  4. Transcriptional Activity of the MADS Box ARLEQUIN/TOMATO AGAMOUS-LIKE1 Gene Is Required for Cuticle Development of Tomato Fruit1

    PubMed Central

    Giménez, Estela; Dominguez, Eva; Pineda, Benito; Heredia, Antonio; Moreno, Vicente; Angosto, Trinidad

    2015-01-01

    Fruit development and ripening entail key biological and agronomic events, which ensure the appropriate formation and dispersal of seeds and determine productivity and yield quality traits. The MADS box gene ARLEQUIN/TOMATO AGAMOUS-LIKE1 (hereafter referred to as TAGL1) was reported as a key regulator of tomato (Solanum lycopersicum) reproductive development, mainly involved in flower development, early fruit development, and ripening. It is shown here that silencing of the TAGL1 gene (RNA interference lines) promotes significant changes affecting cuticle development, mainly a reduction of thickness and stiffness, as well as a significant decrease in the content of cuticle components (cutin, waxes, polysaccharides, and phenolic compounds). Accordingly, overexpression of TAGL1 significantly increased the amount of cuticle and most of its components while rendering a mechanically weak cuticle. Expression of the genes involved in cuticle biosynthesis agreed with the biochemical and biomechanical features of cuticles isolated from transgenic fruits; it also indicated that TAGL1 participates in the transcriptional control of cuticle development mediating the biosynthesis of cuticle components. Furthermore, cell morphology and the arrangement of epidermal cell layers, on whose activity cuticle formation depends, were altered when TAGL1 was either silenced or constitutively expressed, indicating that this transcription factor regulates cuticle development, probably through the biosynthetic activity of epidermal cells. Our results also support cuticle development as an integrated event in the fruit expansion and ripening processes that characterize fleshy-fruited species such as tomato. PMID:26019301

  5. X Linkage of AP3A, a Homolog of the Y-Linked MADS-Box Gene AP3Y in Silene latifolia and S. dioica

    PubMed Central

    Penny, Rebecca H.; Montgomery, Benjamin R.; Delph, Lynda F.

    2011-01-01

    Background The duplication of autosomal genes onto the Y chromosome may be an important element in the evolution of sexual dimorphism.A previous cytological study reported on a putative example of such a duplication event in a dioecious tribe of Silene (Caryophyllaceae): it was inferred that the Y-linked MADS-box gene AP3Y originated from a duplication of the reportedly autosomal orthologAP3A. However, a recent study, also using cytological methods, indicated that AP3A is X-linked in Silenelatifolia. Methodology/Principal Findings In this study, we hybridized S. latifolia and S. dioicato investigate whether the pattern of X linkage is consistent among distinct populations, occurs in both species, and is robust to genetic methods. We found inheritance patterns indicative of X linkage of AP3A in widely distributed populations of both species. Conclusions/Significance X linkage ofAP3A and Y linkage of AP3Yin both species indicates that the genes' ancestral progenitor resided on the autosomes that gave rise to the sex chromosomesand that neither gene has moved between chromosomes since species divergence.Consequently, our results do not support the contention that inter-chromosomal gene transfer occurred in the evolution of SlAP3Y from SlAP3A. PMID:21533056

  6. Evolution of AGL6-like MADS Box Genes in Grasses (Poaceae): Ovule Expression Is Ancient and Palea Expression Is New[W][OA

    PubMed Central

    Reinheimer, Renata; Kellogg, Elizabeth A.

    2009-01-01

    AGAMOUS-like6 (AGL6) genes encode MIKC-type MADS box transcription factors and are closely related to SEPALLATA and AP1/FUL-like genes. Here, we focus on the molecular evolution and expression of the AGL6-like genes in grasses. We have found that AGL6-like genes are expressed in ovules, lodicules (second whorl floral organs), paleas (putative first whorl floral organs), and floral meristems. Each of these expression domains was acquired at a different time in evolution, indicating that each represents a distinct function of the gene product and that the AGL6-like genes are pleiotropic. Expression in the inner integument of the ovule appears to be an ancient expression pattern corresponding to the expression of the gene in the megasporangium and integument in gymnosperms. Expression in floral meristems appears to have been acquired in the angiosperms and expression in second whorl organs in monocots. Early in grass evolution, AGL6-like orthologs acquired a new expression domain in the palea. Stamen expression is variable. Most grasses have a single AGL6-like gene (orthologous to the rice [Oryza sativa] gene MADS6). However, rice and other species of Oryza have a second copy (orthologous to rice MADS17) that appears to be the result of an ancient duplication. PMID:19749151

  7. The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10.

    PubMed

    Agarwal, Pooja; Verzi, Michael P; Nguyen, Thuyen; Hu, Jianxin; Ehlers, Melissa L; McCulley, David J; Xu, Shan-Mei; Dodou, Evdokia; Anderson, Joshua P; Wei, Maria L; Black, Brian L

    2011-06-01

    Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. Here, we establish a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. We identify a transcriptional enhancer of Mef2c that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. We demonstrate that Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, we show that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes. PMID:21610032

  8. Histone acetylation accompanied with promoter sequences displaying differential expression profiles of B-class MADS-box genes for phalaenopsis floral morphogenesis.

    PubMed

    Hsu, Chia-Chi; Wu, Pei-Shan; Chen, Tien-Chih; Yu, Chun-Wei; Tsai, Wen-Chieh; Wu, Keqiang; Wu, Wen-Luan; Chen, Wen-Huei; Chen, Hong-Hwa

    2014-01-01

    Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis. PMID:25501842

  9. Histone Acetylation Accompanied with Promoter Sequences Displaying Differential Expression Profiles of B-Class MADS-Box Genes for Phalaenopsis Floral Morphogenesis

    PubMed Central

    Hsu, Chia-Chi; Wu, Pei-Shan; Chen, Tien-Chih; Yu, Chun-Wei; Tsai, Wen-Chieh; Wu, Keqiang; Wu, Wen-Luan; Chen, Wen-Huei; Chen, Hong-Hwa

    2014-01-01

    Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis. PMID:25501842

  10. ODDSOC2 Is a MADS Box Floral Repressor That Is Down-Regulated by Vernalization in Temperate Cereals1[W][OA

    PubMed Central

    Greenup, Aaron G.; Sasani, Shahryar; Oliver, Sandra N.; Talbot, Mark J.; Dennis, Elizabeth S.; Hemming, Megan N.; Trevaskis, Ben

    2010-01-01

    In temperate cereals, such as wheat (Triticum aestivum) and barley (Hordeum vulgare), the transition to reproductive development can be accelerated by prolonged exposure to cold (vernalization). We examined the role of the grass-specific MADS box gene ODDSOC2 (OS2) in the vernalization response in cereals. The barley OS2 gene (HvOS2) is expressed in leaves and shoot apices but is repressed by vernalization. Vernalization represses OS2 independently of VERNALIZATION1 (VRN1) in a VRN1 deletion mutant of einkorn wheat (Triticum monococcum), but VRN1 is required to maintain down-regulation of OS2 in vernalized plants. Furthermore, barleys that carry active alleles of the VRN1 gene (HvVRN1) have reduced expression of HvOS2, suggesting that HvVRN1 down-regulates HvOS2 during development. Overexpression of HvOS2 delayed flowering and reduced spike, stem, and leaf length in transgenic barley plants. Plants overexpressing HvOS2 showed reduced expression of barley homologs of the Arabidopsis (Arabidopsis thaliana) gene FLOWERING PROMOTING FACTOR1 (FPF1) and increased expression of RNase-S-like genes. FPF1 promotes floral development and enhances cell elongation, so down-regulation of FPF1-like genes might explain the phenotypes of HvOS2 overexpression lines. We present an extended model of the genetic pathways controlling vernalization-induced flowering in cereals, which describes the regulatory relationships between VRN1, OS2, and FPF1-like genes. Overall, these findings highlight differences and similarities between the vernalization responses of temperate cereals and the model plant Arabidopsis. PMID:20431086

  11. ZmSOC1, a MADS-box transcription factor from Zea mays, promotes flowering in Arabidopsis.

    PubMed

    Zhao, Suzhou; Luo, Yanzhong; Zhang, Zhanlu; Xu, Miaoyun; Wang, Weibu; Zhao, Yangmin; Zhang, Lan; Fan, Yunliu; Wang, Lei

    2014-01-01

    Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis. PMID:25372944

  12. ZmSOC1, an MADS-Box Transcription Factor from Zea mays, Promotes Flowering in Arabidopsis

    PubMed Central

    Zhao, Suzhou; Luo, Yanzhong; Zhang, Zhanlu; Xu, Miaoyun; Wang, Weibu; Zhao, Yangmin; Zhang, Lan; Fan, Yunliu; Wang, Lei

    2014-01-01

    Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis. PMID:25372944

  13. Reciprocal Loss of CArG-Boxes and Auxin Response Elements Drives Expression Divergence of MPF2-Like MADS-Box Genes Controlling Calyx Inflation

    PubMed Central

    Khan, Muhammad Ramzan; Hu, Jinyong; Ali, Ghulam Muhammad

    2012-01-01

    Expression divergence is thought to be a hallmark of functional diversification between homologs post duplication. Modification in regulatory elements has been invoked to explain expression divergence after duplication for several MADS-box genes, however, verification of reciprocal loss of cis-regulatory elements is lacking in plants. Here, we report that the evolution of MPF2-like genes has entailed degenerative mutations in a core promoter CArG-box and an auxin response factor (ARF) binding element in the large 1st intron in the coding region. Previously, MPF2-like genes were duplicated into MPF2-like-A and -B through genome duplication in Withania and Tubocapsicum (Withaninae). The calyx of Withania grows exorbitantly after pollination unlike Tubocapsicum, where it degenerates. Besides inflated calyx syndrome formation, MPF2-like transcription factors are implicated in functions both during the vegetative and reproductive development as well as in phase transition. MPF2-like-A of Withania (WSA206) is strongly expressed in sepals, while MPF2-like-B (WSB206) is not. Interestingly, their combined expression patterns seem to replicate the pattern of their closely related hypothetical progenitors from Vassobia and Physalis. Using phylogenetic shadowing, site-directed mutagenesis and motif swapping, we could show that the loss of a conserved CArG-box in MPF2-like-B of Withania is responsible for impeding its expression in sepals. Conversely, loss of an ARE in MPF2-like-A relaxed the constraint on expression in sepals. Thus, the ARE is an active suppressor of MPF2-like gene expression in sepals, which in contrast is activated via the CArG-box. The observed expression divergence in MPF2-like genes due to reciprocal loss of cis-regulatory elements has added to genetic and phenotypic variations in the Withaninae and enhanced the potential of natural selection for the adaptive evolution of ICS. Moreover, these results provide insight into the interplay of floral

  14. Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein-Protein Interactions.

    PubMed

    Bartlett, Madelaine; Thompson, Beth; Brabazon, Holly; Del Gizzi, Robert; Zhang, Thompson; Whipple, Clinton

    2016-06-01

    Protein-protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PI(L)) and AP3-like (AP3(L)) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PI(L) protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PI(L) homodimerization is an anomaly or indicative of broader trends, we characterized PI(L) dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PI(L) homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PI(L) homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PI(L) protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PI(L) dimerization activity. Furthermore, ectopic expression of a Joinvillea PI(L) homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner. PMID:26908583

  15. Aspergillus fumigatus MADS-Box Transcription Factor rlmA Is Required for Regulation of the Cell Wall Integrity and Virulence

    PubMed Central

    Rocha, Marina Campos; Fabri, João Henrique Tadini Marilhano; Franco de Godoy, Krissia; Alves de Castro, Patrícia; Hori, Juliana Issa; Ferreira da Cunha, Anderson; Arentshorst, Mark; Ram, Arthur F. J.; van den Hondel, Cees A. M. J. J.; Goldman, Gustavo Henrique; Malavazi, Iran

    2016-01-01

    The Cell Wall Integrity (CWI) pathway is the primary signaling cascade that controls the de novo synthesis of the fungal cell wall, and in Saccharomyces cerevisiae this event is highly dependent on the RLM1 transcription factor. Here, we investigated the function of RlmA in the fungal pathogen Aspergillus fumigatus. We show that the ΔrlmA strain exhibits an altered cell wall organization in addition to defects related to vegetative growth and tolerance to cell wall-perturbing agents. A genetic analysis indicated that rlmA is positioned downstream of the pkcA and mpkA genes in the CWI pathway. As a consequence, rlmA loss-of-function leads to the altered expression of genes encoding cell wall-related proteins. RlmA positively regulates the phosphorylation of MpkA and is induced at both protein and transcriptional levels during cell wall stress. The rlmA was also involved in tolerance to oxidative damage and transcriptional regulation of genes related to oxidative stress adaptation. Moreover, the ΔrlmA strain had attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Our results suggest that RlmA functions as a transcription factor in the A. fumigatus CWI pathway, acting downstream of PkcA-MpkA signaling and contributing to the virulence of this fungus. PMID:27473315

  16. Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein–Protein Interactions

    PubMed Central

    Bartlett, Madelaine; Thompson, Beth; Brabazon, Holly; Del Gizzi, Robert; Zhang, Thompson; Whipple, Clinton

    2016-01-01

    Protein–protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PIL) and AP3-like (AP3L) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PIL protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PIL homodimerization is an anomaly or indicative of broader trends, we characterized PIL dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PIL homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PIL homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PIL protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PIL dimerization activity. Furthermore, ectopic expression of a Joinvillea PIL homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner. PMID:26908583

  17. The Analysis of the Inflorescence miRNome of the Orchid Orchis italica Reveals a DEF-Like MADS-Box Gene as a New miRNA Target

    PubMed Central

    Aceto, Serena; Sica, Maria; De Paolo, Sofia; D'Argenio, Valeria; Cantiello, Piergiuseppe; Salvatore, Francesco; Gaudio, Luciano

    2014-01-01

    Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the “orchid code” theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs. PMID:24832004

  18. Protein expression and characterization of SEP3 from Arabidopsis thaliana.

    PubMed

    Shi, Q; Zhou, J; Wang, P; Lin, X; Xu, Y

    2015-01-01

    SEPALLATA (SEP) MADS-box genes play crucial roles in the regulation of floral growth and development. They are required for the specification of sepals, petals, stamens, and carpels as well as for floral determinacy. SEPs perform their functions through the formation of homo- or hetero-polymers, which are the molecular basis of floral quartets. In vitro assays indicated that SEP3 forms a tetramer after binding to DNA, but it is unclear whether DNA binding induces the tetramer, because SEP3 is often reported to form a dimer. Here, we analyzed the oligomeric status of SEP3 domains in the absence of the DNA-binding MADS-box domain. The truncated SEP3 was constructed as a fusion protein and expressed in prokaryotic cells. The purified protein fragment displayed as a tetramer in the size exclusion chromatographic column, and a glutaraldehyde cross-linking assay demonstrated that the protein contained a dimer unit. Yeast two-hybrid tests further verified that the fragments form homologous polymers in vivo, and that the K domain is involved in tetramer formation. Current results imply that the SEP3 protein regulates the formation of flower meristems using the tetramer as a unit, and that the DNA-binding MADS-box is dispensable for polymer formation. The C-terminal region does not contribute to homo-tetramer formation, but it may be reserved to glue other proteins. PMID:26505403

  19. Evolutionary Conserved Positions Define Protein Conformational Diversity.

    PubMed

    Saldaño, Tadeo E; Monzon, Alexander M; Parisi, Gustavo; Fernandez-Alberti, Sebastian

    2016-03-01

    Conformational diversity of the native state plays a central role in modulating protein function. The selection paradigm sustains that different ligands shift the conformational equilibrium through their binding to highest-affinity conformers. Intramolecular vibrational dynamics associated to each conformation should guarantee conformational transitions, which due to its importance, could possibly be associated with evolutionary conserved traits. Normal mode analysis, based on a coarse-grained model of the protein, can provide the required information to explore these features. Herein, we present a novel procedure to identify key positions sustaining the conformational diversity associated to ligand binding. The method is applied to an adequate refined dataset of 188 paired protein structures in their bound and unbound forms. Firstly, normal modes most involved in the conformational change are selected according to their corresponding overlap with structural distortions introduced by ligand binding. The subspace defined by these modes is used to analyze the effect of simulated point mutations on preserving the conformational diversity of the protein. We find a negative correlation between the effects of mutations on these normal mode subspaces associated to ligand-binding and position-specific evolutionary conservations obtained from multiple sequence-structure alignments. Positions whose mutations are found to alter the most these subspaces are defined as key positions, that is, dynamically important residues that mediate the ligand-binding conformational change. These positions are shown to be evolutionary conserved, mostly buried aliphatic residues localized in regular structural regions of the protein like β-sheets and α-helix. PMID:27008419

  20. Evolutionary Conserved Positions Define Protein Conformational Diversity

    PubMed Central

    Saldaño, Tadeo E.; Monzon, Alexander M.; Parisi, Gustavo; Fernandez-Alberti, Sebastian

    2016-01-01

    Conformational diversity of the native state plays a central role in modulating protein function. The selection paradigm sustains that different ligands shift the conformational equilibrium through their binding to highest-affinity conformers. Intramolecular vibrational dynamics associated to each conformation should guarantee conformational transitions, which due to its importance, could possibly be associated with evolutionary conserved traits. Normal mode analysis, based on a coarse-grained model of the protein, can provide the required information to explore these features. Herein, we present a novel procedure to identify key positions sustaining the conformational diversity associated to ligand binding. The method is applied to an adequate refined dataset of 188 paired protein structures in their bound and unbound forms. Firstly, normal modes most involved in the conformational change are selected according to their corresponding overlap with structural distortions introduced by ligand binding. The subspace defined by these modes is used to analyze the effect of simulated point mutations on preserving the conformational diversity of the protein. We find a negative correlation between the effects of mutations on these normal mode subspaces associated to ligand-binding and position-specific evolutionary conservations obtained from multiple sequence-structure alignments. Positions whose mutations are found to alter the most these subspaces are defined as key positions, that is, dynamically important residues that mediate the ligand-binding conformational change. These positions are shown to be evolutionary conserved, mostly buried aliphatic residues localized in regular structural regions of the protein like β-sheets and α-helix. PMID:27008419

  1. The MADS domain protein DIANA acts together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules.

    PubMed

    Bemer, Marian; Wolters-Arts, Mieke; Grossniklaus, Ueli; Angenent, Gerco C

    2008-08-01

    MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein-beta-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt. PMID:18713950

  2. The MADS Domain Protein DIANA Acts Together with AGAMOUS-LIKE80 to Specify the Central Cell in Arabidopsis Ovules[W

    PubMed Central

    Bemer, Marian; Wolters-Arts, Mieke; Grossniklaus, Ueli; Angenent, Gerco C.

    2008-01-01

    MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein–β-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt. PMID:18713950

  3. Proteins interacting with cloning scars: a source of false positive protein-protein interactions

    PubMed Central

    Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

    2015-01-01

    A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

  4. Positive modulator of bone morphogenic protein-2

    DOEpatents

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua; Takahashi, Kazuyuki

    2009-01-27

    Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

  5. A microdomain for protein secretion in Gram-positive bacteria.

    PubMed

    Rosch, Jason; Caparon, Michael

    2004-06-01

    Gram-positive bacteria face unique challenges in generating biologically active conformations for their exported proteins because they lack a dedicated compartment for folding secreted polypeptides. We have discovered that protein secretion by way of the general secretory (Sec) pathway in the important human pathogen Streptococcus pyogenes proceeds through a single microdomain. Unlike other mechanisms for asymmetry involving the Sec pathway, proteins destined for secretion are targeted to a single locus distal to either cell pole that has specialized to contain the Sec translocons. This subcellular organization may represent a paradigm for secretion common to other Gram-positive pathogens with profound implications for pathogenesis. PMID:15178803

  6. RCAN 1 and 3 proteins regulate thymic positive selection.

    PubMed

    Serrano-Candelas, Eva; Alemán-Muench, Germán; Solé-Sánchez, Sònia; Aubareda, Anna; Martínez-Høyer, Sergio; Adán, Jaume; Aranguren-Ibáñez, Álvaro; Pritchard, Melanie A; Soldevila, Gloria; Pérez-Riba, Mercè

    2015-05-01

    Cooperation between calcineurin (CN)-NFATc and RAF-MEK-ERK signaling pathways is essential in thymocyte positive selection. It is known that the Regulators of Calcineurin (RCAN) proteins can act either facilitating or suppressing CN-dependent signaling events. Here, we show that RCAN genes are expressed in lymphoid tissues, and address the role of RCAN proteins in T cell development. Overexpression of human RCAN3 and RCAN1 can modulate T cell development by increasing positive selection-related surface markers, as well as the "Erk(hi) competence state" in double positive thymocytes, a characteristic molecular signature of positive selection, without affecting CN activity. We also found that RCAN1/3 interact with RAF kinases and CN in a non-exclusive manner. Our data suggests that the balance of RCAN interactions with CN and/or RAF kinases may influence T cell positive selection. PMID:25783055

  7. Protein secretion and surface display in Gram-positive bacteria

    PubMed Central

    Schneewind, Olaf; Missiakas, Dominique M.

    2012-01-01

    The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

  8. MADS-box out of the black box

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The compelling elegance of using genome-wide scans to detect the signature of selection is difficult to resist, but is countered by the low demonstrated efficacy of pinpointing the actual genes and traits that are the targets of selection in non-model species. While the difficulty of going from a s...

  9. DORMANCY ASSOCIATED MADS-BOX genes: a review

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DAM genes encode transcription factors suspected of regulating bud dormancy in numerous perennials. This chapter discusses the functional genetics and regulation of these genes and summarizes the evidence that these transcription factors play a central role in seasonal bud dormancy induction and mai...

  10. Heparin modifies the immunogenicity of positively charged proteins

    PubMed Central

    Chudasama, Shalini L.; Espinasse, Benjamin; Hwang, Fred; Qi, Rui; Joglekar, Manali; Afonina, Galyna; Wiesner, Mark R.; Welsby, Ian J.; Ortel, Thomas L.

    2010-01-01

    The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our studies indicate that PRT/H and lysozyme/H, like PF4/H, show H-dependent binding over a range of H concentrations and that formation of complexes occurs at distinct stoichiometric ratios. We show that protein/H complexes are capable of eliciting high-titer antigen-specific antibodies in a murine immunization model and that PRT/H antibodies occur in patients undergoing cardiopulmonary bypass surgery. Finally, our studies indicate that protein/H complexes, but not uncomplexed protein, directly activate dendritic cells in vitro leading to interleukin-12 release. Taken together, these studies indicate that H significantly alters the biophysical and biologic properties of positively charged compounds through formation of multimolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo. PMID:20852126

  11. Positive Selection and Centrality in the Yeast and Fly Protein-Protein Interaction Networks

    PubMed Central

    Chakraborty, Sandip

    2016-01-01

    Proteins within a molecular network are expected to be subject to different selective pressures depending on their relative hierarchical positions. However, it is not obvious what genes within a network should be more likely to evolve under positive selection. On one hand, only mutations at genes with a relatively high degree of control over adaptive phenotypes (such as those encoding highly connected proteins) are expected to be “seen” by natural selection. On the other hand, a high degree of pleiotropy at these genes is expected to hinder adaptation. Previous analyses of the human protein-protein interaction network have shown that genes under long-term, recurrent positive selection (as inferred from interspecific comparisons) tend to act at the periphery of the network. It is unknown, however, whether these trends apply to other organisms. Here, we show that long-term positive selection has preferentially targeted the periphery of the yeast interactome. Conversely, in flies, genes under positive selection encode significantly more connected and central proteins. These observations are not due to covariation of genes' adaptability and centrality with confounding factors. Therefore, the distribution of proteins encoded by genes under recurrent positive selection across protein-protein interaction networks varies from one species to another. PMID:27119079

  12. Werner syndrome protein positively regulates XRCC4-like factor transcription

    PubMed Central

    LIU, DONGYUN; DENG, XIAOLI; YUAN, CHONGZHEN; CHEN, LIN; CONG, YUSHENG; XU, XINGZHI

    2014-01-01

    XRCC4-like factor (XLF) is involved in non-homologous end joining-mediated repair of DNA double-strand breaks (DSBs). Mutations in the WRN gene results in the development of Werner syndrome (WS), a rare autosomal recessive disorder characterized by premature ageing and genome instability. In the present study, it was identified that XLF protein levels were lower in WRN-deficient fibroblasts, compared with normal fibroblasts. Depletion of WRN in HeLa cells led to a decrease of XLF mRNA and its promoter activity. Chromatin immunoprecipitation assays demonstrated that WRN was associated with the XLF promoter. Depletion of XLF in normal human fibroblasts increased the percentage of β-galactosidase (β-gal) staining-positive cells, indicating acceleration in cellular senescence. Taken together, the results suggest that XLF is a transcriptional target of WRN and may be involved in the regulation of cellular senescence. PMID:24626809

  13. Automated design of the surface positions of protein helices.

    PubMed Central

    Dahiyat, B. I.; Gordon, D. B.; Mayo, S. L.

    1997-01-01

    Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions. PMID:9194194

  14. Getting into position: the catalytic mechanisms of protein ubiquitylation.

    PubMed Central

    Passmore, Lori A; Barford, David

    2004-01-01

    The role of protein ubiquitylation in the control of diverse cellular pathways has recently gained widespread attention. Ubiquitylation not only directs the targeted destruction of tagged proteins by the 26 S proteasome, but it also modulates protein activities, protein-protein interactions and subcellular localization. An understanding of the components involved in protein ubiquitylation (E1s, E2s and E3s) is essential to understand how specificity and regulation are conferred upon these pathways. Much of what we know about the catalytic mechanisms of protein ubiquitylation comes from structural studies of the proteins involved in this process. Indeed, structures of ubiquitin-activating enzymes (E1s) and ubiquitin-conjugating enzymes (E2s) have provided insight into their mechanistic details. E3s (ubiquitin ligases) contain most of the substrate specificity and regulatory elements required for protein ubiquitylation. Although several E3 structures are available, the specific mechanistic role of E3s is still unclear. This review will discuss the different types of ubiquitin signals and how they are generated. Recent advances in the field of protein ubiquitylation will be examined, including the mechanisms of E1, E2 and E3. In particular, we discuss the complexity of molecular recognition required to impose selectivity on substrate selection and topology of poly-ubiquitin chains. PMID:14998368

  15. NMR spectroscopy of proteins encapsulated in a positively charged surfactant.

    PubMed

    Lefebvre, Brian G; Liu, Weixia; Peterson, Ronald W; Valentine, Kathleen G; Wand, A Joshua

    2005-07-01

    Traditionally, large proteins, aggregation-prone proteins, and membrane proteins have been difficult to examine by modern multinuclear and multidimensional solution NMR spectroscopy. A major limitation presented by these protein systems is that their slow molecular reorientation compromises many aspects of the more powerful solution NMR methods. Several approaches have emerged to deal with the various spectroscopic difficulties arising from slow molecular reorientation. One of these takes the approach of actively seeking to increase the effective rate of molecular reorientation by encapsulating the protein of interest within the protective shell of a reverse micelle and dissolving the resulting particle in a low viscosity fluid. Since the encapsulation is largely driven by electrostatic interactions, the preparation of samples of acidic proteins suitable for NMR spectroscopy has been problematic owing to the paucity of suitable cationic surfactants. Here, it is shown that the cationic surfactant CTAB may be used to prepare samples of encapsulated anionic proteins dissolved in low viscosity solvents. In a more subtle application, it is further shown that this surfactant can be employed to encapsulate a highly basic protein, which is completely denatured upon encapsulation using an anionic surfactant. PMID:15949753

  16. The tomato floral homeotic protein FBP1-like gene, SlGLO1, plays key roles in petal and stamen development.

    PubMed

    Guo, Xuhu; Hu, Zongli; Yin, Wencheng; Yu, Xiaohui; Zhu, Zhiguo; Zhang, Jianling; Chen, Guoping

    2016-01-01

    MADS-box transcription factors play important role in plant growth and development, especially floral organ identities. In our study, a MADS-box gene SlGLO1- tomato floral homeotic protein FBP1-like gene was isolated. Its tissue-specific expression profile analysis showed that SlGLO1 was highly expressed in petals and stamens. RNAi (RNA interference) repression of SlGLO1 resulted in floral organ abnormal phenotypes, including green petals with shorter size, and aberrant carpelloid stamens. SlGLO1-silenced lines are male sterile. Total chlorophyll content was increased and chlorophyll biosynthetic genes were significantly up-regulated in SlGLO1-silenced petals and stamens. Furthermore, B-class genes expression analysis indicated that the repressed function of SlGLO1 led to the enhanced expression of TAP3 and the down-regulation of TPI in the petals and stamens, while the expression of TM6 was reduced in petals and increased in stamens and carpels of SlGLO1-RNAi plants. Additionally, pollen grains of transgenic lines were aberrant and failed to germinate and tomato pollen-specific genes were down-regulated by more than 90% in SlGLO1-silenced lines. These results suggest that SlGLO1 plays important role in regulating plant floral organ and pollen development in tomato. PMID:26842499

  17. The tomato floral homeotic protein FBP1-like gene, SlGLO1, plays key roles in petal and stamen development

    PubMed Central

    Guo, Xuhu; Hu, Zongli; Yin, Wencheng; Yu, Xiaohui; Zhu, Zhiguo; Zhang, Jianling; Chen, Guoping

    2016-01-01

    MADS-box transcription factors play important role in plant growth and development, especially floral organ identities. In our study, a MADS-box gene SlGLO1- tomato floral homeotic protein FBP1-like gene was isolated. Its tissue-specific expression profile analysis showed that SlGLO1 was highly expressed in petals and stamens. RNAi (RNA interference) repression of SlGLO1 resulted in floral organ abnormal phenotypes, including green petals with shorter size, and aberrant carpelloid stamens. SlGLO1-silenced lines are male sterile. Total chlorophyll content was increased and chlorophyll biosynthetic genes were significantly up-regulated in SlGLO1-silenced petals and stamens. Furthermore, B-class genes expression analysis indicated that the repressed function of SlGLO1 led to the enhanced expression of TAP3 and the down-regulation of TPI in the petals and stamens, while the expression of TM6 was reduced in petals and increased in stamens and carpels of SlGLO1-RNAi plants. Additionally, pollen grains of transgenic lines were aberrant and failed to germinate and tomato pollen-specific genes were down-regulated by more than 90% in SlGLO1-silenced lines. These results suggest that SlGLO1 plays important role in regulating plant floral organ and pollen development in tomato. PMID:26842499

  18. Rapid visualization of hydrogen positions in neutron protein crystallography structures

    SciTech Connect

    Blakeley, Matthew P.; Meilleur, Flora; Myles, Dean A A; Weiss, Kevin L; Munshi, Parthapratim; Shang-Lin, Chung

    2012-01-01

    Neutron crystallography is a powerful technique to visualize experimentally the position of light atoms, including hydrogen and its isotope deuterium. Over the last several years, structural biologists have shown an increasing interest for the technique as it uniquely complements X-ray crystallographic data by revealing the position of hydrogen/deuterium atoms in macromolecules. With this regained interest, access to macromolecule neutron crystallography beam lines is becoming a limiting step. In this report we show that rapid data collection could be a valuable alternative to long data collection time when appropriate. Comparison of perdeuterated Rubredoxin structures refined against neutron data sets collected over hours and up to five days shows that rapid neutron data collection in just 14 hours is sufficient to provide the position of 262 hydrogen positions atoms without ambiguity.

  19. Continuum electrostatic approach for evaluating positions and interactions of proteins in a bilayer membrane.

    PubMed

    Supunyabut, Chirayut; Fuklang, Sunit; Sompornpisut, Pornthep

    2015-06-01

    Orientations of proteins in the membranes are crucial to their function and stability. Unfortunately the exact positions of these proteins in the lipid bilayer are mostly undetermined. Here, the spatial orientation of membrane proteins within the lipid membrane was evaluated using a Poisson-Boltzmann solvent continuum approach to calculate the electrostatic free energy of the protein solvation at various orientations in an implicit bilayer. The solvation energy was obtained by computing the difference in electrostatic energies of the protein in water and in lipid/water environments, treating each as an implicit solvent model. The optimal position of transmembrane proteins (TMP) in a lipid bilayer is identified by the minimum in the "downhill" pathway of the solvation energy landscape. The energy landscape pattern was considerably conserved in various TMP classes. Evaluation of the position of 1060 membrane proteins from the orientations of proteins in membranes (OPM) database revealed that most of the polytopic and β-barrel proteins were in good agreement with those of the OPM database. The study provides a useful scheme for estimating the membrane solvation energy made by lipid-exposed amino acids in membrane proteins. In addition, our results tested with the bacterial potassium channel model demonstrated the potential usefulness of the approach in assessing the quality of membrane protein models. The present approach should be applicable for constructing transmembrane proteins-lipid configuration suitable for membrane protein simulations and will have utility for the structural modeling of membrane proteins. PMID:25912455

  20. Positive feedback of protein kinase C proteolytic activation during apoptosis.

    PubMed Central

    Leverrier, Sabrina; Vallentin, Alice; Joubert, Dominique

    2002-01-01

    In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation. PMID:12238950

  1. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  2. Identifying relevant positions in proteins by Critical Variable Selection.

    PubMed

    Grigolon, Silvia; Franz, Silvio; Marsili, Matteo

    2016-06-21

    Evolution in its course has found a variety of solutions to the same optimisation problem. The advent of high-throughput genomic sequencing has made available extensive data from which, in principle, one can infer the underlying structure on which biological functions rely. In this paper, we present a new method aimed at the extraction of sites encoding structural and functional properties from a set of protein primary sequences, namely a multiple sequence alignment. The method, called critical variable selection, is based on the idea that subsets of relevant sites correspond to subsequences that occur with a particularly broad frequency distribution in the dataset. By applying this algorithm to in silico sequences, to the response regulator receiver and to the voltage sensor domain of ion channels, we show that this procedure recovers not only the information encoded in single site statistics and pairwise correlations but also captures dependencies going beyond pairwise correlations. The method proposed here is complementary to statistical coupling analysis, in that the most relevant sites predicted by the two methods differ markedly. We find robust and consistent results for datasets as small as few hundred sequences that reveal a hidden hierarchy of sites that are consistent with the present knowledge on biologically relevant sites and evolutionary dynamics. This suggests that critical variable selection is capable of identifying a core of sites encoding functional and structural information in a multiple sequence alignment. PMID:26974515

  3. Positioning.

    ERIC Educational Resources Information Center

    Conone, Ruth M.

    The key to positioning is the creation of a clear benefit image in the consumer's mind. One positioning strategy is creating in the prospect's mind a position that takes into consideration the company's or agency's strengths and weaknesses as well as those of its competitors. Another strategy is to gain entry into a position ladder owned by…

  4. Genetic Reporter System for Positioning of Proteins at the Bacterial Pole

    PubMed Central

    Fixen, Kathryn R.; Janakiraman, Anuradha; Garrity, Sean; Slade, Daniel J.; Gray, Andrew N.; Karahan, Nilay; Hochschild, Ann; Goldberg, Marcia B.

    2012-01-01

    ABSTRACT Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells. PMID:22375072

  5. OPM database and PPM web server: resources for positioning of proteins in membranes

    PubMed Central

    Lomize, Mikhail A.; Pogozheva, Irina D.; Joo, Hyeon; Mosberg, Henry I.; Lomize, Andrei L.

    2012-01-01

    The Orientations of Proteins in Membranes (OPM) database is a curated web resource that provides spatial positions of membrane-bound peptides and proteins of known three-dimensional structure in the lipid bilayer, together with their structural classification, topology and intracellular localization. OPM currently contains more than 1200 transmembrane and peripheral proteins and peptides from approximately 350 organisms that represent approximately 3800 Protein Data Bank entries. Proteins are classified into classes, superfamilies and families and assigned to 21 distinct membrane types. Spatial positions of proteins with respect to the lipid bilayer are optimized by the PPM 2.0 method that accounts for the hydrophobic, hydrogen bonding and electrostatic interactions of the proteins with the anisotropic water-lipid environment described by the dielectric constant and hydrogen-bonding profiles. The OPM database is freely accessible at http://opm.phar.umich.edu. Data can be sorted, searched or retrieved using the hierarchical classification, source organism, localization in different types of membranes. The database offers downloadable coordinates of proteins and peptides with membrane boundaries. A gallery of protein images and several visualization tools are provided. The database is supplemented by the PPM server (http://opm.phar.umich.edu/server.php) which can be used for calculating spatial positions in membranes of newly determined proteins structures or theoretical models. PMID:21890895

  6. Dietary protein distribution positively influences 24-h muscle protein synthesis in healthy adults.

    PubMed

    Mamerow, Madonna M; Mettler, Joni A; English, Kirk L; Casperson, Shanon L; Arentson-Lantz, Emily; Sheffield-Moore, Melinda; Layman, Donald K; Paddon-Jones, Douglas

    2014-06-01

    The RDA for protein describes the quantity that should be consumed daily to meet population needs and to prevent deficiency. Protein consumption in many countries exceeds the RDA; however, intake is often skewed toward the evening meal, whereas breakfast is typically carbohydrate rich and low in protein. We examined the effects of protein distribution on 24-h skeletal muscle protein synthesis in healthy adult men and women (n = 8; age: 36.9 ± 3.1 y; BMI: 25.7 ± 0.8 kg/m2). By using a 7-d crossover feeding design with a 30-d washout period, we measured changes in muscle protein synthesis in response to isoenergetic and isonitrogenous diets with protein at breakfast, lunch, and dinner distributed evenly (EVEN; 31.5 ± 1.3, 29.9 ± 1.6, and 32.7 ± 1.6 g protein, respectively) or skewed (SKEW; 10.7 ± 0.8, 16.0 ± 0.5, and 63.4 ± 3.7 g protein, respectively). Over 24-h periods on days 1 and 7, venous blood samples and vastus lateralis muscle biopsy samples were obtained during primed (2.0 μmol/kg) constant infusion [0.06 μmol/(kg⋅min)] of l-[ring-(13)C6]phenylalanine. The 24-h mixed muscle protein fractional synthesis rate was 25% higher in the EVEN (0.075 ± 0.006%/h) vs. the SKEW (0.056 ± 0.006%/h) protein distribution groups (P = 0.003). This pattern was maintained after 7 d of habituation to each diet (EVEN vs. SKEW: 0.077 ± 0.006 vs. 0.056 ± 0.006%/h; P = 0.001). The consumption of a moderate amount of protein at each meal stimulated 24-h muscle protein synthesis more effectively than skewing protein intake toward the evening meal. PMID:24477298

  7. [The genetic polymorphism of the blood proteins in RID-positive reacting cows].

    PubMed

    Kivan, M

    1991-01-01

    Cattle herd of Black-and-White, Red and Simmental breeds having positive or negative RID-reaction to leucosis was studied as to the polymorphism of serum blood proteins in three loci: Tf, Am and Cp. One system of polymorphic proteins has been determined as having a higher concentration of homozygotes (Tf) and another one as having a higher concentration of heterozygotes (Am) within one and the same herd among animals with the positive RID-reaction. PMID:1796504

  8. The Genomic Landscape of Position Effects on Protein Expression Level and Noise in Yeast.

    PubMed

    Chen, Xiaoshu; Zhang, Jianzhi

    2016-05-25

    Position effect, the influence of the chromosomal location of a gene on its activity, is a fundamental property of the genome. By placing a GFP gene cassette at 482 different locations across all chromosomes in budding yeast, we quantified the position effects on protein expression level and noise at the genomic scale. The position effects are significant, altering the mean protein expression level by up to 15 times and expression noise by up to 20 times. DNA replication timing, 3D chromosomal conformation, and several histone modifications are major covariates of position effects. Essential genes are enriched in genomic regions with inherently low expression noise, supporting the hypothesis that chromosomal clustering of essential genes results from selection against their expressional stochasticity. Position effects exhibit significant interactions with promoters. Together, our results suggest that position effects have shaped the evolution of chromosome organization and should inform future genome engineering efforts. PMID:27185547

  9. Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins

    PubMed Central

    2010-01-01

    Background Reproductive proteins are central to the continuation of all mammalian species. The evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. Positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. However, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. We have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia. Results We show that selective pressures on reproductive proteins are highly varied. Of the 10 genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. Our analysis of SP56 and Col1a1 are entirely novel and the results show positively selected sites present in each gene. Our findings for the Col1a1 gene are suggestive of a link between positive selection and severe disease type. We find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality. Conclusion Our in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. In those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. This work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. We have shown that positive selection and disease are closely linked in the Col1a1 gene. PMID:20149245

  10. Soluble expression of proteins correlates with a lack of positively-charged surface

    NASA Astrophysics Data System (ADS)

    Chan, Pedro; Curtis, Robin A.; Warwicker, Jim

    2013-11-01

    Prediction of protein solubility is gaining importance with the growing use of protein molecules as therapeutics, and ongoing requirements for high level expression. We have investigated protein surface features that correlate with insolubility. Non-polar surface patches associate to some degree with insolubility, but this is far exceeded by the association with positively-charged patches. Negatively-charged patches do not separate insoluble/soluble subsets. The separation of soluble and insoluble subsets by positive charge clustering (area under the curve for a ROC plot is 0.85) has a striking parallel with the separation that delineates nucleic acid-binding proteins, although most of the insoluble dataset are not known to bind nucleic acid. Additionally, these basic patches are enriched for arginine, relative to lysine. The results are discussed in the context of expression systems and downstream processing, contributing to a view of protein solubility in which the molecular interactions of charged groups are far from equivalent.

  11. Protein domain networks: Scale-free mixing of positive and negative exponents

    NASA Astrophysics Data System (ADS)

    Nacher, J. C.; Hayashida, M.; Akutsu, T.

    2006-07-01

    Many biological studies have been focused on the study of proteins, since proteins are essential for most cell functions. Although proteins are unique, they share certain common properties. For example, well-defined regions within a protein can fold independently from the rest of the protein and have their own function. They are called protein domains, and served as protein building blocks. In this article, we present a theoretical model for studying the protein domain networks, where one node of the network corresponds to one protein and two proteins are connected if they contain the same domain. The resulting distribution of nodes with a given degree, k, shows not only a power-law with negative exponent γ=-1, but it resembles the superposition of two power-law functions, one with a negative exponent and another with a positive exponent β=1. We call this distribution pattern “ scale-free mixing”. To explain the emergence of this superposition of power-laws, we propose a basic model with two main components: (1) mutation and (2) duplication of domains. Precisely, duplication gives rise to complete subgraphs (i.e., cliques) on the network, thus for several values of k a large number of nodes with degree k is produced, which explains the positive power-law branch of the degree distribution. In order to compare our model with experimental data, we generate protein domain networks with data from the UniProt Knowledgebase-Swissprot database for protein sequences and using InterPro, Pfam and Smart for domain databases. Our results indicate that the signal of this scale-free mixing pattern is also observed in the experimental data and it is conserved among organisms as Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens.

  12. Protein Normal Modes: Calculations of Amide Band Positions and Infrared Intensities for Helical Polypeptides and Proteins

    NASA Astrophysics Data System (ADS)

    Reisdorf, William Charles, Jr.

    To understand protein function requires input from a wide variety of techniques. The ability of diffraction and magnetic resonance studies to provide structural models with high atomic resolution are particularly crucial. Increasingly, information on protein dynamics is also sought. Vibrational spectroscopy can contribute information on the conformations of peptides and proteins, but the complexity of proteins makes interpretation of their spectra difficult. For this reason, computational models of protein vibrational modes are expected to play a major role in aiding our comprehension of protein dynamics. A set of FORTRAN programs referred to as 'POLYPEP' has been designed for computing the frequencies (eigenvalues) and normal modes (eigenvectors) of polypeptides of any structure. Model structures can be generated by specifying the backbone dihedral angles and using standard peptide group geometry. Alternatively one can use cartesian coordinates from experimental structures as input. The side chains are modeled as point masses, except for cysteine residues which may participate in disulfide linkages. Hydrogen bonding interactions between backbone groups are also included. The force fields adopted have been developed and refined to accurately reproduce the vibrational modes of alpha-helical and beta -sheet conformations of poly-L-alanine. Preliminary attempts have also been made for modifying selected force constants according to variations in hydrogen bond strength. Dipole derivatives for the peptide group are taken from an ab initio study of hydrogen-bonded N-methylacetamide. Those values, in combination with the calculated eigenvectors and frequencies, allow determination of infrared intensities for selected spectral regions, and the use of transition dipole coupling theory to obtain better frequencies. The present work involves application of this model to studies of model helical polypeptides and proteins. For the model structures (alpha-helix, 3 _{10}-helix

  13. Interaction with the Yes-associated protein (YAP) allows TEAD1 to positively regulate NAIP expression.

    PubMed

    Landin Malt, André; Georges, Adrien; Silber, Joël; Zider, Alain; Flagiello, Domenico

    2013-10-01

    Although the expression of the neuronal apoptosis inhibitory protein (NAIP) gene is considered involved in apoptosis suppression as well as in inflammatory response, the molecular basis of the NAIP gene expression is poorly understood. Here we show that the TEA domain protein 1 (TEAD1) is able to positively activate the transcription of NAIP. We further demonstrate that this regulation is mediated by the presence of the endogenous Yes associated protein (YAP) cofactor, and requires the interaction with YAP. We finally identified an intronic region of the NAIP gene responding to TEAD1/YAP activity, suggesting that regulation of NAIP by TEAD1/YAP is at the transcriptional level. PMID:23994529

  14. Membrane positioning for high- and low-resolution protein structures through a binary classification approach.

    PubMed

    Postic, Guillaume; Ghouzam, Yassine; Guiraud, Vincent; Gelly, Jean-Christophe

    2016-03-01

    The critical importance of algorithms for orienting proteins in the lipid bilayer stems from the extreme difficulty in obtaining experimental data about the membrane boundaries. Here, we present a computational method for positioning protein structures in the membrane, based on the sole alpha carbon coordinates and, therefore, compatible with both high and low structural resolutions. Our algorithm follows a new and simple approach, by treating the membrane assignment problem as a binary classification. Compared with the state-of-the-art algorithms, our method achieves similar accuracy, while being faster. Finally, our open-source software is also capable of processing coarse-grained models of protein structures. PMID:26685702

  15. Computing highly correlated positions using mutual information and graph theory for G protein-coupled receptors.

    PubMed

    Fatakia, Sarosh N; Costanzi, Stefano; Chow, Carson C

    2009-01-01

    G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions) were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families. PMID:19262747

  16. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

    PubMed Central

    Zhang, Hua; Song, Lei; Cong, Haolong

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCE The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection

  17. Role of Positive Selection in Functional Divergence of Mammalian Neuronal Apoptosis Inhibitor Proteins during Evolution

    PubMed Central

    Kong, Fanzhi; Su, Zhaoliang; Zhou, Chenglin; Sun, Caixia; Liu, Yanfang; Zheng, Dong; Yuan, Hongyan; Yin, Jingping; Fang, Jie; Wang, Shengjun; Xu, Huaxi

    2011-01-01

    Neuronal apoptosis inhibitor proteins (NAIPs) are members of Nod-like receptor (NLR) protein family. Recent research demostrated that some NAIP genes were strongly associated with both innate immunity and many inflammatory diseases in humans. However, no similar phenomena have been reported in other mammals. Furthermore, some NAIP genes have undergone pseudogenization or have been lost during the evolution of some higher mammals. We therefore aimed to determine if functional divergence had occurred, and if natural selection had played an important role in the evolution of these genes. The results showed that NAIP genes have undergone pseudogenization and functional divergence, driven by positive selection. Positive selection has also influenced NAIP protein structure, resulting in further functional divergence. PMID:22131819

  18. Nonstructural Proteins Are Preferential Positive Selection Targets in Zika Virus and Related Flaviviruses.

    PubMed

    Sironi, Manuela; Forni, Diego; Clerici, Mario; Cagliani, Rachele

    2016-09-01

    The Flavivirus genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian

  19. Effects of endogenous proteins and microRNA target sequence in a positive feedback system.

    PubMed

    Kanda, Genki N; Togashi, Ryohei; Harashima, Hideyoshi; Kamiya, Hiroyuki

    2012-01-01

    A positive feedback system, using GAL4-vp16 (a fusion protein of yeast GAL4 and herpes simplex virus vp16) as an activator and firefly luciferase as a reporter, maintained luciferase expression for 7 d in mice. However, the luciferase expression decreased after 7 d, and this phenomenon could be caused by immunoreactions against these exogenous proteins. This hypothesis was examined by the following three strategies, designed to avoid the putative immunoreactions: (i) use of the endogenous secreted alkaline phosphatase (SEAP) protein as a reporter, (ii) replacement of vp16 with endogenous transcription factors, and (iii) insertion of the target sequence of microRNA expressed in cells of hematopoietic origin, to suppress GAL4-vp16 expression in antigen-presenting cells. The results obtained in this study suggested that silencing would be induced by mechanism(s) besides immunoreactions against reporter and activator proteins. PMID:22975505

  20. Human self protein CD8+ T cell epitopes are both positively and negatively selected

    PubMed Central

    almani, Michal; Raffaeli, Shai; Vider-Shalit, Tal; Tsaban, Lea; Fishbain, Vered; Louzoun, Yoram

    2009-01-01

    The cellular immune system recognizes self epitopes in the context of MHC-I molecules. The immunological general view presumes that these self epitopes are just a background, both positively and negatively selecting T cells. We here estimate the number of epitopes in each human protein for many frequent HLA alleles, and a score representing over or under presentation of epitopes on these proteins. We further show that there is a clear selection for the presentation of specific self proteins types. Proteins presenting many epitopes include for example AIRE upregulated Tissue specific antigens, immune system receptors and proteins with a high expression level. On the other hand, proteins that may be considered less “useful” for the immune system, such as low expression level proteins, are under presented. We combine our epitope estimate with SNP measures to show that this selection can be directly observed through the fraction of non-synonymous SNPs (replacement fraction), which is significantly higher inside epitopes than outside PMID:19291702

  1. Human self-protein CD8+ T-cell epitopes are both positively and negatively selected.

    PubMed

    Almani, Michal; Raffaeli, Shai; Vider-Shalit, Tal; Tsaban, Lea; Fishbain, Vered; Louzoun, Yoram

    2009-04-01

    The cellular immune system recognizes self-epitopes in the context of MHC-I molecules. The immunological general view presumes that these self-epitopes are just a background, both positively and negatively selecting T cells. We here estimate the number of epitopes in each human protein for many frequent HLA alleles, and a score representing over or under presentation of epitopes on these proteins. We further show that there is a clear selection for the presentation of specific self-protein types. Proteins presenting many epitopes include, for example, autoimmune regulator (AIRE) upregulated tissue-specific antigens, immune system receptors and proteins with a high expression level. On the other hand, proteins that may be considered less "useful" for the immune system, such as low expression level proteins, are under-presented. We combine our epitope estimate with single nucleotide polymorphism (SNP) measures to show that this selection can be directly observed through the fraction of non-synonymous SNP (replacement fraction), which is significantly higher inside epitopes than outside. PMID:19291702

  2. Effects of anions on the positive ion electrospray ionization mass spectra of peptides and proteins.

    PubMed

    Mirza, U A; Chait, B T

    1994-09-15

    Positive ion electrospray ionization mass spectra of polypeptides are usually obtained from solutions that are acidified and therefore contain relatively high concentrations of anions. The present study describes an investigation of the effects of these ubiquitous anions on the positive ion electrospray ionization mass spectra of peptides and proteins. Certain anionic species in the spray solutions were observed to cause a marked decrease in the net average charge of peptide and protein ions in the mass spectra compared to the average charge measured in the absence of these anions. This charge neutralization effect was found to depend solely on the nature of the anionic species and was independent of the source of the anion (acid or salt), with the propensity for neutralization following the order: CCl3COO- > CF3COO- > CH3COO- approximately Cl-. A mechanism for the observed charge reduction effect is proposed that involves two steps. The first step occurs in solution, where an anion pairs with a positively charged basic group on the peptide. The second step occurs during the process of desolvation or in the gas phase, where the ion pair dissociates to yield the neutral acid and the peptide with reduced charge state. The different propensities for charge neutralization of the different anionic species is presumed to reflect the avidity of the anion-peptide interaction. These findings demonstrate that any attempt to correlate the distribution of charge states observed on proteins in the gas phase (by positive ion electrospray ionization mass spectrometry) with the net charge residing on the protein in solution will require that the described anion effect be taken into account. In addition, it appears that some control over the distribution of charge states on peptides and protein ions can be exercised by an appropriate choice of anion in the electrospray solution. PMID:7978296

  3. Isolation and solubilization of gram-positive bacterial cell wall-associated proteins.

    PubMed

    Cole, Jason N; Djordjevic, Steven P; Walker, Mark J

    2008-01-01

    This chapter describes a simple, rapid and reproducible method to prepare bacterial cell wall extracts for two-dimensional gel electrophoresis (2DE). The extraction process uses mutanolysin, an N-acetylmuramidase, to gently solubilize cell wall-associated proteins from Gram-positive prokaryotes. The cells are first washed with buffer and resuspended in a solution containing mutanolysin. Following incubation at 37 degrees C, the sample is centrifuged and the supernatant containing the soluble cell wall-associated proteins is harvested. Following a brief precipitation step, the pellet is solubilized in sample buffer ready for isoelectric focusing and 2DE analysis. PMID:18369905

  4. The 14-3-3 protein PAR-5 regulates the asymmetric localization of the LET-99 spindle positioning protein.

    PubMed

    Wu, Jui-Ching; Espiritu, Eugenel B; Rose, Lesilee S

    2016-04-15

    PAR proteins play important roles in establishing cytoplasmic polarity as well as regulating spindle positioning during asymmetric division. However, the molecular mechanisms by which the PAR proteins generate asymmetry in different cell types are still being elucidated. Previous studies in Caenorhabditis elegans revealed that PAR-3 and PAR-1 regulate the asymmetric localization of LET-99, which in turn controls spindle positioning by affecting the distribution of the conserved force generating complex. In wild-type embryos, LET-99 is localized in a lateral cortical band pattern, via inhibition at the anterior by PAR-3 and at the posterior by PAR-1. In this report, we show that the 14-3-3 protein PAR-5 is also required for cortical LET-99 asymmetry. PAR-5 associated with LET-99 in pull-down assays, and two PAR-5 binding sites were identified in LET-99 using the yeast two-hybrid assay. Mutation of these sites abolished binding in yeast and altered LET-99 localization in vivo: LET-99 was present at the highest levels at the posterior pole of the embryo instead of a band in par-5 embryos. Together the results indicate that PAR-5 acts in a mechanism with PAR-1 to regulate LET-99 cortical localization. PMID:26921457

  5. Changing preferences: deformation of single position amino acid fitness landscapes and evolution of proteins.

    PubMed

    Bazykin, Georgii A

    2015-10-01

    The fitness landscape-the function that relates genotypes to fitness-and its role in directing evolution are a central object of evolutionary biology. However, its huge dimensionality precludes understanding of even the basic aspects of its shape. One way to approach it is to ask a simpler question: what are the properties of a function that assigns fitness to each possible variant at just one particular site-a single position fitness landscape-and how does it change in the course of evolution? Analyses of genomic data from multiple species and multiple individuals within a species have proved beyond reasonable doubt that fitness functions of positions throughout the genome do themselves change with time, thus shaping protein evolution. Here, I will briefly review the literature that addresses these dynamics, focusing on recent genome-scale analyses of fitness functions of amino acid sites, i.e. vectors of fitnesses of 20 individual amino acid variants at a given position of a protein. The set of amino acids that confer high fitness at a particular position changes with time, and the rate of this change is comparable with the rate at which a position evolves, implying that this process plays a major role in evolutionary dynamics. However, the causes of these changes remain largely unclear. PMID:26445980

  6. Proteomic Analysis of Saliva in HIV-Positive Heroin Addicts Reveals Proteins Correlated with Cognition

    PubMed Central

    Ryder, Mark I.; Gritsenko, Marina; Jacobs, Jon M.; Smith, Richard D.

