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Sample records for basic helix-loop-helix proteins

  1. Phylogeny, Functional Annotation, and Protein Interaction Network Analyses of the Xenopus tropicalis Basic Helix-Loop-Helix Transcription Factors

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    Wuyi Liu

    2013-01-01

    Full Text Available The previous survey identified 70 basic helix-loop-helix (bHLH proteins, but it was proved to be incomplete, and the functional information and regulatory networks of frog bHLH transcription factors were not fully known. Therefore, we conducted an updated genome-wide survey in the Xenopus tropicalis genome project databases and identified 105 bHLH sequences. Among the retrieved 105 sequences, phylogenetic analyses revealed that 103 bHLH proteins belonged to 43 families or subfamilies with 46, 26, 11, 3, 15, and 4 members in the corresponding supergroups. Next, gene ontology (GO enrichment analyses showed 65 significant GO annotations of biological processes and molecular functions and KEGG pathways counted in frequency. To explore the functional pathways, regulatory gene networks, and/or related gene groups coding for Xenopus tropicalis bHLH proteins, the identified bHLH genes were put into the databases KOBAS and STRING to get the signaling information of pathways and protein interaction networks according to available public databases and known protein interactions. From the genome annotation and pathway analysis using KOBAS, we identified 16 pathways in the Xenopus tropicalis genome. From the STRING interaction analysis, 68 hub proteins were identified, and many hub proteins created a tight network or a functional module within the protein families.

  2. A Dual Mechanism Controls Nuclear Localization in the Atypical Basic-Helix-Loop-Helix Protein PAR1 of Arabidopsis thaliana

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    Anahit Galstyan; Jordi Bou-Torrent; Irma Roig-Villanova; Jaime F. Martínez-García

    2012-01-01

    PAR1 is an atypical basic-helix-loop-helix (bHLH) protein that negatively regulates the shade avoidance syndrome in Arabidopsis thaliana acting as a transcriptional cofactor.Consistently with this function,PAR1 has to be in the nucleus to display biological activity.Previous structure-function analyses revealed that the N-terminal region of PAR1 drives the protein to the nucleus.However,truncated forms of PAR1 lacking this region still display biological activity,implying that PAR1 has additional mechanisms to localize into the nucleus.In this work,we compared the primary structure of PAR1 and various related and unrelated plant bHLH proteins,which led us to suggest that PAR1 contains a non-canonical nuclear localization signal (NLS) in the N-terminal region.By overexpressing truncated and mutated derivatives of PAR1,we have also investigated the importance of other regions of PAR1,such as the acidic and the extended HLH dimerization domains,for its nuclear localization.We found that,in the absence of the N-terminal region,a functional HLH domain is required for nuclear localization.Our results suggest the existence of a dual mechanism for PAR1 nuclear localization:(1) one mediated by the N-terminal non-consensus NLS and (2) a second one that involves interaction with other proteins via the dimerization domain.

  3. Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice.

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    Sweeney, Megan T; Thomson, Michael J; Pfeil, Bernard E; McCouch, Susan

    2006-02-01

    Rc is a domestication-related gene required for red pericarp in rice (Oryza sativa). The red grain color is ubiquitous among the wild ancestors of O. sativa, in which it is closely associated with seed shattering and dormancy. Rc encodes a basic helix-loop-helix (bHLH) protein that was fine-mapped to an 18.5-kb region on rice chromosome 7 using a cross between Oryza rufipogon (red pericarp) and O. sativa cv Jefferson (white pericarp). Sequencing of the alleles from both mapping parents as well as from two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14-bp deletion within exon 6 that knocked out the bHLH domain of the protein. A premature stop codon was identified in the second mutant stock that had a light red pericarp. RT-PCR experiments confirmed that the Rc gene was expressed in both red- and white-grained rice but that a shortened transcript was present in white varieties. Phylogenetic analysis, supported by comparative mapping in rice and maize (Zea mays), showed that Rc, a positive regulator of proanthocyanidin, is orthologous with INTENSIFIER1, a negative regulator of anthocyanin production in maize, and is not in the same clade as rice bHLH anthocyanin regulators.

  4. Challenges in Targeting a Basic Helix-Loop-Helix Transcription Factor with Hydrocarbon-Stapled Peptides

    NARCIS (Netherlands)

    Edwards, Amanda L; Meijer, Dimphna H; Guerra, Rachel M; Molenaar, Remco J; Alberta, John A; Bernal, Federico; Bird, Gregory H; Stiles, Charles D; Walensky, Loren D

    2016-01-01

    Basic helix-loop-helix (bHLH) transcription factors play critical roles in organism development and disease by regulating cell proliferation and differentiation. Transcriptional activity, whether by bHLH homo- or heterodimerization, is dependent on protein-protein and protein-DNA interactions mediat

  5. Fluorescence Resonance Energy Transfer (FRET as a method to calculate the dimerization strength of basic Helix-Loop-Helix (bHLH proteins

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    Centonze Victoria E.

    2004-01-01

    Full Text Available Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH transcription factor HAND1, we show that HAND1 is phosphorylated during the trophoblast giant cell differentiation on residues residing in Helix I of the bHLH domain. Our hypothesis is that these modifications result in changes in HAND1 dimerization affinities with other bHLH factors. To test this idea, we employed FRET to measure the protein-protein interactions of HAND1 and HAND1 point mutants in HEK293 cells using YFP and CFP fusion proteins and laser scanning confocal microscopy.

  6. Elevated endogenous expression of the dominant negative basic helix-loop-helix protein ID1 correlates with significant centrosome abnormalities in human tumor cells

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    Gutmann Anja

    2010-01-01

    Full Text Available Abstract Background ID proteins are dominant negative inhibitors of basic helix-loop-helix transcription factors that have multiple functions during development and cellular differentiation. Ectopic (over-expression of ID1 extends the lifespan of primary human epithelial cells. High expression levels of ID1 have been detected in multiple human malignancies, and in some have been correlated with unfavorable clinical prognosis. ID1 protein is localized at the centrosomes and forced (over-expression of ID1 results in errors during centrosome duplication. Results Here we analyzed the steady state expression levels of the four ID-proteins in 18 tumor cell lines and assessed the number of centrosome abnormalities. While expression of ID1, ID2, and ID3 was detected, we failed to detect protein expression of ID4. Expression of ID1 correlated with increased supernumerary centrosomes in most cell lines analyzed. Conclusions This is the first report that shows that not only ectopic expression in tissue culture but endogenous levels of ID1 modulate centrosome numbers. Thus, our findings support the hypothesis that ID1 interferes with centrosome homeostasis, most likely contributing to genomic instability and associated tumor aggressiveness.

  7. OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice.

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    Seo, Ju-Seok; Joo, Joungsu; Kim, Min-Jeong; Kim, Yeon-Ki; Nahm, Baek Hie; Song, Sang Ik; Cheong, Jong-Joo; Lee, Jong Seob; Kim, Ju-Kon; Choi, Yang Do

    2011-03-01

    Jasmonates play important roles in development, stress responses and defense in plants. Here, we report the results of a study using a functional genomics approach that identified a rice basic helix-loop-helix domain gene, OsbHLH148, that conferred drought tolerance as a component of the jasmonate signaling module in rice. OsbHLH148 transcript levels were rapidly increased by treatment with methyl jasmonate (MeJA) or abscisic acid, and abiotic stresses including dehydration, high salinity, low temperature and wounding. Transgenic over-expression of OsbHLH148 in rice confers plant tolerance to drought stress. Expression profiling followed by DNA microarray and RNA gel-blot analyses of transgenic versus wild-type rice identified genes that are up-regulated by OsbHLH148 over-expression. These include OsDREB and OsJAZ genes that are involved in stress responses and the jasmonate signaling pathway, respectively. OsJAZ1, a rice ZIM domain protein, interacted with OsbHLH148 in yeast two-hybrid and pull-down assays, but it interacted with the putative OsCOI1 only in the presence of coronatine. Furthermore, the OsJAZ1 protein was degraded by rice and Arabidopsis extracts in the presence of coronatine, and its degradation was inhibited by MG132, a 26S proteasome inhibitor, suggesting 26S proteasome-mediated degradation of OsJAZ1 via the SCF(OsCOI1) complex. The transcription level of OsJAZ1 increased upon exposure of rice to MeJA. These results show that OsJAZ1 could act as a transcriptional regulator of the OsbHLH148-related jasmonate signaling pathway leading to drought tolerance. Thus, our study suggests that OsbHLH148 acts on an initial response of jasmonate-regulated gene expression toward drought tolerance, constituting the OsbHLH148-OsJAZ-OsCOI1 signaling module in rice.

  8. A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis.

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    Bai, Ming-Yi; Fan, Min; Oh, Eunkyoo; Wang, Zhi-Yong

    2012-12-01

    Environmental and endogenous signals, including light, temperature, brassinosteroid (BR), and gibberellin (GA), regulate cell elongation largely by influencing the expression of the paclobutrazol-resistant (PRE) family helix-loop-helix (HLH) factors, which promote cell elongation by interacting antagonistically with another HLH factor, IBH1. However, the molecular mechanism by which PREs and IBH1 regulate gene expression has remained unknown. Here, we show that IBH1 interacts with and inhibits a DNA binding basic helix-loop-helix (bHLH) protein, HBI1, in Arabidopsis thaliana. Overexpression of HBI1 increased hypocotyl and petiole elongation, whereas dominant inactivation of HBI1 and its homologs caused a dwarf phenotype, indicating that HBI1 is a positive regulator of cell elongation. In vitro and in vivo experiments showed that HBI1 directly bound to the promoters and activated two EXPANSIN genes encoding cell wall-loosening enzymes; HBI1's DNA binding and transcriptional activities were inhibited by IBH1, but the inhibitory effects of IBH1 were abolished by PRE1. The results indicate that PREs activate the DNA binding bHLH factor HBI1 by sequestering its inhibitor IBH1. Altering each of the three factors affected plant sensitivities to BR, GA, temperature, and light. Our study demonstrates that PREs, IBH1, and HBI1 form a chain of antagonistic switches that regulates cell elongation downstream of multiple external and endogenous signals.

  9. Biochemical analysis of the basic helix-loop-helix transcription factor Olig2

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    Meijer, D.H.M.

    2014-01-01

    The basic helix-loop-helix (bHLH) transcription factors oligodendrocyte transcription factor 1 (Olig1) and Olig2 are structurally similar and, to a first approximation, coordinately expressed in the developing CNS and postnatal brain. Notwithstanding these similarities, it was apparent from early on

  10. Challenges in Targeting a Basic Helix-Loop-Helix Transcription Factor with Hydrocarbon-Stapled Peptides.

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    Edwards, Amanda L; Meijer, Dimphna H; Guerra, Rachel M; Molenaar, Remco J; Alberta, John A; Bernal, Federico; Bird, Gregory H; Stiles, Charles D; Walensky, Loren D

    2016-11-18

    Basic helix-loop-helix (bHLH) transcription factors play critical roles in organism development and disease by regulating cell proliferation and differentiation. Transcriptional activity, whether by bHLH homo- or heterodimerization, is dependent on protein-protein and protein-DNA interactions mediated by α-helices. Thus, α-helical decoys have been proposed as potential targeted therapies for pathologic bHLH transcription. Here, we developed a library of stabilized α-helices of OLIG2 (SAH-OLIG2) to test the capacity of hydrocarbon-stapled peptides to disrupt OLIG2 homodimerization, which drives the development and chemoresistance of glioblastoma multiforme, one of the deadliest forms of human brain cancer. Although stapling successfully reinforced the α-helical structure of bHLH constructs of varying length, sequence-specific dissociation of OLIG2 dimers from DNA was not achieved. Re-evaluation of the binding determinants for OLIG2 self-association and stability revealed an unanticipated role of the C-terminal domain. These data highlight potential pitfalls in peptide-based targeting of bHLH transcription factors given the liabilities of their positively charged amino acid sequences and multifactorial binding determinants.

  11. Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome.

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    Howard, T D; Paznekas, W A; Green, E D; Chiang, L C; Ma, N; Ortiz de Luna, R I; Garcia Delgado, C; Gonzalez-Ramos, M; Kline, A D; Jabs, E W

    1997-01-01

    Saethre-Chotzen syndrome is one of the most common autosomal dominant disorders of craniosynostosis in humans and is characterized by craniofacial and limb anomalies. The locus for Saethre-Chotzen syndrome maps to chromosome 7p21-p22. We have evaluated TWIST, a basic helix-loop-helix transcription factor, as a candidate gene for this condition because its expression pattern and mutant phenotypes in Drosophila and mouse are consistent with the Saethre-Chotzen phenotype. We mapped TWIST to human chromosome 7p21-p22 and mutational analysis reveals nonsense, missense, insertion and deletion mutations in patients. These mutations occur within the basic DNA binding, helix I and loop domains, or result in premature termination of the protein. Studies in Drosophila indicate that twist may affect the transcription of fibroblast growth factor receptors (FGFRs), another gene family implicated in human craniosynostosis. The emerging cascade of molecular components involved in craniofacial and limb development now includes TWIST, which may function as an upstream regulator of FGFRs.

  12. Molecular recognition in helix-loop-helix and helix-loop-helix-leucine zipper domains. Design of repertoires and selection of high affinity ligands for natural proteins.

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    Ciarapica, Roberta; Rosati, Jessica; Cesareni, Gianni; Nasi, Sergio

    2003-04-04

    Helix-loop-helix (HLH) and helix-loop-helix-leucine zipper (HLHZip) are dimerization domains that mediate selective pairing among members of a large transcription factor family involved in cell fate determination. To investigate the molecular rules underlying recognition specificity and to isolate molecules interfering with cell proliferation and differentiation control, we assembled two molecular repertoires obtained by directed randomization of the binding surface in these two domains. For this strategy we selected the Heb HLH and Max Zip regions as molecular scaffolds for the randomization process and displayed the two resulting molecular repertoires on lambda phage capsids. By affinity selection, many domains were isolated that bound to the proteins Mad, Rox, MyoD, and Id2 with different levels of affinity. Although several residues along an extended surface within each domain appeared to contribute to dimerization, some key residues critically involved in molecular recognition could be identified. Furthermore, a number of charged residues appeared to act as switch points facilitating partner exchange. By successfully selecting ligands for four of four HLH or HLHZip proteins, we have shown that the repertoires assembled are rather general and possibly contain elements that bind with sufficient affinity to any natural HLH or HLHZip molecule. Thus they represent a valuable source of ligands that could be used as reagents for molecular dissection of functional regulatory pathways.

  13. A Classification of Basic Helix-Loop-Helix Transcription Factors of Soybean

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    Karen A. Hudson

    2015-01-01

    Full Text Available The complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits. In particular, the potential functions of regulatory genes are a priority for analysis. The basic helix-loop-helix (bHLH family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality, including photosynthesis, light signalling, pigment biosynthesis, and seed pod development. Using a hidden Markov model search algorithm, 319 genes with basic helix-loop-helix transcription factor domains were identified within the soybean genome sequence. These were classified with respect to their predicted DNA binding potential, intron/exon structure, and the phylogeny of the bHLH domain. Evidence is presented that the vast majority (281 of these 319 soybean bHLH genes are expressed at the mRNA level. Of these soybean bHLH genes, 67% were found to exist in two or more homeologous copies. This dataset provides a framework for future studies on bHLH gene function in soybean. The challenge for future research remains to define functions for the bHLH factors encoded in the soybean genome, which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop.

  14. Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

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    Selote, Devarshi; Samira, Rozalynne; Matthiadis, Anna; Gillikin, Jeffrey W; Long, Terri A

    2015-01-01

    Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::β-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

  15. A single amino acid substitution in IIIf subfamily of basic helix-loop-helix transcription factor AtMYC1 leads to trichome and root hair patterning defects by abolishing its interaction with partner proteins in Arabidopsis.

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    Zhao, Hongtao; Wang, Xiaoxue; Zhu, Dandan; Cui, Sujuan; Li, Xia; Cao, Ying; Ma, Ligeng

    2012-04-20

    Plant trichomes and root hairs are powerful models for the study of cell fate determination. In Arabidopsis thaliana, trichome and root hair initiation requires a combination of three groups of proteins, including the WD40 repeat protein transparent TESTA GLABRA1 (TTG1), R2R3 repeat MYB protein GLABRA1 (GL1), or werewolf (WER) and the IIIf subfamily of basic helix-loop-helix (bHLH) protein GLABRA3 (GL3) or enhancer of GLABRA3 (EGL3). The bHLH component acts as a docking site for TTG1 and MYB proteins. Here, we isolated a mutant showing defects in trichome and root hair patterning that carried a point mutation (R173H) in AtMYC1 that encodes the fourth member of IIIf bHLH family protein. Genetic analysis revealed partial redundant yet distinct function between AtMYC1 and GL3/EGL3. GLABRA2 (GL2), an important transcription factor involved in trichome and root hair control, was down-regulated in Atmyc1 plants, suggesting the requirement of AtMYC1 for appropriate GL2 transcription. Like its homologs, AtMYC1 formed a complex with TTG1 and MYB proteins but did not dimerized. In addition, the interaction of AtMYC1 with MYB proteins and TTG1 was abrogated by the R173H substitution in Atmyc1-1. We found that this amino acid (Arg) is conserved in the AtMYC1 homologs GL3/EGL3 and that it is essential for their interaction with MYB proteins and for their proper functions. Our findings indicate that AtMYC1 is an important regulator of trichome and root hair initiation, and they reveal a novel amino acid necessary for protein-protein interactions and gene function in IIIf subfamily bHLH transcription factors.

  16. A genome-wide survey on basic helix-loop-helix transcription factors in rat and mouse.

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    Zheng, Xiaodong; Zheng, X; Wang, Yong; Wang, Y; Yao, Qin; Yao, Q; Yang, Zhe; Yang, Z; Chen, Keping; Chen, K

    2009-04-01

    The basic helix-loop-helix (bHLH) proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including nematode, fruit fly, and human. Our study identified 114 rat and 14 additional mouse bHLH members in rat and mouse genomes, respectively. Phylogenetic analyses revealed that both rat and mouse had 49, 26, 15, 4, 12, and 4 bHLH members in groups A, B, C, D, E, and F, respectively. Only the rat Mxi1 gene has two copies in the genome. All other rat bHLH genes and all mouse bHLH genes are single-copy genes. The chromosomal distribution pattern of mouse, rat, and human bHLH genes suggests the emergence of some bHLH genes through gene duplication, which probably happened at least before the divergence of vertebrates from invertebrates. The present study provides useful information for future studies using rat as a model animal for mammalian development.

  17. Molecular characterization of cold-responsive basic helix-loop-helix transcription factors MabHLHs that interact with MaICE1 in banana fruit.

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    Peng, Huan-Huan; Shan, Wei; Kuang, Jian-Fei; Lu, Wang-Jin; Chen, Jian-Ye

    2013-11-01

    Basic helix-loop-helix (bHLH) transcription factors (TFs) are ubiquitously involved in the response of higher plants to various abiotic stresses. However, little is known about bHLH TFs involved in the cold stress response in economically important fruits. Here, five novel full-length bHLH genes, designated as MabHLH1-MabHLH5, were isolated and characterized from banana fruit. Gene expression profiles revealed that MabHLH1/2/4 were induced by cold stress and methyl jasmonate (MeJA) treatment. Transient assays in tobacco BY2 protoplasts showed that MabHLH1/2/4 promoters were activated by cold stress and MeJA treatments. Moreover, protein-protein interaction analysis demonstrated that MabHLH1/2/4 not only physically interacted with each other to form hetero-dimers in the nucleus, but also interacted with an important upstream component of cold signaling MaICE1, with different interaction domains at their N-terminus. These results indicate that banana fruit cold-responsive MabHLHs may form a big protein complex in the nucleus with MaICE1. Taken together, our findings advance our understanding of the possible involvement of bHLH TFs in the regulatory network of ICE-CBF cold signaling pathway.

  18. Mutations within or upstream of the basic helix-loop-helix domain of the TWIST gene are specific to Saethre-Chotzen syndrome.

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    El Ghouzzi, V; Lajeunie, E; Le Merrer, M; Cormier-Daire, V; Renier, D; Munnich, A; Bonaventure, J

    1999-01-01

    Saethre-Chotzen syndrome (ACS III) is an autosomal dominant craniosynostosis syndrome recently ascribed to mutations in the TWIST gene, a basic helix-loop-helix (b-HLH) transcription factor regulating head mesenchyme cell development during cranial neural tube formation in mouse. Studying a series of 22 unrelated ACS III patients, we have found TWIST mutations in 16/22 cases. Interestingly, these mutations consistently involved the b-HLH domain of the protein. Indeed, mutant genotypes included frameshift deletions/insertions, nonsense and missense mutations, either truncating or disrupting the b-HLH motif of the protein. This observation gives additional support to the view that most ACS III cases result from loss-of-function mutations at the TWIST locus. The P250R recurrent FGFR 3 mutation was found in 2/22 cases presenting mild clinical manifestations of the disease but 4/22 cases failed to harbour TWIST or FGFR 3 mutations. Clinical re-examination of patients carrying TWIST mutations failed to reveal correlations between the mutant genotype and severity of the phenotype. Finally, since no TWIST mutations were detected in 40 cases of isolated coronal craniosynostosis, the present study suggests that TWIST mutations are specific to Saethre-Chotzen syndrome.

  19. Heterogeneity of myotubes generated by the MyoD and E12 basic helix-loop-helix transcription factors in otherwise non-differentiation growth conditions.

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    Grubišić, Vladimir; Gottipati, Manoj K; Stout, Randy F; Grammer, J Robert; Parpura, Vladimir

    2014-02-01

    We used a synthetic biology approach to produce myotubes from mammalian C2C12 myoblasts in non-differentiation growth conditions using the expression of basic helix-loop-helix transcription factors, MyoD and E12, in various combinations and configurations. Our approach not only recapitulated the basics of muscle development and physiology, as the obtained myotubes showed qualities similar to those seen in striated muscle fibers in vivo, but also allowed for the synthesis of populations of myotubes which assumed distinct morphology, myofibrillar development and Ca(2+) dynamics. This fashioned class of biomaterials is suitable for the building blocks of soft actuators in micro-scale biomimetic robotics. This production line strategy can be embraced in reparative medicine as synthetic human myotubes with predetermined morphological/functional properties could be obtained using this very approach. This methodology can be adopted beyond striated muscle for the engineering of other tissue components/cells whose differentiation is governed by the principles of basic helix-loop-helix transcription factors, as in the case, for example, of neural or immune cell types.

  20. Basic helix-loop-helix transcription factor Bmsage is involved in regulation of fibroin H-chain gene via interaction with SGF1 in Bombyx mori.

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    Zhao, Xiao-Ming; Liu, Chun; Li, Qiong-Yan; Hu, Wen-Bo; Zhou, Meng-Ting; Nie, Hong-Yi; Zhang, Yin-Xia; Peng, Zhang-Chuan; Zhao, Ping; Xia, Qing-You

    2014-01-01

    Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix-loop-helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells.

  1. A genome-wide identification and analysis of the basic helix-loop-helix transcription factors in the ponerine ant, Harpegnathos saltator

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    Liu Ake

    2012-08-01

    Full Text Available Abstract Background The basic helix-loop-helix (bHLH transcription factors and their homologs form a superfamily that plays essential roles in transcriptional networks of multiple developmental processes. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, human and mouse. Result In this study, we conducted a genome-wide survey for bHLH sequences, and identified 57 bHLH sequences encoded in complete genome sequence of the ponerine ant, Harpegnathos saltator. Phylogenetic analysis of the bHLH domain sequences classified these genes into 38 bHLH families with 23, 14, 10, 1, 8 and 1 members in group A, B, C, D, E and F, respectively. The number of PabHLHs (ponerine ant bHLHs with introns is higher than many other insect species, and they are found to have introns with average lengths only inferior to those of pea aphid. In addition, two H. saltator bHLHs named PaCrp1 and PaSide locate on two separate contigs in the genome. Conclusions A putative full set of PabHLH genes is comparable with other insect species and genes encoding Oligo, MyoRb and Figα were not found in genomes of all insect species of which bHLH family members have been identified. Moreover, in-family phylogenetic analyses indicate that the PabHLH genes are more closely related with Apis mellifera than others. The present study will serve as a solid foundation for further investigations into the structure and function of bHLH proteins in the regulation of H. saltator development.

  2. Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA

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    Zixi Wang; Yaling Wu; Lanfen Li; Xiao-Dong Su

    2013-01-01

    CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) are both transcription factors of the circadian core loop in mammals.Recently published mouse CLOCK-BMAL1 bHLH (basic helix-loop-helix)-PAS (period-ARNT-single-minded) complex structure sheds light on the mechanism for heterodimer formation,but the structural details of the protein-DNA recognition mechanisms remain elusive.Here we have elucidated the crystal structure of human CLOCK-BMAL1 bHLH domains bound to a canonical E-box DNA.We demonstrate that CLOCK and BMAL1 bHLH domains can be mutually selected,and that hydrogen-bonding networks mediate their E-box recognition.We identified a hydrophobic contact between BMAL1 Ile80 and a fianking thymine nucleotide,suggesting that CLOCK-BMAL1 actually reads 7-bp DNA and not the previously believed 6-bp DNA.To find potential non-canonical E-boxes that could be recognized by CLOCK-BMAL1,we constructed systematic single-nucleotide mutations on the E-box and measured their relevant affinities.We defined two non-canonical E-box patterns with high affinities,AACGTGA and CATGTGA,in which the flanking A7-T7' base pair is indispensable for recognition.These results will help us to identify functional CLOCK-BMAL1-binding sites in vivo and to search for clock-controlled genes.Furthermore,we assessed the inhibitory role of potential phosphorylation sites in bHLH regions.We found that the phospho-mimicking mutation on BMAL1 Ser78 could efficiently block DNA binding as well as abolish normal circadian oscillation in cells.We propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade.

  3. Functional diversity of human basic helix-loop-helix transcription factor TCF4 isoforms generated by alternative 5' exon usage and splicing.

    Directory of Open Access Journals (Sweden)

    Mari Sepp

    Full Text Available BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2 is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG. While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence

  4. Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus.

    Science.gov (United States)

    Paolocci, Francesco; Robbins, Mark P; Madeo, Laura; Arcioni, Sergio; Martens, Stefan; Damiani, Francesco

    2007-01-01

    Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

  5. Protein (Viridiplantae): 297852830 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available QSAPSSYFSSFGESIEEFLDRPTSPETERILSGFLQTTDTSNNVDSFLHHTFNSDGTEKKPPEVKTEEDETEIPVTVTTME...972:1358 basic helix-loop-helix family protein Arabidopsis lyrata subsp. lyrata MESEFQQHHFLLHDHQHQRPRNSGLIRY

  6. The neurogenic basic helix-loop-helix transcription factor NeuroD6 enhances mitochondrial biogenesis and bioenergetics to confer tolerance of neuronal PC12-NeuroD6 cells to the mitochondrial stressor rotenone

    Energy Technology Data Exchange (ETDEWEB)

    Baxter, Kristin Kathleen; Uittenbogaard, Martine [Department of Anatomy and Regenerative Biology, George Washington University Medical Center, Washington, DC (United States); Chiaramello, Anne, E-mail: achiaram@gwu.edu [Department of Anatomy and Regenerative Biology, George Washington University Medical Center, Washington, DC (United States)

    2012-10-15

    The fundamental question of how and which neuronal specific transcription factors tailor mitochondrial biogenesis and bioenergetics to the need of developing neuronal cells has remained largely unexplored. In this study, we report that the neurogenic basic helix-loop-helix transcription factor NeuroD6 possesses mitochondrial biogenic properties by amplifying the mitochondrial DNA content and TFAM expression levels, a key regulator for mitochondrial biogenesis. NeuroD6-mediated increase in mitochondrial biogenesis in the neuronal progenitor-like PC12-NEUROD6 cells is concomitant with enhanced mitochondrial bioenergetic functions, including increased expression levels of specific subunits of respiratory complexes of the electron transport chain, elevated mitochondrial membrane potential and ATP levels produced by oxidative phosphorylation. Thus, NeuroD6 augments the bioenergetic capacity of PC12-NEUROD6 cells to generate an energetic reserve, which confers tolerance to the mitochondrial stressor, rotenone. We found that NeuroD6 induces an adaptive bioenergetic response throughout rotenone treatment involving maintenance of the mitochondrial membrane potential and ATP levels in conjunction with preservation of the actin network. In conclusion, our results support the concept that NeuroD6 plays an integrative role in regulating and coordinating the onset of neuronal differentiation with acquisition of adequate mitochondrial mass and energetic capacity to ensure energy demanding events, such as cytoskeletal remodeling, plasmalemmal expansion, and growth cone formation. -- Highlights: Black-Right-Pointing-Pointer NeuroD6 induces mitochondrial biogenesis in neuroprogenitor-like cells. Black-Right-Pointing-Pointer NeuroD6 augments the bioenergetic reserve of the neuronal PC12-NeuroD6 cells. Black-Right-Pointing-Pointer NeuroD6 increases the mitochondrial membrane potential and ATP levels. Black-Right-Pointing-Pointer NeuroD6 confers tolerance to rotenone via an adaptive

  7. Dynamic antagonism between phytochromes and PIF family basic helix-loop-helix factors induces selective reciprocal responses to light and shade in a rapidly responsive transcriptional network in Arabidopsis.

    Science.gov (United States)

    Leivar, Pablo; Tepperman, James M; Cohn, Megan M; Monte, Elena; Al-Sady, Bassem; Erickson, Erika; Quail, Peter H

    2012-04-01

    Plants respond to shade-modulated light signals via phytochrome (phy)-induced adaptive changes, termed shade avoidance. To examine the roles of Phytochrome-Interacting basic helix-loop-helix Factors, PIF1, 3, 4, and 5, in relaying such signals to the transcriptional network, we compared the shade-responsive transcriptome profiles of wild-type and quadruple pif (pifq) mutants. We identify a subset of genes, enriched in transcription factor-encoding loci, that respond rapidly to shade, in a PIF-dependent manner, and contain promoter G-box motifs, known to bind PIFs. These genes are potential direct targets of phy-PIF signaling that regulate the primary downstream transcriptional circuitry. A second subset of PIF-dependent, early response genes, lacking G-box motifs, are enriched for auxin-responsive loci, and are thus potentially indirect targets of phy-PIF signaling, mediating the rapid cell expansion induced by shade. Comparing deetiolation- and shade-responsive transcriptomes identifies another subset of G-box-containing genes that reciprocally display rapid repression and induction in response to light and shade signals. These data define a core set of transcriptional and hormonal processes that appear to be dynamically poised to react rapidly to light-environment changes via perturbations in the mutually antagonistic actions of the phys and PIFs. Comparing the responsiveness of the pifq and triple pif mutants to light and shade confirms that the PIFs act with overlapping redundancy on seedling morphogenesis and transcriptional regulation but that each PIF contributes differentially to these responses.

  8. Human variants in the neuronal basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS transcription factor complex NPAS4/ARNT2 disrupt function.

    Directory of Open Access Journals (Sweden)

    David C Bersten

    Full Text Available Neuronal Per-Arnt-Sim homology (PAS Factor 4 (NPAS4 is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K and two variants in ARNT2 (R46W and R107H which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.

  9. Protein: MPA6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA6 SREBBP1c SREBF1 BHLHD1, SREBP1 Sterol regulatory element-binding protein 1 Cla...ss D basic helix-loop-helix protein 1, Sterol regulatory element-binding transcription factor 1 9606 Homo sapiens P36956 6720 1AM9 6720 P36956 ...

  10. Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

    NARCIS (Netherlands)

    Verweij, W.; Spelt, C.E.; Bliek, M.; de Vries, M.; Wit, N.; Faraco, M.; Koes, R.; Quattrocchio, F.

    2016-01-01

    The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) fromArabidopsis thalianaand associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein

  11. The basic helix-loop-helix/leucine zipper transcription factor USF2 integrates serum-induced PAI-1 expression and keratinocyte growth.

    Science.gov (United States)

    Qi, Li; Higgins, Craig E; Higgins, Stephen P; Law, Brian K; Simone, Tessa M; Higgins, Paul J

    2014-10-01

    Plasminogen activator inhibitor type-1 (PAI-1), a major regulator of the plasmin-dependent pericellular proteolytic cascade, is prominently expressed during the tissue response to injury although the factors that impact PAI-1 induction and their role in the repair process are unclear. Kinetic modeling using established biomarkers of cell cycle transit (c-MYC; cyclin D1; cyclin A) in synchronized human (HaCaT) keratinocytes, and previous cytometric assessments, indicated that PAI-1 transcription occurred early after serum-stimulation of quiescent (G0) cells and prior to G1 entry. It was established previously that differential residence of USF family members (USF1→USF2 switch) at the PE2 region E box (CACGTG) characterized the G0  → G1 transition period and the transcriptional status of the PAI-1 gene. A consensus PE2 E box motif (5'-CACGTG-3') at nucleotides -566 to -561 was required for USF/E box interactions and serum-dependent PAI-1 transcription. Site-directed CG → AT substitution at the two central nucleotides inhibited formation of USF/probe complexes and PAI-1 promoter-driven reporter expression. A dominant-negative USF (A-USF) construct or double-stranded PE2 "decoy" attenuated serum- and TGF-β1-stimulated PAI-1 synthesis. Tet-Off induction of an A-USF insert reduced both PAI-1 and PAI-2 transcripts while increasing the fraction of Ki-67(+) cells. Conversely, overexpression of USF2 or adenoviral-delivery of a PAI-1 vector inhibited HaCaT colony expansion indicating that the USF1 → USF2 transition and subsequent PAI-1 transcription are critical events in the epithelial go-or-grow response. Collectively, these data suggest that USF2, and its target gene PAI-1, regulate serum-stimulated keratinocyte growth, and likely the cadence of cell cycle progression in replicatively competent cells as part of the injury repair program.

  12. Marked induction of the helix-loop-helix protein Id3 promotes the gammadelta T cell fate and renders their functional maturation Notch independent

    DEFF Research Database (Denmark)

    Lauritsen, Jens Peter Holst; Wong, Gladys W; Lee, Sang-Yun

    2009-01-01

    alphabeta and gammadelta T cells arise from a common thymocyte progenitor during development in the thymus. Emerging evidence suggests that the pre-T cell receptor (pre-TCR) and gammadelta T cell receptor (gammadeltaTCR) play instructional roles in specifying the alphabeta and gammadelta T-lineag...

  13. SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative

    DEFF Research Database (Denmark)

    Rishi, Vikas; Gal, Jozsef; Krylov, Dmitry

    2004-01-01

    The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A...

  14. A juvenile hormone transcription factor Bmdimm-fibroin H chain pathway is involved in the synthesis of silk protein in silkworm, Bombyx mori.

    Science.gov (United States)

    Zhao, Xiao-Ming; Liu, Chun; Jiang, Li-Jun; Li, Qiong-Yan; Zhou, Meng-Ting; Cheng, Ting-Cai; Mita, Kazuei; Xia, Qing-You

    2015-01-09

    The genes responsible for silk biosynthesis are switched on and off at particular times in the silk glands of Bombyx mori. This switch appears to be under the control of endogenous and exogenous hormones. However, the molecular mechanisms by which silk protein synthesis is regulated by the juvenile hormone (JH) are largely unknown. Here, we report a basic helix-loop-helix transcription factor, Bmdimm, its silk gland-specific expression, and its direct involvement in the regulation of fibroin H-chain (fib-H) by binding to an E-box (CAAATG) element of the fib-H gene promoter. Far-Western blots, enzyme-linked immunosorbent assays, and co-immunoprecipitation assays revealed that Bmdimm protein interacted with another basic helix-loop-helix transcription factor, Bmsage. Immunostaining revealed that Bmdimm and Bmsage proteins are co-localized in nuclei. Bmdimm expression was induced in larval silk glands in vivo, in silk glands cultured in vitro, and in B. mori cell lines after treatment with a JH analog. The JH effect on Bmdimm was mediated by the JH-Met-Kr-h1 signaling pathway, and Bmdimm expression did not respond to JH by RNA interference with double-stranded BmKr-h1 RNA. These data suggest that the JH regulatory pathway, the transcription factor Bmdimm, and the targeted fib-H gene contribute to the synthesis of fibroin H-chain protein in B. mori.

  15. EST Table: FS845099 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available PREDICTED: similar to class b basic helix-loop-helix protein (bhlhb) (differentially expressed in chondrocytes) (mdec) (sharp...lar to class b basic helix-loop-helix protein (bhlhb) (differentially expressed in chondrocytes) (mdec) (sharp) [Tribolium castaneum] FS845099 fner ...

  16. EST Table: FS816912 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available milar to class b basic helix-loop-helix protein (bhlhb) (differentially expressed in chondrocytes) (mdec) (sharp...asic helix-loop-helix protein (bhlhb) (differentially expressed in chondrocytes) (mdec) (sharp) [Tribolium castaneum] FS845099 fmgV ...

  17. Inhibition of Tcf3 binding by I-mfa domain proteins.

    Science.gov (United States)

    Snider, L; Thirlwell, H; Miller, J R; Moon, R T; Groudine, M; Tapscott, S J

    2001-03-01

    We have determined that I-mfa, an inhibitor of several basic helix-loop-helix (bHLH) proteins, and XIC, a Xenopus ortholog of human I-mf domain-containing protein that shares a highly conserved cysteine-rich C-terminal domain with I-mfa, inhibit the activity and DNA binding of the HMG box transcription factor XTcf3. Ectopic expression of I-mfa or XIC in early Xenopus embryos inhibited dorsal axis specification, the expression of the Tcf3/beta-catenin-regulated genes siamois and Xnr3, and the ability of beta-catenin to activate reporter constructs driven by Lef/Tcf binding sites. I-mfa domain proteins can regulate both the Wnt signaling pathway and a subset of bHLH proteins, possibly coordinating the activities of these two critical developmental pathways.

  18. Type I bHLH Proteins Daughterless and Tcf4 Restrict Neurite Branching and Synapse Formation by Repressing Neurexin in Postmitotic Neurons

    Directory of Open Access Journals (Sweden)

    Mitchell D’Rozario

    2016-04-01

    Full Text Available Proneural proteins of the class I/II basic-helix-loop-helix (bHLH family are highly conserved transcription factors. Class I bHLH proteins are expressed in a broad number of tissues during development, whereas class II bHLH protein expression is more tissue restricted. Our understanding of the function of class I/II bHLH transcription factors in both invertebrate and vertebrate neurobiology is largely focused on their function as regulators of neurogenesis. Here, we show that the class I bHLH proteins Daughterless and Tcf4 are expressed in postmitotic neurons in Drosophila melanogaster and mice, respectively, where they function to restrict neurite branching and synapse formation. Our data indicate that Daughterless performs this function in part by restricting the expression of the cell adhesion molecule Neurexin. This suggests a role for these proteins outside of their established roles in neurogenesis.

  19. Direct detection of transcription factors in cotyledons during seedling development using sensitive silicon-substrate photonic crystal protein arrays.

    Science.gov (United States)

    Jones, Sarah I; Tan, Yafang; Shamimuzzaman, Md; George, Sherine; Cunningham, Brian T; Vodkin, Lila

    2015-03-01

    Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts.

  20. Structural basis of nucleic acid recognition by FK506-binding protein 25 (FKBP25), a nuclear immunophilin.

    Science.gov (United States)

    Prakash, Ajit; Shin, Joon; Rajan, Sreekanth; Yoon, Ho Sup

    2016-04-01

    The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25-DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition.

  1. Functional diversification of the potato R2R3 MYB anthocyanin activators AN1, MYBA1, and MYB113 and their interaction with basic helix-loop-helix cofactors.

    Science.gov (United States)

    Liu, Yuhui; Lin-Wang, Kui; Espley, Richard V; Wang, Li; Yang, Hongyu; Yu, Bin; Dare, Andrew; Varkonyi-Gasic, Erika; Wang, Jing; Zhang, Junlian; Wang, Di; Allan, Andrew C

    2016-04-01

    In potato (Solanum tuberosum L.), R2R3 MYBs are involved in the regulation of anthocyanin biosynthesis. We examined sequences of these MYBs in cultivated potatoes, which are more complex than diploid potato due to ploidy and heterozygosity. We found amino acid variants in the C-terminus of the MYB StAN1, termed R0, R1, and R3, due to the presence of a repeated 10-amino acid motif. These variant MYBs showed some expression in both white and pigmented tubers. We found several new alleles or gene family members of R2R3 MYBs,StMYBA1 and StMYB113, which were also expressed in white potato tubers. From functional analysis in tobacco, we showed that the presence of a C-terminal 10-amino acid motif is optimal for activating anthocyanin accumulation. Engineering a motif back into a MYB lacking this sequence enhanced its activating ability. Versions of StMYBA1 and StMYB113 can also activate anthocyanin accumulation in tobacco leaves, with the exception of StMYB113-3, which has a partial R2R3 domain. We isolated five family members of potato StbHLH1, and one StJAF13, to test their ability to interact with MYB variants. The results showed that two alleles of StbHLH1 from white skin and red skin are non-functional, while three other StbHLH1s have different co-regulating abilities, and need to be activated by StJAF13. Combined with expression analysis in potato tuber, results suggest that StbHLH1 and StJAF13a re key co-regulators of anthocyanin biosynthesis, while the transcripts of MYB variants StAN1,StMYBA1, and StMYB113 are well expressed, even in the absence of pigmentation.

  2. Responses of a triple mutant defective in three iron deficiency-induced Basic Helix-Loop-Helix genes of the subgroup Ib(2) to iron deficiency and salicylic acid.

    Science.gov (United States)

    Maurer, Felix; Naranjo Arcos, Maria Augusta; Bauer, Petra

    2014-01-01

    Plants are sessile organisms that adapt to external stress by inducing molecular and physiological responses that serve to better cope with the adverse growth condition. Upon low supply of the micronutrient iron, plants actively increase the acquisition of soil iron into the root and its mobilization from internal stores. The subgroup Ib(2) BHLH genes function as regulators in this response, however their concrete functions are not fully understood. Here, we analyzed a triple loss of function mutant of BHLH39, BHLH100 and BHLH101 (3xbhlh mutant). We found that this mutant did not have any iron uptake phenotype if iron was provided. However, under iron deficiency the mutant displayed a more severe leaf chlorosis than the wild type. Microarray-based transcriptome analysis revealed that this mutant phenotype resulted in the mis-regulation of 198 genes, out of which only 15% were associated with iron deficiency regulation itself. A detailed analysis revealed potential targets of the bHLH transcription factors as well as genes reflecting an exaggerated iron deficiency response phenotype. Since the BHLH genes of this subgroup have been brought into the context of the plant hormone salicylic acid, we investigated whether the 3xbhlh mutant might have been affected by this plant signaling molecule. Although a very high number of genes responded to SA, also in a differential manner between mutant and wild type, we did not find any indication for an association of the BHLH gene functions in SA responses upon iron deficiency. In summary, our study indicates that the bHLH subgroup Ib(2) transcription factors do not only act in iron acquisition into roots but in other aspects of the adaptation to iron deficiency in roots and leaves.

  3. Responses of a triple mutant defective in three iron deficiency-induced Basic Helix-Loop-Helix genes of the subgroup Ib(2 to iron deficiency and salicylic acid.

    Directory of Open Access Journals (Sweden)

    Felix Maurer

    Full Text Available Plants are sessile organisms that adapt to external stress by inducing molecular and physiological responses that serve to better cope with the adverse growth condition. Upon low supply of the micronutrient iron, plants actively increase the acquisition of soil iron into the root and its mobilization from internal stores. The subgroup Ib(2 BHLH genes function as regulators in this response, however their concrete functions are not fully understood. Here, we analyzed a triple loss of function mutant of BHLH39, BHLH100 and BHLH101 (3xbhlh mutant. We found that this mutant did not have any iron uptake phenotype if iron was provided. However, under iron deficiency the mutant displayed a more severe leaf chlorosis than the wild type. Microarray-based transcriptome analysis revealed that this mutant phenotype resulted in the mis-regulation of 198 genes, out of which only 15% were associated with iron deficiency regulation itself. A detailed analysis revealed potential targets of the bHLH transcription factors as well as genes reflecting an exaggerated iron deficiency response phenotype. Since the BHLH genes of this subgroup have been brought into the context of the plant hormone salicylic acid, we investigated whether the 3xbhlh mutant might have been affected by this plant signaling molecule. Although a very high number of genes responded to SA, also in a differential manner between mutant and wild type, we did not find any indication for an association of the BHLH gene functions in SA responses upon iron deficiency. In summary, our study indicates that the bHLH subgroup Ib(2 transcription factors do not only act in iron acquisition into roots but in other aspects of the adaptation to iron deficiency in roots and leaves.

  4. Antagonistic regulation of growth and immunity by the Arabidopsis basic helix-loop-helix transcription factor homolog of brassinosteroid enhanced expression2 interacting with increased leaf inclination1 binding bHLH1

    DEFF Research Database (Denmark)

    Malinovsky, Frederikke Gro; Batoux, Martine; Schwessinger, Benjamin;

    2014-01-01

    Plants need to finely balance resources allocated to growth and immunity to achieve optimal fitness. A tradeoff between pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and brassinosteroid (BR)-mediated growth was recently reported, but more information about the underlying...... to different PAMPs. HBI1 overexpression leads to reduced PAMP-triggered responses. This inhibition correlates with reduced steady-state expression of immune marker genes, leading to increased susceptibility to the bacterium Pseudomonas syringae. Overexpression of the HBI1-related bHLHs brassinosteroid enhanced...... expression2 (BEE2) and cryptochrome-interacting bHLH (CIB1) partially inhibits immunity, indicating that BEE2 and CIB1 may act redundantly with HBI1. In contrast to its expression pattern upon PAMP treatment, HBI1 expression is enhanced by BR treatment. Also, HBI1-overexpressing plants are hyperresponsive...

  5. Bimolecular fluorescence complementation as a tool to study interactions of regulatory proteins in plant protoplasts.

    Science.gov (United States)

    Pattanaik, Sitakanta; Werkman, Joshua R; Yuan, Ling

    2011-01-01

    Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.

  6. Mutations in the basic domain and the loop-helix II junction of TWIST abolish DNA binding in Saethre-Chotzen syndrome.

    Science.gov (United States)

    El Ghouzzi, V; Legeai-Mallet, L; Benoist-Lasselin, C; Lajeunie, E; Renier, D; Munnich, A; Bonaventure, J

    2001-03-09

    Saethre-Chotzen syndrome is an autosomal dominant skull disorder resulting from premature fusion of coronal sutures (craniosynostosis). It is caused by mutations in the TWIST gene encoding a basic Helix-Loop-Helix transcription factor. Here we report on the identification of a novel mutation affecting a highly conserved residue of the basic domain. Unlike nonsense and missense mutations lying within helices, this mutation does not affect protein stability or heterodimerisation of TWIST with its partner E12. However, it does abolish TWIST binding capacity to a target E-box as efficiently as two missense mutations in the loop-helix II junction. By contrast, elongation of the loop through a 7 amino acid insertion appears not to hamper binding to the DNA target. We conclude that loss of TWIST protein function in Saethre-Chotzen patients can occur at three different levels, namely protein stability, dimerisation, and DNA binding and that the loop-helix II junction is essential for effective protein-DNA interaction.

  7. Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

    Directory of Open Access Journals (Sweden)

    Hiu-Kwan Chow

    Full Text Available Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim protein Arnt fused to the leucine zipper (LZ dimerization domain from bZIP (basic region-leucine zipper protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H, transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed, as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d 148.9 nM and 40.2 nM, respectively, but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa. Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

  8. Proneural proteins Achaete and Scute associate with nuclear actin to promote formation of external sensory organs.

    Science.gov (United States)

    Hsiao, Yun-Ling; Chen, Yu-Ju; Chang, Yi-Jie; Yeh, Hsiao-Fong; Huang, Yi-Chun; Pi, Haiwei

    2014-01-01

    Basic helix-loop-helix (bHLH) proneural proteins promote neurogenesis through transcriptional regulation. Although much is known about the tissue-specific regulation of proneural gene expression, how proneural proteins interact with transcriptional machinery to activate downstream target genes is less clear. Drosophila proneural proteins Achaete (Ac) and Scute (Sc) induce external sensory organ formation by activating neural precursor gene expression. Through co-immunoprecipitation and mass spectrometric analyses, we found that nuclear but not cytoplasmic actin associated with the Ac and Sc proteins in Drosophila S2 cells. Daughterless (Da), the common heterodimeric partner of Drosophila bHLH proteins, was observed to associate with nuclear actin through proneural proteins. A yeast two-hybrid assay revealed that the binding specificity between actin and Ac or Sc was conserved in yeast nuclei without the presence of additional Drosophila factors. We further show that actin is required in external sensory organ formation. Reduction in actin gene activity impaired proneural-protein-dependent expression of the neural precursor genes, as well as formation of neural precursors. Furthermore, increased nuclear actin levels, obtained by expression of nucleus-localized actin, elevated Ac-Da-dependent gene transcription as well as Ac-mediated external sensory organ formation. Taken together, our in vivo and in vitro observations suggest a novel link for actin in proneural-protein-mediated transcriptional activation and neural precursor differentiation.

  9. A proteomic study showing differential regulation of stress, redox regulation and peroxidase proteins by iron supply and the transcription factor FER.

    Science.gov (United States)

    Brumbarova, Tzvetina; Matros, Andrea; Mock, Hans-Peter; Bauer, Petra

    2008-04-01

    Plants need to mobilize iron in the soil, and the basic helix-loop-helix transcription factor FER is a central regulator of iron acquisition in tomato roots. FER activity is controlled by iron supply. To analyse to what extent FER influences Fe-regulated protein expression, we investigated the root proteome of wild-type tomato, the fer mutant and a transgenic FER overexpression line under low-iron conditions versus sufficient and generous iron supply. The root proteomes were analysed by two-dimensional gel electrophoresis with three technical and three biological replicates. Statistical analysis identified 39 protein spots that were differentially regulated in selected pairwise comparisons of experimental conditions. Of these, 24 were correlated with expression clusters revealed by principal component analysis. The 39 protein spots were analysed by MALDI-TOF and nanoLC-MS/MS to deduce their possible functions. We investigated the functional representation in the identified expression clusters, and found that loss of FER function in iron-cultured plants mimicked an iron-deficiency status. The largest identified protein expression cluster was upregulated by iron deficiency and in the fer mutant. Two iron-regulated proteins required FER activity for induction by iron deficiency. Few proteins were suppressed by iron deficiency. The differentially expressed proteins belonged predominantly to the functional categories 'stress', 'redox regulation' and 'miscellaneous peroxidases'. Hence, we were able to identify distinct expression clusters of proteins with distinct functions.

  10. Novel Nuclear Protein ALC-INTERACTING PROTEIN1 is Expressed in Vascular and Mesocarp Cells in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Dong-Qiao Shi; Jie Liu; Wei-Cai Yang

    2008-01-01

    Pod shattering is an agronomical trait that is a result of the coordinated action of cell differentiation and separation. In Arabidopsis, pod shattering is controlled by a complex genetic network in which ALCATRAZ (ALC), a member of the basic helix-loop-helix family, is critical for cell separation during fruit dehiscence. Herein, we report the identification of ALC-INTERACTiNG PROTEIN1 (ACI1) via the yeast two-hybrid screen. ACI1 encodes a nuclear protein with a lysine-rich domain and a C-terminal serine-rich domain. ACI1 is mainly expressed in the vascular system throughout the plant and mesocarp of the valve in siliques. Our data showed that ACI1 interacts strongly with the N-terminal portion of ALC in yeast cells and in plant cells in the nucleus as demonstrated by bimolecular fluorescence complementation assay. Both ACl1 and ALC share an overlapping expression pattern, suggesting that they likely function together in planta. However, no detectable phenotype was found in plants with reduced ACI1 expression by RNA interference technology, suggesting that ACI1 may be redundant. Taken together, these data indicate that ALC may interact with ACll and its homologs to control cell separation during fruit dehiscence in Arabidopsis.

  11. Verification at the protein level of the PIF4-mediated external coincidence model for the temperature-adaptive photoperiodic control of plant growth in Arabidopsis thaliana.

    Science.gov (United States)

    Yamashino, Takafumi; Nomoto, Yuji; Lorrain, Séverine; Miyachi, Miki; Ito, Shogo; Nakamichi, Norihito; Fankhauser, Christian; Mizuno, Takeshi

    2013-03-01

    Plant circadian clock controls a wide variety of physiological and developmental events, which include the short-days (SDs)-specific promotion of the elongation of hypocotyls during de-etiolation and also the elongation of petioles during vegetative growth. In A. thaliana, the PIF4 gene encoding a phytochrome-interacting basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this photoperiodic control of plant growth. According to the proposed external coincidence model, the PIF4 gene is transcribed precociously at the end of night specifically in SDs, under which conditions the protein product is stably accumulated, while PIF4 is expressed exclusively during the daytime in long days (LDs), under which conditions the protein product is degraded by the light-activated phyB and also the residual proteins are inactivated by the DELLA family of proteins. A number of previous reports provided solid evidence to support this coincidence model mainly at the transcriptional level of the PIF 4 and PIF4-traget genes. Nevertheless, the diurnal oscillation profiles of PIF4 proteins, which were postulated to be dependent on photoperiod and ambient temperature, have not yet been demonstrated. Here we present such crucial evidence on PIF4 protein level to further support the external coincidence model underlying the temperature-adaptive photoperiodic control of plant growth in A. thaliana.

  12. Conserved Structural Motifs at the C-Terminus of Baculovirus Protein IE0 are Important for its Functions in Transactivation and Supporting hr5-mediated DNA Replication

    Directory of Open Access Journals (Sweden)

    Neta Luria

    2012-05-01

    Full Text Available IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV. IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  13. BARREN STALK FASTIGIATE1 is an AT-hook protein required for the formation of maize ears.

    Science.gov (United States)

    Gallavotti, Andrea; Malcomber, Simon; Gaines, Craig; Stanfield, Sharon; Whipple, Clinton; Kellogg, Elizabeth; Schmidt, Robert J

    2011-05-01

    Ears are the seed-bearing inflorescences of maize (Zea mays) plants and represent a crucial component of maize yield. The first step in the formation of ears is the initiation of axillary meristems in the axils of developing leaves. In the classic maize mutant barren stalk fastigiate1 (baf1), first discovered in the 1950s, ears either do not form or, if they do, are partially fused to the main stalk. We positionally cloned Baf1 and found that it encodes a transcriptional regulator containing an AT-hook DNA binding motif. Single coorthologs of Baf1 are found in syntenic regions of brachypodium (Brachypodium distachyon), rice (Oryza sativa), and sorghum (Sorghum bicolor), suggesting that the gene is likely present in all cereal species. Protein-protein interaction assays suggest that BAF1 is capable of forming homodimers and heterodimers with other members of the AT-hook family. Another transcriptional regulator required for ear initiation is the basic helix-loop-helix protein BARREN STALK1 (BA1). Genetic and expression analyses suggest that Baf1 is required to reach a threshold level of Ba1 expression for the initiation of maize ears. We propose that Baf1 functions in the demarcation of a boundary region essential for the specification of a stem cell niche.

  14. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    Science.gov (United States)

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  15. BARREN STALK FASTIGIATE1 Is an AT-Hook Protein Required for the Formation of Maize Ears[W][OA

    Science.gov (United States)

    Gallavotti, Andrea; Malcomber, Simon; Gaines, Craig; Stanfield, Sharon; Whipple, Clinton; Kellogg, Elizabeth; Schmidt, Robert J.

    2011-01-01

    Ears are the seed-bearing inflorescences of maize (Zea mays) plants and represent a crucial component of maize yield. The first step in the formation of ears is the initiation of axillary meristems in the axils of developing leaves. In the classic maize mutant barren stalk fastigiate1 (baf1), first discovered in the 1950s, ears either do not form or, if they do, are partially fused to the main stalk. We positionally cloned Baf1 and found that it encodes a transcriptional regulator containing an AT-hook DNA binding motif. Single coorthologs of Baf1 are found in syntenic regions of brachypodium (Brachypodium distachyon), rice (Oryza sativa), and sorghum (Sorghum bicolor), suggesting that the gene is likely present in all cereal species. Protein–protein interaction assays suggest that BAF1 is capable of forming homodimers and heterodimers with other members of the AT-hook family. Another transcriptional regulator required for ear initiation is the basic helix-loop-helix protein BARREN STALK1 (BA1). Genetic and expression analyses suggest that Baf1 is required to reach a threshold level of Ba1 expression for the initiation of maize ears. We propose that Baf1 functions in the demarcation of a boundary region essential for the specification of a stem cell niche. PMID:21540434

  16. An effective approach for identification of in vivo protein-DNA binding sites from paired-end ChIP-Seq data

    Directory of Open Access Journals (Sweden)

    Wilson Zoe A

    2010-02-01

    Full Text Available Abstract Background ChIP-Seq, which combines chromatin immunoprecipitation (ChIP with high-throughput massively parallel sequencing, is increasingly being used for identification of protein-DNA interactions in vivo in the genome. However, to maximize the effectiveness of data analysis of such sequences requires the development of new algorithms that are able to accurately predict DNA-protein binding sites. Results Here, we present SIPeS (Site Identification from Paired-end Sequencing, a novel algorithm for precise identification of binding sites from short reads generated by paired-end solexa ChIP-Seq technology. In this paper we used ChIP-Seq data from the Arabidopsis basic helix-loop-helix transcription factor ABORTED MICROSPORES (AMS, which is expressed within the anther during pollen development, the results show that SIPeS has better resolution for binding site identification compared to two existing ChIP-Seq peak detection algorithms, Cisgenome and MACS. Conclusions When compared to Cisgenome and MACS, SIPeS shows better resolution for binding site discovery. Moreover, SIPeS is designed to calculate the mappable genome length accurately with the fragment length based on the paired-end reads. Dynamic baselines are also employed to effectively discriminate closely adjacent binding sites, for effective binding sites discovery, which is of particular value when working with high-density genomes.

  17. Rbms3, an RNA-binding protein, mediates the expression of Ptf1a by binding to its 3'UTR during mouse pancreas development.

    Science.gov (United States)

    Lu, Chung-Kuang; Lai, Yi-Chyi; Chen, Hau-Ren; Chiang, Ming-Ko

    2012-07-01

    The development of the pancreas is a complicated process that is regulated on several levels. Pancreas transcription factor 1, alpha subunit (Ptf1a), also known as p48, is a pancreas-specific basic helix-loop-helix transcription factor that is critical for both exocrine pancreas development and maintenance of acinar cell differentiation. Based on a differential screening assay, we identified Rbms3, a gene encoding a glycine-rich RNA-binding protein, to be specifically expressed in the neural tube and the pancreatic rudiment of e10.5 embryos. The presence of Rbms3 in the early developing pancreas suggests that specific post-transcriptional regulation mechanisms play an important role in controlling pancreas development. In this study, we show that Rbms3 binds to the 3'UTR of Ptf1a mRNA, but not the 3'UTR of Pdx1, which is another pancreatic transcription factor. The ectopic expression of Rbms3 stimulates the translation of a reporter gene carrying the Ptf1a 3'UTR. In addition, when Rbms3 expression is suppressed in the AR42J-B13 pancreatic exocrine cell line, the expression of Ptf1a is also down-regulated. These results suggest that binding of Rbms3 to the 3'UTR of Ptf1a regulates the production of the Ptf1a protein and, thereby, indirectly regulates the expression of the Ptf1a downstream target genes.

  18. Functional and Structural Properties of a Novel Protein and Virulence Factor (sHIP) in Streptococcus pyogenes

    DEFF Research Database (Denmark)

    Wisniewska, Magdalena; Happonen, Lotta; Kahn, Fredrik;

    2014-01-01

    strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG......), and the name sHIP (streptococcal Histidine-rich glycoprotein Interacting Protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody...

