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Sample records for alpha motifs regulate

  1. Calreticulin-independent regulation of the platelet integrin alphaIIbbeta3 by the KVGFFKR alphaIIb-cytoplasmic motif.

    PubMed

    Reilly, Dermot; Larkin, Deirdre; Devocelle, Marc; Fitzgerald, Desmond J; Moran, Niamh

    2004-02-01

    The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets. PMID:14985176

  2. In vivo Regulation of the Allergic Response by the Interleukin 4 Receptor Alpha Chain Immunoreceptor Tyrosine-based Inhibitory Motif

    PubMed Central

    Tachdjian, Raffi; Khatib, Shadi Al; Schwinglshackl, Andreas; Kim, Hong Sook; Chen, Andrew; Blasioli, Julie; Mathias, Clinton; Kim, Hye-Young; Umetsu, Dale T.; Oettgen, Hans C.; Chatila, Talal A.

    2010-01-01

    Background Signaling by IL-4 and IL-13 via the IL-4 receptor alpha chain (IL-4Rα) plays a critical role in the pathology of allergic diseases. The IL-4Rα is endowed with an immunoreceptor tyrosine-based inhibitory motif (ITIM), centered on tyrosine 709 (Y709) in the cytoplasmic domain, that binds a number of regulatory phosphatases. The function of the ITIM in the in vivo regulation of IL-4R signaling remains unknown. Objective To determine the in vivo function of the IL-4Rα ITIM using mice in which the ITIM was inactivated by mutagenesis of the tyrosine Y709 residue into phenylalanine (F709). Methods F709 ITIM mutant mice were derived by knockin mutagenesis. Activation of intracellular signaling cascades by IL-4 and IL-13 was assessed by intracellular staining of phosphorylated signaling intermediates and by gene expression analysis. In vivo responses to allergic sensitization were assessed using models of allergic airway inflammation. Results The F709 mutation increased STAT6 phosphorylation by IL-4 and, disproportionately, by IL-13. This was associated with exaggerated Th2 polarization, enhanced alternative macrophage activation by IL-13, augmented basal and antigen-induced IgE responses and intensified allergen-induced eosinophilic airway inflammation and hyperreactivity. Conclusions These results point to a physiologic negative regulatory role for the Y709 ITIM in signaling via IL-4Rα, especially by IL-13. PMID:20392476

  3. Multiple Binding Modes between HNF4[alpha] and the LXXLL Motifs of PGC-1[alpha] Lead to Full Activation

    SciTech Connect

    Rha, Geun Bae; Wu, Guangteng; Shoelson, Steven E.; Chi, Young-In

    2010-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4{alpha} interacts, peroxisome proliferation-activated receptor {gamma} (PPAR{gamma}) coactivator 1{alpha} (PGC-1{alpha}) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1{alpha} recruitment, we have determined the crystal structure of HNF4{alpha} in complex with a fragment of PGC-1{alpha} containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4{alpha} toward the LXXLL motifs of PGC-1{alpha} could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.

  4. Acidic/IQ Motif Regulator of Calmodulin*

    PubMed Central

    Putkey, John A.; Waxham, M. Neal; Gaertner, Tara R.; Brewer, Kari J.; Goldsmith, Michael; Kubota, Yoshihisa; Kleerekoper, Quinn K.

    2013-01-01

    The small IQ motif proteins PEP-19 (62 amino acids) and RC3 (78 amino acids) greatly accelerate the rates of Ca2+ binding to sites III and IV in the C-domain of calmodulin (CaM). We show here that PEP-19 decreases the degree of cooperativity of Ca2+ binding to sites III and IV, and we present a model showing that this could increase Ca2+ binding rate constants. Comparative sequence analysis showed that residues 28 to 58 from PEP-19 are conserved in other proteins. This region includes the IQ motif (amino acids 39–62), and an adjacent acidic cluster of amino acids (amino acids 28–40). A synthetic peptide spanning residues 28–62 faithfully mimics intact PEP-19 with respect to increasing the rates of Ca2+ association and dissociation, as well as binding preferentially to the C-domain of CaM. In contrast, a peptide encoding only the core IQ motif does not modulate Ca2+ binding, and binds to multiple sites on CaM. A peptide that includes only the acidic region does not bind to CaM. These results show that PEP-19 has a novel acidic/IQ CaM regulatory motif in which the IQ sequence provides a targeting function that allows binding of PEP-19 to CaM, whereas the acidic residues modify the nature of this interaction, and are essential for modulating Ca2+ binding to the C-domain of CaM. PMID:17991744

  5. Cofunctional Subpathways Were Regulated by Transcription Factor with Common Motif, Common Family, or Common Tissue

    PubMed Central

    Su, Fei; Shang, Desi; Xu, Yanjun; Feng, Li; Yang, Haixiu; Liu, Baoquan; Su, Shengyang; Chen, Lina; Li, Xia

    2015-01-01

    Dissecting the characteristics of the transcription factor (TF) regulatory subpathway is helpful for understanding the TF underlying regulatory function in complex biological systems. To gain insight into the influence of TFs on their regulatory subpathways, we constructed a global TF-subpathways network (TSN) to analyze systematically the regulatory effect of common-motif, common-family, or common-tissue TFs on subpathways. We performed cluster analysis to show that the common-motif, common-family, or common-tissue TFs that regulated the same pathway classes tended to cluster together and contribute to the same biological function that led to disease initiation and progression. We analyzed the Jaccard coefficient to show that the functional consistency of subpathways regulated by the TF pairs with common motif, common family, or common tissue was significantly greater than the random TF pairs at the subpathway level, pathway level, and pathway class level. For example, HNF4A (hepatocyte nuclear factor 4, alpha) and NR1I3 (nuclear receptor subfamily 1, group I, member 3) were a pair of TFs with common motif, common family, and common tissue. They were involved in drug metabolism pathways and were liver-specific factors required for physiological transcription. In short, we inferred that the cofunctional subpathways were regulated by common-motif, common-family, or common-tissue TFs. PMID:26688819

  6. A High-Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction

    PubMed Central

    Kimple, Adam J.; Yasgar, Adam; Hughes, Mark; Jadhav, Ajit; Willard, Francis S.; Muller, Robin E.; Austin, Christopher P.; Inglese, James; Ibeanu, Gordon C.; Siderovski, David P.; Simeonov, Anton

    2008-01-01

    The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high-throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Gαi1. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z′-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z′-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC1280 collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively. PMID:18537560

  7. Stable proline box motif at the N-terminal end of alpha-helices.

    PubMed Central

    Viguera, A. R.; Serrano, L.

    1999-01-01

    We describe a novel N-terminal alpha-helix local motif that involves three hydrophobic residues and a Pro residue (Pro-box motif). Database analysis shows that when Pro is the N-cap of an alpha-helix the distribution of amino acids in adjacent positions changes dramatically with respect to the average distribution in an alpha-helix, but not when Pro is at position N1. N-cap Pro residues are usually associated to Ile and Leu, at position N', Val at position N3 and a hydrophobic residue (h) at position N4. The side chain of the N-cap Pro packs against Val, while the hydrophobic residues at positions N' and N4 make favorable interactions. To analyze the role of this putative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic interactions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N-cap and helix formers results in a small overall stabilization of the alpha-helix with respect to other peptides having Gly as the N-cap and Ala at N3 and N4. This suggests that the Pro-box motif will not specially contribute to protein stability but to the specificity of its fold. In fact, 80% of the sequences that contain the fingerprint sequence in the protein database are adopting the described structural motif, and in none of them is the helix extended to place Pro at the more favorable N1 position. PMID:10493574

  8. The PPFLMLLKGSTR motif in globular domain 3 of the human laminin-5 {alpha}3 chain is crucial for integrin {alpha}3{beta}1 binding and cell adhesion

    SciTech Connect

    Kim, Jin-Man; Park, Won Ho; Min, Byung-Moo . E-mail: bmmin@snu.ac.kr

    2005-03-10

    Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin {alpha}3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin {alpha}3{beta}1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin {alpha}3{beta}1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin {alpha}3{beta}1-dependent cell adhesion and spreading.