    2014-01-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last several years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse; however, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+), 11 −negative (HIV−) heroin addicts. In addition, saliva samples were investigated from 11 HIV−, non-heroin addicted healthy controls. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores, implicating disruption of protein quality control pathways by HIV. Notably, only one protein from the HIV− heroin addict cohort showed a significant correlation with cognitive scores, and no proteins correlated with cognitive scores in the healthy control group. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes. PMID:24717448

  7. Mis-expression of a PISTILLATA-like MADS box gene prevents fruit development in grapevine.

    PubMed

    Fernandez, Lucie; Chaïb, Jamila; Martinez-Zapater, José-Miguel; Thomas, Mark R; Torregrosa, Laurent

    2013-03-01

    The FLESHLESS BERRY (Flb) somatic variant identified in the grapevine cultivar Ugni Blanc develops grape berries without flesh, suggesting a role for the altered gene in differentiation of flesh cells. Here we describe identification of the molecular defect responsible for this phenotype. Using a combination of genetic and transcriptomic approaches, we detected the insertion of a miniature inverted-repeat transposable element in the promoter region of the PISTILLATA-like (VvPI) gene, the grapevine homologue of Arabidopsis PISTILLATA. The transposon insertion causes specific ectopic expression of the corresponding VvPI allele during early fruit development, causing expression of genes specific for petal and stamen development within the fruit. A causal relationship between the insertion and the phenotype was demonstrated by phenotypic and molecular analyses of somatic revertants showing that ectopic expression and mutant phenotype were always linked to the presence of the transposon insertion. The various phenotypic effects of the flb mutation on ovary morphology, fruit set and fruit development, depending on the cell lineage affected, are presented for each phenotype, offering new insights into floral and fleshly fruit development. The results highlight the importance of VvPI repression after fertilization to achieve normal fleshy fruit development, and the complex genetic, genomic and cellular interactions required for the flower to fruit transition in grapevine. PMID:23181568

  8. Monolith disk chromatography separates PEGylated protein positional isoforms within minutes at low pressure.

    PubMed

    Isakari, Yu; Podgornik, Ales; Yoshimoto, Noriko; Yamamoto, Shuichi

    2016-01-01

    Although PEGylation makes proteins drugs more effective, the PEGylation reaction must be controlled carefully in order to obtain a desired PEGylated protein form since various different PEGylated forms may be produced during the reaction. For monitoring the PEGylation reaction, a method with monolith disk ion exchange chromatography, which can separate positional isomers as well as PEGmers, has been developed as a process analytical tool (PAT). The method was optimized for separation of randomly PEGylated protein (lysozyme) isoforms based on the number of resolved peaks, peak resolution, analysis time and pressure drop. In order to increase the retention of mono- and di-PEGylated protein isomers the mobile phase was decreased to pH 4.5, where a large number of mono- and di-PEGylated isomers were resolved within a few minutes. Based on the linear gradient elution optimization model, the following values were determined: gradient slope 0.016 M/mL, disk thickness 3 mm (single disk) and flow rate 10 mL/min. Under these optimal conditions, the analysis was completed within ca. 4 min while the pressure drop was below 1 MPa. As the method was successfully applied to monitoring mono and di-PEGylated positional isoforms in the reaction mixture of random PEGylation of lysozyme, it is expected to be an efficient PAT tool. PMID:26626923

  9. Proteomic Analysis of Saliva in HIV-positive Heroin Addicts Reveals Proteins Correlated with Cognition

    SciTech Connect

    Dominy, Stephen; Brown, Joseph N.; Ryder, Mark I.; Gritsenko, Marina A.; Jacobs, Jon M.; Smith, Richard D.

    2014-04-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last few years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse. However, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+) and 11 -negative (HIV-) heroin addicts. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores and that implicate disruption of protein quality control pathways by HIV. Notably, no proteins from the HIV- heroin addict cohort showed significant correlations with cognitive scores. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.

  10. Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

    PubMed Central

    Marraffini, Luciano A.; DeDent, Andrea C.; Schneewind, Olaf

    2006-01-01

    The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis. PMID:16524923

  11. Positive Lysosomal Modulation As a Unique Strategy to Treat Age-Related Protein Accumulation Diseases

    PubMed Central

    Wisniewski, Meagan L.; Butler, David

    2012-01-01

    Abstract Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ1–38 peptide corresponded with decreased levels of Aβ1–42, supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders

  12. Arabidopsis COP1-interacting protein 1 is a positive regulator of ABA response.

    PubMed

    Ren, Chenxia; Zhu, Xili; Zhang, Pingping; Gong, Qingqiu

    2016-09-01

    COP1-interacting protein 1 (CIP1, At5g41790) was the first reported interacting protein for CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) of Arabidopsis; however its physiological function has remained unknown for two decades. Here we show that CIP1 is a positive regulator of abscisic acid (ABA) response. CIP1 is mainly expressed in the photosynthetic cells and the vascular tissue, and its promoter activity can be induced by osmotic stress and ABA. The CIP1 protein is localized to the plasma membrane. A T-DNA insertion mutant cip1-1 was then characterized. The mutant is sensitive to osmotic stress and has ABA insensitive phenotypes. RNA sequencing showed that cip1-1 has lower levels of gene expression in abiotic stress response compared with the wild-type. Meanwhile, transcript levels of ABA biosynthesis genes are higher in cip1-1 than in the wild-type. These results suggested that CIP1 is positively involved in ABA response. PMID:27372427

  13. Positive Charge of “Sticky” Peptides and Proteins Impedes Release From Negatively Charged PLGA Matrices

    PubMed Central

    Balmert, Stephen C.; Zmolek, Andrew C.; Glowacki, Andrew J.; Knab, Timothy D.; Rothstein, Sam N.; Wokpetah, Joseph M.; Fedorchak, Morgan V.; Little, Steven R.

    2015-01-01

    The influence of electrostatic interactions and/or acylation on release of charged (“sticky”) agents from biodegradable polymer matrices was systematically characterized. We hypothesized that release of peptides with positive charge would be hindered from negatively charged poly(lactic-co-glycolic acid) (PLGA) microparticles. Thus, we investigated release of peptides with different degrees of positive charge from several PLGA microparticle formulations, with different molecular weights and/or end groups (acid- or ester-terminated). Indeed, release studies revealed distinct inverse correlations between the amount of positive charge on peptides and their release rates from each PLGA microparticle formulation. Furthermore, we examined the case of peptides with net charge that changes from negative to positive within the pH range observed in degrading microparticles. These charge changing peptides displayed counterintuitive release kinetics, initially releasing faster from slower degrading (less acidic) microparticles, and releasing slower from the faster degrading (more acidic) microparticles. Importantly, trends between agent charge and release rates for model peptides also translated to larger, therapeutically relevant proteins and oligonucleotides. The results of these studies may improve future design of controlled release systems for numerous therapeutic biomolecules exhibiting positive charge, ultimately reducing time-consuming and costly trial and error iterations of such formulations. PMID:26085928

  14. Computationally predicting protein-RNA interactions using only positive and unlabeled examples.

    PubMed

    Cheng, Zhanzhan; Zhou, Shuigeng; Guan, Jihong

    2015-06-01

    Protein-RNA interactions (PRIs) are considerably important in a wide variety of cellular processes, ranging from transcriptional and post-transcriptional regulations of gene expression to the active defense of host against virus. With the development of high throughput technology, large amounts of PRI information is available for computationally predicting unknown PRIs. In recent years, a number of computational methods for predicting PRIs have been developed in the literature, which usually artificially construct negative samples based on verified nonredundant datasets of PRIs to train classifiers. However, such negative samples are not real negative samples, some even may be unknown positive samples. Consequently, the classifiers trained with such training datasets cannot achieve satisfactory prediction performance. In this paper, we propose a novel method PRIPU that employs biased-support vector machine (SVM) for predicting Protein-RNA Interactions using only Positive and Unlabeled examples. To the best of our knowledge, this is the first work that predicts PRIs using only positive and unlabeled samples. We first collect known PRIs as our benchmark datasets and extract sequence-based features to represent each PRI. To reduce the dimension of feature vectors for lowering computational cost, we select a subset of features by a filter-based feature selection method. Then, biased-SVM is employed to train prediction models with different PRI datasets. To evaluate the new method, we also propose a new performance measure called explicit positive recall (EPR), which is specifically suitable for the task of learning positive and unlabeled data. Experimental results over three datasets show that our method not only outperforms four existing methods, but also is able to predict unknown PRIs. Source code, datasets and related documents of PRIPU are available at: http://admis.fudan.edu.cn/projects/pripu.htm . PMID:25790785

  15. Nano-Positioning System for Structural Analysis of Functional Homomeric Proteins in Multiple Conformations

    PubMed Central

    Hyde, H. Clark; Sandtner, Walter; Vargas, Ernesto; Dagcan, Alper; Robertson, Janice L.; Roux, Benoit; Correa, Ana M.; Bezanilla, Francisco

    2012-01-01

    SUMMARY Proteins may undergo multiple conformational changes required for their function. One strategy used to estimate target site positions in unknown structural conformations involves single-pair resonance energy transfer (RET) distance measurements. However, interpretation of inter-residue distances is difficult when applied to three-dimensional structural rearrangements, especially in homomeric systems. We developed a novel method using inverse trilateration/triangulation to map target sites within a homomeric protein in all defined states with simultaneous functional recordings. The procedure accounts for probe diffusion to accurately determine the three-dimensional position and confidence region of lanthanide LRET donors attached to a target site (one/subunit), relative to a single fluorescent acceptor placed in a static site. As a first application, the method is used to determine the position of a functional voltage-gated potassium channel’s voltage sensor. Our results verify the crystal structure relaxed conformation and report on the resting and active conformations for which crystal structures are not available. PMID:23063010

  16. A heparin-mimicking reverse thermal gel for controlled delivery of positively charged proteins.

    PubMed

    Peña, Brisa; Shandas, Robin; Park, Daewon

    2015-06-01

    Positively charged therapeutic proteins have been used extensively for biomedical applications. However, the safety and efficacy of proteins are mostly limited by their physical and chemical instability and short half-lives in physiological conditions. To this end, we created a heparin-mimicking sulfonated reverse thermal gel as a novel protein delivery system by sulfonation of a graft copolymer, poly(serinol hexamethylene urea)-co-poly(N-isopropylacylamide), or PSHU-NIPAAm. The net charge of the sulfonated PSHU-NIPAAm was negative due to the presence of sulfonate groups. The sulfonated PSHU-NIPAAm showed a typical temperature-dependent sol-gel phase transition, where polymer solutions turned to a physical gel at around 32°C and maintained gel status at body temperature. Both in vitro cytotoxicity tests using C2C12 myoblast cells and in vivo cytotoxicity tests by subcutaneous injections demonstrated excellent biocompatibility. In vitro release tests using bovine serum albumin revealed that the release from the sulfonated PSHU-NIPAAm was more sustained than that from the plain PSHU-NIPAAm. Furthermore, this sulfonated PSHU-NIPAAm system did not affect protein structure after 70-day observation periods. PMID:25294242

  17. A heparin-mimicking reverse thermal gel for controlled delivery of positively charged proteins

    PubMed Central

    Peña, Brisa; Shandas, Robin; Park, Daewon

    2014-01-01

    Positively charged therapeutic proteins have been used extensively for biomedical applications. However, the safety and efficacy of proteins are mostly limited by their physical and chemical instability and short half-lives in physiological conditions. To this end, we created a heparin-mimicking sulfonated reverse thermal gel as a novel protein delivery system by sulfonation of a graft copolymer, poly(serinol hexamethylene urea)-co-poly(N-isopropylacylamide), or PSHU-NIPAAm. The net charge of the sulfonated PSHU-NIPAAm was negative due to the presence of sulfonate groups. The sulfonated PSHU-NIPAAm showed a typical temperature-dependent sol-gel phase transition, where polymer solutions turned to a physical gel at around 32°C and maintained gel status at body temperature. Both in vitro cytotoxicity tests using C2C12 myoblast cells and in vivo cytotoxicity tests by subcutaneous injections demonstrated excellent biocompatibility. In vitro release tests using bovine serum albumin (BSA) revealed that the release from the sulfonated PSHU-NIPAAm was more sustained than that from the plain PSHU-NIPAAm. Furthermore, this sulfonated PSHU-NIPAAm system did not affect protein structure after 70-day observation periods. PMID:25294242

  18. Alteration of cardiac glycoside positive inotropic action by modulators of protein synthesis and degradation

    SciTech Connect

    Nosek, T.M.; Adams, R.J.

    1986-03-05

    Numerous membrane bound and cytoplasmic proteins participate in the cardiac expression of the positive inotropic action (PIA) of digitalis glycosides including the Na,K-ATPase (NKA). Exposure of the myocardium to an inhibitor of protein synthesis (cycloheximide, CYC) or of protein degradation (leupeptin, LEU) alters the PIA of ouabain in isolated, paced guinea pig papillary muscles (PM) in opposite ways. In vivo exposure to CYC for 3 hr resulted in a 30% depression of the in vitro PIA of ouabain at 1.7..mu..M compared to control. In vivo exposure to LEU for 1 hr resulted in a 47% enhancement of the in vitro PIA of 1.7..mu..M ouabain. Neither drug had an apparent effect on the ouabain PIA ED50. Neither CYC nor LEU exposure to PM in vitro affect resting or developed tension or the response of skinned PM to calcium. The mechanisms of the PIA alterations by CYC or LEU do not involve a direct effect on the digitalis receptor. Exposure of isolated cardiac sarcolemma enriched in NKA to 10-100..mu..M CYC or LEU did not affect NKA activity or /sup 3/H-ouabain binding. Although direct physicochemical effects of CYC or LEU may be involved in the alterations of the ouabain PIA, it is possible that modulation of the cellular levels or turnover rate of short-lived proteins may affect cardiac regulation of the digitalis PIA.

  19. Positive Regulation of TRAF6-Dependent Innate Immune Responses by Protein Phosphatase PP1-γ

    PubMed Central

    Chiang, Chih-yuan; Nguyen, Quy T.; Maestre, Ana M.; Mulder, Lubbertus C. F.; Secundino, Ismael; De Jesus, Paul D.; König, Renate; Simon, Viviana; Nizet, Victor; MacLeod, Graham; Varmuza, Susannah; Fernandez-Sesma, Ana; Chanda, Sumit K.

    2014-01-01

    Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses. PMID:24586659

  20. Size controlled protein nanoemulsions for active targeting of folate receptor positive cells.

    PubMed

    Loureiro, Ana; Nogueira, Eugénia; Azoia, Nuno G; Sárria, Marisa P; Abreu, Ana S; Shimanovich, Ulyana; Rollett, Alexandra; Härmark, Johan; Hebert, Hans; Guebitz, Georg; Bernardes, Gonçalo J L; Preto, Ana; Gomes, Andreia C; Cavaco-Paulo, Artur

    2015-11-01

    Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy. PMID:26241920

  1. Paediatric group position statement on the use of soya protein for infants.

    PubMed

    2003-01-01

    Breast feeding should be strongly encouraged as providing the safest, most nutritionally adequate form of feeding for most infants. Dietitians should discourage the use of soya protein in children with atopy or cow's milk allergy in the first six months of life to avoid sensitisation to soya protein and exposure to phytoestrogens while organ systems remain at their most vulnerable. This would include soy infant formula and soya products such as desserts etc. When a soy-based infant formula is used, parents should be informed of current findings relating to phytoestrogens and health and on the clinical need for soy formula. Any parent choosing to refuse soya for their infant should be supported in their decision. More research into the long-term effects of phytoestrogen exposure in infants is needed and into whether any adverse effects are dose related. This position statement will be updated as further evidence becomes available. PMID:14528647

  2. Arginine residues at internal positions in a protein are always charged

    PubMed Central

    Harms, Michael J.; Schlessman, Jamie L.; Sue, Gloria R.; García-Moreno E., Bertrand

    2011-01-01

    Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pKa values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pKa values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pKa; they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments. PMID:22080604

  3. The Positive Regulatory Roles of the TIFY10 Proteins in Plant Responses to Alkaline Stress

    PubMed Central

    Zhu, Dan; Li, Rongtian; Liu, Xin; Sun, Mingzhe; Wu, Jing; Zhang, Ning; Zhu, Yanming

    2014-01-01

    The TIFY family is a novel plant-specific protein family, and is characterized by a conserved TIFY motif (TIFF/YXG). Our previous studies indicated the potential roles of TIFY10/11 proteins in plant responses to alkaline stress. In the current study, we focused on the regulatory roles and possible physiological and molecular basis of the TIFY10 proteins in plant responses to alkaline stress. We demonstrated the positive function of TIFY10s in alkaline responses by using the AtTIFY10a and AtTIFY10b knockout Arabidopsis, as evidenced by the relatively lower germination rates of attify10a and attify10b mutant seeds under alkaline stress. We also revealed that ectopic expression of GsTIFY10a in Medicago sativa promoted plant growth, and increased the NADP-ME activity, citric acid content and free proline content but decreased the MDA content of transgenic plants under alkaline stress. Furthermore, expression levels of the stress responsive genes including NADP-ME, CS, H+-ppase and P5CS were also up-regulated in GsTIFY10a transgenic plants under alkaline stress. Interestingly, GsTIFY10a overexpression increased the jasmonate content of the transgenic alfalfa. In addition, we showed that neither GsTIFY10a nor GsTIFY10e exhibited transcriptional activity in yeast cells. However, through Y2H and BiFc assays, we demonstrated that GsTIFY10a, not GsTIFY10e, could form homodimers in yeast cells and in living plant cells. As expected, we also demonstrated that GsTIFY10a and GsTIFY10e could heterodimerize with each other in both yeast and plant cells. Taken together, our results provided direct evidence supporting the positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress. PMID:25375909

  4. Arginine residues at internal positions in a protein are always charged.

    PubMed

    Harms, Michael J; Schlessman, Jamie L; Sue, Gloria R; García-Moreno, Bertrand

    2011-11-22

    Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK(a) values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pK(a) values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pK(a); they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments. PMID:22080604

  5. Robust, tunable genetic memory from protein sequestration combined with positive feedback.

    PubMed

    Shopera, Tatenda; Henson, William R; Ng, Andrew; Lee, Young Je; Ng, Kenneth; Moon, Tae Seok

    2015-10-15

    Natural regulatory networks contain many interacting components that allow for fine-tuning of switching and memory properties. Building simple bistable switches, synthetic biologists have learned the design principles of complex natural regulatory networks. However, most switches constructed so far are so simple (e.g. comprising two regulators) that they are functional only within a limited parameter range. Here, we report the construction of robust, tunable bistable switches in Escherichia coli using three heterologous protein regulators (ExsADC) that are sequestered into an inactive complex through a partner swapping mechanism. On the basis of mathematical modeling, we accurately predict and experimentally verify that the hysteretic region can be fine-tuned by controlling the interactions of the ExsADC regulatory cascade using the third member ExsC as a tuning knob. Additionally, we confirm that a dual-positive feedback switch can markedly increase the hysteretic region, compared to its single-positive feedback counterpart. The dual-positive feedback switch displays bistability over a 10(6)-fold range of inducer concentrations, to our knowledge, the largest range reported so far. This work demonstrates the successful interlocking of sequestration-based ultrasensitivity and positive feedback, a design principle that can be applied to the construction of robust, tunable, and predictable genetic programs to achieve increasingly sophisticated biological behaviors. PMID:26384562

  6. The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase.

    PubMed Central

    Luke, M M; Della Seta, F; Di Como, C J; Sugimoto, H; Kobayashi, R; Arndt, K T

    1996-01-01

    SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that is required for G1 cyclin transcription and for bud formation. SIT4 associates with several high-molecular-mass proteins in a cell cycle-dependent fashion. We purified two SIT4-associated proteins, SAP155 and SAP190, and cloned the corresponding genes. By sequence homology, we isolated two additional SAP genes, SAP185 and SAP4. Through such an association is not yet proven for SAP4, each of SAP155, SAP185, and SAP190 physically associates with SIT4 in separate complexes. The SAPs function positively with SIT4, and by several criteria, the loss of all four SAPs is equivalent to the loss of SIT4. The data suggest that the SAPs are not functional in the absence of SIT4 and likewise that SIT4 is not functional in the absence of the SAPs. The SAPs are hyperphoshorylated in cells lacking SIT4, raising the possibility that the SAPs are substrates of SIT4. By sequence similarity, the SAPs fall into two groups, the SAP4/SAP155 group and the SAP185/SAP190 group. Overexpression of a SAP from one group does not suppress the defects due to the loss of the other group. These findings and others indicate that the SAPs have distinct functions. PMID:8649382

  7. Bayesian Top-Down Protein Sequence Alignment with Inferred Position-Specific Gap Penalties.

    PubMed

    Neuwald, Andrew F; Altschul, Stephen F

    2016-05-01

    We describe a Bayesian Markov chain Monte Carlo (MCMC) sampler for protein multiple sequence alignment (MSA) that, as implemented in the program GISMO and applied to large numbers of diverse sequences, is more accurate than the popular MSA programs MUSCLE, MAFFT, Clustal-Ω and Kalign. Features of GISMO central to its performance are: (i) It employs a "top-down" strategy with a favorable asymptotic time complexity that first identifies regions generally shared by all the input sequences, and then realigns closely related subgroups in tandem. (ii) It infers position-specific gap penalties that favor insertions or deletions (indels) within each sequence at alignment positions in which indels are invoked in other sequences. This favors the placement of insertions between conserved blocks, which can be understood as making up the proteins' structural core. (iii) It uses a Bayesian statistical measure of alignment quality based on the minimum description length principle and on Dirichlet mixture priors. Consequently, GISMO aligns sequence regions only when statistically justified. This is unlike methods based on the ad hoc, but widely used, sum-of-the-pairs scoring system, which will align random sequences. (iv) It defines a system for exploring alignment space that provides natural avenues for further experimentation through the development of new sampling strategies for more efficiently escaping from suboptimal traps. GISMO's superior performance is illustrated using 408 protein sets containing, on average, 235 sequences. These sets correspond to NCBI Conserved Domain Database alignments, which have been manually curated in the light of available crystal structures, and thus provide a means to assess alignment accuracy. GISMO fills a different niche than other MSA programs, namely identifying and aligning a conserved domain present within a large, diverse set of full length sequences. The GISMO program is available at http://gismo.igs.umaryland.edu/. PMID:27192614

  8. False-Positive Rate Determination of Protein Target Discovery using a Covalent Modification- and Mass Spectrometry-Based Proteomics Platform

    NASA Astrophysics Data System (ADS)

    Strickland, Erin C.; Geer, M. Ariel; Hong, Jiyong; Fitzgerald, Michael C.

    2014-01-01

    Detection and quantitation of protein-ligand binding interactions is important in many areas of biological research. Stability of proteins from rates of oxidation (SPROX) is an energetics-based technique for identifying the proteins targets of ligands in complex biological mixtures. Knowing the false-positive rate of protein target discovery in proteome-wide SPROX experiments is important for the correct interpretation of results. Reported here are the results of a control SPROX experiment in which chemical denaturation data is obtained on the proteins in two samples that originated from the same yeast lysate, as would be done in a typical SPROX experiment except that one sample would be spiked with the test ligand. False-positive rates of 1.2-2.2 % and <0.8 % are calculated for SPROX experiments using Q-TOF and Orbitrap mass spectrometer systems, respectively. Our results indicate that the false-positive rate is largely determined by random errors associated with the mass spectral analysis of the isobaric mass tag (e.g., iTRAQ®) reporter ions used for peptide quantitation. Our results also suggest that technical replicates can be used to effectively eliminate such false positives that result from this random error, as is demonstrated in a SPROX experiment to identify yeast protein targets of the drug, manassantin A. The impact of ion purity in the tandem mass spectral analyses and of background oxidation on the false-positive rate of protein target discovery using SPROX is also discussed.

  9. Bayesian Top-Down Protein Sequence Alignment with Inferred Position-Specific Gap Penalties

    PubMed Central

    Neuwald, Andrew F.; Altschul, Stephen F.

    2016-01-01

    We describe a Bayesian Markov chain Monte Carlo (MCMC) sampler for protein multiple sequence alignment (MSA) that, as implemented in the program GISMO and applied to large numbers of diverse sequences, is more accurate than the popular MSA programs MUSCLE, MAFFT, Clustal-Ω and Kalign. Features of GISMO central to its performance are: (i) It employs a “top-down” strategy with a favorable asymptotic time complexity that first identifies regions generally shared by all the input sequences, and then realigns closely related subgroups in tandem. (ii) It infers position-specific gap penalties that favor insertions or deletions (indels) within each sequence at alignment positions in which indels are invoked in other sequences. This favors the placement of insertions between conserved blocks, which can be understood as making up the proteins’ structural core. (iii) It uses a Bayesian statistical measure of alignment quality based on the minimum description length principle and on Dirichlet mixture priors. Consequently, GISMO aligns sequence regions only when statistically justified. This is unlike methods based on the ad hoc, but widely used, sum-of-the-pairs scoring system, which will align random sequences. (iv) It defines a system for exploring alignment space that provides natural avenues for further experimentation through the development of new sampling strategies for more efficiently escaping from suboptimal traps. GISMO’s superior performance is illustrated using 408 protein sets containing, on average, 235 sequences. These sets correspond to NCBI Conserved Domain Database alignments, which have been manually curated in the light of available crystal structures, and thus provide a means to assess alignment accuracy. GISMO fills a different niche than other MSA programs, namely identifying and aligning a conserved domain present within a large, diverse set of full length sequences. The GISMO program is available at http://gismo.igs.umaryland.edu/. PMID

  10. Changes in Morphology, Gene Expression and Protein Content in Chondrocytes Cultured on a Random Positioning Machine

    PubMed Central

    Aleshcheva, Ganna; Sahana, Jayashree; Ma, Xiao; Hauslage, Jens; Hemmersbach, Ruth; Egli, Marcel; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

    2013-01-01

    Tissue engineering of chondrocytes on a Random Positioning Machine (RPM) is a new strategy for cartilage regeneration. Using a three-dimensional RPM, a device designed to simulate microgravity on Earth, we investigated the early effects of RPM exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of RPM exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis (TGF-β1, osteopontin); and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours of RPM exposure disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours on the RPM, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced during RPM culture for 24 h. Taking these results together, we suggest that chondrocytes exposed to the RPM seem to change their extracellular matrix production behaviour while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates. PMID:24244418

  11. HER-2/neu protein-receptor-positive breast carcinoma: an immunologic perspective.

    PubMed

    Brown, R E; Bernath, A M; Lewis, G O

    2000-07-01

    Immunotherapy using a monoclonal antibody against the human epidermal growth factor receptor 2 protein, HER-2/neu, has proven to be clinically efficacious in about one-half of breast cancer patients who exhibit strong (3+) plasmalemmal immunoreactivity for this protein. The tumoricidal effect of this antibody relies in part upon antibody-dependent cell-mediated cytotoxicity. This report provides observations on certain factors or circumstances which could have an impact on this aspect of the therapeutic approach. These include: (1) concurrent medications; (2) the composition (immunophenotype) of peritumoral lymphocytes and the generally limited numbers of intratumoral T-lymphocytes/natural killer (NK) cells, monocytes, and neutrophilic granulocytes; (3) the presence of circulating HER-2/neu antigens which might bind the exogenous antibody and lead to immune complex formation; (4) the variable co-expression in the tumor of cytokines known to downregulate NK cell function (ie, transforming growth factor-beta1 [TGF-beta1] and platelet-derived growth factor [PDGF]-AB); and (5) the tumoral and/or stromal immunoreactivity for angiotensin-converting enzyme, which forms a part of one of the pathways for the activation of latent TGF-beta1 and for the biosynthesis of PDGF-AB. These observations provide an immunologic perspective for the use of monoclonal antibody therapy in HER-2/neu protein-receptor-positive breast carcinoma and suggest a role for the clinical laboratory in identifying potential avenues for additional manipulations of the immune system in individual cases in order to enhance the therapeutic response. PMID:10945564

  12. Expected distributions of root-mean-square positional deviations in proteins.

    PubMed

    Pitera, Jed W

    2014-06-19

    The atom positional root-mean-square deviation (RMSD) is a standard tool for comparing the similarity of two molecular structures. It is used to characterize the quality of biomolecular simulations, to cluster conformations, and as a reaction coordinate for conformational changes. This work presents an approximate analytic form for the expected distribution of RMSD values for a protein or polymer fluctuating about a stable native structure. The mean and maximum of the expected distribution are independent of chain length for long chains and linearly proportional to the average atom positional root-mean-square fluctuations (RMSF). To approximate the RMSD distribution for random-coil or unfolded ensembles, numerical distributions of RMSD were generated for ensembles of self-avoiding and non-self-avoiding random walks. In both cases, for all reference structures tested for chains more than three monomers long, the distributions have a maximum distant from the origin with a power-law dependence on chain length. The purely entropic nature of this result implies that care must be taken when interpreting stable high-RMSD regions of the free-energy landscape as "intermediates" or well-defined stable states. PMID:24655018

  13. OsMADS32 interacts with PI-like proteins and regulates rice flower development.

    PubMed

    Wang, Huanhuan; Zhang, Liang; Cai, Qiang; Hu, Yun; Jin, Zhenming; Zhao, Xiangxiang; Fan, Wei; Huang, Qianming; Luo, Zhijing; Chen, Mingjiao; Zhang, Dabing; Yuan, Zheng

    2015-05-01

    OsMADS32 is a monocot specific MIKC(c) type MADS-box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clarified. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1-3 and identified its function in controlling rice flower development by bioinformatics and protein-protein interaction analysis. The cfo1-3 mutant produces defective flowers, including loss of lodicule identity, formation of ectopic lodicule or hull-like organs and decreased stamen number, mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis-splicing of OsMADS32 transcripts in the cfo1-3 mutant resulted in an extra eight amino acids in the K-domain of OsMADS32 protein. By yeast two hybrid and bimolecular fluorescence complementation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of OsMADS32 affects the interaction between OsMADS32 with PISTILLATA (PI)-like proteins OsMADS2 and OsMADS4. This work provides new insight into the mechanism by which OsMADS32 regulates rice lodicule and stamen identity, by interaction with two PI-like proteins via its K domain. PMID:25081486

  14. Interaction between the transcription factor SPBP and the positive cofactor RNF4. An interplay between protein binding zinc fingers.

    PubMed

    Lyngsø, C; Bouteiller, G; Damgaard, C K; Ryom, D; Sanchez-Muñoz, S; Nørby, P L; Bonven, B J; Jørgensen, P

    2000-08-25

    The activator of stromelysin 1 gene transcription, SPBP, interacts with the RING finger protein RNF4. Both proteins are ubiquitously expressed and localized in the nucleus. RNF4 facilitates accumulation of specific SPBP-DNA complexes in vitro and acts as a positive cofactor in SPBP-mediated transactivation. SPBP harbors an internal zinc finger of the PHD/LAP type. This domain can form intra-chain protein-protein contacts in SPBP resulting in negative modulation of SPBP-RNF4 interaction. PMID:10849425

  15. Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

    PubMed Central

    Janmaat, C. J.; de Rooij, K. E; Locher, H; de Groot, S. C.; de Groot, J. C. M. J.; Frijns, J. H. M.; Huisman, M. A.

    2015-01-01

    In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality. PMID:26678612

  16. Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins.

    PubMed

    Janmaat, C J; de Rooij, K E; Locher, H; de Groot, S C; de Groot, J C M J; Frijns, J H M; Huisman, M A

    2015-01-01

    In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality. PMID:26678612

  17. Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

    PubMed Central

    Navarre, William Wiley; Schneewind, Olaf

    1999-01-01

    The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836

  18. Quantitative evaluation of positive ϕ angle propensity in flexible regions of proteins from three-bond J couplings†

    PubMed Central

    Lee, Jung Ho; Ying, Jinfa

    2015-01-01

    3JHNHα and 3JC′C′ couplings can be readily measured in isotopically enriched proteins and were shown to contain precise information on the backbone torsion angles, ϕ, sampled in disordered regions of proteins. However, quantitative interpretation of these couplings required the population of conformers with positive ϕ angles to be very small. Here, we demonstrate that this restriction can be removed by measurement of 3JC′Hα values. Even though the functional forms of the 3JC′Hα and 3JHNHα Karplus equations are the same, large differences in their coefficients enable accurate determination of the fraction of time that positive ϕ angles are sampled. A four-dimensional triple resonance HACANH[C′] E.COSY experiment is introduced to simultaneously measure 3JC′Hα and 3JHNC′ in the typically very congested spectra of disordered proteins. High resolution in these spectra is obtained by non-uniform sampling (in the 0.1-0.5% range). Application to the intrinsically disordered protein α-synuclein shows that while most residues have close-to-zero positive ϕ angle populations, up to 16% positive ϕ population is observed for Asn residues. Positive ϕ angle populations determined with the new approach agree closely with consensus values from protein coil libraries and prior analysis of a large set of other NMR parameters. The combination of 3JHNC′ and 3JC′C′ provides information about the amplitude of ϕ angle dynamics. PMID:26415896

  19. Quantitative evaluation of positive ϕ angle propensity in flexible regions of proteins from three-bond J couplings.

    PubMed

    Lee, Jung Ho; Ying, Jinfa; Bax, Ad

    2016-02-17

    (3)JHNHα and (3)JC'C' couplings can be readily measured in isotopically enriched proteins and were shown to contain precise information on the backbone torsion angles, ϕ, sampled in disordered regions of proteins. However, quantitative interpretation of these couplings required the population of conformers with positive ϕ angles to be very small. Here, we demonstrate that this restriction can be removed by measurement of (3)JC'Hα values. Even though the functional forms of the (3)JC'Hα and (3)JHNHα Karplus equations are the same, large differences in their coefficients enable accurate determination of the fraction of time that positive ϕ angles are sampled. A four-dimensional triple resonance HACANH[C'] E.COSY experiment is introduced to simultaneously measure (3)JC'Hα and (3)JHNC' in the typically very congested spectra of disordered proteins. High resolution in these spectra is obtained by non-uniform sampling (in the 0.1-0.5% range). Application to the intrinsically disordered protein α-synuclein shows that while most residues have close-to-zero positive ϕ angle populations, up to 16% positive ϕ population is observed for Asn residues. Positive ϕ angle populations determined with the new approach agree closely with consensus values from protein coil libraries and prior analysis of a large set of other NMR parameters. The combination of (3)JHNC' and (3)JC'C' provides information about the amplitude of ϕ angle dynamics. PMID:26415896

  20. The identification of novel PMADS3 interacting proteins indicates a role in post-transcriptional control.

    PubMed

    Li, Xin; Ning, Guogui; Han, Xueping; Liu, Caixian; Bao, Manzhu

    2015-06-10

    PMADS3, a known MADS-box transcriptional factor and a C-class gene for floral development, plays dual roles in controlling the identity of inner floral organs and the termination of flower meristems in petunia. In this study, it was confirmed by bimolecular fluorescence complementation (BiFC) assays that the PMADS3 protein can interact individually with E-class proteins FBP2, FBP5, FBP9 and PMADS12. A yeast two-hybrid cDNA library was screened using the entire PMADS3 as bait, and this identified further potential interaction candidates. Two novel genes, PheIF3f and PhAGO10, were isolated, and suggested to regulate mRNA and translational processes according to the analysis of protein functional domains and subcellular localization predictions. Notably, the PhAGO10 protein belongs to the Argonaute family, members of which are major players in small-RNA-guided gene silencing processes via mRNA cleavage or translational inhibition. The results of yeast two-hybrid and BiFC assays indicated that PheIF3f and PhAGO10 could interact with PMADS3. Our findings indicate that the C-class gene PMADS3 potentially participates in post-transcriptional control, as well as transcriptional regulation. PMID:25827715

  1. FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential.

    PubMed

    Romano, S; Staibano, S; Greco, A; Brunetti, A; Nappo, G; Ilardi, G; Martinelli, R; Sorrentino, A; Di Pace, A; Mascolo, M; Bisogni, R; Scalvenzi, M; Alfano, B; Romano, M F

    2013-01-01

    Melanoma is the most aggressive skin cancer; there is no cure in advanced stages. Identifying molecular participants in melanoma progression may provide useful diagnostic and therapeutic tools. FK506 binding protein 51 (FKBP51), an immunophilin with a relevant role in developmental stages, is highly expressed in melanoma and correlates with aggressiveness and therapy resistance. We hypothesized a role for FKBP51 in melanoma invasive behaviour. FKBP51 promoted activation of epithelial-to-mesenchymal transition (EMT) genes and improved melanoma cell migration and invasion. In addition, FKBP51 induced some melanoma stem cell (MCSC) genes. Purified MCSCs expressed high EMT genes levels, suggesting that genetic programs of EMT and MCSCs overlap. Immunohistochemistry of samples from patients showed intense FKBP51 nuclear signal and cytoplasmic positivity for the stem cell marker nestin in extravasating melanoma cells and metastatic brains. In addition, FKBP51 targeting by small interfering RNA (siRNA) prevented the massive metastatic substitution of liver and lung in a mouse model of experimental metastasis. The present study provides evidence that the genetic programs of cancer stemness and invasiveness overlap in melanoma, and that FKBP51 plays a pivotal role in sustaining such a program. PMID:23559012

  2. FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential

    PubMed Central

    Romano, S; Staibano, S; Greco, A; Brunetti, A; Nappo, G; Ilardi, G; Martinelli, R; Sorrentino, A; Di Pace, A; Mascolo, M; Bisogni, R; Scalvenzi, M; Alfano, B; Romano, M F

    2013-01-01

    Melanoma is the most aggressive skin cancer; there is no cure in advanced stages. Identifying molecular participants in melanoma progression may provide useful diagnostic and therapeutic tools. FK506 binding protein 51 (FKBP51), an immunophilin with a relevant role in developmental stages, is highly expressed in melanoma and correlates with aggressiveness and therapy resistance. We hypothesized a role for FKBP51 in melanoma invasive behaviour. FKBP51 promoted activation of epithelial-to-mesenchymal transition (EMT) genes and improved melanoma cell migration and invasion. In addition, FKBP51 induced some melanoma stem cell (MCSC) genes. Purified MCSCs expressed high EMT genes levels, suggesting that genetic programs of EMT and MCSCs overlap. Immunohistochemistry of samples from patients showed intense FKBP51 nuclear signal and cytoplasmic positivity for the stem cell marker nestin in extravasating melanoma cells and metastatic brains. In addition, FKBP51 targeting by small interfering RNA (siRNA) prevented the massive metastatic substitution of liver and lung in a mouse model of experimental metastasis. The present study provides evidence that the genetic programs of cancer stemness and invasiveness overlap in melanoma, and that FKBP51 plays a pivotal role in sustaining such a program. PMID:23559012

  3. Binding of TATA Binding Protein to a Naturally Positioned Nucleosome Is Facilitated by Histone Acetylation

    PubMed Central

    Sewack, Gerald F.; Ellis, Thomas W.; Hansen, Ulla

    2001-01-01

    The TATA sequence of the human, estrogen-responsive pS2 promoter is complexed in vivo with a rotationally and translationally positioned nucleosome (NUC T). Using a chromatin immunoprecipitation assay, we demonstrate that TATA binding protein (TBP) does not detectably interact with this genomic binding site in MCF-7 cells in the absence of transcriptional stimuli. Estrogen stimulation of these cells results in hyperacetylation of both histones H3 and H4 within the pS2 chromatin encompassing NUC T and the TATA sequence. Concurrently, TBP becomes associated with the pS2 promoter region. The relationship between histone hyperacetylation and the binding of TBP was assayed in vitro using an in vivo-assembled nucleosomal array over the pS2 promoter. With chromatin in its basal state, the binding of TBP to the pS2 TATA sequence at the edge of NUC T was severely restricted, consistent with our in vivo data. Acetylation of the core histones facilitated the binding of TBP to this nucleosomal TATA sequence. Therefore, we demonstrate that one specific, functional consequence of induced histone acetylation at a native promoter is the alleviation of nucleosome-mediated repression of the binding of TBP. Our data support a fundamental role for histone acetylation at genomic promoters in transcriptional activation by nuclear receptors and provide a general mechanism for rapid and reversible transcriptional activation from a chromatin template. PMID:11158325

  4. cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

    SciTech Connect

    Son, Jieun; Lee, Jong-Ho; Kim, Ha-Neui; Ha, Hyunil Lee, Zang Hee

    2010-07-23

    Research highlights: {yields} CREB is highly expressed in advanced breast cancer cells. {yields} Tumor-related factors such as TGF-{beta} further elevate CREB expression. {yields} CREB upregulation stimulates metastatic potential of breast cancer cells. {yields} CREB signaling is required for breast cancer-induced bone destruction. -- Abstract: cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-{beta}. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.

  5. A case of anti-nuclear matrix protein 2 antibody positive myopathy associated with lung cancer.

    PubMed

    Ohta, Shin; Unoda, Ki-Ichi; Nakajima, Hideto; Ikeda, Soichiro; Hamaguchi, Yasuhito; Kimura, Fumiharu

    2016-08-31

    Myositis-specific autoantibodies (MSAs) are associated with myositis. Anti-nuclear matrix protein 2 (NXP-2) antibody was recently identified as a major MSA and was observed mostly in juvenile dermatomyositis. We report the case of a 44-year-old man who presented with myopathy with anti-NXP-2 antibody and large cell carcinoma of the lung. He was hospitalized because of myalgia and edema of limbs. Neurological examination revealed mild proximal-dominant weakness in all four extremities, and laboratory studies showed elevated creatine kinase level (6,432 IU/l). Needle electromyography showed myogenic patterns. MRI of the lower limbs demonstrated inflammatory lesions in the thighs. Biopsied specimen from the left quadriceps femoris muscle showed mild mononuclear inflammatory infiltrate surrounding muscle fibres but no fiber necrosis. He was diagnosed with myopathy based on neurological examinations and clinical symptoms. His chest X-ray and CT showed tumor shadow on the right upper lung field, but CT didn't indicate the findings of interstitial lung disease. This was surgically removed, and a histological diagnosis of non-small cell lung cancer was suspected. He was also treated with definitive chemoradiotherapy before and after operation. His symptoms of myopathy promptly remitted with the preoperative chemotherapy. His serum analysis was positive for the anti-NXP-2. Further investigation and experience of MSAs are necessary to evaluate the therapeutic strategy against cancer-associated myopathy/myositis. PMID:27477574

  6. Pattern Formation in the Arabidopsis Embryo Revealed by Position-Specific Lipid Transfer Protein Gene Expression.

    PubMed Central

    Vroemen, C. W.; Langeveld, S.; Mayer, U.; Ripper, G.; Jurgens, G.; Van Kammen, A.; De Vries, S. C.

    1996-01-01

    During Arabidopsis embryogenesis, the zygote divides asymmetrically in the future apical-basal axis; however, a radial axis is initiated only within the eight-celled embryo. Mutations in the GNOM, KNOLLE, and KEULE genes affect these processes: gnom zygotes tend to divide symmetrically; knolle embryos lack oriented cell divisions that initiate protoderm formation; and in keule embryos, an outer cell layer is present that consists of abnormally enlarged cells from early development. Pattern formation along the two axes is reflected by the position-specific expression of the Arabidopsis lipid transfer protein (AtLTP1) gene. In wild-type embryos, the AtLTP1 gene is expressed in the protoderm and initially in all protodermal cells; later, AtLTP1 expression is confined to the cotyledons and the upper end of the hypocotyl. Analysis of AtLTP1 expression in gnom, knolle, and keule embryos showed that gnom embryos also can have no or reversed apical-basal polarity, whereas radial polarity is unaffected. knolle embryos initially lack but eventually form a radial pattern, and keule embryos are affected in protoderm cell morphology rather than in the establishment of the radial pattern. PMID:12239400

  7. Homologous metalloregulatory proteins from both gram-positive and gram-negative bacteria control transcription of mercury resistance operons

    SciTech Connect

    Helmann, J.D.; Walsh, C.T. ); Wang, Ying; Mahler, I. )

    1989-01-01

    The authors report the overexpression, purification, and properties of the regulatory protein, MerR, for a chromosomally encoded mercury resistance determinant from Bacillus strain RC607. This protein is similar in sequence to the metalloregulatory proteins encoded by gram-negative resistance determinants found on transposons Tn21 and Tn501 and to a predicted gene product of a Staphylococcus aureus resistance determinant. In vitro DNA-binding and transcription experiments were used to demonstrate those purified Bacillus MerR protein controls transcription from a promoter-operator site similar in sequence to that found in the transposon resistance determinants. The Bacillus MerR protein bound in vitro to its promoter-operator region in both the presence and absence of mercuric ion and functioned as a negative and positive regulator of transcription. The MerR protein bound less tightly to its operator region (ca. 50- to 100-fold) in the presence of mercuric ion; this reduced affinity was largely accounted for by an increased rate of dissociation of the MerR protein from the DNA. Despite this reduced DNA-binding affinity, genetic and biochemical evidence support a model in which the MerR protein-mercuric ion complex is a positive regulator of operon transcription. Although the Bacillus MerR protein bound only weakly to the heterologous Tn501 operator region, the Tn501 and Tn21 MerR proteins bound with high affinity to the Bacillus promoter-operator region and exhibited negative, but not positive, transcriptional control.