  19. AcEST: DK950560 [AcEST

    Lifescience Database Archive (English)

    Full Text Available A0|UVRC_SYNWW UvrABC system protein C OS=Syntrophomonas w... 36 0.28 sp|Q9NX45|SOLH2_HUMAN Spermatogenesis- and oogenesis...HUMAN Spermatogenesis- and oogenesis-specific basic helix-loop-helix-containing protein 2 OS=Homo sapiens GN

  20. Interaction of MTG family proteins with NEUROG2 and ASCL1 in the developing nervous system.

    Science.gov (United States)

    Aaker, Joshua D; Patineau, Andrea L; Yang, Hyun-Jin; Ewart, David T; Nakagawa, Yasushi; McLoon, Steven C; Koyano-Nakagawa, Naoko

    2010-04-19

    During neural development, members of MTG family of transcriptional repressors are induced by proneural basic helix-loop-helix (bHLH) transcription factors and in turn inhibit the activity of the bHLH proteins, forming a negative feedback loop that regulates the normal progression of neurogenesis. Three MTG genes, MTG8, MTG16 and MTGR1, are expressed in distinct patterns in the developing nervous system. Various bHLH proteins are also expressed in distinct patterns. We asked whether there is a functional relationship between specific MTG and bHLH proteins in developing chick spinal cord. First, we examined if each MTG gene is induced by specific bHLH proteins. Although expression of NEUROG2, ASCL1 and MTG genes overlapped, the boundaries of gene expression did not match. Ectopic expression analysis showed that MTGR1 and NEUROD4, which show similar expression patterns, are regulated differently by NEUROG2 and ASCL1. Thus, our results show that expression of MTG genes is not regulated by a single upstream bHLH protein, but represents an integration of the activity of multiple regulators. Next, we asked if each MTG protein inhibits specific bHLH proteins. Transcription assay showed that NEUROG2 and ASCL1 are inhibited by MTGR1 and MTG16, and less efficiently by MTG8. Deletion mapping of MTGR1 showed that MTGR1 binds NEUROG2 and ASCL1 using multiple interaction surfaces, and all conserved domains are required for its repressor activity. These results support the model that MTG proteins form a higher-order repressor complex and modulate transcriptional activity of bHLH proteins during neurogenesis.

  1. Maintaining cholesterol homeostasis:Sterol regulatory element-binding proteins

    Institute of Scientific and Technical Information of China (English)

    Lutz W. Weber; Meinrad Boll; Andreas Stampfl

    2004-01-01

    The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins are members of the basic helix-loop-helix-leucine zipper (bHLHZip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP).The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones,cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.

  2. Targeting the bHLH transcriptional networks by mutated E proteins in experimental glioma.

    Science.gov (United States)

    Beyeler, Sarah; Joly, Sandrine; Fries, Michel; Obermair, Franz-Josef; Burn, Felice; Mehmood, Rashid; Tabatabai, Ghazaleh; Raineteau, Olivier

    2014-10-01

    Glioblastomas (GB) are aggressive primary brain tumors. Helix-loop-helix (HLH, ID proteins) and basic HLH (bHLH, e.g., Olig2) proteins are transcription factors that regulate stem cell proliferation and differentiation throughout development and into adulthood. Their convergence on many oncogenic signaling pathways combined with the observation that their overexpression in GB correlates with poor clinical outcome identifies these transcription factors as promising therapeutic targets. Important dimerization partners of HLH/bHLH proteins are E proteins that are necessary for nuclear translocation and DNA binding. Here, we overexpressed a wild type or a dominant negative form of E47 (dnE47) that lacks its nuclear localization signal thus preventing nuclear translocation of bHLH proteins in long-term glioma cell lines and in glioma-initiating cell lines and analyzed the effects in vitro and in vivo. While overexpression of E47 was sufficient to induce apoptosis in absence of bHLH proteins, dnE47 was necessary to prevent nuclear translocation of Olig2 and to achieve similar proapoptotic responses. Transcriptional analyses revealed downregulation of the antiapoptotic gene BCL2L1 and the proproliferative gene CDC25A as underlying mechanisms. Overexpression of dnE47 in glioma-initiating cell lines with high HLH and bHLH protein levels reduced sphere formation capacities and expression levels of Nestin, BCL2L1, and CDC25A. Finally, the in vivo induction of dnE47 expression in established xenografts prolonged survival. In conclusion, our data introduce a novel approach to jointly neutralize HLH and bHLH transcriptional networks activities, and identify these transcription factors as potential targets in glioma.

  3. Identification of interacting proteins with aryl hydrocarbon receptor in scallop Chlamys farreri by yeast two hybrid screening.

    Science.gov (United States)

    Cai, Yuefeng; Pan, Luqing; Miao, Jingjing; Liu, Tong

    2016-11-01

    The aryl hydrocarbon receptor (AhR) belongs to the basic-helix-loop helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors. AhR has been known primarily for its role in the regulation of several drug and xenobiotic metabolizing enzymes, as well as the mediation of the toxicity of certain xenobiotics, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Although the AhR is well-studied as a mediator of the toxicity of certain xenobiotics in marine bivalves, the normal physiological function remains unknown. In order to explore the function of the AhR, the bait protein expression plasmid pGBKT7-CfAhR and the cDNA library of gill from Chlamys farreri were constructed. By yeast two hybrid system, after multiple screening with the high screening rate medium, rotary verification, sequencing and bioinformatics analysis, the interactions of the CfAhR with receptor for activated protein kinase C 1 (RACK1), thyroid peroxidase-like protein (TPO), Toll-like receptor 4(TLR 4), androglobin-like, store-operated Ca(2+) entry (SocE), ADP/ATP carrier protein, cytochrome b, thioesterase, actin, ferritin subunit 1, poly-ubiquitin, short-chain collagen C4-like and one hypothetical protein in gill cells were identified. This study suggests that the CfAhR played fundamental roles in immune system homeostasis, oxidative stress response, and in grow and development of C. farreri. The elucidation of these protein interactions is of much importance both in understanding the normal physiological function of AhR, and as potential targets for further research on protein function in AhR interactions.

  4. The Arabidopsis SET-domain protein ASHR3 is involved in stamen development and interacts with the bHLH transcription factor ABORTED MICROSPORES (AMS).

    Science.gov (United States)

    Thorstensen, Tage; Grini, Paul E; Mercy, Inderjit S; Alm, Vibeke; Erdal, Sigrid; Aasland, Rein; Aalen, Reidunn B

    2008-01-01

    The Arabidopsis thaliana genome contains more than 30 genes encoding SET-domain proteins that are thought to be epigenetic regulators of gene expression and chromatin structure. SET-domain proteins can be divided into subgroups, and members of the Polycomb group (PcG) and trithorax group (trxG) have been shown to be important regulators of development. Both in animals and plants some of these proteins are components of multimeric protein complexes. Here, we have analyzed the Arabidopsis trxG protein ASHR3 which has a SET domain and pre- and post-SET domains similar to that of Ash1 in Drosophila. In addition to the SET domain, a divergent PHD finger is found in the N-terminus of the ASHR3 protein. As expected from SET-domain proteins involved in transcriptional activation, ASHR3 (coupled to GFP) localizes to euchromatin. A yeast two-hybrid screening revealed that the ASHR3 protein interacts with the putative basic helix-loop-helix (bHLH) transcription factor ABORTED MICROSPORES (AMS), which is involved in anther and stamen development in Arabidopsis. Deletion mapping indicated that both the PHD finger and the SET domain mediate the interaction between the two proteins. Overexpression of ASHR3 led in general to growth arrest, and specifically to degenerated anthers and male sterility. Expression analyses demonstrated that ASHR3 like AMS is expressed in the anther and in stamen filaments. We therefore propose that AMS can target ASHR3 to chromatin and regulate genes involved in stamen development and function.

  5. Molecular cloning of a novel human I-mfa domain-containing protein that differently regulates human T-cell leukemia virus type I and HIV-1 expression.

    Science.gov (United States)

    Thébault, S; Gachon, F; Lemasson, I; Devaux, C; Mesnard, J M

    2000-02-18

    Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.

  6. I-mfa domain proteins interact with Axin and affect its regulation of the Wnt and c-Jun N-terminal kinase signaling pathways.

    Science.gov (United States)

    Kusano, Shuichi; Raab-Traub, Nancy

    2002-09-01

    I-mfa has been identified as an inhibitor of myogenic basic helix-loop-helix transcription factors, and a related human I-mfa domain-containing protein (HIC) also has been identified as a protein that regulates Tat- and Tax-mediated expression of viral promoters. HIC and I-mfa represent a family of proteins that share a highly conserved cysteine-rich domain, termed the I-mfa domain. We show here that both I-mfa domain proteins, HIC and I-mfa, interacted in vivo with the Axin complex through their C-terminal I-mfa domains. This interaction inhibited Axin-mediated downregulation of free levels of cytosolic beta-catenin. I-mfa and HIC also both directly interacted with lymphocyte enhancer factor (LEF); however, I-mfa but not HIC significantly inhibited reporter constructs regulated by beta-catenin. The overexpression of HIC but not I-mfa decreased the inhibitory effects of Axin on beta-catenin-regulated reporter constructs, while both HIC and I-mfa decreased Axin-mediated c-Jun N-terminal kinase (JNK) activation. These data reveal for the first time that I-mfa domain proteins interact with the Axin complex and affect Axin regulation of both the Wnt and the JNK activation pathways. Interestingly, HIC differs from I-mfa in that I-mfa affects both Axin function and T-cell factor- or LEF-regulated transcription in the Wnt signaling pathway while HIC affects primarily Axin function.

  7. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

    Science.gov (United States)

    Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

    2011-12-01

    The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology.

  8. The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation.

    Science.gov (United States)

    Sun, Tong; Fu, Junjiang; Shen, Tao; Lin, Xia; Liao, Lan; Feng, Xin-Hua; Xu, Jianming

    2016-05-27

    Twist1 is a basic helix-loop-helix transcription factor that strongly promotes epithelial-to-mesenchymal transition, migration, invasion, and metastasis of cancer cells. The MAPK-phosphorylated Twist1 on its serine 68 (Ser(P)(68)-Twist1) has a significantly enhanced stability and function to drive cancer cell invasion and metastasis. However, the phosphatase that dephosphorylates Ser(P)(68)-Twist1 and destabilizes Twist1 has not been identified and characterized. In this study, we screened a serine/threonine phosphatase cDNA expression library in HEK293T cells with ectopically coexpressed Twist1. We found that the small C-terminal domain phosphatase 1 (SCP1) specifically dephosphorylates Ser(P)(68)-Twist1 in both cell-free reactions and living cells. SCP1 uses its amino acid residues 43-63 to interact with the N terminus of Twist1. Increased SCP1 expression in cells decreased Ser(P)(68)-Twist1 and total Twist1 proteins, whereas knockdown of SCP1 increased Ser(P)(68)-Twist1 and total Twist1 proteins. Furthermore, the levels of SCP1 are negatively correlated with Twist1 protein levels in several cancer cell lines. SCP1-dephosphorylated Twist1 undergoes fast degradation via the ubiquitin-proteasome pathway. Importantly, an increase in SCP1 expression in breast cancer cells with either endogenous or ectopically expressed Twist1 largely inhibits the Twist1-induced epithelial-to-mesenchymal transition phenotype and the migration and invasion capabilities of these cells. These results indicate that SCP1 is the phosphatase that counterregulates the MAPK-mediated phosphorylation of Ser(68)-Twist1. Thus, an increase in SCP1 expression and activity may be a useful strategy for eliminating the detrimental roles of Twist1 in cancer cells.

  9. Solitons and Collapse in the lambda-repressor protein

    CERN Document Server

    Krokhotin, Andrey; Niemi, Antti J

    2012-01-01

    The enterobacteria lambda phage is a paradigm temperate bacteriophage. Its lysogenic and lytic life cycles echo competition between the DNA binding $\\lambda$-repressor (CI) and CRO proteins. Here we scrutinize the structure, stability and folding pathways of the $\\lambda$-repressor protein, that controls the transition from the lysogenic to the lytic state. We first investigate the super-secondary helix-loop-helix composition of its backbone. We use a discrete Frenet framing to resolve the backbone spectrum in terms of bond and torsion angles. Instead of four, there appears to be seven individual loops. We model the putative loops using an explicit soliton Ansatz. It is based on the standard soliton profile of the continuum nonlinear Schr\\"odinger equation. The accuracy of the Ansatz far exceeds the B-factor fluctuation distance accuracy of the experimentally determined protein configuration. We then investigate the folding pathways and dynamics of the $\\lambda$-repressor protein. We introduce a coarse-graine...

  10. Id transcriptional regulators in adipogenesis and adipose tissue metabolism.

    Science.gov (United States)

    Patil, Mallikarjun; Sharma, Bal Krishan; Satyanarayana, Ande

    2014-06-01

    Id proteins (Id1-Id4) are helix-loop-helix (HLH) transcriptional regulators that lack a basic DNA binding domain. They act as negative regulators of basic helix-loop-helix (bHLH) transcription factors by forming heterodimers and inhibit their DNA binding and transcriptional activity. Id proteins are implicated in the regulation of various cellular mechanisms such as cell proliferation, cellular differentiation, cell fate determination, angiogenesis and tumorigenesis. A handful of recent studies also disclosed that Id proteins have critical functions in adipocyte differentiation and adipose tissue metabolism. Here, we reviewed the progress made thus far in understanding the specific functions of Id proteins in adipose tissue differentiation and metabolism. In addition to reviewing the known mechanisms of action, we also discuss possible additional mechanisms in which Id proteins might participate in regulating adipogenic and metabolic pathways.

  11. The P3 domain of eukaryotic RNases P/MRP: making a protein-rich RNA-based enzyme.

    Science.gov (United States)

    Perederina, Anna; Krasilnikov, Andrey S

    2010-01-01

    Nuclear Ribonuclease (RNase) P is a universal essential RNA-based enzyme made of a catalytic RNA component and a protein part; eukaryotic RNase P is closely related to a universal eukaryotic ribonucleoprotein RNase MRP. The protein part of the eukaryotic RNases P/MRP is dramatically more complex than that in bacterial and archaeal RNases P. The increase in the complexity of the protein part in eukaryotic RNases P/MRP was accompanied by the appearance of a novel structural element in the RNA component: an essential and phylogenetically conserved helix-loop-helix P3 RNA domain. The crystal structure of the P3 RNA domain in a complex with protein components Pop6 and Pop7 has been recently solved. Here we discuss the most salient structural features of the P3 domain as well as its possible role in the evolutionary transition to the protein-rich eukaryotic RNases P/MRP.

  12. Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities.

    Science.gov (United States)

    Firulli, Beth A; Krawchuk, Dayana; Centonze, Victoria E; Vargesson, Neil; Virshup, David M; Conway, Simon J; Cserjesi, Peter; Laufer, Ed; Firulli, Anthony B

    2005-04-01

    Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage-dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A- and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A-mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.

  13. Id4 Promotes Senescence and Sensitivity to Doxorubicin-induced Apoptosis in DU145 Prostate Cancer Cells

    OpenAIRE

    Carey, Jason P; Knowell, Ashley Evans; Chinaranagari, Swathi; Chaudhary, Jaideep

    2013-01-01

    Inhibitor of differentiation proteins (Id1, 2, 3 and 4) are dominant negative regulators of basic helix loop helix transcription factors and play dominant roles in cancer cells, spanning several molecular pathways including senescence, invasion, metastasis, proliferation and apoptosis. In contrast to high Id1, Id2 and Id3 expression, the expression of Id4 is epigenetically silenced in prostate cancer. In the present study we demonstrated a novel role of Id4, that of promotion of cellular sene...

  14. Identifying Novel Helix-Loop-Helix Genes in "Caenorhabditis elegans" through a Classroom Demonstration of Functional Genomics

    Science.gov (United States)

    Griffin, Vernetta; McMiller, Tracee; Jones, Erika; Johnson, Casonya M.

    2003-01-01

    A 14-week, undergraduate-level Genetics and Population Biology course at Morgan State University was modified to include a demonstration of functional genomics in the research laboratory. Students performed a rudimentary sequence analysis of the "Caenorhabditis elegans" genome and further characterized three sequences that were predicted to encode…

  15. Analysis of interactions between heterologously produced bHLH and MYB proteins that regulate anthocyanin biosynthesis: quantitative interaction kinetics by Microscale Thermophoresis.

    Science.gov (United States)

    Nemie-Feyissa, Dugassa; Heidari, Behzad; Blaise, Mickael; Lillo, Cathrine

    2015-03-01

    The two Arabidopsis basic-helix-loop-helix transcription factors GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) are positive regulators of anthocyanin biosynthesis, and form protein complexes (MBW complexes) with various R2R3 MYB transcription factors and a WD40 repeat protein TRANSPARENT TESTA GLABROUS1 (TTG1). In earlier studies, GL3, in contrast to EGL3, was shown to be essential for accumulation of anthocyanins in response to nitrogen depletion. This could not be fully explained by the strong induction of GL3 in response to nitrogen depletion because the EGL3 transcripts were constitutively at a relatively high level and transcripts levels of the two genes were similar under nitrogen depletion. Here the GL3 and EGL3 proteins were characterized with respect to their affinities for PRODUCTION OF ANTHOCYANIN PIGMENT2 (PAP2), a R2R3-MYB which is induced by nitrogen depletion and is part of MBW complexes promoting anthocyanin synthesis. GL3 and EGL3 were also tested for their binding to MYBL2, a negative regulator of anthocyanin synthesis and MBW complexes. Using heterologously expressed proteins and Microscale Thermophoresis, GL3 showed binding constants (Kd) of 3.5±1.7 and 22.7±3.7 μM, whereas EGL3 showed binding constants of 7.5±2.3 and 8.9±1.4 μM for PAP2 and MYBL2, respectively. This implies that MYBL2 will not inhibit a MBW complex containing GL3 as easily as for a complex containing EGL3. In transgenic plants where EGL3 reaches high concentrations compared with MYBL2 the equilibrium is shifted and MYBL2 is not likely to be an efficient competitor, hence anthocyanin formation could be restored by either EGL3 or GL3 genes when overexpressed by help of the 35S promoter. The present work underpins that GL3 is essential for anthocyanin accumulation under nitrogen depletion not only due to transcriptional activation, but also because of binding properties to proteins promoting or inhibiting the activity of the MBW complex.

  16. Exploring the repeat protein universe through computational protein design.

    Science.gov (United States)

    Brunette, T J; Parmeggiani, Fabio; Huang, Po-Ssu; Bhabha, Gira; Ekiert, Damian C; Tsutakawa, Susan E; Hura, Greg L; Tainer, John A; Baker, David

    2015-12-24

    A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

  17. Protein Folding: Search for Basic Physical Models

    Directory of Open Access Journals (Sweden)

    Ivan Y. Torshin

    2003-01-01

    Full Text Available How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context of in vivo protein folding (which has been studied only for a few proteins, the roles of the fundamental physical forces in the in vitro folding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces. Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

  18. Functional and structural properties of a novel protein and virulence factor (Protein sHIP) in Streptococcus pyogenes.

    Science.gov (United States)

    Wisniewska, Magdalena; Happonen, Lotta; Kahn, Fredrik; Varjosalo, Markku; Malmström, Lars; Rosenberger, George; Karlsson, Christofer; Cazzamali, Giuseppe; Pozdnyakova, Irina; Frick, Inga-Maria; Björck, Lars; Streicher, Werner; Malmström, Johan; Wikström, Mats

    2014-06-27

    Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.

  19. Gibberellins control fruit patterning in Arabidopsis thaliana.

    Science.gov (United States)

    Arnaud, Nicolas; Girin, Thomas; Sorefan, Karim; Fuentes, Sara; Wood, Thomas A; Lawrenson, Tom; Sablowski, Robert; Østergaard, Lars

    2010-10-01

    The Arabidopsis basic helix-loop-helix (bHLH) proteins INDEHISCENT (IND) and ALCATRAZ (ALC) specify tissues required for fruit opening that have major roles in seed dispersal and plant domestication. Here, we show that synthesis of the phytohormone gibberellin is a direct and necessary target of IND, and that ALC interacts directly with DELLA repressors, which antagonize ALC function but are destabilized by gibberellin. Thus, the gibberellin/DELLA pathway has a key role in patterning the Arabidopsis fruit, and the interaction between DELLA and bHLH proteins, previously shown to connect gibberellin and light responses, is a versatile regulatory module also used in tissue patterning.

  20. Myogenic repressor I-mfa interferes with the function of Zic family proteins.

    Science.gov (United States)

    Mizugishi, Kiyomi; Hatayama, Minoru; Tohmonda, Takahide; Ogawa, Miyuki; Inoue, Takashi; Mikoshiba, Katsuhiko; Aruga, Jun

    2004-07-16

    Zinc finger proteins belonging to the Zic family control several developmental processes such as patterning of the axial skeleton. Here we mapped the transcriptional regulatory domains in Zic2 protein and identified a protein which specifically binds to one of them. In the mapping experiments, an amino-terminal region was identified as transcriptional regulatory domains. A search for proteins binding to the amino terminal domain of Zic2 revealed that inhibitor of MyoD family (I-mfa) protein, which has been identified as a repressor of myogenic helix-loop-helix class transcription factors, can physically interact with the amino terminal domain. When Zic1-3 and I-mfa proteins were co-expressed in cultured cells, nuclear import of the Zic proteins was inhibited. Consequently, I-mfa inhibited transcriptional activation by the Zic proteins in cultured cells. These results suggest that the physical and functional interaction between Zic and I-mfa proteins can play a role in the vertebrate development.

  1. Calciomics:prediction and analysis of EF-hand calcium binding proteins by protein engineering

    Institute of Scientific and Technical Information of China (English)

    YANG; Jenny; Jie

    2010-01-01

    Ca2+ plays a pivotal role in the physiology and biochemistry of prokaryotic and mammalian organisms.Viruses also utilize the universal Ca2+ signal to create a specific cellular environment to achieve coexistence with the host,and to propagate.In this paper we first describe our development of a grafting approach to understand site-specific Ca2+ binding properties of EF-hand proteins with a helix-loop-helix Ca2+ binding motif,then summarize our prediction and identification of EF-hand Ca2+ binding sites on a genome-wide scale in bacteria and virus,and next report the application of the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor(Shr) of Streptococcus pyrogenes and the nonstructural protein 1(nsP1) of Sindbis virus.When methods such as the grafting approach are developed in conjunction with prediction algorithms we are better able to probe continuous Ca2+-binding sites that have been previously underrepresented due to the limitation of conventional methodology.

  2. Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome

    OpenAIRE

    2001-01-01

    The Saethre-Chotzen syndrome is characterized by premature fusion of cranial sutures resulting from mutations in Twist, a basic helix-loop-helix (bHLH) transcription factor. We have identified Twist target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome with a Twist mutation that introduces a stop codon upstream of the bHLH domain. We observed that Twist mRNA and protein levels were reduced in mutant cells and that the Twist mutation increased c...

  3. Yas3p, an Opi1 Family Transcription Factor, Regulates Cytochrome P450 Expression in Response to n-Alkanes in Yarrowia lipolytica*

    OpenAIRE

    Hirakawa, Kiyoshi; Kobayashi, Satoshi; Inoue, Takuro; Endoh-Yamagami, Setsu; Fukuda, Ryouichi; Ohta, Akinori

    2009-01-01

    In the alkane-assimilating yeast Yarrowia lipolytica, the expression of ALK1, a gene encoding cytochrome P450 that catalyzes the first step of n-alkane oxidation, is induced by n-alkanes. We previously demonstrated that two basic helix-loop-helix proteins, Yas1p and Yas2p, activate the transcription of ALK1 in an alkane-dependent manner by forming a heterocomplex and binding to alkane-responsive element 1 (ARE1), a cis-acting element in the ALK1 promoter. Here we i...

  4. Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-hand calcium-binding protein.

    Science.gov (United States)

    Maciejewski, J J; Vacchiano, E J; McCutcheon, S M; Buhse, H E

    1999-01-01

    The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.

  5. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD

    Directory of Open Access Journals (Sweden)

    Yu Wei-Hsuan

    2012-05-01

    Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional “domain” found to date for the matrixin family. Conclusions The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.

  6. Analysis of the DNA-binding and dimerization activities of Neurospora crassa transcription factor NUC-1.

    Science.gov (United States)

    Peleg, Y; Metzenberg, R L

    1994-12-01

    NUC-1, a positive regulatory protein of Neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. The carboxy-terminal end of the NUC-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. Bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of NUC-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. These upstream sequences contain the core sequence, CACGTG, a target for many helix-loop-helix proteins. A large loop region (47 amino acids) separates the helix I and helix II domains. Mutations and deletion within the loop region did not interfere with the in vitro or in vivo functions of the protein. Immediately carboxy-proximal to the helix II domain, the NUC-1 protein contains an atypical zipper domain which is essential for function. This domain consists of a heptad repeat of alanine and methionine rather than leucine residues. Analysis of mutant NUC-1 proteins suggests that the helix II and the zipper domains are essential for the protein dimerization, whereas the basic and the helix I domains are involved in DNA binding. The helix I domain, even though likely to participate in dimer formation while NUC-1 is bound to DNA, is not essential for in vitro dimerization.

  7. A wheat R2R3-MYB protein PURPLE PLANT1 (TaPL1) functions as a positive regulator of anthocyanin biosynthesis.

    Science.gov (United States)

    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.

  8. 15N NMR relaxation studies of calcium-loaded parvalbumin show tight dynamics compared to those of other EF-hand proteins

    DEFF Research Database (Denmark)

    Baldellon, C; Alattia, J R; Strub, M P;

    1998-01-01

    for the rat alpha-parvalbumin calcium-loaded form are (1) the extreme rigidity of the helix-loop-helix EF-hand motifs and the linker segment connecting them, (2) the N and C termini of the protein being restricted in their mobility, (3) a conformational exchange occurring at the kink of helix D, and (4......) the residue at relative position 2 in the Ca2+-binding sites having an enhanced mobility. Comparison of the Ca2+-binding EF-hand domains of alpha-parvalbumin-Ca2+, calbindin-Ca2+, and calmodulin-Ca2+ shows that parvalbumin is probably the most rigid of the EF-hand proteins. It also illustrates the dynamical...... properties which are conserved in the EF-hand domains from different members of this superfamily: (1) a tendency toward higher mobility of NH vectors at relative position 2 in the Ca2+-binding loop, (2) a restricted mobility for the other residues in the binding loop, and (3) an overall rigidity...

  9. Specific interaction of central nervous system myelin basic protein with lipids effects of basic protein on glucose leakage from liposomes

    NARCIS (Netherlands)

    Gould, R.M.; London, Y.

    1972-01-01

    The leakage from liposomes preloaded with glucose was continuously monitored in a Perkin-Elmer Model 356 dual beam spectrophotometer using an enzyme-linked assay system. The central nervous system myelin basic protein (A1 protein) caused a 3–4-fold increase in the rate of leakage from liposomes prep

  10. Mixed lineage kinase phosphorylates transcription factor E47 and inhibits TrkB expression to link neuronal death and survival pathways.

    Science.gov (United States)

    Pedraza, Neus; Rafel, Marta; Navarro, Isis; Encinas, Mario; Aldea, Martí; Gallego, Carme

    2009-11-20

    E47 is a basic helix-loop-helix transcription factor involved in neuronal differentiation and survival. We had previously shown that the basic helix-loop-helix protein E47 binds to E-box sequences within the promoter of the TrkB gene and activates its transcription. Proper expression of the TrkB receptor plays a key role in development and function of the vertebrate nervous system, and altered levels of TrkB have been associated with important human diseases. Here we show that E47 interacts with MLK2, a mixed lineage kinase (MLK) involved in JNK-mediated activation of programmed cell death. MLK2 enhances phosphorylation of the AD2 activation domain of E47 in vivo in a JNK-independent manner and phosphorylates in vitro defined serine and threonine residues within a loop-helix structure of AD2 that also contains a putative MLK docking site. Although these residues are essential for MLK2-mediated inactivation of E47, inhibition of MLKs by CEP11004 causes up-regulation of TrkB at a transcriptional level in cerebellar granule neurons and differentiating neuroblastoma cells. These findings allow us to propose a novel mechanism by which MLK regulates TrkB expression through phosphorylation of an activation domain of E47. This molecular link would explain why MLK inhibitors not only prevent activation of cell death processes but also enhance cell survival signaling as a key aspect of their neuroprotective potential.

  11. Solitons and protein folding: An In Silico experiment

    Science.gov (United States)

    Ilieva, N.; Dai, J.; Sieradzan, A.; Niemi, A.

    2015-10-01

    Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen's dogma states that the native 3D shape of a protein is completely determined by protein's amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolved problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix-loop-helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics.

  12. 酵母双杂交诱饵载体pGBKT7_MYC2构建及表达鉴定%Construction and Expressional Identification of Yeast Two - hybrid Bait Expression Vector pGBKT7_MYC2

    Institute of Scientific and Technical Information of China (English)

    刘武; 肖牧; 阮颖; 刘春林

    2012-01-01

    MYC2是一类含有helix - loop - helix (bHLH)结构域的转录因子.为进一步研究MYC2因子在植物防御抗性中的作用及其参与植物JA,SA等信号途径的作用机制,克隆了拟南芥的MYC2基因,以此构建了pGBKT7_MYC2酵母双杂交载体,通过Western blotting验证表明,该载体能在酵母细胞里正常表达.%MYC2, a basic helix - loop - helix (bHLH) domain - containing TF, acts as a positive regulator of abseisic acid - dependent drought responses and is also induced in JA - mediated responses. Taking the cDNA from Arabidophsis as the template, full - length COS of MYC2 had been cloned and then was ligated into the bait expression vector pG-BKT7. After verified by digestion, the bait vector was transformed into Clod yeast cells, and the expression of MYC2 gene was checked by Western blotting. As a result, the bait expression vector pGBKT7_MYC2 was constructed successfully, which laid the foundation for screening target proteins interacting and mapping the network with the MYC2 protein.

  13. Technical updates to basic proteins focalization using IPG strips

    Directory of Open Access Journals (Sweden)

    Dépagne Jordane

    2012-09-01

    Full Text Available Abstract Background Gel-based proteomic is a popular and versatile method of global protein separation and quantification. However, separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility. Results Three different protocols of protein loading were compared using MCF7 cells proteins. In-gel rehydration, cup-loading and paper-bridge loading were first compared using 6–11 IPG strips, as attempted, in-gel rehydration gave large horizontal steaking; paper-bridge loading displayed an interesting spot resolution, but with a predominant loss of material; cup-loading was selected as the most relevant method, but still needing improvement. Twelve cup-loading protocols were compared with various strip rehydration, and cathodic wick solutions. Destreak appeared as better than DTT for strip rehydration; the use of isopropanol gave no improvement. The best 2DE separation was observed with cathodic wicks filled with rehydration solution complemented with DTT. Paper-bridge loading was finally analyzed using non-limited samples, such as bovine milk. In this case, new spots of basic milk proteins were observed, with or without paper wicks. Conclusion According to this technical study of basic protein focalization with IPG strips, the cup-loading protocol clearly displayed the best resolution and reproducibility: strips were first rehydrated with standard solution, then proteins were cup-loaded with destreak reagent, and focalisation was performed with cathodic wicks filled with rehydration solution and DTT. Paper-bridge loading could be as well used, but preferentially with non-limited samples.

  14. Solitons and collapse in the λ-repressor protein

    Science.gov (United States)

    Krokhotin, Andrey; Lundgren, Martin; Niemi, Antti J.

    2012-08-01

    The enterobacteria lambda phage is a paradigm temperate bacteriophage. Its lysogenic and lytic life cycles echo competition between the DNA binding λ-repressor (CI) and CRO proteins. Here we scrutinize the structure, stability, and folding pathways of the λ-repressor protein, which controls the transition from the lysogenic to the lytic state. We first investigate the supersecondary helix-loop helix composition of its backbone. We use a discrete Frenet framing to resolve the backbone spectrum in terms of bond and torsion angles. Instead of four, there appears to be seven individual loops. We model the putative loops using an explicit soliton Ansatz. It is based on the standard soliton profile of the continuum nonlinear Schrödinger equation. The accuracy of the Ansatz far exceeds the B-factor fluctuation distance accuracy of the experimentally determined protein configuration. We then investigate the folding pathways and dynamics of the λ-repressor protein. We introduce a coarse-grained energy function to model the backbone in terms of the Cα atoms and the side chains in terms of the relative orientation of the Cβ atoms. We describe the folding dynamics in terms of relaxation dynamics and find that the folded configuration can be reached from a very generic initial configuration. We conclude that folding is dominated by the temporal ordering of soliton formation. In particular, the third soliton should appear before the first and second. Otherwise, the DNA binding turn does not acquire its correct structure. We confirm the stability of the folded configuration by repeated heating and cooling simulations.

  15. Synergistic interactions of lipids and myelin basic protein

    Science.gov (United States)

    Hu, Yufang; Doudevski, Ivo; Wood, Denise; Moscarello, Mario; Husted, Cynthia; Genain, Claude; Zasadzinski, Joseph A.; Israelachvili, Jacob

    2004-09-01

    This report describes force measurements and atomic force microscope imaging of lipid-protein interactions that determine the structure of a model membrane system that closely mimics the myelin sheath. Our results suggest that noncovalent, mainly electrostatic and hydrophobic, interactions are responsible for the multilamellar structure and stability of myelin. We find that myelin basic protein acts as a lipid coupler between two apposed bilayers and as a lipid "hole-filler," effectively preventing defect holes from developing. From our protein-mediated-adhesion and force-distance measurements, we develop a simple quantitative model that gives a reasonably accurate picture of the molecular mechanism and adhesion of bilayer-bridging proteins by means of noncovalent interactions. The results and model indicate that optimum myelin adhesion and stability depend on the difference between, rather than the product of, the opposite charges on the lipid bilayers and myelin basic protein, as well as on the repulsive forces associated with membrane fluidity, and that small changes in any of these parameters away from the synergistically optimum values can lead to large changes in the adhesion or even its total elimination. Our results also show that the often-asked question of which membrane species, the lipids or the proteins, are the "important ones" may be misplaced. Both components work synergistically to provide the adhesion and overall structure. A better appreciation of the mechanism of this synergy may allow for a better understanding of stacked and especially myelin membrane structures and may lead to better treatments for demyelinating diseases such as multiple sclerosis. lipid-protein interactions | myelin membrane structure | membrane adhesion | membrane regeneration/healing | demyelinating diseases

  16. Basic Endochitinases Are Major Proteins in Castanea sativa Cotyledons.

    Science.gov (United States)

    Collada, C; Casado, R; Fraile, A; Aragoncillo, C

    1992-10-01

    Basic endochitinases are abundant proteins in Castanea sativa Mill. cotyledons. Three basic chitinases were purified with molecular masses of 25, 26, and 32 kD (Ch1, Ch2, and Ch3) and with isoelectric points between 8 and 9.5. Antibodies raised against Ch1 cross-reacted with Ch2 and Ch3. However, Ch3 showed differences when compared with the other two enzymes, especially in its higher cysteine content. The size, amino acid composition, and N-terminal sequence of Ch1 indicate that it is a class II endochitinase and, therefore, has no cysteine-rich hevein domain. Ch1 inhibits the growth of the fungus Trichoderma viride. The biological role of these endochitinases is discussed.

  17. Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum

    Directory of Open Access Journals (Sweden)

    Tian Shengli

    2009-08-01

    Full Text Available Abstract Background Serine/arginine (SR protein-specific kinases (SRPKs are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379 from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. Results We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS. Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328 was localized in the alkaline Ω-loop of a helix-loop-helix motif (HLHM of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. Conclusion This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Ω-loop structure could play a crucial role in the NLS function of PSRPK.

  18. Design and characterization of a membrane protein unfolding platform in lipid bilayers.

    Directory of Open Access Journals (Sweden)

    Vincent G Nadeau

    Full Text Available Accurate measurement of membrane protein stability--and particularly how it may vary as a result of disease-phenotypic mutations--ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR, wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding--results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.

  19. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

    Science.gov (United States)

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-03-18

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.

  20. Structural, functional, and mutational analysis of the NblA protein provides insight into possible modes of interaction with the phycobilisome.

    Science.gov (United States)

    Dines, Monica; Sendersky, Eleonora; David, Liron; Schwarz, Rakefet; Adir, Noam

    2008-10-31

    The enormous macromolecular phycobilisome antenna complex (>4 MDa) in cyanobacteria and red algae undergoes controlled degradation during certain forms of nutrient starvation. The NblA protein (approximately 6 kDa) has been identified as an essential component in this process. We have used structural, biochemical, and genetic methods to obtain molecular details on the mode of action of the NblA protein. We have determined the three-dimensional structure of the NblA protein from both the thermophilic cyanobacterium Thermosynechococcus vulcanus and the mesophilic cyanobacterium Synechococcus elongatus sp. PCC 7942. The NblA monomer has a helix-loop-helix motif which dimerizes into an open, four-helical bundle, identical to the previously determined NblA structure from Anabaena. Previous studies indicated that mutations to NblA residues near the C terminus impaired its binding to phycobilisome proteins in vitro, whereas the only mutation known to affect NblA function in vivo is located near the protein N terminus. We performed random mutagenesis of the S. elongatus nblA gene which enabled the identification of four additional amino acids crucial for NblA function in vivo. This data shows that essential amino acids are not confined to the protein termini. We also show that expression of the Anabaena nblA gene complements phycobilisome degradation in an S. elongatus NblA-null mutant despite the low homology between NblAs of these cyanobacteria. We propose that the NblA interacts with the phycobilisome via "structural mimicry" due to similarity in structural motifs found in all phycobiliproteins. This suggestion leads to a new model for the mode of NblA action which involves the entire NblA protein.

  1. Structure-Function Analysis of the v-Myc Oncoprotein

    Science.gov (United States)

    1997-06-01

    transcription activation domain (TAD) and a carboxy-terminal basic helix-loop-helix/ leucine zipper (bHLH/LZ) motif (Henriksson and Luscher , 1996). Work by...U. (1996). Active repression mechanisms of eukaryotic transcription repressors. Trends in Genetics 12: 229-234. Henriksson, M. and Luscher , B. (1996

  2. Translational control of TWIST1 expression in MCF-10A cell lines recapitulating breast cancer progression

    DEFF Research Database (Denmark)

    Nairismägi, Maarja-Liisa; Vislovukh, Andrii; Meng, Q

    2012-01-01

    TWIST1 is a highly conserved basic helix-loop-helix transcription factor that promotes epithelial–mesenchymal transition (EMT). Its misregulation has been observed in various types of tumors. Using the MCF-10A-series of cell lines that recapitulate the early stages of breast cancer formation...

  3. Domain Modeling: NP_005797.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_005797.1 chr21 Crystal Structure of the basic-helix-loop-helix domains of the he...terodimer E47/NeuroD1 bound to DNA p2ql2d_ chr21/NP_005797.1/NP_005797.1_holo_110-166.pdb blast 113I,114N,11

  4. The window period of NEUROGENIN3 during human gestation

    NARCIS (Netherlands)

    R.J. Salisbury (Rachel J.); J. Blaylock (Jennifer); A.A. Berry (Andrew A.); R.E. Jennings (Rachel E.); R.R. de Krijger (Ronald); K.P. Hanley (Karen Piper); N.A. Hanley (Neil A)

    2014-01-01

    textabstractThe basic helix-loop-helix transcription factor, NEUROG3, is critical in causing endocrine commitment from a progenitor cell population in the developing pancreas. In human, NEUROG3 has been detected from 8 weeks postconception (wpc). However, the profile of its production and when it ce

  5. AcEST: DK944591 [AcEST

    Lifescience Database Archive (English)

    Full Text Available e-15 sp|P0C7P8|Y1615_ARATH Uncharacterized basic helix-loop-helix pro... 79 2e-14 sp|P49259|PLA2R...TEKQGEKTWICFVVEGQNNKVMHRMDILWSLVQ 725 >sp|P49259|PLA2R_BOVIN Secretory phospholipase A2 receptor OS=Bos taurus GN=PLA2R

  6. Upregulation of the transcription factor TFEB in t(6;11)(p21;q13)-positive renal cell carcinomas due to promoter substitution

    NARCIS (Netherlands)

    Kuiper, RP; Schepens, M; Thijssen, J; van Asseldonk, M; van den Berg, E; Bridge, J; Schuuring, E; Schoenmakers, EFPM; van Kessel, AG

    2003-01-01

    The MITF/TFE subfamily of basic helix-loop-helix leucine-zipper (bHLH-LZ) transcription factors consists of four closely related members, TFE3, TFEB, TFEC and MITF, which can form both homo- and heterodimers. Previously, we demonstrated that in t(X;1)(p11;q21)-positive renal cell carcinomas (RCCs),

  7. Domain Modeling: NP_004307.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_004307.2 chr12 Crystal Structure of the basic-helix-loop-helix domains of the he...terodimer E47/NeuroD1 bound to DNA p2ql2d_ chr12/NP_004307.2/NP_004307.2_holo_119-175.pdb psi-blast 120V,122

  8. Functional domains of the transcriptional activator NUC-1 in Neurospora crassa.

    Science.gov (United States)

    Kang, S

    1993-08-25

    The NUC-1 regulatory protein directly controls the transcription of these genes and how the activity enzymes in Neurospora crassa. To understand how NUC-1 regulates the transcription of these genes and how the activity of NUC-1 is modulated by other regulatory proteins, two putative functional domains of NUC-1 were analysed: the DNA-binding domain and the regulatory domain. The DNA-binding activity of NUC-1 has not been directly demonstrated; however, results of deletion analysis, sequence analysis of the nuc-1 mutant alleles, and strong sequence similarity with the Saccharomyces cerevisiae PHO4 protein strongly suggest that the basic helix-loop-helix motif of NUC-1 forms a DNA-binding domain. Deletion and mutant analyses revealed that 39 amino acid (aa) residues (aa 463 to 501), or fewer, of NUC-1 are interacting with the negative regulatory factor(s), the PREG and/or PGOV proteins.

  9. EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada).

    Science.gov (United States)

    Prasath, Thiruketheeswaran; Greven, Hartmut; D'Haese, Jochen

    2012-06-01

    Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships.

  10. Sequence Classification: 785813 [

    Lifescience Database Archive (English)

    Full Text Available -1, mammalian HIF (hypoxia inducible factor) homolog, Helix Loop Helix containing protein (79.9 kD) (hif-1) || http://www.ncbi.nlm.nih.gov/protein/25153882 ... ...Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|25153882|ref|NP_508008.2| hypoxia Inducible Factor HIF

  11. Separation of basic proteins from Leishmania using a combination of Free flow electrophoresis (FFE) and 2D electrophoresis (2-DE) under basic conditions.

    Science.gov (United States)

    Brotherton, Marie-Christine; Racine, Gina; Ouellette, Marc

    2015-01-01

    Basic proteins, an important class of proteins in intracellular organisms such as Leishmania, are usually underrepresented on 2D gels. This chapter describes a method combining basic proteins fractionation using Free flow electrophoresis in isoelectric focusing mode (IEF-FFE) followed by protein separation using two-dimensional gel electrophoresis (2-DE) in basic conditions. The combination of these two techniques represents a great improvement for the visualization of Leishmania proteins with basic pI using 2D gels.

  12. Translocation of Neurospora crassa transcription factor NUC-1 into the nucleus is induced by phosphorus limitation.

    Science.gov (United States)

    Peleg, Y; Addison, R; Aramayo, R; Metzenberg, R L

    1996-09-01

    NUC-1, a basic helix-loop-helix zipper protein, activates the expression of several genes involved in phosphorus acquisition in Neurospora crassa. In the present study we investigated whether posttranscriptional mechanisms control the activity of NUC-1. The NUC-1 level was higher (up to fivefold) in wild-type cells grown at low external phosphate concentration and in mutant strains expressing the phosphorus acquisition genes constitutively than in a wild-type strain grown at high external phosphate concentration. Using indirect immunofluorescence we demonstrated that NUC-1 is localized at least predominantly in the cytosol when wild-type N. crassa is grown with an adequate supply of phosphate, whereas NUC-1 is largely concentrated in the nucleus upon limitation of external phosphate. In mutant strains expressing the phosphorus acquisition genes constitutively, NUC-1 localization was also primarily in the nucleus. Thus, subcellular compartmentation of regulatory proteins is an important mechanism in regulating gene expression in filamentous fungi.

  13. Hypoxia-inducible factor-1α increased the expression of peroxisome proliferator activated receptor α in lung cancer cell A549

    Institute of Scientific and Technical Information of China (English)

    张惠兰; 张珍祥; 徐永健

    2004-01-01

    @@ Hypoxia plays a fundamental role in many pathologic processes. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric basic helix-loop-helix-per-aryl hydrocarbon receptor ARNT-sim (PAS) domain protein, consisting of α and β subunits and is precisely regulated by cellular oxygen levels.1 The peroxisome proliferator-activated receptors (PPARs) are family nuclear hormone-binding proteins with increasing diverse functions as transcriptional regulators, owning three subtypes (α, β, and γ).2 PPARα plays a critical physiological role as lipid sensors and regulators of proliferation.3 Hypoxia can elicit up-regulation of PPAR-α expression.4 Herein, we report the results of an investigation on the correlation of HIF-1α and PPARα.

  14. The structure of Aquifex aeolicus ribosomal protein S8 reveals a unique subdomain that contributes to an extremely tight association with 16S rRNA.

    Science.gov (United States)

    Menichelli, Elena; Edgcomb, Stephen P; Recht, Michael I; Williamson, James R

    2012-01-20

    The assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from Aquifex aeolicus (AS8) is unique in that there is a 41-residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually high affinity for the 16S ribosomal RNA, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal site on helix 21 occurred at the same nanomolar affinity found for other bacterial species. The additional affinity required the presence of a three-helix junction between helices 20, 21, and 22. The crystal structure of AS8 was solved, revealing the helix-loop-helix geometry of the unique AS8 insertion region, while the core of the molecule is conserved with known S8 structures. The AS8 structure was modeled onto the structure of the 30S ribosomal subunit from E. coli, suggesting the possibility that the unique subdomain provides additional backbone and side-chain contacts between the protein and an unpaired base within the three-way junction of helices 20, 21, and 22. Point mutations in the protein insertion subdomain resulted in a significantly reduced RNA binding affinity with respect to wild-type AS8. These results indicate that the AS8-specific subdomain provides additional interactions with the three-way junction that contribute to the extremely tight binding to ribosomal RNA.

  15. The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments

    Directory of Open Access Journals (Sweden)

    M. Garbuglia

    1999-10-01

    Full Text Available The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix type, S100A1 and S100B, that have been shown to inhibit microtubule (MT protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF subunits, desmin and glial fibrillary acidic protein (GFAP, with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 µM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities.

  16. FHL2 Interacts with and Acts as a Functional Repressor of Id2 in Human Neuroblastoma Cells

    Institute of Scientific and Technical Information of China (English)

    Wei-dong Han; Zhi-qiang Wu; Ya-li Zhao; Yi-ling Si; Xiao-bing Fu

    2009-01-01

    Objective: Id2 is a natural inhibitor of the basic helix-loop-helix(bHLH) transcription factors. Although it is well known that active Id2 prevents differentiation and promotes cell cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated.Methods: Yeast two-hybrid, mammalian two-hybrid, GST-pulldown and immunoprecipitation (CoIP) assays were used to screen and identify novel Id2 interactors. Luciferase assays were used to detect E47-mediated transcription activity. Colony formation and BrdU incorporation assays were used to determine cellular proliferation abilities. Northorn blot, western blot and quantitative PCR methods were used to measure gene expression levels. Electrophoretic mobility shift assays (EMSAs) were performed to investigate protein/DNA binding.Results: The LIM-only protein FHL2 (four-and-a-half-LIM-only protein 2) was identified to be a novel Id2 interactor. The HLH domain within Id2 is not required for its interaction with FHL2. FHL2 antagonizes the inhibitory effect of Id2 on the basic helix-loop-helix protein E47-mediated transcription. FHL2 prevents the formation of Id2-E47 heterdimer, thus releasing E47 to its target DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 is opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2.Conclusion: These results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.

  17. Dose-dependent regulation of target gene expression and cell proliferation by c-Myc levels.

    Science.gov (United States)

    Schuhmacher, Marino; Eick, Dirk

    2013-01-01

    The proto-oncogene c-myc encodes a basic helix-loop-helix leucine zipper transcription factor (c-Myc). c-Myc plays a crucial role in cell growth and proliferation. Here, we examined how expression of c-Myc target genes and cell proliferation depend on variation of c-Myc protein levels. We show that proliferation rates, the number of cells in S-phase, and cell size increased in a dose-dependent manner in response to increasing c-Myc levels. Likewise, the mRNA levels of c-Myc responsive genes steadily increased with rising c-Myc levels. Strikingly, steady-state mRNA levels of c-Myc target genes did not saturate even at highest c-Myc concentrations. These characteristics predestine c-Myc levels as a cellular rheostat for the control and fine-tuning of cell proliferation and growth rates.

  18. Grasses use an alternatively wired bHLH transcription factor network to establish stomatal identity.

    Science.gov (United States)

    Raissig, Michael T; Abrash, Emily; Bettadapur, Akhila; Vogel, John P; Bergmann, Dominique C

    2016-07-19

    Stomata, epidermal valves facilitating plant-atmosphere gas exchange, represent a powerful model for understanding cell fate and pattern in plants. Core basic helix-loop-helix (bHLH) transcription factors regulating stomatal development were identified in Arabidopsis, but this dicot's developmental pattern and stomatal morphology represent only one of many possibilities in nature. Here, using unbiased forward genetic screens, followed by analysis of reporters and engineered mutants, we show that stomatal initiation in the grass Brachypodium distachyon uses orthologs of stomatal regulators known from Arabidopsis but that the function and behavior of individual genes, the relationships among genes, and the regulation of their protein products have diverged. Our results highlight ways in which a kernel of conserved genes may be alternatively wired to produce diversity in patterning and morphology and suggest that the stomatal transcription factor module is a prime target for breeding or genome modification to improve plant productivity.

  19. [Research progress of the bHLH transcription factors involved in genic male sterility in plants].

    Science.gov (United States)

    Yongming, Liu; Ling, Zhang; Jianyu, Zhou; Moju, Cao

    2015-12-01

    Male sterility exists widely in the spermatophytes. It contributes to the study of plant reproductive development and can be used as an effective tool for hybrid seed production in heterosis utilization. Therefore, the study on male sterility is of great value in both theory and application. As one of the largest transcription factor families in plants, basic helix-loop-helix proteins (bHLHs) play a crucial role in regulating plant growth and development. This paper introduces the mechanism of bHLH regulating stamen development in several important model plants. Furthermore, we discuss the molecular mechanisms of genic male sterility resulting from bHLH dysfunction to provide references for crop breeding and theoretical studies.

  20. Circadian transcription factor BMAL1 regulates innate immunity against select RNA viruses.

    Science.gov (United States)

    Majumdar, Tanmay; Dhar, Jayeeta; Patel, Sonal; Kondratov, Roman; Barik, Sailen

    2017-02-01

    BMAL1 (brain and muscle ARNT-like protein 1, also known as MOP3 or ARNT3) belongs to the family of the basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors, and is a key component of the molecular oscillator that generates circadian rhythms. Here, we report that BMAL1-deficient cells are significantly more susceptible to infection by two major respiratory viruses of the Paramyxoviridae family, namely RSV and PIV3. Embryonic fibroblasts from Bmal1(-/-) mice produced nearly 10-fold more progeny virus than their wild type controls. These results were supported by animal studies whereby pulmonary infection of RSV produced a more severe disease and morbidity in Bmal1(-/-)mice. These results show that BMAL1 can regulate cellular innate immunity against specific RNA viruses.

  1. Transcription of a zebrafish gene of the hairy-Enhancer of split family delineates the midbrain anlage in the neural plate.

    Science.gov (United States)

    Müller, M; von Weizsäcker, E; Campos-Ortega, J A

    1996-09-01

    her5 encodes a basic helix-loop-helix (bHLH) protein with all features characteristic of the Drosophila hairy-E(spl) family. her5 is expressed in a band of cells within the neural anlage from about 90% epiboly on to at least 36 h postfertilization (hpf). After completion of brain morphogenesis, her5-expressing cells are located in the caudal region of the midbrain, at the boundary with the rhombencephalon. Labelling of cells within the her5 expression domain in the neural plate by injection of fluorescein-dextran allows their labelled progeny to be localized in the 36-hpf-old embryo using an anti-fluorescein antibody. This shows that the her5 expression domain corresponds to the midbrain primordium, including both the tectum and the tegmentum, in the neural plate. A possible function for her5 in regionalization of the brain and/or control of the midbrain-hindbrain boundary is discussed.

  2. Study of Myelin Basic Protein Associated with Pediatric Systematic Epilepsy

    Institute of Scientific and Technical Information of China (English)

    Yang Sida; He Xin; Yang Yiyu; Zhu Huihua; He Dansha; Deng Weiyi

    2000-01-01

    Objective: To investigate the quantitative myelin basic protein (MBP) in cerebrospinal fluid (CSF) and serum in pediatric systematic epilepsy (SEP), study the relation between SEP and MBP, and the possibility predicating'the injury of myelin and blood-brain barrier (BBB) from pediatric SEP. Background: While tactors induced destroy of cerebral and Myelin, MBP was released out into CSF to increase its concentration. On the other hand, the BBB was involved to make serum MBP increased. The related studies had confirmed these viewpoints above. The test for quantitative MBP was recognized as the specific biochemical index, which diagnose if there is or not organic injury of cerebral and myelin. There was few reports about the studies of quantitative MBP in CSF and serum of EP, not mention to those published in domestic pediatric academia. Methods: 47 cases were studied during one month after the SEP attack, whose MBP in serum were quantitatively and 31 inside in CSF were also tested by easy MBP-ELISA method; the quantitative MBP in serum of 30 control cases and 10 in CSF were tested, too. Results: MBP values in CSF and serum of SEP pediatric patients were 2.95±0.61 ng/ml and 3.17±0.53 ng/ml; whereas 1.41 ±0.19 ng/ml and 1.30±0.04 ng/ml in control group. Both mean valves of MBP in CSF and serum in study group were significantly higher than control group (either P< 0.01). Discussion: In general, electrophysiological evidences supported the issue that epileptic episode was originated from abnormal electrical activities of nervous cells. Pathological studies revealed degeneration and necrosis of nerve existed in temporal epileptic focus, where there was morphological change of myelin. This study showed MBP values in CSF and serum of SEEP, during one month after attack, increased significantly; suggested there was changed component of MBP, while SEP could not be controled. Those above indicated the destroy of myelin, increasing of BBB permeability that induced its

  3. A novel sterol regulatory element-binding protein gene (sreA identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51 regulation.

    Directory of Open Access Journals (Sweden)

    Jing Liu

    Full Text Available Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014 at its carboxyl terminus and a helix-loop-helix (HLH leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA in a prochloraz-resistant strain (PdHS-F6 by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA. A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.

  4. A novel sterol regulatory element-binding protein gene (sreA) identified in penicillium digitatum is required for prochloraz resistance, full virulence and erg11 (cyp51) regulation.

    Science.gov (United States)

    Liu, Jing; Yuan, Yongze; Wu, Zhi; Li, Na; Chen, Yuanlei; Qin, Tingting; Geng, Hui; Xiong, Li; Liu, Deli

    2015-01-01

    Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression.

  5. Urinary proteins of tubular origin: basic immunochemical and clinical aspects.

    Science.gov (United States)

    Scherberich, J E

    1990-01-01

    A variety of tubular marker proteins, as compared to healthy controls, are excreted at an increased rate in the urine of patients with renal damage. Beside cytoplasmic glutathione-S-transferase and lysosomal beta-N-acetyl-glucosaminidase (beta-NAG) the majority of kidney-related urine proteins derives from membrane surface components of the most vulnerable proximal tubule epithelia, among them ala-(leu-gly)-aminopeptidase, gamma-glutamyl transpeptidase (GGT), the tubular portion of angiotensinase A, the major brush border glycoprotein 'SGP-240' and adenosine-deaminase-binding protein. Urinary tissue proteins, e.g. brush border (BB) microvilli, are immunologically identical with those antigens prepared from cell membranes of the human kidney itself. BB antigens are shed into the urine of patients with glomerulonephritis, interstitial nephritis, systemic diseases, e.g. systemic lupus erythematosus (SLE), diabetes mellitus and multiple myeloma, arterial hypertension, infectious diseases (malaria, AIDS) and after operations, renal grafting and administration of X-ray contrast media, aminoglycosides or certain cytostatics (cis-platinum). Tissue proteinuria of tubular proteins is determined by enzyme-kinetic or quantitative immunological assays applying either poly- or monoclonal antikidney antibodies. Clinical, ultrastructural and histochemical studies support the idea that both 'soluble' and high-molecular-weight membrane particles (vacuolar blebs, greater than 10(6) dalton) as well as microfilamental components of the epithelial cytoskeleton contribute to tubular 'histuria' which appears as a sensitive parameter in monitoring tubular damage under clinical conditions at a very early phase.

  6. Protein-membrane interaction: effect of myelin basic protein on the dynamics of oriented lipids

    Energy Technology Data Exchange (ETDEWEB)

    Natali, F.; Relini, A.; Gliozzi, A.; Rolandi, R.; Cavatorta, P.; Deriu, A.; Fasano, A.; Riccio, P

    2003-08-01

    We have studied the effect of physiological amounts of myelin basic protein (MBP) on pure dimyristoyl L-{alpha}-phosphatidic acid (DMPA) oriented membranes. The investigation has been carried out using several complementary experimental methods to provide a detailed characterization of the proteo-lipid complexes. In particular, taking advantage of the power of the quasi-elastic neutron scattering (QENS) technique as optimal probe in biology, a significant effect is suggested to be induced by MBP on the anisotropy of lipid dynamics across the liquid-gel phase transition. Thus, the enhancement of the spatially restricted, vertical translation motion of DMPA is suggested to be the main responsible for the increased contribution of the out of plane lipid dynamics observed at 340 K.

  7. Protein membrane interaction: effect of myelin basic protein on the dynamics of oriented lipids

    Science.gov (United States)

    Natali, F.; Relini, A.; Gliozzi, A.; Rolandi, R.; Cavatorta, P.; Deriu, A.; Fasano, A.; Riccio, P.

    2003-08-01

    We have studied the effect of physiological amounts of myelin basic protein (MBP) on pure dimyristoyl L-α-phosphatidic acid (DMPA) oriented membranes. The investigation has been carried out using several complementary experimental methods to provide a detailed characterization of the proteo-lipid complexes. In particular, taking advantage of the power of the quasi-elastic neutron scattering (QENS) technique as optimal probe in biology, a significant effect is suggested to be induced by MBP on the anisotropy of lipid dynamics across the liquid-gel phase transition. Thus, the enhancement of the spatially restricted, vertical translation motion of DMPA is suggested to be the main responsible for the increased contribution of the out of plane lipid dynamics observed at 340 K.

  8. The complete amino acid sequence of the basic nuclear protein of bull spermatozoa

    NARCIS (Netherlands)

    Coelingh, J.P.; Monfoort, Cornelis H.; Rozijn, Thomas H.; Gevers Leuven, Jan A.; Schiphof, R.; Steyn-Parvé, Elizabeth P.; Braunitzer, Gerhard; Schrank, Barbara; Ruhfus, Annette

    1972-01-01

    The complete amino acid sequence of the basic nuclear protein of bull spermatozoa has been established. The sequence was partially deduced by characterization of peptides isolated from thermolysine and chymotryptic digests of the reduced and S-aminoethylated protein. The complete sequence of the fir

  9. Shark myelin basic protein: amino acid sequence, secondary structure, and self-association.

    Science.gov (United States)

    Milne, T J; Atkins, A R; Warren, J A; Auton, W P; Smith, R

    1990-09-01

    Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.