  9. The noncatalytic triad of alpha-amylases: a novel structural motif involved in conformational stability.

    PubMed

    Marx, Jean-Claude; Poncin, Johan; Simorre, Jean-Pierre; Ramteke, Pramod W; Feller, Georges

    2008-02-01

    Chloride-activated alpha-amylases contain a noncatalytic triad, independent of the glycosidic active site, perfectly mimicking the catalytic triad of serine-proteases and of other active serine hydrolytic enzymes. Mutagenesis of Glu, His, and Ser residues in various alpha-amylases shows that this pattern is a structural determinant of the enzyme conformation that cannot be altered without losing the intrinsic stability of the protein. (1)H-(15)N NMR spectra of a bacterial alpha-amylase reveal proton signals that are identical with the NMR signature of catalytic triads and especially a deshielded proton involving a protonated histidine and displaying properties similar to that of a low barrier hydrogen bond. It is proposed that the H-bond between His and Glu of the noncatalytic triad is an unusually strong interaction, responsible for the observed NMR signal and for the weak stability of the triad mutants. Furthermore, a stringent template-based search of the Protein Data Bank demonstrated that this motif is not restricted to alpha-amylases, but is also found in 80 structures from 33 different proteins, amongst which SH2 domain-containing proteins are the best representatives.

  10. Regulation of GPCR Anterograde Trafficking by Molecular Chaperones and Motifs.

    PubMed

    Young, Brent; Wertman, Jaime; Dupré, Denis J

    2015-01-01

    G protein-coupled receptors (GPCRs) make up a superfamily of integral membrane proteins that respond to a wide variety of extracellular stimuli, giving them an important role in cell function and survival. They have also proven to be valuable targets in the fight against various diseases. As such, GPCR signal regulation has received considerable attention over the last few decades. With the amplitude of signaling being determined in large part by receptor density at the plasma membrane, several endogenous mechanisms for modulating GPCR expression at the cell surface have come to light. It has been shown that cell surface expression is determined by both exocytic and endocytic processes. However, the body of knowledge surrounding GPCR trafficking from the endoplasmic reticulum to the plasma membrane, commonly known as anterograde trafficking, has considerable room for growth. We focus here on the current paradigms of anterograde GPCR trafficking. We will discuss the regulatory role of both the general and "nonclassical private" chaperone systems in GPCR trafficking as well as conserved motifs that serve as modulators of GPCR export from the endoplasmic reticulum and Golgi apparatus. Together, these topics summarize some of the known mechanisms by which the cell regulates anterograde GPCR trafficking. PMID:26055064

  11. Analysis of Genomic Sequence Motifs for Deciphering Transcription Factor Binding and Transcriptional Regulation in Eukaryotic Cells

    PubMed Central

    Boeva, Valentina

    2016-01-01

    Eukaryotic genomes contain a variety of structured patterns: repetitive elements, binding sites of DNA and RNA associated proteins, splice sites, and so on. Often, these structured patterns can be formalized as motifs and described using a proper mathematical model such as position weight matrix and IUPAC consensus. Two key tasks are typically carried out for motifs in the context of the analysis of genomic sequences. These are: identification in a set of DNA regions of over-represented motifs from a particular motif database, and de novo discovery of over-represented motifs. Here we describe existing methodology to perform these two tasks for motifs characterizing transcription factor binding. When applied to the output of ChIP-seq and ChIP-exo experiments, or to promoter regions of co-modulated genes, motif analysis techniques allow for the prediction of transcription factor binding events and enable identification of transcriptional regulators and co-regulators. The usefulness of motif analysis is further exemplified in this review by how motif discovery improves peak calling in ChIP-seq and ChIP-exo experiments and, when coupled with information on gene expression, allows insights into physical mechanisms of transcriptional modulation. PMID:26941778

  12. Analysis of Genomic Sequence Motifs for Deciphering Transcription Factor Binding and Transcriptional Regulation in Eukaryotic Cells.

    PubMed

    Boeva, Valentina

    2016-01-01

    Eukaryotic genomes contain a variety of structured patterns: repetitive elements, binding sites of DNA and RNA associated proteins, splice sites, and so on. Often, these structured patterns can be formalized as motifs and described using a proper mathematical model such as position weight matrix and IUPAC consensus. Two key tasks are typically carried out for motifs in the context of the analysis of genomic sequences. These are: identification in a set of DNA regions of over-represented motifs from a particular motif database, and de novo discovery of over-represented motifs. Here we describe existing methodology to perform these two tasks for motifs characterizing transcription factor binding. When applied to the output of ChIP-seq and ChIP-exo experiments, or to promoter regions of co-modulated genes, motif analysis techniques allow for the prediction of transcription factor binding events and enable identification of transcriptional regulators and co-regulators. The usefulness of motif analysis is further exemplified in this review by how motif discovery improves peak calling in ChIP-seq and ChIP-exo experiments and, when coupled with information on gene expression, allows insights into physical mechanisms of transcriptional modulation.

  13. Network motifs in integrated cellular networks of transcription-regulation and protein-protein interaction

    NASA Astrophysics Data System (ADS)

    Yeger-Lotem, Esti; Sattath, Shmuel; Kashtan, Nadav; Itzkovitz, Shalev; Milo, Ron; Pinter, Ron Y.; Alon, Uri; Margalit, Hanah

    2004-04-01

    Genes and proteins generate molecular circuitry that enables the cell to process information and respond to stimuli. A major challenge is to identify characteristic patterns in this network of interactions that may shed light on basic cellular mechanisms. Previous studies have analyzed aspects of this network, concentrating on either transcription-regulation or protein-protein interactions. Here we search for composite network motifs: characteristic network patterns consisting of both transcription-regulation and protein-protein interactions that recur significantly more often than in random networks. To this end we developed algorithms for detecting motifs in networks with two or more types of interactions and applied them to an integrated data set of protein-protein interactions and transcription regulation in Saccharomyces cerevisiae. We found a two-protein mixed-feedback loop motif, five types of three-protein motifs exhibiting coregulation and complex formation, and many motifs involving four proteins. Virtually all four-protein motifs consisted of combinations of smaller motifs. This study presents a basic framework for detecting the building blocks of networks with multiple types of interactions.

  14. Single-molecule study of thymidine glycol and i-motif through the alpha-hemolysin ion channel

    NASA Astrophysics Data System (ADS)

    He, Lidong

    Nanopore-based devices have emerged as a single-molecule detection and analysis tool for a wide range of applications. Through electrophoretically driving DNA molecules across a nanosized pore, a lot of information can be received, including unfolding kinetics and DNA-protein interactions. This single-molecule method has the potential to sequence kilobase length DNA polymers without amplification or labeling, approaching "the third generation" genome sequencing for around $1000 within 24 hours. alpha-Hemolysin biological nanopores have the advantages of excellent stability, low-noise level, and precise site-directed mutagenesis for engineering this protein nanopore. The first work presented in this thesis established the current signal of the thymidine glycol lesion in DNA oligomers through an immobilization experiment. The thymidine glycol enantiomers were differentiated from each other by different current blockage levels. Also, the effect of bulky hydrophobic adducts to the current blockage was investigated. Secondly, the alpha-hemolysin nanopore was used to study the human telomere i-motif and RET oncogene i-motif at a single-molecule level. In Chapter 3, it was demonstrated that the alpha-hemolysin nanopore can differentiate an i-motif form and single-strand DNA form at different pH values based on the same sequence. In addition, it shows potential to differentiate the folding topologies generated from the same DNA sequence.