  8. Physical activity, sleep, and C-reactive protein as markers of positive health in resilient older men.

    PubMed

    Fields, Alison J; Hoyt, Robert E; Linnville, Steven E; Moore, Jeffery L

    2016-09-01

    This study explored whether physical activity and sleep, combined with the biomarker C-reactive protein, indexed positive health in older men. Many were former prisoners of war, with most remaining psychologically resilient and free of any psychiatric diagnoses. Activity and sleep were recorded through actigraphy in 120 veterans (86 resilient and 34 nonresilient) for 7 days. Resilient men had higher physical activity, significantly lower C-reactive protein levels, and 53 percent had lower cardiac-disease risk compared to nonresilient men. Sleep was adequate and not associated with C-reactive protein. Results suggest continued study is needed in actigraphy and C-reactive protein as means to index positive health. PMID:25673372

  9. Right Place, Right Time: Focalization of Membrane Proteins in Gram-Positive Bacteria.

    PubMed

    Mitra, Sumitra D; Afonina, Irina; Kline, Kimberly A

    2016-08-01

    Membrane proteins represent a significant proportion of total bacterial proteins and perform vital cellular functions ranging from exchanging metabolites and genetic material, secretion and sorting, sensing signal molecules, and cell division. Many of these functions are carried out at distinct foci on the bacterial membrane, and this subcellular localization can be coordinated by a number of factors, including lipid microdomains, protein-protein interactions, and membrane curvature. Elucidating the mechanisms behind focal protein localization in bacteria informs not only protein structure-function correlation, but also how to disrupt the protein function to limit virulence. Here we review recent advances describing a functional role for subcellular localization of membrane proteins involved in genetic transfer, secretion and sorting, cell division and growth, and signaling. PMID:27117048

  10. Positively charged amino acids are essential for electron transfer and protein-protein interactions in the soluble methane monooxygenase complex from Methylococcus capsulatus (Bath).

    PubMed

    Balendra, Suki; Lesieur, Claire; Smith, Thomas J; Dalton, Howard

    2002-02-26

    The soluble methane monooxygenase (sMMO) complex from Methylococcus capsulatus (Bath) catalyses oxygen- and NAD(P)H-dependent oxygenation of methane, propene, and other substrates. Whole-complex sMMO oxygenase activity requires all three sMMO components: the hydroxylase, the reductase, and protein B. Also, in the presence of hydrogen peroxide, the hydroxylase alone catalyzes substrate oxygenation via the peroxide shunt reaction. We investigated the effect of amine cross-linking on hydroxylase activity to probe the role of a gross conformational change that occurs in the hydroxylase upon binding of the other protein components. The cross-linker inhibited hydroxylase activity in the whole complex, but this effect was due to covalent modification of primary amine groups rather than cross-linking. Covalent modification of arginine side-chains on the hydroxylase had a similar effect, but, most remarkably, neither form of modification affected the activity of the hydroxylase via the peroxide shunt reaction. It was shown that covalent modification of positively charged groups on the hydroxylase, which occurred at multiple sites, interfered with its physical and functional interactions with protein B and with the passage of electrons from the reductase. These results indicate that protein B and the reductase of the sMMO complex interact via positively charged groups on the surface of the hydroxylase to induce a conformational change that is necessary for delivery of electrons into the active site of the hydroxylase. Modification of positively charged groups on protein B had no effect on its function, consistent with the hypothesis that positively charged groups on the hydroxylase interact with negative charges on protein B. Thus, we have discovered a means of specifically inactivating the interactions between the sMMO complex while preserving the catalytic activity of the hydroxylase active site which provides a new method of studying intercomponent interactions within s

  11. The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects.

    PubMed Central

    Roseman, R R; Pirrotta, V; Geyer, P K

    1993-01-01

    Mutations in the suppressor of Hairy-wing [su(Hw)] locus reverse the phenotype of a number of tissue-specific mutations caused by insertion of a gypsy retrotransposon. The su(Hw) gene encodes a zinc finger protein which binds to a 430 bp region of gypsy shown to be both necessary and sufficient for its mutagenic effects. su(Hw) protein causes mutations by inactivation of enhancer elements only when a su(Hw) binding region is located between these regulatory sequences and a promoter. To understand the molecular basis of enhancer inactivation, we tested the effects of su(Hw) protein on expression of the mini-white gene. We find that su(Hw) protein stabilizes mini-white gene expression from chromosomal position-effects in euchromatic locations by inactivating negative and positive regulatory elements present in flanking DNA. Furthermore, the su(Hw) protein partially protects transposon insertions from the negative effects of heterochromatin. To explain our current results, we propose that su(Hw) protein alters the organization of chromatin by creating a new boundary in a pre-existing domain of higher order chromatin structure. This separates enhancers and silencers distal to the su(Hw) binding region into an independent unit of gene activity, thereby causing their inactivation. Images PMID:8382607

  12. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    PubMed

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed. PMID:25246127

  13. Serine-rich protein is a novel positive regulator for silicon accumulation in mangrove.

    PubMed

    Sahebi, Mahbod; Hanafi, Mohamed M; Siti Nor Akmar, A; Rafii, Mohd Y; Azizi, Parisa; Idris, A S

    2015-02-10

    Silicon (Si) plays an important role in reducing plant susceptibility against a variety of different biotic and abiotic stresses; and also has an important regulatory role in soil to avoid heavy metal toxicity and providing suitable growing conditions for plants. A full-length cDNAs of 696bp of serine-rich protein was cloned from mangrove plant (Rhizophora apiculata) by amplification of cDNA ends from an expressed sequence tag homologous to groundnut (Arachis hypogaea), submitted to NCBI (KF211374). This serine-rich protein gene encodes a deduced protein of 223 amino acids. The transcript titre of the serine-rich protein was found to be strongly enriched in roots compared with the leaves of two month old mangrove plants and expression level of this serine-rich protein was found to be strongly induced when the mangrove seedlings were exposed to SiO2. Expression of the serine-rich protein transgenic was detected in transgenic Arabidopsis thaliana, where the amount of serine increased from 1.02 to 37.8mg/g. The same trend was also seen in Si content in the roots (14.3%) and leaves (7.4%) of the transgenic A. thaliana compared to the wild-type plants under Si treatment. The biological results demonstrated that the accumulation of the serine amino acid in the vegetative tissues of the transgenic plants enhanced their ability to absorb and accumulate more Si in the roots and leaves and suggests that the serine-rich protein gene has potential for use in genetic engineering of different stress tolerance characteristics. PMID:25479011

  14. Variations in Protein Concentration and Nitrogen Sources in Different Positions of Grain in Wheat

    PubMed Central

    Li, Xiangnan; Zhou, Longjing; Liu, Fulai; Zhou, Qin; Cai, Jian; Wang, Xiao; Dai, Tingbo; Cao, Weixing; Jiang, Dong

    2016-01-01

    The distribution patterns of total protein and protein components in different layers of wheat grain were investigated using the pearling technique, and the sources of different protein components and pearling fractions were identified using 15N isotope tracing methods. It was found that N absorbed from jointing to anthesis (JA) and remobilized to the grain after anthesis was the principal source of grain N, especially in the outer layer. For albumin and globulin, the amount of N absorbed during different stages all showed a decreasing trend from the surface layer to the center part. Whereas, for globulin and glutenin, the N absorbed after anthesis accounted for the main part indicating that for storage protein, the utilization of N assimilated after anthesis is greater than that of the stored N assimilated before anthesis. It is concluded that manipulation of the N application rate during different growth stages could be an effective approach to modulate the distribution of protein fractions in pearled grains for specific end-uses. PMID:27446169

  15. Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms

    PubMed Central

    Erkelenz, Steffen; Mueller, William F.; Evans, Melanie S.; Busch, Anke; Schöneweis, Katrin; Hertel, Klemens J.; Schaal, Heiner

    2013-01-01

    Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5′ splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection. PMID:23175589

  16. Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

    PubMed Central

    2010-01-01

    Background CC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions. In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events. Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML) were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR) gene family. The results of neutral vs. adaptive evolution (positive selection) hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or ω) >1. Results Of the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive selection code for amino acid

  17. Endothelin signaling activates Mef2c expression in the neural crest through a MEF2C-dependent positive-feedback transcriptional pathway.

    PubMed

    Hu, Jianxin; Verzi, Michael P; Robinson, Ashley S; Tang, Paul Ling-Fung; Hua, Lisa L; Xu, Shan-Mei; Kwok, Pui-Yan; Black, Brian L

    2015-08-15

    Endothelin signaling is essential for neural crest development, and dysregulated Endothelin signaling is associated with several neural crest-related disorders, including Waardenburg and other syndromes. However, despite the crucial roles of this pathway in neural crest development and disease, the transcriptional effectors directly activated by Endothelin signaling during neural crest development remain incompletely elucidated. Here, we establish that the MADS box transcription factor MEF2C is an immediate downstream transcriptional target and effector of Endothelin signaling in the neural crest. We show that Endothelin signaling activates Mef2c expression in the neural crest through a conserved enhancer in the Mef2c locus and that CRISPR-mediated deletion of this Mef2c neural crest enhancer from the mouse genome abolishes Endothelin induction of Mef2c expression. Moreover, we demonstrate that Endothelin signaling activates neural crest expression of Mef2c by de-repressing MEF2C activity through a Calmodulin-CamKII-histone deacetylase signaling cascade. Thus, these findings identify a MEF2C-dependent, positive-feedback mechanism for Endothelin induction and establish MEF2C as an immediate transcriptional effector and target of Endothelin signaling in the neural crest. PMID:26160899

  18. SubMito-PSPCP: Predicting Protein Submitochondrial Locations by Hybridizing Positional Specific Physicochemical Properties with Pseudoamino Acid Compositions

    PubMed Central

    Yu, Yuan

    2013-01-01

    Knowing the submitochondrial location of a mitochondrial protein is an important step in understanding its function. We developed a new method for predicting protein submitochondrial locations by introducing a new concept: positional specific physicochemical properties. With the framework of general form pseudoamino acid compositions, our method used only about 100 features to represent protein sequences, which is much simpler than the existing methods. On the dataset of SubMito, our method achieved over 93% overall accuracy, with 98.60% for inner membrane, 93.90% for matrix, and 70.70% for outer membrane, which are comparable to all state-of-the-art methods. As our method can be used as a general method to upgrade all pseudoamino-acid-composition-based methods, it should be very useful in future studies. We implement our method as an online service: SubMito-PSPCP. PMID:24027753

  19. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression.

    PubMed Central

    Wek, R C; Ramirez, M; Jackson, B M; Hinnebusch, A G

    1990-01-01

    GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae. GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression. Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells. Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions. Two of the GCN2(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety. Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro. These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation. Images PMID:2188100

  20. The seirena B Class Floral Homeotic Mutant of California Poppy (Eschscholzia californica) Reveals a Function of the Enigmatic PI Motif in the Formation of Specific Multimeric MADS Domain Protein Complexes[C][W][OA

    PubMed Central

    Lange, Matthias; Orashakova, Svetlana; Lange, Sabrina; Melzer, Rainer; Theißen, Günter; Smyth, David R.; Becker, Annette

    2013-01-01

    The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class–related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences. PMID:23444328

  1. Argonaute2 Protein in Rat Spermatogenic Cells Is Localized to Nuage Structures and LAMP2-Positive Vesicles Surrounding Chromatoid Bodies.

    PubMed

    Fujii, Yuki; Onohara, Yuko; Fujita, Hideaki; Yokota, Sadaki

    2016-04-01

    Localization of Argonaute2 (AGO2) protein-an essential component for the processing of small interfering RNA (siRNA)-directed RNA interference (RNAi) in RNA-induced silencing complex (RISC) in nuage of rat spermatogenic cells-was evaluated by immunofluorescence microscopy (IFM) and immunoelectron microscopy (IEM). AGO2 was shown, for the first time, to be localized to four previously classified types of nuage: irregularly shaped perinuclear granules (ISPGs), intermitochondrial cement (IMC), satellite bodies (SBs), and chromatoid bodies (CBs). Dual IEM staining for AGO2/Maelstrom (MAEL) protein or AGO2/MIWI protein demonstrated that AGO2 is colocalized with MAEL or MIWI proteins in these types of nuage. Dual IFM and IEM staining of AGO2/lysosomal-associated membrane protein 2 (LAMP2) showed that CB in round spermatids are in contact with and surrounded by LAMP2-positive vesicles, whereas nuage in pachytene spermatocytes are not. Taken together, our findings indicate that: (i) AGO2 in pachytene spermatocytes functions in ISPGs, IMC, and SBs; (ii) AGO2 in round spermatids functions in CBs, and that CBs are associated with lysosomal compartments. PMID:27029769

  2. Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

    PubMed Central

    Wan, Xiaohu; Cheerambathur, Dhanya; Gassmann, Reto; Suzuki, Aussie; Lawrimore, Josh; Desai, Arshad; Salmon, E.D.

    2013-01-01

    Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation. PMID:23979716

  3. Tbx Protein Level Critical for Clock-Mediated Somite Positioning Is Regulated through Interaction between Tbx and Ripply

    PubMed Central

    Wanglar, Chimwar; Takahashi, Jun; Yabe, Taijiro; Takada, Shinji

    2014-01-01

    Somitogenesis in vertebrates is a complex and dynamic process involving many sequences of events generated from the segmentation clock. Previous studies with mouse embryos revealed that the presumptive somite boundary is periodically created at the anterior border of the expression domain of Tbx6 protein. Ripply1 and Ripply2 are required for the determination of the Tbx6 protein border, but the mechanism by which this Tbx6 domain is regulated remains unclear. Furthermore, since zebrafish and frog Ripplys are known to be able to suppress Tbx6 function at the transcription level, it is also unclear whether Ripply-mediated mechanism of Tbx6 regulation is conserved among different species. Here, we tested the generality of Tbx6 protein-mediated process in somite segmentation by using zebrafish and further examined the mechanism of regulation of Tbx6 protein. By utilizing an antibody against zebrafish Tbx6/Fss, previously referred to as Tbx24, we found that the anterior border of Tbx6 domain coincided with the presumptive intersomitic boundary even in the zebrafish and it shifted dynamically during 1 cycle of segmentation. Consistent with the findings in mice, the tbx6 mRNA domain was located far anterior to its protein domain, indicating the possibility of posttranscriptional regulation. When both ripply1/2 were knockdown, the Tbx6 domain was anteriorly expanded. We further directly demonstrated that Ripply could reduce the expression level of Tbx6 protein depending on physical interaction between Ripply and Tbx6. Moreover, the onset of ripply1 and ripply2 expression occurred after reduction of FGF signaling at the anterior PSM, but this expression initiated much earlier on treatment with SU5402, a chemical inhibitor of FGF signaling. These results strongly suggest that Ripply is a direct regulator of the Tbx6 protein level for the establishment of intersomitic boundaries and mediates a reduction in FGF signaling for the positioning of the presumptive intersomitic

  4. The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein

    PubMed Central

    Yang, Wei-Cai; Ye, De; Xu, Jian; Sundaresan, Venkatesan

    1999-01-01

    The formation of haploid spores marks the initiation of the gametophytic phase of the life cycle of all vascular plants ranging from ferns to angiosperms. In angiosperms, this process is initiated by the differentiation of a subset of floral cells into sporocytes, which then undergo meiotic divisions to form microspores and megaspores. Currently, there is little information available regarding the genes and proteins that regulate this key step in plant reproduction. We report here the identification of a mutation, SPOROCYTELESS (SPL), which blocks sporocyte formation in Arabidopsis thaliana. Analysis of the SPL mutation suggests that development of the anther walls and the tapetum and microsporocyte formation are tightly coupled, and that nucellar development may be dependent on megasporocyte formation. Molecular cloning of the SPL gene showed that it encodes a novel nuclear protein related to MADS box transcription factors and that it is expressed during microsporogenesis and megasporogenesis. These data suggest that the SPL gene product is a transcriptional regulator of sporocyte development in Arabidopsis. PMID:10465788

  5. Unbound position II in MXCXXC metallochaperone model peptides impacts metal binding mode and reactivity: Distinct similarities to whole proteins.

    PubMed

    Shoshan, Michal S; Dekel, Noa; Goch, Wojciech; Shalev, Deborah E; Danieli, Tsafi; Lebendiker, Mario; Bal, Wojciech; Tshuva, Edit Y

    2016-06-01

    The effect of position II in the binding sequence of copper metallochaperones, which varies between Thr and His, was investigated through structural analysis and affinity and oxidation kinetic studies of model peptides. A first Cys-Cu(I)-Cys model obtained for the His peptide at acidic and neutral pH, correlated with higher affinity and more rapid oxidation of its complex; in contrast, the Thr peptide with the Cys-Cu(I)-Met coordination under neutral conditions demonstrated weaker and pH dependent binding. Studies with human antioxidant protein 1 (Atox1) and three of its mutants where S residues were replaced with Ala suggested that (a) the binding affinity is influenced more by the binding sequence than by the protein fold (b) pH may play a role in binding reactivity, and (c) mutating the Met impacted the affinity and oxidation rate more drastically than did mutating one of the Cys, supporting its important role in protein function. Position II thus plays a dominant role in metal binding and transport. PMID:26901629

  6. Identification of Positively Charged Residues in Enterovirus 71 Capsid Protein VP1 Essential for Production of Infectious Particles

    PubMed Central

    Yuan, Shilin; Li, Guiming; Wang, Ying; Gao, Qianqian; Wang, Yizhuo; Cui, Rui

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children, which can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues in VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine changes were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra- and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to the EV71 entry inhibitor suramin, which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles. IMPORTANCE Infection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are

  7. Structure–function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

    PubMed Central

    Manuse, Sylvie; Jean, Nicolas L.; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M.; VanNieuwenhze, Michael S.; Grangeasse, Christophe; Simorre, Jean-Pierre

    2016-01-01

    Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure–function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division. PMID:27346279

  8. Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ.

    PubMed

    Manuse, Sylvie; Jean, Nicolas L; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M; VanNieuwenhze, Michael S; Grangeasse, Christophe; Simorre, Jean-Pierre

    2016-01-01

    Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division. PMID:27346279

  9. Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

    NASA Astrophysics Data System (ADS)

    Manuse, Sylvie; Jean, Nicolas L.; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M.; Vannieuwenhze, Michael S.; Grangeasse, Christophe; Simorre, Jean-Pierre

    2016-06-01

    Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division.

  10. MIKC* MADS-Protein Complexes Bind Motifs Enriched in the Proximal Region of Late Pollen-Specific Arabidopsis Promoters[W

    PubMed Central

    Verelst, Wim; Saedler, Heinz; Münster, Thomas

    2007-01-01

    The genome of Arabidopsis (Arabidopsis thaliana) encodes over 100 MADS-domain transcription factors, categorized into five phylogenetic subgroups. Most research efforts have focused on just one of these subgroups (MIKCc), whereas the other four remain largely unexplored. Here, we report on five members of the so-called Mδ or Arabidopsis MIKC* (AtMIKC*) subgroup, which are predominantly expressed during the late stages of pollen development. Very few MADS-box genes function in mature pollen, and from this perspective, the AtMIKC* genes are therefore highly exceptional. We found that the AtMIKC* proteins are able to form multiple heterodimeric complexes in planta, and that these protein complexes exhibit a for the MADS-family unusual and high DNA binding specificity in vitro. Compared to their occurrence in promoters genome wide, AtMIKC* binding sites are strongly overrepresented in the proximal region of late pollen-specific promoters. By combining our experimental data with in silico genomics and pollen transcriptomics approaches, we identified a considerable number of putative direct target genes of the AtMIKC* transcription factor complexes in pollen, many of which have known or proposed functions in pollen tube growth. The expression of several of these predicted targets is altered in mutant pollen in which all AtMIKC* complexes are affected, and in vitro germination of this mutant pollen is severely impaired. Our data therefore suggest that the AtMIKC* protein complexes play an essential role in transcriptional regulation during late pollen development. PMID:17071640

  11. STAP-2 Protein Expression in B16F10 Melanoma Cells Positively Regulates Protein Levels of Tyrosinase, Which Determines Organs to Infiltrate in the Body*

    PubMed Central

    Sekine, Yuichi; Togi, Sumihito; Muromoto, Ryuta; Kon, Shigeyuki; Kitai, Yuichi; Yoshimura, Akihiko; Oritani, Kenji; Matsuda, Tadashi

    2015-01-01

    Melanoma is the most serious type of skin cancer, with a highly metastatic phenotype. In this report, we show that signal transducing adaptor protein 2 (STAP-2) is involved in cell migration, proliferation, and melanogenesis as well as chemokine receptor expression and tumorigenesis in B16F10 melanoma cells. This was evident in mice injected with STAP-2 shRNA (shSTAP-2)-expressing B16F10 cells, which infiltrated organs in a completely different pattern from the original cells, showing massive colonization in the liver, kidney, and neck but not in the lung. The most important finding was that STAP-2 expression determined tyrosinase protein content. STAP-2 colocalized with tyrosinase in lysosomes and protected tyrosinase from protein degradation. It is noteworthy that B16F10 cells with knocked down tyrosinase showed similar cell characteristics as shSTAP-2 cells. These results indicated that tyrosinase contributed to some cellular events beyond melanogenesis. Taken together, one possibility is that STAP-2 positively regulates the protein levels of tyrosinase, which determines tumor invasion via controlling chemokine receptor expression. PMID:26023234

  12. Estimation of Position Specific Energy as a Feature of Protein Residues from Sequence Alone for Structural Classification.

    PubMed

    Iqbal, Sumaiya; Hoque, Md Tamjidul

    2016-01-01

    A set of features computed from the primary amino acid sequence of proteins, is crucial in the process of inducing a machine learning model that is capable of accurately predicting three-dimensional protein structures. Solutions for existing protein structure prediction problems are in need of features that can capture the complexity of molecular level interactions. With a view to this, we propose a novel approach to estimate position specific estimated energy (PSEE) of a residue using contact energy and predicted relative solvent accessibility (RSA). Furthermore, we demonstrate PSEE can be reasonably estimated based on sequence information alone. PSEE is useful in identifying the structured as well as unstructured or, intrinsically disordered region of a protein by computing favorable and unfavorable energy respectively, characterized by appropriate threshold. The most intriguing finding, verified empirically, is the indication that the PSEE feature can effectively classify disorder versus ordered residues and can segregate different secondary structure type residues by computing the constituent energies. PSEE values for each amino acid strongly correlate with the hydrophobicity value of the corresponding amino acid. Further, PSEE can be used to detect the existence of critical binding regions that essentially undergo disorder-to-order transitions to perform crucial biological functions. Towards an application of disorder prediction using the PSEE feature, we have rigorously tested and found that a support vector machine model informed by a set of features including PSEE consistently outperforms a model with an identical set of features with PSEE removed. In addition, the new disorder predictor, DisPredict2, shows competitive performance in predicting protein disorder when compared with six existing disordered protein predictors. PMID:27588752

  13. Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

    PubMed Central

    Tapprich, W E; Goss, D J; Dahlberg, A E

    1989-01-01

    A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis. PMID:2662189

  14. Functional divergence within class B MADS-box genes TfGLO and TfDEF in Torenia fournieri Lind

    PubMed Central

    Sasaki, Katsutomo; Aida, Ryutaro; Yamaguchi, Hiroyasu; Shikata, Masahito; Niki, Tomoya; Nishijima, Takaaki

    2010-01-01

    Homeotic class B genes GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) are involved in the development of petals and stamens in Arabidopsis. However, functions of these genes in the development of floral organs in torenia are less well known. Here, we demonstrate the unique floral phenotypes of transgenic torenia formed due to the modification of class B genes, TfGLO and TfDEF. TfGLO-overexpressing plants showed purple-stained sepals that accumulated anthocyanins in a manner similar to that of petals. TfGLO-suppressed plants showed serrated petals and TfDEF-suppressed plants showed partially decolorized petals. In TfGLO-overexpressing plants, cell shapes on the surfaces of sepals were altered to petal-like cell shapes. Furthermore, TfGLO- and TfDEF-suppressed plants partially had sepal-like cells on the surfaces of their petals. We isolated putative class B gene-regulated genes and examined their expression in transgenic plants. Three xyloglucan endo-1,4-beta-d-glucanase genes were up-regulated in TfGLO- and TfDEF-overexpressing plants and down-regulated in TfGLO- and TfDEF-suppressed plants. In addition, 10 anthocyanin biosynthesis-related genes, including anthocyanin synthase and chalcone isomerase, were up-regulated in TfGLO-overexpressing plants and down-regulated in TfGLO-suppressed plants. The expression patterns of these 10 genes in TfDEF transgenic plants were diverse and classified into several groups. HPLC analysis indicated that sepals of TfGLO-overexpressing plants accumulate the same type of anthocyanins and flavones as wild-type plants. The difference in phenotypes and expression patterns of the 10 anthocyanin biosynthesis-related genes between TfGLO and TfDEF transgenic plants indicated that TfGLO and TfDEF have partial functional divergence, while they basically work synergistically in torenia. Electronic supplementary material The online version of this article (doi:10.1007/s00438-010-0574-z) contains supplementary material, which is available to authorized users. PMID:20872230

  15. Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

    PubMed Central

    Isensee, Jörg; Wenzel, Carsten; Buschow, Rene; Weissmann, Robert; Kuss, Andreas W.; Hucho, Tim

    2014-01-01

    Normal and painful stimuli are detected by specialized subgroups of peripheral sensory neurons. The understanding of the functional differences of each neuronal subgroup would be strongly enhanced by knowledge of the respective subgroup transcriptome. The separation of the subgroup of interest, however, has proven challenging as they can hardly be enriched. Instead of enriching, we now rapidly eliminated the subgroup of neurons expressing the heat-gated cation channel TRPV1 from dissociated rat sensory ganglia. Elimination was accomplished by brief treatment with TRPV1 agonists followed by the removal of compromised TRPV1(+) neurons using density centrifugation. By differential microarray and sequencing (RNA-Seq) based expression profiling we compared the transcriptome of all cells within sensory ganglia versus the same cells lacking TRPV1 expressing neurons, which revealed 240 differentially expressed genes (adj. p<0.05, fold-change>1.5). Corroborating the specificity of the approach, many of these genes have been reported to be involved in noxious heat or pain sensitization. Beyond the expected enrichment of ion channels, we found the TRPV1 transcriptome to be enriched for GPCRs and other signaling proteins involved in adenosine, calcium, and phosphatidylinositol signaling. Quantitative population analysis using a recent High Content Screening (HCS) microscopy approach identified substantial heterogeneity of expressed target proteins even within TRPV1-positive neurons. Signaling components defined distinct further subgroups within the population of TRPV1-positive neurons. Analysis of one such signaling system showed that the pain sensitizing prostaglandin PGD2 activates DP1 receptors expressed predominantly on TRPV1(+) neurons. In contrast, we found the PGD2 producing prostaglandin D synthase to be expressed exclusively in myelinated large-diameter neurons lacking TRPV1, which suggests a novel paracrine neuron-neuron communication. Thus, subgroup analysis based

  16. Positive Selection in Bone Morphogenetic Protein 15 Targets a Natural Mutation Associated with Primary Ovarian Insufficiency in Human

    PubMed Central

    Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  17. Positive selection in bone morphogenetic protein 15 targets a natural mutation associated with primary ovarian insufficiency in human.

    PubMed

    Auclair, Sylvain; Rossetti, Raffaella; Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  18. Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

    PubMed Central

    Draberova, Lubica; Bugajev, Viktor; Potuckova, Lucie; Halova, Ivana; Bambouskova, Monika; Polakovicova, Iva; Xavier, Ramnik J.; Seed, Brian

    2014-01-01

    The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved. PMID:25246632

  19. A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes

    PubMed Central

    Thomson-Luque, Richard; Torres, Letícia de Menezes; Gunalan, Karthigayan; Carvalho, Luzia H.

    2016-01-01

    ABSTRACT Erythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71high) Duffy-positive (Fy+) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes. PMID:27555313

  20. Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2008-01-01

    The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the 1La and 1Lb components. The relative intensities of the 0-0 transitions of the 1Lb bands correlate linearly with the solvent polarity values (ETN). A novel strategy, which utilizes a set of the experimental 1Lb bands, was employed to resolve the near-UV CD spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence as well as structural data of TL. Analysis of the 1Lb bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The “DMSO-like” 1Lb band of Trp CD spectra may be used as a “fingerprint” to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side-chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method we call site-directed circular dichroism is broadly applicable to various proteins to obtain the position-specific data. PMID:18047823

  1. Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2008-03-15

    The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the (1)L(a) and (1)L(b) components. The relative intensities of the 0-0 transitions of the (1)L(b) bands correlate linearly with the solvent polarity values (E(T)(N)). A novel strategy that uses a set of the experimental (1)L(b) bands was employed to resolve the near-UV circular dichroism (CD) spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence and structural data of TL. Analysis of the (1)L(b) bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The dimethyl sulfoxide (DMSO)-like (1)L(b) band of Trp CD spectra may be used as a "fingerprint" to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method, which we call site-directed circular dichroism, is broadly applicable to various proteins to obtain the position-specific data. PMID:18047823

  2. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes.

    PubMed

    Gryaznova, Yuliya; Koca Caydasi, Ayse; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. PMID:27159239

  3. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

    PubMed Central

    Gryaznova, Yuliya; Caydasi, Ayse Koca; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001 PMID:27159239

  4. The asymmetric protein expression hypothesis - Explaining the unilaterality of HLA-B27-positive acute anterior uveitides.

    PubMed

    Clarke, Margo S; Plouznikoff, Alexandre; Deschênes, Jean

    2016-03-01

    For reasons still unclear, most HLA-B27-positive acute anterior uveitides occur unilaterally. Building upon the growing literature showing that left-right asymmetry exist at the biomolecular and at the cellular levels, we propose a new hypothesis to explain why HLA-B27-positive acute anterior uveitides tend to affect one eye selectively. We postulate that left and right uveal tissue may present quantitatively and qualitatively different proteins to the immune system, capable to trigger an autoimmune response, and that other variables, including anatomical, cellular and molecular barriers, as well as our own eye-derived immunological tolerance and immune suppressive intraocular microenvironment may also be unequally distributed, and impact differently the immune privileges of the left and right eye. These same quantitative and qualitative differences might also explain why HLA-B27-positive acute anterior uveitides can flip-flop between the left and the right eye, after the first attack. By trying to figure out why one eye is targeted by an autoimmune reaction while the other is clinically unaffected, we might be able to better understand how and why an autoimmune reaction starts. Hopefully, this will help us devise better treatments for ocular autoimmune diseases, and contribute to the management of autoinflammatory conditions with a marked asymmetric clinical presentation in other fields. PMID:26880626

  5. Toll-Interleukin 1 Receptor domain-containing adaptor protein positively regulates BV2 cell M1 polarization.

    PubMed

    Gong, Leilei; Wang, Hanxiang; Sun, Xiaolei; Liu, Chun; Duan, Chengwei; Cai, Rixin; Gu, Xingxing; Zhu, Shunxing

    2016-06-01

    Microglial activation, including classical (M1) and alternative (M2) activation, plays important roles in the development of several central nervous system disorders and promotes tissue reconstruction. Toll-like receptor (TLR)4 is important for microglial polarization. TIR domain-containing adaptor protein (TIRAP) is an intracellular adaptor protein, which is responsible for the early phase of TLR4 activation. The role of TIRAP in BV2 cell M1 polarization is still unknown. In this study, we showed that TIRAP expression is greatly elevated in lipopolysaccharide (LPS)/interferon (IFN)-γ-treated microglia. TIRAP overexpression promoted BV2 microglial M1 polarization by increasing M1-related marker production (inducible nitric oxide synthase, CD86, interleukin-6, interleukin-1β and tumour necrosis factor-α). In contrast, TIRAP knockdown prevented M1-related marker production. Mechanistically, TIRAP could interact with TNF Receptor-Associated Factor 6 (TRAF6) to increase M1-related marker production in TIRAP overexpressed and LPS/IFN-γ-treated BV2 cells. In addition, silencing of TIRAP effectively inhibited the activation of the Transforming Growth Factor-Beta-Activated Kinase 1/I-Kappa-B Kinase /Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B-Cells (TAK1/IKK/NF-κB) signalling pathway and the phosphorylation of Akt and mitogen-activated protein kinases, which were activated by LPS/IFN-γ stimulation. Thus, our results suggest that TIRAP positively regulated BV2 microglial M1 polarization through TLR4-mediated TAK1/IKK/NF-κB, mitogen-activated protein kinases and Akt signalling pathways. PMID:27061018

  6. Phenotypic Consequences Resulting from a Methionine-to-Valine Substitution at Position 48 in the HPr Protein of Streptococcus salivarius

    PubMed Central

    Plamondon, Pascale; Brochu, Denis; Thomas, Suzanne; Fradette, Julie; Gauthier, Lucie; Vaillancourt, Katy; Buckley, Nicole; Frenette, Michel; Vadeboncoeur, Christian

    1999-01-01

    In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His15) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser46) by an ATP-dependent protein kinase (His∼P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His15 by EI nor the phosphorylation at Ser46 by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His∼P) than the wild-type strain, while the levels of free HPr and HPr(His∼P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of α-galactosidase, β-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule. PMID:10559156

  7. Arabidopsis cold shock domain proteins: relationships to floral and silique development

    PubMed Central

    Nakaminami, Kentaro; Hill, Kristine; Perry, Sharyn E.; Sentoku, Naoki; Long, Jeffrey A.; Karlson, Dale T.

    2009-01-01

    Cold shock domain proteins (CSPs) are highly conserved from bacteria to higher plants and animals. Bacterial cold shock proteins function as RNA chaperones by destabilizing RNA secondary structures and promoting translation as an adaptative mechanism to low temperature stress. In animals, cold shock domain proteins exhibit broad functions related to growth and development. In order to understand better the function of CSPs in planta, detailed analyses were performed for Arabidopsis thaliana CSPs (AtCSPs) on the transcript and protein levels using an extensive series of tissue harvested throughout developmental stages within the entire life cycle of Arabidopsis. On both the transcript and protein levels, AtCSPs were enriched in shoot apical meristems and siliques. Although all AtCSPs exhibited similar expression patterns, AtCSP2 was the most abundantly expressed gene. In situ hybridization analyses were also used to confirm that AtCSP2 and AtCSP4 transcripts accumulate in developing embryos and shoot apices. AtCSPs transcripts were also induced during a controlled floral induction study. In vivo ChIP analysis confirmed that an embryo expressed MADS box transcription factor, AGL15, interacts within two AtCSP promoter regions and alters the respective patterns of AtCSP transcription. Comparative analysis of AtCSP gene expression between Landsberg and Columbia ecotypes confirmed a 1000-fold reduction of AtCSP4 gene expression in the Landsberg background. Analysis of the AtCSP4 genomic locus identified multiple polymorphisms in putative regulatory cis-elements between the two ecotypes. Collectively, these data support the hypothesis that AtCSPs are involved in the transition to flowering and silique development in Arabidopsis. PMID:19269998

  8. The Immunologic Properties of Bone Morphogenic Protein Receptor IB Positive Subpopulation before and after Osteogenic Differentiation in Mouse Dermis

    PubMed Central

    Wang, Tao; Xu, Hua; Zhang, Yi; Dong, Jia-Sheng

    2016-01-01

    We have previously reported that human dermal bone morphogenic protein receptor (BMPR) IB positive subpopulation had a high osteogenic differentiation potential and may be a promising cell source for allogeneic bone tissue engineering. In this study, the immunologic properties of dermal BMPR-IB+ subpopulation before and after osteogenic differentiation were reported. The results confirmed that dermal BMPR-IB+ cells possessed a similar osteogenic differentiation potential with bone marrow mesenchymal stromal cells in a mouse model. Furthermore, the expression of immune rejection-related surface antigens such as major histocompatibility class II and co-stimulatory proteins (CD40, CD80, and CD86) were absent on dermal BMPRIB+ cells. Dermal BMPRIB+ cells elicited no proliferation of allogeneic splenocytes and suppressed the proliferation of stimulated immune cells. Interestingly, osteogenic differentiation in vitro had no adverse effect on the immunological features of these cells. Most importantly, inducible NO synthase (iNOS) was involved in immunoregulatory effects by undifferentiated BMPRIB+ fibroblasts, whereas indoleamine 2,3-dioxygenase (IDO) activity was related to mediating immunomodulatory function by osteogenic differentiated BMPRIB+ fibroblasts. In conclusion, dermal BMPRIB+ cells have a low immunogenicity and possess immunosuppressive capacity before and after osteogenic differentiation in vitro, which would facilitate the allotransplantation in the future. However, mechanisms mediating immunoregulatory property between undifferentiated and osteogenic differentiated BMPRIB+ fibroblasts may be different and need further investigation. PMID:27552226

  9. The Immunologic Properties of Bone Morphogenic Protein Receptor IB Positive Subpopulation before and after Osteogenic Differentiation in Mouse Dermis.

    PubMed

    He, Jin-Guang; Wang, Ting-Liang; Wang, Tao; Xu, Hua; Zhang, Yi; Dong, Jia-Sheng

    2016-01-01

    We have previously reported that human dermal bone morphogenic protein receptor (BMPR) IB positive subpopulation had a high osteogenic differentiation potential and may be a promising cell source for allogeneic bone tissue engineering. In this study, the immunologic properties of dermal BMPR-IB+ subpopulation before and after osteogenic differentiation were reported. The results confirmed that dermal BMPR-IB+ cells possessed a similar osteogenic differentiation potential with bone marrow mesenchymal stromal cells in a mouse model. Furthermore, the expression of immune rejection-related surface antigens such as major histocompatibility class II and co-stimulatory proteins (CD40, CD80, and CD86) were absent on dermal BMPRIB+ cells. Dermal BMPRIB+ cells elicited no proliferation of allogeneic splenocytes and suppressed the proliferation of stimulated immune cells. Interestingly, osteogenic differentiation in vitro had no adverse effect on the immunological features of these cells. Most importantly, inducible NO synthase (iNOS) was involved in immunoregulatory effects by undifferentiated BMPRIB+ fibroblasts, whereas indoleamine 2,3-dioxygenase (IDO) activity was related to mediating immunomodulatory function by osteogenic differentiated BMPRIB+ fibroblasts. In conclusion, dermal BMPRIB+ cells have a low immunogenicity and possess immunosuppressive capacity before and after osteogenic differentiation in vitro, which would facilitate the allotransplantation in the future. However, mechanisms mediating immunoregulatory property between undifferentiated and osteogenic differentiated BMPRIB+ fibroblasts may be different and need further investigation. PMID:27552226

  10. Piezometric biosensors for anti-apoptotic protein survivin based on buried positive-potential barrier and immobilized monoclonal antibodies.

    PubMed

    Stobiecka, Magdalena; Chalupa, Agata; Dworakowska, Beata

    2016-10-15

    The anti-apoptotic protein survivin (Sur) plays an important role in the regulation of cell division and inducing the chemotherapeutic drug resistance. The Sur protein and its mRNA have recently been studied as cancer biomarkers and potential targets for cancer therapy. In this work, we have focused on the design of immunosensors for the detection of Sur based on buried positive-potential barrier layer structure and anti-survivin antibody. The modification of solid AuQC piezoelectrodes was monitored by recording the resonance frequency shift and electrochemical measurements during each step of the sensor preparation. Our results indicate that the immunosensor with covalently bound monoclonal anti-survivin antibody can detect Sur with the limit of detection, LOD=1.7nM (S/N=3σ). The immunosensor applicability for the analysis of real samples was assessed by testing samples of cell lysate solutions obtained from human astrocytoma (glioblastoma) U-87MG cell line, with the experiments performed using the standard addition method. The good linearity of the calibration curves for PBS and lysate solutions at low Sur concentrations confirm the high specificity of the proposed biosensor and good discrimination against nonspecific interactions with lysate components. The calculations indicate that there is still room to increase the Sur capture capacity for Sur while miniaturizing the sensor. The important advantage of the sensor is that it can be reused by a simple regeneration procedure. PMID:26507667

  11. High Expression of PTGR1 Promotes NSCLC Cell Growth via Positive Regulation of Cyclin-Dependent Protein Kinase Complex

    PubMed Central

    Zhou, Weihe; Zhang, Yuefeng; Liu, Yong

    2016-01-01

    Lung cancer has been the most common cancer and the main cause of cancer-related deaths worldwide for several decades. PTGR1 (prostaglandin reductase 1), as a bifunctional enzyme, has been involved in the occurrence and progression of cancer. However, its impact on human lung cancer is rarely reported. In this study, we found that PTGR1 was overexpressed in lung cancer based on the analyses of Oncomine. Moreover, lentivirus-mediated shRNA knockdown of PTGR1 reduced cell viability in human lung carcinoma cells 95D and A549 by MTT and colony formation assay. PTGR1 depletion led to G2/M phase cell cycle arrest and increased the proportion of apoptotic cells in 95D cells by flow cytometry. Furthermore, silencing PTGR1 in 95D cells resulted in decreased levels of cyclin-dependent protein kinase complex (CDK1, CDK2, cyclin A2, and cyclin B1) by western blotting and then PTGR1 is positively correlated with cyclin-dependent protein by using the data mining of the Oncomine database. Therefore, our findings suggest that PTGR1 may play a role in lung carcinogenesis through regulating cell proliferation and is a potential new therapeutic strategy for lung cancer. PMID:27429979

  12. Positive Role of Promyelocytic Leukemia Protein in Type I Interferon Response and Its Regulation by Human Cytomegalovirus

    PubMed Central

    Kim, Young-Eui; Ahn, Jin-Hyun

    2015-01-01

    Promyelocytic leukemia protein (PML), a major component of PML nuclear bodies (also known as nuclear domain 10), is involved in diverse cellular processes such as cell proliferation, apoptosis, gene regulation, and DNA damage response. PML also acts as a restriction factor that suppresses incoming viral genomes, therefore playing an important role in intrinsic defense. Here, we show that PML positively regulates type I interferon response by promoting transcription of interferon-stimulated genes (ISGs) and that this regulation by PML is counteracted by human cytomegalovirus (HCMV) IE1 protein. Small hairpin RNA-mediated PML knockdown in human fibroblasts reduced ISG induction by treatment of interferon-β or infection with UV-inactivated HCMV. PML was required for accumulation of activated STAT1 and STAT2, interacted with them and HDAC1 and HDAC2, and was associated with ISG promoters after HCMV infection. During HCMV infection, viral IE1 protein interacted with PML, STAT1, STAT2, and HDACs. Analysis of IE1 mutant viruses revealed that, in addition to the STAT2-binding domain, the PML-binding domain of IE1 was necessary for suppression of interferon-β-mediated ISG transcription, and that IE1 inhibited ISG transcription by sequestering interferon-stimulated gene factor 3 (ISGF3) in a manner requiring its binding of PML and STAT2, but not of HDACs. In conclusion, our results demonstrate that PML participates in type I interferon-induced ISG expression by regulating ISGF3, and that this regulation by PML is counteracted by HCMV IE1, highlighting a widely shared viral strategy targeting PML to evade intrinsic and innate defense mechanisms. PMID:25812002

  13. Arabidopsis Flower and Embryo Developmental Genes are Repressed in Seedlings by Different Combinations of Polycomb Group Proteins in Association with Distinct Sets of Cis-regulatory Elements

    PubMed Central

    Liu, Jian; Zhang, Lei; He, Chongsheng; Shen, Wen-Hui; Jin, Hong; Xu, Lin; Zhang, Yijing

    2016-01-01

    Polycomb repressive complexes (PRCs) play crucial roles in transcriptional repression and developmental regulation in both plants and animals. In plants, depletion of different members of PRCs causes both overlapping and unique phenotypic defects. However, the underlying molecular mechanism determining the target specificity and functional diversity is not sufficiently characterized. Here, we quantitatively compared changes of tri-methylation at H3K27 in Arabidopsis mutants deprived of various key PRC components. We show that CURLY LEAF (CLF), a major catalytic subunit of PRC2, coordinates with different members of PRC1 in suppression of distinct plant developmental programs. We found that expression of flower development genes is repressed in seedlings preferentially via non-redundant role of CLF, which specifically associated with LIKE HETEROCHROMATIN PROTEIN1 (LHP1). In contrast, expression of embryo development genes is repressed by PRC1-catalytic core subunits AtBMI1 and AtRING1 in common with PRC2-catalytic enzymes CLF or SWINGER (SWN). This context-dependent role of CLF corresponds well with the change in H3K27me3 profiles, and is remarkably associated with differential co-occupancy of binding motifs of transcription factors (TFs), including MADS box and ABA-related factors. We propose that different combinations of PRC members distinctively regulate different developmental programs, and their target specificity is modulated by specific TFs. PMID:26760036

  14. Effects of detraining on the temporal expression of positive and negative angioregulatory proteins in skeletal muscle of mice.

    PubMed

    Olenich, Sara A; Audet, Gerald N; Roberts, Kathleen A; Olfert, I Mark

    2014-08-01

    Temporal expression of positive and negative angiogenic factors in response to detraining is poorly understood. We report the protein expression of anti-angiogenic peptides (thrombospondin-1, TSP-1; and endostatin) as well as pro-angiogenic factors (vascular endothelial growth factor, VEGF; matrix metalloproteinases-2 and -9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) in response to detraining in triceps surae muscles of C57BL/6 mice. Male mice were allowed to exercise voluntarily for 21 days, and then basal and acute response to exercise were evaluated at 1, 7, 14 and 28 days detraining (D1, D7, D14, D28, respectively, n = 12/group). As seen in the D1 mice, training resulted in the increased muscle capillary-to-fibre ratio (C/F), increased maximal running time and elevated basal expression of VEGF and matrix metalloproteinase-9 (P < 0.05). After 7 days of detraining (D7), C/F levels were similar to control levels, but both basal VEGF and TSP-1 were elevated (P < 0.05). At D14 and D28, TSP-1 protein was not different compared to baseline levels; however, VEGF was elevated in gastrocnemius (GA), but not the soleus (SOL) or plantaris (PLT) muscles, of D14 mice. Endostatin tended to decrease in D14 and D28 compared to controls. Timing of nucleolin protein expression differed between muscle groups, with increases at D1, D7 and D14 in the PLT, SOL and GA muscles, respectively. The response of VEGF and nucleolin to acute exercise was blunted with training, and remained blunted in the PLT and SOL even after 28 days of detraining, at a time point long after muscle capillarization was observed to be similar to pre-training levels. These data suggest that TSP-1 may be a mediator of capillary regression with detraining, even in the face of elevated VEGF, suggesting that pro-angiogenic regulators may not be able to prevent the regression of skeletal muscle capillaries under physiological conditions. The responses of matrix

  15. Effects of detraining on the temporal expression of positive and negative angioregulatory proteins in skeletal muscle of mice

    PubMed Central

    Olenich, Sara A; Audet, Gerald N; Roberts, Kathleen A; Olfert, I Mark

    2014-01-01

    Temporal expression of positive and negative angiogenic factors in response to detraining is poorly understood. We report the protein expression of anti-angiogenic peptides (thrombospondin-1, TSP-1; and endostatin) as well as pro-angiogenic factors (vascular endothelial growth factor, VEGF; matrix metalloproteinases-2 and -9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) in response to detraining in triceps surae muscles of C57BL/6 mice. Male mice were allowed to exercise voluntarily for 21 days, and then basal and acute response to exercise were evaluated at 1, 7, 14 and 28 days detraining (D1, D7, D14, D28, respectively, n = 12/group). As seen in the D1 mice, training resulted in the increased muscle capillary-to-fibre ratio (C/F), increased maximal running time and elevated basal expression of VEGF and matrix metalloproteinase-9 (P < 0.05). After 7 days of detraining (D7), C/F levels were similar to control levels, but both basal VEGF and TSP-1 were elevated (P < 0.05). At D14 and D28, TSP-1 protein was not different compared to baseline levels; however, VEGF was elevated in gastrocnemius (GA), but not the soleus (SOL) or plantaris (PLT) muscles, of D14 mice. Endostatin tended to decrease in D14 and D28 compared to controls. Timing of nucleolin protein expression differed between muscle groups, with increases at D1, D7 and D14 in the PLT, SOL and GA muscles, respectively. The response of VEGF and nucleolin to acute exercise was blunted with training, and remained blunted in the PLT and SOL even after 28 days of detraining, at a time point long after muscle capillarization was observed to be similar to pre-training levels. These data suggest that TSP-1 may be a mediator of capillary regression with detraining, even in the face of elevated VEGF, suggesting that pro-angiogenic regulators may not be able to prevent the regression of skeletal muscle capillaries under physiological conditions. The responses of matrix

  16. OVATE Family Protein 8 Positively Mediates Brassinosteroid Signaling through Interacting with the GSK3-like Kinase in Rice

    PubMed Central

    He, Yong; Tian, Zhihong; Li, Jianxiong

    2016-01-01

    OVATE gene was first identified as a key regulator of fruit shape in tomato. OVATE family proteins (OFPs) are characterized as plant-specific transcription factors and conserved in Arabidopsis, tomato, and rice. Roles of OFPs involved in plant development and growth are largely unknown. Brassinosteroids (BRs) are a class of steroid hormones involved in diverse biological functions. OsGKS2 plays a critical role in BR signaling by phosphorylating downstream components such as OsBZR1 and DLT. Here we report in rice that OsOFP8 plays a positive role in BR signaling pathway. BL treatment induced the expression of OsOFP8 and led to enhanced accumulation of OsOFP8 protein. The gain-of-function mutant Osofp8 and OsOFP8 overexpression lines showed enhanced lamina joint inclination, whereas OsOFP8 RNAi transgenic lines showed more upright leaf phenotype, which suggest that OsOFP8 is involved in BR responses. Further analyses indicated that OsGSK2 interacts with and phosphorylates OsOFP8. BRZ treatment resulted in the cytoplasmic distribution of OsOFP8, and bikinin treatment reduced the cytoplasmic accumulation of OsOFP8. Phosphorylation of OsOFP8 by OsGSK2 is needed for its nuclear export. The phospphorylated OsOFP8 shuttles to the cytoplasm and is targeted for proteasomal degradation. These results indicate that OsOFP8 is a substrate of OsGSK2 and the function of OsOFP8 in plant growth and development is at least partly through the BR signaling pathway. PMID:27332964

  17. Host Acyl Coenzyme A Binding Protein Regulates Replication Complex Assembly and Activity of a Positive-Strand RNA Virus

    PubMed Central

    Zhang, Jiantao; Diaz, Arturo; Mao, Lan; Ahlquist, Paul

    2012-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly. PMID:22345450

  18. Biochemical markers and protein pattern analysis for canine coagulase-positive staphylococci and their distribution on dog skin.