  10. Basic residues within the ebolavirus VP35 protein are required for its viral polymerase cofactor function.

    Science.gov (United States)

    Prins, Kathleen C; Binning, Jennifer M; Shabman, Reed S; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

    2010-10-01

    The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

  11. Changes in the anisotropy of oriented membrane dynamics induced by myelin basic protein

    Science.gov (United States)

    Natali, F.; Gliozzi, A.; Rolandi, R.; Relini, A.; Cavatorta, P.; Deriu, A.; Fasano, A.; Riccio, P.

    We report recent results showing the evidence of the effect induced by physiological amounts of myelin basic protein (MBP) on the dynamics of dimyristoyl L-a-phosphatidic acid (DMPA) membranes. Incoherent elastic neutron scattering scans, performed over a wide temperature range, have shown that the anisotropy of motions in oriented membranes is significantly enhanced by the presence of MBP.

  12. Changes in the anisotropy of oriented membrane dynamics induced by myelin basic protein

    Energy Technology Data Exchange (ETDEWEB)

    Natali, F. [OGG-INFM, Grenoble (France); Gliozzi, A.; Rolandi, R.; Relini, A. [Dipartimento di Fisica and Istituto Nazionale per la Fisica della Materia, Universita di Genova (Italy); Cavatorta, P.; Deriu, A. [Dipartimento di Fisica and Istituto Nazionale per la Fisica della Materia, Universita di Parma (Italy); Fasano, A. [Dipartimento di Biochimica e Biologia Molecolare, Universita di Bari (Italy); Riccio, P. [Dipartimento di Biologia D.B.A.F., Universita della Basilicata, Potenza (Italy)

    2002-07-01

    We report recent results showing the evidence of the effect induced by physiological amounts of myelin basic protein (MBP) on the dynamics of dimyristoyl L-a-phosphatidic acid (DMPA) membranes. Incoherent elastic neutron scattering scans, performed over a wide temperature range, have shown that the anisotropy of motions in oriented membranes is significantly enhanced by the presence of MBP. (orig.)

  13. The role of basic residues in the adsorption of blood proteins onto the graphene surface

    Science.gov (United States)

    Gu, Zonglin; Yang, Zaixing; Wang, Lingle; Zhou, Hong; Jimenez-Cruz, Camilo A.; Zhou, Ruhong

    2015-06-01

    With its many unique properties, graphene has shown great potential in various biomedical applications, while its biocompatibility has also attracted growing concerns. Previous studies have shown that the formation of protein-graphene corona could effectively reduce its cytotoxicity; however, the underlying molecular mechanism remains not well-understood. Herein, we use extensive molecular dynamics simulations to demonstrate that blood proteins such as bovine fibrinogen (BFG) can absorb onto the graphene surface quickly and tightly to form a corona complex. Aromatic residues contributed significantly during this adsorption process due to the strong π-π stacking interactions between their aromatic rings and the graphene sp2-carbons. Somewhat surprisingly, basic residues like arginine, also played an equally or even stronger role during this process. The strong dispersion interactions between the sidechains of these solvent-exposed basic residues and the graphene surface provide the driving force for a tight binding of these basic residues. To the best of our knowledge, this is the first study with blood proteins to show that, in addition to the aromatic residues, the basic residues also play an important role in the formation of protein-graphene corona complexes.

  14. Ox peripheral nerve myelin membrane. Purification and partial characterization of two basic proteins

    NARCIS (Netherlands)

    London, Y.

    1971-01-01

    Two basic proteins were purified from the peripheral nervous system. The isolation was achieved by (1) delipidation with chloroform-butanol mixtures, dry acetone, and dry ether, (2) acid extraction at pH 2 and then (3) dialysis against distilled water, lyophilization, and solubilization in pH-10.7 b

  15. Kinetics of the interaction of myelin basic protein with phospholipid layers

    Science.gov (United States)

    Facci, Paolo; Cavatorta, Paolo; Cristofolini, Luigi; Fontana, M. P.; Riccio, Paolo

    1999-04-01

    The time dependence of the adsorption of myelin basic protein onto dipalmitoyl phosphatidyl glycerol multilayers has been followed directly, using a novel application of a microgravimetric gauge. Our results, supplemented by other data obtained by FTIR, show the ease and versatility of the quartz microbalance for investigating the interaction processes between proteins and phospholipid layers and show that the protein adsorption is accompanied by structural changes in the proteolipid ensemble and adsorbed liquid water; it is furthermore dependent on the mesoscopic defect morphology of the ensemble.

  16. Effects of diethyldithiocarbamate on myelin basic protein expression in the rat lateral olfactory tract

    Institute of Scientific and Technical Information of China (English)

    Kun Xiong; He Huang; Hui Wang; Yan Cai; Jing Yang; Jufang Huang; Xuegang Luo

    2009-01-01

    BACKGROUND: Dithiocarbamates can cause demyelination of axons in the peripheral nervous system. Its derivate, diethyldithiocarbamate, is cytotoxic, and causes olfactory mucosal damage and atrophy of the olfactory bulb. However, it is still unclear whether the myelin sheath of the lateral olfactory tract is affected by diethyldithiocarbamate.OBJECTIVE: To investigate the effects of diethyldithiocarbamate on the myelin sheath of the rat lateral olfactory tract. This was done by examining changes in myelin basic protein expression after diethyldithiocarbamate treatment.DESIGN, TIME AND SETTING: A randomized, controlled, animal study was performed at the Laboratory of the Department of Human Anatomy and Neurobiology, Xiangya School of Medicine, Central South University, China from July to November 2007.MATERIALS: A total of 72 Sprague Dawley rats were randomly assigned into a diethyldithiocarbamate group (n=32), a solvent control group (n=32), and a blank control group (n=8). The diethyldithiocarbamate and solvent control groups were separately divided into 3-d, 7-d, 14-d and 28-d survival subgroups, with eight rats in each. Diethyldithiocarbamate (Sigma, USA) and goat anti-myelin basic protein polyclonal antibody (Santa Cruz, USA) were used in this study.METHODS: Rats in the diethyldithiocarbamate and solvent control groups were subcutaneously injected with diethyldithiocarbamate (600 mg/kg) and 0.01 mol/L phosphate buffered saline (600 mg/kg) at the posterior neck, respectively. Rats in the blank control group received no treatment.MAIN OUTCOME MEASURES: Immunohistochemical staining and Western blot assay were used to measure myelin basic protein expression in the rat lateral olfactory tract.RESULTS: Following immunohistochemical staining, myelin basic protein was uniformly distributed in the rat lateral olfactory tract in the blank control and solvent control groups. Western blot assay showed 21.5, 18, 17 and 14 ku positive bands. No significant difference was found

  17. Genomic pathways modulated by Twist in breast cancer

    OpenAIRE

    Vesuna, Farhad; Bergman, Yehudit; Raman, Venu

    2017-01-01

    Background The basic helix-loop-helix transcription factor TWIST1 (Twist) is involved in embryonic cell lineage determination and mesodermal differentiation. There is evidence to indicate that Twist expression plays a role in breast tumor formation and metastasis, but the role of Twist in dysregulating pathways that drive the metastatic cascade is unclear. Moreover, many of the genes and pathways dysregulated by Twist in cell lines and mouse models have not been validated against data obtaine...

  18. NOTCH Signaling and ATOH1 in Colorectal Cancers

    OpenAIRE

    2011-01-01

    The Notch receptor signaling pathway regulates expression of the basic helix-loop-helix transcription factor ATOH1 (Math1/Hath1) to determine cell fate in the intestine. In differentiating intestinal stem cells, high levels of Notch activity specify absorptive enterocyte/colonocyte differentiation, whereas high ATOH1 activity specifies secretory (goblet, enteroendocrine, and Paneth) cell differentiation. In colorectal cancer, ATOH1 is a tumor suppressor that is silenced in most tumors, while ...

  19. Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork.

    Science.gov (United States)

    Aggarwal, Shweta; Snaidero, Nicolas; Pähler, Gesa; Frey, Steffen; Sánchez, Paula; Zweckstetter, Markus; Janshoff, Andreas; Schneider, Anja; Weil, Marie-Theres; Schaap, Iwan A T; Görlich, Dirk; Simons, Mikael

    2013-01-01

    Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.

  20. Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork.

    Directory of Open Access Journals (Sweden)

    Shweta Aggarwal

    Full Text Available Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.

  1. Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

    Science.gov (United States)

    Filikov, Anton V.; James, Thomas L.

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  2. Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

    Science.gov (United States)

    Belogurov, Alexey; Kudriaeva, Anna; Kuzina, Ekaterina; Smirnov, Ivan; Bobik, Tatyana; Ponomarenko, Natalia; Kravtsova-Ivantsiv, Yelena; Ciechanover, Aaron; Gabibov, Alexander

    2014-06-20

    The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

  3. Myelin basic protein reduces molecular motions in DMPA, an elastic neutron scattering study

    Science.gov (United States)

    Natali, F.; Gliozzi, A.; Rolandi, R.; Cavatorta, P.; Deriu, A.; Fasano, A.; Riccio, P.

    2001-07-01

    We have studied the effect of physiological amounts of myelin basic protein (MBP) on pure dimyristoyl L- α-phosphatidic acid (DMPA) vesicles using the elastic neutron scattering technique. Elastic scans have been performed in a wide temperature range (20-300 K). In the lower temperature region the behaviour of the integrated elastic intensity was the typical one of harmonic systems. The analysis of the Q and T dependence performed in terms of an asymmetric double well potential clearly showed that the effect of the protein consisted in a significant reduction of the conformational mobility of the DMPA bilayers and in the stabilisation of the membrane.

  4. Myelin basic protein synthesis is regulated by small non-coding RNA 715.

    Science.gov (United States)

    Bauer, Nina M; Moos, Christina; van Horssen, Jack; Witte, Maarten; van der Valk, Paul; Altenhein, Benjamin; Luhmann, Heiko J; White, Robin

    2012-09-01

    Oligodendroglial Myelin Basic Protein (MBP) synthesis is essential for myelin formation in the central nervous system. During oligodendrocyte differentiation, MBP mRNA is kept in a translationally silenced state while intracellularly transported, until neuron-derived signals initiate localized MBP translation. Here we identify the small non-coding RNA 715 (sncRNA715) as an inhibitor of MBP translation. SncRNA715 localizes to cytoplasmic granular structures and associates with MBP mRNA transport granule components. We also detect increased levels of sncRNA715 in demyelinated chronic human multiple sclerosis lesions, which contain MBP mRNA but lack MBP protein.

  5. Hematopoietic progenitors express myelin basic protein and ensheath axons in Shiverer brain.

    Science.gov (United States)

    Goolsby, James; Makar, Tapas; Dhib-Jalbut, Suhayl; Bever, Christopher T; Pessac, Bernard; Trisler, David

    2013-04-15

    Oligodendroglia are cells of the central nervous system (CNS) that form myelin sheath, which insulates neuronal axons. Neuropathologies of the CNS include dysmyelination of axons in multiple sclerosis and CNS trauma. Cell replacement is a promising but largely untested therapy for dysmyelination. Shiverer mouse, a genetic mutant that does not synthesize full-length myelin basic protein (MBP), a critical prerequisite protein in CNS myelin sheath formation, provides an unequivocal model for determining the potential of stem cells to become oligodendroglia. We demonstrate that adult wild-type mouse bone marrow stem cells can express MBP and ensheath axons when transplanted into Shiverer brain.

  6. Structure and function of the proline-rich region of myelin basic protein.

    Science.gov (United States)

    Fraser, P E; Deber, C M

    1985-08-13

    Myelin basic protein (MBP)--the major extrinsic membrane protein of central nervous system myelin--from several species contains a rarely encountered highly conserved triproline segment as residues 99-101 of its 170-residue sequence. Cis peptide bonds are known to arise at X-Pro junctions in proteins and may be of functional significance in protein folding, chain reversal, and/or maintenance of tertiary structure. We have examined the conformation of this proline-rich region using principally 13C nuclear magnetic resonance spectroscopy (125 MHz) both in intact bovine MBP and in several MBP fragment peptides which we synthesized, including octapeptide 97-104 (Arg-Thr-Pro-Pro-Pro-Ser-Gln-Gly). Results suggested an all-trans conformation in aqueous solution for the triproline segment in MBP hexapeptide (99-104), heptapeptide (98-104), and octapeptide. Comparison with the 13C spectrum of intact MBP (125 MHz) suggested that the proline-rich region, as well as all other X-Pro MBP peptide junctures, was also essentially all trans in aqueous solution. Although experiments in which octapeptide 97-104 was bound to a lipid preparation (4:1 dipalmitoylphosphatidylcholine/dimyristoylphosphatidic acid) demonstrated that cis-proline bonds do arise (to the extent of ca. 5%) in the membrane environment, a role of linear chain propagation is suggested for the triproline segment of myelin basic protein.

  7. Myelin basic protein induces neuron-specific toxicity by directly damaging the neuronal plasma membrane.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    Full Text Available The central nervous system (CNS insults may cause massive demyelination and lead to the release of myelin-associated proteins including its major component myelin basic protein (MBP. MBP is reported to induce glial activation but its effect on neurons is still little known. Here we found that MBP specifically bound to the extracellular surface of the neuronal plasma membrane and induced neurotoxicity in vitro. This effect of MBP on neurons was basicity-dependent because the binding was blocked by acidic lipids and competed by other basic proteins. Further studies revealed that MBP induced damage to neuronal membrane integrity and function by depolarizing the resting membrane potential, increasing the permeability to cations and other molecules, and decreasing the membrane fluidity. At last, artificial liposome vesicle assay showed that MBP directly disturbed acidic lipid bilayer and resulted in increased membrane permeability. These results revealed that MBP induces neurotoxicity through its direct interaction with acidic components on the extracellular surface of neuronal membrane, which may suggest a possible contribution of MBP to the pathogenesis in the CNS disorders with myelin damage.

  8. 腺病毒介导的秦川牛固醇调节元件结合蛋白2基因(SREBP2)过表达载体的构建与鉴定%Construction and Identification of Recombinant Adenovirus Vector Specific to Qinchuan Cattle(Bos taurus) Derived Sterol Regulatory Element Binding Protein 2 Gene (SREBP2)

    Institute of Scientific and Technical Information of China (English)

    付常振; 刘扬; 昝林森; 王虹; 成功; 王嘉力

    2013-01-01

    Sterol regulatory element binding protein 2(SERBP2) is a basic-helix-loop-helix-luecine zipper factor which regulates the metabolization process of cholesterol.We cloned the SREBP2 gene from Qinchuan cattle (Bos taurus) and constructed the overexpression adenoviral vector,and packed and amplified the virus for a high titer,as a antecedent work for the further study of cellular level function of SERBP2 gene.Total RNA was extracted from the adipose tissue of Qinchuan cattle and then reversely transcripted to cDNA.A pair of exclusive primers were designed according to the GenBank sequence information of SREBP2 gene(Accession No.NM_001205600) to amplify the complete coding sequence(CDS) area of SREBP2 gene by polymerase chain reaction (PCR).The fragments containing CDS area of SREBP2 gene were inserted into the shuttle vector to construct the pAdTrack-CMV-SREBP2 plasmid.The recombinant plasmid and the blank control pAdTrack-CMV were linearized by digesting with restriction endonuclease Pine Ⅰ and subsequently transformed into Escherichia coli B J5183 containing pAdEasy-1 to homologous recombine and obtain the recombinant adenovirus plasmid pAd-SREBP2 and pAd-CMV.And then,the confirmed recombinant adenovirus plasmid pAd-SREBP2 was digested with Pac Ⅰ and transfected into 293A cell line to package and amplify the recombinant adenovirus Ad-SREBP2 and Ad-CMV,and to collect virus of high titer.The viral titer of AdSREBP2 and Ad-CMV was 7 × 108 and 1.3 × 109 GFU/mL respectively,measured by green fluorescent protein (GFP) labelled method.Qinchuan cattle derived preadipocyte was infected by Ad-SREBP2 and Ad-CMV to verify the availability of the virues.The expression of SREBP2 increased by 102.3 times after infected with the recombinant adenovirus for 48 h,determined by quantitative Real-time PCR.The cloning of SERBP2 gene of Qinchuan cattle obtaining of recombinant adenoviru and virus of high titer are set as foundation for the studies of the gene function on cellular

  9. Putting muscle in DNA methylation

    Institute of Scientific and Technical Information of China (English)

    James P Reddington; Richard R Meehan

    2011-01-01

    Over 25 years ago seminal experiments from the labs of Peter Jones and Harold Weintraub demonstrated that alteration in the DNA modification state underlie the myogenic conversion of fibroblast cell lines [1,2].This paved the way for the identification of myogenic helix-loop-helix (HLH) proteins in muscle differentiation,but the mechanism by which DNA methylation regulates muscle differentiation has remained elusive [3].

  10. Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein.

    Science.gov (United States)

    Jones, A J; Rumsby, M G

    1977-12-01

    The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.

  11. Multiple biological functions of novel basic proteins isolated from duck egg white: duck basic protein small 1 (dBPS1) and 2 (dBPS2).

    Science.gov (United States)

    Naknukool, Supaporn; Hayakawa, Shigeru; Ogawa, Masahiro

    2011-05-11

    Biological functions of duck basic protein small 1 (dBPS(1)) and 2 (dBPS(2)) were investigated by in vitro experiments. Results of agarose gel retardation assay indicated that dBPS(1) and dBPS(2) associate with RNA. Addition of NaCl or urea induced partial dissociation of dBPS(1)/dBPS(2)-RNA complex, implying that electrostatic interaction, hydrophobic interaction, and hydrogen bonds are involved in the association of dBPS(1)/dBPS(2) to RNA. dBPS(1) and dBPS(2) inhibited pancreatic lipase activity with the fifty percent inhibitory concentration (IC(50)) of 250 and 100 μg/mL, respectively. Peptic hydrolysates of dBPS(1) and those of dBPS(2) showed a potent angiotensin I-converting enzyme (ACE) inhibition with an IC(50) of 22.5 and 49.6 mg/L. The most potent ACE-inhibitory peptide was a nanopeptide (EKKGFCAGY) from dBPS(1) and an octapeptide (KYCPKVGY) from dBPS(2). These multiple biological functions of dBPS(1) and dBPS(2) may contribute to reducing the risk of lifestyle diseases.

  12. Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillaries.

    Science.gov (United States)

    Haselberg, Rob; de Jong, Gerhardus J; Somsen, Govert W

    2009-07-01

    The usefulness of a noncovalent, positively charged capillary coating for the efficient analysis of intact basic proteins with CE was studied. Capillaries were coated by subsequent flushing with solutions of 10% w/v Polybrene (PB), 3% w/v dextran sulfate (DS), and again 10% w/v PB. Coating characterization studies showed that stable coatings could be produced which exhibited a pH-independent and highly reproducible EOF. The PB-DS-PB coating was evaluated with Tris phosphate BGEs of various pH using the four basic model proteins: alpha-chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme. Typical migration time RSDs for the proteins were less than 0.85%, and apparent plate numbers were above 125,000 using a capillary length of 40 cm. The high separation efficiency allowed detection of several minor impurities in the model proteins. Using a BGE of medium pH, the CE system with triple-layer coating appeared to be useful for the repeatable profiling of recombinant humanized mouse monoclonal immunoglobulin G(1) showing a characteristic pattern of glycoforms. The CE system was also applied to the characterization of two llama antibodies, which were produced in Saccharomyces cerevisiae, revealing the presence of a side product in one of the antibodies. The high migration time stability allowed the reliable determination of antibody-antigen binding by monitoring migration time shifts. Finally, the feasibility of using the PB-DS-PB coated capillaries for CE with mass spectrometric detection was shown by the characterization of the impure llama antibody sample.

  13. Ionic liquid-polyvinyl chloride ionomer for highly selective isolation of basic proteins.

    Science.gov (United States)

    Shu, Yang; Chen, Xu-Wei; Wang, Jian-Hua

    2010-04-15

    Hydrophilic ionic liquid-polyvinyl chloride (PVC) hybrids were prepared by immobilizing N-methylimidazole (N-mim) to PVC chains in toluene. The NmimCl-PVC hybrids were characterized by FT-IR, (1)H NMR, surface charge analysis and elemental analysis. The immobilization ratio, i.e., the percentage of chloride on PVC chain reacting with N-mim to form the hybrid, varies from 4.3% to 15.1% by increasing the N-mim/PVC molar ratio. The most distinct feature of the hybrid is its excellent selectivity for adsorbing basic proteins by effective suppression of the non-specific protein adsorption by pure PVC, and a higher immobilization ratio facilitates better selectivity. In Tris-HCl buffer, 100 microg mL(-1) of basic proteins, i.e., lysozyme (Lys), cytochrome c (cyt-c) and hemoglobin (Hb), were favorably adsorbed with efficiencies of 97%, 98% and 94% by the hybrid with an immobilization ratio of 15.1%, while the adsorption of acidic proteins, i.e., bovine albumin serum (BSA), transferring (Trf) and immunoglobulin G (IgG) were negligible. The retained Lys, cyt-c and Hb could be readily recovered by elution with phosphate buffer, carbonate buffer and SDS solution with efficiencies of 89%, 87% and 84%, respectively. Another feature of the hybrid is the significant improvement of the biocompatibility characterized by the maintenance of the activity of hemoglobin after adsorption and elution process. The practical usefulness of the hybrid was demonstrated by selective isolation of hemoglobin from human whole blood.

  14. CONTENTS OF SERUM MYELIN BASIC PROTEIN-IGG ANTIBODIES COMPLEXES IN NORMAL PREGNANCY AND GESTOSIS

    Directory of Open Access Journals (Sweden)

    V. G. Levchenko

    2010-01-01

    Full Text Available Serum levels of myelin basic protein (MBP-bound immune complexes were studied in blood sera from women with gestosis, as compared with those in normal pregnancy and non-pregnant woman. The amounts of IgG-MBP complex in blood serum were determined by enzyme immunoassay using isolated anti-МBP-antibodies. The study has shown that about 0.05 mcg of IgG ml of blood serum are associated with myelin basic protein in unpregnant women or in normal pregnancy. Mild gestosis is accompanied by a 2-3-fold increase in MBP immunocomplex concentrations in blood serum. More severe stages of gestosis are characterized by its further rise, thus achieving maximal values of such MBP immune complexes (0.8 mcg/ml in patients with pre-eclampsia and eclampsia. Their amounts were reduced twice after the periods of eclampsia. Serum levels of MBP-bound IgGs may be used to determine severity of gestosis and to predict a risk of eclampsia in pregnant women.

  15. Phosphate cycling on the basic protein of Plodia interpunctella granulosis virus

    Science.gov (United States)

    Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The presence of infected cell-specific phosphoproteins was investigated in Plodia interpunctella granulosis virus (PiGV)-infected fat body using [32P]orthophosphoric acid labeling. One infected cell-specific phosphoprotein had a mobility similar to that of the basic protein (VP12) of PiGV. Further analysis, using immunoblotting and acid-urea gel analysis of infected fat body, confirmed that this phosphoprotein was VP12. However we did not detect phosphorylated VP12 in 32P-labeled nucleocapsids. Phosphoamino acid analysis of 32P-labeled VP12 revealed that phosphoserine was present in the basic protein. Since VP12 is phosphorylated in the infected cell, but not in the nucleocapsid, it appears that dephosphorylation of VP12 is a critical event in the life cycle of the virus. We therefore assayed virus nucleocapsids and infected fat body for the presence of phosphatase activity. Phosphatase activity was not detected in the virus, but the infected fat body had more activity than uninfected fat body. A model for nucleocapsid assembly and uncoating is presented which takes into account the phosphorylation state of VP12, the role of Zn2+ in the nucleocapsid, and the role of the capsid-associated kinase.

  16. Making myelin basic protein -from mRNA transport to localized translation.

    Science.gov (United States)

    Müller, Christina; Bauer, Nina M; Schäfer, Isabelle; White, Robin

    2013-09-27

    In the central nervous system (CNS) of most vertebrates, oligodendrocytes enwrap neuronal axons with extensions of their plasma membrane to form the myelin sheath. Several proteins are characteristically found in myelin of which myelin basic protein (MBP) is the second most abundant one after proteolipid protein. The lack of functional MBP in rodents results in a severe hypomyelinated phenotype in the CNS demonstrating its importance for myelin synthesis. Mbp mRNA is transported from the nucleus to the plasma membrane and is translated locally at the axon-glial contact site. Axonal properties such as diameter or electrical activity influence the degree of myelination. As oligodendrocytes can myelinate many axonal segments with varying properties, localized MBP translation represents an important part of a rapid and axon-tailored synthesis machinery. MBP's ability to compact cellular membranes may be problematic for the integrity of intracellular membranous organelles and can also explain why MBP is transported in oligodendrocytes in the form of an mRNA rather than as a protein. Here we review the recent findings regarding intracellular transport and signaling mechanisms leading to localized translation of Mbp mRNA in oligodendrocytes. More detailed insights into the MBP synthesis pathway are important for a better understanding of the myelination process and may foster the development of remyelination therapies for demyelinating diseases.

  17. Making Myelin Basic Protein -from mRNA transport to localized translation

    Directory of Open Access Journals (Sweden)

    Christina eMüller

    2013-09-01

    Full Text Available In the central nervous system (CNS of most vertebrates, oligodendrocytes enwrap neuronal axons with extensions of their plasma membrane to form the myelin sheath. Several proteins are characteristically found in myelin of which Myelin Basic Protein (MBP is the second most abundant one after Proteolipid Protein (PLP. The lack of functional MBP in rodents results in a severe hypomyelinated phenotype in the CNS demonstrating its importance for myelin synthesis. Mbp mRNA is transported from the nucleus to the plasma membrane and is translated locally at the axon-glial contact site. Axonal properties such as diameter or electrical activity influence the degree of myelination. As oligodendrocytes can myelinate many axonal segments with varying properties, localized MBP translation represents an important part of a rapid and axon-tailored synthesis machinery. MBP’s ability to compact cellular membranes may be problematic for the integrity of intracellular membranous organelles and can also explain why MBP is transported in oligodendrocytes in the form of an mRNA rather than as a protein. Here we review the recent findings regarding intracellular transport and signalling mechanisms leading to localized translation of Mbp mRNA in oligodendrocytes. More detailed insights into the MBP synthesis pathway are important for a better understanding of the myelination process and may foster the development of remyelination therapies for demyelinating diseases.

  18. Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue

    Directory of Open Access Journals (Sweden)

    Jennifer L. Kaplan

    2015-11-01

    Conclusions: This study provides the first in vivo evidence, to our knowledge, that committed AdPCs in VAT are the initial source of obesity-induced MCP-1 and identifies the helix-loop-helix transcription factor Id3 as a critical regulator of p21Cip1 expression, AdPC proliferation, MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation.

  19. Combined effects of soy isoflavones and milk basic protein on bone mineral density in hind-limb unloaded mice.

    Science.gov (United States)

    Matsumoto, Yu; Tousen, Yuko; Nishide, Yoriko; Tadaishi, Miki; Kato, Ken; Ishimi, Yoshiko

    2016-03-01

    We examined whether the combination of isoflavone and milk basic protein both are reported to be effective for bone metabolism, prevents bone loss induced by skeletal hind-limb unloading in mice. Female ddY strain mice, aged 8 weeks, were divided into six groups (n = 6-8 each): (1) normally housed group, (2) loading group, (3) hind-limb unloading group fed a control diet, (4) hind-limb unloading group fed a 0.2% isoflavone conjugates diet, (5) hind-limb unloading group fed a 1.0% milk basic protein diet, and (6) hind-limb unloading group fed a 0.2% isoflavone conjugates and 1.0% milk basic protein diet. After 3 weeks, femoral bone mineral density was markedly reduced in unloading mice. The combination of isoflavone and milk basic protein showed cooperative effects in preventing bone loss and milk basic protein inhibited the increased expression of osteogenic genes in bone marrow cells in unloading mice. These results suggest that the combination of soy isoflavone and milk basic protein may be useful for bone health in subjects with disabling conditions as well as astronauts.

  20. T-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis.

    Science.gov (United States)

    Michaelevski, Izhak; Kirshenbaum, Noam; Sharon, Michal

    2010-07-31

    -MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)(10). We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical Omega values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

  1. Golli myelin basic proteins stimulate oligodendrocyte progenitor cell proliferation and differentiation in remyelinating adult mouse brain.

    Science.gov (United States)

    Paez, Pablo M; Cheli, Veronica T; Ghiani, Cristina A; Spreuer, Vilma; Handley, Vance W; Campagnoni, Anthony T

    2012-07-01

    Golli myelin basic proteins are necessary for normal myelination, acting via voltage and store-dependent Ca(2+) entry at multiple steps during oligodendrocyte progenitor cell (OPC) development. To date nothing is known regarding the role of golli proteins in demyelination or remyelination events. Here the effects of golli ablation and overexpression in myelin loss and recovery were examined using the cuprizone (CPZ) model of demyelination/remyelination. We found severe demyelination in the corpus callosum (CC) of golli-overexpressing mice (JOE) during the CPZ treatment, which was accompanied by an increased number of reactive astrocytes and activation of microglia/macrophages. During demyelination of JOE brains, a significant increase in the number of proliferating OPCs was found in the CC as well as in the subventricular zone, and our data indicate that these progenitors matured and fully remyelinated the CC of JOE animals after CPZ withdrawal. In contrast, in the absence of golli (golli-KO mice) delayed myelin loss associated with a smaller immune response, and a lower number of OPCs was found in these mice during the CPZ treatment. Furthermore, incomplete remyelination was observed after CPZ removal in large areas of the CC of golli-KO mice, reflecting irregular recovery of the oligodendrocyte population and subsequent myelin sheath formation. Our findings demonstrate that golli proteins sensitize mature oligodendrocytes to CPZ-induced demyelination, while at the same time stimulate the proliferation/recruitment of OPCs during demyelination, resulting in accelerated remyelination.

  2. Transcriptional upregulation of myelin components in spontaneous myelin basic protein-deficient mice.

    Science.gov (United States)

    Staats, Kim A; Pombal, Diana; Schönefeldt, Susann; Van Helleputte, Lawrence; Maurin, Hervé; Dresselaers, Tom; Govaerts, Kristof; Himmelreich, Uwe; Van Leuven, Fred; Van Den Bosch, Ludo; Dooley, James; Humblet-Baron, Stephanie; Liston, Adrian

    2015-05-01

    Myelin is essential for efficient signal transduction in the nervous system comprising of multiple proteins. The intricacies of the regulation of the formation of myelin, and its components, are not fully understood. Here, we describe the characterization of a novel myelin basic protein (Mbp) mutant mouse, mbp(jive), which spontaneously occurred in our mouse colony. These mice displayed the onset of a shaking gait before 3 weeks of age and seizure onset before 2 months of age. Due to a progressive increase of seizure intensity, mbp(jive) mice experienced premature lethality at around 3 months of age. Mbp mRNA transcript or protein was undetectable and, accordingly, genetic analysis demonstrated a homozygous loss of exons 3 to 6 of Mbp. Peripheral nerve conductance was mostly unimpaired. Additionally, we observed grave structural changes in white matter predominant structures were detected by T1, T2 and diffusion weighted magnetic resonance imaging. We additionally observed that Mbp-deficiency results in an upregulation of Qkl, Mag and Cnp, suggestive of a regulatory feedback mechanism whereby compensatory increases in Qkl have downstream effects on Mag and Cnp. Further research will clarify the role and specifications of this myelin feedback loop, as well as determine its potential role in therapeutic strategies for demyelinating disorders.

  3. Myelin Basic Protein Citrullination in Multiple Sclerosis: A Potential Therapeutic Target for the Pathology.

    Science.gov (United States)

    Yang, Lei; Tan, Dewei; Piao, Hua

    2016-08-01

    Multiple sclerosis (MS) is a multifactorial demyelinating disease characterized by neurodegenerative events and autoimmune response against myelin component. Citrullination or deimination, a post-translational modification of protein-bound arginine into citrulline, catalyzed by Ca(2+) dependent peptidylarginine deiminase enzyme (PAD), plays an essential role in physiological processes include gene expression regulation, apoptosis and the plasticity of the central nervous system, while aberrant citrullination can generate new epitopes, thus involving in the initiation and/or progression of autoimmune disorder like MS. Myelin basic protein (MBP) is the major myelin protein and is generally considered to maintain the stability of the myelin sheath. This review describes the MBP citrullination and its consequence, as well as offering further support for the "inside-out" hypothesis that MS is primarily a neurodegenerative disease with secondary inflammatory demyelination. In addition, it discusses the role of MBP citrullination in the immune inflammation and explores the potential of inhibition of PAD enzymes as a therapeutic strategy for the disease.

  4. Post-translational Modifications of Chicken Myelin Basic Protein Charge Components

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jeongkwon; Zhang, Rui; Strittmatter, Eric F.; Smith, Richard D.; Zand, Robert

    2008-07-11

    Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP’s. Mammalian MBP’s, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the posttranslational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial.

  5. The proform of eosinophil major basic protein: a new maternal serum marker for adverse pregnancy outcome

    DEFF Research Database (Denmark)

    Pihl, Kasper; Larsen, Torben; Rasmussen, Steen;

    2009-01-01

    MBP median was significantly reduced in pregnancies with SGA (0.81 MoM), spontaneous preterm delivery (0.83 MoM), preeclampsia (0.88 MoM) and gestational hypertension (0.60 MoM). The best screening performance was found for preeclampsia including the covariates proMBP and nulliparity yielding an area under......OBJECTIVE: To establish the first trimester serum levels of the proform of eosinophil major basic protein (proMBP) in pregnancies with adverse outcome. Furthermore, to determine the screening performance using proMBP alone and in combination with other first trimester markers. METHODS: A case......-control study was conducted in a primary hospital setting. The proMBP concentration was measured in cases with small-for-gestational age (SGA) (n = 150), spontaneous preterm delivery (n = 88), preeclampsia (n = 40), gestational hypertension (n = 10) and in controls (n = 500). Concentrations were converted...

  6. A thermodynamic investigation on the binding of mercury ion with myelin basic protein at different temperatures

    Institute of Scientific and Technical Information of China (English)

    G. Rezaei Behbehani; L. Barzegar; A.A. Saboury; S. Ghammami

    2011-01-01

    A thermodynamic study on the interaction of myelin basic protein with mercury ion was studied by using isothermal titration calorimetry, ITC, at 300.15, 310.15 and 320.15 K in Tris buffer solution at pH 7. The enthalpies of MBP + Hg2+ interaction are reported and analysed in terms of the extended solvation model. It was found that MBP has two identical and non-cooperative binding sites for Hg2+ ions. The intrinsic dissociation equilibrium constants are 99.904,112.968 and 126.724 |μmol/L, and the molar enthalpy of binding are -11.634, -10.768 and -10.117 kJ mol 1 at 300.15, 310.15 and 320.15 K, respectively.

  7. Loss of Myelin Basic Protein Function Triggers Myelin Breakdown in Models of Demyelinating Diseases

    Directory of Open Access Journals (Sweden)

    Marie-Theres Weil

    2016-07-01

    Full Text Available Breakdown of myelin sheaths is a pathological hallmark of several autoimmune diseases of the nervous system. We employed autoantibody-mediated animal models of demyelinating diseases, including a rat model of neuromyelitis optica (NMO, to target myelin and found that myelin lamellae are broken down into vesicular structures at the innermost region of the myelin sheath. We demonstrated that myelin basic proteins (MBP, which form a polymer in between the myelin membrane layers, are targeted in these models. Elevation of intracellular Ca2+ levels resulted in MBP network disassembly and myelin vesiculation. We propose that the aberrant phase transition of MBP molecules from their cohesive to soluble and non-adhesive state is a mechanism triggering myelin breakdown in NMO and possibly in other demyelinating diseases.

  8. Loss of Myelin Basic Protein Function Triggers Myelin Breakdown in Models of Demyelinating Diseases.

    Science.gov (United States)

    Weil, Marie-Theres; Möbius, Wiebke; Winkler, Anne; Ruhwedel, Torben; Wrzos, Claudia; Romanelli, Elisa; Bennett, Jeffrey L; Enz, Lukas; Goebels, Norbert; Nave, Klaus-Armin; Kerschensteiner, Martin; Schaeren-Wiemers, Nicole; Stadelmann, Christine; Simons, Mikael

    2016-07-12

    Breakdown of myelin sheaths is a pathological hallmark of several autoimmune diseases of the nervous system. We employed autoantibody-mediated animal models of demyelinating diseases, including a rat model of neuromyelitis optica (NMO), to target myelin and found that myelin lamellae are broken down into vesicular structures at the innermost region of the myelin sheath. We demonstrated that myelin basic proteins (MBP), which form a polymer in between the myelin membrane layers, are targeted in these models. Elevation of intracellular Ca(2+) levels resulted in MBP network disassembly and myelin vesiculation. We propose that the aberrant phase transition of MBP molecules from their cohesive to soluble and non-adhesive state is a mechanism triggering myelin breakdown in NMO and possibly in other demyelinating diseases.

  9. Downregulation of the microtubule associated protein tau impairs process outgrowth and myelin basic protein mRNA transport in oligodendrocytes.

    Science.gov (United States)

    Seiberlich, Veronika; Bauer, Nina G; Schwarz, Lisa; Ffrench-Constant, Charles; Goldbaum, Olaf; Richter-Landsberg, Christiane

    2015-09-01

    Oligodendrocytes, the myelin forming cells of the CNS, are characterized by their numerous membranous extensions, which enwrap neuronal axons and form myelin sheaths. During differentiation oligodendrocytes pass different morphological stages, downregulate the expression of the proteoglycan NG2, and acquire major myelin specific proteins, such as myelin basic proteins (MBP) and proteolipid protein. MBP mRNA is transported in RNA granules along the microtubules (MTs) to the periphery and translated locally. MTs participate in the elaboration and stabilization of the myelin forming extensions and are essential for cellular sorting processes. Their dynamic properties are regulated by microtubule associated proteins (MAPs). The MAP tau is present in oligodendrocytes and involved in the regulation and stabilization of the MT network. To further elucidate the functional significance of tau in oligodendrocytes, we have downregulated tau by siRNA technology and studied the effects on cell differentiation and neuron-glia contact formation. The data show that tau knockdown impairs process outgrowth and leads to a decrease in MBP expression. Furthermore, MBP mRNA transport to distant cellular extensions is impaired and cells remain in the NG2 stage. In myelinating cocultures with dorsal root ganglion neurons, oligodendrocyte precursor cells after tau miR RNA lentiviral knockdown develop into NG2 positive cells with very long and thin processes, contacting axons loosely, but fail to form internodes. This demonstrates that tau is important for MBP mRNA transport and involved in process formation. The disturbance of the balance of tau leads to abnormalities in oligodendrocyte differentiation, neuron-glia contact formation and the early myelination process.

  10. Classic and Golli Myelin Basic Protein have distinct developmental trajectories in human visual cortex.

    Science.gov (United States)

    Siu, Caitlin R; Balsor, Justin L; Jones, David G; Murphy, Kathryn M

    2015-01-01

    Traditionally, myelin is viewed as insulation around axons, however, more recent studies have shown it also plays an important role in plasticity, axonal metabolism, and neuroimmune signaling. Myelin is a complex multi-protein structure composed of hundreds of proteins, with Myelin Basic Protein (MBP) being the most studied. MBP has two families: Classic-MBP that is necessary for activity driven compaction of myelin around axons, and Golli-MBP that is found in neurons, oligodendrocytes, and T-cells. Furthermore, Golli-MBP has been called a "molecular link" between the nervous and immune systems. In visual cortex specifically, myelin proteins interact with immune processes to affect experience-dependent plasticity. We studied myelin in human visual cortex using Western blotting to quantify Classic- and Golli-MBP expression in post-mortem tissue samples ranging in age from 20 days to 80 years. We found that Classic- and Golli-MBP have different patterns of change across the lifespan. Classic-MBP gradually increases to 42 years and then declines into aging. Golli-MBP has early developmental changes that are coincident with milestones in visual system sensitive period, and gradually increases into aging. There are three stages in the balance between Classic- and Golli-MBP expression, with Golli-MBP dominating early, then shifting to Classic-MBP, and back to Golli-MBP in aging. Also Golli-MBP has a wave of high inter-individual variability during childhood. These results about cortical MBP expression are timely because they compliment recent advances in MRI techniques that produce high resolution maps of cortical myelin in normal and diseased brain. In addition, the unique pattern of Golli-MBP expression across the lifespan suggests that it supports high levels of neuroimmune interaction in cortical development and in aging.

  11. Classic and Golli Myelin Basic Protein have distinct developmental trajectories in human visual cortex

    Directory of Open Access Journals (Sweden)

    Caitlin R Siu

    2015-04-01

    Full Text Available Traditionally myelin is viewed as insulation around axons however more recent studies have shown it plays an important role in plasticity, axonal metabolism and neuroimmune signalling. Myelin is a complex multi-protein structure composed of hundreds of proteins, with Myelin Basic Protein (MBP being the most studied. MBP has two families: Classic-MBP that is necessary for activity driven compaction of myelin around axons, and Golli-MBP that is found in neurons, oligodendrocytes, and T cells, and has been called a 'molecular link' between the nervous and immune systems. In visual cortex myelin proteins interact with immune processes to affect experience-dependent plasticity. We studied myelin in human visual cortex using Western blotting to quantify Classic- and Golli-MBP expression in post-mortem tissue samples ranging in age from 20 days to 80 years. We found that Classic- and Golli-MBP have different patterns of change across the lifespan: Classic-MBP gradually increases to 42 years and then declines into aging; Golli-MBP has changes that are coincident with milestones in visual system sensitive period, before gradually increasing into aging. There are 3 stages in the balance between Classic- and Golli-MBP expression, with Golli-MBP dominating early, then shifting to Classic-MBP, and back to Golli-MBP in aging. Also Golli-MBP has a wave of high inter-individual variability during childhood. These results about cortical MBP expression are timely because they compliment recent advances in MRI techniques that produce high resolution maps of cortical myelin in normal and diseased brain. In addition the unique pattern of Golli-MBP expression across the lifespan suggests that it supports high levels of neuroimmune interaction in cortical development and in aging.

  12. Reduced myelin basic protein and actin-related gene expression in visual cortex in schizophrenia.

    Science.gov (United States)

    Matthews, Paul R; Eastwood, Sharon L; Harrison, Paul J

    2012-01-01

    Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17) from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]). Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology.

  13. Reduced myelin basic protein and actin-related gene expression in visual cortex in schizophrenia.

    Directory of Open Access Journals (Sweden)

    Paul R Matthews

    Full Text Available Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17 from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]. Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology.

  14. SncRNA715 Inhibits Schwann Cell Myelin Basic Protein Synthesis.

    Science.gov (United States)

    Müller, Christina; Hochhaus, Nina M; Fontana, Xavier; Luhmann, Heiko J; White, Robin

    2015-01-01

    Myelin basic proteins (MBP) are major constituents of the myelin sheath in the central nervous system (CNS) and the peripheral nervous system (PNS). In the CNS Mbp translation occurs locally at the axon-glial contact site in a neuronal activity-dependent manner. Recently we identified the small non-coding RNA 715 (sncRNA715) as a key inhibitor of Mbp translation during transport in oligodendrocytes. Mbp mRNA localization in Schwann cells has been observed, but has not been investigated in much detail. Here we could confirm translational repression of Mbp mRNA in Schwann cells. We show that sncRNA715 is expressed and its levels correlate inversely with MBP in cultured Schwann cells and in the sciatic nerve in vivo. Furthermore we could reduce MBP protein levels in cultured Schwann cells by increasing the levels of the inhibitory sncRNA715. Our findings suggest similarities in sncRNA715-mediated translational repression of Mbp mRNA in oligodendrocytes and Schwann cells.

  15. Molecular evolution of myelin basic protein, an abundant structural myelin component.

    Science.gov (United States)

    Nawaz, Schanila; Schweitzer, Jörn; Jahn, Olaf; Werner, Hauke B

    2013-08-01

    Rapid nerve conduction in jawed vertebrates is facilitated by the myelination of axons, which evolved in ancient cartilaginous fish. We aim to understand the coevolution of myelin and the major myelin proteins. We found that myelin basic protein (MBP) derived from living cartilaginous fish (sharks and rays) associated with the plasma membrane of glial cells similar to the phosphatidylinositol (4,5)-bisphosphate (PIP₂)-binding marker PH-PLCδ1, and that ionomycin-induced PIP₂-hydrolysis led to its cellular redistribution. We identified two paralogous mbp genes in multiple teleost species, consistent with a genome duplication at the root of the teleost clade. Zebrafish mbpb is organized in a complex transcription unit together with the unrelated gene-of-the-oligodendrocyte-lineage (golli) while mbpa does not encode GOLLI. Moreover, the embryonic expression of mbpa and mbpb differed, indicating functional specialization after duplication. However, both mbpa and mbpb-mRNAs were detected in mature oligodendrocytes and Schwann cells, MBPa and MBPb were mass spectrometrically identified in zebrafish myelin, both associated with the plasma membrane via PIP₂, and the ratio of nonsynonymous to synonymous nucleotide-substitution rates (Ka/Ks) was low. Together, this indicates selective pressure to conserve many aspects of the cellular expression and function of MBP across vertebrate species. We propose that the PIP₂-binding function of MBP is evolutionarily old and that its emergence in ancient gnathostomata provided glial cells with the competence to myelinate.

  16. Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity.

    Science.gov (United States)

    Belogurov, Alexey; Kuzina, Ekaterina; Kudriaeva, Anna; Kononikhin, Alexey; Kovalchuk, Sergey; Surina, Yelena; Smirnov, Ivan; Lomakin, Yakov; Bacheva, Anna; Stepanov, Alexey; Karpova, Yaroslava; Lyupina, Yulia; Kharybin, Oleg; Melamed, Dobroslav; Ponomarenko, Natalia; Sharova, Natalia; Nikolaev, Eugene; Gabibov, Alexander

    2015-05-01

    Recent findings indicate that the ubiquitin-proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain-derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin-independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE-SJL mice is caused by a dramatic shift in the balance between constitutive and β1i(high) immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, β1i is increased in resident CNS cells, whereas β5i is imported by infiltrating lymphocytes through the blood-brain barrier. Peptidyl epoxyketone specifically inhibits brain-derived β1i(high) immunoproteasomes in vitro (kobs/[I] = 240 M(-1)s(-1)), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the β1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.

  17. Lipid domains control myelin basic protein adsorption and membrane interactions between model myelin lipid bilayers.

    Science.gov (United States)

    Lee, Dong Woog; Banquy, Xavier; Kristiansen, Kai; Kaufman, Yair; Boggs, Joan M; Israelachvili, Jacob N

    2014-02-25

    The surface forces apparatus and atomic force microscope were used to study the effects of lipid composition and concentrations of myelin basic protein (MBP) on the structure of model lipid bilayers, as well as the interaction forces and adhesion between them. The lipid bilayers had a lipid composition characteristic of the cytoplasmic leaflets of myelin from "normal" (healthy) and "disease-like" [experimental allergic encephalomyelitis (EAE)] animals. They showed significant differences in the adsorption mechanism of MBP. MBP adsorbs on normal bilayers to form a compact film (3-4 nm) with strong intermembrane adhesion (∼0.36 mJ/m(2)), in contrast to its formation of thicker (7-8 nm) swelled films with weaker intermembrane adhesion (∼0.13 mJ/m(2)) on EAE bilayers. MBP preferentially adsorbs to liquid-disordered submicron domains within the lipid membranes, attributed to hydrophobic attractions. These results show a direct connection between the lipid composition of membranes and membrane-protein adsorption mechanisms that affects intermembrane spacing and adhesion and has direct implications for demyelinating diseases.

  18. Interaction of myelin basic protein isoforms with lipid bilayers studied by FTIR spectroscopy

    Science.gov (United States)

    Jackson, Michael; Choo, Lin-P'ing; Boulias, Christopher; Moscarello, Mario A.; Mantsch, Henry H.

    1993-05-01

    The secondary structure of the naturally occurring isoforms of myelin basic protein (MBP1-8) from human myelin was studied by Fourier transform infrared spectroscopy under a variety of experimental conditions. In aqueous solution each isoform was found to be unstructured. In the presence of negatively charged liquid bilayers MBP1-4 were shown to exhibit an amide I band maximum indicative of the adoption of (alpha) -helical secondary structures. A detailed analysis revealed that significant proportions of (beta) -sheet secondary structure were also present. MBP5 and MBP8, which have significantly less cationic charge than MBP1-4, exhibited an amide I maximum identical to that seen in solution, suggesting that no interaction with the bilayer occurred. Analysis of the lipid CH2 and C equals O stretching vibrations also pointed towards significant interaction of MBP1-4 with the bilayer. The changes in intensity and frequency of these bands which typically accompany the phase transition in the pure bilayer were abolished by addition of the proteins. No such effect was seen for MBP5 and 8, the normal lipid phase transition being apparent. The implications of these results in the aetiology of multiple sclerosis is discussed.

  19. Purification and identification of lactoperoxidase in milk basic proteins as an inhibitor of osteoclastogenesis.

    Science.gov (United States)

    Morita, Y; Ono, A; Serizawa, A; Yogo, K; Ishida-Kitagawa, N; Takeya, T; Ogawa, T

    2011-05-01

    A milk protein fraction with alkaline isoelectric points (milk basic protein, MBP) inhibits both bone resorption and osteoclastogenesis for in vitro models. We previously identified bovine angiogenin as a component of MBP that inhibits bone resorption. However, purified angiogenin had no effect on osteoclastogenesis, suggesting that MBP contains unidentified component(s) that inhibit osteoclast formation. In this study, we purified lactoperoxidase (LPO) as the predominant inhibitor of osteoclastogenesis in MBP. The LPO treatment downregulated levels of reactive oxygen species in osteoclasts. Signaling by receptor activator of NF-kappa-B ligand/receptor activator of NF-kappa-B (RANKL/RANK) was downregulated in LPO-treated cells, and, in particular, the ubiquitination of tumor necrosis factor receptor associate factor 6 (TRAF6) and activation of downstream signaling cascades (JNK, p38, ERK, and NFκB) were suppressed. Ultimately, LPO treatment led to decreased expression of c-Fos and NFAT2. These results suggest that MBP contains at least 2 components that independently suppress bone resorption through a unique mechanism: angiogenin inhibits bone resorption and LPO inhibits RANKL-induced osteoclast differentiation. These data explain many of the positive aspects of milk consumption on bone health.

  20. Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

    Science.gov (United States)

    Zhang, Lei; Hao, Changchun; Feng, Ying; Gao, Feng; Lu, Xiaolong; Li, Junhua; Sun, Runguang

    2016-09-01

    Myelin basic protein (MBP) is an essential structure involved in the generation of central nervous system (CNS) myelin. Myelin shape has been described as liquid crystal structure of biological membrane. The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin. In this paper, we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy (AFM). By analyzing the pressure-area (π-A) and pressure-time (π-T) isotherms, univariate linear regression equation was obtained. In addition, the elastic modulus, surface pressure increase, maximal insertion pressure, and synergy factor of monolayers were detected. These parameters can be used to modulate the monolayers binding of protein, and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3- phosphoserine (DPPS) monolayer, followed by DPPC/DPPS mixed and 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) monolayers via electrostatic and hydrophobic interactions. AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP (5 nM) show a phase separation texture at the surface pressure of 20 mN/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure. MBP is not an integral membrane protein but, due to its positive charge, interacts with the lipid head groups and stabilizes the membranes. The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central

  1. Genome-wide identification, classification and functional analyses of the bHLH transcription factor family in the pig, Sus scrofa.

    Science.gov (United States)

    Liu, Wuyi

    2015-08-01

    The basic helix-loop-helix (bHLH) transcription factors are one of the largest families of gene regulatory proteins and play crucial roles in genetic, developmental and physiological processes in eukaryotes. Here, we conducted a survey of the Sus scrofa genome and identified 109 putative bHLH transcription factor members belonging to super-groups A, B, C, D, E, and F, respectively, while four members were orphan genes. We identified 6 most significantly enriched KEGG pathways and 116 most significant GO annotation categories. Further comprehensive surveys in human genome and other 12 medical databases identified 72 significantly enriched biological pathways with these 113 pig bHLH transcription factors. From the functional protein association network analysis 93 hub proteins were identified and 55 hub proteins created a tight network or a functional module within their protein families. Especially, there were 20 hub proteins found highly connected in the functional interaction network. The present study deepens our understanding and provided insights into the evolution and functional aspects of animal bHLH proteins and should serve as a solid foundation for further for analyses of specific bHLH transcription factors in the pig and other mammals.

  2. Basic science and spine literature document bone morphogenetic protein increases cancer risk

    Directory of Open Access Journals (Sweden)

    Nancy E Epstein

    2014-01-01

    Full Text Available Background: Increasingly, clinical articles document that bone morphogenetic protein (BMP/INFUSE: Medtronic, Memphis, TN, USA and its derivatives utilized in spinal surgery increase the risk of developing cancer. However, there is also a large body of basic science articles that also document that various types of BMP and other members of the TGF-Beta (transforming growth factor beta family promote the growth of different types of cancers. Methods: This review looks at many clinical articles citing BMP/INFUSE′s role, largely "off-label", in contributing to complications encountered during spinal surgery. Next, however, specific attention is given to the clinical and basic science literature regarding how BMP and its derivatives (e.g. members of the TGF-beta family may also impact the development of breast and other cancers. Results: Utilizing BMP/INFUSE in spine surgery increased the risk of cancers/new malignancy as documented in several studies. For example, Carragee et al. found that for single-level instrumented posterolateral fusions (PLF using high-dose rhBMP-2 (239 patients vs. autograft (control group; n = 224, the risks of new cancers at 2 and 5 years postoperatively were increased. In laboratory studies, BMP′s along with other members of the TGF-Beta family also modulated/contributed to the proliferation/differentiation of breast cancer (e.g. bone formation/turnover, breast cancer-related solid tumors, and metastases, lung, adrenal, and colon cancer. Conclusions: BMP/INFUSE when utilized clinically in spinal fusion surgery appears to promote cancer at higher rates than observed in the overall population. Furthermore, BMP and TGF-beta are correlated with increased cancer growth both in the clinic and the laboratory.

  3. Longitudinal changes in C-reactive protein, proform of eosinophil major basic protein, and pregnancy-associated plasma protein-A during weight changes in obese children

    DEFF Research Database (Denmark)

    Lausten-Thomsen, Ulrik; Gamborg, Michael; Bøjsøe, Christine

    2015-01-01

    BACKGROUND: Childhood obesity is associated with several complications, including cardiovascular comorbidity. Several biomarkers, such as high-sensitive C-reactive protein (hs-CRP), proform of eosinophil major basic protein (Pro-MBP) and pregnancy associated plasma protein-A (PAPP-A), have equally...... been linked to increased cardiovascular susceptibility. This study investigates these biomarkers during weight loss and regain in obese children. MATERIALS AND METHODS: A longitudinal study during a 12-week weight loss program with a 28 months follow-up was conducted. Anthropometrics and plasma......), and 2.70 (girls) were included. Ninety children completed the weight loss program and 68 children entered the follow-up program. Pro-MBP and PAPP-A, but not hs-CRP, exhibited individual-specific levels (tracking) during weight loss and regain. The PAPP-A/Pro-MBP correlation was strong, whereas the hs...

  4. Basic Residues within the Ebolavirus VP35 Protein Are Required for Its Viral Polymerase Cofactor Function ▿

    OpenAIRE

    Prins, Kathleen C.; Binning, Jennifer M.; Shabman, Reed S.; Leung, Daisy W.; Amarasinghe, Gaya K.; Christopher F Basler

    2010-01-01

    The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and ...

  5. Protective Effect of Electroacupuncture on Neural Myelin Sheaths is Mediated via Promotion of Oligodendrocyte Proliferation and Inhibition of Oligodendrocyte Death After Compressed Spinal Cord Injury.

    Science.gov (United States)

    Huang, Siqin; Tang, Chenglin; Sun, Shanquan; Cao, Wenfu; Qi, Wei; Xu, Jin; Huang, Juan; Lu, Weitian; Liu, Qian; Gong, Biao; Zhang, Yi; Jiang, Jin

    2015-12-01

    Electroacupuncture (EA) has been used worldwide to treat demyelinating diseases, but its therapeutic mechanism is poorly understood. In this study, a custom-designed model of compressed spinal cord injury (CSCI) was used to induce demyelination. Zusanli (ST36) and Taixi (KI3) acupoints of adult rats were stimulated by EA to demonstrate its protective effect. At 14 days after EA, both locomotor skills and ultrastructural features of myelin sheath were significantly improved. Phenotypes of proliferating cells were identified by double immunolabeling of 5-ethynyl-2'-deoxyuridine with antibodies to cell markers: NG2 [oligodendrocyte precursor cell (OPC) marker], 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) (oligodendrocyte marker), and glial fibrillary acidic protein (GFAP) (astrocyte marker). EA enhanced the proliferation of OPCs and CNPase, as well as the differentiation of OPCs by promoting Olig2 (the basic helix-loop-helix protein) and attenuating Id2 (the inhibitor of DNA binding 2). EA could also improve myelin basic protein (MBP) and protect existing oligodendrocytes from apoptosis by inhibiting caspase-12 (a representative of endoplasmic reticulum stress) and cytochrome c (an apoptotic factor and hallmark of mitochondria). Therefore, our results indicate that the protective effect of EA on neural myelin sheaths is mediated via promotion of oligodendrocyte proliferation and inhibition of oligodendrocyte death after CSCI.

  6. Regulation of the nuclear gene that encodes the alpha-subunit of the mitochondrial F0F1-ATP synthase complex. Activation by upstream stimulatory factor 2.

    Science.gov (United States)

    Breen, G A; Jordan, E M

    1997-04-18

    We have previously identified several positive cis-acting regulatory regions in the promoters of the bovine and human nuclear-encoded mitochondrial F0F1-ATP synthase alpha-subunit genes (ATPA). One of these cis-acting regions contains the sequence 5'-CACGTG-3' (an E-box), to which a number of transcription factors containing a basic helix-loop-helix motif can bind. This E-box element is required for maximum activity of the ATPA promoter in HeLa cells. The present study identifies the human transcription factor, upstream stimulatory factor 2 (USF2), as a nuclear factor that binds to the ATPA E-box and demonstrates that USF2 plays a critical role in the activation of the ATPA gene in vivo. Evidence includes the following. Antiserum directed against USF2 recognized factors present in HeLa nuclear extracts that interact with the ATPA promoter in mobility shift assays. Wild-type USF2 proteins synthesized from expression vectors trans-activated the ATPA promoter through the E-box, whereas truncated USF2 proteins devoid of the amino-terminal activation domains did not. Importantly, expression of a dominant-negative mutant of USF2 lacking the basic DNA binding domain but able to dimerize with endogenous USF proteins significantly reduced the level of activation of the ATPA promoter caused by ectopically coexpressed USF2, demonstrating the importance of endogenous USF2 in activation of the ATPA gene.

  7. MAGED1 is a novel regulator of a select subset of bHLH PAS transcription factors.

    Science.gov (United States)

    Sullivan, Adrienne E; Peet, Daniel J; Whitelaw, Murray L

    2016-09-01

    Transcription factors of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family generally have critical and nonredundant biological roles, but some bHLH PAS proteins compete for common cofactors or recognise similar DNA elements. Identifying factors that regulate function of bHLH PAS proteins, particularly in cells where multiple family members are coexpressed, is important for understanding bHLH PAS factor biology. This study identifies and characterises a novel interaction between melanoma-associated antigen D1 (MAGED1) and select members of the bHLH PAS transcription factor family. MAGED1 binds and positively regulates the transcriptional activity of family members SIM1, SIM2, NPAS4 and ARNT2, but does not interact with AhR, HIF1α and ARNT. This interaction is mediated by PAS repeat regions which also form the interface for bHLH PAS dimerisation, and accordingly MAGED1 is not found in complex with bHLH PAS dimers. We show that MAGED1 does not affect bHLH PAS protein levels and cannot be acting as a coactivator of transcriptionally active heterodimers, but rather appears to interact with nascent bHLH PAS proteins in the cytoplasm to enhance their function prior to nuclear import. As a selective regulator, MAGED1 may play an important role in the biology of these specific factors and in general bHLH PAS protein dynamics.