  15. The PXDLS linear motif regulates circadian rhythmicity through protein–protein interactions

    PubMed Central

    Shalev, Moran; Aviram, Rona; Adamovich, Yaarit; Kraut-Cohen, Judith; Shamia, Tal; Ben-Dor, Shifra; Golik, Marina; Asher, Gad

    2014-01-01

    The circadian core clock circuitry relies on interlocked transcription-translation feedback loops that largely count on multiple protein interactions. The molecular mechanisms implicated in the assembly of these protein complexes are relatively unknown. Our bioinformatics analysis of short linear motifs, implicated in protein interactions, reveals an enrichment of the Pro-X-Asp-Leu-Ser (PXDLS) motif within circadian transcripts. We show that the PXDLS motif can bind to BMAL1/CLOCK and disrupt circadian oscillations in a cell-autonomous manner. Remarkably, the motif is evolutionary conserved in the core clock protein REV-ERBα, and additional proteins implicated in the clock's function (NRIP1, CBP). In this conjuncture, we uncover a novel cross talk between the two principal core clock feedback loops and show that BMAL/CLOCK and REV-ERBα interact and that the PXDLS motif of REV-ERBα participates in their binding. Furthermore, we demonstrate that the PXDLS motifs of NRIP1 and CBP are involved in circadian rhythmicity. Our findings suggest that the PXDLS motif plays an important role in circadian rhythmicity through regulation of protein interactions within the clock circuitry and that short linear motifs can be employed to modulate circadian oscillations. PMID:25260595

  16. Regulation of amyloid precursor protein processing by its KFERQ motif

    PubMed Central

    Park, Ji-Seon; Kim, Dong-Hou; Yoon, Seung-Yong

    2016-01-01

    Understanding of trafficking, processing, and degradation mechanisms of amyloid precursor protein (APP) is important because APP can be processed to produce β-amyloid (Aβ), a key pathogenic molecule in Alzheimer’s disease (AD). Here, we found that APP contains KFERQ motif at its C-terminus, a consensus sequence for chaperone-mediated autophagy (CMA) or microautophagy which are another types of autophagy for degradation of pathogenic molecules in neurodegenerative diseases. Deletion of KFERQ in APP increased C-terminal fragments (CTFs) and secreted N-terminal fragments of APP and kept it away from lysosomes. KFERQ deletion did not abolish the interaction of APP or its cleaved products with heat shock cognate protein 70 (Hsc70), a protein necessary for CMA or microautophagy. These findings suggest that KFERQ motif is important for normal processing and degradation of APP to preclude the accumulation of APP-CTFs although it may not be important for CMA or microautophagy. [BMB Reports 2016;49(6): 337-342] PMID:26779997

  17. The VQ Motif-Containing Protein Family of Plant-Specific Transcriptional Regulators1

    PubMed Central

    Jing, Yanjun; Lin, Rongcheng

    2015-01-01

    The VQ motif-containing proteins (designated as VQ proteins) are a class of plant-specific proteins with a conserved and single short FxxhVQxhTG amino acid sequence motif. VQ proteins regulate diverse developmental processes, including responses to biotic and abiotic stresses, seed development, and photomorphogenesis. In this Update, we summarize and discuss recent advances in our understanding of the regulation and function of VQ proteins and the role of the VQ motif in mediating transcriptional regulation and protein-protein interactions in signaling pathways. Based on the accumulated evidence, we propose a general mechanism of action for the VQ protein family, which likely defines a novel class of transcriptional regulators specific to plants. PMID:26220951

  18. Structural characterization of a capping protein interaction motif defines a family of actin filament regulators

    PubMed Central

    Hernandez-Valladares, Maria; Kim, Taekyung; Kannan, Balakrishnan; Tung, Alvin; Aguda, Adeleke H; Larsson, Mårten; Cooper, John A; Robinson, Robert C

    2011-01-01

    Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments. PMID:20357771

  19. Three distinct motifs within the C-terminus of acid-sensing ion channel 1a regulate its surface trafficking.

    PubMed

    Jing, L; Chu, X-P; Zha, X-M

    2013-09-01

    Various protein motifs play a key role in regulating protein biogenesis and trafficking. Here, we discovered that three distinct motifs regulate the trafficking of acid-sensing ion channel 1a (ASIC1a), the primary neuronal proton receptor which plays critical roles in neurological diseases including stroke, multiple sclerosis and seizures. Mutating the PDZ binding motif of ASIC1a increased its surface expression and current density. In contrast, mutating either a RRGK motif or a KEAKR motif reduced ASIC1a surface expression and acid-activated current density. Mutating or deleting the RRGK motif also reduced pH sensitivity and the rate of desensitization of ASIC1a. These changes were likely due to a change in ASIC1a biogenesis; mutating either the RRGK or KEAKR motif reduced N-glycosylation of ASIC1a while mutating the PDZ binding motif had the opposite effect. Our results demonstrate that these C-terminal motifs are important for ASIC1a trafficking and channel function. In addition, in contrast to multiple previous studies, which all show that K/R containing motifs lead to endoplasmic reticulum (ER) retention, our findings indicate that these motifs can also be required for efficient trafficking.

  20. Feedback loops and reciprocal regulation: recurring motifs in the systems biology of the cell cycle

    PubMed Central

    Ferrell, James E.

    2013-01-01

    The study of eukaryotic cell cycle regulation over the last several decades has led to a remarkably detailed understanding of the complex regulatory system that drives this fundamental process. This allows us to now look for recurring motifs in the regulatory system. Among these are negative feedback loops, which underpin checkpoints and generate cell cycle oscillations; positive feedback loops, which promote oscillations and make cell cycle transitions switch-like and unidirectional; and reciprocal regulation, which can increase the control a key regulator exerts. These simple motifs are found at multiple points in the cell cycle (e.g., S-phase and M-phase control) and are conserved in diverse organisms. These findings argue for an underlying unity in the principles of cell cycle control. PMID:23927869

  1. Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

    SciTech Connect

    Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.; Owens, Timothy R.; Oyesiku, Nelson M.

    2009-11-01

    We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

  2. Regulation of Airway Inflammation by G-protein Regulatory Motif Peptides of AGS3 protein.

    PubMed

    Choi, Il-Whan; Ahn, Do Whan; Choi, Jang-Kyu; Cha, Hee-Jae; Ock, Mee Sun; You, EunAe; Rhee, SangMyung; Kim, Kwang Chul; Choi, Yung Hyun; Song, Kyoung Seob

    2016-01-01

    Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFβ but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment. PMID:27270970

  3. Regulation of Airway Inflammation by G-protein Regulatory Motif Peptides of AGS3 protein

    PubMed Central

    Choi, IL-Whan; Ahn, Do Whan; Choi, Jang-Kyu; Cha, Hee-Jae; Ock, Mee Sun; You, EunAe; Rhee, SangMyung; Kim, Kwang Chul; Choi, Yung Hyun; Song, Kyoung Seob

    2016-01-01

    Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFβ but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment. PMID:27270970

  4. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  5. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

    PubMed

    Céspedes, Nora; Habel, Catherine; Lopez-Perez, Mary; Castellanos, Angélica; Kajava, Andrey V; Servis, Catherine; Felger, Ingrid; Moret, Remy; Arévalo-Herrera, Myriam; Corradin, Giampietro; Herrera, Sócrates

    2014-01-01

    Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

  6. Identification of a pKa-regulating motif stabilizing imidazole-modified double-stranded DNA

    PubMed Central

    Buyst, Dieter; Gheerardijn, Vicky; Fehér, Krisztina; Van Gasse, Bjorn; Van Den Begin, Jos; Martins, José C.; Madder, Annemieke

    2015-01-01

    The predictable 3D structure of double-stranded DNA renders it ideally suited as a template for the bottom-up design of functionalized nucleic acid-based active sites. We here explore the use of a 14mer DNA duplex as a scaffold for the precise and predictable positioning of catalytic functionalities. Given the ubiquitous participation of the histidine-based imidazole group in protein recognition and catalysis events, single histidine-like modified duplexes were investigated. Tethering histamine to the C5 of the thymine base via an amide bond, allows the flexible positioning of the imidazole function in the major groove. The mutual interactions between the imidazole and the duplex and its influence on the imidazolium pKaH are investigated by placing a single modified thymine at four different positions in the center of the 14mer double helix. Using NMR and unrestrained molecular dynamics, a structural motif involving the formation of a hydrogen bond between the imidazole and the Hoogsteen side of the guanine bases of two neighboring GC base pairs is established. The motif contributes to a stabilization against thermal melting of 6°C and is key in modulating the pKaH of the imidazolium group. The general features, prerequisites and generic character of the new pKaH-regulating motif are described. PMID:25520197

  7. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    SciTech Connect

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  8. A TGACGT motif in the 5'-upstream region of alpha-amylase gene from Vigna mungo is a cis-element for expression in cotyledons of germinated seeds.