    PubMed

    Chanchaithong, Pattrarat; Prapasarakul, Nuvee

    2011-08-01

    Coagulase-positive staphylococci (CoPS) including S. pseudintermedius, S. schleiferi subsp. coagulans and S. aureus are etiological agents of dermatitis in companion animals and can be zoonotic pathogens. To date no consensual biochemical marker for routine microbiological identification of these species has been identified. The aim of this study was to evaluate biochemical markers and compare the results with the approved molecular method, multiplex-PCR (M-PCR), and confirm their species-specific phenotypic characteristic by using SDS-PAGE. The distribution and frequency of CoPS species were also determined. Three hundred and thirty-seven canine CoPS isolates were obtained from the nasal mucosa, perineum and groins of 66 healthy dogs and were identified by the M-PCR as S. aureus (n=5), S. pseudintermedius (n=263) and S. schleiferi subsp. coagulans (n=69). Selected biochemical tests including the Voges-Proskauer test, mannitol broth fermentation, the assimilation of maltose, galactose, trahalose and lactose using broth medium, were successfully used to distinguish the three species of canine CoPS from other CoPS species. Additionally, species-specific protein patterns were also found to be useful for phenotypic differentiation, with good agreement with the results of M-PCR and the use of biochemical markers. S. aureus occured infrequently on dog skin while co-colonization with S. pseudintermedius and S. schleiferi subsp. coagulans was observed. We propose the use of consensual biochemical markers of canine CoPS with the presence of the unique protein patterns as an alternative tool for conventional laboratory use. PMID:21586304

  19. PARP1 and phospho-p65 protein expression is increased in human HER2-positive breast cancers

    PubMed Central

    Stanley, Jennifer; Klepczyk, Lisa; Keene, Kimberly; Wei, Shi; Li, Yufeng; Forero, Andres; Grizzle, William; Wielgos, Monica; Brazelton, Jason; LoBuglio, Albert F.; Yang, Eddy S.

    2015-01-01

    Purpose Previous studies have shown that basal breast cancers, which may have an inherent “BRCAness” phenotype and sensitivity to inhibitors of poly (ADP-Ribose) polymerase (PARP), express elevated levels of PARP1. Our lab recently reported that HER2+ breast cancers also exhibit sensitivity to PARP inhibitors (PARPi) by attenuating the NF-kB pathway. In this study, we assessed PARP1 and phospho-p65, a marker of activated NF-kB levels in human breast cancer tissues. Methods PARP1 and PARP2 copy number, mRNA, and protein expression was assessed by interrogating the PAM-50 defined breast cancer patient set from the TCGA using the cBioPortal. PARP1 and phospho-p65 immunohistochemistry and correlation to clinical parameters was conducted using 307 primary breast cancer specimens (132 basal, 82 luminal, 93 HER2+) through univariate and multivariate analyses. Results In the PAM50 breast cancer data set, PARP1 and 2 expression was altered in 24/58 (41%) HER2+, 32/81 (40%) basal, and 75/324 (23%) luminal A/B breast cancer patients. This correlated with a statistically significant increase in PARP1 protein levels in HER2+ and basal but not luminal breast cancers (p=0.003, p=0.027, p=0.289, respectively). No change in PARP2 protein level was observed. Interestingly, using breast cancer specimens from 307 patients, HER2 positivity correlated with elevated PARP1 expression (p<0.0001) and was three times more likely than HER2 negative breast cancers to exhibit high PARP1 levels. No significant differences were noted between race, ER status, or PR status for PARP1 expression. Additionally, we found a significant correlation between HER2 status and phospho-p65 expression (p<0.0001). Lastly, a direct correlation between PARP1 and phospho-p65 (p<0.0001) was noted. Conclusions These results indicate a potential connection between HER2, PARP1, and phospho-p65. Furthermore, these data suggest that the PARPi sensitivity we previously observed in HER2+ breast cancer cells may be due

  20. Protein Phosphatase 2A Holoenzyme Is Targeted to Peroxisomes by Piggybacking and Positively Affects Peroxisomal β-Oxidation1[OPEN

    PubMed Central

    Kataya, Amr R.A.; Heidari, Behzad; Hagen, Lars; Kommedal, Roald; Slupphaug, Geir; Lillo, Cathrine

    2015-01-01

    The eukaryotic, highly conserved serine (Ser)/threonine-specific protein phosphatase 2A (PP2A) functions as a heterotrimeric complex composed of a catalytic (C), scaffolding (A), and regulatory (B) subunit. In Arabidopsis (Arabidopsis thaliana), five, three, and 17 genes encode different C, A, and B subunits, respectively. We previously found that a B subunit, B′θ, localized to peroxisomes due to its C-terminal targeting signal Ser-Ser-leucine. This work shows that PP2A C2, C5, andA2 subunits interact and colocalize with B′θ in peroxisomes. C and A subunits lack peroxisomal targeting signals, and their peroxisomal import depends on B′θ and appears to occur by piggybacking transport. B′θ knockout mutants were impaired in peroxisomal β-oxidation as shown by developmental arrest of seedlings germinated without sucrose, accumulation of eicosenoic acid, and resistance to protoauxins indole-butyric acid and 2,4-dichlorophenoxybutyric acid. All of these observations strongly substantiate that a full PP2A complex is present in peroxisomes and positively affects β-oxidation of fatty acids and protoauxins. PMID:25489022

  1. Trypsin- and low pH-mediated fusogenicity of avian metapneumovirus fusion proteins is determined by residues at positions 100, 101 and 294

    PubMed Central

    Yun, Bingling; Guan, Xiaolu; Liu, Yongzhen; Gao, Yanni; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Li; Li, Kai; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2015-01-01

    Avian metapneumovirus (aMPV) and human metapneumovirus (hMPV) are members of the genus Metapneumovirus in the subfamily Pneumovirinae. Metapneumovirus fusion (F) protein mediates the fusion of host cells with the virus membrane for infection. Trypsin- and/or low pH-induced membrane fusion is a strain-dependent phenomenon for hMPV. Here, we demonstrated that three subtypes of aMPV (aMPV/A, aMPV/B, and aMPV/C) F proteins promoted cell-cell fusion in the absence of trypsin. Indeed, in the presence of trypsin, only aMPV/C F protein fusogenicity was enhanced. Mutagenesis of the amino acids at position 100 and/or 101, located at a putative cleavage region in aMPV F proteins, revealed that the trypsin-mediated fusogenicity of aMPV F proteins is regulated by the residues at positions 100 and 101. Moreover, we demonstrated that aMPV/A and aMPV/B F proteins mediated cell-cell fusion independent of low pH, whereas the aMPV/C F protein did not. Mutagenesis of the residue at position 294 in the aMPV/A, aMPV/B, and aMPV/C F proteins showed that 294G played a critical role in F protein-mediated fusion under low pH conditions. These findings on aMPV F protein-induced cell-cell fusion provide new insights into the molecular mechanisms underlying membrane fusion and pathogenesis of aMPV. PMID:26498473

  2. Trypsin- and low pH-mediated fusogenicity of avian metapneumovirus fusion proteins is determined by residues at positions 100, 101 and 294.

    PubMed

    Yun, Bingling; Guan, Xiaolu; Liu, Yongzhen; Gao, Yanni; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Li; Li, Kai; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2015-01-01

    Avian metapneumovirus (aMPV) and human metapneumovirus (hMPV) are members of the genus Metapneumovirus in the subfamily Pneumovirinae. Metapneumovirus fusion (F) protein mediates the fusion of host cells with the virus membrane for infection. Trypsin- and/or low pH-induced membrane fusion is a strain-dependent phenomenon for hMPV. Here, we demonstrated that three subtypes of aMPV (aMPV/A, aMPV/B, and aMPV/C) F proteins promoted cell-cell fusion in the absence of trypsin. Indeed, in the presence of trypsin, only aMPV/C F protein fusogenicity was enhanced. Mutagenesis of the amino acids at position 100 and/or 101, located at a putative cleavage region in aMPV F proteins, revealed that the trypsin-mediated fusogenicity of aMPV F proteins is regulated by the residues at positions 100 and 101. Moreover, we demonstrated that aMPV/A and aMPV/B F proteins mediated cell-cell fusion independent of low pH, whereas the aMPV/C F protein did not. Mutagenesis of the residue at position 294 in the aMPV/A, aMPV/B, and aMPV/C F proteins showed that 294G played a critical role in F protein-mediated fusion under low pH conditions. These findings on aMPV F protein-induced cell-cell fusion provide new insights into the molecular mechanisms underlying membrane fusion and pathogenesis of aMPV. PMID:26498473

  3. Multiple phosphorylated forms of the Saccharomyces cerevisiae Mcm1 protein include an isoform induced in response to high salt concentrations.

    PubMed Central

    Kuo, M H; Nadeau, E T; Grayhack, E J

    1997-01-01

    The Saccharomyces cerevisiae Mcm1 protein is an essential multifunctional transcription factor which is highly homologous to human serum response factor. Mcm1 protein acts on a large number of distinctly regulated genes: haploid cell-type-specific genes, G2-cell-cycle-regulated genes, pheromone-induced genes, arginine metabolic genes, and genes important for cell wall and cell membrane function. We show here that Mcm1 protein is phosphorylated in vivo. Several (more than eight) isoforms of Mcm1 protein, resolved by isoelectric focusing, are present in vivo; two major phosphorylation sites lie in the N-terminal 17 amino acids immediately adjacent to the conserved MADS box DNA-binding domain. The implications of multiple species of Mcm1, particularly the notion that a unique Mcm1 isoform could be required for regulation of a specific set of Mcm1's target genes, are discussed. We also show here that Mcm1 plays an important role in the response to stress caused by NaCl. G. Yu, R. J. Deschenes, and J. S. Fassler (J. Biol. Chem. 270:8739-8743, 1995) showed that Mcm1 function is affected by mutations in the SLN1 gene, a signal transduction component implicated in the response to osmotic stress. We find that mcm1 mutations can confer either reduced or enhanced survival on high-salt medium; deletion of the N terminus or mutation in the primary phosphorylation site results in impaired growth on high-salt medium. Furthermore, Mcm1 protein is a target of a signal transduction system responsive to osmotic stress: a new isoform of Mcm1 is induced by NaCl or KCl; this result establishes that Mcm1 itself is regulated. PMID:9001236

  4. Body Position Modulates Gastric Emptying and Affects the Post-Prandial Rise in Plasma Amino Acid Concentrations Following Protein Ingestion in Humans

    PubMed Central

    Holwerda, Andrew M.; Lenaerts, Kaatje; Bierau, Jörgen; van Loon, Luc J. C.

    2016-01-01

    Dietary protein digestion and amino acid absorption kinetics determine the post-prandial muscle protein synthetic response. Body position may affect gastrointestinal function and modulate the post-prandial rise in plasma amino acid availability. We aimed to assess the impact of body position on gastric emptying rate and the post-prandial rise in plasma amino acid concentrations following ingestion of a single, meal-like amount of protein. In a randomized, cross-over design, eight healthy males (25 ± 2 years, 23.9 ± 0.8 kg·m−2) ingested 22 g protein and 1.5 g paracetamol (acetaminophen) in an upright seated position (control) and in a −20° head-down tilted position (inversion). Blood samples were collected during a 240-min post-prandial period and analyzed for paracetamol and plasma amino acid concentrations to assess gastric emptying rate and post-prandial amino acid availability, respectively. Peak plasma leucine concentrations were lower in the inversion compared with the control treatment (177 ± 15 vs. 236 ± 15 mmol·L−1, p < 0.05), which was accompanied by a lower plasma essential amino acid (EAA) response over 240 min (31,956 ± 6441 vs. 50,351 ± 4015 AU; p < 0.05). Peak plasma paracetamol concentrations were lower in the inversion vs. control treatment (5.8 ± 1.1 vs. 10.0 ± 0.6 mg·L−1, p < 0.05). Gastric emptying rate and post-prandial plasma amino acid availability are significantly decreased after protein ingestion in a head-down tilted position. Therefore, upright body positioning should be considered when aiming to augment post-prandial muscle protein accretion in both health and disease. PMID:27089362

  5. Increased expression of amyloid precursor protein promotes proliferation and migration of AML1/ETO-positive leukemia cells and be inhibited by panobinostat.

    PubMed

    Wang, C L; Ding, B J; Jiang, L; Yin, C X; Zhong, Q X; Yu, G P; Li, X D; Meng, F Y

    2015-01-01

    Amyloid precursor protein (APP) is a highly conserved integral membrane protein extensively expressed in various types of cells. Previously we found that overexpression of APP in patients with AML1/ETO-positive acute myeloid leukemia (AML) associated with a higher incidence of extramedullary infiltrationin and indicate a poor prognosis. In this study, we attempted to define the roles of APP in AML1/ETO-positive leukemia cells. Western blotting and qRT-PCR analysis showed that protein levels of APP are significantly higher in Kasumi-1, a t(8;21)/ AML1/ETO-positive M2-type AML cell line. Stable knockdown of APP by lentivirus-based RNA interference (RNAi) dramatically impaired colony-formation and migration ability of Kasumi-1 cells, whereas APP knockdown had very little effect on cell viability, apoptosis, cell cycle and differentiation. We further explored whether the pan-histone deacetylase inhibitor panobinostat could deplete the protein levels of APP in Kasumi-1 cells. Treatment with panobinostat caused depletion of APP in Kasumi-1 cells. These findings indicate that overexpression of APP is involved in promoting proliferation and migration of AML1/ETO-positive leukemia cells and can be inhibited by panobinostat, which provide an attractive prospect for treatment of AML1/ETO-positive AML. PMID:26458322

  6. Small-animal PET imaging of human epidermal growth factor receptor positive tumor with a 64Cu labeled affibody protein.

    PubMed

    Miao, Zheng; Ren, Gang; Liu, Hongguang; Jiang, Lei; Cheng, Zhen

    2010-05-19

    Epidermal growth factor receptor (EGFR) has become an attractive target for cancer molecular imaging and therapy. Affibody proteins against EGFR have been reported, and thus, we were interested in evaluating their potential for positron emission tomography (PET) imaging of EGFR positive cancer. An Affibody analogue (Ac-Cys-Z(EGFR:1907)) binding to EGFR was made through conventional solid phase peptide synthesis. The purified protein was site-specifically coupled with the 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA) to produce the bioconjugate, DOTA-Z(EGFR:1907). (64)Cu labeled probe (64)Cu-DOTA-Z(EGFR:1907) displayed a moderate specific activity (5-8 MBq/nmol, 22-35 microCi/microg). Cell uptake assays by pre-incubating without or with 300 times excess unlabeled Ac-Cys-Z(EGFR:1907) showed high EGFR-specific uptake (20% applied activity at 0.5 h) in A431 epidermoid carcinoma cancer cells. The affinity (K(D)) of (64)Cu-DOTA-Z(EGFR:1907) as tested by cell saturation analysis was 20 nM. The serum stability test showed excellent stability of the probe with >95% intact after 4 h of incubation in mouse serum. In vivo small-animal PET imaging showed fast tumor targeting, high tumor accumulation (approximately 10% ID/g at 1 h p.i.), and good tumor-to-normal tissue contrast of (64)Cu-DOTA-Z(EGFR:1907) spiked with a wide dose range of Ac-Cys-Z(EGFR:1907). Bio-distribution studies further demonstrated that the probe had high tumor, blood, liver, and kidney uptakes, while blood radioactivity concentration dropped dramatically at increased spiking doses. Co-injection of the probe with 500 microg of Ac-Cys-Z(EGFR:1907) for blocking significantly reduced the tumor uptake. Thus, (64)Cu-DOTA-Z(EGFR:1907) showed potential as a high tumor contrast EGFR PET imaging reagent. The probe spiked with 50 microg of Ac-Cys-Z(EGFR:1907) improved tumor imaging contrast which may have important clinical applications. PMID:20402512

  7. Major latex protein-like protein 43 (MLP43) functions as a positive regulator during abscisic acid responses and confers drought tolerance in Arabidopsis thaliana

    PubMed Central

    Wang, Yanping; Yang, Li; Chen, Xi; Ye, Tiantian; Zhong, Bao; Liu, Ruijie; Wu, Yan; Chan, Zhulong

    2016-01-01

    Drought stress is one of the disadvantageous environmental conditions for plant growth and reproduction. Given the importance of abscisic acid (ABA) to plant growth and abiotic stress responses, identification of novel components involved in ABA signalling transduction is critical. In this study, we screened numerous Arabidopsis thaliana mutants by seed germination assay and identified a mutant mlp43 (major latex protein-like 43) with decreased ABA sensitivity in seed germination. The mlp43 mutant was sensitive to drought stress while the MLP43-overexpressed transgenic plants were drought tolerant. The tissue-specific expression pattern analysis showed that MLP43 was predominantly expressed in cotyledons, primary roots and apical meristems, and a subcellular localization study indicated that MLP43 was localized in the nucleus and cytoplasm. Physiological and biochemical analyses indicated that MLP43 functioned as a positive regulator in ABA- and drought-stress responses in Arabidopsis through regulating water loss efficiency, electrolyte leakage, ROS levels, and as well as ABA-responsive gene expression. Moreover, metabolite profiling analysis indicated that MLP43 could modulate the production of primary metabolites under drought stress conditions. Reconstitution of ABA signalling components in Arabidopsis protoplasts indicated that MLP43 was involved in ABA signalling transduction and acted upstream of SnRK2s by directly interacting with SnRK2.6 and ABF1 in a yeast two-hybrid assay. Moreover, ABA and drought stress down-regulated MLP43 expression as a negative feedback loop regulation to the performance of MLP43 in ABA and drought stress responses. Therefore, this study provided new insights for interpretation of physiological and molecular mechanisms of Arabidopsis MLP43 mediating ABA signalling transduction and drought stress responses. PMID:26512059

  8. Major latex protein-like protein 43 (MLP43) functions as a positive regulator during abscisic acid responses and confers drought tolerance in Arabidopsis thaliana.

    PubMed

    Wang, Yanping; Yang, Li; Chen, Xi; Ye, Tiantian; Zhong, Bao; Liu, Ruijie; Wu, Yan; Chan, Zhulong

    2016-01-01

    Drought stress is one of the disadvantageous environmental conditions for plant growth and reproduction. Given the importance of abscisic acid (ABA) to plant growth and abiotic stress responses, identification of novel components involved in ABA signalling transduction is critical. In this study, we screened numerous Arabidopsis thaliana mutants by seed germination assay and identified a mutant mlp43 (major latex protein-like 43) with decreased ABA sensitivity in seed germination. The mlp43 mutant was sensitive to drought stress while the MLP43-overexpressed transgenic plants were drought tolerant. The tissue-specific expression pattern analysis showed that MLP43 was predominantly expressed in cotyledons, primary roots and apical meristems, and a subcellular localization study indicated that MLP43 was localized in the nucleus and cytoplasm. Physiological and biochemical analyses indicated that MLP43 functioned as a positive regulator in ABA- and drought-stress responses in Arabidopsis through regulating water loss efficiency, electrolyte leakage, ROS levels, and as well as ABA-responsive gene expression. Moreover, metabolite profiling analysis indicated that MLP43 could modulate the production of primary metabolites under drought stress conditions. Reconstitution of ABA signalling components in Arabidopsis protoplasts indicated that MLP43 was involved in ABA signalling transduction and acted upstream of SnRK2s by directly interacting with SnRK2.6 and ABF1 in a yeast two-hybrid assay. Moreover, ABA and drought stress down-regulated MLP43 expression as a negative feedback loop regulation to the performance of MLP43 in ABA and drought stress responses. Therefore, this study provided new insights for interpretation of physiological and molecular mechanisms of Arabidopsis MLP43 mediating ABA signalling transduction and drought stress responses. PMID:26512059

  9. Comparative Distribution of Relaxin-3 Inputs and Calcium-Binding Protein-Positive Neurons in Rat Amygdala.

    PubMed

    Santos, Fabio N; Pereira, Celia W; Sánchez-Pérez, Ana M; Otero-García, Marcos; Ma, Sherie; Gundlach, Andrew L; Olucha-Bordonau, Francisco E

    2016-01-01

    The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or "hubs") within these key circuits. One such input arises from the nucleus incertus (NI) in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals) within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin (PV), calretinin (CR) and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST) and in the endopiriform nucleus. In

  10. Comparative Distribution of Relaxin-3 Inputs and Calcium-Binding Protein-Positive Neurons in Rat Amygdala

    PubMed Central

    Santos, Fabio N.; Pereira, Celia W.; Sánchez-Pérez, Ana M.; Otero-García, Marcos; Ma, Sherie; Gundlach, Andrew L.; Olucha-Bordonau, Francisco E.

    2016-01-01

    The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or “hubs”) within these key circuits. One such input arises from the nucleus incertus (NI) in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals) within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin (PV), calretinin (CR) and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST) and in the endopiriform nucleus

  11. Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

    PubMed Central

    Otwell, A.E.; Sherwood, R.W.; Zhang, S.; Nelson, O.D.; Li, Z.; Lin, H.; Callister, S.J.; Richardson, R.E.

    2015-01-01

    Summary Understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. Using non-denaturing separations and mass spectrometry identification, in combination with a colorimetric screen for chelated Fe(III)-NTA reduction with NADH as electron donor, we have identified proteins from the D. reducens proteome not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. The proteins identified are NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. This study is the first functional proteomic analysis of D. reducens and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium. PMID:25389064

  12. Effect of amino acid sequence variations at position 149 on the fusogenic activity of the subtype B avian metapneumovirus fusion protein.

    PubMed

    Yun, Bingling; Gao, Yanni; Liu, Yongzhen; Guan, Xiaolu; Wang, Yongqiang; Qi, Xiaole; Gao, Honglei; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Gao, Yulong; Wang, Xiaomei

    2015-10-01

    The entry of enveloped viruses into host cells requires the fusion of viral and cell membranes. These membrane fusion reactions are mediated by virus-encoded glycoproteins. In the case of avian metapneumovirus (aMPV), the fusion (F) protein alone can mediate virus entry and induce syncytium formation in vitro. To investigate the fusogenic activity of the aMPV F protein, we compared the fusogenic activities of three subtypes of aMPV F proteins using a TCSD50 assay developed in this study. Interestingly, we found that the F protein of aMPV subtype B (aMPV/B) strain VCO3/60616 (aMPV/vB) was hyperfusogenic when compared with F proteins of aMPV/B strain aMPV/f (aMPV/fB), aMPV subtype A (aMPV/A), and aMPV subtype C (aMPV/C). We then further demonstrated that the amino acid (aa) residue 149F contributed to the hyperfusogenic activity of the aMPV/vB F protein. Moreover, we revealed that residue 149F had no effect on the fusogenic activities of aMPV/A, aMPV/C, and human metapneumovirus (hMPV) F proteins. Collectively, we provide the first evidence that the amino acid at position 149 affects the fusogenic activity of the aMPV/B F protein, and our findings will provide new insights into the fusogenic mechanism of this protein. PMID:26175070

  13. Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria

    PubMed Central

    Asli, Abdelhamid; Brouillette, Eric; Krause, Kevin M.; Nichols, Wright W.

    2015-01-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets. PMID:26574008

  14. Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Asli, Abdelhamid; Brouillette, Eric; Krause, Kevin M; Nichols, Wright W; Malouin, François

    2016-02-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets. PMID:26574008

  15. Magnesium ions mediate contacts between phosphoryl oxygens at positions 2122 and 2176 of the 23S rRNA and ribosomal protein L1.

    PubMed Central

    Drygin, D; Zimmermann, R A

    2000-01-01

    The complex of ribosomal protein L1 with 23S rRNA from Escherichia coli is of great interest because of the unique structural and functional aspects of this ribonucleoprotein domain. We have minimized the binding site for protein L1 on the 23S rRNA to nt 2120-2129, 2159-2162, and 2167-2178. This RNA fragment consists of two helices as well as an interconnecting loop of unknown structure. RNA molecules corresponding to the minimized L1 binding site, in which G, A, U, or C were individually replaced by their deoxyribo- (dN) or alpha-thio- (rNaS) analogs have been synthesized by T7 transcription in vitro and analyzed for their ability to bind protein L1. It has been demonstrated that the substitution of rNaS at position 2122 or 2176 decreases the affinity of the RNA for the protein in the presence of magnesium five- to tenfold, whereas the same changes have little effect on binding in the presence of manganese. This suggests that Rp oxygens in the phosphates preceding positions 2122 and 2176 are coordinated with Mg2+ and may participate in L1-23S rRNA interaction via magnesium bridges. We have also shown that this interaction is impaired by the presence of dC at position 2122 coupled with the presence of deoxyribonucleotide(s) at other positions in the RNA. This study demonstrates that the ribose-phosphate backbone of the helix encompassing nt 2120-2124/2174-2178 is intimately involved in the interaction of protein L1 with the 23S rRNA. In particular, we suggest that this helix is positioned in the cleft between the two domains of protein L1. PMID:11142372

  16. Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics

    PubMed Central

    Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

    2016-01-01

    The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics. PMID:26732734

  17. Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics.

    PubMed

    Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

    2016-01-01

    The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics. PMID:26732734

  18. Properties of a Streptococcus salivarius spontaneous mutant in which the methionine at position 48 in the protein HPr has been replaced by a valine.

    PubMed Central

    Vadeboncoeur, C; Gauthier, L; Gagnon, G; Leduc, A; Brochu, D; Lapointe, R; Desjardins, B; Frenette, M

    1994-01-01

    HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that participates in the concomitant transport and phosphorylation of sugars in bacteria. In gram-positive bacteria, HPr is also reversibly phosphorylated at a seryl residue at position 46 (Ser-46) by a metabolite-activated ATP-dependent kinase and a Pi-dependent HPr(Ser-P) phosphatase. We report in this article the isolation of a spontaneous mutant (mutant A66) from a streptococcus (Streptococcus salivarius) in which the methionine at position 48 (Met-48) in the protein HPr has been replaced by a valine (Val). The mutation inhibited the phosphorylation of HPr on Ser-46 by the ATP-dependent kinase but did not prevent phosphorylation of HPr by enzyme I or the phosphorylation of enzyme II complexes by HPr(His-P). The results, however, suggested that replacement of Met-48 by Val decreased the affinity of enzyme I for HPr or the affinity of enzyme II proteins for HPr(His-P) or both. Characterization of mutant A66 demonstrated that it has pleiotropic properties, including the lack of IIILman, a specific protein of the mannose PTS; decreased levels of HPr; derepression of some cytoplasmic proteins; reduced growth on PTS as well as on non-PTS sugars; and aberrant growth in medium containing a mixture of sugars. Images PMID:8288549

  19. Tomato 14-3-3 protein 7 (TFT7) positively regulates immunity-associated programmed cell death by enhancing accumulation and signaling ability of MAPKKKalpha

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Programmed cell death (PCD) is triggered when Pto, a serine-threonine protein kinase recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv. tomato. This PCD requires MAPKKKalpha as a positive regulator in tomato and Nicotiana benthamiana. To examine how PCD-eliciting activi...

  20. Localization of the adenovirus E1Aa protein, a positive-acting transcriptional factor, in infected cells infected cells.

    PubMed Central

    Feldman, L T; Nevins, J R

    1983-01-01

    The function of the adenovirus E1Aa protein (the product of the 13S E1A mRNA) during a productive viral infection is to activate transcription of the six early viral transcription units. To study the mechanism of action of this protein, a peptide which was 13 amino acids long and had a sequence unique to the protein product of the adenovirus 13S E1A mRNA (pE1Aa) was coupled to keyhole limpet hemocyanin and used to raise an antibody in rabbits. The resulting antiserum was specific to this protein and did not react with the protein product of the 12S E1A mRNA, which shares considerable sequence with the E1Aa protein. This antiserum was used to probe for the E1Aa protein in situ by indirect immunofluorescence and in extracts of infected HeLa cells. We found that the protein was associated with large cellular structures both in the nucleus and in the cytoplasm. The nuclear form of the protein was analyzed further and was found to purify with the nuclear matrix. Images PMID:6346057

  1. Positive charges on lysine residues of the extrinsic 18 kDa protein are important to its electrostatic interaction with spinach photosystem II membranes.

    PubMed

    Gao, Jin-Peng; Yong, Zhen-Hua; Zhang, Feng; Ruan, Kang-Cheng; Xu, Chun-He; Chen, Gen-Yun

    2005-11-01

    To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes. PMID:16270152

  2. 14-3-3 Proteins Bind to the Brassinosteroid Receptor Kinase, BRI1 and are Positive Regulators of Brassinosteroid Signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple members of the 14-3-3 protein family have been found in all eukaryotes, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. Thus, 14-3-3s are involved in many processes. The plant brassinosteroid (BR) recepto...

  3. Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

    SciTech Connect

    Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng; Nelson, Ornella D.; Li, Zhi; Lin, Hening; Callister, Stephen J.; Richardson, Ruth E.

    2015-01-01

    Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor, we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.

  4. The Achilles' Heel of "Ultrastable" Hyperthermophile Proteins: Submillimolar Concentrations of SDS Stimulate Rapid Conformational Change, Aggregation, and Amyloid Formation in Proteins Carrying Overall Positive Charge.

    PubMed

    Khan, Javed M; Sharma, Prerna; Arora, Kanika; Kishor, Nitin; Kaila, Pallavi; Guptasarma, Purnananda

    2016-07-19

    Low concentrations (<3.0 mM) of the anionic surfactant sodium dodecyl sulfate (SDS) have been shown to induce the formation of amyloid fibers in more than 20 different mesophile-derived proteins in the cationic state. It is not known whether SDS has similar effects on hyperthermophile-derived proteins, which are otherwise thought to be "ultrastable" and inordinately resistant to structural perturbations at room temperature. Here, we show that low (<4.5 mM) concentrations of SDS rapidly induce the formation of aggregates and amyloid fibers in five different ultrastable Pyrococcus furiosus proteins in the cationic state. We also show that amyloid formation is accompanied by the development of a characteristic, negative circular dichroism band at ∼230 nm. These effects are not seen if the proteins have a net negative charge or when higher concentrations of SDS are used (which induce helix formation instead). Our results appear to reveal a potential weakness or "Achilles' heel" in ultrastable proteins from hyperthermophiles. They also provide very strong support for the view that SDS initially interacts with proteins through electrostatic interactions, and not hydrophobic interactions, eliciting similar effects entirely regardless of protein molecular weight, or structural features such as quaternary structure or tertiary structural stability. PMID:27331826

  5. A protein kinase antigenically related to pp60v-src possibly involved in yeast cell cycle control: positive in vivo regulation by sterol.

    PubMed Central

    Dahl, C; Biemann, H P; Dahl, J

    1987-01-01

    The effects of ergosterol, yeast's natural sterol, on cell cycling and a protein kinase antigenically related to pp60v-src were examined in a sterol auxotroph of Saccharomyces cerevisiae. Sterol-depleted cells accumulate in an unbudded, G1 state. Cell budding and proliferation are reinitiated upon addition of nonlimiting ergosterol or cholesterol with trace ergosterol, whereas cholesterol or trace ergosterol alone is less effective. Stimulation of a protein kinase associated with immune complexes of yeast protein and anti-pp60v-src shows a positive correlation with exit from the G1 phase following ergosterol addition. Ergosterol-stimulated cells also demonstrate an increase in phosphatidylinositol kinase activity. The data suggest that hormonal levels of ergosterol (effective concentration, approximately equal to 1 nM) participate in a signaling process associated with a protein kinase possibly involved in yeast cell cycle control. Images PMID:2438691

  6. The function of the PduJ microcompartment shell protein is determined by the genomic position of its encoding gene.

    PubMed

    Chowdhury, Chiranjit; Chun, Sunny; Sawaya, Michael R; Yeates, Todd O; Bobik, Thomas A

    2016-09-01

    Bacterial microcompartments (MCPs) are complex organelles that consist of metabolic enzymes encapsulated within a protein shell. In this study, we investigate the function of the PduJ MCP shell protein. PduJ is 80% identical in amino acid sequence to PduA and both are major shell proteins of the 1,2-propanediol (1,2-PD) utilization (Pdu) MCP of Salmonella. Prior studies showed that PduA mediates the transport of 1,2-PD (the substrate) into the Pdu MCP. Surprisingly, however, results presented here establish that PduJ has no role 1,2-PD transport. The crystal structure revealed that PduJ was nearly identical to that of PduA and, hence, offered no explanation for their differential functions. Interestingly, however, when a pduJ gene was placed at the pduA chromosomal locus, the PduJ protein acquired a new function, the ability to mediate 1,2-PD transport into the Pdu MCP. To our knowledge, these are the first studies to show that that gene location can determine the function of a MCP shell protein. We propose that gene location dictates protein-protein interactions essential to the function of the MCP shell. PMID:27561553

  7. Tail proteins of phage T5: investigation of the effect of the His6-tag position, from expression to crystallisation.

    PubMed

    Noirclerc-Savoye, Marjolaine; Flayhan, Ali; Pereira, Cindy; Gallet, Benoit; Gans, Pierre; Ebel, Christine; Breyton, Cécile

    2015-05-01

    Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality. PMID:25676818

  8. Positively-Charged Semi-Tunnel Is a Structural and Surface Characteristic of Polyphosphate-Binding Proteins: An In-Silico Study

    PubMed Central

    Wei, Zheng Zachory; Vatcher, Greg; Tin, Alvin Hok Yan; Teng, Jun Lin; Wang, Juan; Cui, Qing Hua; Chen, Jian Guo; Yu, Albert Cheung Hoi

    2015-01-01

    Phosphate is essential for all major life processes, especially energy metabolism and signal transduction. A linear phosphate polymer, polyphosphate (polyP), linked by high-energy phosphoanhydride bonds, can interact with various proteins, playing important roles as an energy source and regulatory factor. However, polyP-binding structures are largely unknown. Here we proposed a putative polyP binding site, a positively-charged semi-tunnel (PCST), identified by surface electrostatics analyses in polyP kinases (PPKs) and many other polyP-related proteins. We found that the PCSTs in varied proteins were folded in different secondary structure compositions. Molecular docking calculations revealed a significant value for binding affinity to polyP in PCST-containing proteins. Utilizing the PCST identified in the β subunit of PPK3, we predicted the potential polyP-binding domain of PPK3. The discovery of this feature facilitates future searches for polyP-binding proteins and discovery of the mechanisms for polyP-binding activities. This should greatly enhance the understanding of the many physiological functions of protein-bound polyP and the involvement of polyP and polyP-binding proteins in various human diseases. PMID:25879219

  9. Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function.

    PubMed Central

    Austin, S; Kundrot, C; Dixon, R

    1991-01-01

    A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NTRC-phosphate were similar to those of wild-type NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NTRC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NTRC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation. Images PMID:2041769

  10. Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells

    SciTech Connect

    Sides, Mark D.; Block, Gregory J.; Shan, Bin; Esteves, Kyle C.; Lin, Zhen; Flemington, Erik K.; Lasky, Joseph A.

    2011-06-20

    Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies.

  11. Arsenic mediated disruption of promyelocytic leukemia protein nuclear bodies induces ganciclovir susceptibility in Epstein-Barr positive epithelial cells.

    PubMed

    Sides, Mark D; Block, Gregory J; Shan, Bin; Esteves, Kyle C; Lin, Zhen; Flemington, Erik K; Lasky, Joseph A

    2011-07-20

    Promyelocytic leukemia protein nuclear bodies (PML NBs) have been implicated in host immune response to viral infection. PML NBs are targeted for degradation during reactivation of herpes viruses, suggesting that disruption of PML NB function supports this aspect of the viral life cycle. The Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1) has been shown to suppress EBV reactivation. Our finding that LMP1 induces PML NB immunofluorescence intensity led to the hypothesis that LMP1 may modulate PML NBs as a means of maintaining EBV latency. Increased PML protein and morphometric changes in PML NBs were observed in EBV infected alveolar epithelial cells and nasopharyngeal carcinoma cells. Treatment with low dose arsenic trioxide disrupted PML NBs, induced expression of EBV lytic proteins, and conferred ganciclovir susceptibility. This study introduces an effective modality to induce susceptibility to ganciclovir in epithelial cells with implications for the treatment of EBV associated pathologies. PMID:21605886

  12. Breast Cancer Proteomics – Differences in Protein Expression between Estrogen Receptor-Positive and -Negative Tumors Identified by Tandem Mass Tag Technology

    PubMed Central

    Ruckhäberle, Eugen; Karn, Thomas; Hanker, Lars; Schwarz, Josef; Schulz-Knappe, Peter; Kuhn, Karsten; Böhm, Gitte; Selzer, Stefan; Erhard, Neukum; Engels, Knut; Holtrich, Uwe; Kaufmann, Manfred; Rody, Achim

    2010-01-01

    Background Proteomic analysis has become an effective tool in breast cancer research. In this study, we applied the new gel-free tandem mass tag (TMT) reference method for the first time in breast cancer. Materials and Methods Proteomic analysis was used to compare 10 estrogen receptor (ER)-positive and 10 ER-negative samples. The results of the proteomic approach were validated by Western blot, immunohistochemistry and gene expression analysis. Results 17 proteins with significant differences in expression were identified. 13 proteins were overexpressed in ER-negative tumors and 4 were overexpressed in ER-positive samples. All these proteins were characterized by relatively high cellular abundance. Conclusions Our results demonstrate that the gel-free TMT approach allows the quantification of differences in protein expression levels. Further improvement of the sensitivity by subfractionation of the tissue should allow also the identification of low-abundance proteins and might lead to the use of this method in breast cancer research. PMID:22619634

  13. (1)H, (15)N and (13)C chemical shift assignment of the Gram-positive conjugative transfer protein TraHpIP501.

    PubMed

    Fercher, Christian; Keller, Walter; Zangger, Klaus; Helge Meyer, N

    2016-04-01

    Conjugative transfer of DNA represents the most important transmission pathway in terms of antibiotic resistance and virulence gene dissemination among bacteria. TraH is a putative transfer protein of the type IV secretion system (T4SS) encoded by the Gram-positive (G+) conjugative plasmid pIP501. This molecular machine involves a multi-protein core complex spanning the bacterial envelope thereby serving as a macromolecular secretion channel. Here, we report the near complete (1)H, (13)C and (15)N resonance assignment of a soluble TraH variant comprising the C-terminal domain. PMID:26559076

  14. [Position statement. Protein/creatinine in a randomly obtained urine sample in the diagnosis of proteinuria in pregnant patients with arterial hypertension].

    PubMed

    2012-01-01

    Leaños Miranda and collaborators published that the measurement of protein/creatinine ratio in a single random urine sample is a reliable indicator of significant proteinuria and may be reasonably used as alternative to the 24-hours urine collection method as a diagnostic criteria for urinary protein, and it is also a criterion for identifying the disease severity. This leads us to present this successful result of the investigation as a position statement in the care of pregnant women with hypertension. PMID:23282273

  15. The AT-hook protein D1 is essential for Drosophila melanogaster development and is implicated in position-effect variegation.

    PubMed

    Aulner, Nathalie; Monod, Caroline; Mandicourt, Guillaume; Jullien, Denis; Cuvier, Olivier; Sall, Alhousseynou; Janssen, Sam; Laemmli, Ulrich K; Käs, Emmanuel

    2002-02-01

    We have analyzed the expression pattern of the D1 gene and the localization of its product, the AT hook-bearing nonhistone chromosomal protein D1, during Drosophila melanogaster development. D1 mRNAs and protein are maternally contributed, and the protein localizes to discrete foci on the chromosomes of early embryos. These foci correspond to 1.672- and 1.688-g/cm(3) AT-rich satellite repeats found in the centromeric heterochromatin of the X and Y chromosomes and on chromosomes 3 and 4. D1 mRNA levels subsequently decrease throughout later development, followed by the accumulation of the D1 protein in adult gonads, where two distributions of D1 can be correlated to different states of gene activity. We show that the EP473 mutation, a P-element insertion upstream of D1 coding sequences, affects the expression of the D1 gene and results in an embryonic homozygous lethal phenotype correlated with the depletion of D1 protein during embryogenesis. Remarkably, decreased levels of D1 mRNA and protein in heterozygous flies lead to the suppression of position-effect variegation (PEV) of the white gene in the white-mottled (w(m4h)) X-chromosome inversion. Our results identify D1 as a DNA-binding protein of known sequence specificity implicated in PEV. D1 is the primary factor that binds the centromeric 1.688-g/cm(3) satellite repeats which are likely involved in white-mottled variegation. We propose that the AT-hook D1 protein nucleates heterochromatin assembly by recruiting specialized transcriptional repressors and/or proteins involved in chromosome condensation. PMID:11809812

  16. Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

    NASA Astrophysics Data System (ADS)

    Takayama, Mitsuo; Sekiya, Sadanori; Iimuro, Ryunosuke; Iwamoto, Shinichi; Tanaka, Koichi

    2014-01-01

    Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z'-ions originating from cleavage at the N-Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z'-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a "mobile proton" are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

  17. Floral ontogeny and gene protein localization rules out euanthial interpretation of reproductive units in Lepironia (Cyperaceae, Mapanioideae, Chrysitricheae)

    PubMed Central

    Prychid, C. J.; Bruhl, J. J.

    2013-01-01

    Background and Aims In the sedge subfamily Mapanioideae there are considerable discrepancies between the standard trimerous monocot floral architecture expected and the complex floral and inflorescence morphologies seen. Decades of debate about whether the basic reproductive units are single flowers or pseudanthia have not resolved the question. This paper evaluates current knowledge about Mapaniid reproductive structures and presents an ontogenetic study of the Mapaniid genus Lepironia with the first floral protein expression maps for the family, localizing the products of the APETALA1/FRUITFULL-like (AP1/FUL) MADS-box genes with the aim of shedding light on this conundrum. Methods A range of reproductive developmental stages, from spikelet primordia through to infructescence material, were processed for anatomical and immunohistochemical analyses. Key Results The basic reproductive unit is subtended by a bract and possesses two prophyll-like structures, the first organs to be initiated on the primordium, which grow rapidly, enclosing two whorls of initiating leaf-like structures with intervening stamens and a central gynoecium, formed from an annular primordium. The subtending bract and prophyll-like structures possess very different morphologies from that of the internal leaf-like structures and do not show AP1/FUL-like protein localization, which is otherwise strongly localized in the internal leaf-like structures, stamens and gynoecia. Conclusions Results support the synanthial hypothesis as the evolutionary origin of the reproductive unit. Thus, the basic reproductive unit in Lepironia is an extremely condensed pseudanthium, of staminate flowers surrounding a central terminal pistillate female flower. Early in development the reproductive unit becomes enclosed by a split-prophyll, with the whole structure subtended by a bract. PMID:23723258

  18. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression

    SciTech Connect

    Wek, R.C.; Ramirez, M.; Jackson, B.M.; Hinnebusch, A.G. )

    1990-06-01

    GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast {ital Saccharomyces cerevisiae}. GCN2, a translational activator of {ital GCN4} expression, contains a domain homologous to the catalytic subunit of eukaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating {ital GCN4} expression. Elevated {ital GCN2} gene dosage led to depression of {ital GCN4} under nonstarvation conditions; however, the authors found that {ital GCN2} mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated postranslationally in amino acid-starved cells. Three dominant-constitutive {ital GCN2} point mutations were isolated that led to derepressed {ital GCN4} expression under nonstarvation conditions. Two of the {ital GCN2}(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which the authors suggest might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety.

  19. Alanine Scanning of Cucumber Mosaic Virus (CMV) 2B Protein Identifies Different Positions for Cell-To-Cell Movement and Gene Silencing Suppressor Activity

    PubMed Central

    Nemes, Katalin; Gellért, Ákos; Balázs, Ervin; Salánki, Katalin

    2014-01-01

    The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa) to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA) overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA), and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA), which are not essential for suppressor activity. PMID:25380036

  20. Recent Positive Selection Has Acted on Genes Encoding Proteins with More Interactions within the Whole Human Interactome

    PubMed Central

    Pybus, Marc; Fares, Mario A.; Bertranpetit, Jaume; Laayouni, Hafid

    2015-01-01

    Genes vary in their likelihood to undergo adaptive evolution. The genomic factors that determine adaptability, however, remain poorly understood. Genes function in the context of molecular networks, with some occupying more important positions than others and thus being likely to be under stronger selective pressures. However, how positive selection distributes across the different parts of molecular networks is still not fully understood. Here, we inferred positive selection using comparative genomics and population genetics approaches through the comparison of 10 mammalian and 270 human genomes, respectively. In agreement with previous results, we found that genes with lower network centralities are more likely to evolve under positive selection (as inferred from divergence data). Surprisingly, polymorphism data yield results in the opposite direction than divergence data: Genes with higher centralities are more likely to have been targeted by recent positive selection during recent human evolution. Our results indicate that the relationship between centrality and the impact of adaptive evolution highly depends on the mode of positive selection and/or the evolutionary time-scale. PMID:25840415

  1. MACROCALYX and JOINTLESS Interact in the Transcriptional Regulation of Tomato Fruit Abscission Zone Development1[C][W

    PubMed Central

    Nakano, Toshitsugu; Kimbara, Junji; Fujisawa, Masaki; Kitagawa, Mamiko; Ihashi, Nao; Maeda, Hideo; Kasumi, Takafumi; Ito, Yasuhiro

    2012-01-01

    Abscission in plants is a crucial process used to shed organs such as leaves, flowers, and fruits when they are senescent, damaged, or mature. Abscission occurs at predetermined positions called abscission zones (AZs). Although the regulation of fruit abscission is essential for agriculture, the developmental mechanisms remain unclear. Here, we describe a novel transcription factor regulating the development of tomato (Solanum lycopersicum) pedicel AZs. We found that the development of tomato pedicel AZs requires the gene MACROCALYX (MC), which was previously identified as a sepal size regulator and encodes a MADS-box transcription factor. MC has significant sequence similarity to Arabidopsis (Arabidopsis thaliana) FRUITFULL, which is involved in the regulation of fruit dehiscent zone development. The MC protein interacted physically with another MADS-box protein, JOINTLESS, which is known as a regulator of fruit abscission; the resulting heterodimer acquired a specific DNA-binding activity. Transcriptome analyses of pedicels at the preabscission stage revealed that the expression of the genes involved in phytohormone-related functions, cell wall modifications, fatty acid metabolism, and transcription factors is regulated by MC and JOINTLESS. The regulated genes include homologs of Arabidopsis WUSCHEL, REGULATOR OF AXILLARY MERISTEMS, CUP-SHAPED COTYLEDON, and LATERAL SUPPRESSOR. These Arabidopsis genes encode well-characterized transcription factors regulating meristem maintenance, axillary meristem development, and boundary formation in plant tissues. The tomato homologs were specifically expressed in AZs but not in other pedicel tissues, suggesting that these transcription factors may play key roles in pedicel AZ development. PMID:22106095

  2. AB248. Expression of EphA2 protein is positively associated with age, tumor size and Fuhrman nuclear grade in clear cell renal cell carcinomas

    PubMed Central

    Wang, Longxin; Zhou, Wenquan

    2016-01-01

    Background The receptor tyrosine kinase of EphA2 has been shown frequently overexpressed in various types of human carcinomas, but the relationship between the expression of EphA2 protein in clear cell renal cell carcinoma was not well documented. Methods In the present study, using specific anit-EphA2 polyclonal antibody and immunohistochemistry, we evaluated EphA2 protein expression levels in clear cell RCC specimens surgically resected from 90 patients. Results Our results shows that EphA2 protein was positively expressed in all normal renal tubes of 90 samples (100%, 3+), which was expressed at low levels in renal cortex but high levels in the collecting ducts of the renal medulla and papilla. EphA2 was negatively or weakly expressed in 30 out of 90 samples (33.3%, 0/1+), moderately expressed in 24 samples (26.7%, 2+) and strongly expressed in 36 samples (40%, 3+). Expression of EphA2 was positively associated with age (P=0.029), tumor diameters (P<0.001) and Fuhrman nuclear grade (P<0.001). Conclusions Our results indicate that EphA2 variably expressed in clear cell renal cell carci-nomas. High expression of EphA2 was more often found in big size and high nuclear grade tumors, which indicated EphA2 protein may be used as a new marker for the prognosis of clear cell renal cell carcinoma.

  3. Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450BM-1 in bacillus megaterium.

    PubMed

    Shaw, G C; Sung, C C; Liu, C H; Lin, C H

    1998-04-01

    The Bm1P1 protein was previously proposed to act as a positive transcription factor involved in barbiturate-mediated induction of cytochrome P450BM-1 in Bacillus megaterium. We now report that the bm1P1 gene encodes a protein of 217 amino acids, rather than the 98 amino acids as reported previously. In vitro gel shift assays indicate that the Bm1P1 protein did not interact with probes comprising the regulatory regions of the P450BM-1 gene. Moreover, disruption of the bm1P1 gene did not markedly affect barbiturate induction of P450BM-1 expression. A multicopy plasmid harboring only the P450BM-1 promoter region could increase expression of the chromosome-encoded P450BM-1. The level of expression is comparable with that shown by a multicopy plasmid harboring the P450BM-1 promoter region along with the bm1P1 gene. These results strongly suggest that the Bm1P1 protein is unlikely to act as a positive regulator for barbiturate induction of P450BM-1 expression. Finally, deletion of the Barbie box did not markedly diminish the effect of pentobarbital on expression of a reporter gene transcriptionally fused to the P450BM-1 promoter. This suggests that the Barbie box is unlikely to be a key element in barbiturate-mediated induction of P450BM-1. PMID:9525898

  4. Ligand-Induced Changes of the Apparent Transition-State Position in Mechanical Protein Unfolding

    PubMed Central

    Stigler, Johannes; Rief, Matthias

    2015-01-01

    Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins. PMID:26200872

  5. Serological diagnosis of pneumococcal infection in children with pneumonia using protein antigens: A study of cut-offs with positive and negative controls.