  8. Structure of the leukemia oncogene LMO2: implications for the assembly of a hematopoietic transcription factor complex.

    Science.gov (United States)

    El Omari, Kamel; Hoosdally, Sarah J; Tuladhar, Kapil; Karia, Dimple; Vyas, Paresh; Patient, Roger; Porcher, Catherine; Mancini, Erika J

    2011-02-17

    The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

  9. The execution of the transcriptional axis mutant p53, E2F1 and ID4 promotes tumor neo-angiogenesis.

    Science.gov (United States)

    Fontemaggi, Giulia; Dell'Orso, Stefania; Trisciuoglio, Daniela; Shay, Tal; Melucci, Elisa; Fazi, Francesco; Terrenato, Irene; Mottolese, Marcella; Muti, Paola; Domany, Eytan; Del Bufalo, Donatella; Strano, Sabrina; Blandino, Giovanni

    2009-10-01

    ID4 (inhibitor of DNA binding 4) is a member of a family of proteins that function as dominant-negative regulators of basic helix-loop-helix transcription factors. Growing evidence links ID proteins to cell proliferation, differentiation and tumorigenesis. Here we identify ID4 as a transcriptional target of gain-of-function p53 mutants R175H, R273H and R280K. Depletion of mutant p53 protein severely impairs ID4 expression in proliferating tumor cells. The protein complex mutant p53-E2F1 assembles on specific regions of the ID4 promoter and positively controls ID4 expression. The ID4 protein binds to and stabilizes mRNAs encoding pro-angiogenic factors IL8 and GRO-alpha. This results in the increase of the angiogenic potential of cancer cells expressing mutant p53. These findings highlight the transcriptional axis mutant p53, E2F1 and ID4 as a still undefined molecular mechanism contributing to tumor neo-angiogenesis.

  10. Conformational studies of immunodominant myelin basic protein 1-11 analogues using NMR and molecular modeling.

    Science.gov (United States)

    Laimou, Despina; Lazoura, Eliada; Troganis, Anastassios N; Matsoukas, Minos-Timotheos; Deraos, Spyros N; Katsara, Maria; Matsoukas, John; Apostolopoulos, Vasso; Tselios, Theodore V

    2011-11-01

    Τwo dimensional nuclear magnetic resonance studies complimented by molecular dynamics simulations were conducted to investigate the conformation of the immunodominant epitope of acetylated myelin basic protein residues 1-11 (Ac-MBP(1-11)) and its altered peptide ligands, mutated at position 4 to an alanine (Ac-MBP(1-11)[4A]) or a tyrosine residue (Ac-MBP(1-11)[4Y]). Conformational analysis of the three analogues indicated that they adopt an extended conformation in DMSO solution as no long distance NOE connectivities were observed and seem to have a similar conformation when bound to the active site of the major histocompatibility complex (MHC II). The interaction of each peptide with MHC class II I-A(u) was further investigated in order to explore the molecular mechanism of experimental autoimmune encephalomyelitis induction/inhibition in mice. The present findings indicate that the Gln(3) residue, which serves as a T-cell receptor (TCR) contact site in the TCR/peptide/I-A(u) complex, has a different orientation in the mutated analogues especially in the Ac-MBP(1-11)[4A] peptide. In particular the side chain of Gln(3) is not solvent exposed as for the native Ac-MBP(1-11) and it is not available for interaction with the TCR.

  11. Interleukin-12 suppresses filaria-induced pulmonary eosinophilia, deposition of major basic protein and airway hyperresponsiveness.

    Science.gov (United States)

    Mehlotra, R K; Hall, L R; Higgins, A W; Dreshaj, I A; Haxhiu, M A; Kazura, J W; Pearlman, E

    1998-10-01

    Tropical Pulmonary Eosinophilia (TPE) is a severe form of allergic asthma caused by the host inflammatory response to filarial helminths in the lung microvasculature, and is characterized by pulmonary eosinophilia, increased filarial-specific IgG and IgE antibodies, and airway hyperresponsiveness. The current study examined the effect of IL-12 on pulmonary eosinophilia, deposition of eosinophil major basic protein and airway hyperresponsiveness in mice inoculated i.v. with Brugia malayi microfilariae. Injection of recombinant murine IL-12 modulated the T helper (Th) response in the lungs from Th2- to Th1-like, with elevated IFN-gamma, and decreased IL-4 and IL-5 production. Consistent with this shift in cytokine response, antigen-specific IgG2a was elevated, and IgG1 and total serum IgE were decreased. In addition, eosinophils in BAL fluid from IL-12 treated mice were reduced from 56% to 11%, and there was no detectable MBP on respiratory epithelial cells. Importantly, IL-12 suppressed airway hyperresponsiveness compared with saline-injected control animals. Taken together, these data clearly demonstrate that by modulating Th associated cytokine production, IL-12 down-regulates filaria-induced lung immunopathology.

  12. Uptake and presentation of myelin basic protein by normal human B cells

    DEFF Research Database (Denmark)

    Brimnes, Marie Klinge; Hansen, Bjarke Endel; Nielsen, Leif Kofoed

    2014-01-01

    B cells may play both pathogenic and protective roles in T-cell mediated autoimmune diseases such as multiple sclerosis (MS). These functions relate to the ability of B cells to bind and present antigens. Under serum-free conditions we observed that 3-4% of circulating B cells from healthy donors...... were capable of binding the MS-associated self-antigen myelin basic protein (MBP) and of presenting the immunodominant peptide MBP85-99, as determined by staining with the mAb MK16 recognising the peptide presented by HLA-DR15-positive cells. In the presence of serum, however, the majority of B cells...... bound MBP in a complement-dependent manner, and almost half of the B cells became engaged in presentation of MBP85-99. Even though complement receptor 1 (CR1, CD35) and CR2 (CD21) both contributed to binding of MBP to B cells, only CR2 was important for the subsequent presentation of MBP85-99. A high...

  13. A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum

    Directory of Open Access Journals (Sweden)

    Dyall-Smith Mike

    2011-09-01

    Full Text Available Abstract Background The halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light. Results A new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein. The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions. Conclusions The activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.

  14. Regulation of the Drosophila Hypoxia-Inducible Factor α Sima by CRM1-Dependent Nuclear Export ▿

    Science.gov (United States)

    Romero, Nuria M.; Irisarri, Maximiliano; Roth, Peggy; Cauerhff, Ana; Samakovlis, Christos; Wappner, Pablo

    2008-01-01

    Hypoxia-inducible factor α (HIF-α) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-α protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia. PMID:18332128

  15. Molecular characterization and expression of As-nurp1 gene from Artemia sinica during development and in response to salinity and temperature stress.

    Science.gov (United States)

    Li, Qiuying; Zhang, Qiaozhi; Han, Lulu; Yuan, Zhe; Tan, Jian; Du, Bin; Zou, Xiangyang; Hou, Lin

    2012-06-01

    Nuclear protein 1 (NURP1) is a stress-related protein and closely related to diapause in the development of Artemia. In the present paper, the full-length 568-bp cDNA sequence of the nurp1 homolog of Artemia sinica (As-nurp1) was isolated by RACE technology for the first time. The putative As-nurp1 protein consists of 66 amino acids with a basic helix-loop-helix (bHLH) motif and a bipartite nuclear localization signal (NLS). Multiple sequence alignments revealed that the putative As-nurp1 protein sequence was relatively conserved across species, especially in the bHLH domain. The expression of As-nurp1 is widely distributed during A. sinica development. This is followed by a dramatic downregulation after diapause and is newly upregulated from the larval nauplius stage. Furthermore, As-nurp1 transcripts are highly upregulated under conditions of high salinity and low temperature. These findings suggest that As-nurp1 is stress-related and may act as a protective factor in embryonic development.

  16. Molecular markers of neuronal progenitors in the embryonic cerebellar anlage.

    Science.gov (United States)

    Morales, Daniver; Hatten, Mary E

    2006-11-22

    The cerebellum, like the cerebrum, includes a nuclear structure and an overlying cortical structure. Experiments in the past decade have expanded knowledge beyond the traditional function of the cerebellum to include critical roles in motor learning and memory and sensory discrimination. The initial steps in cerebellar development depend on inductive signaling involving FGF and Wnt proteins produced at the mesencephalic/metencephalic boundary. To address the issue of how individual cerebellar cell fates within the cerebellar territory are specified, we examined the expression of transcription factors, including mammalian homologues of LIM homeodomain-containing proteins, basic helix-loop-helix proteins, and three amino acid loop-containing proteins. The results of these studies show that combinatorial codes of transcription factors define precursors of the cerebellar nuclei, and both Purkinje cells and granule neurons of the cerebellar cortex. Examination of gene expression patterns in several hundred lines of Egfp-BAC (bacterial artificial chromosome) transgenic mice in the GENSAT Project revealed numerous genes with restricted expression in cerebellar progenitor populations, including genes specific for cerebellar nuclear precursors and Purkinje cell precursors. In addition, we identified patterns of gene expression that link granule and Purkinje cells to their precerebellar nuclei. These results identify molecular pathways that offer new insights on the development of the nuclear and cortical structures of the cerebellum, as well as components of the cerebellar circuitry.

  17. Variation in NOD2 augments Th2- and Th17 responses to myelin basic protein in multiple sclerosis

    DEFF Research Database (Denmark)

    Hedegaard, Chris Juul; Enevold, Christian; Sellebjerg, Finn;

    2011-01-01

    cell proliferation elicited by human myelin basic protein (MBP) in blood mononuclear cell (MNC) cultures from 29 patients with MS. No polymorphism was observed at rs5743277. No associations with the rs2066842 polymorphism were found. Concerning rs5743291, none were homozygous for the minor allele...

  18. Uptake and presentation of myelin basic protein by normal human B cells.

    Directory of Open Access Journals (Sweden)

    Marie Klinge Brimnes

    Full Text Available B cells may play both pathogenic and protective roles in T-cell mediated autoimmune diseases such as multiple sclerosis (MS. These functions relate to the ability of B cells to bind and present antigens. Under serum-free conditions we observed that 3-4% of circulating B cells from healthy donors were capable of binding the MS-associated self-antigen myelin basic protein (MBP and of presenting the immunodominant peptide MBP85-99, as determined by staining with the mAb MK16 recognising the peptide presented by HLA-DR15-positive cells. In the presence of serum, however, the majority of B cells bound MBP in a complement-dependent manner, and almost half of the B cells became engaged in presentation of MBP85-99. Even though complement receptor 1 (CR1, CD35 and CR2 (CD21 both contributed to binding of MBP to B cells, only CR2 was important for the subsequent presentation of MBP85-99. A high proportion of MBP85-99 presenting B cells expressed CD27, and showed increased expression of CD86 compared to non-presenting B cells. MBP-pulsed B cells induced a low frequency of IL-10-producing CD4+ T cells in 3 out of 6 donors, indicating an immunoregulatory role of B cells presenting MBP-derived peptides. The mechanisms described here refute the general assumption that B-cell presentation of self-antigens requires uptake via specific B-cell receptors, and may be important for maintenance of tolerance as well as for driving T-cell responses in autoimmune diseases.

  19. IgG reactivity against citrullinated myelin basic protein in multiple sclerosis.

    Science.gov (United States)

    de Seze, J; Dubucquoi, S; Lefranc, D; Virecoulon, F; Nuez, I; Dutoit, V; Vermersch, P; Prin, L

    2001-07-02

    An increased level of citrullinated myelin basic protein (MBP-C8) has been reported in the brains of multiple sclerosis (MS) patients. However, the involvement of the immune response to post-translational modified MBP in the pathophysiology of MS remains speculative. The aim of this study was to compare the levels of immunoglobulin G antibodies to several MBP epitopes, before and after citrullination, in the cerebrospinal fluid (CSF) and sera of MS patients using enzyme-linked immunosorbent assay (ELISA). We analyzed antibody reactivity against various MBP-peptides in the CSF and sera of 60 MS patients, and 30 patients with other neurological diseases (OND) as controls. The peptides tested were: MBP(75-98) (peptide 1), native (peptide 2) and citrullinated (peptide 3) MBP(108-126) (ARG(122)-->Cit(122)), and native (peptide 4) and citrullinated (peptide 5) MBP(151-170) (ARG(159, 170)-->Cit(159, 170)). All selected peptides could support an immune reactivity in CSF and sera of MS and OND patients. A higher reactivity against peptide 4 was found in the CSF of MS patients compared with OND patients (P<0.0001), but not against citrullinated peptides (peptides 3 and 5). However, we observed that the citrullination state of peptide 2 modified the patterns of immune reactivity more markedly in MS patients (P<0.0001) than in OND patients (P<0.02). Although some MBP epitopes could be a potential target in MS, our data did not demonstrate any difference of antibody response to MBP peptides in their citrullinated forms.

  20. Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K

    DEFF Research Database (Denmark)

    Laursen, Lisbeth Schmidt; Chan, Colin W; ffrench-Constant, Charles

    2011-01-01

    Myelination in the central nervous system provides a unique example of how cells establish asymmetry. The myelinating cell, the oligodendrocyte, extends processes to and wraps multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. Local regulation...... translation of a key sheath protein, myelin basic protein (MBP), by reversing the inhibitory effect of the mRNA 3′UTR. During oligodendrocyte differentiation and myelination α6β1-integrin interacts with hnRNP-K, an mRNA-binding protein, which binds to MBP mRNA and translocates from the nucleus to the myelin...

  1. Characterization of MxFIT, an iron deficiency induced transcriptional factor in Malus xiaojinensis.

    Science.gov (United States)

    Yin, Lili; Wang, Yi; Yuan, Mudan; Zhang, Xinzhong; Xu, Xuefeng; Han, Zhenhai

    2014-02-01

    Iron deficiency often results in nutritional disorder in fruit trees. Transcription factors play an important role in the regulation of iron uptake. In this study, we isolated an iron deficiency response transcription factor gene, MxFIT, from an iron-efficient apple genotype of Malus xiaojinensis. MxFIT encoded a basic helix-loop-helix protein and contained a 966 bp open reading frame. MxFIT protein was targeted to the nucleus in onion epidermal cells and showed strong transcriptional activation in yeast cells. Spatiotemporal expression analysis revealed that MxFIT was up-regulated in roots under iron deficiency at both mRNA and protein levels, while almost no expression was detected in leaves irrespective of iron supply. Ectopic expression of MxFIT resulted in enhanced iron deficiency responses in Arabidopsis under iron deficiency and stronger resistance to iron deficiency. Thus, MxFIT might be involved in iron uptake and plays an important role in iron deficiency response.

  2. Regulation of Sterol Biosynthesis in the Human Fungal Pathogen Aspergillus fumigatus: Opportunities for Therapeutic Development

    Science.gov (United States)

    Dhingra, Sourabh; Cramer, Robert A.

    2017-01-01

    Sterols are a major component of eukaryotic cell membranes. For human fungal infections caused by the filamentous fungus Aspergillus fumigatus, antifungal drugs that target sterol biosynthesis and/or function remain the standard of care. Yet, an understanding of A. fumigatus sterol biosynthesis regulatory mechanisms remains an under developed therapeutic target. The critical role of sterol biosynthesis regulation and its interactions with clinically relevant azole drugs is highlighted by the basic helix loop helix (bHLH) class of transcription factors known as Sterol Regulatory Element Binding Proteins (SREBPs). SREBPs regulate transcription of key ergosterol biosynthesis genes in fungi including A. fumigatus. In addition, other emerging regulatory pathways and target genes involved in sterol biosynthesis and drug interactions provide additional opportunities including the unfolded protein response, iron responsive transcriptional networks, and chaperone proteins such as Hsp90. Thus, targeting molecular pathways critical for sterol biosynthesis regulation presents an opportunity to improve therapeutic options for the collection of diseases termed aspergillosis. This mini-review summarizes our current understanding of sterol biosynthesis regulation with a focus on mechanisms of transcriptional regulation by the SREBP family of transcription factors. PMID:28203225

  3. Clusters of basic amino acids contribute to RNA binding and nucleolar localization of ribosomal protein L22.

    Directory of Open Access Journals (Sweden)

    Jennifer L Houmani

    Full Text Available The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80-93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80-93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA.

  4. Basic Tilted Helix Bundle – A new protein fold in human FKBP25/FKBP3 and HectD1

    Energy Technology Data Exchange (ETDEWEB)

    Helander, Sara; Montecchio, Meri [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Lemak, Alexander [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Farès, Christophe [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Almlöf, Jonas [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Li, Yanjun [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Yee, Adelinda [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Arrowsmith, Cheryl H. [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Dhe-Paganon, Sirano [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Sunnerhagen, Maria, E-mail: maria.sunnerhagen@liu.se [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden)

    2014-04-25

    Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP25{sub 1–73}, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.

  5. Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

    2015-01-01

    To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

  6. The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity

    DEFF Research Database (Denmark)

    Hansen, B E; Nielsen, Claus Henrik; Madsen, H O;

    2011-01-01

    Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype...

  7. An ABA-increased interaction of the PYL6 ABA receptor with MYC2 Transcription Factor: A putative link of ABA and JA signaling.

    Science.gov (United States)

    Aleman, Fernando; Yazaki, Junshi; Lee, Melissa; Takahashi, Yohei; Kim, Alice Y; Li, Zixing; Kinoshita, Toshinori; Ecker, Joseph R; Schroeder, Julian I

    2016-06-30

    Abscisic acid (ABA) is a plant hormone that mediates abiotic stress tolerance and regulates growth and development. ABA binds to members of the PYL/RCAR ABA receptor family that initiate signal transduction inhibiting type 2C protein phosphatases. Although crosstalk between ABA and the hormone Jasmonic Acid (JA) has been shown, the molecular entities that mediate this interaction have yet to be fully elucidated. We report a link between ABA and JA signaling through a direct interaction of the ABA receptor PYL6 (RCAR9) with the basic helix-loop-helix transcription factor MYC2. PYL6 and MYC2 interact in yeast two hybrid assays and the interaction is enhanced in the presence of ABA. PYL6 and MYC2 interact in planta based on bimolecular fluorescence complementation and co-immunoprecipitation of the proteins. Furthermore, PYL6 was able to modify transcription driven by MYC2 using JAZ6 and JAZ8 DNA promoter elements in yeast one hybrid assays. Finally, pyl6 T-DNA mutant plants show an increased sensitivity to the addition of JA along with ABA in cotyledon expansion experiments. Overall, the present study identifies a direct mechanism for transcriptional modulation mediated by an ABA receptor different from the core ABA signaling pathway, and a putative mechanistic link connecting ABA and JA signaling pathways.

  8. The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice.

    Science.gov (United States)

    Ko, Swee-Suak; Li, Min-Jeng; Sun-Ben Ku, Maurice; Ho, Yi-Cheng; Lin, Yi-Jyun; Chuang, Ming-Hsing; Hsing, Hong-Xian; Lien, Yi-Chen; Yang, Hui-Ting; Chang, Hung-Chia; Chan, Ming-Tsair

    2014-06-01

    Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.

  9. Neuronal differentiation of human iPS cells induced by baicalin via regulation of bHLH gene expression.

    Science.gov (United States)

    Morita, Akihiro; Soga, Kohei; Nakayama, Hironobu; Ishida, Torao; Kawanishi, Shosuke; Sato, Eisuke F

    2015-09-25

    Efficient differentiation is important for regenerative medicine based on pluripotent stem cells, including treatment of neurodegenerative disorders and trauma. Baicalin promotes neuronal differentiation of neural stem/progenitor cells of rats and mice. To evaluate the suitability of baicalin for neuronal differentiation of human iPS cells, we investigated whether it promotes neuronal differentiation in human iPS cells and monitored basic helix-loop-helix (bHLH) gene expression during neuronal differentiation. Baicalin promoted neuronal differentiation and inhibited glial differentiation, suggesting that baicalin can influence the neuronal fate decision in human iPS cells. Notch signaling, which is upstream of bHLH proteins, was not involved in baicalin-induced neuronal differentiation. Baicalin treatment did not down-regulate Hes1 gene expression, but it reduced Hes1 protein levels and up-regulated Ascl1 gene expression. Thus, baicalin promoted neuronal differentiation via modulation of bHLH transcriptional factors. Therefore, baicalin has potential to be used as a small-molecule drug for regenerative treatment of neurodegenerative disorders.

  10. Hes-1, a known transcriptional repressor, acts as a transcriptional activator for the human acid alpha-glucosidase gene in human fibroblast cells.

    Science.gov (United States)

    Yan, Bo; Raben, Nina; Plotz, Paul H

    2002-03-01

    Hes-1, the mammalian homologue 1 of Drosophila hairy and Enhancer of split proteins, belongs to a family of basic helix-loop-helix proteins that are essential to neurogenesis, myogenesis, hematopoiesis, and sex determination. Hes-1 is a transcriptional repressor for a number of known genes including the human acid alpha-glucosidase (GAA) gene as we have previously shown in Hep G2 cells. The human GAA gene encodes the enzyme for glycogen breakdown in lysosomes, deficiency of which results in Glycogen Storage Disease type II (Pompe syndrome). Using constructs containing the DNA element that demonstrates repressive activity in Hep G2 cells and conditions in which the same transcription factors, Hes-1 and YY1, bind, we have shown that this element functions as an enhancer in human fibroblasts. Site-directed mutagenesis and overexpression of Hes-1 showed that Hes-1 functions as a transcriptional activator. The dual function of Hes-1 we have found is likely to contribute to the subtle tissue-specific control of this housekeeping gene.

  11. A role for Id2 in regulating photic entrainment of the mammalian circadian system.

    Science.gov (United States)

    Duffield, Giles E; Watson, Nathan P; Mantani, Akio; Peirson, Stuart N; Robles-Murguia, Maricela; Loros, Jennifer J; Israel, Mark A; Dunlap, Jay C

    2009-02-24

    Inhibitor of DNA binding genes (Id1-Id4) encode helix-loop-helix (HLH) transcriptional repressors associated with development and tumorigenesis [1, 2], but little is known concerning the function(s) of these genes in normal adult animals. Id2 was identified in DNA microarray screens for rhythmically expressed genes [3-5], and further analysis revealed a circadian pattern of expression of all four Id genes in multiple tissues including the suprachiasmatic nucleus. To explore an in vivo function, we generated and characterized deletion mutations of Id2 and of Id4. Id2(-/-) mice exhibit abnormally rapid entrainment and an increase in the magnitude of the phase shift of the pacemaker. A significant proportion of mice also exhibit disrupted rhythms when maintained under constant darkness. Conversely, Id4(-/-) mice did not exhibit a noticeable circadian phenotype. In vitro studies using an mPer1 and an AVP promoter reporter revealed the potential for ID1, ID2, and ID3 proteins to interact with the canonical basic HLH clock proteins BMAL1 and CLOCK. These data suggest that the Id genes may be important for entrainment and operation of the mammalian circadian system, potentially acting through BMAL1 and CLOCK targets.

  12. FHL2 Antagonizes Id1-Promoted Proliferation and Invasive Capacity of Human MCF-7 Breast Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Wei-dong Han; Zhi-qiang Wu; Ya-li Zhao; Yi-ling Si; Ming-zhou Guo; Xiao-bing Fu

    2010-01-01

    Objective:FHL2 was previously identified to be a novel interacting factor of Id family proteins.The aim of this study was to investigate,the effects of FHL2 on Id1-mediated transcriptional regulation activity and its oncogenic activity in human breast cancer cells.Methods:Cell transfection was performed by Superfect reagent.Id1 stably overexpressed MCF-7 cells was cloned by G418 screening.The protein level of Id1 was detected by western blot analysis.Dual relative luciferase assays were used to measure the effect of E47-mediated transcriptional activity in MCF-7 human breast cancer cells.MTT assay was used to measure cell proliferation.Transwell assay was used to measure the invasive capacity of MCF-7 cancer cells.Results:The basic helix-loop-helix(bHLH)factor E47-mediated transcription activity was markedly repressed by Id1 in MCF-7 cells.This Id1-mediated repression was effectively antagonized by FHL2 transduction.Overexpression of Id1 markedly promoted the proliferation rate and invasive capacity of MCF-7 cells; however,these effects induced by Id1 were significantly suppressed by overexpression of FHL2 in cells.Conclusion:FHL2 can inhibit the proliferation and invasiveness of human breast cancer cells by repressing the functional activity of Id1.These findings provide the basis for further investigating the functional roles of FHL2-Id1 signaling in the carcinogenesis and development of human breast cancer.

  13. A Novel Soybean ERF Transcription Factor, GmERF113, Increases Resistance to Phytophthora sojae Infection in Soybean

    Science.gov (United States)

    Zhao, Yuanling; Chang, Xin; Qi, Dongyue; Dong, Lidong; Wang, Guangjin; Fan, Sujie; Jiang, Liangyu; Cheng, Qun; Chen, Xi; Han, Dan; Xu, Pengfei; Zhang, Shuzhen

    2017-01-01

    Phytophthora root and stem rot of soybean caused by the oomycete Phytophthora sojae, is a destructive disease worldwide. Ethylene response factors (ERFs) play important roles in regulating plant biotic and abiotic stress tolerance. In this study, a new ERF gene, GmERF113, was isolated from the highly resistant soybean ‘Suinong 10.’ Sequence analysis suggested that the protein encoded by GmERF113 contained a conserved AP2/ERF domain of 58 amino acid and belonged to the B-4 subgroup of the ERF subfamily. Expression of GmERF113 was significantly induced by P. sojae, ethylene, and methyl jasmonate. GmERF113 protein localized to the nucleus when transiently expressed in Arabidopsis protoplasts, could bind to the GCC-box, and acted as a transcription activator. In addition, a region of the full-length GmERF113, GmERF113-II, interacted with a basic helix-loop-helix transcription factor (GmbHLH) in yeast cells. Full-length GmERF113 also interacted with GmbHLH in planta. GmERF113-overexpressing transgenic plants in susceptible cultivar ‘Dongnong 50’ soybean exhibited increased resistance to P. sojae and positively regulated the expression of the pathogenesis-related genes, PR1 and PR10-1. These results indicate that GmERF113 may play a crucial role in the defense of soybean against P. sojae infection. PMID:28326092

  14. Immunodominant fragments of myelin basic protein initiate T cell-dependent pain

    Directory of Open Access Journals (Sweden)

    Liu Huaqing

    2012-06-01

    Full Text Available Abstract Background The myelin sheath provides electrical insulation of mechanosensory Aβ-afferent fibers. Myelin-degrading matrix metalloproteinases (MMPs damage the myelin sheath. The resulting electrical instability of Aβ-fibers is believed to activate the nociceptive circuitry in Aβ-fibers and initiate pain from innocuous tactile stimulation (mechanical allodynia. The precise molecular mechanisms, responsible for the development of this neuropathic pain state after nerve injury (for example, chronic constriction injury, CCI, are not well understood. Methods and results Using mass spectrometry of the whole sciatic nerve proteome followed by bioinformatics analyses, we determined that the pathways, which are classified as the Infectious Disease and T-helper cell signaling, are readily activated in the nerves post-CCI. Inhibition of MMP-9/MMP-2 suppressed CCI-induced mechanical allodynia and concomitant TNF-α and IL-17A expression in nerves. MMP-9 proteolysis of myelin basic protein (MBP generated the MBP84-104 and MBP68-86 digest peptides, which are prominent immunogenic epitopes. In agreement, the endogenous MBP69-86 epitope co-localized with MHCII and MMP-9 in Schwann cells and along the nodes of Ranvier. Administration of either the MBP84-104 or MBP68-86 peptides into the naïve nerve rapidly produced robust mechanical allodynia with a concomitant increase in T cells and MHCII-reactive cell populations at the injection site. As shown by the genome-wide expression profiling, a single intraneural MBP84-104 injection stimulated the inflammatory, immune cell trafficking, and antigen presentation pathways in the injected naïve nerves and the associated spinal cords. Both MBP84-104-induced mechanical allodynia and characteristic pathway activation were remarkably less prominent in the T cell-deficient athymic nude rats. Conclusions These data implicate MBP as a novel mediator of pain. Furthermore, the action of MMPs expressed within 1

  15. MyelStones: the executive roles of myelin basic protein in myelin assembly and destabilization in multiple sclerosis.

    Science.gov (United States)

    Vassall, Kenrick A; Bamm, Vladimir V; Harauz, George

    2015-11-15

    The classic isoforms of myelin basic protein (MBP, 14-21.5 kDa) are essential to formation of the multilamellar myelin sheath of the mammalian central nervous system (CNS). The predominant 18.5-kDa isoform links together the cytosolic surfaces of oligodendrocytes, but additionally participates in cytoskeletal turnover and membrane extension, Fyn-mediated signalling pathways, sequestration of phosphoinositides and maintenance of calcium homoeostasis. All MBP isoforms are intrinsically disordered proteins (IDPs) that interact via molecular recognition fragments (MoRFs), which thereby undergo local disorder-to-order transitions. Their conformations and associations are modulated by environment and by a dynamic barcode of post-translational modifications, particularly phosphorylation by mitogen-activated and other protein kinases and deimination [a hallmark of demyelination in multiple sclerosis (MS)]. The MBPs are thus to myelin what basic histones are to chromatin. Originally thought to be merely structural proteins forming an inert spool, histones are now known to be dynamic entities involved in epigenetic regulation and diseases such as cancer. Analogously, the MBPs are not mere adhesives of compact myelin, but active participants in oligodendrocyte proliferation and in membrane process extension and stabilization during myelinogenesis. A central segment of these proteins is pivotal in membrane-anchoring and SH3 domain (Src homology 3) interaction. We discuss in the present review advances in our understanding of conformational conversions of this classic basic protein upon membrane association, including new thermodynamic analyses of transitions into different structural ensembles and how a shift in the pattern of its post-translational modifications is associated with the pathogenesis and potentially onset of demyelination in MS.

  16. Calcium receptor expression and function in oligodendrocyte commitment and lineage progression: potential impact on reduced myelin basic protein in CaR-null mice

    DEFF Research Database (Denmark)

    Chattopadhyay, N.; Espinosa-Jeffrey, A.; Yano, S.;

    2008-01-01

    cellular proliferation. We further observed that high Ca(2+) stimulates the mRNA levels of myelin basic protein in preoligodendrocytes, which is also CaR mediated. Finally, myelin basic protein levels were significantly reduced in the cerebellum of CaR-null mice during development. Our results show that Ca...

  17. Mutual stimulation by phosphatidylinositol-4-phosphate and myelin basic protein of their phosphorylation by the kinases solubilized from rat brain myelin

    Energy Technology Data Exchange (ETDEWEB)

    Deshmukh, D.S.; Kuizon, S.; Brockerhoff, H.

    1984-01-16

    Myelin basic protein and phosphatidylinositol-4-phosphate are phosphorylated in vitro by ATP and solubilized rat brain myelin. When both substrates are present together, the rate of phosphorylation of each is increased about eight-fold. It appears likely that the phosphate turnover of myelin basic protein and of phosphatidylinositol-4-phosphate are coupled in vivo.

  18. Cation-exchange high-performance liquid chromatography: Separation of highly basic proteins using volatile acidic solvents

    NARCIS (Netherlands)

    Eijnden-van Raaij, A.J.M. van den; Koornneef, I.; Oostwaard, Th.M.J.; Laat, S.W. de; Zoelen, E.J.J. van

    1987-01-01

    The chromatographic behavior of a number of globular proteins was studied on a Bio-Sil TSK CM-2-SW weak cation exchange HPLC column under acidic conditions. A linear gradient of O-I M NH₄Ac in I M HOAc, inducing a convex pH gradient from 2.4-4.8, resulted in an excellent separation of highly basic p

  19. Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

    OpenAIRE

    Jacoby, D B; Gleich, G J; Fryer, A. D.

    1993-01-01

    The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of ...

  20. Endogenous interferon-β-inducible gene expression and interferon-β-treatment are associated with reduced T cell responses to myelin basic protein in multiple sclerosis

    DEFF Research Database (Denmark)

    Börnsen, Lars; Christensen, Jeppe Romme; Ratzer, Rikke;

    2015-01-01

    patients with an increased expression of interferon-β-inducible genes in peripheral blood mononuclear cells and interferon-β-treated multiple sclerosis patients had decreased CD4+ T-cell reactivity to the autoantigen myelin basic protein ex vivo. Interferon-β-treated multiple sclerosis patients had...... increased IL10 and IL27 gene expression levels in monocytes in vivo. In vitro, neutralization of interleukin-10 and monocyte depletion increased CD4+ T-cell reactivity to myelin basic protein while interleukin-10, in the presence or absence of monocytes, inhibited CD4+ T-cell reactivity to myelin basic...... protein. Our findings suggest that spontaneous expression of interferon-β-inducible genes in peripheral blood mononuclear cells from untreated multiple sclerosis patients and treatment with interferon-β are associated with reduced myelin basic protein-induced T-cell responses. Reduced myelin basic protein...

  1. Molecular characterisation, evolution and expression of hypoxia-inducible factor in Aurelia sp.1.

    Science.gov (United States)

    Wang, Guoshan; Yu, Zhigang; Zhen, Yu; Mi, Tiezhu; Shi, Yan; Wang, Jianyan; Wang, Minxiao; Sun, Song

    2014-01-01

    The maintenance of physiological oxygen homeostasis is mediated by hypoxia-inducible factor (HIF), a key transcriptional factor of the PHD-HIF system in all metazoans. However, the molecular evolutionary origin of this central physiological regulatory system is not well characterized. As the earliest eumetazoans, Cnidarians can be served as an interesting model for exploring the HIF system from an evolutionary perspective. We identified the complete cDNA sequence of HIF-1α (ASHIF) from the Aurelia sp.1, and the predicted HIF-1α protein (pASHIF) was comprised of 674 amino acids originating from 2,025 bp nucleotides. A Pairwise comparison revealed that pASHIF not only possessed conserved basic helix-loop-helix (bHLH) and Per-Arnt-Sim (PAS) domains but also contained the oxygen dependent degradation (ODD) and the C-terminal transactivation domains (C-TAD), the key domains for hypoxia regulation. As indicated by sequence analysis, the ASHIF gene contains 8 exons interrupted by 7 introns. Western blot analysis indicated that pASHIF that existed in the polyps and medusa of Aurelia. sp.1 was more stable for a hypoxic response than normoxia.

  2. RSL Class I Genes Controlled the Development of Epidermal Structures in the Common Ancestor of Land Plants.

    Science.gov (United States)

    Proust, Hélène; Honkanen, Suvi; Jones, Victor A S; Morieri, Giulia; Prescott, Helen; Kelly, Steve; Ishizaki, Kimitsune; Kohchi, Takayuki; Dolan, Liam

    2016-01-11

    The colonization of the land by plants, sometime before 470 million years ago, was accompanied by the evolution tissue systems [1-3]. Specialized structures with diverse functions-from nutrient acquisition to reproduction-derived from single cells in the outermost layer (epidermis) were important sources of morphological innovation at this time [2, 4, 5]. In extant plants, these structures may be unicellular extensions, such as root hairs or rhizoids [6-9], or multicellular structures, such as asexual propagules or secretory hairs (papillae) [10-12]. Here, we show that a ROOTHAIR DEFECTIVE SIX-LIKE (RSL) class I basic helix-loop-helix transcription factor positively regulates the development of the unicellular and multicellular structures that develop from individual cells that expand out of the epidermal plane of the liverwort Marchantia polymorpha; mutants that lack MpRSL1 function do not develop rhizoids, slime papillae, mucilage papillae, or gemmae. Furthermore, we discovered that RSL class I genes are also required for the development of multicellular axillary hairs on the gametophyte of the moss Physcomitrella patens. Because class I RSL proteins also control the development of rhizoids in mosses and root hairs in angiosperms [13, 14], these data demonstrate that the function of RSL class I genes was to control the development of structures derived from single epidermal cells in the common ancestor of the land plants. Class I RSL genes therefore controlled the generation of adaptive morphological diversity as plants colonized the land from the water.

  3. Physiological loading of tendons induces scleraxis expression in epitenon fibroblasts.

    Science.gov (United States)

    Mendias, Christopher L; Gumucio, Jonathan P; Bakhurin, Konstantin I; Lynch, Evan B; Brooks, Susan V

    2012-04-01

    Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting fibroblast proliferation and matrix synthesis during the embryonic development of tendons. Mice with a targeted inactivation of scleraxis (Scx(-/-)) fail to properly form limb tendons, but the role that scleraxis has in regulating the growth and adaptation of tendons of adult organisms is unknown. To determine if scleraxis expression changes in response to a physiological growth stimulus to tendons, we subjected adult mice that express green fluorescent protein (GFP) under the control of the scleraxis promoter (ScxGFP) to a 6-week-treadmill training program designed to induce adaptive growth in Achilles tendons. Age matched sedentary ScxGFP mice were used as controls. Scleraxis expression was sparsely observed in the epitenon region of sedentary mice, but in response to treadmill training, scleraxis was robustly expressed in fibroblasts that appeared to be emerging from the epitenon and migrating into the superficial regions of tendon fascicles. Treadmill training also led to an increase in scleraxis, tenomodulin, and type I collagen gene expression as measured by qPCR. These results suggest that in addition to regulating the embryonic formation of limb tendons, scleraxis also appears to play an important role in the adaptation of adult tendons to physiological loading.

  4. MicroRNA 146 (Mir146) modulates spermatogonial differentiation by retinoic acid in mice.

    Science.gov (United States)

    Huszar, Jessica M; Payne, Christopher J

    2013-01-01

    Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3' untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation.

  5. MicroRNA 146 (Mir146) Modulates Spermatogonial Differentiation by Retinoic Acid in Mice1

    Science.gov (United States)

    Huszar, Jessica M.; Payne, Christopher J.

    2012-01-01

    ABSTRACT Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3′ untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation. PMID:23221399

  6. The effect of carbon monoxide integrating nitric oxide through auxin signal in Arabidopsis to modulate iron deficiency

    Directory of Open Access Journals (Sweden)

    Liming eYang

    2016-03-01

    Full Text Available Carbon monoxide (CO and nitric oxide (NO are essential modulators that regulate the plant response to iron deficiency (-Fe. Auxin is a phytohormone that plays important roles in plant growth and development. We report here that in Arabidopsis –Fe enhanced heme oxygenase-dependent CO generation and auxin transport through redistribution of PIN1 protein, which subsequently increased NO accumulation; NO signaling regulated the activity of ferric chelate reductase (FCR and the expression of Fe-uptake genes including basic helix-loop-helix transcription factor (FIT and the ferric reduction oxidase 2 (FRO2. Over-expression of HY1 encoding heme oxygenase, or treatment with CO donor enhanced basipetal auxin transport, FCR activity, and the expressions of FIT and FRO2 under –Fe. Such effects were compromised in the mutant aux1-7 impaired in auxin transport or in the mutant noa1 or nia1/nia2 defective in NO biosynthesis. -Fe failed to promote auxin transport and FCR activity in hy1 mutant; such inability was reversed in the double mutant of hy1/yucca1 with elevated auxin production, or in hy1/cue1 mutant with NO over-accumulation. Taken together, our results suggest that CO modulates NO signaling through auxin to cope with Fe deficiency in Arabidopsis.

  7. The mutational spectrum in Waardenburg syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Read, A.P.; Tassabehji, M.; Liu, X.Z. [and others

    1994-09-01

    101 individuals or families with Waardenburg syndrome (WS) or related abnormalities have been screened for mutations in the PAX3 gene. PAX3 mutations were seen in 19 of 35 individuals or families with features of Type I Waardenburg syndrome. None of the 47 Type 2 WS families showed any PAX3 mutation, nor did any of 19 individuals with other neural crest syndromes or pigmentary disturbances. PAX3 mutations included substitutions of highly conserved amino acids, splice site mutations, nonsense mutations and frameshifting deletions or insertions. One patient (with Type 1 WS, mental retardation and growth retardation) had a chromosomal deletion of 7-8 Mb encompassing the PAX3 gene. Mutations were seen in each of exons 2-6, with a concentration in the 5{prime} part of the paired box (exon 2) and the 3{prime} part of the homeobox (exon 6). There was no evident relation between the molecular change and the clinical manifestations in mutation carriers. We conclude that PAX3 dosage effects very specifically produce dystopia canthorum, the distinguishing feature of Type 1 WS, and variably produce the other features of Type 1 WS depending on genetic background or chance events. Two of the Type 2 families showed linkage to markers from 3p14, the location of the MITF gene. MITF encodes a basic helix-loop-helix-zipper protein which is the homologue of the mouse microphthalmia gene product. It is likely that mutations in MITF cause some but not all Type 2 WS.

  8. [Mutation screening of MITF gene in patients with Waardenburg syndrome type 2].

    Science.gov (United States)

    Chen, Jing; Yang, Shu-Zhi; Liu, Jun; Han, Bing; Wang, Guo-Jian; Zhang, Xin; Kang, Dong-Yang; Dai, Pu; Young, Wie-Yen; Yuan, Hui-Jun

    2008-04-01

    Warrgenburg syndrome type 2 (WS2) is the most common autosomal dominantly-inherited syndrome with hearing loss. MITF (microphthalmia associated transcription factor)is a basic-helix-loop-helix-luecine zipper (bHLHZip) factor which regulates expression of tyrosinase, and is involved in melanocyte differentiation. Mutations in MITF associated with WS2 have been identified in some but not all affected families. Here, we report a three-generation Chinese family with a point mutation in the MITF gene causing WS2. The proband exhibits congenital severe sensorineural hearing loss, heterochromia iridis and facial freckles. One of family members manifests sensorineural deafness, and the other patients show premature greying or/and freckles. This mutation, heterozygous deletion c.639delA, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking normal interaction with its target DNA motif. This mutation is a novel mutation and the third case identified in exon 7 of MITF in WS2. Though there is only one base pair distance between this novel mutation and the other two documented cases and similar amino acids change, significant difference is seen in clinical phenotype, which suggests genetic background may play an important role.

  9. Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome.

    Science.gov (United States)

    Yousfi, M; Lasmoles, F; Lomri, A; Delannoy, P; Marie, P J

    2001-05-01

    The Saethre-Chotzen syndrome is characterized by premature fusion of cranial sutures resulting from mutations in Twist, a basic helix-loop-helix (bHLH) transcription factor. We have identified Twist target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome with a Twist mutation that introduces a stop codon upstream of the bHLH domain. We observed that Twist mRNA and protein levels were reduced in mutant cells and that the Twist mutation increased cell growth in mutant osteoblasts compared with control cells. The mutation also caused increased alkaline phosphatase and type I collagen expression independently of cell growth. During in vitro osteogenesis, Twist mutant cells showed increased ability to form alkaline phosphatase-positive bone-like nodular structures associated with increased type I collagen expression. Mutant cells also showed increased collagen synthesis and matrix production when cultured in aggregates, as well as an increased capacity to form a collagenous matrix in vivo when transplanted into nude mice. In contrast, Twist mutant osteoblasts displayed a cell-autonomous reduction of osteocalcin mRNA expression in basal conditions and during osteogenesis. The data show that genetic deletion of Twist causing reduced Twist dosage increases cell growth, collagen expression, and osteogenic capability, but inhibits osteocalcin gene expression. This provides one mechanism that may contribute to the premature cranial ossification induced by deletion of the bHLH Twist domain in Saethre-Chotzen syndrome.

  10. Disease-related growth factor and embryonic signaling pathways modulate an enhancer of TCF21 expression at the 6q23.2 coronary heart disease locus.

    Directory of Open Access Journals (Sweden)

    Clint L Miller

    Full Text Available Coronary heart disease (CHD is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription factor TCF21 gene. TCF21 (Capsulin/Pod1/Epicardin is a member of the basic-helix-loop-helix (bHLH transcription factor family, and regulates cell fate decisions and differentiation in the developing coronary vasculature. Herein, we characterize a cis-regulatory mechanism by which the lead polymorphism rs12190287 disrupts an atypical activator protein 1 (AP-1 element, as demonstrated by allele-specific transcriptional regulation, transcription factor binding, and chromatin organization, leading to altered TCF21 expression. Further, this element is shown to mediate signaling through platelet-derived growth factor receptor beta (PDGFR-β and Wilms tumor 1 (WT1 pathways. A second disease allele identified in East Asians also appears to disrupt an AP-1-like element. Thus, both disease-related growth factor and embryonic signaling pathways may regulate CHD risk through two independent alleles at TCF21.

  11. The aryl hydrocarbon receptor in barrier organ physiology, immunology, and toxicology.

    Science.gov (United States)

    Esser, Charlotte; Rannug, Agneta

    2015-01-01

    The aryl hydrocarbon receptor (AhR) is an evolutionarily old transcription factor belonging to the Per-ARNT-Sim-basic helix-loop-helix protein family. AhR translocates into the nucleus upon binding of various small molecules into the pocket of its single-ligand binding domain. AhR binding to both xenobiotic and endogenous ligands results in highly cell-specific transcriptome changes and in changes in cellular functions. We discuss here the role of AhR for immune cells of the barrier organs: skin, gut, and lung. Both adaptive and innate immune cells require AhR signaling at critical checkpoints. We also discuss the current two prevailing views-namely, 1) AhR as a promiscuous sensor for small chemicals and 2) a role for AhR as a balancing factor for cell differentiation and function, which is controlled by levels of endogenous high-affinity ligands. AhR signaling is considered a promising drug and preventive target, particularly for cancer, inflammatory, and autoimmune diseases. Therefore, understanding its biology is of great importance.

  12. Circadian clock and PIF4-mediated external coincidence mechanism coordinately integrates both of the cues from seasonal changes in photoperiod and temperature to regulate plant growth in Arabidopsis thaliana.

    Science.gov (United States)

    Nomoto, Yuji; Kubozono, Saori; Miyachi, Miki; Yamashino, Takafumi; Nakamichi, Norihito; Mizuno, Takeshi

    2013-02-01

    In Arabidopsis thaliana, the circadian clock regulates the photoperiodic plant growth including the elongation of hypocotyls in a short-days (SDs)-specific manner. The clock-controlled PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) gene encoding a basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this regulation. The SDs-specific elongation of hypocotyls is best explained by accumulation of the active PIF4 proteins at the end of night specifically in SDs due to coincidence between internal (circadian clock) and external (photoperiod) cues. However, this external coincidence model was challenged with the recent finding that the elongation of hypocotyls is markedly promoted at high growth temperature (28˚C) even in long-days (LDs), implying that the model to explain the photoperiodic response of plant architecture appears to be conditional on ambient temperature. With regard to this problem, the results of this and previous studies showed that the model holds under a wide range of ambient temperature conditions (16˚C to 28˚C). We propose that the circadian clock and PIF4-mediated external coincidence mechanism coordinately integrates both of the cues from seasonal changes in photoperiod and temperature to regulate plant growth in natural habitats.

  13. SPATULA links daytime temperature and plant growth rate.

    Science.gov (United States)

    Sidaway-Lee, Kate; Josse, Eve-Marie; Brown, Alanna; Gan, Yinbo; Halliday, Karen J; Graham, Ian A; Penfield, Steven

    2010-08-24

    Plants exhibit a wide variety of growth rates that are known to be determined by genetic and environmental factors, and different plants grow optimally at different temperatures, indicating that this is a genetically determined character. Moderate decreases in ambient temperature inhibit vegetative growth, but the mechanism is poorly understood, although a decrease in gibberellin (GA) levels is known to be required. Here we demonstrate that the basic helix-loop-helix transcription factor SPATULA (SPT), previously known to be a regulator of low temperature-responsive germination, mediates the repression of growth by cool daytime temperatures but has little or no growth-regulating role under warmer conditions. We show that only daytime temperatures affect vegetative growth and that SPT couples morning temperature to growth rate. In seedlings, warm temperatures inhibit the accumulation of the SPT protein, and SPT autoregulates its own transcript abundance in conjunction with diurnal effects. Genetic data show that repression of growth by SPT is independent of GA signaling and phytochrome B, as previously shown for PIF4. Our data suggest that SPT integrates time of day and temperature signaling to control vegetative growth rate.

  14. Molecular cloning and its expression of trachealess gene (As-trh) during development in brine shrimp, Artemia sinica.

    Science.gov (United States)

    Wang, Jia-Qing; Hou, Lin; Yi, Nan; Zhang, Riu-Feng; Zou, Xiang-Yang; Xiao, Qin; Guo, Ran

    2012-02-01

    Basic helix-loop-helix-PAS (bHLH-PAS) family transcription factors are implicated in multiple developmental and physiological regulatory processes. Herein, a full-length cDNA encoding a bHLH-PAS domain transcription factor trachealess gene (designated as As-trh) was cloned and characterized from brine shrimp (Artemia sinica) for the first time. The full-length cDNA of As-trh was 2,698 bp with a 2,319 bp open reading frame encoding a deduced protein of 772 amino acid polypeptide with a calculated molecular mass of 86.02 kDa and an isoelectric point of 5.87. Sequence alignment revealed that As-trh had high homology with other species trh gene, including the D-trh gene in Drosophila melanogaster and Bm-trh in Bombyx mori. The early and persistent expression of As-trh in the naupliar stages by whole-mount embryonic in situ hybridization and immunohistochemistry suggest that As-trh functions very early in the salt gland and may be required continuously in this tissue. Later in development, expression of As-trh begins to decrease and disappear in salt gland of the older nauplius and appears in the thoracic epipods of the sub-adult Artemia. These results indicated that As-trh might play an important role in osmoregulatiory organ development from the larvae stages through adult stages.

  15. Transcription factor ABF-1 suppresses plasma cell differentiation but facilitates memory B cell formation.

    Science.gov (United States)

    Chiu, Yi-Kai; Lin, I-Ying; Su, Shin-Tang; Wang, Kuan-Hsiung; Yang, Shii-Yi; Tsai, Dong-Yan; Hsieh, Yi-Ting; Lin, Kuo-I

    2014-09-01

    Ag-primed B cells that result from an immune response can form either memory B cells or Ab-secreting plasma cells; however, the molecular machinery that controls this cellular fate is poorly understood. In this study, we show that activated B cell factor-1 (ABF-1), which encodes a basic helix-loop-helix transcriptional repressor, participates in this regulation. ABF-1 was prevalently expressed in purified memory B cells and induced by T follicular helper cell-mediated signals. ABF-1 expression declined by the direct repression of B lymphocyte-induced maturation protein-1 during differentiation. Ectopic expression of ABF-1 reduced the formation of Ab-secreting cells in an in vitro differentiation system of human memory B cells. Accordingly, knockdown of ABF-1 potentiates the formation of Ab-secreting cells. A transgenic mouse that expresses inducible ABF-1 in a B cell-specific manner was generated to demonstrate that the formation of germinal center and memory B cells was augmented by induced ABF-1 in an immune response, whereas the Ag-specific plasma cell response was dampened. This effect was associated with the ability of ABF-1 to limit cell proliferation. Together, our results demonstrate that ABF-1 facilitates formation of memory B cells but prevents plasma cell differentiation.

  16. Tissue-Specific Regulation of Gibberellin Signaling Fine-Tunes Arabidopsis Iron-Deficiency Responses.

    Science.gov (United States)

    Wild, Michael; Davière, Jean-Michel; Regnault, Thomas; Sakvarelidze-Achard, Lali; Carrera, Esther; Lopez Diaz, Isabel; Cayrel, Anne; Dubeaux, Guillaume; Vert, Grégory; Achard, Patrick

    2016-04-18

    Iron is an essential element for most living organisms. Plants acquire iron from the rhizosphere and have evolved different biochemical and developmental responses to adapt to a low-iron environment. In Arabidopsis, FIT encodes a basic helix-loop-helix transcription factor that activates the expression of iron-uptake genes in root epidermis upon iron deficiency. Here, we report that the gibberellin (GA)-signaling DELLA repressors contribute substantially in the adaptive responses to iron-deficient conditions. When iron availability decreases, DELLAs accumulate in the root meristem, thereby restraining root growth, while being progressively excluded from epidermal cells in the root differentiation zone. Such DELLA exclusion from the site of iron acquisition relieves FIT from DELLA-dependent inhibition and therefore promotes iron uptake. Consistent with this mechanism, expression of a non-GA-degradable DELLA mutant protein in root epidermis interferes with iron acquisition. Hence, spatial distribution of DELLAs in roots is essential to fine-tune the adaptive responses to iron availability.

  17. TAL-1/SCL and its partners E47 and LMO2 up-regulate VE-cadherin expression in endothelial cells.

    Science.gov (United States)

    Deleuze, Virginie; Chalhoub, Elias; El-Hajj, Rawan; Dohet, Christiane; Le Clech, Mikaël; Couraud, Pierre-Olivier; Huber, Philippe; Mathieu, Danièle

    2007-04-01

    The basic helix-loop-helix TAL-1/SCL essential for hematopoietic development is also required during vascular development for embryonic angiogenesis. We reported that TAL-1 acts positively on postnatal angiogenesis by stimulating endothelial morphogenesis. Here, we investigated the functional consequences of TAL-1 silencing in human primary endothelial cells. We found that TAL-1 knockdown caused the inhibition of in vitro tubulomorphogenesis, which was associated with a dramatic reduction in vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Consistently, silencing of TAL-1 as well as of its cofactors E47 and LMO2 down-regulated VE-cadherin at both the mRNA and the protein level. Endogenous VE-cadherin transcription could be activated in nonendothelial HEK-293 cells by the sole concomitant ectopic expression of TAL-1, E47, and LMO2. Transient transfections in human primary endothelial cells derived from umbilical vein (HUVECs) demonstrated that VE-cadherin promoter activity was dependent on the integrity of a specialized E-box associated with a GATA motif and was maximal with the coexpression of the different components of the TAL-1 complex. Finally, chromatin immunoprecipitation assays showed that TAL-1 and its cofactors occupied the VE-cadherin promoter in HUVECs. Together, these data identify VE-cadherin as a bona fide target gene of the TAL-1 complex in the endothelial lineage, providing a first clue to TAL-1 function in angiogenesis.

  18. Auxin signaling modules regulate maize inflorescence architecture.

    Science.gov (United States)

    Galli, Mary; Liu, Qiujie; Moss, Britney L; Malcomber, Simon; Li, Wei; Gaines, Craig; Federici, Silvia; Roshkovan, Jessica; Meeley, Robert; Nemhauser, Jennifer L; Gallavotti, Andrea

    2015-10-27

    In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes, BARREN INFLORESCENCE1 and BARREN INFLORESCENCE4 (BIF1 and BIF4), that regulate the early steps required for inflorescence formation. BIF1 and BIF4 encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.

  19. The role of barren stalk1 in the architecture of maize.

    Science.gov (United States)

    Gallavotti, Andrea; Zhao, Qiong; Kyozuka, Junko; Meeley, Robert B; Ritter, Matthew K; Doebley, John F; Pè, M Enrico; Schmidt, Robert J

    2004-12-02

    The architecture of higher plants is established through the activity of lateral meristems--small groups of stem cells formed during vegetative and reproductive development. Lateral meristems generate branches and inflorescence structures, which define the overall form of a plant, and are largely responsible for the evolution of different plant architectures. Here, we report the isolation of the barren stalk1 gene, which encodes a non-canonical basic helix-loop-helix protein required for the initiation of all aerial lateral meristems in maize. barren stalk1 represents one of the earliest genes involved in the patterning of maize inflorescences, and, together with the teosinte branched1 gene, it regulates vegetative lateral meristem development. The architecture of maize has been a major target of selection for early agriculturalists and modern farmers, because it influences harvesting, breeding strategies and mechanization. By sampling nucleotide diversity in the barren stalk1 region, we show that two haplotypes entered the maize gene pool from its wild progenitor, teosinte, and that only one was incorporated throughout modern inbreds, suggesting that barren stalk1 was selected for agronomic purposes.

  20. Failure to Target RANKL Signaling Through p38-MAPK Results in Defective Osteoclastogenesis in the Microphthalmia Cloudy-Eyed Mutant.

    Science.gov (United States)

    Carey, Heather A; Bronisz, Agnieszka; Cabrera, Jennifer; Hildreth, Blake E; Cuitiño, Maria; Fu, Qi; Ahmad, Asrar; Toribio, Ramiro E; Ostrowski, Michael C; Sharma, Sudarshana M

    2016-03-01

    The Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of Receptor Activator of NFkB Ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C-terminal end. Here we show that the Mitf(ce) allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitf(ce/ce) mice. The loss of the leucine zipper domain in Mitf(ce/ce) mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitf(ce). This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site-directed mutations in the docking site interfered with the interaction between MITF and its co-factors FUS and BRG1. MITF-ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/p38 MAPK interactions in osteoclast differentiation.

  1. Conserved sequence motifs in the small subunit of human general transcription factor TFIIE.

    Science.gov (United States)

    Sumimoto, H; Ohkuma, Y; Sinn, E; Kato, H; Shimasaki, S; Horikoshi, M; Roeder, R G

    1991-12-05

    A general initiation factor, TFIIE, is essential for transcription initiation by RNA polymerase II in conjunction with other general factors. TFIIE is a heterotetramer containing two subunits of relative molecular mass 57,000 (TFIIE-alpha) and two of 34,000 (TFIIE-beta). TFIIE-beta is required in conjunction with TFIIE-alpha for transcription initiation. Here we report the cloning and expression of a complementary DNA encoding a functional human TFIIE-beta. Recombinant TFIIE-beta could replace the natural TFIIE-beta for transcription in conjunction with TFIIE-alpha. Amino-acid sequence comparisons reveal regions with sequence similarities to: subregion 3 of bacterial sigma factors; a region of RAP30 (the small subunit of TFIIF) with sequence similarity to a sigma-factor subregion implicated in binding to RNA polymerase; and a portion of the basic region-helix-loop-helix motif found in several enhancer-binding proteins. These potential homologies have implications for the role of TFIIE in preinitiation complex assembly and function.

  2. Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.

    Science.gov (United States)

    Friedler, A; Zakai, N; Karni, O; Friedler, D; Gilon, C; Loyter, A

    1999-06-11

    The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import.

  3. Molecular "negativity" may underlie multiple sclerosis: role of the myelin basic protein family in the pathogenesis of MS.

    Science.gov (United States)

    Musse, Abdiwahab A; Harauz, George

    2007-01-01

    Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is responsible for adhesion of these surfaces in the multilayered myelin sheath. The pattern of extensive posttranslational modifications of MBP is dynamic during normal central nervous system development and during myelin degeneration in multiple sclerosis (MS), affecting its interactions with the myelin membranes and other proteins. In particular, the degree of deimination (or citrullination) of MBP is correlated with the severity of MS, and may represent a primary defect that precedes neurodegeneration due to autoimmune attack. That MBP deimination also affects topological accessibility of an otherwise partially buried immunodominant epitope of the protein indicates that this modification may play a major role in the autoimmune pathogenesis of the disease. In this chapter, we describe the structural and functional consequences of MBP deimination in healthy and diseased myelin.

  4. Site-selective protein-modification chemistry for basic biology and drug development

    Science.gov (United States)

    Krall, Nikolaus; da Cruz, Filipa P.; Boutureira, Omar; Bernardes, Gonçalo J. L.

    2016-02-01

    Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

  5. Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule.

    Science.gov (United States)

    Majava, Viivi; Wang, Chaozhan; Myllykoski, Matti; Kangas, Salla M; Kang, Sung Ung; Hayashi, Nobuhiro; Baumgärtel, Peter; Heape, Anthony M; Lubec, Gert; Kursula, Petri

    2010-06-01

    Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP-CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein-protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP-CaM interaction, including a 3D model of the complex between full-length proteins.

  6. Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes.