    PubMed

    Yamauchi, D

    2001-06-01

    Alpha-amylase is expressed at high levels in cotyledons of germinated seeds of Vigna mungo. The mRNA for alpha-amylase appeared in cotyledons of the seeds at 1 d after imbibition started (DAI). Two TGACGT motifs at -445 and at -125 in the promoter region of the gene interacted with nuclear proteins from cotyledons of dry seeds and the activities were detected until 3 DAI. A transient assay with particle bombardment showed that the downstream region from -135 in the promoter was required for high level expression in the cotyledons and the activity was reduced by mutation of the TGACGT motif at -125. The activities to bind the TGACGT motifs were detected in the axes of the seeds at 1 DAI but disappeared at 4 DAI, although the mRNA for alpha-amylase in the axes appeared at 4 DAI and increased in level by 6 DAI. A transient assay experiment showed that a positive regulatory element for the expression in the axes was located in the region from -630 to -453. These results indicated that the TGACGT motif at -125 was required for high level expression of the gene in the cotyledons of the germinated seeds.

  9. An Alpha Motif at Tas3C Terminus Mediates RITS Cis Spreading and Promotes Heterochromatic Gene Silencing

    SciTech Connect

    Li, H.; Motamedi, M; Yip, C; Wang, Z; Walz, T; Patel, D; Moazed, D

    2009-01-01

    RNA interference (RNAi) plays a pivotal role in the formation of heterochromatin at the fission yeast centromeres. The RNA-induced transcriptional silencing (RITS) complex, composed of heterochromatic small interfering RNAs (siRNAs), the siRNA-binding protein Ago1, the chromodomain protein Chp1, and the Ago1/Chp1-interacting protein Tas3, provides a physical tether between the RNAi and heterochromatin assembly pathways. Here, we report the structural and functional characterization of a C-terminal Tas3 {alpha}-helical motif (TAM), which self-associates into a helical polymer and is required for cis spreading of RITS in centromeric DNA regions. Site-directed mutations of key residues within the hydrophobic monomer-monomer interface disrupt Tas3-TAM polymeric self-association in vitro and result in loss of gene silencing, spreading of RITS, and a dramatic reduction in centromeric siRNAs in vivo. These results demonstrate that, in addition to the chromodomain of Chp1 and siRNA-loaded Ago1, Tas3 self-association is required for RITS spreading and efficient heterochromatic gene silencing at centromeric repeat regions.

  10. Mitogen-activated protein kinase 4-like carrying an MEY motif instead of a TXY motif is involved in ozone tolerance and regulation of stomatal closure in tobacco.

    PubMed

    Yanagawa, Yuki; Yoda, Hiroshi; Osaki, Kohei; Amano, Yuta; Aono, Mitsuko; Seo, Shigemi; Kuchitsu, Kazuyuki; Mitsuhara, Ichiro

    2016-05-01

    The mitogen-activated protein kinases (MAPKs/MPKs) are important factors in the regulation of signal transduction in response to biotic and abiotic stresses. Previously, we characterized a MAPK from tobacco, Nicotiana tabacum MPK4 (NtMPK4). Here, we found a highly homologous gene, NtMPK4-like (NtMPK4L), in tobacco as well as other species in Solanaceae and Gramineae. Deduced amino acid sequences of their translation products carried MEY motifs instead of conserved TXY motifs of the MAPK family. We isolated the full length NtMPK4L gene and examined the physiological functions of NtMPK4L. We revealed that NtMPK4L was activated by wounding, like NtMPK4. However, a constitutively active salicylic acid-induced protein kinase kinase (SIPKK(EE)), which phosphorylates NtMPK4, did not phosphorylate NtMPK4L. Moreover, a tyrosine residue in the MEY motif was not involved in NtMPK4L activation. We also found that NtMPK4L-silenced plants showed rapid transpiration caused by remarkably open stomata. In addition, NtMPK4L-silenced plants completely lost the ability to close stomata upon ozone treatment and were highly sensitive to ozone, suggesting that this atypical MAPK plays a role in ozone tolerance through stomatal regulation. PMID:27126796

  11. An Amino Terminal Phosphorylation Motif Regulates Intranuclear Compartmentalization of Olig2 in Neural Progenitor Cells

    PubMed Central

    Meijer, Dimphna H.; Sun, Yu; Liu, Tao; Kane, Michael F.; Alberta, John A.; Adelmant, Guillaume; Kupp, Robert; Marto, Jarrod A.; Rowitch, David H.; Nakatani, Yoshihiro

    2014-01-01

    The bHLH transcription factor Olig2 is expressed in cycling neural progenitor cells but also in terminally differentiated, myelinating oligodendrocytes. Sustained expression of Olig2 is counterintuitive because all known functions of the protein in expansion of neural progenitors and specification of oligodendrocyte progenitors are completed with the formation of mature white matter. How are the biological functions of Olig2 suppressed in terminally differentiated oligodendrocytes? In previous studies, we have shown that a triple serine motif in the amino terminus of Olig2 is phosphorylated in cycling neural progenitors but not in their differentiated progeny. We now show that phosphorylation of the triple serine motif regulates intranuclear compartmentalization of murine Olig2. Phosphorylated Olig2 is preferentially localized to a transcriptionally active “open” chromatin compartment together with coregulator proteins essential for regulation of gene expression. Unphosphorylated Olig2, as seen in mature white matter, is localized mainly within a transcriptionally inactive, chromatin fraction characterized by condensed and inaccessible DNA. Of special note is the observation that the p53 tumor suppressor protein is confined to the open chromatin fraction. Proximity ligation assays show that phosphorylation brings Olig2 within 30 nm of p53 within the open chromatin compartment. The data thus shed light on previously noted promitogenic functions of phosphorylated Olig2, which reflect, at least in part, an oppositional relationship with p53 functions. PMID:24948806

  12. An amino terminal phosphorylation motif regulates intranuclear compartmentalization of Olig2 in neural progenitor cells.