    PubMed

    Andrade, Dafne Carvalho; Borges, Igor Carmo; Ivaska, Lauri; Peltola, Ville; Meinke, Andreas; Barral, Aldina; Käyhty, Helena; Ruuskanen, Olli; Nascimento-Carvalho, Cristiana Maria

    2016-06-01

    The etiological diagnosis of infection by Streptococcus pneumoniae in children is difficult, and the use of indirect techniques is frequently warranted. We aimed to study the use of pneumococcal proteins for the serological diagnosis of pneumococcal infection in children with pneumonia. We analyzed paired serum samples from 13 Brazilian children with invasive pneumococcal pneumonia (positive control group) and 23 Finnish children with viral pharyngitis (negative control group), all aged <5years-old. Children with pharyngitis were evaluated for oropharyngeal colonization, and none of them carried S. pneumoniae. We used a multiplex bead-based assay with eight proteins: Ply, CbpA, PspA1 and 2, PcpA, PhtD, StkP and PcsB. The optimal cut-off for increase in antibody level for the diagnosis of pneumococcal infection was determined for each antigen by ROC curve analysis. The positive control group had a significantly higher rate of ≥2-fold rise in antibody levels against all pneumococcal proteins, except Ply, compared to the negative controls. The cut-off of ≥2-fold increase in antibody levels was accurate for pneumococcal infection diagnosis for all investigated antigens. However, there was a substantial increase in the accuracy of the test with a cut-off of ≥1.52-fold rise in antibody levels for PcpA. When using the investigated protein antigens for the diagnosis of pneumococcal infection, the detection of response against at least one antigen was highly sensitive (92.31%) and specific (91.30%). The use of serology with pneumococcal proteins is a promising method for the diagnosis of pneumococcal infection in children with pneumonia. The use of a ≥2-fold increase cut-off is adequate for most pneumococcal proteins. PMID:26928648

  6. Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system.

    PubMed

    Moon, Y H; Kang, H G; Jung, J Y; Jeon, J S; Sung, S K; An, G

    1999-08-01

    A MADS family gene, OsMADS6, was isolated from a rice (Oryza sativa L.) young flower cDNA library using OsAMDS1 as a probe. With this clone, various MADS box genes that encode for protein-to-protein interaction partners of the OsMADS6 protein were isolated by the yeast two-hybrid screening method. On the basis of sequence homology, OsMADS6 and the selected partners can be classified in the APETALA1/AGAMOUS-LIKE9 (AP1/AGL9) family. One of the interaction partners, OsMADS14, was selected for further study. Both genes began expression at early stages of flower development, and their expression was extended into the later stages. In mature flowers the OsMADS6 transcript was detectable in lodicules and also weakly in sterile lemmas and carpels, whereas the OsMADS14 transcript was detectable in sterile lemmas, paleas/lemmas, stamens, and carpels. Using the yeast two-hybrid system, we demonstrated that the region containing of the 109th to 137th amino acid residues of OsMADS6 is indispensable in the interaction with OsMADS14. Site-directed mutation analysis revealed that the four periodical leucine residues within the region are essential for this interaction. Furthermore, it was shown that the 14 amino acid residues located immediately downstream of the K domain enhance the interaction, and that the two leucine residues within this region play an important role in that enhancement. PMID:10444103

  7. Functional Requirement for a Highly Conserved Charged Residue at Position 75 in the Gap Junction Protein Connexin 32*

    PubMed Central

    Abrams, Charles K.; Islam, Mahee; Mahmoud, Rola; Kwon, Taekyung; Bargiello, Thaddeus A.; Freidin, Mona M.

    2013-01-01

    Charcot Marie Tooth disease (CMT) is a group of inherited disorders characterized clinically by exclusively or predominantly peripheral nerve dysfunction. CMT1X, the most common form of X-linked CMT is caused by mutations in connexin 32 (Cx32). In this work, we used dual whole cell patch clamp recording to examine the functional effects of mutations at the Arg75 position. This residue is highly conserved among members of the connexin family, and disease-causing mutations have been identified at this (or the corresponding) position in Cx26, Cx43, and Cx46. Thus, a better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of a number of different human diseases. All three mutants associated with CMT1X (R75P, R75Q, and R75W) showed very low levels of coupling similar to those of the cells transfected with vector alone. Heterotypic pairing with Cx32 WT showed that the absence of coupling for these mutants in the homotypic configuration could be explained by shifts in their hemichannel Gj-Vj relations. Examination of the expression levels and gating characteristics of seven additional mutants (R75A, R75D, R75E, R75H, R75K, R75L, and R75V) at this position suggest that the positive charge at position 75 in Cx32 is required for normal channel function but not for gap junction assembly. Our studies also suggest that disease treatment strategies for CMT1X, which correct trafficking abnormalities in Cx32, may be ineffective for the group of mutations also conferring changes in gating properties of Cx32 channels. PMID:23209285

  8. Characterization of the Pathway-Specific Positive Transcriptional Regulator for Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2) as a DNA-Binding Protein

    PubMed Central

    Arias, Paloma; Fernández-Moreno, Miguel A.; Malpartida, Francisco

    1999-01-01

    The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the act cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type actII-ORF4 gene but not with the actII-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified from Escherichia coli cultures; both showed specific DNA-binding activity for the actVI-ORF1–ORFA and actIII-actI intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the −35 regions of the corresponding promoters, showing the consensus sequence 5′-TCGAG-3′. Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the act genes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the act biosynthetic genes. PMID:10559161

  9. Discovery and verification of protein differences between Er positive/Her2/neu negative breast tumor tissue and matched adjacent normal breast tissue.

    PubMed

    Weitzel, Lindsay-Rae B; Byers, Tim; Allen, Jenna; Finlayson, Christina; Helmke, Steve M; Hokanson, John E; Hunsucker, Stephen W; Murphy, James R; Newell, Keri; Queensland, Kelly M; Singh, Meenakshi; Wischmeyer, Paul E; Duncan, Mark W; Elias, Anthony

    2010-11-01

    This study was designed to quantify and identify differences in protein levels between tumor and adjacent normal breast tissue from the same breast in 18 women with stage I/II ER positive/Her2/neu negative invasive breast cancer. Eighteen separate difference gel electrophoresis (DIGE) gels were run (1 gel per patient). Relative quantification was based on DIGE analysis. After excision and tryptic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass mapping were used to identify protein spots. Two hundred and forty-three spots were differentially abundant between normal and cancer tissues. Fifty spots were identified: 41 were over abundant and nine were less abundant in cancers than in normal breast tissue. Western blotting provided independent confirmation for three of the most biologically and statistically interesting proteins. All 18 gels were replicated by another technician and 32% of the differentially abundant proteins were verified by the duplicate analysis. Follow-up studies are now examining these proteins as biomarkers in blood. PMID:20087651

  10. A sporulation-specific, sigF-dependent protein, SspA, affects septum positioning in Streptomyces coelicolor

    PubMed Central

    Tzanis, Angelos; Dalton, Kate A; Hesketh, Andrew; den Hengst, Chris D; Buttner, Mark J; Thibessard, Annabelle; Kelemen, Gabriella H

    2014-01-01

    The RNA polymerase sigma factor SigF controls late development during sporulation in the filamentous bacterium Streptomyces coelicolor. The only known SigF-dependent gene identified so far, SCO5321, is found in the biosynthetic cluster encoding spore pigment synthesis. Here we identify the first direct target for SigF, the gene sspA, encoding a sporulation-specific protein. Bioinformatic analysis suggests that SspA is a secreted lipoprotein with two PepSY signature domains. The sspA deletion mutant exhibits irregular sporulation septation and altered spore shape, suggesting that SspA plays a role in septum formation and spore maturation. The fluorescent translational fusion protein SspA–mCherry localized first to septum sites, then subsequently around the surface of the spores. Both SspA protein and sspA transcription are absent from the sigF null mutant. Moreover, in vitro transcription assay confirmed that RNA polymerase holoenzyme containing SigF is sufficient for initiation of transcription from a single sspA promoter. In addition, in vivo and in vitro experiments showed that sspA is a direct target of BldD, which functions to repress sporulation genes, including whiG, ftsZ and ssgB, during vegetative growth, co-ordinating their expression during sporulation septation. PMID:24261854

  11. Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Cohen, Adiel; Kupiec, Martin; Weisman, Ronit

    2014-01-01

    The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling. PMID:24928510

  12. Genetic Diversity and Positive Selection Analysis of Classical Swine Fever Virus Envelope Protein Gene E2 in East China under C-Strain Vaccination

    PubMed Central

    Hu, Dongfang; Lv, Lin; Gu, Jinyuan; Chen, Tongyu; Xiao, Yihong; Liu, Sidang

    2016-01-01

    Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs worldwide. C-strain vaccination is one of the most effective ways to contain this disease. Since 2014, sporadic CSF outbreaks have been occurring in some C-strain vaccinated provinces of China. To decipher the disease etiology, 25 CSFV E2 genes from 169 clinical samples were cloned and sequenced. Phylogenetic analyses revealed that all 25 isolates belonged to subgenotype 2.1. Twenty-three of the 25 isolates were clustered in a newly defined subgenotype, 2.1d, and shared some consistent molecular characteristics. To determine whether the complete E2 gene was under positive selection pressure, we used a site-by-site analysis to identify specific codons that underwent evolutionary selection, and seven positively selected codons were found. Three positively selected sites (amino acids 17, 34, and 72) were identified in antigenicity-relevant domains B/C of the amino-terminal half of the E2 protein. In addition, another positively selected site (amino acid 200) exhibited a polarity change from hydrophilic to hydrophobic, which may change the antigenicity and virulence of CSFV. The results indicate that the circulating CSFV strains in Shandong province were mostly clustered in subgenotype 2.1d. Moreover, the identification of these positively selected sites could help to reveal molecular determinants of virulence or pathogenesis, and to clarify the driving force of CSFV evolution in East China. PMID:26903966

  13. Genetic Diversity and Positive Selection Analysis of Classical Swine Fever Virus Envelope Protein Gene E2 in East China under C-Strain Vaccination.

    PubMed

    Hu, Dongfang; Lv, Lin; Gu, Jinyuan; Chen, Tongyu; Xiao, Yihong; Liu, Sidang

    2016-01-01

    Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs worldwide. C-strain vaccination is one of the most effective ways to contain this disease. Since 2014, sporadic CSF outbreaks have been occurring in some C-strain vaccinated provinces of China. To decipher the disease etiology, 25 CSFV E2 genes from 169 clinical samples were cloned and sequenced. Phylogenetic analyses revealed that all 25 isolates belonged to subgenotype 2.1. Twenty-three of the 25 isolates were clustered in a newly defined subgenotype, 2.1d, and shared some consistent molecular characteristics. To determine whether the complete E2 gene was under positive selection pressure, we used a site-by-site analysis to identify specific codons that underwent evolutionary selection, and seven positively selected codons were found. Three positively selected sites (amino acids 17, 34, and 72) were identified in antigenicity-relevant domains B/C of the amino-terminal half of the E2 protein. In addition, another positively selected site (amino acid 200) exhibited a polarity change from hydrophilic to hydrophobic, which may change the antigenicity and virulence of CSFV. The results indicate that the circulating CSFV strains in Shandong province were mostly clustered in subgenotype 2.1d. Moreover, the identification of these positively selected sites could help to reveal molecular determinants of virulence or pathogenesis, and to clarify the driving force of CSFV evolution in East China. PMID:26903966

  14. Triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes by down-regulating expression of a viral protein LMP1

    SciTech Connect

    Zhou, Heng; Guo, Wei; Long, Cong; Wang, Huan; Wang, Jingchao; Sun, Xiaoping

    2015-01-16

    Highlights: • Triptolide inhibits proliferation of EBV-positive lymphoma cells in vitro and in vivo. • Triptolide reduces expression of LMP1 by decreasing its transcription level. • Triptolide inhibits ED-L1 promoter activity. - Abstract: Epstein–Barr virus (EBV) infects various types of cells and mainly establishes latent infection in B lymphocytes. The viral latent membrane protein 1 (LMP1) plays important roles in transformation and proliferation of B lymphocytes infected with EBV. Triptolide is a compound of Tripterygium extracts, showing anti-inflammatory, immunosuppressive, and anti-cancer activities. In this study, it is determined whether triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes. The CCK-8 assays were performed to examine cell viabilities of EBV-positive B95-8 and P3HR-1 cells treated by triptolide. The mRNA and protein levels of LMP1 were examined by real time-PCR and Western blotting, respectively. The activities of two LMP1 promoters (ED-L1 and TR-L1) were determined by Dual luciferase reportor assay. The results showed that triptolide inhibited the cell viability of EBV-positive B lymphocytes, and the over-expression of LMP1 attenuated this inhibitory effect. Triptolide decreased the LMP1 expression and transcriptional levels in EBV-positive B cells. The activity of LMP1 promoter ED-L1 in type III latent infection was strongly suppressed by triptolide treatment. In addition, triptolide strongly reduced growth of B95-8 induced B lymphoma in BALB/c nude mice. These results suggest that triptolide decreases proliferation of EBV-induced B lymphocytes possibly by a mechanism related to down-regulation of the LMP1 expression.

  15. TaTypA, a Ribosome-Binding GTPase Protein, Positively Regulates Wheat Resistance to the Stripe Rust Fungus

    PubMed Central

    Liu, Peng; Myo, Thwin; Ma, Wei; Lan, Dingyun; Qi, Tuo; Guo, Jia; Song, Ping; Guo, Jun; Kang, Zhensheng

    2016-01-01

    Tyrosine phosphorylation protein A (TypA/BipA) belongs to the ribosome-binding GTPase superfamily. In many bacterial species, TypA acts as a global stress and virulence regulator and also mediates resistance to the antimicrobial peptide bactericidal permeability-increasing protein. However, the function of TypA in plants under biotic stresses is not known. In this study, we isolated and functionally characterized a stress-responsive TypA gene (TaTypA) from wheat, with three copies located on chromosomes 6A, 6B, and 6D, respectively. Transient expression assays indicated chloroplast localization of TaTypA. The transcript levels of TaTypA were up-regulated in response to treatment with methyl viologen, which induces reactive oxygen species (ROS) in chloroplasts through photoreaction, cold stress, and infection by an avirulent strain of the stripe rust pathogen. Knock down of the expression of TaTypA through virus-induced gene silencing decreased the resistance of wheat to stripe rust accompanied by weakened ROS accumulation and hypersensitive response, an increase in TaCAT and TaSOD expression, and an increase in pathogen hyphal growth and branching. Our findings suggest that TaTypA contributes to resistance in an ROS-dependent manner. PMID:27446108

  16. The budding yeast Cdc48(Shp1) complex promotes cell cycle progression by positive regulation of protein phosphatase 1 (Glc7).

    PubMed

    Böhm, Stefanie; Buchberger, Alexander

    2013-01-01

    The conserved, ubiquitin-selective AAA ATPase Cdc48 regulates numerous cellular processes including protein quality control, DNA repair and the cell cycle. Cdc48 function is tightly controlled by a multitude of cofactors mediating substrate specificity and processing. The UBX domain protein Shp1 is a bona fide substrate-recruiting cofactor of Cdc48 in the budding yeast S. cerevisiae. Even though Shp1 has been proposed to be a positive regulator of Glc7, the catalytic subunit of protein phosphatase 1 in S. cerevisiae, its cellular functions in complex with Cdc48 remain largely unknown. Here we show that deletion of the SHP1 gene results in severe growth defects and a cell cycle delay at the metaphase to anaphase transition caused by reduced Glc7 activity. Using an engineered Cdc48 binding-deficient variant of Shp1, we establish the Cdc48(Shp1) complex as a critical regulator of mitotic Glc7 activity. We demonstrate that shp1 mutants possess a perturbed balance of Glc7 phosphatase and Ipl1 (Aurora B) kinase activities and show that hyper-phosphorylation of the kinetochore protein Dam1, a key mitotic substrate of Glc7 and Ipl1, is a critical defect in shp1. We also show for the first time a physical interaction between Glc7 and Shp1 in vivo. Whereas loss of Shp1 does not significantly affect Glc7 protein levels or localization, it causes reduced binding of the activator protein Glc8 to Glc7. Our data suggest that the Cdc48(Shp1) complex controls Glc7 activity by regulating its interaction with Glc8 and possibly further regulatory subunits. PMID:23418575

  17. Prolyl isomerase Pin1 is highly expressed in Her2-positive breast cancer and regulates erbB2 protein stability

    PubMed Central

    Lam, Prudence B; Burga, Laura N; Wu, Bryan P; Hofstatter, Erin W; Lu, Kun Ping; Wulf, Gerburg M

    2008-01-01

    Overexpression of HER-2/Neu occurs in about 25–30% of breast cancer patients and is indicative of poor prognosis. While Her2/Neu overexpression is primarily a result of erbB2 amplification, it has recently been recognized that erbB2 levels are also regulated on the protein level. However, factors that regulate Her2/Neu protein stability are less well understood. The prolyl isomerase Pin1 catalyzes the isomerization of specific pSer/Thr-Pro motifs that have been phosphorylated in response to mitogenic signaling. We have previously reported that Pin1-catalyzed post-phosphorylational modification of signal transduction modulates the oncogenic pathways downstream from c-neu. The goal of this study was to examine the expression of prolyl isomerase Pin1 in human Her2+ breast cancer, and to study if Pin1 affects the expression of Her2/Neu itself. Methods Immunohistochemistry for Her2 and Pin1 were performed on two hundred twenty-three human breast cancers, with 59% of the specimen from primary cancers and 41% from metastatic sites. Pin1 inhibition was achieved using siRNA in Her2+ breast cancer cell lines, and its effects were studied using cell viability assays, immunoblotting and immunofluorescence. Results Sixty-four samples (28.7%) stained positive for Her2 (IHC 3+), and 54% (122/223) of all breast cancers stained positive for Pin1. Of the Her2-positive cancers 40 (62.5%) were also Pin1-positive, based on strong nuclear or nuclear and cytoplasmic staining. Inhibition of Pin1 via RNAi resulted in significant suppression of Her2-positive tumor cell growth in BT474, SKBR3 and AU565 cells. Pin1 inhibition greatly increased the sensitivity of Her2-positive breast cancer cells to the mTOR inhibitor Rapamycin, while it did not increase their sensitivity to Trastuzumab, suggesting that Pin1 might act on Her2 signaling. We found that Pin1 interacted with the protein complex that contains ubiquitinated erbB2 and that Pin1 inhibition accelerated erbB2 degradation, which could

  18. RNA–protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog

    PubMed Central

    Costas, Celina; Yuriev, Elizabeth; Meyer, Karen L.; Guion, Tina S.; Hanna, Michelle M.

    2000-01-01

    A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase. This analog, 8-[(4-azidophenacyl)thio]adenosine 5′-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring. This position is not involved in the normal Watson–Crick base pairing needed for specific hybridization. Although 8-APAS-ATP could not replace ATP as a substrate for transcription initiation, once stable elongation complexes were formed, 8-APAS-AMP could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal positions in the RNA. Irradiation of transcription elongation complexes in which the RNA contained the analog exclusively at the 3′ end of an RNA 22mer, or a 23mer with the analog 1 nt from the 3′ end, produced RNA crosslinks to the RNA polymerase subunits that form the RNA 3′ end binding site (β,β′). Both 8-APAS-AMP and the related 8-azido-AMP were subjected to conformational modeling as nucleoside monophosphates and in DNA–RNA hybrids. Surprisingly, the lowest energy conformation for 8-APAS-AMP was found to be syn, while that of 8-azido-AMP was anti, suggesting that the conformational properties and transcription substrate properties of 8-azido-ATP should be re-evaluated. Although the azide and linker together are larger in 8-APAS-ATP than in 8-N3-ATP, the flexibility of the linker itself allows this analog to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase. PMID:10756182

  19. RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog.

    PubMed

    Costas, C; Yuriev, E; Meyer, K L; Guion, T S; Hanna, M M

    2000-05-01

    A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase. This analog, 8-[(4-azidophenacyl)thio]adenosine 5'-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring. This position is not involved in the normal Watson-Crick base pairing needed for specific hybridization. Although 8-APAS-ATP could not replace ATP as a substrate for transcription initiation, once stable elongation complexes were formed, 8-APAS-AMP could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal positions in the RNA. Irradiation of transcription elongation complexes in which the RNA contained the analog exclusively at the 3' end of an RNA 22mer, or a 23mer with the analog 1 nt from the 3' end, produced RNA crosslinks to the RNA polymerase subunits that form the RNA 3' end binding site (beta, beta'). Both 8-APAS-AMP and the related 8-azido-AMP were subjected to conformational modeling as nucleoside monophosphates and in DNA-RNA hybrids. Surprisingly, the lowest energy conformation for 8-APAS-AMP was found to be syn, while that of 8-azido-AMP was anti, suggesting that the conformational properties and transcription substrate properties of 8-azido-ATP should be re-evaluated. Although the azide and linker together are larger in 8-APAS-ATP than in 8-N(3)-ATP, the flexibility of the linker itself allows this analog to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase. PMID:10756182

  20. Dual Positive Feedback Regulation of Protein Degradation of an Extra-cytoplasmic Function σ Factor for Cell Differentiation in Streptomyces coelicolor *

    PubMed Central

    Mao, Xu-Ming; Sun, Ning; Wang, Feng; Luo, Shuai; Zhou, Zhan; Feng, Wei-Hong; Huang, Fang-Liang; Li, Yong-Quan

    2013-01-01

    Here we report that in Streptomyces coelicolor, the protein stability of an ECF σ factor SigT, which is involved in the negative regulation of cell differentiation, was completely dependent on its cognate anti-σ factor RstA. The degradation of RstA caused a ClpP/SsrA-dependent degradation of SigT during cell differentiation. This was consistent with the delayed morphological development or secondary metabolism in the ΔclpP background after rstA deletion or sigT overexpression. Meanwhile, SigT negatively regulated clpP/ssrA expression by directly binding to the clpP promoter (clpPp). The SigT-clpPp interaction could be disrupted by secondary metabolites, giving rise to the stabilized SigT protein and retarded morphological development in a non-antibiotic-producing mutant. Thus a novel regulatory mechanism was revealed that the protein degradation of the ECF σ factor was initiated by the degradation of its anti-σ factor, and was accelerated in a dual positive feedback manner, through regulation by secondary metabolites, to promote rapid and irreversible development of the secondary metabolism. This ingenious cooperation of intracellular components can ensure economical and exquisite control of the ECF σ factor protein level for the proper cell differentiation in Streptomyces. PMID:24014034

  1. Co-dependent positive regulation of the ansB promoter of Escherichia coli by CRP and the FNR protein: a molecular analysis.

    PubMed

    Jennings, M P; Beacham, I R

    1993-07-01

    Transcription of the ansB gene, encoding L-asparaginase II, is positively regulated by cAMP receptor protein (CRP) and by the product of the fnr gene, the FNR protein. These global regulatory proteins mediate the expression of ansB in Escherichia coli in response to carbon source and to anaerobiosis, respectively, and are required concurrently for optimal ansB expression. The mechanism whereby CRP and FNR interact co-operatively with the ansB promoter to achieve transcription has not previously been established. We have utilized an ansB'-'lacZ fusion, in conjunction with deletion analysis and site-directed mutagenesis, to identify two sites which interact with these regulatory proteins in the ansB promoter. The first is an FNR site, centred 41.5 bp upstream of the major transcriptional start site. The second site, located 28 bp upstream of the FNR site, is the site of CRP regulation. This site is homologous to both the CRP and FNR binding-site consensus sequences and may respond to both CRP and FNR. The concurrent requirement for CRP and FNR for optimal expression of ansB may be explained if, first, essentially no transcription occurs unless the FNR is bound at the downstream site, and, second, the level of transcription when FNR alone is present is enhanced when CRP binds at the upstream site. PMID:8412660

  2. Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses.

    PubMed

    Kim, Eun Yu; Park, Ki Youl; Seo, Young Sam; Kim, Woo Taek

    2016-04-01

    Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed. PMID:26903535

  3. Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses1[OPEN

    PubMed Central

    Kim, Eun Yu; Park, Ki Youl; Seo, Young Sam; Kim, Woo Taek

    2016-01-01

    Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed. PMID:26903535

  4. Mutation in P0, a dual function ribosomal protein/apurinic/apyrimidinic endonuclease, modifies gene expression and position effect variegation in Drosophila.

    PubMed Central

    Frolov, M V; Birchler, J A

    1998-01-01

    In a search for modifiers of gene expression with the white eye color gene as a target, a third chromosomal P-element insertion mutant l(3)01544 has been identified that exhibits a strong pigment increase in a white-apricot background. Molecular analysis shows that the P-element insertion is found in the first intron of the gene surrounding the insertion site. Sequencing both the cDNA and genomic fragments revealed that the identified gene is identical to one encoding ribosomal protein P0/apurinic/apyrimidinic endonuclease. The P-element-induced mutation, l(3)01544, affects the steady-state level of white transcripts and transcripts of some other genes. In addition, l(3)01544 suppresses the variegated phenotypes of In(1)wm4h and In(1)y3P, suggesting a potential involvement of the P0 protein in modifying position effect variegation. The revertant generated by the precise excision of the P element has lost all mutant phenotypes. Recent work revealed that Drosophila ribosomal protein P0 contains an apurinic/apyrimidinic endonuclease activity. Our results suggest that this multifunctional protein is also involved in regulation of gene expression in Drosophila. PMID:9832526

  5. Role of positively charged residues of the second transmembrane domain in the ion transport activity and conformation of human uncoupling protein-2.

    PubMed

    Hoang, Tuan; Matovic, Tijana; Parker, James; Smith, Matthew D; Jelokhani-Niaraki, Masoud

    2015-04-14

    Residing at the inner mitochondrial membrane, uncoupling protein-2 (UCP2) mediates proton transport from the intermembrane space (IMS) to the mitochondrial matrix and consequently reduces the rate of ATP synthesis in the mitochondria. The ubiquitous expression of UCP2 in humans can be attributed to the protein's multiple physiological roles in tissues, including its involvement in protective mechanisms against oxidative stress, as well as glucose and lipid metabolisms. Currently, the structural properties and ion transport mechanism of UCP2 and other UCP homologues remain poorly understood. UCP2-mediated proton transport is activated by fatty acids and inhibited by di- and triphosphate purine nucleotides. UCP2 also transports chloride and some other small anions. Identification of key amino acid residues of UCP2 in its ion transport pathway can shed light on the protein's ion transport function. On the basis of our previous studies, the second transmembrane helix segment (TM2) of UCP2 exhibited chloride channel activity. In addition, it was suggested that the positively charged residues on TM2 domains of UCPs 1 and 2 were important for their chloride transport activity. On this basis, to further understand the role of these positively charged residues on the ion transport activity of UCP2, we recombinantly expressed four TM2 mutants: R76Q, R88Q, R96Q, and K104Q. The wild type UCP2 and its mutants were purified and reconstituted into liposomes, and their conformation and ion (proton and chloride) transport activity were studied. TM2 Arg residues at the matrix interface of UCP2 proved to be crucial for the protein's anion transport function, and their absence resulted in highly diminished Cl(-) transport rates. On the other hand, the two other positively charged residues of TM2, located at the UCP2-IMS interface, could participate in the salt-bridge formation in the protein and promote the interhelical tight packing in the UCP2. Absence of these residues did not

  6. The onset of the progression of acute phase response mechanisms induced by extreme impacts can be followed by the decrease in blood levels of positive acute phase proteins.

    NASA Astrophysics Data System (ADS)

    Larina, Olga; Bekker, Anna

    Studies performed at space flights and earth-based simulation models detected the plasma indices of acute phase reaction (APR), i.e. the increase of APR cytokine mediators and alterations in the production of blood acute phase proteins (APP) at the initial stages of adaptation to altered gravity conditions. Acute phase response is the principal constituent of the functional activity of innate immunity system. Changes in plasma APPs contents are considered to serve the restoration of homeostasis state. According to trends of their concentration shifts at the evolving of acute phase reaction APPs are denoted as positive, neutral, or negative. Plasma concentrations of positive acute phase proteins α1-acid glycoprotein (α1-AGP), α1-antitrypsin (α1-AT), and neutral α2-macroglobulin (α2-M) were measured in human study at 12-hour antiorthostatic position (AOP) with 15° head down tilt and hypoxia experiments at 14% oxygen in pressure chamber. Both of these impacts were shown to produce alterations in the APP levels indicative for acute phase response. Nevertheless, in AOP experiment noticeable decrease in α1-AGP concentration occurred by hour 12, and even more pronounced decline of α1-AGP and α1-AT were found on hypoxia hours 12 and 36. Acute phase proteins α1-AGP and α2-M possess the features of proteinase inhibitors. This function is implemented by the formation of complexes with the molecules of proteolytic enzymes which subsequently are removed from the blood flow. Transient decrease in plasma concentrations of protease inhibitors on early phases of APR development was reported to result from the growth of plasma protease activity due to cathepsin release from activated leukocytes, which had not yet been compensated by enhanced APP synthesis. Being a carrier protein for positively charged and neutral substances, α1-AGP shows pronounced elevation in its blood content during APR development. As assumed, it is required for the transportation of the increased

  7. Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

    PubMed Central

    Tejeda-Muñoz, Nydia; González-Aguilar, Héctor; Santoyo-Ramos, Paula; Castañeda-Patlán, M. Cristina

    2015-01-01

    The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells. PMID:26711256

  8. Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

    SciTech Connect

    Son, Ora; Kim, Sunghan; Shin, Yun-jeong; Kim, Woo-Young; Koh, Hee-Jong; Cheon, Choong-Ill

    2015-09-18

    The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription.

  9. A critical role of two positively charged amino acids in the Jas motif of Arabidopsis JAZ proteins in mediating coronatine- and jasmonoyl isoleucine-dependent interaction with the COI1 F-box protein

    PubMed Central

    Melotto, Maeli; Mecey, Christy; Niu, Yajie; Chung, Hoo Sun; Katsir, Leron; Yao, Jian; Zeng, Weiqing; Thines, Bryan; Staswick, Paul; Browse, John; Howe, Gregg; He, Sheng Yang

    2009-01-01

    Summary Coronatine is an important virulence factor produced by several pathovars of the bacterial pathogen Pseudomonas syringae. The structure of coronatine is similar to a class of plant hormones called jasmonates (JAs). An important step in JA signaling is the SCFCOI1 E3 ubiquitin ligase-dependent degradation of JAZ repressor proteins. We recently showed that jasmonoyl-isoleucine (JA-Ile) could promote physical interaction between Arabidopsis JAZ1 and COI1 (the F-box component of SCFCOI1) proteins, and that the JA-Ile-dependent COI1-JAZ1 interaction could be reconstituted in yeast cells (i.e., in the absence of other plant proteins). Here, we show that coronatine, but not its two biosynthetic precursors, could also promote interaction between Arabidopsis COI1 and multiple JAZ proteins. The carboxyl terminal Jas motif, but not the N-terminal (NT) domain or central ZIM domain of JAZ proteins, is critical for JA-Ile/coronatine-dependent interaction with COI1. Two positively charged amino acid residues in the Jas domain were identified as being essential for coronatine-dependent COI1-JAZ interactions. These two mutations did not affect the ability of JAZ1 and JAZ9 to interact with the transcription factor AtMYC2. Importantly, transgenic Arabidopsis plants expressing JAZ1 carrying these two mutations exhibited JA-insensitive phenotypes, including male sterility and enhanced resistance to P. syringae infection. These results not only suggest that coronatine and JA-Ile target the physical interaction between COI1 and the Jas domain of JAZ repressors, but also illustrate a critical role of positively charged amino acids in the Jas domain in mediating JA-Ile/coronatine-dependent JAZ interaction with COI1. PMID:18547396

  10. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  11. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  12. Myofibrillar myopathy with abnormal foci of desmin positivity. II. Immunocytochemical analysis reveals accumulation of multiple other proteins.

    PubMed

    De Bleecker, J L; Engel, A G; Ertl, B B

    1996-05-01

    The two major types of lesions in myofibrillar myopathy consist of hyaline spheroidal structures composed of compacted myofibrillar residues, and nonhyaline lesions that comprise foci of myofibrillar destruction. We employed immunocytochemical analysis to further characterize these abnormalities. The nonhyaline lesions are depleted of actin, alpha-actinin, myosin, and, less consistently, of titin and nebulin. Thus, each major component of the myofibrils is lost or decreased. These lesions also react strongly for both NCAM and desmin. By contrast, the hyaline structures are highly enriched in actin, are immunoreactive for fast and slow myosin, and show increased expression of titin, nebulin, and alpha-actinin. They fail to react for NCAM and react variably for desmin. Both types of lesion react, but with differing intensities, for gelsolin, dystrophin, beta-amyloid precursor protein (beta APP) epitopes amino-terminal to the alpha-secretase site, alpha 1-antichymotrypsin, and ubiquitin, and both can be congophilic. The increased expressions of desmin, dystrophin and gelsolin in muscle are also confirmed by immunoblot studies. The results, in harmony with the ultrastructural findings described in the companion paper, suggest that myofibrillar myopathy is conditioned by abnormal activation of a degradative process that primarily affects the myofibrils. A structural abnormality of desmin alone may not be sufficient to disrupt the myofibrillar architecture, but abnormal activation of a phosphorylating process could account for dissolution of the myofibrils. The cause and significance of the ectopic overexpression of desmin, dystrophin, NCAM, and beta APP components, and the chemical basis of the congophilia remain unknown. PMID:8627347

  13. Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket.

    PubMed Central

    Belduz, A O; Lee, E J; Harman, J G

    1993-01-01

    The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP. PMID:8388097

  14. Mycobacterium tuberculosis SigM positively regulates Esx secreted protein and nonribosomal peptide synthetase genes and down regulates virulence-associated surface lipid synthesis.

    PubMed

    Raman, Sahadevan; Puyang, Xiaoling; Cheng, Tan-Yun; Young, David C; Moody, D Branch; Husson, Robert N

    2006-12-01

    The Mycobacterium tuberculosis genome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigated M. tuberculosis SigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in a sigM mutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and the kasA-kasB operon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in a sigM-disrupted strain and is undetectable in a sigM overexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions. PMID:17028284

  15. A Set of miRNAs, Their Gene and Protein Targets and Stromal Genes Distinguish Early from Late Onset ER Positive Breast Cancer

    PubMed Central

    Bastos, E. P.; Brentani, H.; Pereira, C. A. B.; Polpo, A.; Lima, L.; Puga, R. D.; Pasini, F. S.; Osorio, C. A. B. T.; Roela, R. A.; Achatz, M. I.; Trapé, A. P.; Gonzalez-Angulo, A. M.; Brentani, M. M.

    2016-01-01

    Breast cancer (BC) in young adult patients (YA) has a more aggressive biological behavior and is associated with a worse prognosis than BC arising in middle aged patients (MA). We proposed that differentially expressed miRNAs could regulate genes and proteins underlying aggressive phenotypes of breast tumors in YA patients when compared to those arising in MA patients. Objective: Using integrated expression analyses of miRs, their mRNA and protein targets and stromal gene expression, we aimed to identify differentially expressed profiles between tumors from YA-BC and MA-BC. Methodology and Results: Samples of ER+ invasive ductal breast carcinomas, divided into two groups: YA-BC (35 years or less) or MA-BC (50–65 years) were evaluated. Screening for BRCA1/2 status according to the BOADICEA program indicated low risk of patients being carriers of these mutations. Aggressive characteristics were more evident in YA-BC versus MA-BC. Performing qPCR, we identified eight miRs differentially expressed (miR-9, 18b, 33b, 106a, 106b, 210, 518a-3p and miR-372) between YA-BC and MA-BC tumors with high confidence statement, which were associated with aggressive clinicopathological characteristics. The expression profiles by microarray identified 602 predicted target genes associated to proliferation, cell cycle and development biological functions. Performing RPPA, 24 target proteins differed between both groups and 21 were interconnected within a network protein-protein interactions associated with proliferation, development and metabolism pathways over represented in YA-BC. Combination of eight mRNA targets or the combination of eight target proteins defined indicators able to classify individual samples into YA-BC or MA-BC groups. Fibroblast-enriched stroma expression profile analysis resulted in 308 stromal genes differentially expressed between YA-BC and MA-BC. Conclusion: We defined a set of differentially expressed miRNAs, their mRNAs and protein targets and stromal

  16. An Intriguing Shift Occurs in the Novel Protein Phosphatase 1 Binding Partner, TCTEX1D4: Evidence of Positive Selection in a Pika Model

    PubMed Central

    Korrodi-Gregório, Luís; Margarida Lopes, Ana; Esteves, Sara L. C.; Afonso, Sandra; Lemos de Matos, Ana; Lissovsky, Andrey A.; da Cruz e Silva, Odete A. B.; Esteves, Pedro José; Fardilha, Margarida

    2013-01-01

    T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) contains the canonical phosphoprotein phosphatase 1 (PPP1) binding motif, composed by the amino acid sequence RVSF. We identified and validated the binding of TCTEX1D4 to PPP1 and demonstrated that indeed this protein is a novel PPP1 interacting protein. Analyses of twenty-one mammalian species available in public databases and seven Lagomorpha sequences obtained in this work showed that the PPP1 binding motif 90RVSF93 is present in all of them and is flanked by a palindromic sequence, PLGS, except in three species of pikas (Ochotona princeps, O. dauurica and O. pusilla). Furthermore, for the Ochotona species an extra glycosylation site, motif 96NLS98, and the loss of the palindromic sequence were observed. Comparison with other lagomorphs suggests that this event happened before the Ochotona radiation. The dN/dS for the sequence region comprising the PPP1 binding motif and the flanking palindrome highly supports the hypothesis that for Ochotona species this region has been evolving under positive selection. In addition, mutational screening shows that the ability of pikas TCTEX1D4 to bind to PPP1 is maintained, although the PPP1 binding motif is disrupted, and the N- and C-terminal surrounding residues are also abrogated. These observations suggest pika as an ideal model to study novel PPP1 complexes regulatory mechanisms. PMID:24130861

  17. The role of mRNA and protein stability in the function of coupled positive and negative feedback systems in eukaryotic cells.

    PubMed

    Moss Bendtsen, Kristian; Jensen, Mogens H; Krishna, Sandeep; Semsey, Szabolcs

    2015-01-01

    Oscillators and switches are important elements of regulation in biological systems. These are composed of coupling negative feedback loops, which cause oscillations when delayed, and positive feedback loops, which lead to memory formation. Here, we examine the behavior of a coupled feedback system, the Negative Autoregulated Frustrated bistability motif (NAF). This motif is a combination of two previously explored motifs, the frustrated bistability motif (FBM) and the negative auto regulation motif (NAR), which both can produce oscillations. The NAF motif was previously suggested to govern long term memory formation in animals, and was used as a synthetic oscillator in bacteria. We build a mathematical model to analyze the dynamics of the NAF motif. We show analytically that the NAF motif requires an asymmetry in the strengths of activation and repression links in order to produce oscillations. We show that the effect of time delays in eukaryotic cells, originating from mRNA export and protein import, are negligible in this system. Based on the reported protein and mRNA half-lives in eukaryotic cells, we find that even though the NAF motif possesses the ability for oscillations, it mostly promotes constant protein expression at the biologically relevant parameter regimes. PMID:26365394

  18. The role of mRNA and protein stability in the function of coupled positive and negative feedback systems in eukaryotic cells

    PubMed Central

    Moss Bendtsen, Kristian; Jensen, Mogens H.; Krishna, Sandeep; Semsey, Szabolcs

    2015-01-01

    Oscillators and switches are important elements of regulation in biological systems. These are composed of coupling negative feedback loops, which cause oscillations when delayed, and positive feedback loops, which lead to memory formation. Here, we examine the behavior of a coupled feedback system, the Negative Autoregulated Frustrated bistability motif (NAF). This motif is a combination of two previously explored motifs, the frustrated bistability motif (FBM) and the negative auto regulation motif (NAR), which both can produce oscillations. The NAF motif was previously suggested to govern long term memory formation in animals, and was used as a synthetic oscillator in bacteria. We build a mathematical model to analyze the dynamics of the NAF motif. We show analytically that the NAF motif requires an asymmetry in the strengths of activation and repression links in order to produce oscillations. We show that the effect of time delays in eukaryotic cells, originating from mRNA export and protein import, are negligible in this system. Based on the reported protein and mRNA half-lives in eukaryotic cells, we find that even though the NAF motif possesses the ability for oscillations, it mostly promotes constant protein expression at the biologically relevant parameter regimes. PMID:26365394

  19. γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP)

    PubMed Central

    Lessard, Christian B.; Cottrell, Barbara A.; Maruyama, Hiroko; Suresh, Suraj; Golde, Todd E.; Koo, Edward H.

    2015-01-01

    The relative increase in Aβ42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid β-protein (Aβ) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aβ peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aβ42/Aβ40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aβ42 generation by altering processivity but not ε-cleavage site utilization. PMID:26678856

  20. OsCCD1, a novel small calcium-binding protein with one EF-hand motif, positively regulates osmotic and salt tolerance in rice.

    PubMed

    Jing, Pei; Zou, Juanzi; Kong, Lin; Hu, Shiqi; Wang, Biying; Yang, Jun; Xie, Guosheng

    2016-06-01

    Calcium-binding proteins play key roles in the signal transduction in the growth and stress response in eukaryotes. However, a subfamily of proteins with one EF-hand motif has not been fully studied in higher plants. Here, a novel small calcium-binding protein with a C-terminal centrin-like domain (CCD1) in rice, OsCCD1, was characterized to show high similarity with a TaCCD1 in wheat. As a result, OsCCD1 can bind Ca(2+) in the in vitro EMSA and the fluorescence staining calcium-binding assays. Transient expression of green fluorescent protein (GFP)-tagged OsCCD1 in rice protoplasts showed that OsCCD1 was localized in the nucleus and cytosol of rice cells. OsCCD1 transcript levels were transiently induced by osmotic stress and salt stress through the calcium-mediated ABA signal. The rice seedlings of T-DNA mutant lines showed significantly less tolerance to osmotic and salt stresses than wild type plants (p<0.01). Conversely, its overexpressors can significantly enhance the tolerance to osmotic and salt stresses than wild type plants (p<0.05). Semi-quantitative RT-PCR analysis revealed that, OsDREB2B, OsAPX1 and OsP5CS genes are involved in the rice tolerance to osmotic and salt stresses. In sum, OsCCD1 gene probably affects the DREB2B and its downstream genes to positively regulate osmotic and salt tolerance in rice seedlings. PMID:27095404

  1. MADS Box Transcription Factor Mbx2/Pvg4 Regulates Invasive Growth and Flocculation by Inducing gsf2+ Expression in Fission Yeast

    PubMed Central

    Matsuzawa, Tomohiko; Yoritsune, Ken-ichi

    2012-01-01

    The fission yeast Schizosaccharomyces pombe exhibits invasive growth and nonsexual flocculation in response to nitrogen limitation. Gsf2, a flocculin of fission yeast, is required not only for nonsexual flocculation but also for invasive growth through the recognition of galactose residues on cell surface glycoconjugates. We found that pyruvylation negatively regulates nonsexual flocculation by capping the galactose residues of N-linked galactomannan. We investigated whether pyruvylation also regulates invasive growth. The pvg4+ gene originally was isolated as a multicopy suppressor of a pvg4 mutant defective in the pyruvylation of N-linked oligosaccharides. However, we did not detect a defect in cell surface pyruvylation in the pvg4/mbx2 deletion mutant, as assessed by alcian blue staining and a Q-Sepharose binding assay. Instead, the deletion prevented invasive growth under conditions of low nitrogen and high glucose, and it reduced the adhesion and flocculation of otherwise flocculent mutants by reducing gsf2+ expression. mbx2+-overexpressing strains exhibited nonsexual and calcium-dependent aggregation, which was inhibited in the presence of galactose but mediated by the induction of gsf2+. These findings indicate that Mbx2 mediates invasive growth and flocculation via the transcriptional activation of gsf2+ in fission yeast. In addition, we found that fission yeast Mbx2 induces the nonsexual flocculation of budding yeast by the activation of FLO1. PMID:22180499

  2. The MIK region rather than the C-terminal domain of AP3-like class B floral homeotic proteins determines functional specificity in the development and evolution of petals.

    PubMed

    Su, Kunmei; Zhao, Suzhen; Shan, Hongyan; Kong, Hongzhi; Lu, Wenliang; Theissen, Günter; Chen, Zhiduan; Meng, Zheng

    2008-01-01

    In core eudicots, euAP3-type MADS-box genes encode a PISTILLATA (PI)-derived motif, as well as a C-terminal euAP3 motif that originated from a paleoAP3 motif of an ancestral APETALA3 (AP3)-like protein through a translational frameshift mutation. To determine the functional and evolutionary relevance of these motifs, a series of point mutation and domain-swap constructs were generated, involving CsAP3, a paleoAP3-type gene from the basal angiosperm Chloranthus spicatus encoding a truncated paleoAP3 motif, and AtAP3, a euAP3-type gene from the core eudicot Arabidopsis thaliana. The chimeric constructs were expressed in A. thaliana under the control of the AP3 promoter or the CaMV 35S promoter in an ap3 mutant or wild-type background, respectively. Significant recovery of AP3 function was obtained in both complementation and ectopic expression experiments whenever the region upstream of the C-terminal motifs (MIK region) from A. thaliana was taken, even when the PI-derived motif and the truncated paleoAP3 motif of CsAP3 substituted for the corresponding sequences from AtAP3. However, no or very weak complementation or gain-of-function was seen when the MIK region was from CsAP3. Our data suggest that changes in the MIK region rather than mutations in the C-terminal domain were of crucial importance for the evolution of the functional specificity of euAP3-type proteins in stamen and petal development. PMID:18298432

  3. The euAP1 Protein MPF3 Represses MPF2 to Specify Floral Calyx Identity and Displays Crucial Roles in Chinese Lantern Development in Physalis[C][W

    PubMed Central

    Zhao, Jing; Tian, Ying; Zhang, Ji-Si; Zhao, Man; Gong, Pichang; Riss, Simone; Saedler, Rainer; He, Chaoying

    2013-01-01

    The Chinese lantern phenotype or inflated calyx syndrome (ICS) is a postfloral morphological novelty in Physalis. Its origin is associated with the heterotopic expression of the MADS box gene 2 from Physalis floridana (MPF2) in floral organs, yet the process underlying its identity remains elusive. Here, we show that MPF3, which is expressed specifically in floral tissues, encodes a core eudicot APETALA1-like (euAP1) MADS-domain protein. MPF3 was primarily localized to the nucleus, and it interacted with MPF2 and some floral MADS-domain proteins to selectively bind the CC-A-rich-GG (CArG) boxes in the MPF2 promoter. Downregulating MPF3 resulted in a dramatic elevation in MPF2 in the calyces and androecium, leading to enlarged and leaf-like floral calyces; however, the postfloral lantern was smaller and deformed. Starch accumulation in pollen was blocked. MPF3 MPF2 double knockdowns showed normal floral calyces and more mature pollen than those found in plants in which either MPF3 or MPF2 was downregulated. Therefore, MPF3 specifies calyx identity and regulates ICS formation and male fertility through interactions with MPF2/MPF2. Furthermore, both genes were found to activate Physalis floridana invertase gene 4 homolog, which encodes an invertase cleaving Suc, a putative key gene in sugar partitioning. The novel role of the MPF3-MPF2 regulatory circuit in male fertility is integral to the origin of ICS. Our results shed light on the evolution and development of ICS in Physalis and on the functional evolution of euAP1s in angiosperms. PMID:23792370

  4. Serum Zinc Concentration and C-Reactive Protein in Individuals with Human Immunodeficiency Virus Infection: the Positive Living with HIV (POLH) Study.

    PubMed

    Poudel, Krishna C; Bertone-Johnson, Elizabeth R; Poudel-Tandukar, Kalpana

    2016-05-01

    Low zinc levels and chronic inflammation are common in individuals infected with human immunodeficiency virus (HIV). Zinc deficiency may promote systemic inflammation, but research on the role of zinc in inflammation among HIV-positive individuals taking account of anti-retroviral therapy is lacking. We assessed the association between serum zinc and C-reactive protein (CRP) concentration in a cohort of HIV-positive individuals. A cross-sectional survey was conducted among 311 HIV-positive individuals (177 men and 134 women) aged 18-60 years residing in Kathmandu, Nepal. High-sensitive or regular serum CRP concentrations were measured by the latex agglutination nephelometry or turbidimetric method, and zinc concentrations were measured by the atomic absorption method. Relationships were assessed using multiple linear regression analysis. The geometric means of zinc in men and women were 73.83 and 71.93 ug/dL, respectively, and of CRP were 1.64 and 0.96 mg/L, respectively. Mean serum CRP concentration was significantly decreased with increasing serum zinc concentration across zinc tertiles (P for trend = 0.010), with mean serum CRP concentration in the highest tertile of serum zinc concentration was 44.2 % lower than that in the lowest tertile. The mean serum CRP concentrations in men and women in the highest tertile of serum zinc concentrations were 30 and 35.9 % lower, respectively, than that in the lowest tertile (P for trend = 0.263 and 0.162, respectively). We found a significant inverse relation between log zinc and log CRP concentrations (beta for 1 unit change in log zinc; β = -1.79, p = 0.0003). Serum zinc concentration may be inversely associated with serum CRP concentration in HIV-positive individuals. PMID:26429417

  5. Regulation of glycoprotein D synthesis: does alpha 4, the major regulatory protein of herpes simplex virus 1, regulate late genes both positively and negatively?