    Science.gov (United States)

    Aseeva, Elena; Ossenbühl, Friederich; Sippel, Claudia; Cho, Won K; Stein, Bernhard; Eichacker, Lutz A; Meurer, Jörg; Wanner, Gerhard; Westhoff, Peter; Soll, Jürgen; Vothknecht, Ute C

    2007-02-01

    Vipp1 (vesicle inducing protein in plastids 1) is found in cyanobacteria and chloroplasts where it is essential for thylakoid formation. Arabidopsis thaliana mutant plants with a reduction of Vipp1 to about 20% of wild type content become albinotic at an early stage. We propose that this drastic phenotype results from an inability of the remaining Vipp1 protein to assemble into a homo-oligomeric complex, indicating that oligomerization is a prerequisite for Vipp1 function. A Vipp1-ProteinA fusion protein, expressed in the Deltavipp1 mutant background, is able to reinstate oligomerization and restore photoautotrophic growth. Plants containing Vipp1-ProteinA in amounts comparable to Vipp1 in the wild type exhibit a wild type phenotype. However, plants with a reduced amount of Vipp1-ProteinA protein are growth-retarded and significantly paler than the wild type. This phenotype is caused by a decrease in thylakoid membrane content and a concomitant reduction in photosynthetic activity. To the extent that thylakoid membranes are made in these plants they are properly assembled with protein-pigment complexes and are photosynthetically active. This strongly supports a function of Vipp1 in basic thylakoid membrane formation and not in the functional assembly of thylakoid protein complexes. Intriguingly, electron microscopic analysis shows that chloroplasts in the mutant plants are not equally affected by the Vipp1 shortage. Indeed, a wide range of different stages of thylakoid development ranging from wild-type-like chloroplasts to plastids nearly devoid of thylakoids can be observed in organelles of one and the same cell.

  7. A Basic Protein Comparative Three-Dimensional Modeling Methodological Workflow Theory and Practice.

    Science.gov (United States)

    Bitar, Mainá; Franco, Glória Regina

    2014-01-01

    When working with proteins and studying its properties, it is crucial to have access to the three-dimensional structure of the molecule. If experimentally solved structures are not available, comparative modeling techniques can be used to generate useful protein models to subsidize structure-based research projects. In recent years, with Bioinformatics becoming the basis for the study of protein structures, there is a crescent need for the exposure of details about the algorithms behind the softwares and servers, as well as a need for protocols to guide in silico predictive experiments. In this article, we explore different steps of the comparative modeling technique, such as template identification, sequence alignment, generation of candidate structures and quality assessment, its peculiarities and theoretical description. We then present a practical step-by-step workflow, to support the Biologist on the in silico generation of protein structures. Finally, we explore further steps on comparative modeling, presenting perspectives to the study of protein structures through Bioinformatics. We trust that this is a thorough guide for beginners that wish to work on the comparative modeling of proteins.

  8. The M Protein of SARS-CoV: Basic Structural and Immunological Properties

    Institute of Scientific and Technical Information of China (English)

    Yongwu Hu; Jianping Shi; Xiangjun Tian; Feng Jiang; Xiaoqian Zhao; Jun Wang; Siqi Liu; Changqing Zeng; Jian Wang; Huanming Yang; Jie Wen; Lin Tang; Haijun Zhang; Xiaowei Zhang; Yan Li; Jing Wang; Yujun Han; Guoqing Li

    2003-01-01

    We studied structural and immunological properties of the SARS-CoV M (mem-brane) protein, based on comparative analyses of sequence features, phylogeneticinvestigation, and experimental results. The M protein is predicted to contain atriple-spanning transmembrane (TM) region, a single N-glycosylation site near itsN-terminus that is in the exterior of the virion, and a long C-terminal region inthe interior. The M protein harbors a higher substitution rate (0.6% correlated toits size) among viral open reading frames (ORFs) from published data. The foursubstitutions detected in the M protein, which cause non-synonymous changes,can be classified into three types. One of them results in changes of pI (isoelectricpoint) and charge, affecting antigenicity. The second changes hydrophobicity of theTM region, and the third one relates to hydrophilicity of the interior structure.Phylogenetic tree building based on the variations of the M protein appears tosupport the non-human origin of SARS-CoV. To investigate its immunogenicity,we synthesized eight oligopeptides covering 69.2% of the entire ORF and screenedthem by using ELISA (enzyme-linked immunosorbent assay) with sera from SARSpatients. The results confirmed our predictions on antigenic sites.

  9. PCR typing of two short tandem repeat (STR) structures upstreams of the human myelin basic protein (MBP) gene

    DEFF Research Database (Denmark)

    Nellemann, L J; Frederiksen, J; Morling, N

    1995-01-01

    We investigated two short tandem tetranucleotide (TGGA) repeat polymorphisms upstreams of the myelin basic protein (MBP) gene. The region was amplified by the polymerase chain reaction (PCR) and the two repeat systems were separated by cutting with the restriction enzyme NlaIII. The lengths...... of the DNA fragments were analyzed by vertical electrophoresis in polyacrylamide gels followed by silver staining. We compared the DNA fragment frequencies of the two MBP regions in 34 patients suffering from multiple sclerosis and in 78 suffering from monosymptomatic idiopathic optic neuritis to those...

  10. Neuron-specific Enclose and Myelin Basic Protein in Cerebrospinal Fluid of Patients with First Episode Schizophrenia

    Institute of Scientific and Technical Information of China (English)

    LI Shuying; WU Hanrong; GUO Huirong; ZHAO Zheng

    2006-01-01

    In order to study whether patients with schizophrenia have cerebral injury, neuron-specific enolase (NSE) and myelin basic protein (MBP)in cerebrospinal fluid (CSF) of 33 patients with first episode schizophrenia and 9 from the control group were determined by double antibody sandwich enzyme immunoassay method. The results showed that there was significant difference in the NSE contents between the experimental group and control group (P<0.01). The NSE contents in CSF in the experimental group were positively correlated with MBP in schizophrenia patients (P<0.05). These findings suggested that patients with schizophrenia had cerebral injury.

  11. [Diagnostic and prognostic significance of the antibodies to the myelin basic protein in acute neuroinfections in children].

    Science.gov (United States)

    Petrukhin, A S; Idrisova, Zh R; Vorov'eva, N L; Gervazieva, V B; Dekonenko, E P

    2001-01-01

    The development of severe CNS damages including encephalitis is highly probable in some respiratory and exanthemata viral infections (measles, rubella, parotitis). A high level of IgG antibodies to the myelin basic protein was found in patients with parotitis meningitis and rubella encephalitis but it was not high in 80% of patients with encephalitis of the unclear etiology and in 25% of cases with rubella encephalitis. More accurate analysis of clinical, neurovisual and immunologic data revealed a link of appearance of such complications with both the presence of more pronounced demyelinization and prolongation of the disease.

  12. Thermodynamic study of the binding of calcium and magnesium ions with myelin basic protein using the extended solvation theory

    Institute of Scientific and Technical Information of China (English)

    G. Rezaei Behbehani; A.A. Saboury; A. Divsalar

    2008-01-01

    The interaction of myelin basic protein (MBP) from the bovine central nervous system with Ca2+ and Mg2+ ions, named as M2+, was studied by isothermal titration calorimetry at 27℃ in aqueous solution. The extended solvation model was used to reproduce the enthaipies of MBP+M2+ interactions.The solvation parameters recovered from the extended solvation model were attributed to the structural change of MBP due to the metal ion interaction. It was found that there is a set of two identical and noninteracting binding sites for Ca2+ and Mg2+ ions.

  13. Golli Myelin Basic Proteins Modulate Voltage-Operated Ca(++) Influx and Development in Cortical and Hippocampal Neurons.

    Science.gov (United States)

    Vt, Cheli; DA, Santiago González; V, Spreuer; V, Handley; At, Campagnoni; Pm, Paez

    2016-10-01

    The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca(++) channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca(++) influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca(++) entry and processes outgrowth in cortical neurons, and pharmacological activation of L-type Ca(++) channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca(++) influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca(++) alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders.

  14. Purification and Crystallization of Flammulin, a Basic Protein with Anti-tumor Activities from Flammulina Velutipes

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Flammulin, an anti-tumor protein, was purified from the aqueous extract of basidiomes of Flammulina Velutipes to electrophoretic homogeneity and crystallized by microdialysis against a polyethylene glycol- sodium phosphate buffer. The purified product was found to have marked antitumor effects and be able to affect the tumor cells directly.

  15. Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure

    Directory of Open Access Journals (Sweden)

    Hayashi Nobuhiro

    2008-02-01

    Full Text Available Abstract Background The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. Results The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC in different temperatures, and Kd was observed to be in the low μM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. Conclusion Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure

  16. Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimura, T.; Kobayashi, T.; Shinnoh, N.; Goto, I. (Kyushu Univ., Fukuoka (Japan))

    1990-10-01

    In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous (32P)ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using (3H)methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the (3H) methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.

  17. S-layer proteins as basic building blocks in a biomolecular construction kit

    Science.gov (United States)

    Pum, Dietmar; Neubauer, Angela; Györvary, Erika; Sára, Margit; Sleytr, Uwe B.

    2000-06-01

    Crystalline bacterial cell surface layer (S-layer) proteins have been optimized during billions of years of biological evolution as constituent elements of one of the simplest self-assembly systems. Isolated S-layer proteins possess the intrinsic property of being able to recrystallize into two-dimensional arrays at a broad spectrum of surfaces (e.g. silicon) and interfaces (e.g. air-water interface or planar lipid films). The well-defined arrangement of functional groups on S-layer lattices allows the binding of molecules and particles in defined regular arrays. S-layers recrystallized on solid supports can be patterned in the submicrometre range using standard optical lithography. S-layers also represent templates for the formation of inorganic nanocrystal superlattices (e.g. CdS, Au, Ni, Pt, or Pd) as required for molecular electronics and nonlinear optics.

  18. Basic evidence for class A G-protein-coupled receptor heteromerization

    Directory of Open Access Journals (Sweden)

    Rafael eFranco

    2016-03-01

    Full Text Available Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs seem an exception to the general rule of receptor-receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backwards instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery.

  19. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization

    Science.gov (United States)

    Franco, Rafael; Martínez-Pinilla, Eva; Lanciego, José L.; Navarro, Gemma

    2016-01-01

    Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor–receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery. PMID:27065866

  20. Twist1 activity thresholds define multiple functions in limb development

    OpenAIRE

    Krawchuk, Dayana; Weiner, Shoshana J; Chen, You-Tzung; Lu, Benson; Costantini, Frank; Behringer, Richard R.; Laufer, Ed

    2010-01-01

    The basic helix-loop-helix transcription factor Twist1 is essential for normal limb development. Twist1−/− embryos die at midgestation. However, studies on early limb buds found that Twist1−/− mutant limb mesenchyme has an impaired response to FGF signaling from the apical ectodermal ridge, which disrupts the feedback loop between the mesenchyme and AER, and reduces and shifts anteriorly Shh expression in the zone of polarizing activity. We have combined Twist1 null, hypomorph and conditional...

  1. Progress of transcription factor Twist expression in breast cancer and its biological effect

    Institute of Scientific and Technical Information of China (English)

    Tian Qian

    2016-01-01

    Breast cancer is the most common malignant tumor in women and the pathogenesis is not fully elucidated. Proliferation, invasion, epithelial-mesenchymal transition and angiogenesis are the links closely related to the occurrence and development of breast cancer. Twist is a type of basic helix-loop-helix transcription factor that can affect cell proliferation and invasion process, epithelial-mesenchymal transition process and angiogenesis process through regulating the transcription of downstream target genes. In the research, the study of transcription factor Twist expression in breast cancer and its biological effect is reviewed.

  2. High performance aptamer affinity chromatography for single-step selective extraction and screening of basic protein lysozyme.

    Science.gov (United States)

    Han, Bin; Zhao, Chao; Yin, Junfa; Wang, Hailin

    2012-08-15

    A DNA aptamer based high-performance affinity chromatography is developed for selective extraction and screening of a basic protein lysozyme. First, a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic column was synthesized in situ by thermally initiated radical polymerization, and then an anti-lysozyme DNA aptamer was covalently immobilized on the surface of the monolith through a 16-atom spacer arm. The target protein lysozyme but non-target proteins can be trapped by the immobilized anti-lysozyme DNA aptamer. In contrast, lysozyme cannot be trapped by the immobilized oligodeoxynucleotide that does not contain the sequence of the anti-lysozyme DNA aptamer. The study clearly demonstrates the trapping of lysozyme by the immobilized anti-lysozyme DNA aptamer is mainly due to specific recognition rather than simple electrostatic interaction of positively charged protein and the negatively charged DNA. The inter-day precision was determined as 0.8% for migration time and 4.2% for peak area, respectively. By the use of aptamer affinity monolith, a screening strategy is developed to selectively extract lysozyme from chicken egg white, showing the advantages of high efficiency, low cost and ease-of-operation.

  3. Determination of specificity influencing residues for key transcription factor families

    DEFF Research Database (Denmark)

    Patel, Ronak Y.; Garde, Christian; Stormo, Gary D.

    2015-01-01

    -dimensional structure of protein. Structural restraints on the evolution of the amino-acid sequence lead to identification of false SIRs. In this manuscript we extended three methods (direct information, PSICOVand adjusted mutual information) that have been used to disentangle spurious indirect protein residue......-residue contacts from direct contacts, to identify SIRs from joint alignments of amino-acids and specificity. We predicted SIRs for homeodomain (HD), helix-loop-helix, LacI and GntR families of TFs using these methods and compared to MI. Using various measures, we show that the performance of these three methods...

  4. The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein.

    Science.gov (United States)

    Kao, C Cheng; Ni, Peng; Hema, Masarapu; Huang, Xinlei; Dragnea, Bogdan

    2011-05-01

    The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.

  5. Purification and characterization of elicitor protein from Phytophthora colocasiae and basic resistance in Colocasia esculenta.

    Science.gov (United States)

    Mishra, Ajay Kumar; Sharma, Kamal; Misra, Raj Shekhar

    2009-01-01

    An elicitor was identified in the fungus Phytophthora colocasiae. The molecular weight of the purified elicitor was estimated by means of gel filtration chromatography and SDS-PAGE and was estimated as 15kDa. Protease treatment severely reduced its activity, allowing the conclusion that the elicitor is proteinaceous. Infiltration of a few nanograms of this proteinaceous elicitor into taro leaves caused the formation of lesions that closely resemble hypersensitive response lesions. The elicitation of the cells was effective in the induction of the activity of lipoxygenase. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, after the infiltration of the elicitor protein. After few days, systemic acquired resistance was also induced. Thus, taro plant cells that perceived the glycoprotein generated a cascade of signals acting at local, short, and long distances, and causing the coordinate expression of specific defence. The obtained results give important information regarding the plant-pathogen interactions, mainly as subsidy for taro improvement against Phytophthora leaf blight.

  6. N- and O-linked glycosylation site profiling of the human basic salivary proline-rich protein 3M.

    Science.gov (United States)

    Manconi, Barbara; Cabras, Tiziana; Sanna, Monica; Piras, Valentina; Liori, Barbara; Pisano, Elisabetta; Iavarone, Federica; Vincenzoni, Federica; Cordaro, Massimo; Faa, Gavino; Castagnola, Massimo; Messana, Irene

    2016-05-01

    In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.

  7. Effects of active immunisation with myelin basic protein and myelin-derived altered peptide ligand on pain hypersensitivity and neuroinflammation.

    Science.gov (United States)

    Perera, Chamini J; Lees, Justin G; Duffy, Samuel S; Makker, Preet G S; Fivelman, Brett; Apostolopoulos, Vasso; Moalem-Taylor, Gila

    2015-09-15

    Neuropathic pain is a debilitating condition in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Specific myelin basic protein (MBP) peptides are encephalitogenic, and myelin-derived altered peptide ligands (APLs) are capable of preventing and ameliorating EAE. We investigated the effects of active immunisation with a weakly encephalitogenic epitope of MBP (MBP87-99) and its mutant APL (Cyclo-87-99[A(91),A(96)]MBP87-99) on pain hypersensitivity and neuroinflammation in Lewis rats. MBP-treated rats exhibited significant mechanical and thermal pain hypersensitivity associated with infiltration of T cells, MHC class II expression and microglia activation in the spinal cord, without developing clinical signs of paralysis. Co-immunisation with APL significantly decreased pain hypersensitivity and neuroinflammation emphasising the important role of neuroimmune crosstalk in neuropathic pain.

  8. A tale of two citrullines--structural and functional aspects of myelin basic protein deimination in health and disease.

    Science.gov (United States)

    Harauz, George; Musse, Abdiwahab A

    2007-02-01

    Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is responsible for adhesion of these surfaces in the multilayered myelin sheath. The pattern of extensive post-translational modifications of MBP is dynamic during normal central nervous system (CNS) development and during myelin degeneration in multiple sclerosis (MS), affecting its interactions with the myelin membranes and with other molecules. In particular, the degree of deimination (or citrullination) of MBP is correlated with the severity of MS, and may represent a primary defect that precedes neurodegeneration due to autoimmune attack. That the degree of MBP deimination is also high in early CNS development indicates that this modification plays major physiological roles in myelin assembly. In this review, we describe the structural and functional consequences of MBP deimination in healthy and diseased myelin.

  9. Regulation of protein kinase Cmu by basic peptides and heparin. Putative role of an acidic domain in the activation of the kinase.

    Science.gov (United States)

    Gschwendt, M; Johannes, F J; Kittstein, W; Marks, F

    1997-08-15

    Protein kinase Cmu is a novel member of the protein kinase C (PKC) family that differs from the other isoenzymes in structural and enzymatic properties. No substrate proteins of PKCmu have been identified as yet. Moreover, the regulation of PKCmu activity remains obscure, since a structural region corresponding to the pseudosubstrate domains of other PKC isoenzymes has not been found for PKCmu. Here we show that aldolase is phosphorylated by PKCmu in vitro. Phosphorylation of aldolase and of two substrate peptides by PKCmu is inhibited by various proteins and peptides, including typical PKC substrates such as histone H1, myelin basic protein, and p53. This inhibitory activity seems to depend on clusters of basic amino acids in the protein/peptide structures. Moreover, in contrast to other PKC isoenzymes PKCmu is activated by heparin and dextran sulfate. Maximal activation by heparin is about twice and that by dextran sulfate four times as effective as maximal activation by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate, the conventional activators of c- and nPKC isoforms. We postulate that PKCmu contains an acidic domain, which is involved in the formation and stabilization of an active state and which, in the inactive enzyme, is blocked by an intramolecular interaction with a basic domain. This intramolecular block is thought to be released by heparin and possibly also by 12-O-tetradecanoylphorbol-13-acetate/phosphatidylserine, whereas basic peptides and proteins inhibit PKCmu activity by binding to the acidic domain of the active enzyme.

  10. Perturbation of myelin basic protein (Mbp) splice variant expression in developing rat cerebellum following perinatal exposure to methylmercury.

    Science.gov (United States)

    Padhi, Bhaja K; Pelletier, Guillaume

    2012-09-18

    Myelin sheaths surrounding axons are essential for saltatory conduction of nerve impulse in the central nervous system. A major protein constituent of myelin sheaths is produced by the myelin basic protein (Mbp) gene, whose expression in oligodendrocytes is conserved across vertebrates. In rat, five Mbp splice variants resulting from alternative splicing of exons 2, 5 and/or 6 are characterized. We developed a PCR-based strategy to quantify individual Mbp splice variants and characterized a sixth Mbp splice variant lacking only exon 5. This newly identified splice variant is predominantly expressed in developing rat brain and has orthologs in mouse and human. Many neurotoxic chemicals can perturb myelination and Mbp gene expression. Regulation of Mbp gene expression at the post-transcriptional level was assessed following perinatal exposure to neurotoxic methylmercury (2 mg/kg b.w./day). Similar reductions in total and individual Mbp splice variant mRNA levels suggest that methylmercury-induced perturbation in Mbp gene expression occurred as a consequence of decreased oligodendrocyte cell population in absence of a significant impact on its post-transcriptional regulation.

  11. Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro.

    Science.gov (United States)

    Boggs, Joan M; Rangaraj, Godha; Dicko, Awa

    2012-09-01

    Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium-calmodulin dissociated actin from the MBP-lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.

  12. Comparison of antibodies hydrolyzing myelin basic protein from the cerebrospinal fluid and serum of patients with multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Visilii B Doronin

    Full Text Available It was found that antibodies (Abs against myelin basic protein (MBP are the major components of the antibody response in multiple sclerosis (MS patients. We have recently shown that IgGs from sera of MS patients are active in the hydrolysis of MBP. However, in literature there are no available data concerning possible MBP-hydrolyzing Abs in cerebrospinal fluid (CSF of MS patients. We have shown that the average content of IgGs in their sera is about 195-fold higher than that in their CSF. Here we have compared, for the first time, the average content of lambda- and kappa-IgGs as well as IgGs of four different subclasses (IgG1-IgG4 in CSF and sera of MS patients. The average relative content of lambda-IgGs and kappa -IgGs in the case of CSFs (8.0 and 92.0% and sera (12.3 and 87.7% are comparable, while IgG1, IgG2, IgG3, and IgG4: CSF - 40.4, 49.0, 8.2, and 2.5% of total IgGs, respectively and the sera - 53.6, 36.0, 5.6, and 4.8%, decreased in different order. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF efficiently hydrolyze MBP and that their average specific catalytic activity is unpredictably ∼54-fold higher than that of Abs from sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that anti-MBP abzymes of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and in MS pathogenesis development.

  13. Functional interaction of CCAAT/enhancer-binding-proteinbasic region mutants with E2F transcription factors and DNA.

    Science.gov (United States)

    Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim

    2016-07-01

    The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα.

  14. Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

    Science.gov (United States)

    Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

    2011-02-01

    While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

  15. Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification

    DEFF Research Database (Denmark)

    Nawrocki, A; Larsen, Martin Røssel; Podtelejnikov, A V;

    1998-01-01

    Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI......-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross...

  16. An elevated level of circulating galanin promotes developmental expression of myelin basic protein in the mouse brain.

    Science.gov (United States)

    Lyubetska, H; Zhang, L; Kong, J; Vrontakis, M

    2015-01-22

    Myelinogenesis is a scheduled process that is regulated by the intrinsic properties of the cell and extracellular signals. Galanin (GAL) is a bioactive neuropeptide that is widely distributed throughout the nervous system. Chronic increase in circulating GAL levels protects the demyelination processes. Furthermore, GAL is synthesized in myelin-producing glial cells, such as oligodendrocytes and its expression level is at its highest between postnatal days 10 and 40. In the present study, we use our GAL transgenic mouse model to examine the effects of GAL on postnatal myelinogenesis in the CNS. Although we observed no difference in the proliferation of oligodendrocyte precursor cells, we found that GAL has a strong pro-myelinating effect. The transgenic mice at postnatal day 10 appeared to undergo myelinogenesis at an accelerated rate, as demonstrated by the increase in myelin basic protein (MBP) synthesis. The immunohistochemical results are consistent with our preliminary findings that suggest that GAL is a regulator of myelination and may be one of the myelination promoters. This finding is especially important for studies focusing on endogenous molecules for treating myelin-related diseases, such as multiple sclerosis and other leukodystrophies.

  17. Adrenomedullin promotes differentiation of oligodendrocyte precursor cells into myelin-basic-protein expressing oligodendrocytes under pathological conditions in vitro.

    Science.gov (United States)

    Maki, Takakuni; Takahashi, Yoko; Miyamoto, Nobukazu; Liang, Anna C; Ihara, Masafumi; Lo, Eng H; Arai, Ken

    2015-07-01

    Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs). However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM) promotes the in vitro differentiation of OPCs under pathological conditions. Primary OPCs were prepared from neonatal rat brains, and differentiated into myelin-basic-protein expressing oligodendrocytes over time. This in vitro OPC differentiation was inhibited by prolonged chemical hypoxic stress induced by non-lethal CoCl(2) treatment. However, AM promoted the OPC differentiation under the hypoxic stress conditions, and the AM receptor antagonist AM(22-52) canceled the AM-induced OPC differentiation. In addition, AM treatment increased the phosphorylation level of Akt in OPC cultures, and correspondingly, the PI3K/Akt inhibitor LY294002 blocked the AM-induced OPC differentiation. Taken together, AM treatment rescued OPC maturation under pathological conditions via an AM-receptor-PI3K/Akt pathway. Oligodendrocytes play critical roles in white matter by forming myelin sheath. Therefore, AM signaling may be a promising therapeutic target to boost oligodendrocyte regeneration in CNS disorders.

  18. Variation in NOD2 augments Th2- and Th17 responses to myelin basic protein in multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Chris Juul Hedegaard

    Full Text Available Variations in the gene for the nucleotide-binding oligomerisation domain (NOD 2 have been associated with Crohn's disease but not multiple sclerosis (MS. Here we investigate the effect of three polymorphisms in the NOD2 gene (rs5743277, rs2066842 and rs5743291 on cytokine production and CD4+ T cell proliferation elicited by human myelin basic protein (MBP in blood mononuclear cell (MNC cultures from 29 patients with MS. No polymorphism was observed at rs5743277. No associations with the rs2066842 polymorphism were found. Concerning rs5743291, none were homozygous for the minor allele. Seven of 29 (24% patients were heterozygous, and five of these (71% exhibited increased MBP-induced CD4+ T cell proliferation versus four of 22 (18%, who were homozygous for the major allele (p<0.04. Interleukin (IL-5 was induced by MBP in MNC from the same five carriers versus two (9% homozygotes (p<0.004; four carriers (57% versus three non-carriers (14% exhibited IL-17 responses to MBP (p<0.04. By contrast, we found no association between the polymorphisms investigated and interferon-gamma-, tumor necrosis factor-alpha-, IL-2, -4- or IL-10 responses to MBP. These results indicate that the rs5743291 polymorphism influences T helper (Th cell 2- and Th17 cell responses in MNC from MS patients.

  19. Diffusion tensor imaging measures of white matter compared to myelin basic protein immunofluorescence in tissue cleared intact brains

    Directory of Open Access Journals (Sweden)

    Eric H. Chang

    2017-02-01

    Full Text Available We provide datasets from combined ex vivo diffusion tensor imaging (DTI and Clear Lipid-exchanged, Anatomically Rigid, Imaging/immunostaining compatible, Tissue hYdrogel (CLARITY performed on intact mouse brains. DTI-derived measures of fractional anisotropy (FA, radial diffusivity (RD, and axial diffusivity (AD were compared to antibody-based labeling of myelin basic protein (MBP, as measured by fluorescence microscopy. We used a customized CLARITY hydrogel solution to facilitate whole brain tissue clearing and subsequent immunolabeling. We describe how CLARITY was made compatible with magnetic resonance imaging with the intention of facilitating future multimodal imaging studies that may combine noninvasive imaging with 3D immunohistochemistry. These data and methods are related to the accompanying research article entitled, ‘The role of myelination in measures of white matter integrity: Combination of diffusion tensor imaging and two-photon microscopy of CLARITY intact brains’ (E.H. Chang, M. Argyelan, M. Aggarwal, T-S. Chandon, K.H. Karlsgodt, S. Mori, A.K. Malhotra, 2016 [1].

  20. Major basic protein from eosinophils and myeloperoxidase from neutrophils are required for protective immunity to Strongyloides stercoralis in mice.

    Science.gov (United States)

    O'Connell, Amy E; Hess, Jessica A; Santiago, Gilberto A; Nolan, Thomas J; Lok, James B; Lee, James J; Abraham, David

    2011-07-01

    Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity.

  1. Characterization and expression analysis of AH receptors in aquatic mammals and birds

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun-Young [Ehime Prefectural Institute of Public Health and Environmental Science, Matsuyama (Japan); Yasui, Tomoko; Hisato, Iwata; Shinsuke, Tanabe [Ehime Univ., Matsuyama (Japan)

    2004-09-15

    The magnitude of the risk that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) pose to the health of aquatic birds and mammals is uncertain, because of the lack of direct information on the sensitivity and toxicity to these chemicals. Exposure to PHAHs is speculated to produce toxicity through changes in the expression of genes involved in the control of cell growth and differentiation. These changes are initiated by the binding to the aryl hydrocarbon receptor (AHR), a ligand-dependent transcription factor. The AHR and its dimerization partner ARNT belong to the basic-helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulation proteins. The bHLH domain was involved in protein-DNA and protein-protein interactions, and the PAS domain forms a secondary dimerization surface for heteromeric interactions between AHR and ARNT. Although the presence and basic function of AHR are known to be conserved in most vertebrates, only a limited number of studies on the structure and functional diversity of AHR in aquatic mammals and birds have been reported, in spite of their high exposure to dioxins and other related chemicals. To understand the molecular mechanism of susceptibility to dioxin exposure and toxic effects that PHAHs pose in wild animals, we investigated the molecular and functional characterization of AHRs from aquatic mammals and birds. Initially, the AHR cDNAs from the livers of Baikal seal (Pusa sibirica), black-footed albatross (Diomedea nigripes) and common cormorant (Phalacrocorax carbo) were cloned and sequenced. We also clarified the tissue-specific expression pattern of AHR mRNA and the relationships among PHAHs, AHR and CYP expression levels in the liver of Baikal seals and common cormorants.

  2. Basic and clinical research on the regulation of the intestinal barrier by Lactobacillus and its active protein components: a review with experience of one center.

    Science.gov (United States)

    Liu, Zhi-Hua; Kang, Liang; Wang, Jian-Ping

    2014-12-01

    Probiotics got protective effects on the intestinal barrier. Our present study is to review the basic and clinical progress on the regulation of the intestinal barrier by Lactobacillus and its active protein components, combing the study of our center. Our study have isolated the active component of micro integral membrane protein (MIMP) within the media place of the integral membrane protein of Lactobacillus plantarum, which was verified about the protective effects against the intestinal epithelial dysfunction. On the other hand, we also found the effects of perioperative use of probiotics in the prevention and treatment of postoperative intestinal barrier dysfunction, and reduction of the postoperative infective complications. In this review, we would like to report the founding of our center, involving in the basic and clinical research progress of regulation of intestinal barrier by Lactobacillus and its active protein component MIMP. Furthermore, we may also promote our following studies about the MIMP and its clinical verification.

  3. Myelin management by the 18.5-kDa and 21.5-kDa classic myelin basic protein isoforms.

    Science.gov (United States)

    Harauz, George; Boggs, Joan M

    2013-05-01

    The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5-kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two-dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3-domains, participation in Fyn-mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post-translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5-kDa MBP's protein-membrane and protein-protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5-kDa MBP has significant roles beyond membrane adhesion. The full-length, early-developmental 21.5-kDa splice isoform is predominantly karyophilic due to a non-traditional P-Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.

  4. Immunolocalization of alpha-keratins and associated beta-proteins in lizard epidermis shows that acidic keratins mix with basic keratin-associated beta-proteins.

    Science.gov (United States)

    Alibardi, Lorenzo

    2014-07-01

    The differentiation of the corneous layers of lizard epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against alpha-keratins and keratin associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins). Both beta-cells and alpha-cells of the corneous layer derive from the same germinal layer. An acidic type I alpha-keratin is present in basal and suprabasal layers, early differentiating clear, oberhautchen, and beta-cells. Type I keratin apparently disappears in differentiated beta- and alpha-layers of the mature corneous layers. Conversely, a basic type II alpha-keratin rich in glycine is absent or very scarce in basal and suprabasal layers and this keratin likely does not pair with type I keratin to form intermediate filaments but is weakly detected in the pre-corneous and corneous alpha-layer. Single and double labeling experiments show that in differentiating beta-cells, basic KAbetaPs are added and replace type-I keratin to form the hard beta-layer. Epidermal alpha-keratins contain scarce cysteine (0.2-1.4 %) that instead represents 4-19 % of amino acids present in KAbetaPs. Possible chemical bonds formed between alpha-keratins and KAbetaPs may derive from electrostatic interactions in addition to cross-linking through disulphide bonds. Both the high content in glycine of keratins and KAbetaPs may also contribute to increase the hydrophobicy of the beta- and alpha-layers and the resistance of the corneous layer. The increase of gly-rich KAbetaPs amount and the bonds to the framework of alpha-keratins give rise to the inflexible beta-layer while the cys-rich KAbetaPs produce a pliable alpha-layer.

  5. Analyses of loss-of-function mutations of the MITF gene suggest that haploinsufficiency is a cause of Waardenburg syndrome type 2A

    Energy Technology Data Exchange (ETDEWEB)

    Nobukuni, Yoshitaka; Watanabe, A.; Takeda, Kazushisa; Skarka, Hana; Tachibana, Masayoshi [National Inst. of Health, Bethesda, MD (United States)

    1996-07-01

    Waardenburg syndrome type 2 (WS2) is a dominantly inherited disorder characterized by a pigmentation anomaly and hearing impairment due to lack of melanocyte. Previous work has linked a subset of families with WS2 (WS2A) to the MITF gene that encodes a transcription factor with a basic-helix-loop-helix-leucine zipper (bHLH-Zip) motif and that is involved in melanocyte differentiation. Several splice-site and missense mutations have been reported in individuals affected with WS2A. In this report, we have identified two novel point mutations in the MITF gene in affected individuals from two different families with WS2A. The two mutations (C760{r_arrow}T and C895{r_arrow}T) create stop codons in exons 7 and 8, respectively. Corresponding mutant alleles predict the truncated proteins lacking HLH-Zip or Zip structure. To understand how these mutations cause WS2 in heterozygotes, we generated mutant MITF cDNAs and used them for DNA-binding and luciferase reporter assays. The mutated MITF proteins lose the DNA-binding activity and fail to transactivate the promoter of tyrosinase, a melanocyte-specific enzyme. However, these mutated proteins do not appear to interfere with the activity of wild-type MITF protein in these assays, indicating that they do not show a dominant-negative effect. These findings suggest that the phenotypes of the two families with WS2A in the present study are caused by loss-of-function mutations in one of the two alleles of the MITF gene, resulting in haploinsufficiency of the MITF protein, the protein necessary for normal development of melanocytes. 37 refs., 4 figs.

  6. An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis

    Directory of Open Access Journals (Sweden)

    Sitakanta ePattanaik

    2014-06-01

    Full Text Available Trichomes are specialized epidermal cells located on aerial parts of plants and are associated with a wide array of biological processes. Trichomes protect plants from adverse conditions including UV light and herbivore attack and are also an important source of a number of phytochemicals. The simple unicellular trichomes of Arabidopsis serve as an excellent model to study molecular mechanism of cell differentiation and pattern formation in plants. The emerging picture suggests that the developmental process is controlled by a transcriptional network involving three major groups of transcription factors: the R2R3 MYB, basic helix-loop-helix (bHLH and WD40 repeat (WDR protein. These regulatory proteins form a trimeric activator complex that positively regulates trichome development. The single repeat R3 MYBs act as negative regulators of trichome development. They compete with the R2R3 MYBs to bind the bHLH factor and form a repressor complex. In addition to activator-repressor mechanism, a depletion mechanism may operate in parallel during trichome development. In this mechanism, the bHLH factor traps the WDR protein which results in depletion of WDR protein in neighboring cells. Consequently, the cells with high levels of bHLH and WDR proteins are developed into trichomes. A group of C2H2 zinc finger TFs has also been implicated in trichome development. Phytohormones, including gibberellins and jasmonic acid, play significant roles in this developmental process. Recently, microRNAs have been shown to be involved in trichome development. Furthermore, it has been demonstrated that the activities of the key regulatory proteins involved in trichome development are controlled by the 26S/ubiquitin proteasome system (UPS, highlighting the complexity of the regulatory network controlling this developmental process. To complement several excellent recent relevant reviews, this review focuses on the transcriptional network and hormonal interplay

  7. Peripheral nerve P2 basic protein and the Guillain-Barre syndrome : In vitro demonstration of P2-specific antibody-secreting cells

    NARCIS (Netherlands)

    Luijten, J.A.F.M.; Jong, W.A.C. de; Demel, R.A.; Heijnen, C.J.; Ballieux, R.E.

    1984-01-01

    An immune response to the peripheral nerve basic protein P2 may be operative in the pathogenesis of the Guillain-Barré syndrome (GBS). A method is described for the purification of P2 of human origin. Purified P2 was used to investigate whether lymphocytes derived from peripheral blood of GBS patien

  8. T helper cell type 1 (Th1), Th2 and Th17 responses to myelin basic protein and disease activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Hedegaard, Chris J; Krakauer, Martin; Bendtzen, Klaus

    2008-01-01

    Autoreactive T cells are thought to play an essential role in the pathogenesis of multiple sclerosis (MS). We examined the stimulatory effect of human myelin basic protein (MBP) on mononuclear cell (MNC) cultures from 22 patients with MS and 22 sex-matched and age-matched healthy individuals, and...

  9. Further Analysis of the Function of AtBHLH29 in Regulating the Iron Uptake Process in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Juan Zhang; Hui-Fen Zhu; Hui Liang; Kun-Fan Liu; Ai-Min Zhang; Hong-Qing Ling; Dao-Wen Wang

    2006-01-01

    Using T-DNA insertion and chemical mutants, two recent studies have shown that AtBHLH29, encoding a putative basic helix-loop-helix (BHLH) protein, is involved in regulating the iron uptake process in Arabidopsis thaliana. Herein, we report that RNA interference (RNAi) mutants can be used to reveal more accurately the genetic function of AtBHLH29. We compared the iron deficiency responses of seven RNAi strains that contained decreasing amounts of AtBHLH29transcripts. Under high iron conditions (50 μmol/L iron), only in the most severe RNAi strains (R101, R111, and R119) was plant growth significantly retarded. However, these mutants could still survive and produce seeds. This suggests that the function of AtBHLH29 is beneficial, but not absolutely essential, to plant growth when iron supply is not limiting. Under low iron conditions (less than 10 μmol/L iron), the R111 and R119 strains died prematurely, demonstrating that AtBHLH29 is absolutely necessary for plant survival when iron supply is restricted. The transcription of AtBHLH29 was essential for the expression of AtFRO2 (encoding the ferric chelate reductase). In contrast, the expression of AtlRT1(encoding the high-affinity iron transporter) was not so strongly dependent upon the transcription of AtBHLH29.By transient expression, we found that the AtBHLH29-GUS fusion protein was targeted specifically to the nucleus in plant cells. Interestingly, the nuclear localization of AtBHLH29-GUS was abolished when the four consecutive arginine residues located in the basic region of the putative AtBHLH29 protein were replaced by alanine residues by mutagenesis. The implications of our findings on further studies of the mechanism underlying the function of AtBHLH29 are discussed.

  10. Down-regulation of ubiquitin ligase Cbl induced by twist haploinsufficiency in Saethre-Chotzen syndrome results in increased PI3K/Akt signaling and osteoblast proliferation.

    Science.gov (United States)

    Guenou, Hind; Kaabeche, Karim; Dufour, Cécilie; Miraoui, Hichem; Marie, Pierre J

    2006-10-01

    Genetic mutations of Twist, a basic helix-loop-helix transcription factor, induce premature fusion of cranial sutures in Saethre-Chotzen syndrome (SCS). We report here a previously undescribed mechanism involved in the altered osteoblastogenesis in SCS. Cranial osteoblasts from an SCS patient with a Twist mutation causing basic helix-loop-helix deletion exhibited decreased expression of E3 ubiquitin ligase Cbl compared with wild-type osteoblasts. This was associated with decreased ubiquitin-mediated degradation of phosphatidyl inositol 3 kinase (PI3K) and increased PI3K expression and PI3K/Akt signaling. Increased PI3K immunoreactivity was also found in osteoblasts in histological sections of affected cranial sutures from SCS patients. Transfection with Twist or Cbl abolished the increased PI3K/Akt signaling in Twist mutant osteoblasts. Forced overexpression of Cbl did not correct the altered expression of osteoblast differentiation markers in Twist mutant cells. In contrast, pharmacological inhibition of PI3K/Akt, but not ERK signaling, corrected the increased cell growth in Twist mutant osteoblasts. The results show that Twist haploinsufficiency results in decreased Cbl-mediated PI3K degradation in osteoblasts, causing PI3K accumulation and activation of PI3K/Akt-dependent osteoblast growth. This provides genetic and biochemical evidence for a role for Cbl-mediated PI3K signaling in the altered osteoblast phenotype induced by Twist haploinsufficiency in SCS.

  11. Experimental determination of the evolvability of a transcription factor.

    Science.gov (United States)

    Maerkl, Sebastian J; Quake, Stephen R

    2009-11-03

    Sequence-specific binding of a transcription factor to DNA is the central event in any transcriptional regulatory network. However, relatively little is known about the evolutionary plasticity of transcription factors. For example, the exact functional consequence of an amino acid substitution on the DNA-binding specificity of most transcription factors is currently not predictable. Furthermore, although the major structural families of transcription factors have been identified, the detailed DNA-binding repertoires within most families have not been characterized. We studied the sequence recognition code and evolvability of the basic helix-loop-helix transcription factor family by creating all possible 95 single-point mutations of five DNA-contacting residues of Max, a human helix-loop-helix transcription factor and measured the detailed DNA-binding repertoire of each mutant. Our results show that the sequence-specific repertoire of Max accessible through single-point mutations is extremely limited, and we are able to predict 92% of the naturally occurring diversity at these positions. All naturally occurring basic regions were also found to be accessible through functional intermediates. Finally, we observed a set of amino acids that are functional in vitro but are not found to be used naturally, indicating that functionality alone is not sufficient for selection.

  12. Endogenous interferon-β-inducible gene expression and interferon-β-treatment are associated with reduced T cell responses to myelin basic protein in multiple sclerosis.

    Science.gov (United States)

    Börnsen, Lars; Romme Christensen, Jeppe; Ratzer, Rikke; Hedegaard, Chris; Søndergaard, Helle B; Krakauer, Martin; Hesse, Dan; Nielsen, Claus H; Sorensen, Per S; Sellebjerg, Finn

    2015-01-01

    Autoreactive CD4+ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used for treatment of multiple sclerosis, and some untreated multiple sclerosis patients have increased expression levels of type I interferon-inducible genes in immune cells. The role of endogenous type I interferons in multiple sclerosis is controversial: some studies found an association of high expression levels of interferon-β-inducible genes with an increased expression of interleukin-10 and a milder disease course in untreated multiple sclerosis patients, whereas other studies reported an association with a poor response to treatment with interferon-β. In the present study, we found that untreated multiple sclerosis patients with an increased expression of interferon-β-inducible genes in peripheral blood mononuclear cells and interferon-β-treated multiple sclerosis patients had decreased CD4+ T-cell reactivity to the autoantigen myelin basic protein ex vivo. Interferon-β-treated multiple sclerosis patients had increased IL10 and IL27 gene expression levels in monocytes in vivo. In vitro, neutralization of interleukin-10 and monocyte depletion increased CD4+ T-cell reactivity to myelin basic protein while interleukin-10, in the presence or absence of monocytes, inhibited CD4+ T-cell reactivity to myelin basic protein. Our findings suggest that spontaneous expression of interferon-β-inducible genes in peripheral blood mononuclear cells from untreated multiple sclerosis patients and treatment with interferon-β are associated with reduced myelin basic protein-induced T-cell responses. Reduced myelin basic protein-induced CD4+ T-cell autoreactivity in interferon-β-treated multiple sclerosis patients may be mediated by monocyte-derived interleukin-10.

  13. Notch signaling induces rapid degradation of achaete-scute homolog 1.

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    Sriuranpong, Virote; Borges, Michael W; Strock, Christopher L; Nakakura, Eric K; Watkins, D Neil; Blaumueller, Christine M; Nelkin, Barry D; Ball, Douglas W

    2002-05-01

    In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes which leads to transcriptional repression of neural determination and differentiation genes. We now report that in addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, we showed that the initial appearance of Notch1 coincided with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone was capable of down-regulating hASH1 mRNA but could not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 was coinfected with Notch1, we still observed a dramatic and abrupt loss of the exogenous hASH1 protein, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreased the apparent half-life of the adenoviral hASH1 protein and increased the fraction of hASH1 which was polyubiquitinylated. The proteasome inhibitor MG132 reversed the Notch1-induced degradation. The Notch RAM domain was dispensable but a lack of the OPA and PEST domains inactivated this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 could protect hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer.

  14. Gene expression in the spinal cord in female lewis rats with experimental autoimmune encephalomyelitis induced with myelin basic protein.

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    Hayley R Inglis

    Full Text Available BACKGROUND: Experimental autoimmune encephalomyelitis (EAE, the best available model of multiple sclerosis, can be induced in different animal strains using immunization with central nervous system antigens. EAE is associated with inflammation and demyelination of the nervous system. Micro-array can be used to investigate gene expression and biological pathways that are altered during disease. There are few studies of the changes in gene expression in EAE, and these have mostly been done in a chronic mouse EAE model. EAE induced in the Lewis with myelin basic protein (MBP-EAE is well characterised, making it an ideal candidate for the analysis of gene expression in this disease model. METHODOLOGY/PRINCIPAL FINDINGS: MBP-EAE was induced in female Lewis rats by inoculation with MBP and adjuvants. Total RNA was extracted from the spinal cords and used for micro-array analysis using AffimetrixGeneChip Rat Exon 1.0 ST Arrays. Gene expression in the spinal cords was compared between healthy female rats and female rats with MBP-EAE. Gene expression in the spinal cord of rats with MBP-EAE differed from that in the spinal cord of normal rats, and there was regulation of pathways involved with immune function and nervous system function. For selected genes the change in expression was confirmed with real-time PCR. CONCLUSIONS/SIGNIFICANCE: EAE leads to modulation of gene expression in the spinal cord. We have identified the genes that are most significantly regulated in MBP-EAE in the Lewis rat and produced a profile of gene expression in the spinal cord at the peak of disease.

  15. Elucidation of the structural determinants responsible for the specific formation of heterodimeric Mxd1/Max b-HLH-LZ and its binding to E-box sequences.

    Science.gov (United States)

    Montagne, Martin; Naud, Jean-François; Lavigne, Pierre

    2008-02-08

    The proteins of the Mxd family (formally known as Mad) are antagonists of the oncoprotein c-Myc. They compete with c-Myc for their obligate partner Max to prevent the c-Myc/Max heterodimer from binding to E-box sequences in the target gene promoters. In cancer cells, where Myc is overexpressed, the expression of Mxd proteins is usually insufficient or abrogated. However, the reintroduction of Mxd1 expression in these cells prevents growth and proliferation. While the antagonism of c-Myc functions by Mxd proteins is of potential relevance for the development of cancer treatment strategies, the structural determinants responsible for the specific heterodimerization between the Mxd and the Max b-helix-loop-helix-leucine zippers are not fully understood. Moreover, whether the heterodimer is assembled on DNA or in the nucleoplasm prior to DNA binding is under debate. In this article, we demonstrate that Mxd1 D112a and Max N78a and H81d, which are located in the leucine zippers of the proteins, can dictate the specificity of heterodimerization and whether or not the Mxd1/Max/DNA complex forms. Our results also indicate that additional specific determinants exist in the helix-loop-helix domains of Max and Mxd1. Finally, we provide evidence that heterodimerization must precede DNA binding in vivo.

  16. Truncation of merozoite surface protein 3 disrupts its trafficking and that of acidic-basic repeat protein to the surface of Plasmodium falciparum merozoites.

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    Mills, Kerry E; Pearce, J Andrew; Crabb, Brendan S; Cowman, Alan F

    2002-03-01

    Merozoite surface protein 3 (MSP3), an important vaccine candidate, is a soluble polymorphic antigen associated with the surface of Plasmodium falciparum merozoites. The MSP3 sequence contains three blocks of heptad repeats that are consistent with the formation of an intramolecular coiled-coil. MSP3 also contains a glutamic acid-rich region and a putative leucine zipper sequence at the C-terminus. We have disrupted the msp3 gene by homologous recombination, resulting in the expression of a truncated form of MSP3 that lacks the putative leucine zipper sequence but retains the glutamic acid-rich region and the heptad repeats. Here, we show that truncated MSP3, lacking the putative leucine zipper region, does not localize to the parasitophorous vacuole or interact with the merozoite surface. Furthermore, the acidic-basic repeat antigen (ABRA), which is present on the merozoite surface, also was not localized to the merozoite surface in parasites expressing the truncated form of MSP3. The P. falciparum merozoites lacking MSP3 and ABRA on the surface show reduced invasion into erythrocytes. These results suggest that MSP3 is not absolutely essential for blood stage growth and that the putative leucine zipper region is required for the trafficking of both MSP3 and ABRA to the parasitophorous vacuole.

  17. Comprehensive Analysis of Expressed Sequence Tags from the Pulp of the Red Mutant 'Cara Cara' Navel Orange(Citrus sinensis Osbeck)

    Institute of Scientific and Technical Information of China (English)

    Jun-Li Ye; An-Dan Zhu; Neng-Guo Tao; Qiang Xu; Juan Xu; Xiu-Xin Deng

    2010-01-01

    Expressed sequence tag(EST)analysis of the pulp of the red-fleshed mutant 'Cara Cara' navel orange provided a starting point for gene discovery and transcriptome survey during citrus fruit maturation. Interpretation of the EST datasets revealed that the mutant pulp transcriptome held a high section of stress responses related genes,such as the type Ⅲ metallothionein-like gene(6.0%),heat shock protein(2.8%),Cu/Zn superoxide dismutase(0.8%),late embryogenesis abundant protein 5(0.8%),etc. 133transcripts were detected to be differentially expressed between the red mutant and its orange-color wild genotype 'Washington' via digital expression analysis. Among them,genes involved in metabolism,defense/stress and signal transduction were statistical overrepresented. Fifteen transcription factors,composed of NAM,ATAF,and CUC transcription factor(NAC); myeloblastosis(MYB); myelocytomatosis(MYC); basic helix-loop-helix(bHLH); basic leucine zipper(bZIP)domain members,were also included. The data reflected the distinct expression profile and the unique regulatory module associated with these two genotypes. Eight differently expressed genes analyzed in digital were validated by quantitative realtime polymerase chain reaction. For structural polymorphism,both simple sequence repeats and single nucleotide polymorphisms(SNP)loci were surveyed; dinucleotide presentation revealed a bias toward AG/GA/TC/CT repeats(52.5%),against GC/CG repeats(0%). SNPs analysis found that transitions(73%)outnumbered transversions(27%). Seventeen potential cultivar-specific and 387 heterozygous SNP loci were detected from 'Cara Cara' and 'Washington' EST pool.

  18. Delayed onset of experimental autoimmune encephalomyelitis in Olig1 deficient mice.

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    Xiaoli Guo

    Full Text Available BACKGROUND: Olig1 is a basic helix-loop-helix (bHLH transcription factor that is essential for oligodendrogenesis and efficient remyelination. However, its role in neurodegenerative disorders has not been well-elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the effects of Olig1 deficiency on experimental autoimmune encephalomyelitis (EAE, an animal model of multiple sclerosis (MS. We show that the mean disease onset of myelin oligodendrocyte glycoprotein (MOG-induced EAE in Olig1(-/- mice is significantly slower than wide-type (WT mice (19.8 ± 2.2 in Olig1(-/- mice and 9.5 ± 0.3 days in WT mice. In addition, 10% of Olig1(-/- mice did not develop EAE by the end of the observation periods (60 days. The severity of EAE, the extent of demyelination, and the activation of microglial cells and astrocytes in spinal cords, were significantly milder in Olig1(-/- mice compared with WT mice in the early stage. Moreover, the visual function, as assessed by the second-kernel of multifocal electroretinograms, was better preserved, and the number of degenerating axons in the optic nerve was significantly reduced in Olig1(-/- mice. Interestingly, Olig1 deficiency had no effect on T cell response capability, however, it reduced the expression of myelin proteins such as MOG, myelin basic protein (MBP and myelin-associated glycoprotein (MAG. The expression of Olig2 remained unchanged in the optic nerve and brain, and it was reduced in the spinal cord of Olig1(-/- mice. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the Olig1 signaling pathways may be involved in the incidence rate and the severity of neurological symptoms in MS.

  19. CtBP and associated LSD1 are required for transcriptional activation by NeuroD1 in gastrointestinal endocrine cells.

    Science.gov (United States)

    Ray, Subir K; Li, H Joyce; Metzger, Eric; Schüle, Roland; Leiter, Andrew B

    2014-06-01

    Gene expression programs required for differentiation depend on both DNA-bound transcription factors and surrounding histone modifications. Expression of the basic helix-loop-helix (bHLH) protein NeuroD1 is restricted to endocrine cells in the gastrointestinal (GI) tract, where it is important for endocrine differentiation. RREB1 (RAS-responsive element binding protein 1), identified as a component of the CtBP corepressor complex, binds to nearby DNA elements to associate with NeuroD and potentiate transcription of a NeuroD1 target gene. Transcriptional activation by RREB1 depends on recruitment of CtBP with its associated proteins, including LSD1, through its PXDLS motifs. The mechanism of transcriptional activation by CtBP has not been previously characterized. Here we found that activation was dependent on the histone H3 lysine 9 (H3K9) demethylase activity of LSD1, which removes repressive methyl marks from dimethylated H3K9 (H3K9Me2), to facilitate subsequent H3K9 acetylation by the NeuroD1-associated histone acetyltransferase, P300/CBP-associated factor (PCAF). The secretin, β-glucokinase, insulin I, and insulin II genes, four known direct targets of NeuroD1 in intestinal and pancreatic endocrine cells, all show similar promoter occupancy by CtBP-associated proteins and PCAF, with acetylation of H3K9. This work may indicate a mechanism for selective regulation of transcription by CtBP and LSD1 involving their association with specific transcription factors and cofactors to drive tissue-specific transcription.

  20. Cloning and functional characterisation of avian transcription factor E2A

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    Meyer Kerstin B

    2004-06-01

    Full Text Available Abstract Background During B lymphocyte development the E2A gene is a critical regulator of cell proliferation and differentiation. With regards to the immunoglobulin genes the E2A proteins contribute to the regulation of gene rearrangement, expression and class switch recombination. We are now using the chicken cell line DT40 as a model system to further analyse the function of E2A. Results Here we report the cloning and functional analysis of the transcription factor E2A from chicken. Using RACE PCR on the chicken lymphoma cell line DT40 we have isolated full-length clones for the two E2A splice variants E12 and E47. Sequence conservation between the human and chicken proteins is extensive: the basic-helix-loop-helix DNA binding domain of human and chicken E47 and E12 are 93% and 92% identical, respectively. In addition high levels of conservation are seen in activation domain I, the potential NLS and the ubiquitin ligase interaction domain. E2A is expressed in a variety of tissues in chicken, with higher levels of expression in organs rich in immune cells. We demonstrate that chicken E12 and E47 proteins are strong transcriptional activators whose function depends on the presence of activation domain I. As in mammals, the dominant negative proteins Id1 and Id3 can inhibit the function of chicken E47. Conclusions The potential for homologous recombination in DT40 allows the genetic dissection of biochemical pathways in somatic cells. With the cloning of avian E2A and the recent description of an in vitro somatic hypermutation assay in this cell line, it should now be possible to dissect the potential role of E2A in the regulation of somatic hypermutation and gene conversion.

  1. ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) Interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) Linking Iron Deficiency and Oxidative Stress Responses.

    Science.gov (United States)

    Le, Cham Thi Tuyet; Brumbarova, Tzvetina; Ivanov, Rumen; Stoof, Claudia; Weber, Eva; Mohrbacher, Julia; Fink-Straube, Claudia; Bauer, Petra

    2016-01-01

    Plants grown under iron (Fe)-deficient conditions induce a set of genes that enhance the efficiency of Fe uptake by the roots. In Arabidopsis (Arabidopsis thaliana), the central regulator of this response is the basic helix-loop-helix transcription factor FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). FIT activity is regulated by protein-protein interactions, which also serve to integrate external signals that stimulate and possibly inhibit Fe uptake. In the search of signaling components regulating FIT function, we identified ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12), an abiotic stress-induced transcription factor. ZAT12 interacted with FIT, dependent on the presence of the ethylene-responsive element-binding factor-associated amphiphilic repression motif. ZAT12 protein was found expressed in the root early differentiation zone, where its abundance was modulated in a root layer-specific manner. In the absence of ZAT12, FIT expression was upregulated, suggesting a negative effect of ZAT12 on Fe uptake. Consistently, zat12 loss-of-function mutants had higher Fe content than the wild type at sufficient Fe. We found that under Fe deficiency, hydrogen peroxide (H2O2) levels were enhanced in a FIT-dependent manner. FIT protein, in turn, was stabilized by H2O2 but only in the presence of ZAT12, showing that H2O2 serves as a signal for Fe deficiency responses. We propose that oxidative stress-induced ZAT12 functions as a negative regulator of Fe acquisition. A model where H2O2 mediates the negative regulation of plant responses to prolonged stress might be applicable to a variety of stress conditions.

  2. A novel Zea mays ssp. mexicana L. MYC-type ICE-like transcription factor gene ZmmICE1, enhances freezing tolerance in transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Lu, Xiang; Yang, Lei; Yu, Mengyuan; Lai, Jianbin; Wang, Chao; McNeil, David; Zhou, Meixue; Yang, Chengwei

    2017-04-01

    The annual Zea mays ssp. mexicana L., a member of the teosinte group, is a close wild relative of maize and thus can be effectively used in maize improvement. In this study, an ICE-like gene, ZmmICE1, was isolated from a cDNA library of RNA-Seq from cold-treated seedling tissues of Zea mays ssp. mexicana L. The deduced protein of ZmmICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE-like proteins. The ZmmICE1 protein localizes to the nucleus and shows sumoylation when expressed in an Escherichia coli reconstitution system. In addition, yeast one hybrid assays indicated that ZmmICE1 has transactivation activities. Moreover, ectopic expression of ZmmICE1 in the Arabidopsis ice1-2 mutant increased freezing tolerance. The ZmmICE1 overexpressed plants showed lower electrolyte leakage (EL), reduced contents of malondialdehyde (MDA). The expression of downstream cold related genes of Arabidopsis C-repeat-binding factors (AtCBF1, AtCBF2 and AtCBF3), cold-responsive genes (AtCOR15A and AtCOR47), kinesin-1 member gene (AtKIN1) and responsive to desiccation gene (AtRD29A) was significantly induced when compared with wild type under low temperature treatment. Taken together, these results indicated that ZmmICE1 is the homolog of Arabidopsis inducer of CBF expression genes (AtICE1/2) and plays an important role in the regulation of freezing stress response.

  3. Hippocalcin Is Required for Astrocytic Differentiation through Activation of Stat3 in Hippocampal Neural Precursor Cells.

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    Min-Jeong Kang

    2016-10-01

    Full Text Available Hippocalcin (Hpca is a neuronal calcium sensor protein expressed in the mammalian brain. However, its function in neural stem/precursor cells has not yet been studied. Here, we clarify the function of Hpca in astrocytic differentiation in hippocampal neural precursor cells (HNPCs. When we overexpressed Hpca in HNPCs in the presence or absence of bFGF, expression levels of nerve-growth factors such as neurotrophin-3 (NT-3, neurotrophin-4/5 (NT-4/5 and brain-derived neurotrophic factor (BDNF, together with the proneural basic helix loop helix (bHLH transcription factors neuroD and neurogenin 1 (ngn1, increased significantly. In addition, there was an increase in the number of cells expressing glial fibrillary acidic protein (GFAP, an astrocyte marker, and in dendrite outgrowth, indicating astrocytic differentiation of the HNPCs. Downregulation of Hpca by transfection with Hpca siRNA reduced expression of NT-3, NT-4/5, BDNF, neuroD and ngn1 as well as levels of GFAP protein. Furthermore, overexpression of Hpca increased the phosphorylation of STAT3 (Ser727, and this effect was abolished by treatment with a STAT3 inhibitor (S3I-201, suggesting that STAT3 (Ser727 activation is involved in Hpca-mediated astrocytic differentiation. As expected, treatment with Stat3 siRNA or STAT3 inhibitor caused a complete inhibition of astrogliogenesis induced by Hpca overexpression. Taken together, this is the first report to show that Hpca, acting through Stat3, has an important role in the expression of neurotrophins and proneural bHLH transcription factors, and that it is an essential regulator of astrocytic differentiation and dendrite outgrowth in HNPCs.