    PubMed

    Meijer, Dimphna H; Sun, Yu; Liu, Tao; Kane, Michael F; Alberta, John A; Adelmant, Guillaume; Kupp, Robert; Marto, Jarrod A; Rowitch, David H; Nakatani, Yoshihiro; Stiles, Charles D; Mehta, Shwetal

    2014-06-18

    The bHLH transcription factor Olig2 is expressed in cycling neural progenitor cells but also in terminally differentiated, myelinating oligodendrocytes. Sustained expression of Olig2 is counterintuitive because all known functions of the protein in expansion of neural progenitors and specification of oligodendrocyte progenitors are completed with the formation of mature white matter. How are the biological functions of Olig2 suppressed in terminally differentiated oligodendrocytes? In previous studies, we have shown that a triple serine motif in the amino terminus of Olig2 is phosphorylated in cycling neural progenitors but not in their differentiated progeny. We now show that phosphorylation of the triple serine motif regulates intranuclear compartmentalization of murine Olig2. Phosphorylated Olig2 is preferentially localized to a transcriptionally active "open" chromatin compartment together with coregulator proteins essential for regulation of gene expression. Unphosphorylated Olig2, as seen in mature white matter, is localized mainly within a transcriptionally inactive, chromatin fraction characterized by condensed and inaccessible DNA. Of special note is the observation that the p53 tumor suppressor protein is confined to the open chromatin fraction. Proximity ligation assays show that phosphorylation brings Olig2 within 30 nm of p53 within the open chromatin compartment. The data thus shed light on previously noted promitogenic functions of phosphorylated Olig2, which reflect, at least in part, an oppositional relationship with p53 functions. PMID:24948806

  13. Mutation of FVS1, encoding a protein with a sterile alpha motif domain, affects asexual reproduction in the fungal plant pathogen Fusarium oxysporum.

    PubMed

    Iida, Yuichiro; Fujiwara, Kazuki; Yoshioka, Yosuke; Tsuge, Takashi

    2014-02-01

    Fusarium oxysporum produces three kinds of asexual spores: microconidia, macroconidia and chlamydospores. We previously analysed expressed sequence tags during vegetative growth and conidiation in F. oxysporum and found 42 genes that were markedly upregulated during conidiation compared to vegetative growth. One of the genes, FVS1, encodes a protein with a sterile alpha motif (SAM) domain, which functions in protein-protein interactions that are involved in transcriptional or post-transcriptional regulation and signal transduction. Here, we made FVS1-disrupted mutants from the melon wilt pathogen F. oxysporum f. sp. melonis. Although the mutants produced all three kinds of asexual spores with normal morphology, they formed markedly fewer microconidia and macroconidia than the wild type. The mutants appeared to have a defect in the development of the conidiogenesis cells, conidiophores and phialides, required for the formation of microconidia and macroconidia. In contrast, chlamydospore formation was dramatically promoted in the mutants. The growth rates of the mutants on media were slightly reduced, indicating that FVS1 is also involved in, but not essential for, vegetative growth. We also observed that mutation of FVS1 caused defects in conidial germination and virulence, suggesting that the Fvs1 has pleiotropic functions in F. oxysporum.

  14. Regulation of PPAR{gamma} function by TNF-{alpha}

    SciTech Connect

    Ye Jianping

    2008-09-26

    The nuclear receptor PPAR{gamma} is a lipid sensor that regulates lipid metabolism through gene transcription. Inhibition of PPAR{gamma} activity by TNF-{alpha} is involved in pathogenesis of insulin resistance, atherosclerosis, inflammation, and cancer cachexia. PPAR{gamma} activity is regulated by TNF-{alpha} at pre-translational and post-translational levels. Activation of serine kinases including IKK, ERK, JNK, and p38 may be involved in the TNF-regulation of PPAR{gamma}. Of the four kinases, IKK is a dominant signaling molecule in the TNF-regulation of PPAR{gamma}. IKK acts through at least two mechanisms: inhibition of PPAR{gamma} expression and activation of PPAR{gamma} corepressor. In this review article, literature is reviewed with a focus on the mechanisms of PPAR{gamma} inhibition by TNF-{alpha}.

  15. CDR3β sequence motifs regulate autoreactivity of human invariant NKT cell receptors.

    PubMed

    Chamoto, Kenji; Guo, Tingxi; Imataki, Osamu; Tanaka, Makito; Nakatsugawa, Munehide; Ochi, Toshiki; Yamashita, Yuki; Saito, Akiko M; Saito, Toshiki I; Butler, Marcus O; Hirano, Naoto

    2016-04-01

    Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid ligands presented by monomorphic CD1d. Human iNKT T cell receptor (TCR) is largely composed of invariant Vα24 (Vα24i) TCRα chain and semi-variant Vβ11 TCRβ chain, where complementarity-determining region (CDR)3β is the sole variable region. One of the characteristic features of iNKT cells is that they retain autoreactivity even after the thymic selection. However, the molecular features of human iNKT TCR CDR3β sequences that regulate autoreactivity remain unknown. Since the numbers of iNKT cells with detectable autoreactivity in peripheral blood is limited, we introduced the Vα24i gene into peripheral T cells and generated a de novo human iNKT TCR repertoire. By stimulating the transfected T cells with artificial antigen presenting cells (aAPCs) presenting self-ligands, we enriched strongly autoreactive iNKT TCRs and isolated a large panel of human iNKT TCRs with a broad range autoreactivity. From this panel of unique iNKT TCRs, we deciphered three CDR3β sequence motifs frequently encoded by strongly-autoreactive iNKT TCRs: a VD region with 2 or more acidic amino acids, usage of the Jβ2-5 allele, and a CDR3β region of 13 amino acids in length. iNKT TCRs encoding 2 or 3 sequence motifs also exhibit higher autoreactivity than those encoding 0 or 1 motifs. These data facilitate our understanding of the molecular basis for human iNKT cell autoreactivity involved in immune responses associated with human disease. PMID:26748722

  16. MoD Tools: regulatory motif discovery in nucleotide sequences from co-regulated or homologous genes.

    PubMed

    Pavesi, Giulio; Mereghetti, Paolo; Zambelli, Federico; Stefani, Marco; Mauri, Giancarlo; Pesole, Graziano

    2006-07-01

    Understanding the complex mechanisms regulating gene expression at the transcriptional and post-transcriptional levels is one of the greatest challenges of the post-genomic era. The MoD (MOtif Discovery) Tools web server comprises a set of tools for the discovery of novel conserved sequence and structure motifs in nucleotide sequences, motifs that in turn are good candidates for regulatory activity. The server includes the following programs: Weeder, for the discovery of conserved transcription factor binding sites (TFBSs) in nucleotide sequences from co-regulated genes; WeederH, for the discovery of conserved TFBSs and distal regulatory modules in sequences from homologous genes; RNAProfile, for the discovery of conserved secondary structure motifs in unaligned RNA sequences whose secondary structure is not known. In this way, a given gene can be compared with other co-regulated genes or with its homologs, or its mRNA can be analyzed for conserved motifs regulating its post-transcriptional fate. The web server thus provides researchers with different strategies and methods to investigate the regulation of gene expression, at both the transcriptional and post-transcriptional levels. Available at http://www.pesolelab.it/modtools/ and http://www.beacon.unimi.it/modtools/.

  17. Motif co-regulation and co-operativity are common mechanisms in transcriptional, post-transcriptional and post-translational regulation.

    PubMed

    Van Roey, Kim; Davey, Norman E

    2015-01-01

    A substantial portion of the regulatory interactions in the higher eukaryotic cell are mediated by simple sequence motifs in the regulatory segments of genes and (pre-)mRNAs, and in the intrinsically disordered regions of proteins. Although these regulatory modules are physicochemically distinct, they share an evolutionary plasticity that has facilitated a rapid growth of their use and resulted in their ubiquity in complex organisms. The ease of motif acquisition simplifies access to basal housekeeping functions, facilitates the co-regulation of multiple biomolecules allowing them to respond in a coordinated manner to changes in the cell state, and supports the integration of multiple signals for combinatorial decision-making. Consequently, motifs are indispensable for temporal, spatial, conditional and basal regulation at the transcriptional, post-transcriptional and post-translational level. In this review, we highlight that many of the key regulatory pathways of the cell are recruited by motifs and that the ease of motif acquisition has resulted in large networks of co-regulated biomolecules. We discuss how co-operativity allows simple static motifs to perform the conditional regulation that underlies decision-making in higher eukaryotic biological systems. We observe that each gene and its products have a unique set of DNA, RNA or protein motifs that encode a regulatory program to define the logical circuitry that guides the life cycle of these biomolecules, from transcription to degradation. Finally, we contrast the regulatory properties of protein motifs and the regulatory elements of DNA and (pre-)mRNAs, advocating that co-regulation, co-operativity, and motif-driven regulatory programs are common mechanisms that emerge from the use of simple, evolutionarily plastic regulatory modules. PMID:26626130

  18. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators

    PubMed Central

    Hagting, Anja; Izawa, Daisuke; Mansfeld, Jörg; Gibson, Toby J.; Pines, Jonathon

    2016-01-01

    The APC/C is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the Spindle Assembly Checkpoint (SAC). How the APC/C recognises its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in Cyclin A, BUBR1, BUB1 and Acm1, and show that it binds to the APC/C co-activator CDC20. The ABBA motif in Cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC. PMID:25669885

  19. Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1

    PubMed Central

    Boddu, Ravindra; Yang, Chaozhe; O’Connor, Amber K.; Hendrickson, Robert Curtis; Boone, Braden; Cui, Xiangqin; Garcia-Gonzalez, Miguel; Igarashi, Peter; Onuchic, Luiz F.; Germino, Gregory G.