    PubMed Central

    Arsenakis, M; Campadelli-Fiume, G; Roizman, B

    1988-01-01

    Earlier studies have described the alpha 4/c113 baby hamster kidney cell line which constitutively expresses the alpha 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1). Introduction of the HSV-1 glycoprotein B (gB) gene, regulated as a gamma 1 gene, into these cells yielded a cell line which constitutively expressed both the alpha 4 and gamma 1 gB genes. The expression of the gB gene was dependent on the presence of functional alpha 4 protein. In this article we report that we introduced into the alpha 4/c113 and into the parental BHK cells, the HSV-1 BamHI J fragment, which encodes the domains of four genes, including those of glycoproteins D, G, and I (gD, gG, and gI), and most of the coding sequences of the glycoprotein E (gE) gene. In contrast to the earlier studies, we obtained significant constitutive expression of gD (also a gamma 1 gene) in a cell line (BJ) derived from parental BHK cells, but not in a cell line (alpha 4/BJ) which expresses functional alpha 4 protein. RNA homologous to the gD gene was present in significant amounts in the BJ cell line; smaller amounts of this RNA were detected in the alpha 4/BJ cell line. RNA homologous to gE, presumed to be polyadenylated from signals in the vector sequences, was present in the BJ cells but not in the alpha 4/BJ cells. The expression of the HSV-1 gD and gE genes was readily induced in the alpha 4/BJ cells by superinfection with HSV-2. The BJ cell line was, in contrast, resistant to expression of HSV-1 and HSV-2 genes. The BamHI J DNA fragment copy number was approximately 1 per BJ cell genome equivalent and 30 to 50 per alpha 4/BJ cell genome equivalent. We conclude that (i) the genes specifying gD and gB belong to different viral regulatory gene subsets, (ii) the gD gene is subject to both positive and negative regulation, (iii) both gD and gE mRNAs are subject to translational controls although they may be different, and (iv) the absence of expression of gD in the alpha 4/BJ

  6. Extensive protein hydrolysate formula effectively reduces regurgitation in infants with positive and negative challenge tests for cow’s milk allergy

    PubMed Central

    Vandenplas, Y; De Greef, E

    2014-01-01

    Aim Cow’s milk protein allergy (CMPA) is treated using an elimination diet with an extensive protein hydrolysate. We explored whether a thickened or nonthickened version was best for infants with suspected CMPA, which commonly causes regurgitation/vomiting. Methods Diagnosis of CMPA was based on a positive challenge test. We compared the efficacy of two casein extensive hydrolysates (eCH), a nonthickened version (NT-eCH) and a thickened version (T-eCH), using a symptom-based score covering regurgitation, crying, stool consistency, eczema, urticarial and respiratory symptoms. Results A challenge was performed in 52/72 infants with suspected CMPA and was positive in 65.4%. All confirmed CMPA cases tolerated eCH. The symptom-based score decreased significantly in all infants within a month, and the highest reduction was in those with confirmed CMPA. Regurgitation was reduced in all infants (6.4 ± 3.2–2.8 ± 2.9, p < 0.001), but fell more with the T-eCH (−4.2 ± 3.2 regurgitations/day vs. −3.0 ± 4.5, ns), especially in infants with a negative challenge (−3.9 ± 4.0 vs. −1.9 ± 3.4, ns). Conclusion eCH fulfilled the criteria for a hypoallergenic formula, and the NT-eCH and T-eCH formulas both reduced CMPA symptoms. The symptom-based score is useful for evaluating how effective dietary treatments are for CMPA. PMID:24575806

  7. CO2-Responsive CONSTANS, CONSTANS-Like, and Time of Chlorophyll a/b Binding Protein Expression1 Protein Is a Positive Regulator of Starch Synthesis in Vegetative Organs of Rice1[OPEN

    PubMed Central

    Sugino, Miho; Hatanaka, Tomoko; Misoo, Shuji

    2015-01-01

    A unique CO2-Responsive CONSTANS, CONSTANS-like, and Time of Chlorophyll a/b Binding Protein1 (CCT) Protein (CRCT) containing a CCT domain but not a zinc finger motif is described, which is up-regulated under elevated CO2 in rice (Oryza sativa). The expression of CRCT showed diurnal oscillation peaked at the end of the light period and was also increased by sugars such as glucose and sucrose. Promoter β-glucuronidase analysis showed that CRCT was highly expressed in the phloem of various tissues such as leaf blade and leaf sheath. Overexpression or RNA interference knockdown of CRCT had no appreciable effect on plant growth and photosynthesis except that tiller angle was significantly increased by the overexpression. More importantly, starch content in leaf sheath, which serves as a temporary storage organ for photoassimilates, was markedly increased in overexpression lines and decreased in knockdown lines. The expressions of several genes related to starch synthesis, such as ADP-glucose pyrophospholylase and α-glucan phospholylase, were significantly changed in transgenic lines and positively correlated with the expression levels of CRCT. Given these observations, we suggest that CRCT is a positive regulator of starch accumulation in vegetative tissues, regulating coordinated expression of starch synthesis genes in response to the levels of photoassimilates. PMID:25717036

  8. Changes in gene expression, protein content and morphology of chondrocytes cultured on a 3D Random Positioning Machine and 2D rotating clinostat

    NASA Astrophysics Data System (ADS)

    Aleshcheva, Ganna; Hauslage, Jens; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela; Sahana, Jayashree

    Chondrocytes are the only cell type found in human cartilage consisting of proteoglycans and type II collagen. Several studies on chondrocytes cultured either in Space or on a ground-based facility for simulation of microgravity revealed that these cells are very resistant to adverse effects and stress induced by altered gravity. Tissue engineering of chondrocytes is a new strategy for cartilage regeneration. Using a three-dimensional Random Positioning Machine and a 2D rotating clinostat, devices designed to simulate microgravity on Earth, we investigated the early effects of microgravity exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis; and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β1 was enhanced for 24 h. Based on the results achieved, we suggest that chondrocytes exposed to simulated microgravity seem to change their extracellular matrix production behavior while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.

  9. A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter

    PubMed Central

    Kamthan, Ayushi; Kamthan, Mohan; Kumar, Avinash; Sharma, Pratima; Ansari, Sekhu; Thakur, Sarjeet Singh; Chaudhuri, Abira; Datta, Asis

    2015-01-01

    Oxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of 45Ca2+, heat stability and Ca2+ dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca2+ binding transcriptional regulators. PMID:26455820

  10. Protein Tyrosine Phosphatase N2 Is a Positive Regulator of Lipopolysaccharide Signaling in Raw264.7 Cell through Derepression of Src Tyrosine Kinase.

    PubMed

    Ha Thi, Huyen Trang; Choi, Seo-Won; Kim, Young-Mi; Kim, Hye-Youn; Hong, Suntaek

    2016-01-01

    T cell protein tyrosine phosphatase N2 (PTPN2) is a phosphotyrosine-specific nonreceptor phosphatase and is ubiquitously expressed in tissues. Although PTPN2 functions as an important regulator in different signaling pathways, it is still unclear what is specific target protein of PTPN2 and how is regulated in lipopolysaccharide (LPS)-induced inflammatory signaling pathway. Here, we found that PTPN2 deficiency downregulated the expression of LPS-mediated pro-inflammtory cytokine genes. Conversely, overexpression of PTPN2 in Raw264.7 cells enhanced the expression and secretion of those cytokines. The activation of MAPK and NF-κB signaling pathways by LPS was reduced in PTPN2-knockdowned cells and ectopic expression of PTPN2 reversed these effects. Furthermore, we found that PTNP2 directly interacted with Src and removed the inhibitory Tyr527 phosphorylation of Src to enhance the activatory phosphorylation of Tyr416 residue. These results suggested that PTPN2 is a positive regulator of LPS-induced inflammatory response by enhancing the activity of Src through targeting the inhibitory phosphor-tyrosine527 of Src. PMID:27611995

  11. NHE-RF, a Merlin-Interacting Protein, Is Primarily Expressed in Luminal Epithelia, Proliferative Endometrium, and Estrogen Receptor-Positive Breast Carcinomas

    PubMed Central

    Stemmer-Rachamimov, Anat O.; Wiederhold, Thorsten; Nielsen, G. Petur; James, Marianne; Pinney-Michalowski, Denise; Roy, Jennifer E.; Cohen, Wendy A.; Ramesh, Vijaya; Louis, David N.

    2001-01-01

    NHE-RF, a regulatory cofactor for NHE (Na+-H+ exchanger) type 3, interacts with ion transporters and receptors through its PDZ domains and with the MERM proteins (merlin, ezrin, radixin and moesin) via its carboxyl terminus. Thus, NHE-RF may act as a multifunctional adaptor protein and play a role in the assembly of signal transduction complexes, linking ion channels and receptors to the actin cytoskeleton. NHE-RF expression is up-regulated in response to estrogen in estrogen receptor-positive breast carcinoma cell lines, suggesting that it may be involved in estrogen signaling. To further understand NHE-RF function and its possible role in estrogen signaling, we analyzed NHE-RF expression in normal human tissues, including cycling endometrium, and in breast carcinomas, tissues in which estrogen plays an important role in regulating cell growth and proliferation. NHE-RF is expressed in many epithelia, especially in cells specialized in ion transport or absorption, and is often localized to apical (luminal) membranes. NHE-RF expression varies markedly in proliferative versus secretory endometrium, with high expression in proliferative (estrogen-stimulated) endometrium. Furthermore, estrogen receptor status and NHE-RF expression correlate closely in breast carcinoma specimens. These findings support a role for NHE-RF in estrogen signaling. PMID:11141479

  12. A maize mitogen-activated protein kinase kinase, ZmMKK1, positively regulated the salt and drought tolerance in transgenic Arabidopsis.

    PubMed

    Cai, Guohua; Wang, Guodong; Wang, Li; Liu, Yang; Pan, Jiaowen; Li, Dequan

    2014-07-15

    Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction modules in animals, plants and yeast. MAPK cascades are complicated networks and play vital roles in signal transduction pathways involved in biotic and abiotic stresses. In this study, a maize MAPKK gene, ZmMKK1, was characterized. Quantitative real time PCR (qRT-PCR) analysis demonstrated that ZmMKK1 transcripts were induced by diverse stresses and ABA signal molecule in maize root. Further study showed that the ZmMKK1-overexpressing Arabidopsis enhanced the tolerance to salt and drought stresses. However, seed germination, post-germination growth and stomatal aperture analysis demonstrated that ZmMKK1 overexpression was sensitive to ABA in transgenic Arabidopsis. Molecular genetic analysis revealed that the overexpression of ZmMKK1 in Arabidopsis enhanced the expression of ROS scavenging enzyme- and ABA-related genes, such as POD, CAT, RAB18 and RD29A under salt and drought conditions. In addition, heterologous overexpression of ZmMKK1 in yeast (Saccharomyces cerevisiae) improved the tolerance to salt and drought stresses. These results suggested that ZmMKK1 might act as an ABA- and ROS-dependent protein kinase in positive modulation of salt and drought tolerance. Most importantly, ZmMKK1 interacted with ZmMEKK1 as evidenced by yeast two-hybrid assay, redeeming a deficiency of MAPK interaction partners in maize. PMID:24974327

  13. RB1CC1 Protein Positively Regulates Transforming Growth Factor-β Signaling through the Modulation of Arkadia E3 Ubiquitin Ligase Activity*

    PubMed Central

    Koinuma, Daizo; Shinozaki, Masahiko; Nagano, Yoshiko; Ikushima, Hiroaki; Horiguchi, Kana; Goto, Kouichiro; Chano, Tokuhiro; Saitoh, Masao; Imamura, Takeshi; Miyazono, Kohei; Miyazawa, Keiji

    2011-01-01

    Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-β signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-β signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-β signaling, and knockdown of endogenous RB1CC1 attenuated TGF-β-induced expression of target genes as well as TGF-β-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-β signaling by restricting substrate specificity of Arkadia. PMID:21795712

  14. Ectopic expression of the HAM59 gene causes homeotic transformations of reproductive organs in sunflower (Helianthus annuus L.).

    PubMed

    Shulga, O A; Neskorodov, Ya B; Shchennikova, A V; Gaponenko, A K; Skryabin, K G

    2015-01-01

    The function of the HAM59 MADS-box gene in sunflower (Helianthus annuus L.) was studied to clarify homeotic C activity in the Asteraceae plant family. For the first time, transgenic sunflower plants with a modified pattern of HAM59 expression were obtained. It was shown that the HAM59 MADS-box transcription factor did mediate C activity in sunflower. In particular, it participated in termination of the floral meristem, repression of the cadastral function of A-activity, and together with other C-type sunflower protein HAM45-in the specification of the identity of stamens and pistils. PMID:25937227

  15. Positive immunostaining of Sal-like protein 4 is associated with poor patient survival outcome in the large and undifferentiated Korean hepatocellular carcinoma

    PubMed Central

    Jung, Yun Kyung; Jang, Kiseok; Paik, Seung Sam; Kwon, Yong Jin; Kim, Han Jun; Lee, Kyeong Geun; Park, Hwon Kyum

    2016-01-01

    Purpose Previous studies have shown the role of Sal-like protein 4 (SALL4) as a biomarker in hepatocellular carcinoma (HCC), and some studies have shown the relationship between SALL4 and prognosis. Given the debates in study groups differences in terms of etiologic causes between Western and Asian HCC and detection methods, we attempted to verify the features of SALL4 immunoreactivity and its clinical correlation in Korean HCC patients. Methods Immunohistochemical staining of SALL4 of tissue microarrays (TMAs) consisting of 213 surgically resected HCC patients' tissue were scored in a semiquantitative scoring system with immunoreactive score and the results analyzed with clinical outcome, in addition to general demographics and clinical characteristics. Results SALL4 immunoreactivity was expressed in 50 cases. Relevance between SALL4 and α-FP correlated significantly (P = 0.002). Also, the SALL4-positive patients had considerably higher tumor grade (P < 0.001). The survival analysis showed negative correlation with SALL4 immunoreactivity in all HCC patient groups, but SALL4 immunoreactivity in T3 and T4 HCC correlated with poor prognosis. Conclusion Here, we found that positive immunostaining of SALL4 is correlated with poor patient survival outcome in large and undifferentiated Korean HCC. SALL4 expression showed close relationship with clinical outcomes of HCCs in Korean patients. PMID:27433461

  16. The Hydroxyl at Position C1 of Genipin Is the Active Inhibitory Group that Affects Mitochondrial Uncoupling Protein 2 in Panc-1 Cells

    PubMed Central

    Hou, Jianwei; Ding, Yue; Zhang, Tong; Zhang, Yong; Wang, Jianying; Shi, Chenchen; Fu, Wenwei; Cai, Zhenzhen

    2016-01-01

    Genipin (GNP) effectively inhibits uncoupling protein 2 (UCP2), which regulates the leakage of protons across the inner mitochondrial membrane. UCP2 inhibition may induce pancreatic adenocarcinoma cell death by increasing reactive oxygen species (ROS) levels. In this study, the hydroxyls at positions C10 (10-OH) and C1 (1-OH) of GNP were hypothesized to be the active groups that cause these inhibitory effects. Four GNP derivatives in which the hydroxyl at position C10 or C1 was replaced with other chemical groups were synthesized and isolated. Differences in the inhibitory effects of GNP and its four derivatives on pancreatic carcinoma cell (Panc-1) proliferation were assessed. The effects of GNP and its derivatives on apoptosis, UCP2 inhibition and ROS production were also studied to explore the relationship between GNP’s activity and its structure. The derivatives with 1-OH substitutions, geniposide (1-GNP1) and 1-ethyl-genipin (1-GNP2) lacked cytotoxic effects, while the other derivatives that retained 1-OH, 10-piv-genipin (10-GNP1) and 10-acetic acid-genipin (10-GNP2) exerted biological effects similar to those of GNP, even in the absence of 10-OH. Thus, 1-OH is the key functional group in the structure of GNP that is responsible for GNP’s apoptotic effects. These cytotoxic effects involve the induction of Panc-1 cell apoptosis through UCP2 inhibition and subsequent ROS production. PMID:26771380

  17. Carboxyl-Terminal Modulator Protein Positively Acts as an Oncogenic Driver in Head and Neck Squamous Cell Carcinoma via Regulating Akt phosphorylation.

    PubMed

    Chang, Jae Won; Jung, Seung-Nam; Kim, Ju-Hee; Shim, Geun-Ae; Park, Hee Sung; Liu, Lihua; Kim, Jin Man; Park, Jongsun; Koo, Bon Seok

    2016-01-01

    The exact regulatory mechanisms of carboxyl-terminal modulator protein (CTMP) and its downstream pathways in cancer have been controversial and are not completely understood. Here, we report a new mechanism of regulation of Akt serine/threonine kinase, one of the most important dysregulated signals in head and neck squamous cell carcinoma (HNSCC) by the CTMP pathway and its clinical implications. We find that HNSCC tumor tissues and cell lines had relatively high levels of CTMP expression. Clinical data indicate that CTMP expression was significantly associated with positive lymph node metastasis (OR = 3.8, P = 0.033) and correlated with poor prognosis in patients with HNSCC. CTMP was also positively correlated with Akt/GSK-3β phosphorylation, Snail up-regulation and E-cadherin down-regulation, which lead to increased proliferation and epithelial-to-mesenchymal transition, suggesting that CTMP expression results in enhanced tumorigenic and metastatic properties of HNSCC cells. Moreover, CTMP suppression restores sensitivity to cisplatin chemotherapy. Intriguingly, all the molecular responses to CTMP regulation are identical regardless of p53 status in HNSCC cells. We conclude that CTMP promotes Akt phosphorylation and functions as an oncogenic driver and prognostic marker in HNSCC irrespective of p53. PMID:27328758

  18. Carboxyl-Terminal Modulator Protein Positively Acts as an Oncogenic Driver in Head and Neck Squamous Cell Carcinoma via Regulating Akt phosphorylation

    PubMed Central

    Chang, Jae Won; Jung, Seung-Nam; Kim, Ju-Hee; Shim, Geun-Ae; Park, Hee Sung; Liu, Lihua; Kim, Jin Man; Park, Jongsun; Koo, Bon Seok

    2016-01-01

    The exact regulatory mechanisms of carboxyl-terminal modulator protein (CTMP) and its downstream pathways in cancer have been controversial and are not completely understood. Here, we report a new mechanism of regulation of Akt serine/threonine kinase, one of the most important dysregulated signals in head and neck squamous cell carcinoma (HNSCC) by the CTMP pathway and its clinical implications. We find that HNSCC tumor tissues and cell lines had relatively high levels of CTMP expression. Clinical data indicate that CTMP expression was significantly associated with positive lymph node metastasis (OR = 3.8, P = 0.033) and correlated with poor prognosis in patients with HNSCC. CTMP was also positively correlated with Akt/GSK-3β phosphorylation, Snail up-regulation and E-cadherin down-regulation, which lead to increased proliferation and epithelial-to-mesenchymal transition, suggesting that CTMP expression results in enhanced tumorigenic and metastatic properties of HNSCC cells. Moreover, CTMP suppression restores sensitivity to cisplatin chemotherapy. Intriguingly, all the molecular responses to CTMP regulation are identical regardless of p53 status in HNSCC cells. We conclude that CTMP promotes Akt phosphorylation and functions as an oncogenic driver and prognostic marker in HNSCC irrespective of p53. PMID:27328758

  19. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

    SciTech Connect

    Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

  20. Role for the banana AGAMOUS-like gene MaMADS7 in regulation of fruit ripening and quality.

    PubMed

    Liu, Juhua; Liu, Lin; Li, Yujia; Jia, Caihong; Zhang, Jianbin; Miao, Hongxia; Hu, Wei; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

    2015-11-01

    MADS-box transcription factors play important roles in organ development. In plants, most studies on MADS-box genes have mainly focused on flower development and only a few concerned fruit development and ripening. A new MADS-box gene named MaMADS7 was isolated from banana fruit by rapid amplification of cDNA ends (RACE) based on a MADS-box fragment obtained from a banana suppression subtractive hybridization (SSH) cDNA library. MaMADS7 is an AGAMOUS-like MADS-box gene that is preferentially expressed in the ovaries and fruits and in tobacco its protein product localizes to the nucleus. This study found that MaMADS7 expression can be induced by exogenous ethylene. Ectopic expression of MaMADS7 in tomato resulted in broad ripening phenotypes. The expression levels of seven ripening and quality-related genes, ACO1, ACS2, E4, E8, PG, CNR and PSY1 in MaMADS7 transgenic tomato fruits were greatly increased while the expression of the AG-like MADS-box gene TAGL1 was suppressed. Compared with the control, the contents of β-carotene, lycopene, ascorbic acid and organic acid in transformed tomato fruits were increased, while the contents of glucose and fructose were slightly decreased. MaMADS7 interacted with banana 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene 1 (MaACO1) and tomato phytoene synthase gene (LePSY1) promoters. Our results indicated that MaMADS7 plays an important role in initiating endogenous ethylene biosynthesis and fruit ripening. PMID:25980771

  1. A novel approach for targeted elimination of CSPG4-positive triple-negative breast cancer cells using a MAP tau-based fusion protein.

    PubMed

    Amoury, Manal; Mladenov, Radoslav; Nachreiner, Thomas; Pham, Anh-Tuan; Hristodorov, Dmitrij; Di Fiore, Stefano; Helfrich, Wijnand; Pardo, Alessa; Fey, Georg; Schwenkert, Michael; Thepen, Theophilus; Kiessling, Fabian; Hussain, Ahmad F; Fischer, Rainer; Kolberg, Katharina; Barth, Stefan

    2016-08-15

    Chondroitin sulfate proteoglycan 4 (CSPG4) has been identified as a highly promising target antigen for immunotherapy of triple-negative breast cancer (TNBC). TNBC represents a highly aggressive heterogeneous group of tumors lacking expression of estrogen, progesterone and human epidermal growth factor receptor 2. TNBC is particularly prevalent among young premenopausal women. No suitable targeted therapies are currently available and therefore, novel agents for the targeted elimination of TNBC are urgently needed. Here, we present a novel cytolytic fusion protein (CFP), designated αCSPG4(scFv)-MAP, that consists of a high affinity CSPG4-specific single-chain antibody fragment (scFv) genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. Our data indicate that αCSPG4(scFv)-MAP efficiently targets CSPG4(+) TNBC-derived cell lines MDA-MB-231 and Hs 578T and potently inhibits their growth with IC50 values of ∼200 nM. Treatment with αCSPG(scFv)-MAP resulted in induction of the mitochondrial stress pathway by activation of caspase-9 as well as endonuclease G translocation to the nucleus, while induction of the caspase-3 apoptosis pathway was not detectable. Importantly, in vivo studies in mice bearing human breast cancer xenografts revealed efficient targeting to and accumulation of αCSPG4(scFv)-MAP at tumor sites resulting in prominent tumor regression. Taken together, this preclinical proof of concept study confirms the potential clinical value of αCSPG4(scFv)-MAP as a novel targeted approach for the elimination of CSPG4-positive TNBC. PMID:27037627

  2. High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

    PubMed Central

    Dobrijevic, Dragana; Di Liberto, Gaetana; Tanaka, Kosei; de Wouters, Tomas; Dervyn, Rozenn; Boudebbouze, Samira; Binesse, Johan; Blottière, Hervé M.; Jamet, Alexandre; Maguin, Emmanuelle; van de Guchte, Maarten

    2013-01-01

    Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems. PMID

  3. The Bel1 protein of human foamy virus contains one positive and two negative control regions which regulate a distinct activation domain of 30 amino acids.

    PubMed Central

    Lee, C W; Chang, J; Lee, K J; Sung, Y C

    1994-01-01

    The Bel1 transactivator is essential for the replication of human foamy virus (HFV). To define the functional domains of HFV Bel1, we generated random missense mutations throughout the entire coding sequence of Bel1. Functional analyses of 24 missense mutations have revealed the presence of at least two functional domains in Bel1. One domain corresponds to a basic amino acid-rich motif which acts as a bipartite nuclear targeting sequence. A second, central domain corresponds to a presumed effector region which, when mutated, leads to dominant-negative mutants and/or lacks transactivating ability. In addition, deletion analyses and domain-swapping experiments further showed that Bel1 protein contains a strong carboxy-terminal activation domain. The activating region is also capable of functioning as a transcription-activating domain in yeast cells, although it does not bear any significant sequence homology to the well-characterized acidic activation domain which is known to function only in yeast and mammalian cells. We also demonstrated that the regions of Bel1 from residues 1 to 76 and from residues 153 to 225 repressed transcriptional activation exerted by the Bel1 activation domain. In contrast, the region from residues 82 to 150 appears to overcome an inhibitory effect. These results indicate that Bel1 contains one positive and two negative regulatory domains that modulate a distinct activation domain of Bel1. These regulatory domains of Bel1 cannot affect the function of the VP16 activation domain, suggesting that these domains specifically regulate the activation domain of Bel1. Furthermore, in vivo competition experiments showed that the positive regulatory domain acts in trans. Thus, our results demonstrate that Bel1-mediated transactivation appears to undergo a complex regulatory pathway which provides a novel mode of regulation for a transcriptional activation domain. Images PMID:8139046

  4. The value of serum Wisteria floribunda agglutinin-positive human Mac-2-binding protein as a predictive marker for hepatitis C virus-related complications after systemic chemotherapy.

    PubMed

    Totani, Haruhito; Kusumoto, Shigeru; Tanaka, Yasuhito; Suzuki, Nana; Hagiwara, Shinya; Kinoshita, Shiori; Iio, Etsuko; Ito, Asahi; Ri, Masaki; Ishida, Takashi; Komatsu, Hirokazu; Iida, Shinsuke

    2016-09-01

    Wisteria floribunda agglutinin-positive human Mac-2-binding protein (WFA(+)-M2BP) was developed recently as a predictive marker of progression to liver fibrosis and hepatocellular carcinoma (HCC) in patients seropositive for hepatitis C virus (HCV). We retrospectively analyzed 16 HCV-seropositive patients who received systemic chemotherapy for hematologic malignancies to evaluate the usefulness of WFA(+)-M2BP for predicting HCV-related complications. These were defined as the onset of significant liver damage (LD) with increased HCV RNA levels, leading to interrupted or discontinued chemotherapy or the occurrence of HCC after chemotherapy. Baseline WFA(+)-M2BP levels were determined using preserved serum samples. The median level of WFA(+)-M2BP was 1.59 [cutoff index (C.O.I.) value range 0.38-6.66]. With a median follow-up of 623 days (range 120-2404), LD and HCC were observed in three and two patients, respectively. Detectable HCV RNA and WFA(+)-M2BP ≥2.0 C.O.I. at baseline were identified as risk factors for these HCV-related complications (P = 0.034 and P = 0.005, respectively). The sensitivity, specificity, and positive and negative predictive values of the WFA(+)-M2BP level (cutoff point: 2.0 C.O.I.) for the occurrence of HCV-related complications were 100.0, 81.8, 71.4, and 100.0 %, respectively. WFA(+)-M2BP may be a useful marker for the prediction of HCV-related complications in HCV-seropositive patients following systemic chemotherapy. PMID:27255233

  5. High-Content Positional Biosensor Screening Assay for Compounds to Prevent or Disrupt Androgen Receptor and Transcriptional Intermediary Factor 2 Protein–Protein Interactions

    PubMed Central

    Hua, Yun; Shun, Tong Ying; Strock, Christopher J.

    2014-01-01

    Abstract The androgen receptor–transcriptional intermediary factor 2 (AR-TIF2) positional protein–protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) “prey” and TIF2-green fluorescent protein (GFP) “bait” components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence. In unstimulated cells, AR-RFP was localized predominantly to the cytoplasm and TIF2-GFP was localized to nucleoli. Dihydrotestosterone (DHT) treatment induced AR-RFP translocation into the nucleus where the PPIs between AR and TIF2 resulted in the colocalization of both biosensors within the nucleolus. We adapted the translocation enhanced image analysis module to quantify the colocalization of the AR-RFP and TIF2-GFP biosensors in images acquired on the ImageXpress platform. DHT induced a concentration-dependent AR-TIF2 colocalization and produced a characteristic condensed punctate AR-RFP PPI nucleolar distribution pattern. The heat-shock protein 90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and antiandrogens flutamide and bicalutamide inhibited DHT-induced AR-TIF2 PPI formation with 50% inhibition concentrations (IC50s) of 88.5±12.5 nM, 7.6±2.4 μM, and 1.6±0.4 μM, respectively. Images of the AR-RFP distribution phenotype allowed us to distinguish between 17-AAG and flutamide, which prevented AR translocation, and bicalutamide, which blocked AR-TIF2 PPIs. We screened the Library of Pharmacologically Active

  6. Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket.

    PubMed Central

    Lee, E J; Glasgow, J; Leu, S F; Belduz, A O; Harman, J G

    1994-01-01

    The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute cysteine or glycine for serine 83; cysteine, glycine, isoleucine, or serine for threonine 127; and threonine or alanine for serine 128. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to support lacP activation in vitro. CRP structure was assessed by measurement of sensitivity to protease and DTNB-mediated subunit crosslinking. The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and have little effect on cAMP binding. Amino acid substitutions that introduce hydrophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP. Finally, cAMP-induced CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threonine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wild-type CRP:cAMP complex. Images PMID:8065899

  7. HD-Zip Proteins GL2 and HDG11 Have Redundant Functions in Arabidopsis Trichomes, and GL2 Activates a Positive Feedback Loop via MYB23[W

    PubMed Central

    Khosla, Aashima; Paper, Janet M.; Boehler, Allison P.; Bradley, Amanda M.; Neumann, Titus R.; Schrick, Kathrin

    2014-01-01

    The class IV homeodomain leucine zipper transcription factor GLABRA2 (GL2) acts in a complex regulatory circuit that regulates the differentiation of trichomes in Arabidopsis thaliana. We describe a genetic interaction with HOMEODOMAIN GLABROUS11 (HDG11), previously identified as a negative regulator of trichome branching. gl2 hdg11 double mutants display enhanced trichome cell-type differentiation defects. Transgenic expression of HDG11 using the GL2 promoter partially suppresses gl2 trichome phenotypes. Vice versa, expression of GL2 under the control of its native promoter partially complements hdg11 ectopic branching. Since gl2 hdg11 and gl2 myb23 double mutants and the triple mutant display similar trichome differentiation defects, we investigated a connection to the R2R3-MYB transcription factor MYB23. We show that MYB23 transcript levels are significantly reduced in shoots from gl2 mutants and that GL2 can drive the expression of a MYB23-promoter fusion to green fluorescent protein. Yeast one-hybrid, chromatin immunoprecipitation, and in planta reporter gene experiments indicate that an L1-box in the MYB23 promoter acts as a GL2 binding site. Taken together, our findings reveal a functional redundancy between GL2 and HDG11, two homeodomain leucine zipper transcription factors previously thought to mediate opposing functions in trichome morphogenesis. A model is proposed in which GL2 transcript levels are maintained through a positive feedback loop involving GL2 activation of MYB23. PMID:24824485

  8. Iron-Regulated Protein HupB of Mycobacterium tuberculosis Positively Regulates Siderophore Biosynthesis and Is Essential for Growth in Macrophages

    PubMed Central

    Pandey, Satya Deo; Choudhury, Mitali; Yousuf, Suhail; Wheeler, Paul R.; Gordon, Stephen V.; Ranjan, Akash

    2014-01-01

    Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe3+-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 μg Fe ml−1), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo. PMID:24610707

  9. The plasma membrane protein Rch1 is a negative regulator of cytosolic calcium homeostasis and positively regulated by the calcium/calcineurin signaling pathway in budding yeast.

    PubMed

    Zhao, Yunying; Yan, Hongbo; Happeck, Ricardo; Peiter-Volk, Tina; Xu, Huihui; Zhang, Yan; Peiter, Edgar; van Oostende Triplet, Chloë; Whiteway, Malcolm; Jiang, Linghuo

    2016-01-01

    Saccharomyces cerevisiae Rch1 is structurally similar to both the vertebrate solute carrier SLC10A7 and Candida albicans Rch1. We show here that ScRCH1 is a functional homolog of CaRCH1. In S. cerevisiae, overexpression of ScRCH1 suppresses, but deletion of ScRCH1 does not affect, the lithium and rapamycin tolerance of pmr1 cells. Overexpression of ScRCH1 reduces expression of ENA1, prevents sustained accumulation of cytosolic calcium and reduces the activation level of calcium/calcineurin signaling in pmr1 cells. Therefore, similar to the situation in the pathogen C. albicans, ScRch1 negatively regulates the cytosolic homeostasis in response to high levels of extracellular calcium. ScRch1 proteins distribute as multiple foci in the plasma membrane prior to cell division, move toward and concentrate at the bud neck as the bud grows in size, and disperse again along the plasma membrane immediately prior to cytokinesis. Furthermore, our genetic and biochemical data also demonstrate that transcriptional expression of RCH1 is positively regulated by calcium/calcineurin signaling through the sole CDRE element in its promoter. PMID:26832117

  10. Lysine at position 222 of the goat prion protein inhibits the binding of monoclonal antibody F99/97.6.1.

    PubMed

    Mazza, Maria; Guglielmetti, Chiara; Pagano, Marianna; Sciuto, Simona; Ingravalle, Francesco; Martucci, Francesca; Caramelli, Maria; Acutis, Pier Luigi

    2012-09-01

    Prion protein (PrP) is encoded by the PRNP gene, which is highly polymorphic in goats, with polymorphisms encoding amino acid substitutions at the protein level. In the current study, the reactivity of monoclonal antibody (mAb) F99/97.6.1 in binding PrP from goats polymorphic at PRNP codon 222 was investigated. Nervous tissue from 30 scrapie-negative goats with 3 different genotypes (222Q/Q, 222Q/K, and 222K/K) was analyzed by Western blot using mAbs P4 and F99/97.6.1. Although PrP was detected in all 30 samples by mAb P4, detection of PrP by mAb F99/97.6.1 was limited to 222Q/Q (12/12). No PrP was detected by mAb F99/97.6.1 in the 222K/K samples (n = 6), and the signal intensity of mAb F99/97.6.1 for PrP was lower for the 222Q/K samples (12/12 samples). To further investigate these results, additional Western blot analyses were performed, and the PrP signals detected by mAbs F99/97.6.1 and SAF84 were then quantified. The mean F99/SAF84 ratio (± standard deviation) calculated for the 222Q/Q group was 0.73 ± 1.26, and the mean for the 222Q/K group was 0.27 ± 1.31. Statistical analysis of these values evidenced statistically significant differences between the 222Q/Q and 222Q/K samples. The results of the study thus revealed an inhibition by lysine at position 222 on the binding of mAb F99/97.6.1 to goat PrP. This has implications for the use of mAb F99/97.6.1 for diagnostic purposes. Because the 222K allele could be a target for genetic selection in goats, the differential reactivity of mAb F99/97.6.1 could be exploited with a genotyping test setup. PMID:22914824

  11. EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngeal carcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop.

    PubMed

    Yang, C-F; Yang, G-D; Huang, T-J; Li, R; Chu, Q-Q; Xu, L; Wang, M-S; Cai, M-D; Zhong, L; Wei, H-J; Huang, H-B; Huang, J-L; Qian, C-N; Huang, B-J

    2016-06-30

    Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44(+/High) stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, which subsequently stimulated expression of CSC markers, development of side population and tumor sphere formation. This suggests that PI3K/AKT pathway has an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44(+/High) cells. In addition, LMP1 greatly increased expression of miR-21 and downregulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by an miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and upregulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to the development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and have a key role in LMP1-induced CSCs in NPC. PMID:26568302

  12. The pineapple AcMADS1 promoter confers high level expression in tomato and arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of e...

  13. Acyl Carrier Protein Synthases from Gram-Negative, Gram-Positive, and Atypical Bacterial Species: Biochemical and Structural Properties and Physiological Implications

    PubMed Central

    McAllister, Kelly A.; Peery, Robert B.; Zhao, Genshi

    2006-01-01

    Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli, Streptococcus pneumoniae, and Mycoplasma pneumoniae, which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae, like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally, our biochemical studies show that M. pneumoniae AcpS is kinetically a very sluggish enzyme compared with those from E. coli and S. pneumoniae. Together, the results of these studies show that the AcpS enzymes from different bacterial species exhibit different native structures and substrate specificities with regard to the utilization of CoA and its derivatives. These findings suggest that AcpS from different microorganisms plays a different role in cellular physiology. PMID:16788183

  14. The albinism of the feral Asinara white donkeys (Equus asinus) is determined by a missense mutation in a highly conserved position of the tyrosinase (TYR) gene deduced protein.

    PubMed

    Utzeri, V J; Bertolini, F; Ribani, A; Schiavo, G; Dall'Olio, S; Fontanesi, L

    2016-02-01

    A feral donkey population (Equus asinus), living in the Asinara National Park (an island north-west of Sardinia, Italy), includes a unique white albino donkey subpopulation or colour morph that is a major attraction of this park. Disrupting mutations in the tyrosinase (TYR) gene are known to cause recessive albinisms in humans (oculocutaneous albinism Type 1; OCA1) and other species. In this study, we analysed the donkey TYR gene as a strong candidate to identify the causative mutation of the albinism of these donkeys. The TYR gene was sequenced from 13 donkeys (seven Asinara white albino and six coloured animals). Seven single nucleotide polymorphisms were identified. A missense mutation (c.604C>G; p.His202Asp) in a highly conserved amino acid position (even across kingdoms), which disrupts the first copper-binding site (CuA) of functional protein, was identified in the homozygous condition (G/G or D/D) in all Asinara white albino donkeys and in the albino son of a trio (the grey parents had genotype C/G or H/D), supporting the recessive mode of inheritance of this mutation. Genotyping 82 donkeys confirmed that Asinara albino donkeys had genotype G/G whereas all other coloured donkeys had genotype C/C or C/G. Across-population association between the c.604C>G genotypes and the albino coat colour was highly significant (P = 6.17E-18). The identification of the causative mutation of the albinism in the Asinara white donkeys might open new perspectives to study the dynamics of this putative deleterious allele in a feral population and to manage this interesting animal genetic resource. PMID:26763160

  15. Glutamate supply positively affects serum cholesterol concentrations without increases in total protein and urea around the onset of puberty in goats.

    PubMed

    Meza-Herrera, C A; Calderón-Leyva, G; Soto-Sanchez, M J; Serradilla, J M; García-Martinez, A; Mellado, M; Veliz-Deras, F G

    2014-06-30

    Different neurotransmitter and neuromodulatory systems regulate synthesis and secretion of GnRH. Whereas the endocrine and neural systems are activated in response to the metabolic status and the circulating levels of specific blood metabolites, glutamate receptors have been reported at hepatic level. This study evaluated the possible effect of glutamate supplementation upon changes in serum concentrations across time for total protein (TP), urea (UR) and cholesterol (CL) around the onset of puberty in goats. Prepuberal female goats (n=18) were randomly assigned to: (1) excitatory amino acids group, GLUT, n=10; 16.52±1.04kg live weight (LW), 3.4±0.12 body condition score (BCS) receiving an i.v. infusion of 7mgkg(-1) LW of l-glutamate, and (2) Control group, CONT, n=8; 16.1±1.04kg LW, 3.1±0.12 BCS. General averages for LW (23.2±0.72kg), BCS (3.37±0.10 units), serum TP (65.28±2.46mgdL(-1)), UR (23.42±0.95mgdL(-1)), CL (77.89±1.10mgdL(-1)) as well as the serum levels for TP and UR across time did not differ (P>0.05) between treatments. However, while GLUT positively affected (P<0.05) both the onset (207±9 vs. 225±12 d) and the percentage (70 vs. 25%) of females showing puberty, a treatment×time interaction effect (P<0.05) was observed in the GLUT group, with increases in serum cholesterol, coincident with the onset of puberty. Therefore, in peripuberal glutamate supplemented goats, serum cholesterol profile could act as a metabolic modulator for the establishment of puberty, denoting also a potential role of glutamate as modulator of lipid metabolism. PMID:24811839

  16. Proteolysis of insulin-like growth factor-binding protein-3 in human immunodeficiency virus-positive children who fail to thrive.

    PubMed

    Frost, R A; Nachman, S A; Lang, C H; Gelato, M C

    1996-08-01

    Failure to thrive is a common manifestation of human immunodeficiency virus (HIV) infection in children. Given the role of insulin-like growth factor I (IGF-I) in stimulating postnatal growth, we have examined whether HIV-infected pediatric patients with growth failure have lower serum concentrations of IGF-I than age-matched control subjects. IGF-I was measured in 16 HIV-infected children and 13 HIV-negative controls. Ten of the HIV-infected children failed to thrive based on height and linear growth that was below the National Center for Health Statistics 10th percentile. IGF-I levels were significantly lower in children who failed to thrive compared to those in age-matched controls (20 vs. 60 micrograms/L; P < 0.001). Children who failed to thrive also displayed lower IGF-I levels than HIV-positive children, who exhibited normal growth velocity (20 vs. 91 micrograms/L; P < 0.001). Failure to thrive was associated with a significant reduction in circulating levels of IGF-binding protein-3 (IGFBP-3), as determined by ligand and Western blotting (P < 0.001), enhanced IGFBP-3 proteolysis (P < 0.001), and a decrease in the serum concentration of the acid-labile subunit of the IGFBP-3 ternary complex (P < 0.005). IGFBP-3 proteolysis was negatively correlated with IGF-I (r = 0.78) and IGFBP-3 levels (r = 0.70). Failure to thrive was associated with a reduction in the formation of the ternary complex, but the ternary complex could be restored by the addition of an excess of IGFBP-3 to serum. These results indicate that low levels of IGF-I, IGFBP-3, and acid-labile subunit are associated with a failure to thrive in HIV-infected children. PMID:8768858

  17. Serum Levels of Vascular Endothelial Growth Factor and Insulin-like Growth Factor Binding Protein-3 in Obstructive Sleep Apnea Patients: Effect of Continuous Positive Airway Pressure Treatment

    PubMed Central

    Archontogeorgis, Kostas; Nena, Evangelia; Papanas, Nikolaos; Xanthoudaki, Maria; Hatzizisi, Olga; Kyriazis, Georgios; Tsara, Venetia; Maltezos, Efstratios; Froudarakis, Marios; Steiropoulos, Paschalis

    2015-01-01

    Background and Aim: Hypoxia, a major feature of obstructive sleep apnea (OSA), modifies Vascular Endothelial Growth Factor (VEGF) and Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) levels, which contribute to atherogenesis and occurrence of cardiovascular (CV) events. We assessed and compared serum levels of VEGF and IGFBP-3 in newly diagnosed OSA patients and controls, to explore associations with anthropometric and sleep parameters and to study the effect of continuous positive airway pressure (CPAP) treatment on these levels. Materials and Methods: Serum levels of VEGF and IGFBP-3 were measured in 65 OSA patients and 31 age- and body mass index- matched controls. In OSA patients, measurements were repeated after 6 months of CPAP therapy. All participants were non-smokers, without any comorbidities or systemic medication use. Results: At baseline, serum VEGF levels in OSA patients were higher compared with controls (p<0.001), while IGFBP-3 levels were lower (1.41±0.56 vs. 1.61±0.38 μg/ml, p=0.039). VEGF levels correlated with apnea-hypopnea index (r=0.336, p=0.001) and oxygen desaturation index (r=0.282, p=0.007). After 6 months on CPAP treatment, VEGF levels decreased in OSA patients (p<0.001), while IGFBP-3 levels increased (p<0.001). Conclusion: In newly diagnosed OSA patients, serum levels of VEGF are elevated, while IGFBP-3 levels are low. After 6 months of CPAP treatment these levels change. These results may reflect an increased CV risk in untreated OSA patients, which is ameliorated after CPAP therapy. PMID:27006717

  18. Autoregulation of PhoP/PhoQ and positive regulation of the cyclic AMP receptor protein-cyclic AMP complex by PhoP in Yersinia pestis.

    PubMed

    Zhang, Yiquan; Wang, Li; Han, Yanping; Yan, Yanfeng; Tan, Yafang; Zhou, Lei; Cui, Yujun; Du, Zongmin; Wang, Xiaoyi; Bi, Yujing; Yang, Huiying; Song, Yajun; Zhang, Pingping; Zhou, Dongsheng; Yang, Ruifu

    2013-03-01

    Yersinia pestis is one of the most dangerous bacterial pathogens. PhoP and cyclic AMP receptor protein (CRP) are global regulators of Y. pestis, and they control two distinct regulons that contain multiple virulence-related genes. The PhoP regulator and its cognate sensor PhoQ constitute a two-component regulatory system. The regulatory activity of CRP is triggered only by binding to its cofactor cAMP, which is synthesized from ATP by adenylyl cyclase (encoded by cyaA). However, the association between the two regulatory systems PhoP/PhoQ and CRP-cAMP is still not understood for Y. pestis. In the present work, the four consecutive genes YPO1635, phoP, phoQ, and YPO1632 were found to constitute an operon, YPO1635-phoPQ-YPO1632, transcribed as a single primary RNA, whereas the last three genes comprised another operon, phoPQ-YPO1632, transcribed with two adjacent transcriptional starts. Through direct PhoP-target promoter association, the transcription of these two operons was stimulated and repressed by PhoP, respectively; thus, both positive autoregulation and negative autoregulation of PhoP/PhoQ were detected. In addition, PhoP acted as a direct transcriptional activator of crp and cyaA. The translational/transcriptional start sites, promoter -10 and -35 elements, PhoP sites, and PhoP box-like sequences were determined for these PhoP-dependent genes, providing a map of the PhoP-target promoter interaction. The CRP and PhoP regulons have evolved to merge into a single regulatory cascade in Y. pestis because of the direct regulatory association between PhoP/PhoQ and CRP-cAMP. PMID:23264579

  19. Release of Positive Transcription Elongation Factor b (P-TEFb) from 7SK Small Nuclear Ribonucleoprotein (snRNP) Activates Hexamethylene Bisacetamide-inducible Protein (HEXIM1) Transcription*

    PubMed Central

    Liu, Pingyang; Xiang, Yanhui; Fujinaga, Koh; Bartholomeeusen, Koen; Nilson, Kyle A.; Price, David H.; Peterlin, B. Matija

    2014-01-01

    By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis. PMID:24515107

  20. Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.

    PubMed Central

    Wilson, P T; Gershoni, J M; Hawrot, E; Lentz, T L

    1984-01-01

    Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor. Images PMID:6371817

  1. Tamm-Horsfall protein in recurrent calcium kidney stone formers with positive family history: abnormalities in urinary excretion, molecular structure and function.

    PubMed

    Jaggi, Markus; Nakagawa, Yasushi; Zipperle, Ljerka; Hess, Bernhard

    2007-04-01

    Tamm-Horsfall protein (THP) powerfully inhibits calcium oxalate crystal aggregation, but structurally abnormal THPs from recurrent calcium stone formers may promote crystal aggregation. Therefore, increased urinary excretion of abnormal THP might be of relevance in nephrolithiasis. We studied 44 recurrent idiopathic calcium stone formers with a positive family history of stone disease (RCSF(fam)) and 34 age- and sex-matched healthy controls (C). Twenty-four-hour urinary THP excretion was measured by enzyme linked immunosorbent assay. Structural properties of individually purified THPs were obtained from analysis of elution patterns from a Sepharose 4B column. Sialic acid (SA) contents of native whole 24-h urines, crude salt precipitates of native urines and individually purified THPs were measured. THP function was studied by measuring inhibition of CaOx crystal aggregation in vitro (pH 5.7, 200 mM sodium chloride). Twenty-four-hour urine excretion of THP was higher in RCSF(fam) (44.0 +/- 4.0 mg/day) than in C (30.9 +/- 2.2 mg/day, P = 0.015). Upon salt precipitation and lyophilization, elution from a Sepharose 4B column revealed one major peak (peak A, cross-reacting with polyclonal anti-THP antibody) and a second minor peak (peak B, not cross-reacting). THPs from RCSF(fam) eluted later than those from C (P = 0.021), and maximum width of THP peaks was higher in RCSF(fam )than in C (P = 0.024). SA content was higher in specimens from RCSF(fam) than from C, in native 24-h urines (207.5 +/- 20.4 mg vs. 135.2 +/- 16.1 mg, P = 0.013) as well as in crude salt precipitates of 24-h urines (10.4 +/- 0.5 mg vs. 7.4 +/- 0.9 mg, P = 0.002) and in purified THPs (75.3 +/- 9.3 microg/mg vs. 48.8 +/- 9.8 microg/mg THP, P = 0.043). Finally, inhibition of calcium oxalate monohydrate crystal aggregation by 40 mg/L of THP was lower in RCSF(fam) (6.1 +/- 5.5%, range -62.0 to +84.2%) than in C (24.9 +/- 6.0%, range -39.8 to +82.7%), P = 0.022, and only 25 out of 44 (57%) THPs from RCSF

  2. Structural Analysis of Respiratory Syncytial Virus Reveals the Position of M2-1 between the Matrix Protein and the Ribonucleoprotein Complex

    PubMed Central

    Kiss, Gabriella; Holl, Jens M.; Williams, Grant M.; Alonas, Eric; Vanover, Daryll; Lifland, Aaron W.; Gudheti, Manasa; Guerrero-Ferreira, Ricardo C.; Nair, Vinod; Yi, Hong; Graham, Barney S.; Santangelo, Philip J.

    2014-01-01

    ABSTRACT Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. IMPORTANCE hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable

  3. Nursing Positions

    MedlinePlus

    ... Story" 5 Things to Know About Zika & Pregnancy Nursing Positions KidsHealth > For Parents > Nursing Positions Print A ... and actually needs to feed. Getting Comfortable With Breastfeeding Nursing can be one of the most challenging ...

  4. Positive Psychology

    ERIC Educational Resources Information Center

    Peterson, Christopher

    2009-01-01

    Positive psychology is a deliberate correction to the focus of psychology on problems. Positive psychology does not deny the difficulties that people may experience but does suggest that sole attention to disorder leads to an incomplete view of the human condition. Positive psychologists concern themselves with four major topics: (1) positive…

  5. Obg-like ATPase 1 regulates global protein serine/threonine phosphorylation in cancer cells by suppressing the GSK3β-inhibitor 2-PP1 positive feedback loop

    PubMed Central

    Xu, Dong; Song, Renduo; Wang, Guohui; Jeyabal, Prince V.S.; Weiskoff, Amanda M.; Ding, Kefeng; Shi, Zheng-Zheng

    2016-01-01

    OLA1 is an Obg family P-loop NTPase that possesses both GTP- and ATP-hydrolyzing activities. Here we report that OLA1 is a GSK3β interacting protein, and through its ATPase activity, inhibits the GSK3β-mediated activation of protein serine/threonine phosphatase 1 (PP1). It is hypothesized that GSK3β phosphorylates inhibitor 2 (I-2) of PP1 at Thr-72 and activates the PP1 · I-2 complex, which in turn dephosphorylates and stimulates GSK3β, thus forming a positive feedback loop. We revealed that the positive feedback loop is normally suppressed by OLA1, and becomes over-activated under OLA1 deficiency, resulting in increased cellular PP1 activity and dephosphorylation of multiple Ser/Thr phosphoproteins, and more strikingly, decreased global protein threonine phosphorylation. Furthermore, using xenograft models of colon cancer (H116) and ovarian cancer (SKOV3), we established a correlation among downregulation of OLA1, over-activation of the positive feedback loop as indicated by under-phosphorylation of I-2, and more aggressive tumor growth. This study provides the first evidence for the existence of a GSK3β-I-2-PP1 positive feedback loop in human cancer cells, and identifies OLA1 as an endogenous suppressor of this signaling motif. PMID:26655089

  6. Positional plagiocephaly

    PubMed Central

    Cummings, Carl

    2011-01-01

    Cranial asymmetry occurring as a result of forces that deform skull shape in the supine position is known as deformational plagiocephaly. The risk of plagiocephaly may be modified by positioning the baby on alternate days with the head to the right or the left side, and by increasing time spent in the prone position during awake periods. When deformational plagiocephaly is already present, physiotherapy (including positioning equivalent to the preventive positioning, and exercises as needed for torticollis and positional preference) has been shown to be superior to counselling about preventive positioning only. Helmet therapy (moulding therapy) to reduce skull asymmetry has some drawbacks: it is expensive, significantly inconvenient due to the long hours of use per day and associated with skin complications. There is evidence that helmet therapy may increase the initial rate of improvement of asymmetry, but there is no evidence that it improves the final outcome for patients with moderate or severe plagiocephaly. PMID:23024590

  7. Interactome Analysis of the NS1 Protein Encoded by Influenza A H1N1 Virus Reveals a Positive Regulatory Role of Host Protein PRP19 in Viral Replication.