  4. Rapid mitogen-activated protein kinase by basic fibroblast growth factor in rat intestin after ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Xiao-Bing Fu; Yin-Hui Yang; Tong-Zhu Sun; Wei Chen; Jun-You Li; Zhi-Yong Sheng

    2003-01-01

    AIM: Previous studies showed that exogenous basic fibroblast growth factor (bFGF or FGF-2) could improve physiological dysfunction after intestinal ischemia/reperfusion (I/R) injury. However, the mechanisms of this protective effect of bFGF are still unclear. The present study was to detect the effect of bFGF on the activities of mitogen-activated protein kinase (MlAPK) signaling pathway in rat intestine after I/R injury, and to investigate the protective mechanisms of bFGF on intestinal ischemia injury. METttODS: Rat intestinal I/R injury was produced by clamping the superior mesenteric artery (SMA) for 45minutes and followed by repeffusion for 48 hours. Seventyeight Wistar rats were used and divided randomly into sham-operated group (A), normal saline control group (B),bFGF antibody pre-treated group (C), and bFGF treated group (D). Tn group A, SMA was separated without occlusion. In groups B, C and D, SMA was separated and occluded for 45 minutes, then, released for reperfusion for 48 hours. After the animals were sacrificed, blood and tissue samples were taken from the intestine 45 minutes after ischemia in group A and 2, 6, 24, and 48 hours after reperfusion in the other groups. Phosphorylated forms of p42/p44 MAPK, p38 MAPK and stress activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) were measured by immunohistochemistry. Plasma levels of D-lactate were examined and histological changes were observed under the light microscope. RESULTS: Intestinal I/R injury induced the expression of p42/p44 MAPK, p38 MAPK, and SAPK/JNK pathways and exogenous bFGF stimulated the early activation of p42/p44 MAPK and p38 MlAPK pathways. The expression of phosphorylated forms of p42/p44 MAPK was primarily localized in the nuclei of crypt cells and in the cytoplasm and nuclei of villus cells. The positive expression of p38MAPK was localized mainly in the nuclei of crypt cells, very few in villus cells. The activities of p42/p44 MAPK and p38MAPK peaked 6 hours after

  5. Regulatory Role of a Receptor-Like Kinase in Specifying Anther Cell Identity1[OPEN

    Science.gov (United States)

    Yang, Li; Qian, Xiaoling; Chen, Mingjiao

    2016-01-01

    In flowering plants, sequential formation of anther cell types is a highly ordered process that is essential for successful meiosis and sexual reproduction. Differentiation of meristematic cells and cell-cell communication are proposed to coordinate anther development. Among the proposed mechanisms of cell fate specification are cell surface-localized Leu-rich repeat receptor-like kinases (LRR-RLKs) and their putative ligands. Here, we present the genetic and biochemical evidence that a rice (Oryza sativa) LRR-RLK, MSP1 (MULTIPLE SPOROCYTE1), interacts with its ligand OsTDL1A (TPD1-like 1A), specifying the cell identity of anther wall layers and microsporocytes. An in vitro assay indicates that the 21-amino acid peptide of OsTDL1A has a physical interaction with the LRR domain of MSP1. The ostdl1a msp1 double mutant showed the defect in lacking middle layers and tapetal cells and having an increased number of microsporocytes similar to the ostdl1a or msp1 single mutant, indicating the same pathway of OsTDL1A-MSP1 in regulating anther development. Genome-wide expression profiles showed the altered expression of genes encoding transcription factors, particularly basic helix-loop-helix and basic leucine zipper domain transcription factors in ostdl1a and msp1. Among these reduced expressed genes, one putatively encodes a TGA (TGACGTCA cis-element-binding protein) factor OsTGA10, and another one encodes a plant-specific CC-type glutaredoxin OsGrx_I1. OsTGA10 was shown to interact with OsGrx_I1, suggesting that OsTDL1A-MSP1 signaling specifies anther cell fate directly or indirectly affecting redox status. Collectively, these data point to a central role of the OsTDL1A-MSP1 signaling pathway in specifying somatic cell identity and suppressing overproliferation of archesporial cells in rice. PMID:27208278

  6. Role of Very-late Antigen-4 (VLA-4) in Myelin Basic Protein-primed T Cell Contact-induced Expression of Proinflammatory Cytokines in Microglial Cells*

    OpenAIRE

    Dasgupta, Subhajit; Jana, Malabendu; Liu, Xiaojuan; Pahan, Kalipada

    2003-01-01

    The presence of neuroantigen-primed T cells recognizing self-myelin antigens within the CNS is necessary for the development of demyelinating autoimmune disease like multiple sclerosis. This study was undertaken to investigate the role of myelin basic protein (MBP)-primed T cells in the expression of proinflammatory cytokines in microglial cells. MBP-primed T cells alone induced specifically the microglial expression of interleukin (IL)-1β, IL-1α tumor necrosis factor α, and IL-6, proinflamma...

  7. Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation.

    Science.gov (United States)

    Obayashi, Shinya; Tabunoki, Hiroko; Kim, Seung U; Satoh, Jun-ichi

    2009-05-01

    Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

  8. Expression and cellular localization of the transcription factor NeuroD1 in the developing and adult rat pineal gland.

    Science.gov (United States)

    Castro, Analía E; Benitez, Sergio G; Farias Altamirano, Luz E; Savastano, Luis E; Patterson, Sean I; Muñoz, Estela M

    2015-05-01

    Circadian rhythms govern many aspects of mammalian physiology. The daily pattern of melatonin synthesis and secretion is one of the classic examples of circadian oscillations. It is mediated by a class of neuroendocrine cells known as pinealocytes which are not yet fully defined. An established method to evaluate functional and cytological characters is through the expression of lineage-specific transcriptional regulators. NeuroD1 is a basic helix-loop-helix transcription factor involved in the specification and maintenance of both endocrine and neuronal phenotypes. We have previously described developmental and adult regulation of NeuroD1 mRNA in the rodent pineal gland. However, the transcript levels were not influenced by the elimination of sympathetic input, suggesting that any rhythmicity of NeuroD1 might be found downstream of transcription. Here, we describe NeuroD1 protein expression and cellular localization in the rat pineal gland during development and the daily cycle. In embryonic and perinatal stages, protein expression follows the mRNA pattern and is predominantly nuclear. Thereafter, NeuroD1 is mostly found in pinealocyte nuclei in the early part of the night and in cytoplasm during the day, a rhythm maintained into adulthood. Additionally, nocturnal nuclear NeuroD1 levels are reduced after sympathetic disruption, an effect mimicked by the in vivo administration of α- and β-adrenoceptor blockers. NeuroD1 phosphorylation at two sites, Ser(274) and Ser(336) , associates with nuclear localization in pinealocytes. These data suggest that NeuroD1 influences pineal phenotype both during development and adulthood, in an autonomic and phosphorylation-dependent manner.

  9. The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate specification.

    Science.gov (United States)

    Hallam, S; Singer, E; Waring, D; Jin, Y

    2000-10-01

    The basic helix-loop-helix transcription factor NeuroD (Neurod1) has been implicated in neuronal fate determination, differentiation and survival. Here we report the expression and functional analysis of cnd-1, a C. elegans NeuroD homolog. cnd-1 expression was first detected in neuroblasts of the AB lineage in 14 cell embryos and maintained in many neuronal descendants of the AB lineage during embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1 reporter genes were expressed in the precursors of the embryonic ventral cord motor neurons and their progeny. A loss-of-function mutant, cnd-1(ju29), exhibited multiple defects in the ventral cord motor neurons. First, the number of motor neurons was reduced, possibly caused by the premature withdrawal of the precursors from mitotic cycles. Second, the strict correlation between the fate of a motor neuron with respect to its lineage and position in the ventral cord was disrupted, as manifested by the variable expression pattern of motor neuron fate specific markers. Third, motor neurons also exhibited defects in terminal differentiation characteristics including axonal morphology and synaptic connectivity. Finally, the expression patterns of three neuronal type-specific transcription factors, unc-3, unc-4 and unc-30, were altered. Our data suggest that cnd-1 may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1 appears to have combined the functions of several vertebrate neurogenic bHLH proteins and may represent an ancestral form of this protein family.

  10. Twist-1 Up-Regulation in Carcinoma Correlates to Poor Survival

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    Alimujiang Wushou

    2014-11-01

    Full Text Available Epithelial-to-mesenchymal transition (EMT facilitates tumor metastasis. Twist is a basic helix-loop-helix protein that modulates many target genes through E-box-responsive elements. There are two twist-like proteins, Twist-1 and Twist-2, sharing high structural homology in mammals. Twist-1 was found to be a key factor in the promotion of metastasis of cancer cells, and is known to induce EMT. Twist-1 participation in carcinoma progression and metastasis has been reported in a variety of tumors. However, controversy exists concerning the correlation between Twist-1 and prognostic value with respect to carcinoma. A systematic review and meta-analysis were performed to determine whether the expression of Twist-1 was associated with the prognosis of carcinoma patients. This analysis included 17 studies: four studies evaluated lung cancer, three evaluated head and neck cancer, two evaluated breast cancer, two evaluated esophageal cancer, two evaluated liver cancer and one each evaluated osteosarcoma, bladder, cervical and ovarian cancer. A total of 2006 patients were enrolled in these studies, and the median trial sample size was 118 patients. Twist-1 expression was associated with worse overall survival (OS at both 3 years (hazard ratio “HR” for death = 2.13, 95% CI = 1.86 to 2.45, p < 0.001 and 5 years (HR for death = 2.01, 95% CI = 1.76 to 2.29, p < 0.001. Expression of Twist-1 is associated with worse survival in carcinoma.

  11. Adenosine Triphosphate (ATP Is a Candidate Signaling Molecule in the Mitochondria-to-Nucleus Retrograde Response Pathway

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    Zhengchang Liu

    2013-03-01

    Full Text Available Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to and inactivates Mks1, allowing for activation of Rtg1/3 and the RTG pathway. When the pathway is inactive, Mks1 has dissociated from Rtg2 and bound to Bmh1/2, preventing activation of Rtg1/3. What signals association or disassociation of Mks1 and Rtg2 is unknown. Here, we show that ATP at physiological concentrations dissociates Mks1 from Rtg2 in a highly cooperative fashion. We report that ATP-mediated dissociation of Mks1 from Rtg2 is conserved in two other fungal species, K. lactis and K. waltii. Activation of Rtg1/3 upregulates expression of genes encoding enzymes catalyzing the first three reactions of the Krebs cycle, which is coupled to ATP synthesis through oxidative phosphorylation. Therefore, we propose that the retrograde response is an ATP homeostasis pathway coupling ATP production with ATP-mediated repression of the retrograde response by releasing Mks1 from Rtg2.

  12. Early genetic responses in rat vascular tissue after simulated diving.

    Science.gov (United States)

    Eftedal, Ingrid; Jørgensen, Arve; Røsbjørgen, Ragnhild; Flatberg, Arnar; Brubakk, Alf O

    2012-12-18

    Diving causes a transient reduction of vascular function, but the mechanisms behind this are largely unknown. The aim of this study was therefore to analyze genetic reactions that may be involved in acute changes of vascular function in divers. Rats were exposed to 709 kPa of hyperbaric air (149 kPa Po(2)) for 50 min followed by postdive monitoring of vascular bubble formation and full genome microarray analysis of the aorta from diving rats (n = 8) and unexposed controls (n = 9). Upregulation of 23 genes was observed 1 h after simulated diving. The differential gene expression was characteristic of cellular responses to oxidative stress, with functions of upregulated genes including activation and fine-tuning of stress-responsive transcription, cytokine/cytokine receptor signaling, molecular chaperoning, and coagulation. By qRT-PCR, we verified increased transcription of neuron-derived orphan receptor-1 (Nr4a3), plasminogen activator inhibitor 1 (Serpine1), cytokine TWEAK receptor FN14 (Tnfrsf12a), transcription factor class E basic helix-loop-helix protein 40 (Bhlhe40), and adrenomedullin (Adm). Hypoxia-inducible transcription factor HIF1 subunit HIF1-α was stabilized in the aorta 1 h after diving, and after 4 h there was a fivefold increase in total protein levels of the procoagulant plasminogen activator inhibitor 1 (PAI1) in blood plasma from diving rats. The study did not have sufficient power for individual assessment of effects of hyperoxia and decompression-induced bubbles on postdive gene expression. However, differential gene expression in rats without venous bubbles was similar to that of all the diving rats, indicating that elevated Po(2) instigated the observed genetic reactions.

  13. Angiopoietin-2 is a direct transcriptional target of TAL1, LYL1 and LMO2 in endothelial cells.

    Science.gov (United States)

    Deleuze, Virginie; El-Hajj, Rawan; Chalhoub, Elias; Dohet, Christiane; Pinet, Valérie; Couttet, Philippe; Mathieu, Danièle

    2012-01-01

    The two related basic helix-loop-helix, TAL1 and LYL1, and their cofactor LIM-only-2 protein (LMO2) are present in blood and endothelial cells. While their crucial role in early hematopoiesis is well established, their function in endothelial cells and especially in angiogenesis is less understood. Here, we identified ANGIOPOIETIN-2 (ANG-2), which encodes a major regulator of angiogenesis, as a direct transcriptional target of TAL1, LYL1 and LMO2. Knockdown of any of the three transcription factors in human blood and lymphatic endothelial cells caused ANG-2 mRNA and protein down-regulation. Transient transfections showed that the full activity of the ANG-2 promoter required the integrity of a highly conserved Ebox-GATA composite element. Accordingly, chromatin immunoprecipitation assays demonstrated that TAL1, LYL1, LMO2 and GATA2 occupied this region of ANG-2 promoter in human endothelial cells. Furthermore, we showed that LMO2 played a central role in assembling TAL1-E47, LYL1-LYL1 or/and LYL1-TAL1 dimers with GATA2. The resulting complexes were able to activate endogenous ANG-2 expression in endothelial cells as well as in non-endothelial cells. Finally, we showed that ANG-2 gene activation during angiogenesis concurred with the up-regulation of TAL1 and LMO2. Altogether, we identified ANG-2 as a bona fide target gene of LMO2-complexes with TAL1 and/or LYL1, highlighting a new function of the three hematopoietic factors in the endothelial lineage.

  14. Characterization of progenitor domains in the developing mouse thalamus.

    Science.gov (United States)

    Vue, Tou Yia; Aaker, Joshua; Taniguchi, Aya; Kazemzadeh, Christina; Skidmore, Jennifer M; Martin, Donna M; Martin, James F; Treier, Mathias; Nakagawa, Yasushi

    2007-11-01

    To understand the molecular basis of the specification of thalamic nuclei, we analyzed the expression patterns of various transcription factors and defined progenitor cell populations in the embryonic mouse thalamus. We show that the basic helix-loop-helix (bHLH) transcription factor Olig3 is expressed in the entire thalamic ventricular zone and the zona limitans intrathalamica (ZLI). Next, we define two distinct progenitor domains within the thalamus, which we name pTH-R and pTH-C, located caudal to the ZLI. pTH-R is immediately caudal to the ZLI and expresses Nkx2.2, Mash1, and Olig3. pTH-C is caudal to pTH-R and expresses Ngn1, Ngn2, and Olig3. Short-term lineage analysis of Olig3-, Mash1-, Ngn1-, and Ngn2-expressing progenitor cells as well as tracing the Pitx2 cell lineage suggests that pTH-C is the only major source of thalamic nuclei containing neurons that project to the cerebral cortex, whereas pTH-R and ZLI are likely to produce distinct postmitotic populations outside of the cortex-projecting part of the thalamus. To determine if pTH-C is composed of subdomains, we characterized expression of the homeodomain protein Dbx1 and the bHLH protein Olig2. We show that Dbx1 is expressed in caudodorsal-high to rostroventral-low gradient within pTH-C. Analysis of heterozygous Dbx1(nlslacZ) knockin mice demonstrated that Dbx1-expressing progenitors preferentially give rise to caudodorsal thalamic nuclei. Olig2 is expressed in an opposite gradient within pTH-C to that of Dbx1. These results establish the molecular heterogeneity within the progenitor cells of the thalamus, and suggest that such heterogeneity contributes to the specification of thalamic nuclei.

  15. Cellular and developmental adaptations to hypoxia: a Drosophila perspective.

    Science.gov (United States)

    Romero, Nuria Magdalena; Dekanty, Andrés; Wappner, Pablo

    2007-01-01

    The fruit fly Drosophila melanogaster, a widely utilized genetic model, is highly resistant to oxygen starvation and is beginning to be used for studying physiological, developmental, and cellular adaptations to hypoxia. The Drosophila respiratory (tracheal) system has features in common with the mammalian circulatory system so that an angiogenesis-like response occurs upon exposure of Drosophila larvae to hypoxia. A hypoxia-responsive system homologous to mammalian hypoxia-inducible factor (HIF) has been described in the fruit fly, where Fatiga is a Drosophila oxygen-dependent HIF prolyl hydroxylase, and the basic helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins Sima and Tango are, respectively, the Drosophila homologues of mammalian HIF-alpha (alpha) and HIF-beta (beta). Tango is constitutively expressed regardless of oxygen tension and, like in mammalian cells, Sima is controlled at the level of protein degradation and subcellular localization. Sima is critically required for development in hypoxia, but, unlike mammalian model systems, it is dispensable for development in normoxia. In contrast, fatiga mutant alleles are all lethal; however, strikingly, viability to adulthood is restored in fatiga sima double mutants, although these double mutants are not entirely normal, suggesting that Fatiga has Sima-independent functions in fly development. Studies in cell culture and in vivo have revealed that Sima is activated by the insulin receptor (InR) and target-of-rapamycin (TOR) pathways. Paradoxically, Sima is a negative regulator of growth. This suggests that Sima is engaged in a negative feedback loop that limits growth upon stimulation of InR/TOR pathways.

  16. Introducing Pitt-Hopkins syndrome-associated mutations of TCF4 to Drosophila daughterless

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    Laura Tamberg

    2015-12-01

    Full Text Available Pitt-Hopkins syndrome (PTHS is caused by haploinsufficiency of Transcription factor 4 (TCF4, one of the three human class I basic helix-loop-helix transcription factors called E-proteins. Drosophila has a single E-protein, Daughterless (Da, homologous to all three mammalian counterparts. Here we show that human TCF4 can rescue Da deficiency during fruit fly nervous system development. Overexpression of Da or TCF4 specifically in adult flies significantly decreases their survival rates, indicating that these factors are crucial even after development has been completed. We generated da transgenic fruit fly strains with corresponding missense mutations R578H, R580W, R582P and A614V found in TCF4 of PTHS patients and studied the impact of these mutations in vivo. Overexpression of wild type Da as well as human TCF4 in progenitor tissues induced ectopic sensory bristles and the rough eye phenotype. By contrast, overexpression of DaR580W and DaR582P that disrupt DNA binding reduced the number of bristles and induced the rough eye phenotype with partial lack of pigmentation, indicating that these act dominant negatively. Compared to the wild type, DaR578H and DaA614V were less potent in induction of ectopic bristles and the rough eye phenotype, respectively, suggesting that these are hypomorphic. All studied PTHS-associated mutations that we introduced into Da led to similar effects in vivo as the same mutations in TCF4 in vitro. Consequently, our Drosophila models of PTHS are applicable for further studies aiming to unravel the molecular mechanisms of this disorder.

  17. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.

    Science.gov (United States)

    Zhou, Hui; Lin-Wang, Kui; Wang, Huiliang; Gu, Chao; Dare, Andrew P; Espley, Richard V; He, Huaping; Allan, Andrew C; Han, Yuepeng

    2015-04-01

    Anthocyanin pigmentation is an important consumer trait in peach (Prunus persica). In this study, the genetic basis of the blood-flesh trait was investigated using the cultivar Dahongpao, which shows high levels of cyanidin-3-glucoside in the mesocarp. Elevation of anthocyanin levels in the flesh was correlated with the expression of an R2R3 MYB transcription factor, PpMYB10.1. However, PpMYB10.1 did not co-segregate with the blood-flesh trait. The blood-flesh trait was mapped to a 200-kb interval on peach linkage group (LG) 5. Within this interval, a gene encoding a NAC domain transcription factor (TF) was found to be highly up-regulated in blood-fleshed peaches when compared with non-red-fleshed peaches. This NAC TF, designated blood (BL), acts as a heterodimer with PpNAC1 which shows high levels of expression in fruit at late developmental stages. We show that the heterodimer of BL and PpNAC1 can activate the transcription of PpMYB10.1, resulting in anthocyanin pigmentation in tobacco. Furthermore, silencing the BL gene reduces anthocyanin pigmentation in blood-fleshed peaches. The transactivation activity of the BL-PpNAC1 heterodimer is repressed by a SQUAMOSA promoter-binding protein-like TF, PpSPL1. Low levels of PpMYB10.1 expression in fruit at early developmental stages is probably attributable to lower levels of expression of PpNAC1 plus the presence of high levels of repressors such as PpSPL1. We present a mechanism whereby BL is the key gene for the blood-flesh trait in peach via its activation of PpMYB10.1 in maturing fruit. Partner TFs such as basic helix-loop-helix proteins and NAC1 are required, as is the removal of transcriptional repressors.

  18. bHLH106 Integrates Functions of Multiple Genes through Their G-Box to Confer Salt Tolerance on Arabidopsis.

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    Aftab Ahmad

    Full Text Available An activation-tagging methodology was applied to dedifferentiated calli of Arabidopsis to identify new genes involved in salt tolerance. This identified salt tolerant callus 8 (stc8 as a gene encoding the basic helix-loop-helix transcription factor bHLH106. bHLH106-knockout (KO lines were more sensitive to NaCl, KCl, LiCl, ABA, and low temperatures than the wild-type. Back-transformation of the KO line rescued its phenotype, and over-expression (OX of bHLH106 in differentiated plants exhibited tolerance to NaCl. Green fluorescent protein (GFP fused with bHLH106 revealed that it was localized to the nucleus. Prepared bHLH106 protein was subjected to electrophoresis mobility shift assays against E-box sequences (5'-CANNTG-3'. The G-box sequence 5'-CACGTG-3' had the strongest interaction with bHLH106. bHLH106-OX lines were transcriptomically analyzed, and resultant up- and down-regulated genes selected on the criterion of presence of a G-box sequence. There were 198 genes positively regulated by bHLH106 and 36 genes negatively regulated; these genes possessed one or more G-box sequences in their promoter regions. Many of these genes are known to be involved in abiotic stress response. It is concluded that bHLH106 locates at a branching point in the abiotic stress response network by interacting directly to the G-box in genes conferring salt tolerance on plants.

  19. Identification of anthocyanin biosynthesis related microRNAs in a distinctive Chinese radish (Raphanus sativus L.) by high-throughput sequencing.

    Science.gov (United States)

    Sun, Yuyan; Qiu, Yang; Duan, Mengmeng; Wang, Jinglei; Zhang, Xiaohui; Wang, Haiping; Song, Jiangping; Li, Xixiang

    2017-02-01

    Anthocyanins are widely distributed water-soluble phytochemical pigments belonging to the flavonoid group. To date, limited knowledge is available about the regulatory roles of miRNAs in anthocyanin biosynthesis in plants. To identify the miRNAs associated with anthocyanin biosynthesis in radish, five small RNA (sRNA) libraries constructed from 'Xinlimei' radish roots at 11, 21, 44, 56 and 73 days (d) were examined using high-throughput sequencing technology. A total of 102.02 million (M) clean reads were generated, from which 483 known and 1415 novel miRNAs were identified. Combined with target prediction and annotation, 72 differentially expressed miRNAs (52 known and 20 novel miRNAs) were more likely to participate in anthocyanin biosynthesis. Several target genes for these miRNAs encode a few transcription factors, including Myb domain (MYB), basic helix-loop-helix (bHLH), WD40 repeat, squamosa promoter binding protein like (SPL), auxin response factor (ARF), ethylene insensitive 3 (EIN3), WRKY and MADS-box proteins. Furthermore, the expression patterns of some anthocyanin biosynthesis related miRNAs and their corresponding targets were validated by RT-qPCR. Based on the characterization of anthocyanin biosynthesis related miRNAs and their target genes, a putative miRNA-target module regulating anthocyanin biosynthesis was proposed. This study represents the first genome-wide identification of miRNAs associated with anthocyanin biosynthesis in radish, and provides insights into the molecular mechanisms underlying regulation of anthocyanin biosynthesis in radish and other crops.

  20. Effects of hypoxia-inducible factor 1 on ischemic cerebrovascular disease

    Institute of Scientific and Technical Information of China (English)

    Yongjie Luo; Xiaoping Wang; Hongbin Sun

    2008-01-01

    Hypoxia-inducible factor I, a nuclear transcription factor, is induced by hypoxia. Hypoxia-inducible factor I, a heterodimeric DNA-binding protein, is composed of hypoxia-inducible factor 1α and hypoxia-inducible factor 1 β subunits, which are family members of the basic helix-loop-helix-PER, ARNT, SIM (PAS) protein. O2 concentration regulates hypoxia-inducible factor 1 activity via this subunit. Hypoxia-inducible factor 1α plays a major role in response to hypoxia and transcriptional activation, as well as in the target gene specificity of the DNA enhancer. Hypoxia-inducible factor 1β cannot be induced by hypoxia. This effect may be due to hypoxia-inducible factor 1 stability and activated conformation due to dimerization. Previous studies have shown that hypoxia-inducible factor 1 mRNA expression increases in the penumbra following ischemia/hypoxia. Hypoxia-inducible factor 1 plays an important role in brain tissue injury alter ischemia by affecting a series of target genes, elevating tolerance to hypoxia, and ensuring survival of neural cells. This article summarizes the structure, function, expression, regulatory mechanisms, biological effects, and significance of hypoxia-inducible factor 1 in patients with ischemic cerebrovascular disease. As a transcriptional activator, hypoxia- inducible factor 1 plays a key role in hypoxic responses by stabilizing the internal environment. It also has been shown to regulate the expression of several genes. The regulatory effects of hypoxia-inducible factor 1 in patients with ischemic cerebrovascular disease have been described. The present review re-examined the concept of brain protection at the level of gene regulation.

  1. A large insertion in bHLH transcription factor BrTT8 resulting in yellow seed coat in Brassica rapa.

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    Xia Li

    Full Text Available Yellow seed is a desirable quality trait of the Brassica oilseed species. Previously, several seed coat color genes have been mapped in the Brassica species, but the molecular mechanism is still unknown. In the present investigation, map-based cloning method was used to identify a seed coat color gene, located on A9 in B. rapa. Blast analysis with the Arabidopsis genome showed that there were 22 Arabidopsis genes in this region including at4g09820 to at4g10620. Functional complementation test exhibited a phenotype reversion in the Arabidopsis thaliana tt8-1 mutant and yellow-seeded plant. These results suggested that the candidate gene was a homolog of TRANSPARENT TESTA8 (TT8 locus. BrTT8 regulated the accumulation of proanthocyanidins (PAs in the seed coat. Sequence analysis of two alleles revealed a large insertion of a new class of transposable elements, Helitron in yellow sarson. In addition, no mRNA expression of BrTT8 was detected in the yellow-seeded line. It indicated that the natural transposon might have caused the loss in function of BrTT8. BrTT8 encodes a basic/helix-loop-helix (bHLH protein that shares a high degree of similarity with other bHLH proteins in the Brassica. Further expression analysis also revealed that BrTT8 was involved in controlling the late biosynthetic genes (LBGs of the flavonoid pathway. Our present findings provided with further studies could assist in understanding the molecular mechanism involved in seed coat color formation in Brassica species, which is an important oil yielding quality trait.

  2. Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5

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    Lorca Thierry

    2005-12-01

    Full Text Available Abstract Background The two myogenic regulatory factors Myf5 and MyoD are basic helix-loop-helix muscle transcription factors undergoing differential cell cycle dependent proteolysis in proliferating myoblasts. This regulated degradation results in the striking expression of these two factors at distinct phases of the cell cycle, and suggests that their precise and alternated disappearance is an important feature of myoblasts, maybe connected to the maintenance of the proliferative status and/or commitment to the myogenic lineage of these cells. One way to understand the biological function(s of the cyclic expression of these proteins is to specifically alter their degradation, and to analyze the effects of their stabilization on cells. To this aim, we undertook the biochemical analysis of the mechanisms governing Myf5 mitotic degradation, using heterologous systems. Results We show here that mitotic degradation of Myf5 is conserved in non-myogenic cells, and is thus strictly under the control of the cell cycle apparatus. Using Xenopus egg extracts as an in vitro system to dissect the main steps of Myf5 mitotic proteolysis, we show that (1 Myf5 stability is regulated by a complex interplay of phosphorylation/dephosphorylation, probably involving various kinases and phosphatases, (2 Myf5 is ubiquitylated in mitotic extracts, and this is a prerequisite to its degradation by the proteasome and (3 at least in the Xenopus system, the E3 responsible for its mitotic degradation is not the APC/C (the major E3 during mitosis. Conclusion Altogether, our data strongly suggest that the mitotic degradation of Myf5 by the ubiquitin-proteasome system is precisely controlled by multiple phosphorylation of the protein, and that the APC/C is not involved in this process.

  3. Decoding c-Myc networks of cell cycle and apoptosis regulated genes in a transgenic mouse model of papillary lung adenocarcinomas.

    Science.gov (United States)

    Ciribilli, Yari; Singh, Prashant; Spanel, Reinhard; Inga, Alberto; Borlak, Jürgen

    2015-10-13

    The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays.Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies.

  4. Molecular characterization of hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) from Taiwan voles (Microtus kikuchii).

    Science.gov (United States)

    Jiang, Yi-Fan; Chou, Chung-Hsi; Lin, En-Chung; Chiu, Chih-Hsien

    2011-02-01

    Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that senses and adapts cells to hypoxic environmental conditions. HIF-1 is composed of an oxygen-regulated α subunit (HIF-1α) and a constitutively expressed β subunit (HIF-1β). Taiwan voles (Microtus kikuchii) are an endemic species in Taiwan, found only in mountainous areas greater than 2000m above sea level. In this study, the full-length HIF-1α cDNA was cloned and sequenced from liver tissues of Taiwan voles. We found that HIF-1α of Taiwan voles had high sequence similarity to HIF-1α of other species. Sequence alignment of HIF-1α functional domains indicated basic helix-loop-helix (bHLH), PER-ARNT-SIM (PAS) and C-terminal transactivation (TAD-C) domains were conserved among species, but sequence variations were found between the oxygen-dependent degradation domains (ODDD). To measure Taiwan vole HIF-1α responses to hypoxia, animals were challenged with cobalt chloride, and HIF-1α mRNA and protein expression in brain, lung, heart, liver, kidney, and muscle was assessed by quantitative RT-PCR and Western blot analysis. Upon induction of hypoxic stress with cobalt chloride, an increase in HIF-1α mRNA levels was detected in lung, heart, kidney, and muscle tissue. In contrast, protein expression levels showed greater variation between individual animals. These results suggest that the regulation of HIF-1α may be important to the Taiwan vole under cobalt chloride treatments. But more details regarding the evolutionary effect of environmental pressure on HIF-1α primary sequence, HIF-1α function and regulation in Taiwan voles remain to be identified.

  5. In-capillary enrichment, proteolysis and separation using capillary electrophoresis with discontinuous buffers: application on proteins with moderately acidic and basic isoelectric points.

    Science.gov (United States)

    Nesbitt, Chandra A; Yeung, Ken K-C

    2009-01-01

    Advances in mass spectrometry and capillary-format separation continue to improve the sensitivity of protein analysis. Of equal importance is the miniaturization of sample pretreatment such as enrichment and proteolysis. In a previous report (Nesbitt et al., Electrophoresis, 2008, 29, 466-474), nanoliter-volume protein enrichment, tryptic digestion, and partial separation was demonstrated in capillary electrophoresis followed by MALDI mass spectral analysis. A discontinuous buffer system, consisting of ammonium (pH 10) and acetate (pH 4), was used to create a pH junction inside the capillary, trapping a protein with a neutral isoelectric point, myoglobin (pI 7.2). Moreover, co-enrichment of myoglobin with trypsin led to an in-capillary digestion. In this paper, the ability of this discontinuous buffer system to perform similar in-capillary sample pretreatment on proteins with moderately acidic and basic pI was studied and reported. Lentil lectin (pI 8.6) and a multi-phosphorylated protein, beta-casein (pI 5.1), were selected as model proteins. In addition to the previously shown tryptic digestion, proteolysis with endoproteinase Asp-N was also performed. Digestion of these acidic and basic pI proteins produced a few peptides with extreme pI values lying outside the trapping range of the discontinuous buffer. An alteration in the peptide trapping procedure was made to accommodate these analytes. Offline MALDI mass spectral analysis confirmed the presence of the expected peptides. The presented miniaturized sample pretreatment methodology was proven to be applicable on proteins with a moderately wide range of pI. Flexibility in the choice of protease was also evident.

  6. Asthma Basics

    Science.gov (United States)

    ... Old Feeding Your 1- to 2-Year-Old Asthma Basics KidsHealth > For Parents > Asthma Basics A A ... Asthma Categories en español Asma: aspectos fundamentales About Asthma Asthma is a common lung condition in kids ...

  7. Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

    Science.gov (United States)

    Friedrich, Michael G; Hancock, Sarah E; Raftery, Mark J; Truscott, Roger J W

    2016-08-12

    Multiple sclerosis (MS) is associated with breakdown of the myelin sheath that coats neurons in the central nervous system. The cause of MS is not known, although the pathogenesis involves destruction of myelin by the immune system. It was the aim of this study to examine the abundant myelin protein, myelin basic protein (MBP), to determine if there are sites of modification that may be characteristic for MS. MBP from the cerebellum was examined from controls and MS patients across the age range using mass spectrometry and amino acid analysis. Amino acid racemization data indicated that myelin basic protein is long-lived and proteomic analysis of MBP showed it to be highly modified. A common modification of MBP was racemization of Asp and this was significantly greater in MS patients. In long-lived proteins, L-Asp and L-Asn can racemize to three other isomers, D-isoAsp, L-isoAsp and D-Asp and this is significant because isoAsp formation in peptides renders them immunogenic.Proteomic analysis revealed widespread modifications of MBP with two surface regions that are altered in MS. In particular, isoAsp was significantly elevated at these sites in MS patients. The generation of isoAsp could be responsible for eliciting an immune response to modified MBP and therefore be implicated in the etiology of MS.

  8. Differential gene expression and mitotic cell analysis of the drought tolerant soybean (Glycine max L. Merrill Fabales, Fabaceae cultivar MG/BR46 (Conquista under two water deficit induction systems

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    Polyana K. Martins

    2008-01-01

    Full Text Available Drought cause serious yield losses in soybean (Glycine max, roots being the first plant organ to detect the water-stress signals triggering defense mechanisms. We used two drought induction systems to identify genes differentially expressed in the roots of the drought-tolerant soybean cultivar MG/BR46 (Conquista and characterize their expression levels during water deficit. Soybean plants grown in nutrient solution hydroponically and in sand-pots were submitted to water stress and gene expression analysis was conducted using the differential display (DD and real time polymerase chain reaction (PCR techniques. Three differentially expressed mRNA transcripts showed homology to the Antirrhinum majus basic helix-loop-helix transcription factor bHLH, the Arabidopsis thaliana phosphatidylinositol transfer protein PITP and the auxin-independent growth regulator 1 (axi 1. The hydroponic experiments showed that after 100 min outside the nutrient solution photosynthesis completely stopped, stomata closed and leaf temperature rose. Both stress induction treatments produced significant decrease in the mitotic indices of root cells. Axi 1, PITP and bHLH were not only differentially expressed during dehydration in the hydroponics experiments but also during induced drought in the pot experiments. Although, there were differences between the two sets of experiments in the time at which up or down regulation occurred, the expression pattern of all three transcripts was related. Similar gene expression and cytological analysis results occurred in both systems, suggesting that hydroponics could be used to simulate drought detection by roots growing in soil and thus facilitate rapid and easy root sampling.

  9. Down-regulation of AP-4 inhibits proliferation, induces cell cycle arrest and promotes apoptosis in human gastric cancer cells.

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    Xinghua Liu

    Full Text Available BACKGROUND: AP-4 belongs to the basic helix-loop-helix leucine-zipper subgroup; it controls target gene expression, regulates growth, development and cell apoptosis and has been implicated in tumorigenesis. Our previous studies indicated that AP-4 was frequently overexpressed in gastric cancers and may be associated with the poor prognosis. The purpose of this study is to examine whether silencing of AP-4 can alter biological characteristics of gastric cancer cells. METHODS: Two specific siRNAs targeting AP-4 were designed, synthesized, and transfected into gastric cancer cell lines and human normal mucosa cells. AP-4 expression was measured with real-time quantitative PCR and Western blot. Cell proliferation and chemo-sensitivity were detected by CCK-8 assay. Cell cycle assay and apoptosis assay were performed by flow cytometer, and relative expression of cell cycle regulators were detected by real-time quantitative PCR and Western blot, expression of the factors involved in the apoptosis pathway were examined in mRNA and protein level. RESULTS: The expression of AP-4 was silenced by the siRNAs transfection and the effects of AP-4 knockdown lasted 24 to 96 hrs. The siRNA-mediated silencing of AP-4 suppressed the cellular proliferation, induced apoptosis and sensitized cancer cells to anticancer drugs. In addition, the expression level of p21, p53 and Caspase-9 were increased when AP-4 was knockdown, but the expression of cyclin D1, Bcl-2 and Bcl-x(L was inhibited. It didn't induce cell cycle arrest when AP-4 was knockdown in p53 defect gastric cancer cell line Kato-III. CONCLUSIONS: These results illustrated that gene silencing of AP-4 can efficiently inhibited cell proliferation, triggered apoptosis and sensitized cancer cells to anticancer drugs in vitro, suggesting that AP-4 siRNAs mediated silencing has a potential value in the treatment of human gastric cancer.

  10. Evolving gene regulatory networks into cellular networks guiding adaptive behavior: an outline how single cells could have evolved into a centralized neurosensory system.

    Science.gov (United States)

    Fritzsch, Bernd; Jahan, Israt; Pan, Ning; Elliott, Karen L

    2015-01-01

    Understanding the evolution of the neurosensory system of man, able to reflect on its own origin, is one of the major goals of comparative neurobiology. Details of the origin of neurosensory cells, their aggregation into central nervous systems and associated sensory organs and their localized patterning leading to remarkably different cell types aggregated into variably sized parts of the central nervous system have begun to emerge. Insights at the cellular and molecular level have begun to shed some light on the evolution of neurosensory cells, partially covered in this review. Molecular evidence suggests that high mobility group (HMG) proteins of pre-metazoans evolved into the definitive Sox [SRY (sex determining region Y)-box] genes used for neurosensory precursor specification in metazoans. Likewise, pre-metazoan basic helix-loop-helix (bHLH) genes evolved in metazoans into the group A bHLH genes dedicated to neurosensory differentiation in bilaterians. Available evidence suggests that the Sox and bHLH genes evolved a cross-regulatory network able to synchronize expansion of precursor populations and their subsequent differentiation into novel parts of the brain or sensory organs. Molecular evidence suggests metazoans evolved patterning gene networks early, which were not dedicated to neuronal development. Only later in evolution were these patterning gene networks tied into the increasing complexity of diffusible factors, many of which were already present in pre-metazoans, to drive local patterning events. It appears that the evolving molecular basis of neurosensory cell development may have led, in interaction with differentially expressed patterning genes, to local network modifications guiding unique specializations of neurosensory cells into sensory organs and various areas of the central nervous system.

  11. SREBP-2 negatively regulates FXR-dependent transcription of FGF19 in human intestinal cells.

    Science.gov (United States)

    Miyata, Masaaki; Hata, Tatsuya; Yamazoe, Yasushi; Yoshinari, Kouichi

    2014-01-10

    Sterol regulatory element-binding protein-2 (SREBP-2) is a basic helix-loop-helix-leucine zipper transcription factor that positively regulates transcription of target genes involved in cholesterol metabolism. In the present study, we have investigated a possible involvement of SREBP-2 in human intestinal expression of fibroblast growth factor (FGF)19, which is an endocrine hormone involved in the regulation of lipid and glucose metabolism. Overexpression of constitutively active SREBP-2 decreased FGF19 mRNA levels in human colon-derived LS174T cells. In reporter assays, active SREBP-2 overexpression suppressed GW4064/FXR-mediated increase in reporter activities in regions containing the IR-1 motif (+848 to +5200) in the FGF19 gene. The suppressive effect disappeared in reporter activities in the region containing the IR-1 motif when the mutation was introduced into the IR-1 motif. In electrophoretic mobility shift assays, binding of the FXR/retinoid X receptor α heterodimer to the IR-1 motif was attenuated by adding active SREBP-2, but SREBP-2 binding to the IR-1 motif was not observed. In chromatin immunoprecipitation assays, specific binding of FXR to the IR-1-containing region of the FGF19 gene (+3214 to +3404) was increased in LS174T cells by treatment with cholesterol and 25-hydroxycholesterol. Specific binding of SREBP-2 to FXR was observed in glutathione-S-transferase (GST) pull-down assays. These results suggest that SREBP-2 negatively regulates the FXR-mediated transcriptional activation of the FGF19 gene in human intestinal cells.

  12. The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis.

    Science.gov (United States)

    Zhang, Jie; Liu, Bing; Li, Mengshu; Feng, Dongru; Jin, Honglei; Wang, Peng; Liu, Jun; Xiong, Feng; Wang, Jinfa; Wang, Hong-Bin

    2015-03-01

    Iron (Fe) is an indispensable micronutrient for plant growth and development. The regulation of Fe homeostasis in plants is complex and involves a number of transcription factors. Here, we demonstrate that a basic helix-loop-helix (bHLH) transcription factor, bHLH104, belonging to the IVc subgroup of bHLH family, acts as a key component positively regulating Fe deficiency responses. Knockout of bHLH104 in Arabidopsis thaliana greatly reduced tolerance to Fe deficiency, whereas overexpression of bHLH104 had the opposite effect and led to accumulation of excess Fe in soil-grown conditions. The activation of Fe deficiency-inducible genes was substantially suppressed by loss of bHLH104. Further investigation showed that bHLH104 interacted with another IVc subgroup bHLH protein, IAA-LEUCINE RESISTANT3 (ILR3), which also plays an important role in Fe homeostasis. Moreover, bHLH104 and ILR3 could bind directly to the promoters of Ib subgroup bHLH genes and POPEYE (PYE) functioning in the regulation of Fe deficiency responses. Interestingly, genetic analysis showed that loss of bHLH104 could decrease the tolerance to Fe deficiency conferred by the lesion of BRUTUS, which encodes an E3 ligase and interacts with bHLH104. Collectively, our data support that bHLH104 and ILR3 play pivotal roles in the regulation of Fe deficiency responses via targeting Ib subgroup bHLH genes and PYE expression.

  13. Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color.

    Science.gov (United States)

    Ballester, Ana-Rosa; Molthoff, Jos; de Vos, Ric; Hekkert, Bas te Lintel; Orzaez, Diego; Fernández-Moreno, Josefina-Patricia; Tripodi, Pasquale; Grandillo, Silvana; Martin, Cathie; Heldens, Jos; Ykema, Marieke; Granell, Antonio; Bovy, Arnaud

    2010-01-01

    The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit

  14. Smad mediated regulation of inhibitor of DNA binding 2 and its role in phenotypic maintenance of human renal proximal tubule epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mangalakumar Veerasamy

    Full Text Available The basic-Helix-Loop-Helix family (bHLH of transcriptional factors plays a major role in regulating cellular proliferation, differentiation and phenotype maintenance. The downregulation of one of the members of bHLH family protein, inhibitor of DNA binding 2 (Id2 has been shown to induce de-differentiation of epithelial cells. Opposing regulators of epithelial/mesenchymal phenotype in renal proximal tubule epithelial cells (PTEC, TGFβ1 and BMP7 also have counter-regulatory effects in models of renal fibrosis. We investigated the regulation of Id2 by these growth factors in human PTECs and its implication in the expression of markers of epithelial versus myofibroblastic phenotype. Cellular Id2 levels were reduced by TGFβ1 treatment; this was prevented by co-incubation with BMP7. BMP7 alone increased cellular levels of Id2. TGFβ1 and BMP7 regulated Id2 through Smad2/3 and Smad1/5 dependent mechanisms respectively. TGFβ1 mediated Id2 suppression was essential for α-SMA induction in PTECs. Although Id2 over-expression prevented α-SMA induction, it did not prevent E-cadherin loss under the influence of TGFβ1. This suggests that the loss of gate keeper function of E-cadherin alone may not necessarily result in complete EMT and further transcriptional re-programming is essential to attain mesenchymal phenotype. Although BMP7 abolished TGFβ1 mediated α-SMA expression by restoring Id2 levels, the loss of Id2 was not sufficient to induce α-SMA expression even in the context of reduced E-cadherin expression. Hence, a reduction in Id2 is critical for TGFβ1-induced α-SMA expression in this model of human PTECs but is not sufficient in it self to induce α-SMA even in the context of reduced E-cadherin.

  15. Metabolic Profiling of Retrograde Pathway Transcription Factors Rtg1 and Rtg3 Knockout Yeast

    Directory of Open Access Journals (Sweden)

    Zanariah Hashim

    2014-07-01

    Full Text Available Rtg1 and Rtg3 are two basic helix-loop-helix (bHLH transcription factors found in yeast Saccharomyces cerevisiae that are involved in the regulation of the mitochondrial retrograde (RTG pathway. Under RTG response, anaplerotic synthesis of citrate is activated, consequently maintaining the supply of important precursors necessary for amino acid and nucleotide synthesis. Although the roles of Rtg1 and Rtg3 in TCA and glyoxylate cycles have been extensively reported, the investigation of other metabolic pathways has been lacking. Characteristic dimer formation in bHLH proteins, which allows for combinatorial gene expression, and the link between RTG and other regulatory pathways suggest more complex metabolic signaling involved in Rtg1/Rtg3 regulation. In this study, using a metabolomics approach, we examined metabolic alteration following RTG1 and RTG3 deletion. We found that apart from TCA and glyoxylate cycles, which have been previously reported, polyamine biosynthesis and other amino acid metabolism were significantly altered in RTG-deficient strains. We revealed that metabolic alterations occurred at various metabolic sites and that these changes relate to different growth phases, but the difference can be detected even at the mid-exponential phase, when mitochondrial function is repressed. Moreover, the effect of metabolic rearrangements can be seen through the chronological lifespan (CLS measurement, where we confirmed the role of the RTG pathway in extending the yeast lifespan. Through a comprehensive metabolic profiling, we were able to explore metabolic phenotypes previously unidentified by other means and illustrate the possible correlations of Rtg1 and Rtg3 in different pathways.

  16. Transcription factors, sucrose, and sucrose metabolic genes interact to regulate potato phenylpropanoid metabolism.

    Science.gov (United States)

    Payyavula, Raja S; Singh, Rajesh K; Navarre, Duroy A

    2013-11-01

    Much remains unknown about how transcription factors and sugars regulate phenylpropanoid metabolism in tuber crops like potato (Solanum tuberosum). Based on phylogeny and protein similarity to known regulators of phenylpropanoid metabolism, 15 transcription factors were selected and their expression was compared in white, yellow, red, and purple genotypes with contrasting phenolic and anthocyanin profiles. Red and purple genotypes had increased phenylalanine ammonia lyase (PAL) enzyme activity, markedly higher levels of phenylpropanoids, and elevated expression of most phenylpropanoid structural genes, including a novel anthocyanin O-methyltransferase. The transcription factors Anthocyanin1 (StAN1), basic Helix Loop Helix1 (StbHLH1), and StWD40 were more strongly expressed in red and purple potatoes. Expression of 12 other transcription factors was not associated with phenylpropanoid content, except for StMYB12B, which showed a negative relationship. Increased expression of AN1, bHLH1, and WD40 was also associated with environmentally mediated increases in tuber phenylpropanoids. Treatment of potato plantlets with sucrose induced hydroxycinnamic acids, flavonols, anthocyanins, structural genes, AN1, bHLH1, WD40, and genes encoding the sucrose-hydrolysing enzymes SUSY1, SUSY4, and INV2. Transient expression of StAN1 in tobacco leaves induced bHLH1, structural genes, SUSY1, SUSY4, and INV1, and increased phenylpropanoid amounts. StAN1 infiltration into tobacco leaves decreased sucrose and glucose concentrations. In silico promoter analysis revealed the presence of MYB and bHLH regulatory elements on sucrolytic gene promoters and sucrose-responsive elements on the AN1 promoter. These findings reveal an interesting dynamic between AN1, sucrose, and sucrose metabolic genes in modulating potato phenylpropanoids.

  17. ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

    Directory of Open Access Journals (Sweden)

    Dawoon Chung

    2014-11-01

    Full Text Available The Aspergillus fumigatus sterol regulatory element binding protein (SREBP SrbA belongs to the basic Helix-Loop-Helix (bHLH family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA. How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

  18. Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver.

    Directory of Open Access Journals (Sweden)

    Guillaume Rey

    2011-02-01

    Full Text Available The mammalian circadian clock uses interlocked negative feedback loops in which the heterodimeric basic helix-loop-helix transcription factor BMAL1/CLOCK is a master regulator. While there is prominent control of liver functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Using chromatin immunoprecipitation combined with deep sequencing we obtained a time-resolved and genome-wide map of BMAL1 binding in mouse liver, which allowed us to identify over 2,000 binding sites, with peak binding narrowly centered around Zeitgeber time 6. Annotation of BMAL1 targets confirms carbohydrate and lipid metabolism as the major output of the circadian clock in mouse liver. Moreover, transcription regulators are largely overrepresented, several of which also exhibit circadian activity. Genes of the core circadian oscillator stand out as strongly bound, often at promoter and distal sites. Genomic sequence analysis of the sites identified E-boxes and tandem E1-E2 consensus elements. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution centered at Zeitgeber time 10. Importantly, sites with E1-E2 elements showed tighter phases both in binding and mRNA accumulation. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and post-transcriptional regulation contribute differentially to circadian expression phase. Together, our analysis of a dynamic protein-DNA interactome uncovered how genes of the core circadian oscillator crosstalk and drive phase-specific circadian output programs in a complex tissue.

  19. Expression of epithelial-mesenchymal transition regulators SNAI2 and TWIST1 in thyroid carcinomas.

    Science.gov (United States)

    Buehler, Darya; Hardin, Heather; Shan, Weihua; Montemayor-Garcia, Celina; Rush, Patrick S; Asioli, Sofia; Chen, Herbert; Lloyd, Ricardo V

    2013-01-01

    Epithelial-mesenchymal transition is an important mechanism of epithelial tumor progression, local invasion and metastasis. The E-cadherin (CDH1) repressor SLUG (SNAI2) and the basic helix-loop-helix transcription factor TWIST1 inhibit CDH1 expression in poorly differentiated malignancies as inducers of epithelial-mesenchymal transition. Epithelial-mesenchymal transition has been implicated in progression from well to poorly differentiated/anaplastic thyroid carcinoma but the expression of SNAI2 and TWIST1 proteins and their phenotypic association in human thyroid cancers has not been extensively studied. We examined the expression of SNAI2, TWIST1 and CDH1 by immunohistochemistry in a panel of well-differentiated and anaplastic thyroid cancers and by qRT-PCR in thyroid cell lines. Ten normal thyroids, 33 follicular adenomas, 56 papillary thyroid carcinomas including 28 follicular variants, 27 follicular carcinomas and 10 anaplastic thyroid carcinomas were assembled on a tissue microarray and immunostained for SNAI2, TWIST1 and CDH1. Most (8/10) anaplastic thyroid carcinomas demonstrated strong nuclear immunoreactivity for SNAI2 with associated absence of CDH1 in 6/8 cases (75%). TWIST1 was expressed in 5/10 anaplastic thyroid carcinomas with absence of CDH1 in 3/5 (60%) cases. These findings were confirmed in whole sections of all anaplastic thyroid carcinomas and in a separate validation set of 10 additional anaplastic thyroid carcinomas. All normal thyroids, follicular adenomas, papillary and follicular thyroid carcinomas were negative for SNAI2 and TWIST1 (Pcarcinoma and two anaplastic thyroid carcinoma cell lines tested, but the highest levels of CDH1 mRNA were detected in the normal thyroid cell line while the anaplastic thyroid carcinoma cell line demonstrated the highest levels of SNAI2 and TWIST1 mRNA. Our findings support the role of epithelial-mesenchymal transition in the development of anaplastic thyroid carcinoma.

  20. MLK-3: identification of a widely-expressed protein kinase bearing an SH3 domain and a leucine zipper-basic region domain.

    Science.gov (United States)

    Ing, Y L; Leung, I W; Heng, H H; Tsui, L C; Lassam, N J

    1994-06-01

    We have identified a novel protein kinase, designated MLK-3, from human thymus using RT-PCR and cDNA library screening. The deduced open reading frame, derived from sequencing a 3.5 kb MLK-3 cDNA, encodes a protein of 847 amino acids with several interesting structural features. These include an SH3 domain in the absence of an SH2 domain, a region containing two leucine zippers with an adjacent carboxy-terminal basic region, and a proline rich region. This kinase shows homology with the mixed-lineage family of protein kinases (MLK) and shares the unusual leucine zipper-basic motif found in previously identified MLK kinases. By northern analysis, MLK-3 mRNA was detected in a wide variety of normal and transformed human cell lines and tissue specimens. The gene encoding MLK-3 has been mapped using fluorescence in situ hybridization to human chromosome 11 q13.1-13.3, a region frequently altered in human malignancies.

  1. The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein

    Science.gov (United States)

    Lipps, Georg; Ibanez, Pablo; Stroessenreuther, Thomas; Hekimian, Katya; Krauss, Gerhard

    2001-01-01

    The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by ∼60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein–DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins. PMID:11812827

  2. One step physically adsorbed coating of silica capillary with excellent stability for the separation of basic proteins by capillary zone electrophoresis.

    Science.gov (United States)

    Guo, Xiao-Feng; Guo, Xiao-Mei; Wang, Hong; Zhang, Hua-Shan

    2015-11-01

    The coating of capillary inner surface is considered to be an effective approach to suppress the adsorption of proteins on capillary inner surface in CE. However, most of coating materials reported are water-soluble, which may dissolve in BGE during the procedure of electrophoresis. In this study, a novel strategy for selection of physically coating materials has been illustrated to get coating layer with excellent stability using materials having poor solubility in commonly used solvents. Taking natural chitin as example (not hydrolyzed water soluble chitosan), a simple one step coating method using chitin solution in hexafluoroisopropanol was adopted within only 21 min with good coating reproducibility (RSDs of EOF for within-batch coated capillaries of 1.55% and between-batch coated capillaries of 2.31%), and a separation of four basic proteins on a chitin coated capillary was performed to evaluate the coating efficacy. Using chitin coating, the adsorption of proteins on capillary inner surface was successfully suppressed with reversed and stable EOF, and four basic proteins including lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A were baseline separated within 16 min with satisfied separation efficiency using 20 mM pH 2.0 H3PO4-Na2HPO4 as back ground electrolyte and 20 kV as separation voltage. What is more important, the chitin coating layer could be stable for more than two months during this study, which demonstrates that chitin is an ideal material for preparing semi-permanent coating on bare fused silica capillary inner wall and has hopeful potential in routine separation of proteins with CE.

  3. Translational control of myelin basic protein expression by ERK2 MAP kinase regulates timely remyelination in the adult brain.

    Science.gov (United States)

    Michel, Kelly; Zhao, Tianna; Karl, Molly; Lewis, Katherine; Fyffe-Maricich, Sharyl L

    2015-05-20

    Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS.

  4. Capillary electrophoresis-mass spectrometry of intact basic proteins using Polybrene-dextran sulfate-Polybrene-coated capillaries: system optimization and performance.

    Science.gov (United States)

    Haselberg, Rob; de Jong, Gerhardus J; Somsen, Govert W

    2010-09-23

    A capillary electrophoresis-mass spectrometry (CE-MS) method using sheath liquid electrospray ionization interfacing was studied and optimized for the analysis of intact basic proteins. To prevent protein adsorption, capillaries with a noncovalent positively charged coating were utilized. Capillaries were coated by subsequent rinsing with solutions of Polybrene, dextran sulfate and Polybrene. The coating proved to be fully compatible with MS detection, causing no background signals and ionization suppression. The composition of the sheath liquid and BGE was optimized using the model proteins α-chymotrypsinogen A, ribonuclease A, lysozyme and cytochrome c. A sheath liquid of isopropanol-water-acetic acid (75:25:0.1, v/v/v) at 2 μL min(-1) resulted in optimal signal intensities for most proteins, but caused dissociation of the heme group of cytochrome c. Optimum protein responses were obtained with a BGE of 50 mM acetic acid (pH 3.0), which allowed a baseline separation of the test protein mixture. Several minor impurities present in the mixture could be detected and provisionally identified using accurate mass and a protein modification database. The selectivity of the CE-MS system was investigated by the analysis of acetylated lysozyme. Eight highly related species, identified as non-acetylated lysozyme and lysozyme acetylated in various degrees, could be distinguished. The CE-MS system showed good reproducibility yielding interday (three weeks period) RSDs for migration time and peak area within 2% and 10%, respectively. With the CE-MS system, determination coefficients (R(2)) for protein concentration and peak area were higher than 0.996, whereas detection limits were between 11 and 19 nM.

  5. Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I;

    2001-01-01

    subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure....

  6. Structural insight into the function of myelin basic protein as a ligand for integrin αMβ2

    DEFF Research Database (Denmark)

    Stapulionis, Romualdas; Oliveira, Cristiano; Gjelstrup, Mikkel Carstensen;

    2008-01-01

    Multiple sclerosis (MS) is an inflammatory disease where phagocytic cells infiltrate the nerve tissue and act as terminal agents in destruction of the myelin sheath. However, the mechanism that triggers the ability of these cells to recognize myelin remains obscure. We show that myelin basic...... protein (MBP), a major autoantigen in MS, is a potent and specific ligand for the integrin αMβ2 (Mac-1, CD11b/CD18) expressed mainly on phagocytic cells. MBP undergoes a dramatic conformational change when liberated from the lipid-rich environment of the myelin sheath. The MS drug glatiramer acetate...

  7. The effect of beta-interferon therapy on myelin basic protein-elicited CD4+ T cell proliferation and cytokine production in multiple sclerosis

    DEFF Research Database (Denmark)

    Hedegaard, Chris J; Krakauer, Martin; Bendtzen, Klaus

    2008-01-01

    Interferon (IFN)-beta therapy has well-established clinical benefits in multiple sclerosis (MS), but the underlying modulation of cytokine responses to myelin self-antigens remains poorly understood. We analysed the CD4+ T cell proliferation and cytokine responses elicited by myelin basic protein...... (MBP) and a foreign recall antigen, tetanus toxoid (TT), in mononuclear cell cultures from fourteen MS patients undergoing IFN-beta therapy. The MBP-elicited IFN-gamma-, TNF-alpha- and IL-10 production decreased during therapy (p...

  8. Basic electrotechnology

    CERN Document Server

    Ashen, R A

    2013-01-01

    BASIC Electrotechnology discusses the applications of Beginner's All-purpose Symbolic Instruction Code (BASIC) in engineering, particularly in solving electrotechnology-related problems. The book is comprised of six chapters that cover several topics relevant to BASIC and electrotechnology. Chapter 1 provides an introduction to BASIC, and Chapter 2 talks about the use of complex numbers in a.c. circuit analysis. Chapter 3 covers linear circuit analysis with d.c. and sinusoidal a.c. supplies. The book also discusses the elementary magnetic circuit theory. The theory and performance of two windi

  9. Basic domains target protein subunits of the RNase MRP complex to the nucleolus independently of complex association.

    NARCIS (Netherlands)

    Eenennaam, H. van; Heijden, A.G. van der; Janssen, R.J.T.; Venrooij, W.J.W. van; Pruijn, G.J.M.