    2014-01-01

    Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD. PMID:24984783

  20. Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif.

    PubMed

    Voelzmann, André; Wulf, Anna-Lena; Eckardt, Franka; Thielisch, Melanie; Brondolin, Mirco; Pesch, Yanina-Yasmin; Sociale, Mariangela; Bauer, Reinhard; Hoch, Michael

    2016-04-01

    Drosophila Ceramide Synthase (CerS) Schlank regulates both ceramide synthesis and fat metabolism. Schlank contains a catalytic lag1p motif and, like many CerS in other species, a homeodomain of unknown function. Here, we show that the Drosophila CerS Schlank is imported into the nucleus and requires two nuclear localization signals (NLSs) within its homeodomain and functional Importin-β import machinery. Expression of Schlank variants containing the homeodomain without functional lag1p motif rescued the fat metabolism phenotype of schlank mutants whereas a variant with a mutated NLS site did not rescue. Thus, the homeodomain of Schlank is involved in the regulation of lipid metabolism independent of the catalytic lag1p motif.

  1. Matrix metalloproteinase-12 gene regulation by a PPAR alpha agonist in human monocyte-derived macrophages

    SciTech Connect

    Souissi, Imen Jguirim; Billiet, Ludivine; Cuaz-Perolin, Clarisse; Rouis, Mustapha

    2008-11-01

    MMP-12, a macrophage-specific matrix metalloproteinase with large substrate specificity, has been reported to be highly expressed in mice, rabbits and human atherosclerotic lesions. Increased MMP-12 from inflammatory macrophages is associated with several degenerative diseases such as atherosclerosis. In this manuscript, we show that IL-1{beta}, a proinflammatory cytokine found in atherosclerotic plaques, increases both mRNA and protein levels of MMP-12 in human monocyte-derived macrophages (HMDM). Since peroxisome proliferator-activated receptors (PPARs), such as PPAR{alpha} and PPAR{gamma}, are expressed in macrophages and because PPAR activation exerts an anti-inflammatory effect on vascular cells, we have investigated the effect of PPAR{alpha} and {gamma} isoforms on MMP-12 regulation in HMDM. Our results show that MMP-12 expression (mRNA and protein) is down regulated in IL-1{beta}-treated macrophages only in the presence of a specific PPAR{alpha} agonist, GW647, in a dose-dependent manner. In contrast, this inhibitory effect was abolished in IL-1{beta}-stimulated peritoneal macrophages isolated from PPAR{alpha}{sup -/-} mice and treated with the PPAR{alpha} agonist, GW647. Moreover, reporter gene transfection experiments using different MMP-12 promoter constructs showed a reduction of the promoter activities by {approx} 50% in IL-1{beta}-stimulated PPAR{alpha}-pre-treated cells. However, MMP-12 promoter analysis did not reveal the presence of a PPRE response element. The IL-1{beta} effect is known to be mediated through the AP-1 binding site. Mutation of the AP-1 site, located at - 81 in the MMP-12 promoter region relative to the transcription start site, followed by transfection analysis, gel shift and ChIP experiments revealed that the inhibitory effect was the consequence of the protein-protein interaction between GW 647-activated PPAR{alpha} and c-Fos or c-Jun transcription factors, leading to inhibition of their binding to the AP-1 motif. These studies

  2. Age dependent regulation of bone-mass and renal function by the MEPE ASARM-motif

    PubMed Central

    Zelenchuk, Lesya V; Hedge, Anne-Marie; Rowe, Peter S N

    2015-01-01

    Context Mice with null mutations in Matrix Extracellular Phosphoglycoprotein (MEPE) have increased bone mass, increased trabecular density and abnormal cancellous bone (MN-mice). These defects worsen with age and MEPE over expression induces opposite effects. Also, Genome Wide Association studies show MEPE plays a major role in bone mass. We hypothesized the conserved C-terminal MEPE ASARM-motif is chiefly responsible for regulating bone mass and trabecular structure. Design To test our theory we over expressed C-terminal ASARM-peptide in MN-mice using the Col1α1 promoter (MNAt-mice). We then compared the bone and renal phenotypes of the MNAt-mouse with the MN-mouse and the X-linked hypophosphatemic rickets mouse (HYP). The HYP mouse over expresses ASARM-peptides and is defective for the PHEX gene. Results The MN-mouse developed increased bone mass, bone strength and trabecular abnormalities that worsened markedly with age. Defects in bone formation were chiefly responsible with suppressed sclerostin and increased active β-catenin. Increased uric acid levels also suggested abnormalities in purine-metabolism and a reduced fractional excretion of uric acid signaled additional renal transport changes. The MN mouse developed a worsening hyperphosphatemia and reduced FGF23 with age. An increase in the fractional excretion of phosphate (FEP) despite the hyperphosphatemia confirms an imbalance in kidney-intestinal phosphate regulation. Also, the MN mice showed an increased creatinine clearance suggesting hyperfiltration. A reversal of the MN bone-renal phenotype changes occurred with the MNAt mice including the apparent hyperfiltration. The MNAt mice also developed localized hypomineralization, hypophosphatemia and increased FGF23. Conclusions The C-terminal ASARM-motif plays a major role in regulating bone–mass and cancellous structure as mice age. In healthy mice, the processing and release of free ASARM-peptide is chiefly responsible for preserving normal bone and

  3. Extracting regulator activity profiles by integration of de novo motifs and expression data: characterizing key regulators of nutrient depletion responses in Streptomyces coelicolor

    PubMed Central

    Iqbal, Mudassar; Mast, Yvonne; Amin, Rafat; Hodgson, David A.; Wohlleben, Wolfgang; Burroughs, Nigel J.