    PubMed

    Kuo, Rei-Lin; Li, Zong-Hua; Li, Li-Hsin; Lee, Kuo-Ming; Tam, Ee-Hong; Liu, Helene M; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-05-01

    Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1-associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC-MS/MS. By employing software to build biological process and protein-protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNA-processing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication. PMID:27096427

  8. A comparative study of protein patterns of human estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines.

    PubMed

    Flodrova, Dana; Toporova, Lucia; Macejova, Dana; Lastovickova, Marketa; Brtko, Julius; Bobalova, Janette

    2016-07-01

    In the present study, we analyzed the cell lysates of human tumour cell lines representing two major clinically different types of breast cancer. Our main goal was to show the differences between them on proteomic level. Gel electrophoresis followed by MALDI-TOF MS analysis was used for proteins determination. Exactly 98 proteins were unequivocally identified and 60 of them were expressed differentially between MDA-MB-231 and MCF-7 cell lines. Among the proteins reported here, some well-known breast cancer markers (e.g., annexin A1, annexin A2 and vimentin) were identified in the MDA-MB-231 cell line and thus we were able to distinguish both cell lines sufficiently. PMID:27174898

  9. Positive Psychotherapy

    ERIC Educational Resources Information Center

    Seligman, Martin E. P.; Rashid, Tayyab; Parks, Acacia C.

    2006-01-01

    Positive psychotherapy (PPT) contrasts with standard interventions for depression by increasing positive emotion, engagement, and meaning rather than directly targeting depressive symptoms. The authors have tested the effects of these interventions in a variety of settings. In informal student and clinical settings, people not uncommonly reported…

  10. A maize calcium-dependent protein kinase gene, ZmCPK4, positively regulated abscisic acid signaling and enhanced drought stress tolerance in transgenic Arabidopsis.

    PubMed

    Jiang, Shanshan; Zhang, Dan; Wang, Li; Pan, Jiaowen; Liu, Yang; Kong, Xiangpei; Zhou, Yan; Li, Dequan

    2013-10-01

    Calcium-dependent protein kinases (CDPKs) play essential roles in calcium-mediated signal transductions in plant response to abiotic stress. Several members have been identified to be regulators for plants response to abscisic acid (ABA) signaling. Here, we isolated a subgroup I CDPK gene, ZmCPK4, from maize. Quantitative real time PCR (qRT-PCR) analysis revealed that the ZmCPK4 transcripts were induced by various stresses and signal molecules. Transient and stable expression of the ZmCPK4-GFP fusion proteins revealed ZmCPK4 localized to the membrane. Moreover, overexpression of ZmCPK4 in the transgenic Arabidopsis enhanced ABA sensitivity in seed germination, seedling growth and stomatal movement. The transgenic plants also enhanced drought stress tolerance. Taken together, the results suggest that ZmCPK4 might be involved in ABA-mediated regulation of stomatal closure in response to drought stress. PMID:23911729

  11. Arf GTPase-activating Protein ASAP1 Interacts with Rab11 Effector FIP3 and Regulates Pericentrosomal Localization of Transferrin Receptor–positive Recycling Endosome

    PubMed Central

    Inoue, Hiroki; Ha, Vi Luan; Prekeris, Rytis

    2008-01-01

    ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6- and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment. PMID:18685082

  12. Nucleosome Positioning

    PubMed Central

    Nishida, Hiromi

    2012-01-01

    Nucleosome positioning is not only related to genomic DNA compaction but also to other biological functions. After the chromatin is digested by micrococcal nuclease, nucleosomal (nucleosome-bound) DNA fragments can be sequenced and mapped on the genomic DNA sequence. Due to the development of modern DNA sequencing technology, genome-wide nucleosome mapping has been performed in a wide range of eukaryotic species. Comparative analyses of the nucleosome positions have revealed that the nucleosome is more frequently formed in exonic than intronic regions, and that most of transcription start and translation (or transcription) end sites are located in nucleosome linker DNA regions, indicating that nucleosome positioning influences transcription initiation, transcription termination, and gene splicing. In addition, nucleosomal DNA contains guanine and cytosine (G + C)-rich sequences and a high level of cytosine methylation. Thus, the nucleosome positioning system has been conserved during eukaryotic evolution.

  13. Positive Proof.

    ERIC Educational Resources Information Center

    Auty, Geoffrey

    1988-01-01

    Presents experiments which show that in electrostatics there are logical reasons for describing charged materials as positive or negative. Indicates that static and current electricity are not separate areas of physics. Diagrams of experiments and circuits are included. (RT)

  14. Concomitant Detection of HER2 Protein and Gene Alterations by Immunohistochemistry (IHC) and Silver Enhanced In Situ Hybridization (SISH) Identifies HER2 Positive Breast Cancer with and without Gene Amplification

    PubMed Central

    Varga, Zsuzsanna; Tubbs, Raymond R.; Moch, Holger

    2014-01-01

    Introduction HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number. Methods Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis. Results Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification. Conclusions The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells. PMID:25153153

  15. Repression of global protein synthesis by Eif1a-like genes that are expressed specifically in the two-cell embryos and the transient Zscan4-positive state of embryonic stem cells.

    PubMed

    Hung, Sandy S C; Wong, Raymond C B; Sharov, Alexei A; Nakatake, Yuhki; Yu, Hong; Ko, Minoru S H

    2013-08-01

    Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state. PMID:23649898

  16. Selaginella Genome Analysis – Entering the “Homoplasy Heaven” of the MADS World

    PubMed Central

    Gramzow, Lydia; Barker, Elizabeth; Schulz, Christian; Ambrose, Barbara; Ashton, Neil; Theißen, Günter; Litt, Amy

    2012-01-01

    In flowering plants, arguably the most significant transcription factors regulating development are MADS-domain proteins, encoded by Type I and Type II MADS-box genes. Type II genes are divided into the MIKCC and MIKC* groups. In angiosperms, these types and groups play distinct roles in the development of female gametophytes, embryos, and seeds (Type I); vegetative and floral tissues in sporophytes (MIKCC); and male gametophytes (MIKC*), but their functions in other plants are largely unknown. The complete set of MADS-box genes has been described for several angiosperms and a moss, Physcomitrella patens. Our examination of the complete genome sequence of a lycophyte, Selaginella moellendorffii, revealed 19 putative MADS-box genes (13 Type I, 3 MIKCC, and 3 MIKC*). Our results suggest that the most recent common ancestor of vascular plants possessed at least two Type I and two Type II genes. None of the S. moellendorffii MIKCC genes were identified as orthologs of any floral organ identity genes. This strongly corroborates the view that the clades of floral organ identity genes originated in a common ancestor of seed plants after the lineage that led to lycophytes had branched off, and that expansion of MIKCC genes in the lineage leading to seed plants facilitated the evolution of their unique reproductive organs. The number of MIKC* genes and the ratio of MIKC* to MIKCC genes is lower in S. moellendorffii and angiosperms than in P. patens, correlated with reduction of the gametophyte in vascular plants. Our data indicate that Type I genes duplicated and diversified independently within lycophytes and seed plants. Our observations on MADS-box gene evolution echo morphological evolution since the two lineages of vascular plants appear to have arrived independently at similar body plans. Our annotation of MADS-box genes in S. moellendorffii provides the basis for functional studies to reveal the roles of this crucial gene family in basal vascular plants. PMID

  17. Positive cross talk between protein kinase D and β-catenin in intestinal epithelial cells: impact on β-catenin nuclear localization and phosphorylation at Ser552.

    PubMed

    Wang, Jia; Han, Liang; Sinnett-Smith, James; Han, Li-Li; Stevens, Jan V; Rozengurt, Nora; Young, Steven H; Rozengurt, Enrique

    2016-04-01

    Given the fundamental role of β-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with β-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous β-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation (P< 0.01), peaked at 4 h (P< 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in β-catenin-regulated genes and phosphorylation at Ser(552) in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in β-catenin nuclear localization and phosphorylation at Ser(552) in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and β-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of β-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and β-catenin in intestinal epithelial cells, both in vitro and in vivo. PMID:26739494

  18. Molecular characterization of human neogenin, a DCC-related protein, and the mapping of its gene (NEO1) to chromosomal position 15q22.3-q23

    SciTech Connect

    Vielmetter, J.; Miskevich, F.; Lane, R.P.

    1997-05-01

    Neogenin was first identified in the chick embryo, and like a number of cell surface proteins of the immunoglobulin (Ig) superfamily, including N-CAM and L{sub 1} (generally called cell adhesion molecules or CAMs), it is expressed on growing nerve cells in the developing nervous system of vertebrate embryos. Neogenin is also expressed in other embryonic tissues, suggesting a more general role in developmental processes such as tissue growth regulation, cell-cell recognition, and cell migration. Neogenin, unlike the CAMs, is closely related to a unique tumor suppressor candidate molecule, deleted in colorectal carcinoma (DCC). Like DCC, the neogenin protein consists of four immunoglobulin-like (Ig-like) domains followed by six fibronectin type III domains, a transmembrane domain, and an intracellular domain. We now report the cloning and sequencing of cDNA clones coding for the human neogenin protein. Human neogenin shares 87% identity with its chicken homolog, and like its chicken counterpart it is expressed in at least two different isoforms derived from alternative splicing in the intracellular domain. Northern blot analysis revealed two mRNA species of about 5 and 7 kb. The chromosomal location of the human neogenin gene (HGMW-approved symbol NEO1) was determined as 15q22.3-q23, using fluorescence in situ hybridization. The gene therefore maps in the vicinity of a locus associated with Bardet-Biedl syndrome. The identification of human neogenin and its chromosomal location provides a basis for studying its involvement in genetic disorders or diseases. 26 refs., 4 figs.

  19. Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

    PubMed

    Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

    2016-01-01

    Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country. PMID:27622622

  20. Vascular variant of prion protein cerebral amyloidosis with tau-positive neurofibrillary tangles: the phenotype of the stop codon 145 mutation in PRNP.

    PubMed Central

    Ghetti, B; Piccardo, P; Spillantini, M G; Ichimiya, Y; Porro, M; Perini, F; Kitamoto, T; Tateishi, J; Seiler, C; Frangione, B; Bugiani, O; Giaccone, G; Prelli, F; Goedert, M; Dlouhy, S R; Tagliavini, F

    1996-01-01

    Deposition of PrP amyloid in cerebral vessels in conjunction with neurofibrillary lesions is the neuropathologic hallmark of the dementia associated with a stop mutation at codon 145 of PRNP, the gene encoding the prion protein (PrP). In this disorder, the vascular amyloid in tissue sections and the approximately 7.5-kDa fragment extracted from amyloid are labeled by antibodies to epitopes located in the PrP sequence including amino acids 90-147. Amyloid-laden vessels are also labeled by antibodies against the C terminus, suggesting that PrP from the normal allele is involved in the pathologic process. Abundant neurofibrillary lesions are present in the cerebral gray matter. They are composed of paired helical filaments, are labeled with antibodies that recognize multiple phosphorylation sites in tau protein, and are similar to those observed in Alzheimer disease. A PrP cerebral amyloid angiopathy has not been reported in diseases caused by PRNP mutations or in human transmissible spongiform encephalopathies; we propose to name this phenotype PrP cerebral amyloid angiopathy (PrP-CAA). Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8570627

  1. Position indicator

    DOEpatents

    Tanner, David E.

    1981-01-01

    A nuclear reactor system is described in which a position indicator is provided for detecting and indicating the position of a movable element inside a pressure vessel. The movable element may be a valve element or similar device which moves about an axis. Light from a light source is transmitted from a source outside the pressure vessel to a first region inside the pressure vessel in alignment with the axis of the movable element. The light is redirected by a reflector prism to a second region displaced radially from the first region. The reflector prism moves in response to movement of the movable element about its axis such that the second region moves arcuately with respect to the first region. Sensors are arrayed in an arc corresponding to the arc of movement of the second region and signals are transmitted from the sensors to the exterior of the reactor vessel to provide indication of the position of the movable element.

  2. [Positive psychiatry].

    PubMed

    Timmerby, Nina; Austin, Stephen; Bech, Per

    2016-02-01

    Positive psychiatry (PP) is a field within psychiatry with a particular focus on promoting well-being in people who already have or are at high risk of developing mental or physical illness. PP should be considered a supplement to trad-tional psychiatry and a call for therapists in psychiatry to focus on the person as a whole rather than just as a patient. PP is in line with current national and international health policy focus on promoting positive mental health. PMID:26857411

  3. Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

    PubMed

    Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

    2016-02-12

    The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. PMID:26637356

  4. Positive expression of protein chromosome 9 open reading frame 86 (C9orf86) correlated with poor prognosis in non-small cell lung cancer patients

    PubMed Central

    Peng, Gui-Lin; Tao, Ya-Lan; Wu, Qi-Nian; Zhang, Yu

    2016-01-01

    Background Chromosome 9 open reading frame 86 (C9orf86) is a novel subfamily of GTPases. Previous studies have implicated C9orf86 as a potential oncogene. Methods C9orf86 expression was detected in non-small cell lung cancer (NSCLC) cell lines and human bronchial epithelial (16HBE) cell lines by RT-PCR and western blotting. Immunohistochemistry (IHC) was used to detect 180 consecutive NSCLC specimens and 16 normal lung tissues. The correlation between C9orf86 expression and clinicopathological parameters was evaluated. Kaplan-Meier survival analysis and Cox hazards ratio models were used to estimate the effect of C9orf86 expression on survival. Results C9orf86 was expressed in the cytoplasm in 74 of 180 (41.11%) NSCLC specimens. In clinical pathology analysis, C9orf86 expression significantly correlated with lymph node metastasis and clinical stage significantly (P<0.05). Multivariable analysis confirmed that C9orf86 expression increased the risk of death after adjusting for other clinicopathological factors (P<0.01). Overall survival (OS) and disease-free survival (DFS) were significantly prolonged in the C9orf86 negative group compared to the C9orf86 positive group (P<0.001). Adjuvant chemotherapy prolonged OS and DFS in resected NSCLC patients with C9orf86 negative expression (P<0.001) but not C9orf86 positive. Conclusions Positive expression of C9orf86 is an independent prognostic factor for NSCLC patients, and C9orf86 may serve as a prognostic biomarker for patients with NSCLC. PMID:27499931

  5. Anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor α-positive breast cancer.

    PubMed

    Andruska, N; Zheng, X; Yang, X; Helferich, W G; Shapiro, D J

    2015-07-01

    In response to cell stress, cancer cells often activate the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR). Little was known about the potential role in cancer of a different mode of UPR activation, anticipatory activation of the UPR prior to accumulation of unfolded protein or cell stress. We show that estrogen, acting via estrogen receptor α (ERα), induces rapid anticipatory activation of the UPR, resulting in increased production of the antiapoptotic chaperone BiP/GRP78, preparing cancer cells for the increased protein production required for subsequent estrogen-ERα-induced cell proliferation. In ERα-containing cancer cells, the estrogen, 17β-estradiol (E2) activates the UPR through a phospholipase C γ (PLCγ)-mediated opening of EnR IP3R calcium channels, enabling passage of calcium from the lumen of the EnR into the cytosol. siRNA knockdown of ERα blocked the estrogen-mediated increase in cytosol calcium and UPR activation. Knockdown or inhibition of PLCγ, or of IP3R, strongly inhibited the estrogen-mediated increases in cytosol calcium, UPR activation and cell proliferation. E2-ERα activates all three arms of the UPR in breast and ovarian cancer cells in culture and in a mouse xenograft. Knockdown of ATF6α, which regulates UPR chaperones, blocked estrogen induction of BiP and strongly inhibited E2-ERα-stimulated cell proliferation. Mild and transient UPR activation by estrogen promotes an adaptive UPR response that protects cells against subsequent UPR-mediated apoptosis. Analysis of data from ERα(+) breast cancers demonstrates elevated expression of a UPR gene signature that is a powerful new prognostic marker tightly correlated with subsequent resistance to tamoxifen therapy, reduced time to recurrence and poor survival. Thus, as an early component of the E2-ERα proliferation program, the mitogen estrogen, drives rapid anticipatory activation of the UPR. Anticipatory activation of the UPR is a new role for

  6. P16 protein expression in primary cutaneous melanoma with positive and negative lymph node biopsies: Particular aspects of a study performed at the Hospital de Clinicas de Porto Alegre, Brazil

    PubMed Central

    Fauri, JAC; Ricardi, F; Diehl, ES; Cartell, A; Furian, R; Bakos, L; Edelweiss, MI

    2011-01-01

    BACKGROUND: Cutaneous melanoma dermal invasion, identified through measurement of maximum tumour thickness and sentinel lymph node (SLN) biopsy, is important to establish melanoma prognosis and progression. P16 protein expression has been shown to be a predictive factor for melanoma evolution and prognosis. OBJECTIVE: To investigate p16 protein expression in cutaneous melanomas with and without SLN metastasis. PATIENTS AND METHODS: Sixty-seven paraffin-embedded cutaneous melanoma specimens of patients who had undergone SLN investigation were evaluated from 1995 to 2007. SLN biopsy was negative for metastasis in 34 of these patients (controls); in the remaining 33 patients, SLN biopsy was positive (cases). The expression of p16 protein in the primary tumour was measured using an immunohistochemical assay. The samples were classified according to their nuclear expression. RESULTS: P16 nuclear expression was absent in 14 cases and in 15 controls; P=0.812. There was no statistically significant difference in p16 nuclear expression between cases and controls. CONCLUSIONS: The present study does not support the findings of other studies that suggest p16 protein expression is important in the prognosis of cutaneous melanoma. PMID:22942654

  7. Positive psychotherapy.

    PubMed

    Seligman, Martin E P; Rashid, Tayyab; Parks, Acacia C

    2006-11-01

    Positive psychotherapy (PPT) contrasts with standard interventions for depression by increasing positive emotion, engagement, and meaning rather than directly targeting depressive symptoms. The authors have tested the effects of these interventions in a variety of settings. In informal student and clinical settings, people not uncommonly reported them to be "life-changing." Delivered on the Web, positive psychology exercises relieved depressive symptoms for at least 6 months compared with placebo interventions, the effects of which lasted less than a week. In severe depression, the effects of these Web exercises were particularly striking. This address reports two preliminary studies: In the first, PPT delivered to groups significantly decreased levels of mild-to-moderate depression through 1-year follow-up. In the second, PPT delivered to individuals produced higher remission rates than did treatment as usual and treatment as usual plus medication among outpatients with major depressive disorder. Together, these studies suggest that treatments for depression may usefully be supplemented by exercises that explicitly increase positive emotion, engagement, and meaning. ((c) 2006 APA, all rights reserved). PMID:17115810

  8. GSK3 protein positively regulates type I insulin-like growth factor receptor through forkhead transcription factors FOXO1/3/4.

    PubMed

    Huo, Xiaodong; Liu, Shu; Shao, Ting; Hua, Hui; Kong, Qingbin; Wang, Jiao; Luo, Ting; Jiang, Yangfu

    2014-09-01

    Glycogen synthase kinase-3 (GSK3) has either tumor-suppressive roles or pro-tumor roles in different types of human tumors. A number of GSK3 targets in diverse signaling pathways have been uncovered, such as tuberous sclerosis complex subunit 2 and β-catenin. The O subfamily of forkhead/winged helix transcription factors (FOXO) is known as tumor suppressors that induce apoptosis. In this study, we find that FOXO binds to type I insulin-like growth factor receptor (IGF-IR) promoter and stimulates its transcription. GSK3 positively regulates the transactivation activity of FOXO and stimulates IGF-IR expression. Although kinase-dead GSK3β cannot up-regulate IGF-IR, the constitutively active GSK3β induces IGF-IR expression in a FOXO-dependent manner. Serum starvation or Akt inhibition leads to an increase in IGF-IR expression, which could be blunted by GSK3 inhibition. GSK3β knockdown or GSK3 inhibitor suppresses IGF-I-induced IGF-IR, Akt, and ERK1/2 phosphorylation. Moreover, knockdown of GSK3β or FOXO1/3/4 leads to a decrease in cellular proliferation and abrogates IGF-I-induced hepatoma cell proliferation. These results suggest that GSK3 and FOXO may positively regulate IGF-I signaling and hepatoma cell proliferation. PMID:25053419

  9. GSK3 Protein Positively Regulates Type I Insulin-like Growth Factor Receptor through Forkhead Transcription Factors FOXO1/3/4

    PubMed Central

    Huo, Xiaodong; Liu, Shu; Shao, Ting; Hua, Hui; Kong, Qingbin; Wang, Jiao; Luo, Ting; Jiang, Yangfu

    2014-01-01

    Glycogen synthase kinase-3 (GSK3) has either tumor-suppressive roles or pro-tumor roles in different types of human tumors. A number of GSK3 targets in diverse signaling pathways have been uncovered, such as tuberous sclerosis complex subunit 2 and β-catenin. The O subfamily of forkhead/winged helix transcription factors (FOXO) is known as tumor suppressors that induce apoptosis. In this study, we find that FOXO binds to type I insulin-like growth factor receptor (IGF-IR) promoter and stimulates its transcription. GSK3 positively regulates the transactivation activity of FOXO and stimulates IGF-IR expression. Although kinase-dead GSK3β cannot up-regulate IGF-IR, the constitutively active GSK3β induces IGF-IR expression in a FOXO-dependent manner. Serum starvation or Akt inhibition leads to an increase in IGF-IR expression, which could be blunted by GSK3 inhibition. GSK3β knockdown or GSK3 inhibitor suppresses IGF-I-induced IGF-IR, Akt, and ERK1/2 phosphorylation. Moreover, knockdown of GSK3β or FOXO1/3/4 leads to a decrease in cellular proliferation and abrogates IGF-I-induced hepatoma cell proliferation. These results suggest that GSK3 and FOXO may positively regulate IGF-I signaling and hepatoma cell proliferation. PMID:25053419

  10. Binding of the wheat basic leucine zipper protein EmBP-1 to nucleosomal binding sites is modulated by nucleosome positioning.

    PubMed Central

    Niu, X; Adams, C C; Workman, J L; Guiltinan, M J

    1996-01-01

    To investigate interactions of the basic leucine zipper transcription factor EmBP-1 with its recognition sites in nucleosomal DNA, we reconstituted an abscisic acid response element and a high-affinity binding site for EmBP-1 into human and wheat nucleosome cores in vitro. DNA binding studies demonstrated that nucleosomal elements can be bound by EmBP-1 at reduced affinities relative to naked DNA. EmBP-1 affinity was lowest when the recognition sites were positioned near the center of the nucleosome. Binding was achieved with a truncated DNA binding domain; however, binding of full-length EmBP-1 caused additional strong DNase I hypersensitivity flanking the binding sites. Similar results were observed with nucleosomes reconstituted with either human or wheat histones, demonstrating a conserved mechanism of transcription factor-nucleosome interactions. We conclude that positioning of recognition sequences on a nucleosome may play an important role in regulating interactions of EmBP-1 with its target sites in plant cells. PMID:8837510

  11. The Zebrafish Period2 Protein Positively Regulates the Circadian Clock through Mediation of Retinoic Acid Receptor (RAR)-related Orphan Receptor α (Rorα)*

    PubMed Central

    Wang, Mingyong; Zhong, Zhaomin; Zhong, Yingbin; Zhang, Wei; Wang, Han

    2015-01-01

    We report the characterization of a null mutant for zebrafish circadian clock gene period2 (per2) generated by transcription activator-like effector nuclease and a positive role of PER2 in vertebrate circadian regulation. Locomotor experiments showed that per2 mutant zebrafish display reduced activities under light-dark and 2-h phase delay under constant darkness, and quantitative real time PCR analyses showed up-regulation of cry1aa, cry1ba, cry1bb, and aanat2 but down-regulation of per1b, per3, and bmal1b in per2 mutant zebrafish, suggesting that Per2 is essential for the zebrafish circadian clock. Luciferase reporter assays demonstrated that Per2 represses aanat2 expression through E-box and enhances bmal1b expression through the Ror/Rev-erb response element, implicating that Per2 plays dual roles in the zebrafish circadian clock. Cell transfection and co-immunoprecipitation assays revealed that Per2 enhances bmal1b expression through binding to orphan nuclear receptor Rorα. The enhancing effect of mouse PER2 on Bmal1 transcription is also mediated by RORα even though it binds to REV-ERBα. Moreover, zebrafish Per2 also appears to have tissue-specific regulatory roles in numerous peripheral organs. These findings help define the essential functions of Per2 in the zebrafish circadian clock and in particular provide strong evidence for a positive role of PER2 in the vertebrate circadian system. PMID:25544291

  12. Hu antigen R (HuR) is a positive regulator of the RNA-binding proteins TDP-43 and FUS/TLS: implications for amyotrophic lateral sclerosis.

    PubMed

    Lu, Liang; Zheng, Lei; Si, Ying; Luo, Wenyi; Dujardin, Gwendal; Kwan, Thaddaeus; Potochick, Nicholas R; Thompson, Sunnie R; Schneider, David A; King, Peter H

    2014-11-14

    Posttranscriptional gene regulation is governed by a network of RNA-binding proteins (RBPs) that interact with regulatory elements in the mRNA to modulate multiple molecular processes, including splicing, RNA transport, RNA stability, and translation. Mounting evidence indicates that there is a hierarchy within this network whereby certain RBPs cross-regulate other RBPs to coordinate gene expression. HuR, an RNA-binding protein we linked previously to aberrant VEGF mRNA metabolism in models of SOD1-associated amyotrophic lateral sclerosis, has been identified as being high up in this hierarchy, serving as a regulator of RNA regulators. Here we investigated the role of HuR in regulating two RBPs, TDP-43 and FUS/TLS, that have been linked genetically to amyotrophic lateral sclerosis. We found that HuR promotes the expression of both RBPs in primary astrocytes and U251 cells under normal and stressed (hypoxic) conditions. For TDP-43, we found that HuR binds to the 3' untranslated region (UTR) and regulates its expression through translational efficiency rather than RNA stability. With HuR knockdown, there was a shift of TDP-43 and FUS mRNAs away from polysomes, consistent with translational silencing. The TDP-43 splicing function was attenuated upon HuR knockdown and could be rescued by ectopic TDP-43 lacking the 3' UTR regulatory elements. Finally, conditioned medium from astrocytes in which HuR or TDP-43 was knocked down produced significant motor neuron and cortical neuron toxicity in vitro. These findings indicate that HuR regulates TDP-43 and FUS/TLS expression and that loss of HuR-mediated RNA processing in astrocytes can alter the molecular and cellular landscape to produce a toxic phenotype. PMID:25239623

  13. Mapping of T cell epitopes of the 30-kDa {alpha} antigen of Mycobacterium bovis strain bacillus Calmette-Guerin in Purified Protein Derivative (PPD)-positive individuals

    SciTech Connect

    Silver, R.F.; Wallis, R.S.; Ellner, J.J.

    1995-05-01

    The fibronectin-binding 30-kDa {alpha} Ag is a major secretory protein of growing mycobacteria that stimulates in vitro lymphocyte blastogenesis in most healthy purified protein derivative-positive individuals, but only a minority of patients with active tuberculosis. T cell epitopes of the {alpha} Ag were assessed using blastogenic responses of PBMC from 12 healthy purified protein derivative-positive subjects to a set of synthetic peptides based on the 325-amino acid sequence of the {alpha} Ag of Mycobacterium bovis BCG. Because epitope-specific precursor cells are infrequent and randomly distributed, we used Poisson analysis to determine positive responses to 10 {mu}g/ml of each peptide in 12 replicate culture wells. Seven immunodominant regions of the {alpha} Ag were identified. Each subject responded to at least one of the two most dominant epitopes, which correspond to amino acids 131-155 and 233-257 (from N terminus). Peptides of these two epitopes induced production of IFN-{gamma} by sorted CD4{sup +} T cells. The immuno-dominant peptides may have use as components of a vaccine and as tools to study the evolution of the immune response to M. tuberculosis. The two most dominant epitopes both occur in regions of the {alpha} Ag that differ from those of the atypical pathogens M. avium and M. kansasii. In addition, the M. bovis epitope of amino acids 133-155 differs from that of M. tuberculosis by a single amino acid. It may be possible to exploit the sequence differences for development of diagnostic tests with increased specificity. 39 refs., 4 figs., 1 tab.

  14. Serum Antibodies from a Subset of Horses Positive for Babesia caballi by Competitive Enzyme-Linked Immunosorbent Assay Demonstrate a Protein Recognition Pattern That Is Not Consistent with Infection

    PubMed Central

    Awinda, Peter O.; Mealey, Robert H.; Williams, Laura B. A.; Conrad, Patricia A.; Packham, Andrea E.; Reif, Kathryn E.; Grause, Juanita F.; Pelzel-McCluskey, Angela M.; Chung, Chungwon; Bastos, Reginaldo G.; Kappmeyer, Lowell S.; Howe, Daniel K.; Ness, SallyAnne L.; Knowles, Donald P.

    2013-01-01

    Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)–competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1–cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi. PMID:24049108

  15. The DNA-binding domain of the transcriptional activator protein NifA resides in its carboxy terminus, recognises the upstream activator sequences of nif promoters and can be separated from the positive control function of NifA.

    PubMed Central

    Morett, E; Cannon, W; Buck, M

    1988-01-01

    The positive control protein NifA activates transcription of nitrogen fixation promoters in Klebsiella pneumoniae. NifA is believed to bind to specific sites, the upstream activator sequences (UAS's), of the nif promoters which it activates. We have now shown by mutation of the carboxy terminus of NifA that this is the DNA-binding domain and that the DNA-binding and positive activator functions of NifA can be separated. Mutational analysis of the nifH UAS and in vivo methylation protection analysis of the interaction of NifA with the nifH promoter demonstrates that the UAS is recognised by the carboxy terminus of NifA. The UAS's of K. pneumoniae nif promoters are also required for activation by the Rhizobium meliloti NifA indicating that this activator also possesses DNA-binding activity. Images PMID:3062575

  16. Position sensor

    NASA Technical Reports Server (NTRS)

    Auer, Siegfried (Inventor)

    1988-01-01

    A radiant energy angle sensor is provided wherein the sensitive portion thereof comprises a pair of linear array detectors with each detector mounted normal to the other to provide X and Y channels and a pair of slits spaced from the pair of linear arrays with each of the slits positioned normal to its associated linear array. There is also provided electrical circuit means connected to the pair of linear array detectors and to separate X and Y axes outputs.

  17. Lack of cross-reactivity between the Bacillus thuringiensis derived protein Cry1F in maize grain and dust mite Der p7 protein with human sera positive for Der p7-IgE.

    PubMed

    Ladics, Gregory S; Bardina, Luda; Cressman, Robert F; Mattsson, Joel L; Sampson, Hugh A

    2006-03-01

    Cry1F protein, derived from Bacillus thuringiensis, is effective at controlling lepidopteran pests and a synthetic Cry1F transgene was transferred into maize. For the safety assessment of genetically modified food crops, the allergenic potential of the introduced novel trait(s) is evaluated. Because no single parameter is currently predictive of allergic potential, a 'weight of evidence' approach has been proposed. As part of this assessment, the amino acid (aa) sequence of the Cry1F protein was compared to a database of known allergens using recommended criteria. The Cry1F protein did not show significant similarity or a match of eight contiguous identical aa with any allergen. However, a single six contiguous aa match was identified between Cry1F and the Der p7 protein of the dust mite, Dermatophagoides pteronyssinus. To investigate whether Cry1F was cross-reactive with Der p7, sera from 10 dust mite allergic patients containing Der p 7-specific IgE antibody were used to compare IgE-specific binding. No evidence of cross-reactivity was observed between Cry1F and Der p7. This study provides in vitro IgE sera screening data, that when considered in the context of other bioinformatic data [Hileman R.E., Silvanovich, A., Goodman R.E., Rice E.A., Holleschak G., Astwood J.D., Hefle S.L., 2002. Bioinformatic methods for allergenicity assessment using a comprehensive allergen database. Int. Arch. Allergy Immunol. 128, 280-291; Stadler, M.B., Stadler, B.M., 2003. Allergenicity prediction by protein sequence. FASEB J. 17, 1141-1143.], adds further evidence arguing against the use of a six contiguous identical amino acid search to identify potential cross-reactive allergens. Cry1F is heat labile, rapidly hydrolyzed in an in vitro pepsin resistance assay, not glycosylated and not from an allergenic source. Taken together, these data indicate a lack of allergenic concern for Cry1F. PMID:16406630

  18. Positive Psychologists on Positive Constructs

    ERIC Educational Resources Information Center

    Lyubomirsky, Sonja

    2012-01-01

    Comments on the original article by McNulty and Fincham (see record 2011-15476-001). In their article, the authors offered compelling evidence that constructs such as forgiveness and optimism can have both beneficial and adverse consequences, depending on the context. Their caution about labeling particular psychological processes as "positive" is…

  19. Living positively as HIV positive.

    PubMed

    Garraty, Sarah J

    2011-01-01

    A nursing student records a brief biography of a Zambian nurse and certified midwife living with HIV/AIDS while shadowing the nurse during an undergraduate cross-cultural course in Macha, Zambia in January 2009. The nurse strives to live positively, educating, encouraging, and empowering others. PMID:21294466

  20. Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2

    PubMed Central

    Wu, Yifen; Li, Rong; Zhang, Junyi; Wang, Gang; Liu, Bin; Huang, Xiaofang; Zhang, Tao; Luo, Rongcheng

    2015-01-01

    Background Trastuzumab resistance in HER-2 positive breast cancer cells is closely related to overexpression of both epidermal growth factor receptor (EGFR) and human epidermal receptor (HER-2). SHP-1 has been demonstrated to downregulate tyrosine kinase activity including EGFR via its phosphatase function, but its effect on HER-2 activity is still unknown. Here, we examined the hypothesis that SHP-1 enhances the anticancer efficacy of trastuzumab in EGFR/HER-2 positive breast cancer cells through combining dual inhibition of EGFR and HER-2. Methods Trastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3cells in the presence of trastuzumab. The SHP-1 was ectopically expressed by stable transfection. The activity and expression of EGFR, HER-2, and downstream signaling pathways were tested by Western blot. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was examined by flow cytometry. The binding between SHP-1 and EGFR/HER-2 was evaluated by immunoprecipitation assay and bimolecular fluorescence complementation. The effects of SHP-1 on tumorigenicity and trastuzumab sensitivity were confirmed via in vivo xenograft model. Results Trastuzumab-resistant SKBr-3 cells showed aberrant co-expression of EGFR and HER-2. Introduction of wild-type SHP-1 inhibited cell proliferation, clone formation, and promoted the apoptosis induced by trastuzumab. Meanwhile, SHP-1 overexpression reduced phosphorylation levels of EGFR and HER-2 both in parental and trastuzumab-resistant SKBr-3 cells. In vivo study showed an increased antitumor effect of trastuzumab in SHP-1 overexpressed xenografts. At last, we discovered that SHP-1 can make complexes with both EGFR and HER-2, and both phospho-EGFR and phosphor-HER-2 levels in wild-type SHP-1 immunoprecipitates were less than those in phosphatase-inactive SHP-1 (C453S) immunoprecipitates, indicating that EGFR and HER-2 are

  1. A wheat R2R3-MYB protein PURPLE PLANT1 (TaPL1) functions as a positive regulator of anthocyanin biosynthesis.

    PubMed

    Shin, Dong Ho; Choi, Myoung-Goo; Kang, Chon-Sik; Park, Chul-Soo; Choi, Sang-Bong; Park, Youn-Il

    2016-01-15

    Transcriptional activation of anthocyanin biosynthesis genes in vegetative tissues of monocotyledonous plants is mediated by cooperative activity of one component from each of the following two transcription factor families: MYB encoded by PURPLE PLANT1/COLORED ALEURONE1 (PL1/C1), and basic helix-loop-helix (bHLH) encoded by RED/BOOSTER (R1/B1). In the present study, putative PL cDNA was cloned from the wheat (Triticum aestivum) cultivar Iksan370, which preferentially expresses anthocyanins in coleoptiles. Phylogenetic tree analysis of deduced amino acid sequences showed that a putative TaPL1 is highly homologous to barley (Hordeum vulgare) HvPL1, but is distinct from wheat TaC1. Transgenic Arabidopsis thaliana stably expressing putative TaPL1 accumulated anthocyanin pigments in leaves and up-regulated structural genes involved in both early and late anthocyanin biosynthesis steps. TaPL1 transcript levels in Iksan370 were more prominent in vegetative tissues such as young coleoptiles than in reproductive tissues such as spikelets. TaPL1 expression was significantly up-regulated by environmental stresses including cold, salt, and light, which are known to induce anthocyanin accumulation. These combined results suggest that TaPL1 is an active positive regulator of anthocyanin biosynthesis in wheat coleoptiles. PMID:26692488

  2. Discrimination of the prochiral hydrogens at the C-2 position of n-alkanes by the methane/ammonia monooxygenase family proteins.

    PubMed

    Miyaji, Akimitsu; Miyoshi, Teppei; Motokura, Ken; Baba, Toshihide

    2015-08-14

    The selectivity of ammonia monooxygenase from Nitrosomonas europaea (AMO-Ne) for the oxidation of C4-C8n-alkanes to the corresponding alcohol isomers was examined to show the ability of AMO-Ne to recognize the n-alkane orientation within the catalytic site. AMO-Ne in whole cells produces 1- and 2-alcohols from C4-C8n-alkanes, and the regioselectivity is dependent on the length of the carbon chain. 2-Alcohols produced from C4-C7n-alkanes were predominantly either the R- or S-enantiomers, while 2-octanol produced from n-octane was racemic. These results indicate that AMO-Ne can discriminate between the prochiral hydrogens at the C-2 position, with the degree of discrimination varying according to the n-alkane. Compared to the particulate methane monooxygenase (pMMO) of Methylococcus capsulatus (Bath) and that of Methylosinus trichosporium OB3b, AMO-Ne showed a distinct ability to discriminate between the orientation of n-butane and n-pentane in the catalytic site. PMID:26138087

  3. Multivitamin and protein supplement use is associated with positive mood states and health behaviors in US Military and Coast Guard personnel.

    PubMed

    Austin, Krista G; McGraw, Susan M; Lieberman, Harris R

    2014-10-01

    Approximately 60% of Armed Forces personnel regularly consume dietary supplements (DSs). We investigated the association of mood and health behaviors with multiple classes of DSs in military and Coast Guard personnel (N = 5536). Participants completed a survey of DS use and the Quick Mood Scale to assess mood domains of wakeful-drowsiness, relaxed-anxious, cheerful-depressed, friendly-aggression, clearheaded-confused, and well coordinated-clumsy. Supplements were categorized as multivitamin/minerals (MVM), individual vitamin/minerals, protein/amino acid supplements (PS), combination products (C), herbals (H), purported steroid analogs, (S) and other (O). One-way analyses of covariance assessed associations of DSs and perceived health behavior with mood controlling for age. Logistic regression determined associations between DS use and health behavior. Users of MVM and PS reported feeling significantly (P < 0.05) more awake, relaxed, cheerful, clearheaded, and coordinated. Participants using PS and S reported feeling less friendly (more aggressive, P < 0.02). Users of MVM and PS were more likely to report their general health, eating habits, and fitness level as excellent/good (P < 0.05). Participants reporting health behaviors as excellent/good were more (P < 0.01) awake, relaxed, cheerful, friendly, clearheaded, and coordinated. As no known biological mechanisms can explain such diverse effects of MVM and PS use on multiple mood states, health, eating habits, and fitness, we hypothesize these associations are not causal, and DS intake does not alter these parameters per se. Preexisting differences in mood and other health-related behaviors and outcomes between users versus nonusers of DSs could be a confounding factor in studies of DSs. PMID:25122181

  4. Multivitamin and Protein Supplement Use Is Associated With Positive Mood States and Health Behaviors in US Military and Coast Guard Personnel

    PubMed Central

    Austin, Krista G.; McGraw, Susan M.; Lieberman, Harris R.

    2014-01-01

    Abstract Approximately 60% of Armed Forces personnel regularly consume dietary supplements (DSs). We investigated the association of mood and health behaviors with multiple classes of DSs in military and Coast Guard personnel (N = 5536). Participants completed a survey of DS use and the Quick Mood Scale to assess mood domains of wakeful-drowsiness, relaxed-anxious, cheerful-depressed, friendly-aggression, clearheaded-confused, and well coordinated–clumsy. Supplements were categorized as multivitamin/minerals (MVM), individual vitamin/minerals, protein/amino acid supplements (PS), combination products (C), herbals (H), purported steroid analogs, (S) and other (O). One-way analyses of covariance assessed associations of DSs and perceived health behavior with mood controlling for age. Logistic regression determined associations between DS use and health behavior. Users of MVM and PS reported feeling significantly (P < 0.05) more awake, relaxed, cheerful, clearheaded, and coordinated. Participants using PS and S reported feeling less friendly (more aggressive, P < 0.02). Users of MVM and PS were more likely to report their general health, eating habits, and fitness level as excellent/good (P < 0.05). Participants reporting health behaviors as excellent/good were more (P < 0.01) awake, relaxed, cheerful, friendly, clearheaded, and coordinated. As no known biological mechanisms can explain such diverse effects of MVM and PS use on multiple mood states, health, eating habits, and fitness, we hypothesize these associations are not causal, and DS intake does not alter these parameters per se. Preexisting differences in mood and other health-related behaviors and outcomes between users versus nonusers of DSs could be a confounding factor in studies of DSs. PMID:25122181

  5. Expression of S100 Protein in CD4-positive T-cell Lymphomas Is Often Associated With T-cell Prolymphocytic Leukemia.

    PubMed

    Aggarwal, Nidhi; Pongpruttipan, Tawatchai; Patel, Snehal; Bayerl, Michael G; Alkan, Serhan; Nathwani, Bharat; Surti, Urvashi; Kitahara, Sumire; Chinthammitr, Yingyong; Swerdlow, Steven H

    2015-12-01

    S100 T-cell lymphomas are infrequent, and except 1 all have been CD4 negative. On the basis of an index case of CD4 S100 T-cell prolymphocytic leukemia (T-PLL), we studied S100 protein expression in 19 additional T-PLLs and 56 other T-cell lymphomas that are usually CD4, including 15 angioimmunoblastic T-cell lymphomas, 24 anaplastic large cell lymphomas (16 ALK and 8 ALK), 7 mycosis fungoides/Sézary syndrome, and 10 peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). Two additional S100 CD4 PTCL, NOS cases were also reviewed. Thirty percent (6/20) of T-PLLs were S100 compared with 0/56 other T-cell lymphomas with previously unstudied S100 reactivity (40 CD4, 2 CD8, 11 CD4/CD8, 3 unknown) (P=0.0007). There were no significant differences between the S100 and S100 T-PLLs with regard to the male:female ratio (2:1 vs. 1:1), age (71.6±7.7 vs. 65.4±9.3), peripheral blood lymphocyte count (67.2±116.6 vs. 101.1±159.7×10/L), or median survival (463 vs. 578 d, where known). The 2 S100 PTCL, NOS cases occurred in a 7-year-old boy and a 45-year-old woman. Both had involvement of the bone marrow and peripheral blood but were morphologically unlike T-PLL and lacked TCL1 gene rearrangement. These results demonstrate that S100 T-cell lymphomas include a subset that are CD4 and most often, but not exclusively, are T-PLL. Although having diagnostic implications, there were no documented clinical differences between the S100 and S100 T-PLLs. PMID:26379148

  6. Positioning apparatus

    DOEpatents

    Vogel, Max A.; Alter, Paul

    1986-05-06

    An apparatus for precisely positioning materials test specimens within the optimum neutron flux path emerging from a neutron source located in a housing. The test specimens are retained in a holder mounted on the free end of a support pivotably mounted and suspended from a movable base plate. The support is gravity biased to urge the holder in a direction longitudinally of the flux path against the housing. Means are provided for moving the base plate in two directions to effect movement of the holder in two mutually perpendicular directions normal to the axis of the flux path.

  7. Positioning apparatus

    DOEpatents

    Vogel, Max A.; Alter, Paul

    1986-01-01

    An apparatus for precisely positioning materials test specimens within the optimum neutron flux path emerging from a neutron source located in a housing. The test specimens are retained in a holder mounted on the free end of a support pivotably mounted and suspended from a movable base plate. The support is gravity biased to urge the holder in a direction longitudinally of the flux path against the housing. Means are provided for moving the base plate in two directions to effect movement of the holder in two mutually perpendicular directions normal to the axis of the flux path.

  8. POSITIONING DEVICE

    DOEpatents

    Wall, R.R.; Peterson, D.L.

    1959-09-15

    A positioner is described for a vertical reactor-control rod. The positioner comprises four grooved friction rotatable members that engage the control rod on all sides and shift it longitudinally. The four friction members are drivingly interconnected for conjoint rotation and comprise two pairs of coaxial members. The members of each pair are urged toward one another by hydraulic or pneumatic pressure and thus grip the control rod so as to hold it in any position or adjust it. Release of the by-draulic or pneumatic pressure permits springs between the friction members of each pair to force them apart, whereby the control rod moves quickly by gravity into the reactor.

  9. Positioning apparatus

    DOEpatents

    Vogel, M.A.; Alter, P.

    1983-07-07

    An apparatus is provided for precisely adjusting the position of an article relative to a beam emerging from a neutron source disposed in a housing. The apparatus includes a support pivotably mounted on a movable base plate and freely suspended therefrom. The support is gravity biased toward the housing and carries an article holder movable in a first direction longitudinally of the axis of said beam and normally urged into engagement against said housing. Means are provided for moving the base plate in two directions to effect movement of the suspended holder in two mutually perpendicular directions, respectively, normal to the axis of the beam.

  10. Association of Valine and Leucine at HLA–DRB1 Position 11 With Radiographic Progression in Rheumatoid Arthritis, Independent of the Shared Epitope Alleles but Not Independent of Anti–Citrullinated Protein Antibodies

    PubMed Central

    van Steenbergen, H. W.; Raychaudhuri, S.; Rodríguez-Rodríguez, L.; Rantapää-Dahlqvist, S.; Berglin, E.; Toes, R. E. M.; Huizinga, T. W. J.; Fernández-Gutiérrez, B.; Gregersen, P. K.; van der Helm-van Mil, A. H. M.

    2015-01-01

    Objective For decades it has been known that the HLA–DRB1 shared epitope (SE) alleles are associated with an increased risk of development and progression of rheumatoid arthritis (RA). Recently, the following variations in the peptide-binding grooves of HLA molecules that predispose to RA development have been identified: Val and Leu at HLA–DRB1 position 11, Asp at HLA–B position 9, and Phe at HLA–DPB1 position 9. This study was undertaken to investigate whether these variants are also associated with radiographic progression in RA, independent of SE and anti–citrullinated protein antibody (ACPA) status. Methods A total of 4,911 radiograph sets from 1,878 RA patients included in the Leiden Early Arthritis Clinic (The Netherlands), Umeå (Sweden), Hospital Clinico San Carlos–Rheumatoid Arthritis (Spain), and National Data Bank for Rheumatic Diseases (US) cohorts were studied. HLA was imputed using single-nucleotide polymorphism data from an Immunochip, and the amino acids listed above were tested in relation to radiographic progression per cohort using an additive model. Results from the 4 cohorts were combined in inverse-variance weighted meta-analyses using a fixed-effects model. Analyses were conditioned on SE and ACPA status. Results Val and Leu at HLA–DRB1 position 11 were associated with more radiographic progression (meta-analysis P = 5.11 × 10−7); this effect was independent of SE status (meta-analysis P = 0.022) but not independent of ACPA status. Phe at HLA–DPB1 position 9 was associated with more severe radiographic progression (meta-analysis P = 0.024), though not independent of SE status. Asp at HLA–B position 9 was not associated with radiographic progression. Conclusion Val and Leu at HLA–DRB1 position 11 conferred a risk of a higher rate of radiographic progression independent of SE status but not independent of ACPA status. These findings support the relevance of these amino acids at position 11. PMID:25580908

  11. A composite score combining procalcitonin, C-reactive protein and temperature has a high positive predictive value for the diagnosis of intensive care-acquired infections

    PubMed Central

    2013-01-01

    Background Nosocomial infection diagnosis in the intensive care unit (ICU) remains a challenge. We compared routine measurements of procalcitonin (PCT), C-reactive protein (CRP), white blood cell count (WBC) and temperature in the detection of ICU-acquired infections. Method Prospective observational cohort study in a University hospital Medicosurgical ICU. All patients admitted to the ICU ≥ 5 days (n = 141) were included into two groups, either infected (documented infection, n = 25) or non-infected (discharged from the ICU without diagnosis of infection, n = 88). Results PCT, CRP, WBC and temperature progression from day −4 (D-4) to day 0 (D0) (day of infection diagnosis or ICU discharge) was analysed. Differences (Δ) were calculated as D0 levels minus the lowest preceding value. D0 PCT and CRP were significantly increased in infected compared to non-infected patients (median, 1st and 3rd quartiles): 3.6 ng/mL (0.92-25) for PCT, 173 mg/L (126–188) for CRP versus 0.02 ng/mL (0.1-0.9) and 57 mg/mL (31–105) respectively (p < 0.0001). In multivariate analysis, D0 temperature > 38.6°C, PCT > 1.86 ng/mL, and CRP > 88 mg/L, performed well (AUCs of 0.88, 0.84, and 0.88 respectively). The sensitivity/specificity profiles of each marker (76%/94% for temperature, 68%/91% for PCT, and 92%/70% for CRP) led to a composite score (0.068 × D0 PCT + 0.005 × D0 CRP + 0.7 × temperature) more highly specific than each component (AUC of 0.90 and sensitivity/specificity of 80%/97%). Conclusion Combining CRP, PCT and temperature is an approach which may increase of nosocomial infection detection in the ICU. PMID:23547830

  12. Identification of Insulin-Like Growth Factor-I Receptor (IGF-IR) Gene Promoter-Binding Proteins in Estrogen Receptor (ER)-Positive and ER-Depleted Breast Cancer Cells

    PubMed Central

    Sarfstein, Rive; Belfiore, Antonino; Werner, Haim

    2010-01-01

    The insulin-like growth factor I receptor (IGF-IR) has been implicated in the etiology of breast cancer. Overexpression of the IGF-IR gene is a typical feature of most primary breast cancers, whereas low IGF-IR levels are seen at advanced stages. Hence, evaluation of IGF-IR levels might be important for assessing prognosis. In the present study, we employed a proteomic approach based on DNA affinity chromatography followed either by mass spectroscopy (MS) or Western blot analysis to identify transcription factors that may associate with the IGF-IR promoter in estrogen receptor (ER)-positive and ER-depleted breast cancer cells. A biotinylated IGF-IR promoter fragment was bound to streptavidin magnetic beads and incubated with nuclear extracts of breast cancer cells. IGF-IR promoter-binding proteins were eluted with high salt and analyzed by MS and Western blots. Among the proteins that were found to bind to the IGF-IR promoter we identified zinc finger transcription factors Sp1 and KLF6, ER-α, p53, c-jun, and poly (ADP-ribosylation) polymerase. Furthermore, chromatin immune-precipitation (ChIP) analysis confirmed the direct in vivo binding of some of these transcription factors to IGF-IR promoter DNA. The functional relevance of binding data was assessed by cotransfection experiments with specific expression vectors along with an IGF-IR promoter reporter. In summary, we identified nuclear proteins that are potentially responsible for the differential expression of the IGF-IR gene in ER-positive and ER-depleted breast cancer cells. PMID:24281069

  13. Retinoid- and sodium-butyrate-induced decrease in heat shock protein 70 membrane-positive tumor cells is associated with reduced sensitivity to natural killer cell lysis, growth delay, and altered growth morphology.