    2001-01-01

    The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both R

  10. Classic 18.5- and 21.5-kDa myelin basic protein isoforms associate with cytoskeletal and SH3-domain proteins in the immortalized N19-oligodendroglial cell line stimulated by phorbol ester and IGF-1.

    Science.gov (United States)

    Smith, Graham S T; Homchaudhuri, Lopamudra; Boggs, Joan M; Harauz, George

    2012-06-01

    The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca²⁺-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct 'membrane-ruffled' regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.

  11. Catalytic autoantibodies against myelin basic protein (MBP) isolated from serum of autistic children impair in vitro models of synaptic plasticity in rat hippocampus.

    Science.gov (United States)

    Gonzalez-Gronow, Mario; Cuchacovich, Miguel; Francos, Rina; Cuchacovich, Stephanie; Blanco, Angel; Sandoval, Rodrigo; Gomez, Cristian Farias; Valenzuela, Javier A; Ray, Rupa; Pizzo, Salvatore V

    2015-10-15

    Autoantibodies from autistic spectrum disorder (ASD) patients react with multiple proteins expressed in the brain. One such autoantibody targets myelin basic protein (MBP). ASD patients have autoantibodies to MBP of both the IgG and IgA classes in high titers, but no autoantibodies of the IgM class. IgA autoantibodies act as serine proteinases and degrade MBP in vitro. They also induce a decrease in long-term potentiation in the hippocampi of rats either perfused with or previously inoculated with this IgA. Because this class of autoantibody causes myelin sheath destruction in multiple sclerosis (MS), we hypothesized a similar pathological role for them in ASD.

  12. Redirecting Therapeutic T Cells against Myelin-Specific T Lymphocytes Using a Humanized Myelin Basic Protein-HLA-DR2-{zeta} Chimeric Receptor

    DEFF Research Database (Denmark)

    Moisini, Ioana; Nguyen, Phuong; Fugger, Lars

    2008-01-01

    Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors...... to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP(84-102) epitope to HLA-DR2 and either incorporate or lack a TCRzeta signaling domain. The Ag-MHC domain serves...... as a bait, binding the TCR of MBP-specific target cells. The zeta signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic (alpha, beta, green fluorescent protein) retroviral expression system...

  13. Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA synthetase) and myelin basic protein: a possible mechanism of nerve damage in leprosy.

    Science.gov (United States)

    Singh, Itu; Yadav, Asha Ram; Mohanty, Keshar Kunja; Katoch, Kiran; Sharma, Prashant; Mishra, Bishal; Bisht, Deepa; Gupta, U D; Sengupta, Utpal

    2015-04-01

    Autoantibodies against various components of host are known to occur in leprosy. Nerve damage is the primary cause of disability associated with leprosy. The aim of this study was to detect the level of autoantibodies and lympho-proliferative response against myelin basic protein (MBP) in leprosy patients (LPs) and their correlation with clinical phenotypes of LPs. Further, probable role of molecular mimicry in nerve damage of LPs was investigated. We observed significantly high level of anti-MBP antibodies in LPs across the spectrum and a positive significant correlation between the level of anti-MBP antibodies and the number of nerves involved in LPs. We report here that 4 B cell epitopes of myelin A1 and Mycobacterium leprae proteins, 50S ribosomal L2 and lysyl tRNA synthetase are cross-reactive. Further, M. leprae sonicated antigen hyperimmunization was responsible for induction of autoantibody response in mice which could be adoptively transferred to naive mice. For the first time our findings suggest the role of molecular mimicry in nerve damage in leprosy.

  14. Brain Basics

    Medline Plus

    Full Text Available ... Basics will introduce you to some of this science, such as: How the brain develops How genes and the environment affect the brain The basic structure of the brain How different parts of the brain communicate and work with each other How changes in the brain ...

  15. Two basic (hydrophilic) regions in the movement protein of Parietaria mottle virus have RNA binding activity and are required for cell-to-cell transport.

    Science.gov (United States)

    Martínez, Carolina; Coll-Bonfill, Nuria; Aramburu, Jose; Pallás, Vicente; Aparicio, Frederic; Galipienso, Luis

    2014-05-12

    The movement protein (MP) of parietaria mottle virus (PMoV) is required for virus cell-to-cell movement. Bioinformatics analysis identified two hydrophilic non-contiguous regions (R1 and R2) rich in the basic amino acids lysine and arginine and with the predicted secondary structure of an α-helix. Different approaches were used to determine the implication of the R1 and R2 regions in RNA binding, plasmodesmata (PD) targeting and cell-to-cell movement. EMSA (Electrophoretic Mobility Shift Assay) showed that both regions have RNA-binding activity whereas that mutational analysis reported that either deletion of any of these regions, or loss of the basic amino acids, interfered with the viral intercellular movement. Subcellular localization studies showed that PMoV MP locates at PD. Mutants designed to impeded cell-to-cell movement failed to accumulate at PD indicating that basic residues in both R1 and R2 are critical for binding the MP at PD.

  16. A highly basic sequence at the N-terminal region is essential for targeting the DNA replication protein ORC1 to the nucleus in Leishmania donovani.

    Science.gov (United States)

    Kumar, Devanand; Kumar, Diwakar; Saha, Swati

    2012-07-01

    The conserved eukaryotic DNA replication protein ORC1 is one of the constituents of pre-replication complexes that assemble at or very near origins prior to replication initiation. ORC1 has been shown to be constitutively nuclear in Leishmania major. This study investigates the sequences involved in nuclear localization of ORC1 in Leishmania donovani, the causative agent of visceral leishmaniasis. Nuclear localization signals (NLSs) have been reported in only a few Leishmania proteins. Functional analyses have delineated NLSs to regions of ~60 amino acids in length in the tyrosyl DNA phosphodiesterase I and type II DNA topoisomerase of L. donovani, and in the L. major kinesin KIN13-1. Using a panel of site-directed mutations we have identified a sequence essential for nuclear import of LdORC1. This sequence at the N terminus of the protein comprises residues 2-5 (KRSR), with K2, R3 and R5 being crucial. Independent mutation of the K2 residue causes exclusion of the protein from the nucleus, while mutating the R5 residue leads to diffusion of the protein throughout the cell. This sequence, however, is insufficient for targeting a heterologous protein (β-galactosidase) to the nucleus. Analysis of additional ORC1 mutations and reporter constructs reveals that while the highly basic tetra-amino acid sequence at the N terminus is essential for nuclear localization, the ORC1 NLS in its entirety is more complex, and of a distributive character. Our results suggest that nuclear localization signalling sequences in Leishmania nuclear proteins are more complex than what is typically seen in higher eukaryotes.

  17. The AAA+ proteins Pontin and Reptin enter adult age: From understanding their basic biology to the identification of selective inhibitors

    Directory of Open Access Journals (Sweden)

    Pedro M Matias

    2015-05-01

    Full Text Available Pontin and Reptin are related partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities family. They are implicated in multiple and seemingly unrelated processes encompassing the regulation of gene transcription, the remodeling of chromatin, DNA damage sensing and repair, and the assembly of protein and ribonucleoprotein complexes, among others. The 2nd International Workshop on Pontin and Reptin took place at the Instituto de Tecnologia Química e Biológica António Xavier in Oeiras, Portugal on October 10-12, 2014, and reported significant new advances on the mechanisms of action of these two AAA+ ATPases. The major points under discussion were related to the mechanisms through which these proteins regulate gene transcription, their roles as co-chaperones, and their involvement in pathophysiology, especially in cancer and ciliary biology and disease. Finally, they may become anticancer drug targets since small chemical inhibitors were shown to produce anti-tumor effects in animal models.

  18. The AAA+ proteins Pontin and Reptin enter adult age: from understanding their basic biology to the identification of selective inhibitors.

    Science.gov (United States)

    Matias, Pedro M; Baek, Sung Hee; Bandeiras, Tiago M; Dutta, Anindya; Houry, Walid A; Llorca, Oscar; Rosenbaum, Jean

    2015-01-01

    Pontin and Reptin are related partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They are implicated in multiple and seemingly unrelated processes encompassing the regulation of gene transcription, the remodeling of chromatin, DNA damage sensing and repair, and the assembly of protein and ribonucleoprotein complexes, among others. The 2nd International Workshop on Pontin and Reptin took place at the Instituto de Tecnologia Química e Biológica António Xavier in Oeiras, Portugal on October 10-12, 2014, and reported significant new advances on the mechanisms of action of these two AAA+ ATPases. The major points under discussion were related to the mechanisms through which these proteins regulate gene transcription, their roles as co-chaperones, and their involvement in pathophysiology, especially in cancer and ciliary biology and disease. Finally, they may become anticancer drug targets since small chemical inhibitors were shown to produce anti-tumor effects in animal models.

  19. Basic hydraulics

    CERN Document Server

    Smith, P D

    1982-01-01

    BASIC Hydraulics aims to help students both to become proficient in the BASIC programming language by actually using the language in an important field of engineering and to use computing as a means of mastering the subject of hydraulics. The book begins with a summary of the technique of computing in BASIC together with comments and listing of the main commands and statements. Subsequent chapters introduce the fundamental concepts and appropriate governing equations. Topics covered include principles of fluid mechanics; flow in pipes, pipe networks and open channels; hydraulic machinery;

  20. Brain Basics

    Medline Plus

    Full Text Available ... depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit of ... but sometimes give rise to disabilities or diseases. neural circuit —A network of neurons and their interconnections. ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a ... blues" from time to time. In contrast, major depression is a serious disorder that lasts for weeks. ...

  2. Schizophrenia Basics

    Science.gov (United States)

    ... I know with schizophrenia? For More Information Share Schizophrenia Basics Download PDF Download ePub Order a free hardcopy What is schizophrenia? Schizophrenia is a serious mental disorder that affects ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... News About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain ... to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are ...

  4. Brain Basics

    Science.gov (United States)

    ... News About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain ... to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are ...

  5. Fluoridation Basics

    Science.gov (United States)

    ... Page Basic Information About Fluoride Benefits: Strong Teeth History of Fluoride in Water Cost: Saves Money, Saves Teeth Fluoride in the Water Today The mineral fluoride occurs naturally on earth and is released from rocks into the soil, ...

  6. Basic Finance

    Science.gov (United States)

    Vittek, J. F.

    1972-01-01

    A discussion of the basic measures of corporate financial strength, and the sources of the information is reported. Considered are: balance sheet, income statement, funds and cash flow, and financial ratios.

  7. Brain Basics

    Medline Plus

    Full Text Available ... science, such as: How the brain develops How genes and the environment affect the brain The basic ... that with brain development in people mental disorders. Genes and environmental cues both help to direct this ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot effectively coordinate the billions ... the basic working unit of the brain and nervous system. These cells are highly specialized for the function ...

  9. Responsiveness to 6-n-propylthiouracil (PROP) is associated with salivary levels of two specific basic proline-rich proteins in humans.

    Science.gov (United States)

    Cabras, Tiziana; Melis, Melania; Castagnola, Massimo; Padiglia, Alessandra; Tepper, Beverly J; Messana, Irene; Tomassini Barbarossa, Iole

    2012-01-01

    Thiourea tasting can be predictive of individual differences in bitter taste responses, general food preferences and eating behavior, and could be correlated with saliva chemical composition. We investigated the possible relationship between PROP bitter taste responsiveness and the salivary proteome in subjects genotyped for TAS2R38 and gustin gene polymorphisms. Taste perception intensity evoked by PROP and NaCl solutions was measured in sixty-three volunteers (21 males, 42 females, age 25±3 y) to establish their PROP taster status, and 24 PROP super-tasters and 21 nontasters were selected to participate in the study. TAS2R38 and gustin gene molecular analysis were performed using PCR techniques. Qualitative and quantitative determination of salivary proteins was performed by HPLC-ESI-MS before and after PROP taste stimulation. PROP super-tastings was strongly associated with the 'taster' variant (PAV haplotype) of TAS2R38 and the A allele of rs2274333 polymorphism in the gustin gene and nontasting was associated with the minor alleles at both loci. ANOVA revealed that basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in nontaster un-stimulated saliva, and that PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. These data show for the first time that responsiveness to PROP is associated with salivary levels of II-2 peptide and Ps-1 protein, which are products of the PRB1 gene. These findings suggest that PRB1, in addition to TAS2R38 and gustin, could contribute to individual differences in thiourea sensitivity, and the expression of the PROP phenotype as a complex genetic trait.

  10. Substitutions mimicking deimination and phosphorylation of 18.5-kDa myelin basic protein exert local structural effects that subtly influence its global folding.

    Science.gov (United States)

    Vassall, Kenrick A; Bamm, Vladimir V; Jenkins, Andrew D; Velte, Caroline J; Kattnig, Daniel R; Boggs, Joan M; Hinderberger, Dariush; Harauz, George

    2016-06-01

    Intrinsically-disordered proteins (IDPs) present a complex interplay of conformational variability and multifunctionality, modulated by environment and post-translational modifications. The 18.5-kDa myelin basic protein (MBP) is essential to the formation of the myelin sheath of the central nervous system and is exemplary in this regard. We have recently demonstrated that the unmodified MBP-C1 component undergoes co-operative global conformational changes in increasing concentrations of trifluoroethanol, emulating the decreasing dielectric environment that the protein encounters upon adsorption to the oligodendrocyte membrane [K.A. Vassall et al., Journal of Molecular Biology, 427, 1977-1992, 2015]. Here, we extended this study to the pseudo-deiminated MBP-C8 charge component, one found in greater proportion in developing myelin and in multiple sclerosis. A similar tri-conformational distribution as for MBP-C1 was observed with slight differences in Gibbs free energy. A more dramatic difference was observed by cathepsin D digestion of the protein in both aqueous and membrane environments, which showed significantly greater accessibility of the F42-F43 cut site of MBP-C8, indicative of a global conformational change. In contrast, this modification caused little change in the protein's density of packing on myelin-mimetic membranes as ascertained by double electron-electron resonance spectroscopy [D.R. Kattnig et al., Biochimica et Biophysica Acta (Biomembranes), 1818, 2636-2647, 2012], or in its affinity for Ca(2+)-CaM. Site-specific threonyl pseudo-phosphorylation at residues T92 and/or T95 did not appreciably affect any of the thermodynamic mechanisms of conformational transitions, susceptibility to cathepsin D, or affinity for Ca(2+)-CaM, despite previously having been shown to affect local structure and disposition on the membrane surface.

  11. The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Graham S.T.; Seymour, Lauren V. [Molecular and Cellular Biology, University of Guelph, Guelph, Ontario (Canada); Boggs, Joan M. [Molecular Structure and Function, Research Institute, Hospital for Sick Children, and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario (Canada); Harauz, George, E-mail: gharauz@uoguelph.ca [Molecular and Cellular Biology, University of Guelph, Guelph, Ontario (Canada)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipid membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.

  12. Responsiveness to 6-n-propylthiouracil (PROP is associated with salivary levels of two specific basic proline-rich proteins in humans.

    Directory of Open Access Journals (Sweden)

    Tiziana Cabras

    Full Text Available Thiourea tasting can be predictive of individual differences in bitter taste responses, general food preferences and eating behavior, and could be correlated with saliva chemical composition. We investigated the possible relationship between PROP bitter taste responsiveness and the salivary proteome in subjects genotyped for TAS2R38 and gustin gene polymorphisms. Taste perception intensity evoked by PROP and NaCl solutions was measured in sixty-three volunteers (21 males, 42 females, age 25±3 y to establish their PROP taster status, and 24 PROP super-tasters and 21 nontasters were selected to participate in the study. TAS2R38 and gustin gene molecular analysis were performed using PCR techniques. Qualitative and quantitative determination of salivary proteins was performed by HPLC-ESI-MS before and after PROP taste stimulation. PROP super-tastings was strongly associated with the 'taster' variant (PAV haplotype of TAS2R38 and the A allele of rs2274333 polymorphism in the gustin gene and nontasting was associated with the minor alleles at both loci. ANOVA revealed that basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs family, were significantly higher in PROP super-taster than in nontaster un-stimulated saliva, and that PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. These data show for the first time that responsiveness to PROP is associated with salivary levels of II-2 peptide and Ps-1 protein, which are products of the PRB1 gene. These findings suggest that PRB1, in addition to TAS2R38 and gustin, could contribute to individual differences in thiourea sensitivity, and the expression of the PROP phenotype as a complex genetic trait.

  13. Brain Basics

    Medline Plus

    Full Text Available ... segment of DNA that contains codes to make proteins and other important body chemicals. DNA also includes ... the gene to code for a slightly different protein. Some mutations are harmless, some can be helpful, ...

  14. Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1 Are Essential for the Binding of Clathrin Light Chain

    Directory of Open Access Journals (Sweden)

    Joel A. Ybe

    2009-01-01

    Full Text Available The interaction between HIP family proteins (HIP1 and HIP12/1R and clathrin is fundamental to endocytosis. We used circular dichroism (CD to study the stability of an HIP1 subfragment (aa468-530 that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83C∘. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494. The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae, HIP1 from D. melanogaster, and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.

  15. UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lu, M.L.; Sato, Mitsuhiro; Cao, Boliang; Richie, J.P. [Harvard Medical School, Boston, MA (United States)

    1996-08-20

    UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirements for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-{alpha}-and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis. 40 refs., 5 figs.

  16. Basic electronics

    CERN Document Server

    Holbrook, Harold D

    1971-01-01

    Basic Electronics is an elementary text designed for basic instruction in electricity and electronics. It gives emphasis on electronic emission and the vacuum tube and shows transistor circuits in parallel with electron tube circuits. This book also demonstrates how the transistor merely replaces the tube, with proper change of circuit constants as required. Many problems are presented at the end of each chapter. This book is comprised of 17 chapters and opens with an overview of electron theory, followed by a discussion on resistance, inductance, and capacitance, along with their effects on t

  17. Ethanol Basics

    Energy Technology Data Exchange (ETDEWEB)

    None

    2015-01-30

    Ethanol is a widely-used, domestically-produced renewable fuel made from corn and other plant materials. More than 96% of gasoline sold in the United States contains ethanol. Learn more about this alternative fuel in the Ethanol Basics Fact Sheet, produced by the U.S. Department of Energy's Clean Cities program.

  18. Body Basics

    Science.gov (United States)

    ... more about how the body works, what basic human anatomy is, and what happens when parts of the body don't function properly. Blood Bones, Muscles, and Joints Brain and Nervous System Digestive System Endocrine System Eyes Female Reproductive System Heart and Circulatory System Immune ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit ... final destination. Chemical signals from other cells guide neurons in forming various brain structures. Neighboring neurons make connections with each other ...

  20. Insulin Basics

    Science.gov (United States)

    ... Honor Become a Member En Español Type 1 Type 2 About Us Online Community Meal Planning Sign In Search: Search More Sites Search ≡ Are You At Risk? Diabetes Basics Living with Diabetes Food & Fitness In My ... Diabetes and Learning About Prediabetes Type 2 Diabetes Risk Test Lower Your Risk Healthy ...

  1. Focal cerebral ischemia induces increased myelin basic protein and growth-associated protein-43 gene transcription in peri-infarct areas in the rat brain

    DEFF Research Database (Denmark)

    Gregersen, R; Christensen, Thomas; Lehrmann, E;

    2001-01-01

    , in peri-infarct areas in adult rat brain after transient middle cerebral artery occlusion (MCAO) and correlated it to the expression of the growth-associated protein-43 (GAP-43), a marker for axonal regeneration and sprouting, using non-radioactive in situ hybridization techniques. Within the infarct, MBP......, corresponding to the appearance of process-bearing MBP and occasional MOG-immunoreactive oligodendrocytes in parallel sections. Quantitative analysis revealed significant increases in the density of oligodendrocytes (up to 7.6-fold) and in the level of MBP mRNA expressed by individual cells. Parallel sections...... showed that increased expression of GAP-43 mRNA in neurons was concomitant to MBP mRNA upregulation in oligodendrocytes. While the mechanisms regulating oligodendrocyte survival and myelination signals are not clear at this point, axonal sprouting could putatively serve as a stimulus for the upregulation...

  2. Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

    Directory of Open Access Journals (Sweden)

    Przemysław Karpowicz

    Full Text Available The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.

  3. Wavelet basics

    CERN Document Server

    Chan, Y T

    1995-01-01

    Since the study of wavelets is a relatively new area, much of the research coming from mathematicians, most of the literature uses terminology, concepts and proofs that may, at times, be difficult and intimidating for the engineer. Wavelet Basics has therefore been written as an introductory book for scientists and engineers. The mathematical presentation has been kept simple, the concepts being presented in elaborate detail in a terminology that engineers will find familiar. Difficult ideas are illustrated with examples which will also aid in the development of an intuitive insight. Chapter 1 reviews the basics of signal transformation and discusses the concepts of duals and frames. Chapter 2 introduces the wavelet transform, contrasts it with the short-time Fourier transform and clarifies the names of the different types of wavelet transforms. Chapter 3 links multiresolution analysis, orthonormal wavelets and the design of digital filters. Chapter 4 gives a tour d'horizon of topics of current interest: wave...

  4. The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein.

    Directory of Open Access Journals (Sweden)

    Kenrick A Vassall

    Full Text Available The classic isoforms of myelin basic protein (MBP are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99- containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90 upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107 with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012. Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the

  5. The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein.

    Science.gov (United States)

    Vassall, Kenrick A; Bessonov, Kyrylo; De Avila, Miguel; Polverini, Eugenia; Harauz, George

    2013-01-01

    The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99-) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure

  6. Targeted toxicological screening for acidic, neutral and basic substances in postmortem and antemortem whole blood using simple protein precipitation and UPLC-HR-TOF-MS.

    Science.gov (United States)

    Telving, Rasmus; Hasselstrøm, Jørgen Bo; Andreasen, Mette Findal

    2016-09-01

    A broad targeted screening method based on broadband collision-induced dissociation (bbCID) ultra-performance liquid chromatography high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS) was developed and evaluated for toxicological screening of whole blood samples. The acidic, neutral and basic substances covered by the method were identified in postmortem and antemortem whole blood samples from forensic autopsy cases, clinical forensic cases and driving under the influence of drugs (DUID) cases by a reverse target database search. The screening method covered 467 substances. Validation was performed on spiked whole blood samples and authentic postmortem and antemortem whole blood samples. For most of the basic drugs, the established cut-off limits were very low, ranging from 0.25ng/g to 50ng/g. The established cut-off limits for most neutral and acidic drugs, were in the range from 50ng/g to 500ng/g. Sample preparation was performed using simple protein precipitation of 300μL of whole blood with acetonitrile and methanol. Ten microliters of the reconstituted extract were injected and separated within a 13.5min UPLC gradient reverse-phase run. Positive electrospray ionization (ESI) was used to generate the ions in the m/z range of 50-1000. Fragment ions were generated by bbCID. Identification was based on retention time, accurate mass, fragment ion(s) and isotopic pattern. A very sensitive broad toxicological screening method using positive electrospray ionization UPLC-HR-TOF-MS was achieved in one injection. This method covered basic substances, substances traditionally analyzed in negative ESI (e.g., salicylic acid), small highly polar substances such as beta- and gamma-hydroxybutyric acid (BHB and GHB, respectively) and highly non-polar substances such as amiodarone. The new method was shown to combine high sensitivity with a very broad scope that has not previously been reported in toxicological whole blood screening when using only one injection.

  7. Brain Basics

    Medline Plus

    Full Text Available ... that contains codes to make proteins and other important body chemicals. DNA also includes information to control ... cells required for normal function and plays an important role during early brain development. It may also ...

  8. Basic electronics

    CERN Document Server

    Tayal, DC

    2010-01-01

    The second edition of this book incorporates the comments and suggestions of my friends and students who have critically studied the first edition. In this edition the changes and additions have been made and subject matter has been rearranged at some places. The purpose of this text is to provide a comprehensive and up-to-date study of the principles of operation of solid state devices, their basic circuits and application of these circuits to various electronic systems, so that it can serve as a standard text not only for universities and colleges but also for technical institutes. This book

  9. Regression Basics

    CERN Document Server

    Kahane, Leo H

    2007-01-01

    Using a friendly, nontechnical approach, the Second Edition of Regression Basics introduces readers to the fundamentals of regression. Accessible to anyone with an introductory statistics background, this book builds from a simple two-variable model to a model of greater complexity. Author Leo H. Kahane weaves four engaging examples throughout the text to illustrate not only the techniques of regression but also how this empirical tool can be applied in creative ways to consider a broad array of topics. New to the Second Edition Offers greater coverage of simple panel-data estimation:

  10. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

    Science.gov (United States)

    Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

    2015-10-01

    Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

  11. Role of very-late antigen-4 (VLA-4) in myelin basic protein-primed T cell contact-induced expression of proinflammatory cytokines in microglial cells.

    Science.gov (United States)

    Dasgupta, Subhajit; Jana, Malabendu; Liu, Xiaojuan; Pahan, Kalipada

    2003-06-20

    The presence of neuroantigen-primed T cells recognizing self-myelin antigens within the CNS is necessary for the development of demyelinating autoimmune disease like multiple sclerosis. This study was undertaken to investigate the role of myelin basic protein (MBP)-primed T cells in the expression of proinflammatory cytokines in microglial cells. MBP-primed T cells alone induced specifically the microglial expression of interleukin (IL)-1beta, IL-1alpha tumor necrosis factor alpha, and IL-6, proinflammatory cytokines that are primarily involved in the pathogenesis of MS. This induction was primarily dependent on the contact between MBP-primed T cells and microglia. The activation of microglial NF-kappaB and CCAAT/enhancer-binding protein beta (C/EBPbeta) by MBP-primed T cell contact and inhibition of contact-mediated microglial expression of proinflammatory cytokines by dominant-negative mutants of p65 and C/EBPbeta suggest that MBP-primed T cells induce microglial expression of cytokines through the activation of NF-kappaB and C/EBPbeta. In addition, we show that MBP-primed T cells express very late antigen-4 (VLA-4), and functional blocking antibodies to alpha4 chain of VLA-4 (CD49d) inhibited the ability of MBP-primed T cells to induce microglial proinflammatory cytokines. Interestingly, the blocking of VLA-4 impaired the ability of MBP-primed T cells to induce microglial activation of only C/EBPbeta but not that of NF-kappaB. This study illustrates a novel role of VLA-4 in regulating neuroantigen-primed T cell-induced activation of microglia through C/EBPbeta

  12. Functional interconnection of MYC2 and SPA1 in the photomorphogenic seedling development of Arabidopsis.

    Science.gov (United States)

    Gangappa, Sreeramaiah N; Prasad, V Babu Rajendra; Chattopadhyay, Sudip

    2010-11-01

    MYC2 is a basic helix-loop-helix transcription factor that cross talks with light, abscisic acid (ABA), and jasmonic acid (JA) signaling pathways. Here, we have shown that Arabidopsis (Arabidopsis thaliana) MYC2 directly binds to the G-box present in the SUPPRESSOR OF PHYTOCHROME A1 (SPA1) promoter and that it controls the expression of SPA1 in a COP1-dependent manner. Analyses of atmyc2 spa1 double mutants suggest that whereas MYC2 and SPA1 act redundantly to suppress photomorphogenic growth in the dark, they function synergistically for the suppression of photomorphogenic growth in the light. Our studies have also revealed that MYC2-mediated ABA and JA responses are further modulated by SPA1. Taken together, this study demonstrates the molecular and physiological interrelations of MYC2 and SPA1 in light, ABA, and JA signaling pathways.

  13. Transcriptional control of GABAergic neuron development in the dorsal spinal cord

    Institute of Scientific and Technical Information of China (English)

    Huang Jing; Wu Shengxi

    2008-01-01

    GABAergic neurons are the major inhibitory interneurons that powerfully control the function of spinal neuronalnet works. In recent years, tremendous progresses have been made in understanding the transcriptional control of GABAergic neuron development in the dorsal spinal cord. New experimental approaches provide a relatively high throughput way to study the molecular regulation of subgroup fate determination. Our understanding of the molecular mechanisms on GABAergic neuron development in the dorsal spinal cord is rapidly expanding. Recent studies have defined several transcription factors that play essential roles in GABAergic neuron development in the spinal dorsal horn. Here, we review results of very recent analyses of the mechanisms that specify the GABAergic neuron development in the dorsal spinal cord, especially the progresses in the homeodomain (HD) and basic-helix-loop-helix(bHLH) containing transcription factors.

  14. Network theory inspired analysis of time-resolved expression data reveals key players guiding P. patens stem cell development.

    Science.gov (United States)

    Busch, Hauke; Boerries, Melanie; Bao, Jie; Hanke, Sebastian T; Hiss, Manuel; Tiko, Theodhor; Rensing, Stefan A

    2013-01-01

    Transcription factors (TFs) often trigger developmental decisions, yet, their transcripts are often only moderately regulated and thus not easily detected by conventional statistics on expression data. Here we present a method that allows to determine such genes based on trajectory analysis of time-resolved transcriptome data. As a proof of principle, we have analysed apical stem cells of filamentous moss (P. patens) protonemata that develop from leaflets upon their detachment from the plant. By our novel correlation analysis of the post detachment transcriptome kinetics we predict five out of 1,058 TFs to be involved in the signaling leading to the establishment of pluripotency. Among the predicted regulators is the basic helix loop helix TF PpRSL1, which we show to be involved in the establishment of apical stem cells in P. patens. Our methodology is expected to aid analysis of key players of developmental decisions in complex plant and animal systems.

  15. Feedback regulation of NEUROG2 activity by MTGR1 is required for progression of neurogenesis.

    Science.gov (United States)

    Aaker, Joshua D; Patineau, Andrea L; Yang, Hyun-Jin; Ewart, David T; Gong, Wuming; Li, Tongbin; Nakagawa, Yasushi; McLoon, Steven C; Koyano-Nakagawa, Naoko

    2009-12-01

    The sequential steps of neurogenesis are characterized by highly choreographed changes in transcription factor activity. In contrast to the well-studied mechanisms of transcription factor activation during neurogenesis, much less is understood regarding how such activity is terminated. We previously showed that MTGR1, a member of the MTG family of transcriptional repressors, is strongly induced by a proneural basic helix-loop-helix transcription factor, NEUROG2 in developing nervous system. In this study, we describe a novel feedback regulation of NEUROG2 activity by MTGR1. We show that MTGR1 physically interacts with NEUROG2 and represses transcriptional activity of NEUROG2. MTGR1 also prevents DNA binding of the NEUROG2/E47 complex. In addition, we provide evidence that proper termination of NEUROG2 activity by MTGR1 is necessary for normal progression of neurogenesis in the developing spinal cord. These results highlight the importance of feedback regulation of proneural gene activity in neurodevelopment.

  16. RNA profiling and chromatin immunoprecipitation-sequencing reveal that PTF1a stabilizes pancreas progenitor identity via the control of MNX1/HLXB9 and a network of other transcription factors

    DEFF Research Database (Denmark)

    Thompson, Nancy; Gésina, Emilie; Scheinert, Peter;

    2012-01-01

    Pancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion, but their interactions and the downstream targets mediating their activity are poorly understood. Among...... those factors, PTF1a, a basic helix-loop-helix (bHLH) transcription factor which controls pancreas exocrine cell differentiation, maintenance, and functionality, is also needed for the early specification of pancreas progenitors. We used RNA profiling and chromatin immunoprecipitation (ChIP) sequencing...... to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1, and Onecut1 are also direct PTF1a targets whose expression is promoted...

  17. Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene.

    Science.gov (United States)

    Tassabehji, M; Newton, V E; Read, A P

    1994-11-01

    Waardenburg syndrome type 2 (WS2) is a dominantly inherited syndrome of hearing loss and pigmentary disturbances. We recently mapped a WS2 gene to chromosome 3p12.3-p14.1 and proposed as a candidate gene MITF, the human homologue of the mouse microphthalmia (mi) gene. This encodes a putative basic-helix-loop-helix-leucine zipper transcription factor expressed in adult skin and in embryonic retina, otic vesicle and hair follicles. Mice carrying mi mutations show reduced pigmentation of the eyes and coat, and with some alleles, microphthalmia, hearing loss, osteopetrosis and mast cell defects. Here we show that affected individuals in two WS2 families have mutations affecting splice sites in the MITF gene.

  18. Iron assimilation and transcription factor controlled synthesis of riboflavin in plants.

    Science.gov (United States)

    Vorwieger, A; Gryczka, C; Czihal, A; Douchkov, D; Tiedemann, J; Mock, H-P; Jakoby, M; Weisshaar, B; Saalbach, I; Bäumlein, H

    2007-06-01

    Iron homeostasis is vital for many cellular processes and requires a precise regulation. Several iron efficient plants respond to iron starvation with the excretion of riboflavin and other flavins. Basic helix-loop-helix transcription factors (TF) are involved in the regulation of many developmental processes, including iron assimilation. Here we describe the isolation and characterisation of two Arabidopsis bHLH TF genes, which are strongly induced under iron starvation. Their heterologous ectopic expression causes constitutive, iron starvation independent excretion of riboflavin. The results show that both bHLH TFs represent an essential component of the regulatory pathway connecting iron deficiency perception and riboflavin excretion and might act as integrators of various stress reactions.

  19. Time to pump iron: iron-deficiency-signaling mechanisms of higher plants.

    Science.gov (United States)

    Walker, Elsbeth L; Connolly, Erin L

    2008-10-01

    Iron is an essential nutrient for plants, yet it often limits plant growth. On the contrary, overaccumulation of iron within plant cells leads to oxidative stress. As a consequence, iron-uptake systems are carefully regulated to ensure that iron homeostasis is maintained. In response to iron limitation, plants induce expression of sets of activities that function at the root-soil interface to solubilize iron and subsequently transfer it across the plasma membrane of root cells. Recent advances have revealed key players in the signaling pathways that function to induce these iron-uptake responses. Transcription factors belonging to the basic helix-loop-helix, ABI3/VP1(B3), and NAC families appear to function either directly or indirectly in the upregulation of iron deficiency responses.

  20. The Transcriptional Coregulator LEUNIG_HOMOLOG Inhibits Light-Dependent Seed Germination in Arabidopsis.

    Science.gov (United States)

    Lee, Nayoung; Park, Jeongmoo; Kim, Keunhwa; Choi, Giltsu

    2015-08-01

    PHYTOCHROME-INTERACTING FACTOR1 (PIF1) is a basic helix-loop-helix transcription factor that inhibits light-dependent seed germination in Arabidopsis thaliana. However, it remains unclear whether PIF1 requires other factors to regulate its direct targets. Here, we demonstrate that LEUNIG_HOMOLOG (LUH), a Groucho family transcriptional corepressor, binds to PIF1 and coregulates its targets. Not only are the transcriptional profiles of the luh and pif1 mutants remarkably similar, more than 80% of the seeds of both genotypes germinate in the dark. We show by chromatin immunoprecipitation that LUH binds a subset of PIF1 targets in a partially PIF1-dependent manner. Unexpectedly, we found LUH binds and coregulates not only PIF1-activated targets but also PIF1-repressed targets. Together, our results indicate LUH functions with PIF1 as a transcriptional coregulator to inhibit seed germination.

  1. A lack of association between hyperserotonemia and the increased frequency of serum anti-myelin basic protein auto-antibodies in autistic children

    Directory of Open Access Journals (Sweden)

    AL-Ayadhi Laila

    2011-06-01

    Full Text Available Abstract Background One of the most consistent biological findings in autism is the elevated blood serotonin levels. Immune abnormalities, including autoimmunity with production of brain specific auto-antibodies, are also commonly observed in this disorder. Hyperserotonemia may be one of the contributing factors to autoimmunity in some patients with autism through the reduction of T-helper (Th 1-type cytokines. We are the first to investigate the possible role of hyperserotonemia in the induction of autoimmunity, as indicated by serum anti-myelin-basic protein (anti-MBP auto-antibodies, in autism. Methods Serum levels of serotonin and anti-MBP auto-antibodies were measured, by ELISA, in 50 autistic patients, aged between 5 and 12 years, and 30 healthy-matched children. Results Autistic children had significantly higher serum levels of serotonin and anti-MBP auto-antibodies than healthy children (P Conclusions Hyperserotonemia may not be one of the contributing factors to the increased frequency of serum anti-MBP auto-antibodies in some autistic children. These data should be treated with caution until further investigations are performed. However, inclusion of serum serotonin levels as a correlate may be useful in other future immune studies in autism to help unravel the long-standing mystery of hyperserotonemia and its possible role in the pathophysiology of this disorder.

  2. Effect of nerve growth factor on changes of myelin basic protein and functional repair of peripheral nerve following sciatic nerve injury in rats

    Institute of Scientific and Technical Information of China (English)

    邵阳; 马海涵; 伍亚民; 陈恒胜; 曾琳; 李民; 龙在云; 李应玉; 杨恒文

    2002-01-01

    To investigate the therapeutic effect of nerve growth factor ( NGF ) on changes of myelin basic protein (MBP) and functional repair of sensory and motor nerve following sciatic nerve injury. Methods: The sciatic nerves of rats were injured by sectioning with shaver, and divided into 3 groups: NGF group ( Group A ), group of normal saline solution ( Group B), untreated group (Group C). The time point of observation was at the 4th week after operation. Sensory evoked potential (SEP) and motor evoked potential (MEP) were detected by Model WD-4000 nerve potential working diagnosis system. Immunohistochemical analysis was used for identification of MBP. Results: The latency of SEP in the Group A at the 4th week after operation was shorter than that in the Group B ( P < 0.05). The MEP was elicited in 76 % of the Group A and was higher than that in the Group B. Results of immunohistochemistry showed that there were less MBP-positive cells in the Group A than in the Group B in one and four weeks respectively. Conclusions: NGF can improve the conductive function of injured peripheral nerve and facilitate regeneration of nerve.

  3. Psychiatric disorder in a familial 15;18 translocation and sublocalization of myelin basic protein to 18q22.3

    Energy Technology Data Exchange (ETDEWEB)

    Calzolari, E.; Aiello, V.; Palazzi, P.; Sensi, A. [Universita Ferrara (Italy)] [and others

    1996-04-09

    Two related patients with similar clinical features consisting of a few dysmorphic signs and psychiatric disturbance were reported to have a partial trisomy of chromosomes 15(pter-q13.3) and 18(q23-qter) deriving from a familial translocation t(15;18). One patient is affected by bipolar disorder and the other by schizoaffective disorder. Both cases have a predominantly affective course; nevertheless, a clear diagnosis is difficult in the first patient, who is 15 years of age, and only a longitudinal course will allow us to establish a definite diagnosis. The possibility that these two pathologies belong to a single category is discussed, and the presence of a susceptibility locus on chromosome 18 is hypothesized. Cytogenetic data, FISH, and DNA studies indicate that the myelin basic protein (MPB) gene is not involved in the translocation, and localize it centromeric to the breakpoint on chromosome 18(q22.3). Thus, it is unlikely to be involved in the disease. 58 refs., 8 figs.

  4. Ameliorative effects of human adipose tissue-derived mesenchymal stem cells on myelin basic protein-induced experimental autoimmune encephalomyelitis in Lewis rats

    Institute of Scientific and Technical Information of China (English)

    Myung-Soon Ko; Hyeong-geun Park; Young-Min Yun; Jeong Chan Ra; Taekyun Shin; Kyoung-Kap Lee

    2011-01-01

    Mesenchymal stem cells have been previously shown to exert an immunomodulatory function. The present study sought to investigate the effects of multipotential human adipose tissue-derived mesenchymal stem cells (hAdMSCs) on disease progression and cytokine expression in Lewis rats with experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein. The duration of EAE paralysis in the group treated on day 7 postimmunization with 5 × 106 hAdMSCs was significantly reduced compared with the vehicle-treated controls and the 1 × 106 hAdMSC- treated group. The duration of EAE paralysis in the groups treated with 5 × 106 hAdMSCs on both day 1 and day 7 postimmunization was significantly reduced compared with the vehicle-treated controls and the groups treated with 5 × 106 hAdMSCs on both day 7 and day 10 postimmunization. The mRNA expression of interleukin-10 and indoleamine 2, 3-dioxygenase was significantly decreased in the hAdMSC-treated group compared with the vehicle-treated group. These findings suggest that the ameliorative effects of hAdMSCs on EAE symptoms operate in a dose- and time-dependent manner and can be mediated in part by the ample production of anti-inflammatory cytokines.

  5. Brain Basics

    Medline Plus

    Full Text Available ... life," contains all the information inherited from our parents that helps to define who we are, such as our looks and certain abilities, such as a good singing voice. A gene is a segment of DNA that contains codes to make proteins and other important body chemicals. DNA also ...

  6. emc has a role in dorsal appendage fate formation in Drosophila oogenesis.

    Science.gov (United States)

    Papadia, Sofia; Tzolovsky, George; Zhao, Debiao; Leaper, Kevin; Clyde, Dorothy; Taylor, Paul; Asscher, Eva; Kirk, Graeme; Bownes, Mary

    2005-09-01

    extramacrochaetae (emc) functions during many developmental processes in Drosophila, such as sensory organ formation, sex determination, wing vein differentiation, regulation of eye photoreceptor differentiation, cell proliferation and development of the Malpighian tubules, trachea and muscles in the embryo. It encodes a Helix-Loop-Helix transcription factor that negatively regulates bHLH proteins. We show here that emc mRNA and protein are present throughout oogenesis in a dynamic expression pattern and that emc is involved in the regulation of chorionic appendage formation during late oogenesis. Expression of sense and antisense emc constructs as well as emc follicle cell clones leads to eggs with shorter, thicker dorsal appendages that are closer together at base than in the wild type. We demonstrate that emc lies downstream of fs(1)K10, gurken and EGFR in the Grk/EGFR signalling pathway and that it participates in controlling Broad-Complex expression at late stages of oogenesis.

  7. Biochemical characterization of novel lignans isolated from the wood of Taxus yunnanensis as effective stimulators for glycogen synthase kinase-3β and the phosphorylation of basic brain proteins by the kinase in vitro.

    Science.gov (United States)

    Ohtsuki, Kenzo; Miyai, Sayaka; Yamaguchi, Akira; Morikawa, Kouhei; Okano, Tetsuroh

    2012-01-01

    The stimulatory and inhibitory effects of several compounds and lignans isolated from the water extract of Taxus yunnanensis on the phosphorylation of three functional brain proteins (bovine myelin basic protein (bMBP), recombinant human tau protein (rhTP) and rat collapsin response mediator protein-2 (rCRMP-2)) by glycogen synthase kinase-3β (GSK-3β) were quantitatively compared in vitro, using (-)-epigallocatechin-3-gallate [(-)EGCG] as a positive control. We found that (i) three selected Taxus lignans [(3S,4R)-4'-hydroxy-6,3'-dimethoxyisoflavan-4-ol,(7R)-7-hydroxytaxiresinol and tanegool] highly stimulated the autophosphorylation of GSK-3β and the GSK-3β-mediated phosphorylation of two basic brain proteins [bMBP (pI=11.3) and rhTP (pI=8.2)], but inhibited dose-dependently the phosphorylation of an acidic protein (rCRMP-2, pI=6.0) by the kinase; (ii) these three Taxus lignans showed binding-affinities with bMBP as well as rhTP, but had low affinities with rCRMP-2; (iii) the binding of tanegool and (7R)-7-hydroxytaxiresinol to these two basic proteins induced their novel potent phosphorylation sites for GSK-3β; and (iv) these three Taxus lignans, but not EGCG, induced Tyr-phosphorylation of GSK-3β in vitro. These results provided here suggest that (i) these three Taxus lignans act as novel effective activators for GSK-3β and the GSK-3β-mediated phosphorylation of their binding basic proteins (rhTP and bMBP); and (ii) tanegool (IC(50)=1 μM) is an effective inhibitor for the phosphorylation of rCRMP-2 by the kinase in vitro.

  8. 细胞功能调控的重要转录因子TFEB%Function and regulation of the transcription factor TFEB

    Institute of Scientific and Technical Information of China (English)

    陈太琪; 姜丛; 王平; 刘宁

    2013-01-01

    TFEB is a member of the MiTF/TFE (microphthalmia-transcription factor E) subfamily of bHLH-LZ (basic-helix-loop-helix leucine-zipper) factors. And TFEB-related pathway is involved in some of cellular physiological processes and its regulating aberration has been known to contribute to the pathogenesis of several human diseases, such as placenta angiogenesis and renal cell carcinoma. Recent studies have shown that TFEB could regulate autophagy and lysosome function through regulating the expression of the related genes. Future study on the function and mechanism of TFEB will help better understand the pathological process and provide new theory basis and clues for the treatment of TFEB-related diseases.%转录因子TFEB (transcription factor EB)属于亮氨酸拉链bHLH-LZ (basic-helix-loop-helix leucine-zipper)类转录因子中的MiTF/TFE (microphthalmia-transcription factor E)家族成员,参与调控许多重要的细胞生理过程,例如胎盘血管新生、肾癌的发生等.最近研究表明,TFEB能通过调控细胞自噬和溶酶体相关的基因表达而调控细胞自噬以及溶酶体功能.因此,对于TFEB的生物学功能及其相关调控机制的研究,将为进一步阐释其生理病理发生过程及相关疾病的治疗提供重要的线索及理论依据.

  9. Inflation Basics

    Energy Technology Data Exchange (ETDEWEB)

    Green, Dan [Fermi National Accelerator Lab. (FNAL), Batavia, IL (United States)

    2014-03-01

    inflation since metrical fluctuations, both scalar and tensor, are also produced in inflationary models. Thus, the time appears to be appropriate for a very basic and simple exposition of the inflationary model written from a particle physics perspective. Only the simplest scalar model will be explored because it is easy to understand and contains all the basic elements of the inflationary model.

  10. Inflation Basics

    Energy Technology Data Exchange (ETDEWEB)

    Green, Dan [Fermi National Accelerator Lab. (FNAL), Batavia, IL (United States)

    2014-03-01

    inflation since metrical fluctuations, both scalar and tensor, are also produced in inflationary models. Thus, the time appears to be appropriate for a very basic and simple exposition of the inflationary model written from a particle physics perspective. Only the simplest scalar model will be explored because it is easy to understand and contains all the basic elements of the inflationary model.

  11. Computational modeling of the bHLH domain of the transcription factor TWIST1 and R118C, S144R and K145E mutants

    Directory of Open Access Journals (Sweden)

    Maia Amanda M

    2012-07-01

    Full Text Available Abstract Background Human TWIST1 is a highly conserved member of the regulatory basic helix-loop-helix (bHLH transcription factors. TWIST1 forms homo- or heterodimers with E-box proteins, such as E2A (isoforms E12 and E47, MYOD and HAND2. Haploinsufficiency germ-line mutations of the twist1 gene in humans are the main cause of Saethre-Chotzen syndrome (SCS, which is characterized by limb abnormalities and premature fusion of cranial sutures. Because of the importance of TWIST1 in the regulation of embryonic development and its relationship with SCS, along with the lack of an experimentally solved 3D structure, we performed comparative modeling for the TWIST1 bHLH region arranged into wild-type homodimers and heterodimers with E47. In addition, three mutations that promote DNA binding failure (R118C, S144R and K145E were studied on the TWIST1 monomer. We also explored the behavior of the mutant forms in aqueous solution using molecular dynamics (MD simulations, focusing on the structural changes of the wild-type versus mutant dimers. Results The solvent-accessible surface area of the homodimers was smaller on wild-type dimers, which indicates that the cleft between the monomers remained more open on the mutant homodimers. RMSD and RMSF analyses indicated that mutated dimers presented values that were higher than those for the wild-type dimers. For a more careful investigation, the monomer was subdivided into four regions: basic, helix I, loop and helix II. The basic domain presented a higher flexibility in all of the parameters that were analyzed, and the mutant dimer basic domains presented values that were higher than the wild-type dimers. The essential dynamic analysis also indicated a higher collective motion for the basic domain. Conclusions Our results suggest the mutations studied turned the dimers into more unstable structures with a wider cleft, which may be a reason for the loss of DNA binding capacity observed for in vitro

  12. ERT basics

    Energy Technology Data Exchange (ETDEWEB)

    Butters, M. [MBC Energy and Environment, Ottawa, ON (Canada)]|[National Round Table on the Environment and the Economy, Ottawa, ON (Canada)

    2002-07-01

    ERT is an economic instrument which helps power companies achieve emission reduction compliance cost-effectively. This paper presents the basics of ERT with reference to trading concepts, types of systems and types of emissions. The paper also describes the state of the Canadian energy market regarding greenhouse gases (GHG), nitrogen oxides, sulphur dioxide and volatile organic compounds. The association between ERT and district energy is also explained. By 2010, the global market for GHG trading is expected to be worth $10 billion to $3 trillion U.S. Canada has committed to reducing its GHG to 6 per cent below 1990 levels by 2012, but currently emits 705 Mt per year. This is expected to increase to 770 Mt by 2010. Therefore, in order to meet its commitment, GHGs will have to be reduced 200 Mt per year. Canada is currently considering ratifying the Kyoto agreement and a trading system is being developed. There are several abatement technologies currently under consideration for district energy systems, including adding scrubbers, improving efficiency, and fuel switching. The marginal cost of abatement was also discussed. tabs., figs.

  13. The effect of cytosolic extract of Alternaria aternata fungus on Monocyte-derived dendritic cell maturation and T-lymphocyte polarization in the presence of myelin basic protein

    Directory of Open Access Journals (Sweden)

    Loghmanni A

    2013-03-01

    Full Text Available Background: Multiple Sclerosis (MS is an autoimmune disease with impairment in function of central nervous system. Macrophages and dendritic cells play important roles in alleviating or progression of the disease. These cells can cause inflammation and damage to the myelin of nerve cells by realizing of harmful substances when these cells get matured. We studied the effect of Alternaria alternata extract on maturation of monocyte- derived dendritic cell (modc and T-cell responses in the presence of Myelin Basic Protein (MBP as a laboratory model of multiple sclerosis (MS. The purpose of this study is suitable dendritic cells production for usage in MS immunotherapy.Methods: For this study plastic adherent monocytes were cultured with granulocyte/ macrophage- colony stimulating factor (GM-CSF and interleukin -4 for converting these cells to modc and pulsed with MBP and matured in the presence of monocyte-conditioned medium (MCM in control group and MCM + Alternaria alternata extract in treatment groups. Anti-CD14, anti-CD83, anti-human leukocyte antigen-DR (anti HLA-DR monoclonal antibody were carried out for phenotyping. Autologos T cell responses and cytokine production were evaluated.Results: The results showed that the expression of CD14 decreased and CD83, HLA-DR increased in treatment groups in comparison with control groups. The production amount of IL-10 overcame IL-12 and in T cell the production of cytokines, IL-17 and Interferon-γ (IFN-γ decreased and IL-4 was increased (P<0.05. These effects escalated with increasing of dosage from 50 to 100 (mg/ml (P<0.001.Conclusion: Alternaria alternata extract can cause maturation of MBP-pulsed modc and skewing of T- lymphocyte toward Th2 and thereby can evolve into a new strategy in immunotherapy of MS.

  14. Differential effects of myelin basic protein-activated Th1 and Th2 cells on the local immune microenvironment of injured spinal cord.

    Science.gov (United States)

    Hu, Jian-Guo; Shi, Ling-Ling; Chen, Yue-Juan; Xie, Xiu-Mei; Zhang, Nan; Zhu, An-You; Jiang, Zheng-Song; Feng, Yi-Fan; Zhang, Chen; Xi, Jin; Lü, He-Zuo

    2016-03-01

    Myelin basic protein (MBP) activated T cells (MBP-T) play an important role in the damage and repair process of the central nervous system (CNS). However, whether these cells play a beneficial or detrimental role is still a matter of debate. Although some studies showed that MBP-T cells are mainly helper T (Th) cells, their subtypes are still not very clear. One possible explanation for MBP-T immunization leading to conflicting results may be the different subtypes of T cells are responsible for distinct effects. In this study, the Th1 and Th2 type MBP-T cells (MBP-Th1 and -Th2) were polarized in vitro, and their effects on the local immune microenvironment and tissue repair of spinal cord injury (SCI) after adoptive immunization were investigated. In MBP-Th1 cell transferred rats, the high levels of pro-inflammatory cells (Th1 cells and M1 macrophages) and cytokines (IFN-γ, TNF-α, -β, IL-1β) were detected in the injured spinal cord; however, the anti-inflammatory cells (Th2 cells, regulatory T cells, and M2 macrophages) and cytokines (IL-4, -10, and -13) were found in MBP-Th2 cell transferred animals. MBP-Th2 cell transfer resulted in decreased lesion volume, increased myelination of axons, and preservation of neurons. This was accompanied by significant locomotor improvement. These results indicate that MBP-Th2 adoptive transfer has beneficial effects on the injured spinal cord, in which the increased number of Th2 cells may alter the local microenvironment from one primarily populated by Th1 and M1 cells to another dominated by Th2, Treg, and M2 cells and is conducive for SCI repair.

  15. Effect of basic fibroblast growth factor on the expression of glial fibrillary acidic protein after tractive spinal cord injury in rats

    Institute of Scientific and Technical Information of China (English)

    LIU Lei; L(U) Bo; TU Chong-qi; CHI Lei-ting; WANG Guang-lin; PEI Fu-xing

    2005-01-01

    Objective: To investigate the effects of basic fibroblast growth factor (bFGF) on the expression of glial fibrillary acidic protein (GFAP) after tractive spinal cord injury in rats and to explore the recovery of spinal cord function.Methods: The rats were subjected to tractive spinal cord injury at T13-L2. Cortical somatosensory-evoked potential (CSEP) was closely monitored and when P1-N1 wave amplitude decreased to 70% of that before operation, a small-bore catheter was inserted below the injured plane through subarachnoid cavity. In the treatment groups, 20 μl of bFGF solution (containing 20 μg of bFGF) was injected through the catheter right after the operation and 1,2, 3, 4, 8, 12 and 24 h postoperatively. In the control group, same volume of normal saline was injected and every four rats were killed at 1, 4, 7, 14 and 21 d after the operation. Combined behavior score (CBS) and electro-physiological examination were adopted to evaluate function recovery. Expression of GFAP was observed by immuno-histochemical staining and was analyzed quantitatively by computer image analysis.Results: There was statistically significant difference in GFAP-positive cells between bFGF treatment group and the control group (P<0.01). Similar tendency was indicated by the results of CBS and CSEP.Conclusions: bFGF can induce large expression of GFAP after tractive spinal cord injury in rats and promote spinal function recovery, which is highly important for spinal cord regeneration.

  16. Epitope specificity and V gene expression of cerebrospinal fluid T cells specific for intact versus cryptic epitopes of myelin basic protein.

    Science.gov (United States)

    Satyanarayana, K; Chou, Y K; Bourdette, D; Whitham, R; Hashim, G A; Offner, H; Vandenbark, A A

    1993-04-01

    Recent evidence supports the possible involvement of myelin basic protein (BP) as one of the target autoantigens in multiple sclerosis (MS), including elevated frequencies of MS blood and cerebrospinal fluid (CSF) T cells, and the presence in MS plaque tissue of V beta gene sequences and CDR3 motifs characteristic of BP-reactive T cells. Because of its proximity to the target organ, the CSF has long been thought to harbor T cells involved in the pathogenic process. In order to evaluate their frequency and response characteristics, BP-reactive T cells were isolated by limiting dilution from the CSF of patients with MS and other neurological diseases (OND) for quantitation and determination of epitope specificity and V alpha and V beta gene expression. In addition to isolates responsive to intact BP epitopes that were present at a significantly higher frequency in MS versus OND CSF, we here describe a second clonotype responsive to 'cryptic' BP epitopes that is present at approximately equal frequencies in MS and OND patients. In spite of their difference in recognition of intact versus 'cryptic' BP determinants, both clonotypes predominantly recognized epitopes in the N terminal half of human BP, using a similar V gene repertoire that included biased use of V alpha 2 and to a lesser degree V beta 7 and V beta 18. These V gene biases were not related to the epitope specificity of the T cells, indicating that V gene selection is not epitope-driven. These data suggest that there is differential recognition of intact versus 'cryptic' BP determinants in MS versus OND patients that may be related to the processing and presentation of BP to the immune system.

  17. Evidence for a Role of the Transcriptional Regulator Maid in Tumorigenesis and Aging.

    Directory of Open Access Journals (Sweden)

    Koichi Fujisawa

    Full Text Available Maid is a helix-loop-helix protein that is involved in cell proliferation. In order to further elucidate its physiological functions, we studied Maid activity in two small fish model systems. We found that Maid expression was greatest in zebrafish liver and that it increased following partial hepatectomy. Maid levels were also high in hepatic preneoplastic foci induced by treatment of zebrafish with diethylnitrosamine (DEN, but low in hepatocellular carcinomas (HCC, mixed tumors, and cholangiocarcinomas developing in these animals. In DEN-treated transgenic medaka overexpressing Maid, hepatic BrdU uptake and proliferation were reduced. After successive breedings, Maid transgenic medaka exhibited decreased movement and a higher incidence of abnormal spine curvature, possibly due to the senescence of spinal cord cells. Taken together, our results suggest that Maid levels can influence the progression of liver cancer. In conclusion, we found that Maid is important regulator of hepatocarconogenesis and aging.

  18. Water soluble chlorophyll binding protein of higher plants: a most suitable model system for basic analyses of pigment-pigment and pigment-protein interactions in chlorophyll protein complexes.

    Science.gov (United States)

    Renger, G; Pieper, J; Theiss, C; Trostmann, I; Paulsen, H; Renger, T; Eichler, H J; Schmitt, F-J

    2011-08-15

    This short review paper describes spectroscopic studies on pigment-pigment and pigment-protein interactions of chlorophyll (Chl) a and b bound to the recombinant protein of class IIa water soluble chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl-protein complexes. Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets. Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments. The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.

  19. Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.

    Science.gov (United States)

    Yousfi, Malika; Lasmoles, Francoise; El Ghouzzi, Vincent; Marie, Pierre J

    2002-02-15

    Saethre-Chotzen syndrome (SCS) is a human autosomal dominant disorder characterized by premature fusion of cranial sutures caused by mutations of the Twist gene encoding a basic helix-loop-helix (bHLH) transcription factor. We previously showed that Twist haploinsufficiency caused by a Y103X nonsense mutation in SCS alters both proliferation and osteoblast gene expression in human calvarial osteoblasts, indicating that Twist is an important regulator of osteoblast differentiation. Here we show that Twist haploinsufficiency alters osteoblast apoptosis in SCS. Analysis of terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) demonstrated increased osteoblast and osteocyte apoptosis in coronal sutures from two SCS patients with nonsense mutations (Y103X and Q109X) that result in the synthesis of bHLH-truncated proteins, and one patient with a missense mutation in the basic domain (R118C) that abolishes Twist DNA binding. To assess the mechanisms involved, we studied osteoblast apoptosis in mutant (M-Tw) calvarial cells bearing the Y103X mutation resulting in decreased Twist mRNA and protein levels. M-Tw cells cultured in low serum conditions showed enhanced DNA fragmentation compared to normal (Nl) age-matched calvarial cells. Biochemical analysis showed increased activity of initiator caspases-2 and -8 and downstream effector caspases-3, -6 and -7 in mutant osteoblasts. Caspase-2 was upstream of caspase-8 and effector caspases-3, -6 and -7 because their activities were suppressed by a specific caspase-2 inhibitor. M-Tw osteoblasts also showed increased cytochrome c release from the mitochondria. However, the activity of the downstream effector caspase-9 was not increased due to overexpression of the antagonist protein Hsp70. Detection of differentially expressed genes using cDNA expression array revealed increased Bax and TNFalpha mRNA levels in M-Tw compared to Nl cells, a finding confirmed by RT-PCR and western blot analyses. Neutralization of

  20. Myelin basic protein as a novel genetic risk factor in rheumatoid arthritis--a genome-wide study combined with immunological analyses.