    2012-01-01

    Determining transcriptional regulator activities is a major focus of systems biology, providing key insight into regulatory mechanisms and co-regulators. For organisms such as Escherichia coli, transcriptional regulator binding site data can be integrated with expression data to infer transcriptional regulator activities. However, for most organisms there is only sparse data on their transcriptional regulators, while their associated binding motifs are largely unknown. Here, we address the challenge of inferring activities of unknown regulators by generating de novo (binding) motifs and integrating with expression data. We identify a number of key regulators active in the metabolic switch, including PhoP with its associated directed repeat PHO box, candidate motifs for two SARPs, a CRP family regulator, an iron response regulator and that for LexA. Experimental validation for some of our predictions was obtained using gel-shift assays. Our analysis is applicable to any organism for which there is a reasonable amount of complementary expression data and for which motifs (either over represented or evolutionary conserved) can be identified in the genome. PMID:22406834

  4. Binding motifs in bacterial gene promoters modulate transcriptional effect of global regulators

    SciTech Connect

    Leuze, Michael Rex; Karpinets, Tatiana V; Syed, Mustafa H; Beliaev, Alexander S; Uberbacher, Edward C

    2012-01-01

    Bacterial gene regulation involves transcription factors (TFs) that influence the expression of many genes. Global regulators, including CRP (cAMP Receptor Protein), ArcA, and FNR, can modulate the transcriptional activity of multiple operons. The similarity of a regulatory element s sequence to a TF s consensus binding site (BS) and the position of the regulatory element in an operon promoter are considered the most important determinants of this TF s regulatory influence. In this study we explore the hypothesis that the number of TFBS half-sites (where a half-site is one half of the palindromic BS consensus sequence, which we shall refer to as a binding motif or a BM) of a global regulator in an operon s promoter plays an important role in the operon s transcriptional regulation. We examine empirical data from transcriptional profiling of the CRP regulon in Shewanella oneidenses MR 1 and Escherichia coli, and of the ArcA regulon in S. oneidenses MR 1. We compare the power of CRP BM counts and of full, symmetrical CRP TFBS characteristics, namely similarity to consensus and location, to predict CRP-induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full-length TFBS quality or location. Regression analysis indicates that IHF (Integration Host Factor) and ArcA have synergistic effects on CRP-induced gene transcription, positive and negative, respectively. Based on these results, we propose that the fine-tuning of bacterial transcriptional activity by CRP may involves not only the bending of the operon promoter, facilitated by CRP in cooperation with the histone-like protein IHF, but also the cumulative binding affinity of multiple weak BMs.

  5. Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators.

    PubMed

    Hosoda, Kazuo; Imamura, Aya; Katoh, Etsuko; Hatta, Tomohisa; Tachiki, Mari; Yamada, Hisami; Mizuno, Takeshi; Yamazaki, Toshimasa

    2002-09-01

    The B motif is a signature of type-B response regulators (ARRs) involved in His-to-Asp phosphorelay signal transduction systems in Arabidopsis. Homologous motifs occur widely in the GARP family of plant transcription factors. To gain general insight into the structure and function of B motifs (or GARP motifs), we characterized the B motif derived from a representative ARR, ARR10, which led to a number of intriguing findings. First, the B motif of ARR10 (named ARR10-B and extending from Thr-179 to Ser-242) possesses a nuclear localization signal, as indicated by the intracellular localization of a green fluorescent protein-ARR10-B fusion protein in onion epidermal cells. Second, the purified ARR10-B molecule binds specifically in vitro to DNA with the core sequence AGATT. This was demonstrated by several in vitro approaches, including PCR-assisted DNA binding site selection, gel retardation assays, and surface plasmon resonance analysis. Finally, the three-dimensional structure of ARR10-B in solution was determined by NMR spectroscopy, showing that it contains a helix-turn-helix structure. Furthermore, the mode of interaction between ARR10-B and the target DNA was assessed extensively by NMR spectroscopy. Together, these results lead us to propose that the mechanism of DNA recognition by ARR10-B is essentially the same as that of homeodomains. We conclude that the B motif is a multifunctional domain responsible for both nuclear localization and DNA binding and suggest that these insights could be applicable generally to the large GARP family of plant transcription factors. PMID:12215502

  6. Tumor Necrosis Factor alpha (TNF{alpha}) regulates CD40 expression through SMAR1 phosphorylation

    SciTech Connect

    Singh, Kamini; Sinha, Surajit; Malonia, Sunil Kumar; Chattopadhyay, Samit

    2010-01-08

    CD40 plays an important role in mediating inflammatory response and is mainly induced by JAK/STAT phosphorylation cascade. TNF{alpha} is the key cytokine that activates CD40 during inflammation and tumorigenesis. We have earlier shown that SMAR1 can repress the transcription of Cyclin D1 promoter by forming a HDAC1 dependent repressor complex. In this study, we show that SMAR1 regulates the transcription of NF-{kappa}B target gene CD40. SMAR1 recruits HDAC1 and forms a repressor complex on CD40 promoter and keeps its basal transcription in check. Further, we show that TNF{alpha} stimulation induces SMAR1 phosphorylation at Ser-347 and promotes its cytoplasmic translocation, thus releasing its negative effect. Concomitantly, TNF{alpha} induced phosphorylation of STAT1 at Tyr-701 by JAK1 facilitates its nuclear translocation and activation of CD40 through p300 recruitment and core Histone-3 acetylation. Thus, TNF{alpha} mediated regulation of CD40 expression occurs by dual phosphorylation of SMAR1 and STAT1.

  7. Lamin A reassembly at the end of mitosis is regulated by its SUMO-interacting motif.

    PubMed

    Moriuchi, Takanobu; Kuroda, Masaki; Kusumoto, Fumiya; Osumi, Takashi; Hirose, Fumiko

    2016-03-01

    Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

  8. Genome-Wide Analysis of Ethylene-Responsive Element Binding Factor-Associated Amphiphilic Repression Motif-Containing Transcriptional Regulators in Arabidopsis1[W][OA

    PubMed Central

    Kagale, Sateesh; Links, Matthew G.; Rozwadowski, Kevin

    2010-01-01

    The ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif is a transcriptional regulatory motif identified in members of the ethylene-responsive element binding factor, C2H2, and auxin/indole-3-acetic acid families of transcriptional regulators. Sequence comparison of the core EAR motif sites from these proteins revealed two distinct conservation patterns: LxLxL and DLNxxP. Proteins containing these motifs play key roles in diverse biological functions by negatively regulating genes involved in developmental, hormonal, and stress signaling pathways. Through a genome-wide bioinformatics analysis, we have identified the complete repertoire of the EAR repressome in Arabidopsis (Arabidopsis thaliana) comprising 219 proteins belonging to 21 different transcriptional regulator families. Approximately 72% of these proteins contain a LxLxL type of EAR motif, 22% contain a DLNxxP type of EAR motif, and the remaining 6% have a motif where LxLxL and DLNxxP are overlapping. Published in vitro and in planta investigations support approximately 40% of these proteins functioning as negative regulators of gene expression. Comparative sequence analysis of EAR motif sites and adjoining regions has identified additional preferred residues and potential posttranslational modification sites that may influence the functionality of the EAR motif. Homology searches against protein databases of poplar (Populus trichocarpa), grapevine (Vitis vinifera), rice (Oryza sativa), and sorghum (Sorghum bicolor) revealed that the EAR motif is conserved across these diverse plant species. This genome-wide analysis represents the most extensive survey of EAR motif-containing proteins in Arabidopsis to date and provides a resource enabling investigations into their biological roles and the mechanism of EAR motif-mediated transcriptional regulation. PMID:20097792

  9. Oncodevelopmental and hormonal regulation of alpha 1-fetoprotein gene expression.

    PubMed

    Belanger, L; Baril, P; Guertin, M; Gingras, M C; Gourdeau, H; Anderson, A; Hamel, D; Boucher, J M

    1983-01-01

    The main features of the oncodevelopmental biology of alpha 1-fetoprotein (AFP) are reviewed. Progress made in the molecular biology of AFP gene regulation is discussed and we present our recent data on the mechanisms of AFP suppression by glucocorticoid hormones. The relationship between AFP gene transcription and cell replication is examined, and it is suggested that the degree of methylation of the AFP gene (or of co-methylated regulatory DNA sequences) conditions its response to hormones.

  10. Transcriptional activation of human adult alpha-globin genes by hypersensitive site-40 enhancer: function of nuclear factor-binding motifs occupied in erythroid cells.