    PubMed

    Gehrmann, Mathias; Schönberger, Johann; Zilch, Tanja; Rossbacher, Lydia; Thonigs, Gerald; Eilles, Christoph; Multhoff, Gabriele

    2005-01-01

    Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX-) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 microM, 1 microM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX- tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology. PMID:16038410

  14. Retinoid- and sodium-butyrate– induced decrease in heat shock protein 70 membrane-positive tumor cells is associated with reduced sensitivity to natural killer cell lysis, growth delay, and altered growth morphology

    PubMed Central

    Gehrmann, Mathias; Schönberger, Johann; Zilch, Tanja; Rossbacher, Lydia; Thonigs, Gerald; Eilles, Christoph; Multhoff, Gabriele

    2005-01-01

    Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX−) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 μM, 1 μM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX− tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology. PMID:16038410

  15. The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway

    PubMed Central

    Abdelhamed, Zakia A.; Natarajan, Subaashini; Wheway, Gabrielle; Inglehearn, Christopher F.; Toomes, Carmel; Johnson, Colin A.; Jagger, Daniel J.

    2015-01-01

    ABSTRACT Ciliopathies are a group of developmental disorders that manifest with multi-organ anomalies. Mutations in TMEM67 (MKS3) cause a range of human ciliopathies, including Meckel-Gruber and Joubert syndromes. In this study we describe multi-organ developmental abnormalities in the Tmem67tm1Dgen/H1 knockout mouse that closely resemble those seen in Wnt5a and Ror2 knockout mice. These include pulmonary hypoplasia, ventricular septal defects, shortening of the body longitudinal axis, limb abnormalities, and cochlear hair cell stereociliary bundle orientation and basal body/kinocilium positioning defects. The basal body/kinocilium complex was often uncoupled from the hair bundle, suggesting aberrant basal body migration, although planar cell polarity and apical planar asymmetry in the organ of Corti were normal. TMEM67 (meckelin) is essential for phosphorylation of the non-canonical Wnt receptor ROR2 (receptor-tyrosine-kinase-like orphan receptor 2) upon stimulation with Wnt5a-conditioned medium. ROR2 also colocalises and interacts with TMEM67 at the ciliary transition zone. Additionally, the extracellular N-terminal domain of TMEM67 preferentially binds to Wnt5a in an in vitro binding assay. Cultured lungs of Tmem67 mutant mice failed to respond to stimulation of epithelial branching morphogenesis by Wnt5a. Wnt5a also inhibited both the Shh and canonical Wnt/β-catenin signalling pathways in wild-type embryonic lung. Pulmonary hypoplasia phenotypes, including loss of correct epithelial branching morphogenesis and cell polarity, were rescued by stimulating the non-canonical Wnt pathway downstream of the Wnt5a-TMEM67-ROR2 axis by activating RhoA. We propose that TMEM67 is a receptor that has a main role in non-canonical Wnt signalling, mediated by Wnt5a and ROR2, and normally represses Shh signalling. Downstream therapeutic targeting of the Wnt5a-TMEM67-ROR2 axis might, therefore, reduce or prevent pulmonary hypoplasia in ciliopathies and other congenital

  16. Detection of ST772 Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (Bengal Bay clone) and ST22 S. aureus isolates with a genetic variant of elastin binding protein in Nepal

    PubMed Central

    Pokhrel, R.H.; Aung, M.S.; Thapa, B.; Chaudhary, R.; Mishra, S.K.; Kawaguchiya, M.; Urushibara, N.; Kobayashi, N.

    2016-01-01

    Genetic characteristics were analysed for recent clinical isolates of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA respectively) in Kathmandu, Nepal. MRSA isolates harbouring Panton-Valentine leukocidin (PVL) genes were classified into ST1, ST22 and ST88 with SCCmec-IV and ST772 with SCCmec-V (Bengal Bay clone), while PVL-positive MSSA into ST22, ST30 and ST772. ST22 isolates (PVL-positive MRSA and MSSA, PVL-negative MRSA) possessed a variant of elastin binding protein gene (ebpS) with an internal deletion of 180 bp, which was similar to that reported for ST121 S. aureus previously outside Nepal. Phylogenetic analysis indicated that the ebpS variant in ST22 might have occurred independently of ST121 strains. This is the first report of ST772 PVL-positive MRSA in Nepal and detection of the deletion variant of ebpS in ST22 S. aureus. PMID:27014464

  17. Smoking interacts with HLA-DRB1 shared epitope in the development of anti-citrullinated protein antibody-positive rheumatoid arthritis: results from the Malaysian Epidemiological Investigation of Rheumatoid Arthritis (MyEIRA)

    PubMed Central

    2012-01-01

    Introduction Rheumatoid arthritis (RA) is a multifactorial autoimmune disease in which genetic and environmental factors interact in the etiology. In this study, we investigated whether smoking and HLA-DRB1 shared-epitope (SE) alleles interact differently in the development of the two major subgroups of rheumatoid arthritis (RA), anti-citrullinated proteins antibody (ACPA)-positive and ACPA-negative disease, in a multiethnic population of Asian descent. Methods A case-control study comprising early diagnosed RA cases was carried out in Malaysia between 2005 and 2009. In total, 1,076 cases and 1,612 matched controls participated in the study. High-resolution HLA-DRB1 genotyping was performed for shared-epitope (SE) alleles. All participants answered a questionnaire on a broad range of issues, including smoking habits. The odds ratio (OR) of developing ACPA-positive and ACPA-negative disease was calculated for smoking and the presence of any SE alleles separately. Potential interaction between smoking history (defined as "ever" and "never" smoking) and HLA-DRB1 SE alleles also was calculated. Results In our multiethnic study, both the SE alleles and smoking were associated with an increased risk of developing ACPA-positive RA (OR SE alleles, 4.7; 95% confidence interval (CI), 3.6 to 6.2; OR smoking, 4.1; 95% CI, 1.9 to 9.2). SE-positive smokers had an odds ratio of ACPA-positive RA of 25.6 (95% CI, 10.4 to 63.4), compared with SE-negative never-smokers. The interaction between smoking and SE alleles was significant (attributable proportion due to interaction (AP) was 0.7 (95% CI, 0.5 to 1.0)). The HLA-DRB1*04:05 SE allele, which is common in Asian populations, but not among Caucasians, was associated with an increased risk of ACPA-positive RA, and this allele also showed signs of interaction with smoking (AP, 0.4; 95% CI, -0.1 to 0.9). Neither smoking nor SE alleles nor their combination was associated with an increased risk of ACPA-negative RA. Conclusions The risk

  18. Fad24, a Positive Regulator of Adipogenesis, Is Required for S Phase Re-entry of C2C12 Myoblasts Arrested in G0 Phase and Involved in p27(Kip1) Expression at the Protein Level.

    PubMed

    Ochiai, Natsuki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2016-05-01

    Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level. PMID:26902224

  19. Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

    PubMed

    Ido, Yasuo

    2016-07-01

    Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3. PMID:27181414

  20. Dietary Magnesium Is Positively Associated With Skeletal Muscle Power and Indices of Muscle Mass and May Attenuate the Association Between Circulating C-Reactive Protein and Muscle Mass in Women.

    PubMed

    Welch, Ailsa A; Kelaiditi, Eirini; Jennings, Amy; Steves, Claire J; Spector, Tim D; MacGregor, Alexander

    2016-02-01

    Age-related loss of skeletal muscle mass and strength are risk factors for sarcopenia, osteoporosis, falls, fractures, frailty, and mortality. Dietary magnesium (Mg) could play a role in prevention of age-related loss of skeletal muscle mass, power, and strength directly through physiological mechanisms or indirectly through an impact on chronic low-grade inflammation, itself a risk factor for loss of skeletal muscle mass and strength. In a cross-sectional study of 2570 women aged 18 to 79 years, we examined associations between intakes of Mg, estimated using a food-frequency questionnaire (FFQ), dual-energy X-ray absorptiometry (DXA)-derived measures of muscle mass (fat-free mass as a percentage of body weight [FFM%], fat-free mass index [FFMI, kg/m(2) ]), leg explosive power (LEP), and grip strength (n = 949 only). We also examined associations between circulating hs-CRP (C-reactive protein) and muscle mass and LEP, and explored the potential attenuation of these relationships by Mg. We compared our findings with those of age and protein intake. Endpoints were calculated by quintile of Mg and adjusted for relevant confounders. Significant positive associations were found between a higher Mg and indices of skeletal muscle mass and LEP, and also with hs-CRP, after adjustment for covariates. Contrasting extreme quintiles of Mg intake showed differences of 2.6% for FFM% (p trend < 0.001), 0.4 kg/m(2) for FFMI (p trend = 0.005), and 19.6 watts/kg for LEP (p trend < 0.001). Compared with protein, these positive associations were 7 times greater for FFM% and 2.5 times greater for LEP. We also found that higher hs-CRP was negatively associated with skeletal muscle mass and, in statistical modeling, that a higher dietary Mg attenuated this negative relationship by 6.5%, with greater attenuation in women older than 50 years. No association was found between Mg and grip strength. Our results suggest that dietary magnesium may aid conservation of age

  1. The molecular cloning and characterization of BM1P1 and BM1P2 proteins, putative positive transcription factors involved in barbiturate-mediated induction of the genes encoding cytochrome P450BM-1 of Bacillus megaterium.

    PubMed

    He, J S; Liang, Q; Fulco, A J

    1995-08-01

    Analysis of a 1.3-kilobase segment of 5'-flanking DNA from the barbiturate-inducible P450BM-1 gene (CYP106) of Bacillus megaterium revealed two open reading frames. One, BM1P1, encodes 98 amino acids and is located 267 base pairs upstream from the sequence encoding cytochrome P450BM-1 but in the opposite orientation. The second, BM1P2 (88 amino acids), is 892 base pairs upstream from the P450BM-1 coding sequence and in the same coding strand. The expression of BM1P1 and BM1P2 was strongly stimulated in cells grown in the presence of pentobarbital, and the BM1P1 gene product exerted positive control on expression of P450BM-1. When a 177-base pair fragment encompassing the overlapping promoter regions of the P450BM-1 and BM1P1 genes was used as a probe in DNA binding assays, the BM1P1 and BM1P2 gene products and Bm3R1 (the repressor protein regulating the barbiturate-mediated expression of P450BM-3) could bind individually, but the addition of BM1P1 or BM1P2 to a binding mixture containing Bm3R1 completely prevented the appearance of a Bm3R1 binding band. When a 208-base pair fragment containing a Barbie box sequence and located upstream of the 177-base pair fragment was used as a probe, only a Bm3R1 binding band was detected. Although neither BM1P1 and BM1P2 appeared to bind to this 208-base pair fragment, their presence strongly inhibited the binding of Bm3R1 to the same probe. The evidence suggests that BM1P1 and BM1P2 may, in part, act as positive regulatory proteins involved in the expression of the P450BM-1 gene by interfering with the binding of the repressor protein, Bm3R1, to the regulatory regions of P450BM-1. PMID:7629192

  2. The RNA Binding Motif Protein 15B (RBM15B/OTT3) Is a Functional Competitor of Serine-Arginine (SR) Proteins and Antagonizes the Positive Effect of the CDK11p110-Cyclin L2α Complex on Splicing*

    PubMed Central

    Loyer, Pascal; Busson, Adeline; Trembley, Janeen H.; Hyle, Judith; Grenet, Jose; Zhao, Wei; Ribault, Catherine; Montier, Tristan; Kidd, Vincent J.; Lahti, Jill M.

    2011-01-01

    Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11p110 binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11p110, cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11p110, cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing both in vitro and in vivo. PMID:21044963

  3. The RNA binding motif protein 15B (RBM15B/OTT3) is a functional competitor of serine-arginine (SR) proteins and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing.

    PubMed

    Loyer, Pascal; Busson, Adeline; Trembley, Janeen H; Hyle, Judith; Grenet, Jose; Zhao, Wei; Ribault, Catherine; Montier, Tristan; Kidd, Vincent J; Lahti, Jill M

    2011-01-01

    Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo. PMID:21044963

  4. Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution

    PubMed Central

    Yu, Xianxian; Duan, Xiaoshan; Zhang, Rui; Fu, Xuehao; Ye, Lingling; Kong, Hongzhi; Xu, Guixia; Shan, Hongyan

    2016-01-01

    AP1/FUL, SEP, AGL6, and FLC subfamily genes play important roles in flower development. The phylogenetic relationships among them, however, have been controversial, which impedes our understanding of the origin and functional divergence of these genes. One possible reason for the controversy may be the problems caused by changes in the exon-intron structure of genes, which, according to recent studies, may generate non-homologous sites and hamper the homology-based sequence alignment. In this study, we first performed exon-by-exon alignments of these and three outgroup subfamilies (SOC1, AG, and STK). Phylogenetic trees reconstructed based on these matrices show improved resolution and better congruence with species phylogeny. In the context of these phylogenies, we traced evolutionary changes of exon-intron structures in each subfamily. We found that structural changes have occurred frequently following gene duplication and speciation events. Notably, exons 7 and 8 (if present) suffered more structural changes than others. With the knowledge of exon-intron structural changes, we generated more reasonable alignments containing all the focal subfamilies. The resulting trees showed that the SEP subfamily is sister to the monophyletic group formed by AP1/FUL and FLC subfamily genes and that the AGL6 subfamily forms a sister group to the three abovementioned subfamilies. Based on this topology, we inferred the evolutionary history of exon-intron structural changes among different subfamilies. Particularly, we found that the eighth exon originated before the divergence of AP1/FUL, FLC, SEP, and AGL6 subfamilies and degenerated in the ancestral FLC-like gene. These results provide new insights into the origin and evolution of the AP1/FUL, FLC, SEP, and AGL6 subfamilies. PMID:27200066

  5. Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators

    NASA Technical Reports Server (NTRS)

    Belaguli, N. S.; Sepulveda, J. L.; Nigam, V.; Charron, F.; Nemer, M.; Schwartz, R. J.

    2000-01-01

    Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

  6. A novel intronic splice site deletion of the IL-2 receptor common gamma chain results in expression of a dysfunctional protein and T-cell-positive X-linked Severe combined immunodeficiency.

    PubMed

    Gray, P E A; Logan, G J; Alexander, I E; Poulton, S; Roscioli, T; Ziegler, J

    2015-02-01

    X-linked severe combined immunodeficiency is caused by mutations in the IL-2 receptor common gamma chain and classically presents in the first 6 months of life with predisposition to bacterial, viral and fungal infections. In most instances, affected individuals are lymphopenic with near complete absence of T cells and NK cells. We report a boy who presented at 12 months of age with Pneumocystis jiroveci pneumonia and a family history consistent with X-linked recessive inheritance. He had a normal lymphocyte count including the presence of T cells and a broad T-cell-receptor diversity, as well as normal surface expression of the common gamma chain (CD132) protein. He however had profound hypogammaglobulinaemia, and IL-2-induced STAT5 phosphorylation was absent. Sequencing of IL-2RG demonstrated a 12-base pair intronic deletion close to the canonical splice site of exon 5, which resulted in a variety of truncated IL2RG mRNA species. A review of the literature identified 4 other patients with T-cell-positive X-SCID, with the current patient being the first associated with an mRNA splicing defect. This case raises the question of how a dysfunctional protein incapable of mediating STAT5 phosphorylation might nonetheless support T-cell development. Possible explanations are that STAT5-mediated signal transduction may be less relevant to IL7-receptor-mediated T-cell development than are other IL7R-induced intracellular transduction pathways or that a low level of STAT5 phosphorylation, undetectable in the laboratory, may be sufficient to support some T-cell development. PMID:25443657

  7. Calcium-dependent protein kinase CPK6 positively functions in induction by yeast elicitor of stomatal closure and inhibition by yeast elicitor of light-induced stomatal opening in Arabidopsis.

    PubMed

    Ye, Wenxiu; Muroyama, Daichi; Munemasa, Shintaro; Nakamura, Yoshimasa; Mori, Izumi C; Murata, Yoshiyuki

    2013-10-01

    Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca(2+)-dependent signaling pathway. A Ca(2+)-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca(2+)-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca(2+) concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K(+) channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress. PMID:23922271

  8. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

    PubMed

    Solberg, L B; Stang, E; Brorson, S-H; Andersson, G; Reinholt, F P

    2015-02-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis. PMID:25201349

  9. Aptamer BC007 for neutralization of pathogenic autoantibodies directed against G-protein coupled receptors: A vision of future treatment of patients with cardiomyopathies and positivity for those autoantibodies.

    PubMed

    Wallukat, Gerd; Müller, Johannes; Haberland, Annekathrin; Berg, Sabine; Schulz, Angela; Freyse, Ernst-Joachim; Vetter, Roland; Salzsieder, Eckhard; Kreutz, Reinhold; Schimke, Ingolf

    2016-01-01

    Cardiomyopathies such as idiopathic dilated cardiomyopathy (DCM), Chagas' cardiomyopathy and Peripartum cardiomyopathy present with autoantibodies against G-protein coupled receptors (GPCR-AABs) that agonistically activate their receptors. For the treatment of "agonistic autoantibody diseases" and in particular DCM, the removal of the GPCR-AABs by immunoadsorption (IA) has been studied with convincing patient benefit. To overcome cost and logistics problems of IA, the application of the aptamer BC007 for in vivo neutralization of GPCR-AABs could help. We demonstrate here, that the aptamer neutralized, in vitro, the presently known cardiovascular-pathogenic GPCR-AABs. In spontaneously hypertensive rats, the aptamer demonstrated its GPCR-AAB neutralizing potency in vivo. In the serum of DCM patients, the same GPCR-AAB reduction was achieved when patients were either immunoadsorbed or patient's serum was ex vivo treated with the aptamer. In our view, aptamer BC007 treatment in GPCR-AAB-positive patients would have a comparable benefit as that seen after IA. Not knowing all that interfering with our idea of aptamer-dependent neutralization of GPCR-AABs, the first preliminary steps have been taken for bringing the idea closer to patients. PMID:26584137

  10. Amyloid precursor protein cooperates with c-KIT mutation/overexpression to regulate cell apoptosis in AML1-ETO-positive leukemia via the PI3K/AKT signaling pathway.

    PubMed

    Yu, Guopan; Yin, Changxin; Jiang, Ling; Zheng, Zhongxin; Wang, Zhixiang; Wang, Chunli; Zhou, Hongsheng; Jiang, Xuejie; Liu, Qifa; Meng, Fanyi

    2016-09-01

    It has been reported that amyloid precursor protein (APP) promotes cell proliferation and metastasis in various types of solid cancers. In our previous study, we showed that APP is highly expressed and regulates leukemia cell migration in AML1‑ETO-positive (AE) leukemia. Whether APP is involved in the regulation of AE leukemia cell proliferation or apoptosis is unclear. In the present study we focused on the correlation of APP with c-KIT mutation/overexpression and cell proliferation and apoptosis in AE leukemia. APP and c-KIT expression detected by quantitative real-time (qPCR) method, and c-KIT mutations screened using PCR in bone marrow cells from 65 patients with AE leukemia before their first chemotherapy, were simultaneously assessed. Furthermore, the Kasumi-1 cell line was chosen as the cell model, and the APP gene was knocked down using siRNA technology. The correlation of cell cycle distribution and apoptosis and c-Kit expression with APP expression levels, as well as the regulation of the PI3K/AKT signaling pathway by APP were analyzed in the Kasumi-1 cell line. The results showed that peripheral white blood cell counts (P=0.008) and bone marrow cellularity (P=0.031), but not bone marrow blasts, were correlated with APP expression. Moreover, the patients with APP high expression had a significantly higher incidence of c-KIT mutations (P<0.001) and increased levels of c-KIT expression (P=0.001) and poorer disease outcome. In the Kasumi-1 cell line, as compared with the wild-type and negative control cells, cell apoptosis, both early (P<0.001) and late (P<0.001), was significantly increased when the APP gene was knocked down, concomitant with reduced levels of anti-apoptotic protein Bcl-2 and increased levels of caspase-3 and -9, however, no apparent change was observed in the cell cycle distribution (P>0.05). Moreover, the knockdown of APP markedly decreased c-KIT expression at both the transcription (as evidenced by qPCR analysis) and translation

  11. Life Course Socioeconomic Position and C-Reactive Protein: Mediating Role of Health-Risk Behaviors and Metabolic Alterations. The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)

    PubMed Central

    Camelo, Lidyane V.; Giatti, Luana; Neves, Jorge Alexandre Barbosa; Lotufo, Paulo A.; Benseñor, Isabela M.; Chor, Dóra; Griep, Rosane Härter; da Fonseca, Maria de Jesus Mendes; Vidigal, Pedro Guatimosim; Kawachi, Ichiro; Schmidt, Maria Inês; Barreto, Sandhi Maria

    2014-01-01

    Background Chronic inflammation has been postulated to be one mediating mechanism explaining the association between low socioeconomic position (SEP) and cardiovascular disease (CVD). We sought to examine the association between life course SEP and C-reactive protein (CRP) levels in adulthood, and to evaluate the extent to which health-risk behaviors and metabolic alterations mediate this association. Additionally, we explored the possible modifying influence of gender. Methods and Findings Our analytical sample comprised 13,371 participants from ELSA-Brasil baseline, a multicenter prospective cohort study of civil servants. SEP during childhood, young adulthood, and adulthood were considered. The potential mediators between life course SEP and CRP included clusters of health-risk behaviors (smoking, low leisure time physical activity, excessive alcohol consumption), and metabolic alterations (obesity, hypertension, low HDL, hypertriglyceridemia, and diabetes). Linear regression models were performed and structural equation modeling was used to evaluate mediation. Although lower childhood SEP was associated with higher levels of CRP in adult life, this association was not independent of adulthood SEP. However, CRP increased linearly with increasing number of unfavorable social circumstances during the life course (p trend <0.001). The metabolic alterations were the most important mediator between cumulative SEP and CRP. This mediation path accounted for 49.5% of the total effect of cumulative SEP on CRP among women, but only 20.2% among men. In consequence, the portion of the total effect of cumulative SEP on CRP that was mediated by risk behaviors and metabolic alterations was higher among women (55.4%) than among men (36.8%). Conclusions Cumulative SEP across life span was associated with elevated systemic inflammation in adulthood. Although health-risk behaviors and metabolic alterations were important mediators of this association, a sizable fraction of this

  12. A phase I/II trial of the safety and clinical activity of a HER2-protein based immunotherapeutic for treating women with HER2-positive metastatic breast cancer.

    PubMed

    Curigliano, Giuseppe; Romieu, Gilles; Campone, Mario; Dorval, Thierry; Duck, Lionel; Canon, Jean-Luc; Roemer-Becuwe, Celia; Roselli, Mario; Neciosup, Silvia; Burny, Wivine; Callegaro, Andrea; de Sousa Alves, Pedro Miguel; Louahed, Jamila; Brichard, Vincent; Lehmann, Frédéric F

    2016-04-01

    The objectives of this phase I/II study (NCT00140738) were to evaluate the safety and clinical activity of a cancer immunotherapeutic agent (recombinant HER2 protein (dHER2) and the immunostimulant AS15) in patients with HER2-overexpressing metastatic breast cancer (MBC). Forty HER2-positive MBC patients received up to 18 doses (12q2w, 6q3w) of dHER2 immunotherapeutic, as first- or second-line therapy following response to trastuzumab-based treatment as maintenance. Toxicity was graded by the Common Terminology Criteria for Adverse Events (CTCAE) and clinical activity was evaluated by target lesion assessment according to the Response Evaluation Criteria in Solid Tumors (RECIST). Immunogenicity was assessed. The dHER2 immunotherapeutic was well tolerated: grade 1/2 adverse events (AEs) were most common. No cardiac events were observed and one patient experienced an asymptomatic decrease of left ventricular ejection fraction below the normal range (47 %). Both humoral and cellular immunogenicity to the dHER2 antigen was observed. No patient discontinued the immunizations because of AEs but 35/40 withdrew prematurely, 34 because of disease progression (24/34 before or at the tumor assessment after dose 6). One patient achieved a complete response lasting 11 months and one patient had a partial response lasting 3.5 months. Ten patients experienced stable disease ≥26 weeks with 4/10 still in stable disease at the last tumor assessment after 47 weeks. Immunization of MBC patients with the dHER2 immunotherapeutic was associated with minimal toxicity and no cardiac events. Clinical activity was observed with two objective responses and prolonged stable disease for 10/40 patients. PMID:26975189

  13. Positive position control of robotic manipulators

    NASA Technical Reports Server (NTRS)

    Baz, A.; Gumusel, L.

    1989-01-01

    The present, simple and accurate position-control algorithm, which is applicable to fast-moving and lightly damped robot arms, is based on the positive position feedback (PPF) strategy and relies solely on position sensors to monitor joint angles of robotic arms to furnish stable position control. The optimized tuned filters, in the form of a set of difference equations, manipulate position signals for robotic system performance. Attention is given to comparisons between this PPF-algorithm controller's experimentally ascertained performance characteristics and those of a conventional proportional controller.

  14. Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili

    PubMed Central

    Futamura, Norihiro; Totoki, Yasushi; Toyoda, Atsushi; Igasaki, Tomohiro; Nanjo, Tokihiko; Seki, Motoaki; Sakaki, Yoshiyuki; Mari, Adriano; Shinozaki, Kazuo; Shinohara, Kenji

    2008-01-01

    Background Cryptomeria japonica D. Don is one of the most commercially important conifers in Japan. However, the allergic disease caused by its pollen is a severe public health problem in Japan. Since large-scale analysis of expressed sequence tags (ESTs) in the male strobili of C. japonica should help us to clarify the overall expression of genes during the process of pollen development, we constructed a full-length enriched cDNA library that was derived from male strobili at various developmental stages. Results We obtained 36,011 expressed sequence tags (ESTs) from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. The 19,437 cDNA clones corresponded to 10,463 transcripts. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis. An analysis of homologies between ESTs from C. japonica male strobili and known pollen allergens in the Allergome Database revealed that products of 180 transcripts exhibited significant homology. Approximately 2% of the transcripts appeared to encode transcription factors. We identified twelve genes for MADS-box proteins among these transcription factors. The twelve MADS-box genes were classified as DEF/GLO/GGM13-, AG-, AGL6-, TM3- and TM8-like MIKCC genes and type I MADS-box genes. Conclusion Our full-length enriched cDNA library derived from C. japonica male strobili provides information on expression of genes during the development of male reproductive organs. We provided potential allergens in C. japonica. We also provided new information about transcription factors including MADS-box genes expressed in male strobili of C. japonica. Large-scale gene discovery using full-length cDNAs is a valuable tool for studies of gymnosperm species. PMID:18691438

  15. Functional characterization of GhSOC1 and GhMADS42 homologs from upland cotton (Gossypium hirsutum L.).

    PubMed

    Zhang, Xiaohong; Wei, Jianghui; Fan, Shuli; Song, Meizhen; Pang, Chaoyou; Wei, Hengling; Wang, Chengshe; Yu, Shuxun

    2016-01-01

    In Arabidopsis flowering pathway, MADS-box genes encode transcription factors, with their structures and functions highly conserved in many species. In our study, two MADS-box genes GhSOC1 and GhMADS42 (Gossypium hirsutum L.) were cloned from upland cotton CCRI36 and transformed into Arabidopsis. GhSOC1 was additionally transformed into upland cotton. Comparative analysis demonstrated sequence conservation between GhSOC1 and GhMADS42 and genes of other plant species. Tissue-specific expression analysis of GhSOC1 and GhMADS42 revealed spatiotemporal expression patterns involving high transcript levels in leaves, shoot apical buds, and flowers. In addition, overexpression of both GhSOC1 and GhMADS42 in Arabidopsis accelerated flowering, with GhMADS42 transgenic plants showing abnormal floral organ phenotypes. Overexpression of GhSOC1 in upland cotton also produced variations in floral organs. Furthermore, chromatin immunoprecipitation assay demonstrated that GhSOC1 could regulate GhMADS41 and GhMADS42, but not FLOWERING LOCUS T, by directly binding to the genes promoter. Finally, yeast two-hybrid and bimolecular fluorescence complementation approaches were undertaken to better understand the interaction of GhSOC1 and other MADS-box factors. These experiments showed that GhSOC1 can interact with APETALA1/FRUITFULL-like proteins in cotton. PMID:26566835

  16. Positional Plagiocephaly (Flattened Head)

    MedlinePlus

    ... Simple practices like changing a baby's sleep position, holding your baby, and providing lots of "tummy time" ... devices to keep your baby in one position. Alternate positions in the crib. Consider how you lay ...

  17. Want Positive Behavior? Use Positive Language

    ERIC Educational Resources Information Center

    Wood, Chip; Freeman-Loftis, Babs

    2012-01-01

    Positive adult language is the professional use of words and tone of voice to enable students to learn in an engaged, active way. This includes learning social skills. To guide children toward choosing and maintaining positive behaviors, adults need to carefully choose the words and tone of voice used when speaking to them. Learning to use…

  18. Positive Classroom Environments = Positive Academic Results

    ERIC Educational Resources Information Center

    Wilson-Fleming, LaTerra; Wilson-Younger, Dylinda

    2012-01-01

    This article discusses the effects of a positive classroom environment and its impact on student behavior and achievement. It also provides strategies for developing expectations for student achievement and the importance of parental involvement. A positive classroom environment is essential in keeping behavior problems to a minimum. There are a…

  19. Positive Education: Positive Psychology and Classroom Interventions

    ERIC Educational Resources Information Center

    Seligman, Martin E. P.; Ernst, Randal M.; Gillham, Jane; Reivich, Karen; Linkins, Mark

    2009-01-01

    Positive education is defined as education for both traditional skills and for happiness. The high prevalence worldwide of depression among young people, the small rise in life satisfaction, and the synergy between learning and positive emotion all argue that the skills for happiness should be taught in school. There is substantial evidence from…

  20. A Cluster of Ring Stage–specific Genes Linked to a Locus Implicated in Cytoadherence in Plasmodium falciparum Codes for PEXEL-negative and PEXEL-positive Proteins Exported into the Host Cell

    PubMed Central

    Hawthorne, Paula L.; Dixon, Matthew W.A.; Hannemann, Mandy; Klotz, Kathleen; Kemp, David J.; Klonis, Nectarios; Tilley, Leann; Trenholme, Katharine R.

    2006-01-01

    Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasite-induced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures. PMID:16760427

  1. Transformation of gram positive bacteria by sonoporation

    DOEpatents

    Yang, Yunfeng; Li, Yongchao

    2014-03-11

    The present invention provides a sonoporation-based method that can be universally applied for delivery of compounds into Gram positive bacteria. Gram positive bacteria which can be transformed by sonoporation include, for example, Bacillus, Streptococcus, Acetobacterium, and Clostridium. Compounds which can be delivered into Gram positive bacteria via sonoporation include nucleic acids (DNA or RNA), proteins, lipids, carbohydrates, viruses, small organic and inorganic molecules, and nano-particles.

  2. Evolutionarily Conserved Regulatory Motifs in the Promoter of the Arabidopsis Clock Gene LATE ELONGATED HYPOCOTYL[C][W

    PubMed Central

    Spensley, Mark; Kim, Jae-Yean; Picot, Emma; Reid, John; Ott, Sascha; Helliwell, Chris; Carré, Isabelle A.

    2009-01-01

    The transcriptional regulation of the LATE ELONGATED HYPOCOTYL (LHY) gene is key to the structure of the circadian oscillator, integrating information from multiple regulatory pathways. We identified a minimal region of the LHY promoter that was sufficient for rhythmic expression. Another upstream sequence was also required for appropriate waveform of transcription and for maximum amplitude of oscillations under both diurnal and free-running conditions. We showed that two classes of protein complexes interact with a G-box and with novel 5A motifs; mutation of these sites reduced the amplitude of oscillation and broadened the peak of expression. A genome-wide bioinformatic analysis showed that these sites were enriched in phase-specific clusters of rhythmically expressed genes. Comparative genomic analyses showed that these motifs were conserved in orthologous promoters from several species. A position-specific scoring matrix for the 5A sites suggested similarity to CArG boxes, which are recognized by MADS box transcription factors. In support of this, the FLOWERING LOCUS C (FLC) protein was shown to interact with the LHY promoter in planta. This suggests a mechanism by which FLC might affect circadian period. PMID:19789276

  3. Many or most genes in Arabidopsis transposed after the origin of the order Brassicales

    PubMed Central

    Freeling, Michael; Lyons, Eric; Pedersen, Brent; Alam, Maqsudul; Ming, Ray; Lisch, Damon

    2008-01-01

    Previous to this work, typical genes were thought to move from one position to another infrequently. On the contrary, we now estimate that between one-fourth and three-fourths of the genes in Arabidopsis transposed in the Brassicales. We used the CoGe comparative genomics system to perform and visualize multiple orthologous chromosomal alignments. Using this tool, we found large differences between different categories of genes. Ten of the gene families examined, including genes in most transcription factor families, exhibited a median frequency of 5% transposed genes. In contrast, other gene families were composed largely of transposed genes: NB-LRR disease-resistance genes, genes encoding MADS-box and B3 transcription factors, and genes encoding F-box proteins. A unique method involving transposition-rich regions of genome allowed us to obtain an indirect estimate of the positional stability of the average gene. The observed differences between gene families raise important questions concerning the causes and consequences of gene transposition. PMID:18836034

  4. The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression

    PubMed Central

    Garay-Arroyo, Adriana; Ortiz-Moreno, Enrique; de la Paz Sánchez, María; Murphy, Angus S; García-Ponce, Berenice; Marsch-Martínez, Nayelli; de Folter, Stefan; Corvera-Poiré, Adriana; Jaimes-Miranda, Fabiola; Pacheco-Escobedo, Mario A; Dubrovsky, Joseph G; Pelaz, Soraya; Álvarez-Buylla, Elena R

    2013-01-01

    Elucidating molecular links between cell-fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell-fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem-cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS-box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem-cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS-domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4-dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning. PMID:24121311

  5. Adenomatous polyposis coli (APC) membrane recruitment 3, a member of the APC membrane recruitment family of APC-binding proteins, is a positive regulator of Wnt-β-catenin signalling.

    PubMed

    Brauburger, Katharina; Akyildiz, Senem; Ruppert, Jan G; Graeb, Michael; Bernkopf, Dominic B; Hadjihannas, Michel V; Behrens, Jürgen

    2014-02-01

    The adenomatous polyposis coli (APC) membrane recruitment (Amer) family proteins Amer1/Wilms tumour gene on the X chromosome and Amer2 are binding partners of the APC tumour suppressor protein, and act as negative regulators in the Wnt signalling cascade. So far, nothing has been known about the third member of the family, Amer3. Here we show that Amer3 binds to the armadillo repeat domain of APC, similarly to Amer1 and Amer2. Amer3 also binds to the Wnt pathway regulator conductin/axin2. Furthermore, we identified Amer1 as binding partner of Amer3. Whereas Amer1 and Amer2 are linked to the plasma membrane by an N-terminal membrane localization domain, Amer3 lacks this domain. Amer3 localizes to the cytoplasm and nucleus of epithelial cells, and this is dependent on specific nuclear import and export sequences. Functionally, exogenous Amer3 enhances the expression of a β-catenin/T-cell factor-dependent reporter gene, and knockdown of endogenous Amer3 reduces Wnt target gene expression in colorectal cancer cells. Thus, Amer3 acts as an activator of Wnt signalling, in contrast to Amer1 and Amer2, which are inhibitors, suggesting a nonredundant role of Amer proteins in the regulation of this pathway. Our data, together with those of previous studies, provide a comprehensive picture of similarities and differences within the Amer protein family. PMID:24251807

  6. Sharing a Faculty Position.

    ERIC Educational Resources Information Center

    O'Kane, Patricia K.; Meyer, Mary

    1982-01-01

    Describes the experience of two nursing faculty members who shared an assistant professor of nursing position. Discusses positive and negative aspects of the experience and notes that a unified and creative approach must be taken for it to succeed. (JOW)

  7. Positive battery plate

    NASA Technical Reports Server (NTRS)

    Rowlette, John R. (Inventor)

    1985-01-01

    The power characteristics of a lead acid battery are improved by incorporating a dispersion of 1 to 10% by weight of a thermodynamically stable conductivity additive, such as conductive tin oxide coated glass fibers (34) of filamentary glass wool (42) in the positive active layer (32) carried on the grid (30) of the positive plate (16). Positive plate potential must be kept high enough to prevent reduction of the tin oxide to tin by utilizing an oversized, precharged positive paste.

  8. The Positivity Scale

    ERIC Educational Resources Information Center

    Caprara, Gian Vittorio; Alessandri, Guido; Eisenberg, Nancy; Kupfer, A.; Steca, Patrizia; Caprara, Maria Giovanna; Yamaguchi, Susumu; Fukuzawa, Ai; Abela, John

    2012-01-01

    Five studies document the validity of a new 8-item scale designed to measure "positivity," defined as the tendency to view life and experiences with a positive outlook. In the first study (N = 372), the psychometric properties of Positivity Scale (P Scale) were examined in accordance with classical test theory using a large number of college…

  9. Prone positioning for surgery.

    PubMed

    Bowers, Mark

    2012-05-01

    The role of the registered perioperative practitioner (Operating Department Practitioner or Registered Nurse) includes the responsibility for safely positioning patients for surgery. The prone position is in common use for a variety of surgical procedures. The formal term for this surgical position is ventral decubitus (meaning laying face down). PMID:22720505

  10. Non-covalent association of protein and capsular polysaccharide on bacteria-sized latex beads as a model for polysaccharide-specific humoral immunity to intact Gram-positive extracellular bacteria1

    PubMed Central

    Colino, Jesus; Duke, Leah; Snapper, Clifford M.

    2013-01-01

    Intact Streptococcus pneumoniae, expressing type 14 capsular polysaccharide (PPS14) and type III Streptococcus agalactiae containing a PPS14 core capsule identical to PPS14, exhibit non-covalent associations of PPS14 and bacterial protein, in contrast to soluble covalent conjugates of these respective antigens. Both bacteria and conjugates induce murine PPS14-specific IgG responses dependent on CD4+ T cells. Further, secondary immunization with conjugate and S. agalactiae, although not S. pneumoniae, results in a boosted response. However, in contrast to conjugate, PPS14-specific IgG responses to bacteria lack affinity maturation, utilize the 44.1-idiotype and are dependent on marginal zone B cells. To better understand the mechanism underlying this dichotomy we developed a minimal model of intact bacteria in which PPS14 and pneumococcal surface protein A (PspA) were stably attached to 1 μm (bacteria-sized) latex beads, but not directly linked to each other, in contrast to PPS14-PspA conjugate. PPS14+[PspA] beads, similar to conjugate, induced in mice boosted PPS14-specific IgG secondary responses, dependent on T cells and ICOS-dependent costimulation, and in which priming could be achieved with PspA alone. In contrast to conjugate, but similar to intact bacteria, the primary PPS14-specific IgG response to PPS14+[PspA] beads peaked rapidly, with the secondary response highly enriched for the 44.1-idiotype and lacking affinity maturation. These results demonstrate that non-covalent association in a particle, of polysaccharide and protein, recapitulates essential immunologic characteristics of intact bacteria that are distinct from soluble covalent conjugates of these respective antigens. PMID:23926322

  11. Beam position monitor

    DOEpatents

    Alkire, Randy W.; Rosenbaum, Gerold; Evans, Gwyndaf

    2003-07-22

    An apparatus for determining the position of an x-ray beam relative to a desired beam axis. Where the apparatus is positioned along the beam path so that a thin metal foil target intersects the x-ray beam generating fluorescent radiation. A PIN diode array is positioned so that a portion of the fluorescent radiation is intercepted by the array resulting in an a series of electrical signals from the PIN diodes making up the array. The signals are then analyzed and the position of the x-ray beam is determined relative to the desired beam path.

  12. Interval polynomial positivity

    NASA Technical Reports Server (NTRS)

    Bose, N. K.; Kim, K. D.

    1989-01-01

    It is shown that a univariate interval polynomial is globally positive if and only if two extreme polynomials are globally positive. It is shown that the global positivity property of a bivariate interval polynomial is completely determined by four extreme bivariate polynomials. The cardinality of the determining set for k-variate interval polynomials is 2k. One of many possible generalizations, where vertex implication for global positivity holds, is made by considering the parameter space to be the set dual of a boxed domain.

  13. The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs

    PubMed Central

    Borja, Lairton Souza; Tuy, Pétala Gardênia da Silva Estrela; Bastos, Leila Andrade; Solcà, Manuela da Silva; Amorim, Leila Denise Alves Ferreira; Veras, Patrícia Sampaio Tavares

    2016-01-01

    Visceral Leishmaniasis (VL) has spread to many urban centers worldwide. Dogs are considered the main reservoir of VL, because canine cases often precede the occurrence of human cases. Detection and euthanasia of serologically positive dogs is one of the primary VL control measures utilized in some countries, including Brazil. Using accurate diagnostic tests can minimize one undesirable consequence of this measure, culling false-positive dogs, and reduce the maintenance of false-negative dogs in endemic areas. In December 2011, the Brazilian Ministry of Health replaced the ELISA (EIE CVL) screening method and Indirect Immunofluorescence Test (IFI CVL) confirmatory method with a new protocol using the rapid DPP CVL screening test and EIE CVL confirmatory test. A study of diagnostic accuracy of these two protocols was done by comparing their performance using serum samples collected from a random sample of 780 dogs in an endemic area of VL. All samples were evaluated by culture and real time PCR; 766 out of the 780 dogs were tested using the previous protocol (IFI CVL + EIE CVL) and all 780 were tested using the current protocol (DPP CVL + EIE CVL). Performances of both diagnostic protocols were evaluated using a latent class variable as the gold standard. The current protocol had a higher specificity (0.98 vs. 0.95) and PPV (0.83 vs. 0.70) than the previous protocol, although sensitivity of these two protocols was similar (0.73). When tested using sera from asymptomatic animals, the current protocol had a much higher PPV (0.63 vs. 0.40) than the previous protocol (although the sensitivity of either protocol was the same, 0.71). Considering a range of theoretical CVL prevalences, the projected PPVs were higher for the current protocol than for the previous protocol for each theoretical prevalence value. The findings presented herein show that the current protocol performed better than previous protocol primarily by reducing false-positive results. PMID:26731098

  14. The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs.

    PubMed

    Fraga, Deborah Bittencourt Mothé; Pacheco, Luciano Vasconcellos; Borja, Lairton Souza; Tuy, Pétala Gardênia da Silva Estrela; Bastos, Leila Andrade; Solcà, Manuela da Silva; Amorim, Leila Denise Alves Ferreira; Veras, Patrícia Sampaio Tavares

    2016-01-01

    Visceral Leishmaniasis (VL) has spread to many urban centers worldwide. Dogs are considered the main reservoir of VL, because canine cases often precede the occurrence of human cases. Detection and euthanasia of serologically positive dogs is one of the primary VL control measures utilized in some countries, including Brazil. Using accurate diagnostic tests can minimize one undesirable consequence of this measure, culling false-positive dogs, and reduce the maintenance of false-negative dogs in endemic areas. In December 2011, the Brazilian Ministry of Health replaced the ELISA (EIE CVL) screening method and Indirect Immunofluorescence Test (IFI CVL) confirmatory method with a new protocol using the rapid DPP CVL screening test and EIE CVL confirmatory test. A study of diagnostic accuracy of these two protocols was done by comparing their performance using serum samples collected from a random sample of 780 dogs in an endemic area of VL. All samples were evaluated by culture and real time PCR; 766 out of the 780 dogs were tested using the previous protocol (IFI CVL + EIE CVL) and all 780 were tested using the current protocol (DPP CVL + EIE CVL). Performances of both diagnostic protocols were evaluated using a latent class variable as the gold standard. The current protocol had a higher specificity (0.98 vs. 0.95) and PPV (0.83 vs. 0.70) than the previous protocol, although sensitivity of these two protocols was similar (0.73). When tested using sera from asymptomatic animals, the current protocol had a much higher PPV (0.63 vs. 0.40) than the previous protocol (although the sensitivity of either protocol was the same, 0.71). Considering a range of theoretical CVL prevalences, the projected PPVs were higher for the current protocol than for the previous protocol for each theoretical prevalence value. The findings presented herein show that the current protocol performed better than previous protocol primarily by reducing false-positive results. PMID:26731098

  15. Nuclear envelope: positioning nuclei and organizing synapses

    PubMed Central

    Razafsky, David; Hodzic, Didier

    2015-01-01

    The nuclear envelope plays an essential role in nuclear positioning within cells and tissues. This review highlights advances in understanding the mechanisms of nuclear positioning during skeletal muscle and central nervous system development. New findings, particularly about Atype lamins and Nesprin1, may link nuclear envelope integrity to synaptic integrity. Thus synaptic defects, rather than nuclear mispositioning, may underlie human pathologies associated with mutations of nuclear envelope proteins. PMID:26079712

  16. "Positive Discipline": Beginnings Workshop.

    ERIC Educational Resources Information Center

    Stephens, Karen; And Others

    1992-01-01

    Four articles in this special section discuss (1) ideas for making discipline a positive experience for child care staff and children (Karen Stephens); (2) scenarios illustrating the power of adult messages to children (Karen Stephens); (3) classroom environments that facilitate positive discipline (Marjorie K. Kostelnik); and (4) strategies for…

  17. Positioning and locking apparatus

    DOEpatents

    Hayward, M.L.; Harper, W.H.

    1985-06-19

    A positioning and locking apparatus including a fixture having a rotatable torque ring provided with a plurality of cam segments for automatically guiding a container into a desired location within the fixture. Rotation of the ring turns the container into a final position in pressure sealing relation against a hatch member.

  18. Positioning and locking apparatus

    DOEpatents

    Hayward, M.L.; Harper, W.H.

    1987-06-30

    A positioning and locking apparatus are disclosed including a fixture having a rotatable torque ring provided with a plurality of cam segments for automatically guiding a container into a desired location within the fixture. Rotation of the ring turns the container into a final position in pressure sealing relation against a hatch member. 6 figs.

  19. Positioning and locking apparatus

    DOEpatents

    Hayward, Milton L.; Harper, William H.

    1987-01-01

    A positioning and locking apparatus including a fixture having a rotatable torque ring provided with a plurality of cam segments for automatically guiding a container into a desired location within the fixture. Rotation of the ring turns the container into a final position in pressure sealing relation against a hatch member.

  20. Target Genes of the MADS Transcription Factor SEPALLATA3: Integration of Developmental and Hormonal Pathways in the Arabidopsis Flower

    PubMed Central

    Kaufmann, Kerstin; Muiño, Jose M; Jauregui, Ruy; Airoldi, Chiara A; Smaczniak, Cezary; Krajewski, Pawel; Angenent, Gerco C

    2009-01-01

    The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals, and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs. PMID:19385720

  1. The Structure of the RLIP76 RhoGAP-Ral Binding Domain Dyad: Fixed Position of the Domains Leads to Dual Engagement of Small G Proteins at the Membrane

    PubMed Central

    Rajasekar, Karthik V.; Campbell, Louise J.; Nietlispach, Daniel; Owen, Darerca; Mott, Helen R.

    2013-01-01

    Summary RLIP76 is an effector for Ral small GTPases, which in turn lie downstream of the master regulator Ras. Evidence is growing that Ral and RLIP76 play a role in tumorigenesis, invasion, and metastasis. RLIP76 contains both a RhoGAP domain and a Ral binding domain (GBD) and is, therefore, a node between Ras and Rho family signaling. The structure of the RhoGAP-GBD dyad reveals that the RLIP76 RhoGAP domain adopts a canonical RhoGAP domain structure and that the linker between the two RLIP76 domains is structured, fixing the orientation of the two domains and allowing RLIP76 to interact with Rho-family GTPases and Ral simultaneously. However, the juxtaposed domains do not influence each other functionally, suggesting that the RLIP76-Ral interaction controls cellular localization and that the fixed orientation of the two domains orientates the RhoGAP domain with respect to the membrane, allowing it to be perfectly poised to engage its target G proteins. PMID:24207123

  2. CRANE POSITIONING APPARATUS

    DOEpatents

    Landsiedel, F.W.; Wolff, H.

    1960-06-28

    An apparatus is described for automatically accomplishing the final accurate horizontal positioning of a crane after the latter has been placed to within 1/8 in. of its selected position. For this purpose there is provided a tiltable member on the crane mast for lowering into contact with a stationary probe. Misalignment of the tiltable member, with respect to the probe as the member is lowered, causes tilting of the latter to actuate appropriate switches that energize motors for bringing the mast into proper position. When properly aligned the member is not tilted and a central switch is actuated to indicate the final alignment of the crane.

  3. Consensus protein design.

    PubMed

    Porebski, Benjamin T; Buckle, Ashley M

    2016-07-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering. PMID:27274091

  4. Consensus protein design

    PubMed Central

    Porebski, Benjamin T.; Buckle, Ashley M.

    2016-01-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino ac