    Directory of Open Access Journals (Sweden)

    Chikashi Terao

    Full Text Available Rheumatoid arthritis (RA is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2, followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4. SNPs showing p<0.005 in the first two collections and p<10(-4 by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP at chromosome 18q23 showed strong association with RA (p = 2.7×10(-8, OR 1.23, 95% CI: 1.14-1.32. The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001. We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001 and patients with other connective tissue disorders (p<0.001. ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease.

  1. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4.

    Science.gov (United States)

    Wu, Peï-Yu; Frit, Philippe; Meesala, SriLakshmi; Dauvillier, Stéphanie; Modesti, Mauro; Andres, Sara N; Huang, Ying; Sekiguchi, JoAnn; Calsou, Patrick; Salles, Bernard; Junop, Murray S

    2009-06-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  2. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    Energy Technology Data Exchange (ETDEWEB)

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

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

    Directory of Open Access Journals (Sweden)

    Biao eLai

    2016-02-01

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

  4. Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers

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    Luo Hongmei

    2011-12-01

    Full Text Available Abstract Background Panax notoginseng (Burk F.H. Chen is important medicinal plant of the Araliacease family. Triterpene saponins are the bioactive constituents in P. notoginseng. However, available genomic information regarding this plant is limited. Moreover, details of triterpene saponin biosynthesis in the Panax species are largely unknown. Results Using the 454 pyrosequencing technology, a one-quarter GS FLX titanium run resulted in 188,185 reads with an average length of 410 bases for P. notoginseng root. These reads were processed and assembled by 454 GS De Novo Assembler software into 30,852 unique sequences. A total of 70.2% of unique sequences were annotated by Basic Local Alignment Search Tool (BLAST similarity searches against public sequence databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG assignment discovered 41 unique sequences representing 11 genes involved in triterpene saponin backbone biosynthesis in the 454-EST dataset. In particular, the transcript encoding dammarenediol synthase (DS, which is the first committed enzyme in the biosynthetic pathway of major triterpene saponins, is highly expressed in the root of four-year-old P. notoginseng. It is worth emphasizing that the candidate cytochrome P450 (Pn02132 and Pn00158 and UDP-glycosyltransferase (Pn00082 gene most likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis were discovered from 174 cytochrome P450s and 242 glycosyltransferases by phylogenetic analysis, respectively. Putative transcription factors were detected in 906 unique sequences, including Myb, homeobox, WRKY, basic helix-loop-helix (bHLH, and other family proteins. Additionally, a total of 2,772 simple sequence repeat (SSR were identified from 2,361 unique sequences, of which, di-nucleotide motifs were the most abundant motif. Conclusion This study is the first to present a large-scale EST dataset for P. notoginseng root acquired by next

  5. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part C: Protein Synthesis and Post-Translational Processing in Eukaryotic Cells

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available The translation of mRNA constitutes the first step in the synthesis of a functional protein. The polypeptide chain is subsequently folded into the appropriate three-dimensional configuration and undergoes a variety of processing steps before being converted into its active form. These processing steps are intimately related to the cellular events that occur in the endoplasmic reticulum and Golgi compartments, and determine the sorting and transport of different proteins to their appropriate destinations within the cell. While the regulation of gene expression occurs primarily at the level of transcription, the expression of many genes can also be controlled at the level of translation. Most proteins can be regulated in response to extracellular signals. In addition, intracellular protein levels can be controlled by differential rates of protein degradation. Thus, the regulation of both the amounts and activities of intracellular proteins ultimately determines all aspects of cell behaviour.

  6. Conformational choreography of a molecular switch region in myelin basic protein--molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments.

    Science.gov (United States)

    Polverini, Eugenia; Coll, Eoin P; Tieleman, D Peter; Harauz, George

    2011-03-01

    The 18.5 kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5 kDa myelin basic protein (residues Glu80-Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80-Val91 and extended poly-proline type II from Thr92-Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the

  7. Primary structure of a 14 kDa basic structural protein (Lm-76) from the cuticle of the migratory locust, Locusta migratoria

    DEFF Research Database (Denmark)

    Andersen, Jens S.; Andersen, S O; Højrup, P

    1993-01-01

    The complete amino acid sequence of a 14 kDa structural protein (LM-76) isolated from pharate cuticle of the locust, Locusta migratoria, was determined by Edman degradation of the intact protein and enzymatically derived peptides. Plasma desorption and electrospray mass spectrometry was used...... region surrounded by two hydrophobic regions with 7 repeats of a (Tyr)-Ala-Ala-Pro-Ala/Val motif. The conservation around the prolyl residues within this sequence motif is demonstrated for the hitherto sequenced presumptive exocuticle proteins from L. migratoria. The N-terminal region of protein Lm-76...

  8. Transcriptomic analysis of the underground renewal buds during dormancy transition and release in 'Hangbaishao' peony (Paeonia lactiflora.

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    Jiaping Zhang

    Full Text Available Paeonia lactiflora is one of the most famous species of herbaceous peonies with gorgeous flowers. Bud dormancy is a crucial developmental process that allows P. lactiflora to survive unfavorable environmental conditions. However, little information is available on the molecular mechanism of the bud dormancy in P. lactiflora. We performed de novo transcriptome sequencing using the Illumina RNA sequencing platform for the underground renewal buds of P. lactiflora 'Hangbaishao' to study the molecular mechanism underlying its bud dormancy transition (the period from endodormancy to ecodormancy and release (the period from ecodormancy to bud elongation and sprouting. Approximately 300 million high-quality clean reads were generated and assembled into 207,827 (mean length = 828 bp and 51,481 (mean length = 1250 bp unigenes using two assembly methods named "Trinity" and "Trinity+PRICE", respectively. Based on the data obtained by the latter method, 32,316 unigenes were annotated by BLAST against various databases. Approximately 1,251 putative transcription factors were obtained, of which the largest number of unique transcripts belonged to the basic helix-loop-helix protein (bHLH transcription factor family, and five of the top ten highly expressed transcripts were annotated as dehydrin (DHN. A total of 17,705 simple sequence repeat (SSR motifs distributed in 13,797 sequences were obtained. The budbreak morphology, levels of indole-3-acetic acid (IAA and abscisic acid (ABA, and activities of guaiacol peroxidase (POD and catalase (CAT were observed. The expression of 20 interested unigenes, which annotated as DHN, heat shock protein (HSP, histone, late elongated hypocotyl (LHY, and phytochrome (PHY, and so on, were also analyzed. These studies were based on morphological, physiological, biochemical, and molecular levels and provide comprehensive insight into the mechanism of dormancy transition and release in P. lactiflora. Transcriptome dataset can be

  9. Phactr3/scapinin, a member of protein phosphatase 1 and actin regulator (phactr family, interacts with the plasma membrane via basic and hydrophobic residues in the N-terminus.

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    Akihiro Itoh

    Full Text Available Proteins that belong to the protein phosphatase 1 and actin regulator (phactr family are involved in cell motility and morphogenesis. However, the mechanisms that regulate the actin cytoskeleton are poorly understood. We have previously shown that phactr3, also known as scapinin, localizes to the plasma membrane, including lamellipodia and membrane ruffles. In the present study, experiments using deletion and point mutants showed that the basic and hydrophobic residues in the N-terminus play crucial roles in the localization to the plasma membrane. A BH analysis (http://helixweb.nih.gov/bhsearch is a program developed to identify membrane-binding domains that comprise basic and hydrophobic residues in membrane proteins. We applied this program to phactr3. The results of the BH plot analysis agreed with the experimentally determined region that is responsible for the localization of phactr3 to the plasma membrane. In vitro experiments showed that the N-terminal itself binds to liposomes and acidic phospholipids. In addition, we showed that the interaction with the plasma membrane via the N-terminal membrane-binding sequence is required for phactr3-induced morphological changes in Cos7 cells. The membrane-binding sequence in the N-terminus is highly conserved in all members of the phactr family. Our findings may provide a molecular basis for understanding the mechanisms that allow phactr proteins to regulate cell morphogenesis.

  10. Specificity of Baculorivus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation

    NARCIS (Netherlands)

    Wang, M.; Tuladhar, E.; Shen, S.; Wang, H.; Oers, van M.M.; Vlak, J.M.; Westenberg, M.

    2010-01-01

    The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 b

  11. Broadening Horizons and Teaching Basic Biology through Cell-Free Synthesis of Green Fluorescent Protein in a High School Laboratory Course

    Science.gov (United States)

    Albayrak, Cem; Jones, K. C.; Swartz, James R.

    2013-01-01

    Cell-free protein synthesis (CFPS) has emerged as a practical method for producing a broad variety of proteins. In addition, the direct accessibility to the reaction environment makes CFPS particularly suitable as a learning vehicle for fundamental biological concepts. Here, we describe its implementation as a teaching tool for a high school…

  12. UV-Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore.

    Science.gov (United States)

    Antosiewicz, Jan M; Shugar, David

    Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV-Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.

  13. Measurement and analysis of myelin basic protein and antibodies to myelin basic protein in serum and CSF of patients with heroin spongiform leukoencephalopathy%海洛因海绵状白质脑病的脑脊液和血清髓鞘碱性蛋白及其抗体检测及意义

    Institute of Scientific and Technical Information of China (English)

    尹恝; 陆兵勋; 李伟; 周亮

    2003-01-01

    目的通过对海洛因海绵状白质脑病(Heroin Spongiform Leukoencephalopathy,HSLE)患者血清和脑脊液髓鞘碱性蛋白(myelin basic protein,MBP)及其抗体(Anti-MBP)检测,探讨MBP与HSLE的关系.方法采用酶联免疫吸附法(ELISA)检测18例HSLE患者、23例Heroin成瘾者(吸毒但无HSLE临床症状者),17例多发性硬化患者(MS组)、对照组20例(NC组)的血清以及脑脊液(CSF)中MBP及Anti-MBP水平.结果 HSLE组、MS组CSF和血清MBP均值明显高于NC组和Heroin成瘾组(P0.05),Heroin成瘾组血清和CSF MBP均值与NC组间无统计学差异(P>0.05),4组血清和CSF的Anti-MBP含量差异不显著(P>0.05).结论 HSLE患者CSF及血清中MBP含量增高,MBP上升水平与髓鞘损伤程度有关,该病的髓鞘存在病理性损害,以及血脑屏障(blood- brain barrier,BBB)的通透性改变.MBP检测可作为HSLE诊断及与海洛因成瘾者鉴别诊断的重要参数,它在HSLE病理过程中的作用机制有待进一步研究.

  14. Could Intrathymic Injection of Myelin Basic Protein Suppress Inflammatory Response After Co-culture of T Lymphocytes and BV-2 Microglia Cells?

    Institute of Scientific and Technical Information of China (English)

    Zhan-Qun Cui; Bao-Long Liu; Qiao-Li Wu; Ying Cai; Wei-Jia Fan; Ming-Chao Zhang; Wei-Liang Ding

    2016-01-01

    Background:The interaction between activated microglia and T lymphocytes can yield abundant pro-inflammatory cytokines.Our previous study proved that thymus immune tolerance could alleviate the inflammatory response.This study aimed to investigate whether intrathymic injection of myelin basic protein (MBP) in mice could suppress the inflammatory response after co-culture ofT lymphocytes and BV-2 microglia cells.Methods:Totally,72 male C57BL/6 mice were randomly assigned to three groups (n =24 in each):Group A:intrathymic injection of 100 μl MBP (1 mg/ml);Group B:intrathymic injection of 100 μl phosphate-buffered saline (PBS);and Group C:sham operation group.Every eight mice in each group were sacrificed to obtain the spleen at postoperative days 3,7,and 14,respectively.T lymphocytes those were extracted and purified from the spleens were then co-cultured with activated BV-2 microglia cells at a proportion of 1:2 in the medium containing MBP for 3 days.After identified the T lymphocytes by CD3,surface antigens of T lymphocytes (CD4,CD8,CD152,and CD154) and BV-2 microglia cells (CD45 and CD54) were detected by flow cytometry.The expressions of pro-inflammatory factors ofBV-2 microglia cells (interleukin [IL]-1β,tumor necrosis factor-α [TNF-α],and inducible nitric oxide synthase [iNOS]) were detected by quantitative real-time polymerase chain reaction (PCR).One-way analysis of variance (ANOVA) and the least significant difference test were used for data analysis.Results:The levels of CD152 in Group A showed an upward trend from the 3rd to 7th day,with a downward trend from the 7th to 14th day (20.12 ± 0.71%,30.71 ± 1.14%,13.50 ± 0.71% at postoperative days 3,7,and 14,respectively,P < 0.05).The levels of CD154 in Group A showed a downward trend from the 3rd to 7th day,with an upward trend from the 7th to 14th day (10.00 ± 0.23%,5.28 ± 0.69%,14.67 ± 2.71% at postoperative days 3,7,and 14,respectively,P < 0.05).The ratio ofCD4+/CD8 + T in Group

  15. Contents of myelin-basic protein and S-100 in serum and brain tissue of neonatal rats with intrauterine infection-caused brain injury

    Institute of Scientific and Technical Information of China (English)

    Xiaojie Li; Hongying Li; Zhihai Lu

    2006-01-01

    BACKGROUND: The change of the content of myelin basic protein (MBP) in serum and brain tissue is the bio chemical diadynamic index of amyelination. S-100 is a specific and sensitive marker of central nervous system (CNS) injury. Whether or not the content of S-100 and MBP in blood and brain tissue can be used as the quan titative index for early diagnosing the intrauterine infection-caused brain injury still needs investigation. OBJECTIVE: To observe whether or not MBP and S-100 detection can be used as the biochemical indexes for early diagnosing the intrauterine infection-caused brain injury. DESIGN: Randomized controlled animal experiment. SETTING: Laboratory of Pediatric Neuro-rehabilitation, Medical College of Rehabilitation, Jiamusi University. MATERIALS: Sixty female and thirty male common Wistar rats, weighing from 180 to 240 g, were provided by the Experimental Animal Center of Jiamusi University. Reagent: Lipopolysaccharide(LPS, serological type 055: B5, SIGMA Company of USA); MBP enzyme linked immunosobent assay (ELISA) immunoreagent kit (Preclinicai Recombination DNA Laboratory, Chengdu Huaxi Medical Center, Sichuan Province); S-100 ELISA immunoreagent kit ( Department of Physiology, the Fourth Military Medical University of Chinese PLA) and bovine serum albumin(Haitaike Biotechnology Co.,Ltd.).METHODS: This experiment was carried out in the Laboratory of Pediatric Neuro-Rehabilitation, Experimental Animal Center, Department of Pathology and Central Laboratory of Jiamusi University from July 2005 to March 2006. ① Preparation of models and grouping: The female and male rats were placed in one cage at 2: 1 at 17:00 o'clock. Vaginal smear was checked at 8:00 on the next morning. Sperm was found and 0 day of pregnancy was recorded. Pregnant rats were bred in another cage. The pregnant 47 rats were randomly divided into 2 groups: control group (n =10) and experimental group (n =37). The experimental pregnant rats were intraperitoneally injected with LPS

  16. Stem Cell Basics

    Science.gov (United States)

    ... Tips Info Center Research Topics Federal Policy Glossary Stem Cell Information General Information Clinical Trials Funding Information Current ... Basics » Stem Cell Basics I. Back to top Stem Cell Basics I. Introduction: What are stem cells, and ...

  17. Basics of SCI Rehabilitation

    Science.gov (United States)

    ... Donate Experts \\ The Basics of Spinal Cord Injury Rehabilitation Topics Adult Injuries Spinal Cord Injury 101 Spinal ... Injury 101 The Basics of Spinal Cord Injury Rehabilitation The Basics of Spinal Cord Injury Rehabilitation Preventing ...

  18. Organic cation transporter-mediated ergothioneine uptake in mouse neural progenitor cells suppresses proliferation and promotes differentiation into neurons.

    Directory of Open Access Journals (Sweden)

    Takahiro Ishimoto

    cellular proliferation via regulation of oxidative stress, and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action.

  19. Coronary heart disease-associated variation in TCF21 disrupts a miR-224 binding site and miRNA-mediated regulation.

    Directory of Open Access Journals (Sweden)

    Clint L Miller

    2014-03-01

    Full Text Available Genome-wide association studies (GWAS have identified chromosomal loci that affect risk of coronary heart disease (CHD independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3' untranslated region (3'-UTR of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3' UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β and platelet derived growth factor (PDGF signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in

  20. NPAS3 Regulates Transcription and Expression of VGF: Implications for Neurogenesis and Psychiatric Disorders

    Science.gov (United States)

    Yang, Dongxue; Zhang, Wenbo; Padhiar, Arshad; Yue, Yao; Shi, Yonghui; Zheng, Tiezheng; Davis, Kaspar; Zhang, Yu; Huang, Min; Li, Yuyuan; Sha, Li

    2016-01-01

    Neuronal PAS domain protein 3 (NPAS3) and VGF (VGF Nerve Growth Factor (NGF) Inducible) are important for neurogenesis and psychiatric disorders. Previously, we have demonstrated that NPAS3 regulates VGF at the transcriptional level. In this study, VGF (non-acronymic) was found regulated by NPAS3 in neuronal stem cells. However, the underlying mechanism of this regulation remains unclear. The aim of this study was to explore the correlation of NPAS3 and VGF, and their roles in neural cell proliferation, in the context of psychiatric illnesses. First, we focused on the structure of NPAS3, to identify the functional domain of NPAS3. Truncated NPAS3 lacking transactivation domain was also found to activate VGF, which suggested that not only transactivation domain but other structural motifs were also involved in the regulation. Second, Mutated enhancer box (E-box) of VGF promoter showed a significant response to this basic helix-loop-helix (bHLH) transcription factor, which suggested an indirect regulatory mechanism for controlling VGF expression by NPAS3. κB site within VGF promoter was identified for VGF activation induced by NPAS3, apart from direct binding to E-box. Furthermore, ectopically expressed NPAS3 in PC12 cells produced parallel responses for nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB (P65)] expression, which specifies that NPAS3 regulates VGF through the NF-κB signaling pathway. Over-expression of NPAS3 also enhances the cell proliferation, which can be blocked by knockdown of VGF. Finally, NPAS3 was found to influence proliferation of neural cells through VGF. Therefore, downstream signaling pathways that are responsible for NPAS3-VGF induced proliferation via glutamate receptors were explored. Combining this work and published literature, a potential network composed by NPAS3, NF-κB, Brain-Derived Neurotrophic Factor (BDNF), NGF and VGF, was proposed. This network collectively detailed how NPAS3 connects with VGF and

  1. NPAS 3基因在精神分裂症中的研究进展

    Institute of Scientific and Technical Information of China (English)

    徐志忠; 章家新

    2014-01-01

    NPAS3 is a member of the basic helix-loop-helix family of transcription factors expressed in the brain. The gene is located at 14q13 and a reciprocal balanced translocation between chromosomes 9 and 14 was identified in schizophrenia. A number of behavioural abnormalities associated with schizophrenia were identified in Npas3-⁄ - mice including locomotor hyperactivity, subtle gait defects, impairment of prepulse inhibition of acoustic startle. Correlative evidence suggests that neurogenesis may play an important role in schizophrenia, NPAS3 gene may regulates the hippocampal neurogenesis through the FGF signal pathway. The transcription regulation mechanisms of NPAS3 protein expression level might be involved dominant negative, haploinsufficiency and posttranscriptional regulation.%NPAS3是一种主要存在于大脑中的转录因子,属于碱性螺旋-环-螺旋超家族。NPAS3基因位于14号染色体(14q13),在精神分裂症患者中发现,NPAS 3基因在染色体9与14间[t(9;14)(q 34;q 13)]具有平衡易位现象;NPAS 3基因敲除小鼠具有多动、步态有缺陷、前脉冲抑制功能减弱等行为活动,这与精神分裂症症状相类似;NPAS 3基因有可能通过FGF信号通路调节海马区的神经发生,神经发生在精神分裂症的发生发展中具有重要的作用;显性负效、单倍剂量不足效应、转录后调控等有可能是调节NPAS 3蛋白表达水平的不同机制。

  2. Multiple upstream modules regulate zebrafish myf5 expression

    Directory of Open Access Journals (Sweden)

    Weng Chih-Wei

    2007-01-01

    Full Text Available Abstract Background Myf5 is one member of the basic helix-loop-helix family of transcription factors, and it functions as a myogenic factor that is important for the specification and differentiation of muscle cells. The expression of myf5 is somite- and stage-dependent during embryogenesis through a delicate regulation. However, this complex regulatory mechanism of myf5 is not clearly understood. Results We isolated a 156-kb bacterial artificial chromosome clone that includes an upstream 80-kb region and a downstream 70-kb region of zebrafish myf5 and generated a transgenic line carrying this 156-kb segment fused to a green fluorescent protein (GFP reporter gene. We find strong GFP expression in the most rostral somite and in the presomitic mesoderm during segmentation stages, similar to endogenous myf5 expression. Later, the GFP signals persist in caudal somites near the tail bud but are down-regulated in the older, rostral somites. During the pharyngula period, we detect GFP signals in pectoral fin buds, dorsal rostral myotomes, hypaxial myotomes, and inferior oblique and superior oblique muscles, a pattern that also corresponds well with endogenous myf5 transcripts. To characterize the specific upstream cis-elements that regulate this complex and dynamic expression pattern, we also generated several transgenic lines that harbor various lengths within the upstream 80-kb segment. We find that (1 the -80 kb/-9977 segment contains a fin and cranial muscle element and a notochord repressor; (2 the -9977/-6213 segment contains a strong repressive element that does not include the notochord-specific repressor; (3 the -6212/-2938 segment contains tissue-specific elements for bone and spinal cord; (4 the -2937/-291 segment contains an eye enhancer, and the -2937/-2457 segment is required for notochord and myocyte expression; and (5 the -290/-1 segment is responsible for basal transcription in somites and the presomitic mesoderm. Conclusion We suggest

  3. Promoted neuronal differentiation after activation of alpha4/beta2 nicotinic acetylcholine receptors in undifferentiated neural progenitors.

    Directory of Open Access Journals (Sweden)

    Takeshi Takarada

    Full Text Available BACKGROUND: Neural progenitor is a generic term used for undifferentiated cell populations of neural stem, neuronal progenitor and glial progenitor cells with abilities for proliferation and differentiation. We have shown functional expression of ionotropic N-methyl-D-aspartate (NMDA and gamma-aminobutyrate type-A receptors endowed to positively and negatively regulate subsequent neuronal differentiation in undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of nicotinic acetylcholine receptor (nAChR by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains. METHODOLOGY/PRINCIPAL FINDINGS: Reverse transcription polymerase chain reaction analysis revealed mRNA expression of particular nAChR subunits in undifferentiated rat and mouse progenitors prepared before and after the culture with epidermal growth factor under floating conditions. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction activity at a concentration range of 1 µM to 1 mM without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells under adherent conditions. Both effects of nicotine were significantly prevented by the heteromeric α4β2 nAChR subtype antagonists dihydro-β-erythroidine and 4-(5-ethoxy-3-pyridinyl-N-methyl-(3E-3-buten-1-amine, but not by the homomeric α7 nAChR subtype antagonist methyllycaconitine, in murine progenitors. Sustained exposure to nicotine preferentially increased the expression of Math1 among different basic helix-loop-helix proneural genes examined. In undifferentiated progenitors from embryonic mice

  4. Biophysical characterization of the b-HLH-LZ of ΔMax, an alternatively spliced isoform of Max found in tumor cells: Towards the validation of a tumor suppressor role for the Max homodimers

    Science.gov (United States)

    Maltais, Loïka; Montagne, Martin; Bédard, Mikaël; Tremblay, Cynthia; Soucek, Laura

    2017-01-01

    It is classically recognized that the physiological and oncogenic functions of Myc proteins depend on specific DNA binding enabled by the dimerization of its C-terminal basic-region-Helix-Loop-Helix-Leucine Zipper (b-HLH-LZ) domain with that of Max. However, a new paradigm is emerging, where the binding of the c-Myc/Max heterodimer to non-specific sequences in enhancers and promoters drives the transcription of genes involved in diverse oncogenic programs. Importantly, Max can form a stable homodimer even in the presence of c-Myc and bind DNA (specific and non-specific) with comparable affinity to the c-Myc/Max heterodimer. Intriguingly, alterations in the Max gene by germline and somatic mutations or changes in the gene product by alternative splicing (e.g. ΔMax) were recently associated with pheochromocytoma and glioblastoma, respectively. This has led to the proposition that Max is, by itself, a tumor suppressor. However, the actual mechanism through which it exerts such an activity remains to be elucidated. Here, we show that contrary to the WT motif, the b-HLH-LZ of ΔMax does not homodimerize in the absence of DNA. In addition, although ΔMax can still bind the E-box sequence as a homodimer, it cannot bind non-specific DNA in that form, while it can heterodimerize with c-Myc and bind E-box and non-specific DNA as a heterodimer with high affinity. Taken together, our results suggest that the WT Max homodimer is important for attenuating the binding of c-Myc to specific and non-specific DNA, whereas ΔMax is unable to do so. Conversely, the splicing of Max into ΔMax could provoke an increase in overall chromatin bound c-Myc. According to the new emerging paradigm, the splicing event and the stark reduction in homodimer stability and DNA binding should promote tumorigenesis impairing the tumor suppressor activity of the WT homodimer of Max. PMID:28350847

  5. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  6. Targeted toxicological screening for acidic, neutral and basic substances in postmortem and antemortem whole blood using simple protein precipitation and UPLC-HR-TOF-MS

    DEFF Research Database (Denmark)

    Telving, Rasmus; Hasselstrøm, Jørgen Bo; Andreasen, Mette Findal

    2016-01-01

    -HR-TOF-MS was achieved in one injection. This method covered basic substances, substances traditionally analyzed in negative ESI (e.g., salicylic acid), small highly polar substances such as beta- and gamma-hydroxybutyric acid (BHB and GHB, respectively) and highly non-polar substances such as amiodarone. The new method......A broad targeted screening method based on broadband collision-induced dissociation (bbCID) ultra-performance liquid chromatography high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS) was developed and evaluated for toxicological screening of whole blood samples. The acidic, neutral...... was performed on spiked whole blood samples and authentic postmortem and antemortem whole blood samples. For most of the basic drugs, the established cut-off limits were very low, ranging from 0.25ng/g to 50ng/g. The established cut-off limits for most neutral and acidic drugs, were in the range from 50ng...

  7. Formation of high-molecular-weight angiotensinogen during pregnancy is a result of competing redox reactions with the proform of eosinophil major basic protein

    DEFF Research Database (Denmark)

    Kløverpris, Søren; Skov, Louise Lind; Glerup, Simon

    2013-01-01

    compared to monomeric AGT and the proMBP-AGT complex. Furthermore, we have used recombinant proteins to analyse the formation of the proMBP-PAPP-A and the proMBP-AGT complexes, and we demonstrate that they are competing reactions, depending on the same cysteine residue of proMBP, but differentially...... on the redox potential, potentially important for the relative amounts of the complexes in vivo. These findings may be important physiologically, since the biochemical properties of the proteins change as a consequence of complex formation....

  8. Basic Pentacysteine Proteins Repress Abscisic Acid Insensitive4 Expression via Direct Recruitment of the Polycomb-Repressive Complex 2 in Arabidopsis Root Development.

    Science.gov (United States)

    Mu, Ying; Zou, Meijuan; Sun, Xuwu; He, Baoye; Xu, Xiumei; Liu, Yini; Zhang, Lixin; Chi, Wei

    2017-01-30

    Plant transcription factors generally act in complex regulatory networks that function at multiple levels to govern plant developmental programs. Dissection of the interconnections among different classes of transcription factors can elucidate these regulatory networks and thus improve our understanding of plant development. Here, we investigated the molecular and functional relationships of the transcription factors ABSCISIC ACID INSENSITIVE 4 (ABI4) and members of the BASIC PENTACYSTEINE (BPC) family in lateral root (LR) development of Arabidopsis thaliana Genetic analysis showed that BPCs promote LR development by repressing ABI4 expression. Molecular analysis showed that BPCs bind to the ABI4 promoter and repress ABI4 transcription in roots. BPCs directly recruit the Polycomb Repressive Complex 2 (PRC2) to the ABI4 locus and epigenetically repress ABI4 expression by catalyzing the trimethylation of histone H3 at lysine 27. In addition, BPCs and ABI4 coordinate their activities to fine-tune the levels of PIN-FORMED1, a component of the auxin signaling pathway, and thus modulate LR formation. These results establish a functional relationship between two universal and multiple-role transcription factors and provide insight into the mechanisms of the transcriptional regulatory networks that affect Arabidopsis organogenesis.

  9. Multiple sites of the cleavage of 21- and 25-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP by specific anti-MBP antibodies from patients with systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Anna M Timofeeva

    Full Text Available IgGs from patients with multiple sclerosis and systemic lupus erythematosus (SLE purified on MBP-Sepharose in contrast to canonical proteases hydrolyze effectively only myelin basic protein (MBP, but not many other tested proteins. Here we have shown for the first time that anti-MBP SLE IgGs hydrolyze nonspecific tri- and tetrapeptides with an extreme low efficiency and cannot effectively hydrolyze longer 20-mer nonspecific oligopeptides corresponding to antigenic determinants (AGDs of HIV-1 integrase. At the same time, anti-MBP SLE IgGs efficiently hydrolyze oligopeptides corresponding to AGDs of MBP. All sites of IgG-mediated proteolysis of 21-and 25-mer encephalytogenic oligopeptides corresponding to two known AGDs of MBP were found by a combination of reverse-phase chromatography, TLC, and MALDI spectrometry. Several clustered major, moderate, and minor sites of cleavage were revealed in the case of 21- and 25-mer oligopeptides. The active sites of anti-MBP abzymes are localised on their light chains, while heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high affinity to MBP and specificity of this protein hydrolysis. The affinity of anti-MBP abzymes for intact MBP is approximately 1000-fold higher than for the oligopeptides. The data suggest that all oligopeptides interact mainly with the light chains of different monoclonal abzymes of total pool of IgGs, which possesses a lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific than globular protein and can occur in several sites.

  10. Basic Research Firing Facility

    Data.gov (United States)

    Federal Laboratory Consortium — The Basic Research Firing Facility is an indoor ballistic test facility that has recently transitioned from a customer-based facility to a dedicated basic research...

  11. Body Basics Library

    Science.gov (United States)

    ... of Healthy Breakfasts Shyness About the Body Basics Library KidsHealth > For Teens > About the Body Basics Library A A A Did you ever wonder what ... system, part, and process works. Use this medical library to find out about basic human anatomy, how ...

  12. Body Basics Library

    Science.gov (United States)

    ... of Healthy Breakfasts Shyness About the Body Basics Library KidsHealth > For Teens > About the Body Basics Library Print A A A Did you ever wonder ... system, part, and process works. Use this medical library to find out about basic human anatomy, how ...

  13. Basic Cake Decorating Workbook.

    Science.gov (United States)

    Bogdany, Mel

    Included in this student workbook for basic cake decorating are the following: (1) Drawings of steps in a basic way to ice a layer cake, how to make a paper cone, various sizes of flower nails, various sizes and types of tin pastry tubes, and special rose tubes; (2) recipes for basic decorating icings (buttercream, rose paste, and royal icing);…

  14. New methods for measuring macromolecular interactions in solution via static light scattering: basic methodology and application to nonassociating and self-associating proteins.

    Science.gov (United States)

    Attri, Arun K; Minton, Allen P

    2005-02-01

    A method for rapid detection and characterization of reversible associations of macromolecules in solution is presented. A programmable dual-syringe infusion pump is used to introduce a solution of time-varying composition into parallel flow cells for concurrent measurement of laser light scattering at multiple angles and ultraviolet-visible absorbance. An experiment lasting less than 15 min produces a large and information-rich set of data, consisting of several thousand values of the Rayleigh ratio as a function of solute concentration(s) and scattering angle. Using a novel treatment of the data, the entire data set may be equally rapidly analyzed in the context of models for self-association. Validation experiments conducted on previously characterized nonassociating and self-associating proteins yielded robust values for molecular weights in the range 10-330 kDa and equilibrium association constants for dimer formation in the range 2 x 10(3)-6 x 10(5) M(-1).

  15. Myelin Basic Protein-Induced Production of Tumor Necrosis Factor-α and Interleukin-6, and Presentation of the Immunodominant Peptide MBP85-99 by B Cells from Patients with Relapsing-Remitting Multiple Sclerosis

    DEFF Research Database (Denmark)

    Nielsen, Claus H; Börnsen, Lars; Sellebjerg, Finn

    2016-01-01

    to study cytokine production by B cells, but here we used the physiologically relevant self-antigen myelin basic protein (MBP) to stimulate B cells from untreated patients with RRMS and healthy donors. Moreover, we took advantage of the unique ability of the monoclonal antibody MK16 to recognize...... the immunodominant peptide MBP85-99 presented on HLA-DR15, and used it as a probe to directly study B-cell presentation of self-antigenic peptide. The proportions of B cells producing TNF-α or IL-6 after stimulation with MBP were higher in RRMS patients than in healthy donors, indicating a pro-inflammatory profile...... with reduced ability of B cells to produce IL-10 after stimulation with MBP, indicative of diminished B-cell immune regulatory function in patients with the most severe disease. Moreover, EDSS correlated positively with the frequencies of TNF-α, IL-6 and IL-10 producing B cells after polyclonal stimulation...

  16. Basics of Bayesian Learning - Basically Bayes

    DEFF Research Database (Denmark)

    Larsen, Jan

    Tutorial presented at the IEEE Machine Learning for Signal Processing Workshop 2006, Maynooth, Ireland, September 8, 2006. The tutorial focuses on the basic elements of Bayesian learning and its relation to classical learning paradigms. This includes a critical discussion of the pros and cons...

  17. Basic molecular spectroscopy

    CERN Document Server

    Gorry, PA

    1985-01-01

    BASIC Molecular Spectroscopy discusses the utilization of the Beginner's All-purpose Symbolic Instruction Code (BASIC) programming language in molecular spectroscopy. The book is comprised of five chapters that provide an introduction to molecular spectroscopy through programs written in BASIC. The coverage of the text includes rotational spectra, vibrational spectra, and Raman and electronic spectra. The book will be of great use to students who are currently taking a course in molecular spectroscopy.

  18. Basic digital signal processing

    CERN Document Server

    Lockhart, Gordon B

    1985-01-01

    Basic Digital Signal Processing describes the principles of digital signal processing and experiments with BASIC programs involving the fast Fourier theorem (FFT). The book reviews the fundamentals of the BASIC program, continuous and discrete time signals including analog signals, Fourier analysis, discrete Fourier transform, signal energy, power. The text also explains digital signal processing involving digital filters, linear time-variant systems, discrete time unit impulse, discrete-time convolution, and the alternative structure for second order infinite impulse response (IIR) sections.

  19. Phase separation of myelin sheath in Triton X-114 solution: predominant localization of the 21.5-kDa isoform of myelin basic protein in the lipid raft-associated domain.

    Science.gov (United States)

    Uruse, Michihiro; Yamamoto, Masahiro; Sugawa, Makoto; Matsuura, Keiko; Sato, Yurie; Seiwa, Chika; Watanabe, Kenji; Aiso, Sadakazu; Asou, Hiroaki

    2014-04-01

    Myelin basic protein (MBP) isoforms in the myelin sheath are known to have distinct intracellular expression patterns, which are profoundly related to functional specificity. Determining the differential localization of MBP isoforms is therefore important for understanding their pathophysiological roles. In this study, we have developed a new method for phase separation of myelin. The non-ionic detergent Triton X-114 is used to solubilize myelin sheath which then undergoes phase separation to yield four fractions. The lipid raft-associated proteins and lipids in each fraction were analysed by immunoblotting and lipid analysis, respectively. The present method gives two lipid raft-enriched fractions, one of them was found to contain only lipid raft-associated galactocerebroside and cholesterol as the major lipids. The 21.5-kDa MBP isoforms (21.5 MBP), both unphosphorylated and phosphorylated, were exclusively contained in this fraction. Phosphorylated 21.5 MBP (21.5 pMBP) has been shown to specifically disappear from demyelinated loci. The present analytical method clearly indicated that disappearance of 21.5 pMBP corresponded to demyelination and its reappearance corresponded to prevention of demyelination. Demyelination was also associated with aging and was prevented by the myelin-protecting herbal medicine, Chinpi, a type of dried citrus peel.

  20. Basic Research Objectives Reaffirmed

    Institute of Scientific and Technical Information of China (English)

    Guo Haiyan; Zhao Baohua

    2002-01-01

    @@ As a national institution for scientific research and a component of the national innovation system, CAS should and must make key contributions to the great national rejuvenation of the country. Keeping this in mind, CAS has developed four developmental targets for its basic research. This was revealed at a CAS conference on basic research held June 11-12 in Beijing.

  1. Cycles in basic innovations

    NARCIS (Netherlands)

    Groot, de E.A. (Bert); Franses, P.H.P.H.

    2005-01-01

    Basic innovations are often believed to be the drivers of economic growth. It has been widely documented that economic growth follows cyclical patterns of varying length. In this paper we examine if such patterns are also present in basic innovations. For an annual time series of count data covering

  2. Basic Science Training Program.

    Science.gov (United States)

    Brummel, Clete

    These six learning modules were developed for Lake Michigan College's Basic Science Training Program, a workshop to develop good study skills while reviewing basic science. The first module, which was designed to provide students with the necessary skills to study efficiently, covers the following topics: time management; an overview of a study…

  3. Basic principle of superconductivity

    OpenAIRE

    De Cao, Tian

    2007-01-01

    The basic principle of superconductivity is suggested in this paper. There have been two vital wrong suggestions on the basic principle, one is the relation between superconductivity and the Bose-Einstein condensation (BEC), and another is the relation between superconductivity and pseudogap.

  4. Correlation between myelin basic protein contents and cerebral parenchyma damages in cerebral infarction patients%脑梗死患者血清髓鞘碱性蛋白含量与脑实质损害的相关性

    Institute of Scientific and Technical Information of China (English)

    张国元; 王晓明; 唐中; 郭晓兰; 龙存国; 廖涛

    2004-01-01

    目的:通过检测脑梗死患者及正常人的血清髓鞘碱性蛋白(myelin basic protein,MBP)的含量,探讨脑梗死患者血清髓鞘碱性蛋白含量与脑实质损害的相关性.方法:采用双抗体夹心酶联免疫吸附法对32位正常人及30例脑梗死患者血清MBP含量进行了检测.结果:脑梗死患者和对照组血清其MBP含量分别为(5.63±3.56),(1.10±0.45)μg/L,差异有极显著性(t=7.145,P<0.001),且血清MBP含量与脑梗死体积有一定的关系.结论:脑梗死时血清MBP含量明显升高,且与脑实质损害程度有一定的关系.%AIM: To discuss the correlation between myelin basic protein(MBP) contents and cerebral parenchyma damages in cerebral infarction patients through the assay of MBP contents in both cerebral infarction patients and healthy subjects.METHODS: The MBP serous contents of 32 healthy subjects and 30 cerebral infarction patients were detected by double-antibody-filling ELISA.RESULTS: The serous MBP contents of healthy subjects and cerebral infarction patients were(5.63 + 3.56) and(1.10 + 0.45) μg/L respectively.There was significance between the patient group and the control group ( t = 7. 145, P < 0. 001 ) . There was a certain relationship between the serous MBP content and the volume of cerebral infarct area.CONCLUSION: Serous MBP content markedly increases in cerebral infarction patients, and moreover, there is a certain relationship with the severity of cerebral parenchyma damage.

  5. Basic stress analysis

    CERN Document Server

    Iremonger, M J

    1982-01-01

    BASIC Stress Analysis aims to help students to become proficient at BASIC programming by actually using it in an important engineering subject. It also enables the student to use computing as a means of learning stress analysis because writing a program is analogous to teaching-it is necessary to understand the subject matter. The book begins by introducing the BASIC approach and the concept of stress analysis at first- and second-year undergraduate level. Subsequent chapters contain a summary of relevant theory, worked examples containing computer programs, and a set of problems. Topics c

  6. Quantum electronics basic theory

    CERN Document Server

    Fain, V M; Sanders, J H

    1969-01-01

    Quantum Electronics, Volume 1: Basic Theory is a condensed and generalized description of the many research and rapid progress done on the subject. It is translated from the Russian language. The volume describes the basic theory of quantum electronics, and shows how the concepts and equations followed in quantum electronics arise from the basic principles of theoretical physics. The book then briefly discusses the interaction of an electromagnetic field with matter. The text also covers the quantum theory of relaxation process when a quantum system approaches an equilibrium state, and explai

  7. Video Screen Capture Basics

    Science.gov (United States)

    Dunbar, Laura

    2014-01-01

    This article is an introduction to video screen capture. Basic information of two software programs, QuickTime for Mac and BlueBerry Flashback Express for PC, are also discussed. Practical applications for video screen capture are given.

  8. HIV Treatment: The Basics

    Science.gov (United States)

    HIV Treatment HIV Treatment: The Basics (Last updated 2/24/2017; last reviewed 2/24/2017) Key Points Antiretroviral therapy (ART) ... reduces the risk of HIV transmission . How do HIV medicines work? HIV attacks and destroys the infection- ...

  9. Kidney Disease Basics

    Science.gov (United States)

    ... Links Take the first step Alternate Language URL Kidney Disease Basics Page Content Your kidneys filter extra ... blood pressure are the most common causes of kidney disease. ​These conditions can slowly damage the kidneys ...

  10. Health Literacy Basics

    Science.gov (United States)

    ... have the capacity to obtain, process, and understand basic health information and services needed to make appropriate health decisions. 1 Health literacy is dependent on individual and systemic factors: Communication skills of lay persons and professionals Lay and professional ...

  11. Basic Financial Accounting

    DEFF Research Database (Denmark)

    Wiborg, Karsten

    This textbook on Basic Financial Accounting is targeted students in the economics studies at universities and business colleges having an introductory subject in the external dimension of the company's economic reporting, including bookkeeping, etc. The book includes the following subjects...

  12. Basic Concurrency Theory

    DEFF Research Database (Denmark)

    Løvengreen, Hans Henrik

    2002-01-01

    In this set of notes, we present some of the basic theory underlying the discipline of programming with concurrent processes/threads. The notes are intended to supplement a standard textbook on concurrent programming.......In this set of notes, we present some of the basic theory underlying the discipline of programming with concurrent processes/threads. The notes are intended to supplement a standard textbook on concurrent programming....

  13. HPLC-ESI-MS and MS/MS structural characterization of multifucosylated N-glycoforms of the basic proline-rich protein IB-8a CON1+ in human saliva.

    Science.gov (United States)

    Cabras, Tiziana; Boi, Roberto; Pisano, Elisabetta; Iavarone, Federica; Fanali, Chiara; Nemolato, Sonia; Faa, Gavino; Castagnola, Massimo; Messana, Irene

    2012-05-01

    This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1(+), a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1(+) and the variant IB-8a CON1(-), lacking of the glycosylation site, have been also detected in human saliva.

  14. CTF4 (CHL15) mutants exhibit defective DNA metabolism in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Kouprina, N; Kroll, E; Bannikov, V; Bliskovsky, V; Gizatullin, R; Kirillov, A; Shestopalov, B; Zakharyev, V; Hieter, P; Spencer, F

    1992-12-01

    We have analyzed the CTF4 (CHL15) gene, earlier identified in two screens for yeast mutants with increased rates of mitotic loss of chromosome III and artificial circular and linear chromosomes. Analysis of the segregation properties of circular minichromosomes and chromosome fragments indicated that sister chromatid loss (1:0 segregation) is the predominant mode of chromosome destabilization in ctf4 mutants, though nondisjunction events (2:0 segregation) also occur at an increased rate. Both inter- and intrachromosomal mitotic recombination levels are elevated in ctf4 mutants, whereas spontaneous mutation to canavanine resistance was not elevated. A genomic clone of CTF4 was isolated and used to map its physical and genetic positions on chromosome XVI. Nucleotide sequence analysis of CTF4 revealed a 2.8-kb open reading frame with a 105-kDa predicted protein sequence. The CTF4 DNA sequence is identical to that of POB1, characterized as a gene encoding a protein that associates in vitro with DNA polymerase alpha. At the N-terminal region of the protein sequence, zinc finger motifs which define potential DNA-binding domains were found. The C-terminal region of the predicted protein displayed similarity to sequences of regulatory proteins known as the helix-loop-helix proteins. Data on the effects of a frameshift mutation suggest that the helix-loop-helix domain is essential for CTF4 function. Analysis of sequences upstream of the CTF4 open reading frame revealed the presence of a hexamer element, ACGCGT, a sequence associated with many DNA metabolism genes in budding yeasts. Disruption of the coding sequence of CTF4 did not result in inviability, indicating that the CTF4 gene is nonessential for mitotic cell division. However, ctf4 mutants exhibit an accumulation of large budded cells with the nucleus in the neck. ctf4 rad52 double mutants grew very slowly and produced extremely high levels (50%) of inviable cell division products compared with either single mutant

  15. Basic Electromagnetism and Materials

    CERN Document Server

    Moliton, André

    2007-01-01

    Basic Electromagnetism and Materials is the product of many years of teaching basic and applied electromagnetism. This textbook can be used to teach electromagnetism to a wide range of undergraduate science majors in physics, electrical engineering or materials science. However, by making lesser demands on mathematical knowledge than competing texts, and by emphasizing electromagnetic properties of materials and their applications, this textbook is uniquely suited to students of materials science. Many competing texts focus on the study of propagation waves either in the microwave or optical domain, whereas Basic Electromagnetism and Materials covers the entire electromagnetic domain and the physical response of materials to these waves. Professor André Moliton is Director of the Unité de Microélectronique, Optoélectronique et Polymères (Université de Limoges, France), which brings together three groups studying the optoelectronics of molecular and polymer layers, micro-optoelectronic systems for teleco...

  16. Nuclear multifragmentation: Basic concepts

    Indian Academy of Sciences (India)

    G Chaudhuri; S Mallik; S Das Gupta

    2014-05-01

    We present a brief overview of nuclear multifragmentation reaction. Basic formalism of canonical thermodynamical model based on equilibrium statistical mechanics is described. This model is used to calculate basic observables of nuclear multifragmentation like mass distribution, fragment multiplicity, isotopic distribution and isoscaling. Extension of canonical thermodynamical model to a projectile fragmentation model is outlined. Application of the projectile fragmentation model for calculating average number of intermediate mass fragments and the average size of the largest cluster at different bound, differential charge distribution and cross-section of neutron-rich nuclei of different projectile fragmentation reactions at different energies are described. Application of nuclear multifragmentation reaction in basic research as well as in other domains is outlined.

  17. Decontamination: back to basics.

    Science.gov (United States)

    Meredith, Susan J; Sjorgen, Geoff

    2008-07-01

    My invitation from this Journal's Editor, Felicia Cox, to provide a paper for this themed issue, included the sentence 'I was wondering if you or a colleague would like to contribute a back to basics article on the relevant standards and guidelines for decontamination, including what is compliance?'. The reason it is so interesting to me is that the term 'back to basics' implies reverting to a simpler time in life - when by just sticking to the rules, life became easier. However, with decontamination this is not actually true.

  18. Comprehensive basic mathematics

    CERN Document Server

    Veena, GR

    2005-01-01

    Salient Features As per II PUC Basic Mathematics syllabus of Karnataka. Provides an introduction to various basic mathematical techniques and the situations where these could be usefully employed. The language is simple and the material is self-explanatory with a large number of illustrations. Assists the reader in gaining proficiency to solve diverse variety of problems. A special capsule containing a gist and list of formulae titled ''REMEMBER! Additional chapterwise arranged question bank and 3 model papers in a separate section---''EXAMINATION CORNER''.

  19. Basic set theory

    CERN Document Server

    Levy, Azriel

    2002-01-01

    An advanced-level treatment of the basics of set theory, this text offers students a firm foundation, stopping just short of the areas employing model-theoretic methods. Geared toward upper-level undergraduate and graduate students, it consists of two parts: the first covers pure set theory, including the basic motions, order and well-foundedness, cardinal numbers, the ordinals, and the axiom of choice and some of it consequences; the second deals with applications and advanced topics such as point set topology, real spaces, Boolean algebras, and infinite combinatorics and large cardinals. An

  20. Basic research championed

    Science.gov (United States)

    Friebele, Elaine

    In April, the Office of National Science and Technology Policy released its biennial report to Congress. Science and Technology: Shaping the Twenty-First Century addresses the President's policy for maintaining U.S. leadership in science and technology, significant developments, and important national issues in science, and opportunities to use science and technology in federal programs and national goals. The administration strongly supports basic research as a sound investment and an inspiration to society. As corporate laboratories increasingly favor applied R&D projects, the federal government is becoming the dominant sponsor of long-term, basic research.

  1. Basic properties of semiconductors

    CERN Document Server

    Landsberg, PT

    2013-01-01

    Since Volume 1 was published in 1982, the centres of interest in the basic physics of semiconductors have shifted. Volume 1 was called Band Theory and Transport Properties in the first edition, but the subject has broadened to such an extent that Basic Properties is now a more suitable title. Seven chapters have been rewritten by the original authors. However, twelve chapters are essentially new, with the bulk of this work being devoted to important current topics which give this volume an almost encyclopaedic form. The first three chapters discuss various aspects of modern band theory and the

  2. Basic Financial Accounting

    DEFF Research Database (Denmark)

    Wiborg, Karsten

    This textbook on Basic Financial Accounting is targeted students in the economics studies at universities and business colleges having an introductory subject in the external dimension of the company's economic reporting, including bookkeeping, etc. The book includes the following subjects: busin......: business entities, the transformation process, types of businesses, stakeholders, legislation, the annual report, the VAT system, double-entry bookkeeping, inventories, and year-end cast flow analysis.......This textbook on Basic Financial Accounting is targeted students in the economics studies at universities and business colleges having an introductory subject in the external dimension of the company's economic reporting, including bookkeeping, etc. The book includes the following subjects...

  3. 面神经修复中再生室内髓鞘碱性蛋白的作用初探%Preliminary study of the facial nerve regeneration in the chamber: the influence of myelin basic protein

    Institute of Scientific and Technical Information of China (English)

    骆文龙; 林代诚; 李永懋

    2001-01-01

    Objective To study the role of exogenous myelin basic protein(MBP) in neural repairment. Methods Adult New Zealand rabbits were employed in vivo preparation. A 12 μL nerve growth chamber was created by suturing the proximal and distal stumps of a transected facial never (FN) trunk into a tube. The regenerated nerves within the chambers were dissected and fixed for histological studies with light microscope at 4,6 and 8 weeks respectively following the surgery. Results Morphological analysis of nerves showed no difference between the MBP and control group in the size of the regeneration FN within the chambers, diameters of myelinated axons, thickness of myelin sheath and number of myelin axons grew into the distal end of chamber at 4 weeks. At 6 and 8 weeks after operation, the MBP group showed a more mature-appearance regenerative nerve comparing to control group. Especially, the enhancement of maturation in the regeneration axons was very noticeable at 6 weeks. Conclusion The study showed that pharmacological administration of exogenous MBP within a chamber at the time of entubational nerve repair enhances regeneration of myelinated axons across the sectioned ends of FN.%目的探讨外源性髓鞘碱性蛋白(myelin basic protein, MBP)在家兔面神经再生室修复中的作用。方法将33只家兔横断的面神经干近、远端缝于硅胶管壁上,形成约12 μL大小的神经再生室。一侧为实验组,将MBP注入再生室内;对侧为对照组不注任何物质。分别在术后4、6、8周处死动物, 切取标本,在光镜下行组织形态学观察。结果形态学分析表明术后4周2组再生室内再生面神经的有髓轴突直径、髓鞘厚度及长入再生室远端有髓轴突数差异无显著性 (P>0.05),随着时间的延长(术后6、8周),MBP组较对照组再生面神经显得更为成熟,6周时再生轴突成熟程度差异更明显。结论 MBP有促进家兔面神经再生修复的作用,但

  4. The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

    Science.gov (United States)

    Gettins, Peter G W; Dolmer, Klavs

    2016-01-08

    Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.

  5. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

    Energy Technology Data Exchange (ETDEWEB)

    Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D. [Centenary Institute of Cancer Medicine and Cell Biology, Sydney (Australia)] [and others

    1995-05-01

    Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.

  6. PDGF-alpha receptor and myelin basic protein mRNAs are not coexpressed by oligodendrocytes in vivo: a double in situ hybridization study in the anterior medullary velum of the neonatal rat.

    Science.gov (United States)

    Butt, A M; Hornby, M F; Ibrahim, M; Kirvell, S; Graham, A; Berry, M

    1997-01-01

    Platelet-derived growth factor (PDGF) is a growth-regulatory dimer with A and B subunits. PDGF-AA, acting via PDGF receptors of the alpha-unit subtype (PDGF-alphaR), is implicated in the differentiation of oligodendrocyte precursors and in the survival of newly formed oligodendrocytes, which gradually lose expression of PDGF-alphaR. However, it is unclear whether terminally differentiated oligodendrocytes express PDGF-alphaR in vivo. To address this question, and to help clarify the role of PDGF-AA in late oligodendrocyte differentiation, we have used double in situ hybridization with digoxigenin- and fluorescein-labeled riboprobes to relate PDGF-alphaR mRNA and myelin basic protein (MBP) mRNA expression in the isolated intact anterior medullary velum (AMV) of rats ages Postnatal Day (P) 10-12 and P30-32. In parallel experiments, AMV were immunolabeled with the oligodendrocyte-specific monoclonal antibody Rip to provide information on oligodendrocyte development and the extent of myelination. At P10, the AMV contained tracts in which axons ranged from unmyelinated to fully myelinated, whereas myelination was complete in P30-32 AMV. The first oligodendrocytes to express MBP mRNA or Rip were promyelinating oligodendrocytes, which had a "star-burst" morphology and had not yet begun to form myelin sheaths. As myelination proceeded, MBP mRNA became dispersed throughout oligodendrocyte units, comprising cell somata, processes, and internodal myelin sheaths. By P30-32, MBP mRNA had been redistributed to the myelin sheaths only, reflecting a change in the site of protein synthesis in mature myelinated axon tracts. At no stage of oligodendrocyte differentiation did we observe cellular coexpression of mRNA for PDGFalphaR and MBP. Our results indicated that oligodendrocytes lost the expression of PDGFalphaR prior to gaining that of myelin gene products, and preclude an action of PDGF-AA on Rip+/MBP+ star-burst promyelinating oligodendrocytes. The spatial and temporal

  7. Ethanol Basics (Fact Sheet)

    Energy Technology Data Exchange (ETDEWEB)

    2015-01-01

    Ethanol is a widely-used, domestically-produced renewable fuel made from corn and other plant materials. More than 96% of gasoline sold in the United States contains ethanol. Learn more about this alternative fuel in the Ethanol Basics Fact Sheet, produced by the U.S. Department of Energy's Clean Cities program.

  8. Korean Basic Course.

    Science.gov (United States)

    Defense Language Inst., Washington, DC.

    These 11 volumes of the Korean Basic Course comprise 112 lesson units designed to train native English language speakers to Level 3 proficiency in comprehension and speaking and Level 2 proficiency in reading and writing Korean. (Level 5 on this scale is native-speaker level.) Intended for classroom use in the Defense Language Institute intensive…

  9. Basic physics for all

    CERN Document Server

    Kumar, B N

    2012-01-01

    This is a simple, concise book for both student and non-physics students, presenting basic facts in straightforward form and conveying fundamental principles and theories of physics. This book will be helpful as a supplement to class teaching and to aid those who have difficulty in mastering concepts and principles.

  10. Vaccine Basics (Smallpox)

    Science.gov (United States)

    ... this page: About CDC.gov . Smallpox About Smallpox History of Smallpox Spread and Eradication of Smallpox Transmission Signs and Symptoms Prevention and Treatment Smallpox Vaccine Basics Vaccine Safety Side Effects of Vaccination Who Should Get a Smallpox Vaccination? Bioterrorism The ...

  11. FULA BASIC COURSE.

    Science.gov (United States)

    SWIFT, LLOYD B.; AND OTHERS

    THIS BEGINNING COURSE IS AN INTRODUCTION TO FULA (KNOWN VARIOUSLY AS FULANI, FUL, PEUL, OR PHEUL), A NIGER-CONGO LANGUAGE SPOKEN THROUGHOUT THE GRASSLAND AREAS OF WEST AFRICA FROM THE ATLANTIC TO CAMEROUN. THE TEXT IS ONE OF A SERIES OF SHORT BASIC COURSES IN SELECTED AFRICAN LANGUAGES BEING PREPARED BY THE FOREIGN SERVICE INSTITUTE. IT IS…

  12. Basic Library List.

    Science.gov (United States)

    Duren, William L., Jr.

    Reported is an initial attempt to define a minimal college mathematics library. Included is a list of some 300 books, from which approximately 170 are to be chosen to form a basic library in undergraduate mathematics. The areas provided for in this list include Algebra, Analysis, Applied Mathematics, Geometry, Topology, Logic, Foundations and Set…

  13. Lippincott Basic Reading Program.

    Science.gov (United States)

    Monterey Peninsula Unified School District, Monterey, CA.

    This program, included in "Effective Reading Programs...," serves 459 students in grades 1-3 at 15 elementary schools. The program employs a diagnostic-prescriptive approach to instruction in a nongraded setting through the use of the Lippincott Basic Reading program. When a child enters the program, he is introduced to a decoding…

  14. Basic Drafting: Book Two.

    Science.gov (United States)

    Davis, Ronald; And Others

    The second of a two-book course in drafting, this manual consists of 12 topics in the following units: sketching techniques, geometric constructions, orthographic views, dimensioning procedures, basic tolerancing, auxiliary views, sectional views, inking tools and techniques, axonometrics, oblique, perspective, and computer-aided drafting.…

  15. Health Insurance Basics

    Science.gov (United States)

    ... members at a lower cost. The four basic types of managed care plans are: HMO (Health Maintenance Organization). When you join an HMO, you choose a ... may have to pay more. EPO (Exclusive Provider Organization). An EPO is like a PPO, only ... Health Plan (CDHP) This type of plan is fairly new. It lets you ...

  16. Basic bioreactor design.

    NARCIS (Netherlands)

    Riet, van 't K.; Tramper, J.

    1991-01-01

    Based on a graduate course in biochemical engineering, provides the basic knowledge needed for the efficient design of bioreactors and the relevant principles and data for practical process engineering, with an emphasis on enzyme reactors and aerated reactors for microorganisms. Includes exercises.

  17. Basic Nuclear Physics.

    Science.gov (United States)

    Bureau of Naval Personnel, Washington, DC.

    Basic concepts of nuclear structures, radiation, nuclear reactions, and health physics are presented in this text, prepared for naval officers. Applications to the area of nuclear power are described in connection with pressurized water reactors, experimental boiling water reactors, homogeneous reactor experiments, and experimental breeder…

  18. Canadian Adult Basic Education.

    Science.gov (United States)

    Brooke, W. Michael, Comp.

    "Trends," a publication of the Canadian Association for Adult Education, is a collection of abstracts on selected subjects affecting adult education; this issue is on adult basic education (ABE). It covers teachers and teacher training, psychological factors relating to the ABE teacher and students, manuals for teachers, instructional…

  19. Basic Microfluidics Theory

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith

    2015-01-01

    ,000 m−1, which is a huge difference and has a large impact on flow behavior. In this chapter the basic microfluidic theory will be presented, enabling the reader to gain a comprehensive understanding of how liquids behave at the microscale, enough to be able to engage in design of micro systems...

  20. Basic Tuberculosis Facts

    Centers for Disease Control (CDC) Podcasts

    2012-03-12

    In this podcast, Dr. Kenneth Castro, Director of the Division of Tuberculosis Elimination, discusses basic TB prevention, testing, and treatment information.  Created: 3/12/2012 by National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP).   Date Released: 3/12/2012.