    PubMed Central

    Rombel, I; Hu, K Y; Zhang, Q; Papayannopoulou, T; Stamatoyannopoulos, G; Shen, C K

    1995-01-01

    The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element. Images Fig. 2 Fig. 3 Fig. 5 Fig. 6 PMID:7604012

  11. Divergent Protein Motifs Direct Elongation Factor P-Mediated Translational Regulation in Salmonella enterica and Escherichia coli

    PubMed Central

    Hersch, Steven J.; Wang, Mengchi; Zou, S. Betty; Moon, Kyung-Mee; Foster, Leonard J.; Ibba, Michael; Navarre, William Wiley

    2013-01-01

    ABSTRACT Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the Δefp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include both PPP and PPG but also additional motifs, such as APP and YIRYIR, which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing polyproline motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement for EF-P. This work expands the known repertoire of EF-P target motifs and implicates factors beyond polyproline motifs that are required for EF-P-mediated regulation. PMID:23611909

  12. Transcription Factor Tfe3 Directly Regulates Pgc-1alpha in Muscle.

    PubMed

    Salma, Nunciada; Song, Jun S; Arany, Zoltan; Fisher, David E

    2015-10-01

    The microphthalmia (MiT) family of transcription factors is an important mediator of metabolism. Family members Mitf and Tfeb directly regulate the expression of the master regulator of metabolism, peroxisome-proliferator activated receptor gamma coactivator-1 alpha (Pgc-1alpha), in melanomas and in the liver, respectively. Pgc-1alpha is enriched in tissues with high oxidative capacity and plays an important role in the regulation of mitochondrial biogenesis and cellular metabolism. In skeletal muscle, Pgc-1alpha affects many aspects of muscle functionally such as endurance, fiber-type switching, and insulin sensitivity. Tfe3 also regulates muscle metabolic genes that enhance insulin sensitivity in skeletal muscle. Tfe3 has not yet been shown to regulate Pgc-1alpha expression. Our results reported here show that Tfe3 directly regulates Pgc-1alpha expression in myotubes. Tfe3 ectopic expression induces Pgc-1alpha, and Tfe3 silencing suppresses Pgc-1alpha expression. This regulation is direct, as shown by Tfe3's binding to E-boxes on the Pgc-1alpha proximal promoter. We conclude that Tfe3 is a critical transcription factor that regulates Pgc-1alpha gene expression in myotubes. Since Pgc-1alpha coactivates numerous biological programs in diverse tissues, the regulation of its expression by upstream transcription factors such Tfe3 implies potential opportunities for the treatment of diseases where modulation of Pgc-1alpha expression may have important clinical outcomes.

  13. Binding Motifs in Bacterial Gene Promoters Modulate Transcriptional Effects of Global Regulators CRP and ArcA

    PubMed Central

    Leuze, Michael R.; Karpinets, Tatiana V.; Syed, Mustafa H.; Beliaev, Alexander S.; Uberbacher, Edward C.

    2012-01-01

    Bacterial gene regulation involves transcription factors (TF) that bind to DNA recognition sequences in operon promoters. These recognition sequences, many of which are palindromic, are known as regulatory elements or transcription factor binding sites (TFBS). Some TFs are global regulators that can modulate the expression of hundreds of genes. In this study we examine global regulator half-sites, where a half-site, which we shall call a binding motif (BM), is one half of a palindromic TFBS. We explore the hypothesis that the number of BMs plays an important role in transcriptional regulation, examining empirical data from transcriptional profiling of the CRP and ArcA regulons. We compare the power of BM counts and of full TFBS characteristics to predict induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full TFBS quality or location. PMID:22701314

  14. Binding motifs in bacterial gene promoters modulate transcriptional effects of global regulators CRP and ArcA

    SciTech Connect

    Leuze, Mike; Karpinets, Tatiana V.; Syed, Mustafa H.; Beliaev, Alex S.; Uberbacher, Edward

    2012-05-30

    Bacterial gene regulation involves transcription factors (TF) that bind to DNA recognition sequences in operon promoters. These recognition sequences, many of which are palindromic, are known as regulatory elements or transcription factor binding sites (TFBS). Some TFs are global regulators that can modulate the expression of hundreds of genes. In this study we examine global regulator half-sites, where a half-site, which we shall call a binding motif (BM), is one half of a palindromic TFBS. We explore the hypothesis that the number of BMs plays an important role in transcriptional regulation, examining empirical data from transcriptional profiling of the CRP and ArcA regulons. We compare the power of BM counts and of full TFBS characteristics to predict induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full TFBS quality or location.

  15. Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics.

    PubMed

    Brodsky, F M; Hill, B L; Acton, S L; Näthke, I; Wong, D H; Ponnambalam, S; Parham, P

    1991-06-01

    Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain. PMID:1909824

  16. Prediction of DNA binding motifs from 3D models of transcription factors; identifying TLX3 regulated genes

    PubMed Central

    Pujato, Mario; Kieken, Fabien; Skiles, Amanda A.; Tapinos, Nikos; Fiser, Andras

    2014-01-01

    Proper cell functioning depends on the precise spatio-temporal expression of its genetic material. Gene expression is controlled to a great extent by sequence-specific transcription factors (TFs). Our current knowledge on where and how TFs bind and associate to regulate gene expression is incomplete. A structure-based computational algorithm (TF2DNA) is developed to identify binding specificities of TFs. The method constructs homology models of TFs bound to DNA and assesses the relative binding affinity for all possible DNA sequences using a knowledge-based potential, after optimization in a molecular mechanics force field. TF2DNA predictions were benchmarked against experimentally determined binding motifs. Success rates range from 45% to 81% and primarily depend on the sequence identity of aligned target sequences and template structures, TF2DNA was used to predict 1321 motifs for 1825 putative human TF proteins, facilitating the reconstruction of most of the human gene regulatory network. As an illustration, the predicted DNA binding site for the poorly characterized T-cell leukemia homeobox 3 (TLX3) TF was confirmed with gel shift assay experiments. TLX3 motif searches in human promoter regions identified a group of genes enriched in functions relating to hematopoiesis, tissue morphology, endocrine system and connective tissue development and function. PMID:25428367

  17. An Unexpected Duo: Rubredoxin Binds Nine TPR Motifs to Form LapB, an Essential Regulator of Lipopolysaccharide Synthesis.

    PubMed

    Prince, Chelsy; Jia, Zongchao

    2015-08-01

    Lipopolysaccharide (LPS) synthesis and export are essential pathways for bacterial growth, proliferation, and virulence. The essential protein LapB from Escherichia coli has recently been identified as a regulator of LPS synthesis. We have determined the crystal structure of LapB (without the N-terminal transmembrane helix) at 2 Å resolution using zinc single-wavelength anomalous diffraction phasing derived from a single bound zinc atom. This structure demonstrates the presence of nine tetratricopeptide repeats (TPR) motifs, including two TPR folds that were not predicted from sequence, and a rubredoxin-type metal binding domain. The rubredoxin domain is bound intimately to the TPR motifs, which has not been previously observed or predicted. Mutations in the rubredoxin/TPR interface inhibit in vivo cell growth, and in vitro studies indicate that these modifications cause local displacement of rubredoxin from its binding site without changing the secondary structure of LapB. LapB is the first reported structure to contain both a rubredoxin domain and TPR motifs.

  18. The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets.

    PubMed

    Wee, Janet L; Jackson, Denise E

    2005-12-01

    Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect. PMID:16081692

  19. A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.

    PubMed

    Mughal, W; Nguyen, L; Pustylnik, S; da Silva Rosa, S C; Piotrowski, S; Chapman, D; Du, M; Alli, N S; Grigull, J; Halayko, A J; Aliani, M; Topham, M K; Epand, R M; Hatch, G M; Pereira, T J; Kereliuk, S; McDermott, J C; Rampitsch, C; Dolinsky, V W; Gordon, J W

    2015-01-01

    Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology. PMID:26512955

  20. Phosphoinositides differentially regulate alpha-actinin flexibility and function.

    PubMed

    Corgan, Anne Marie; Singleton, CoreyAyne; Santoso, Cynthia B; Greenwood, Jeffrey A

    2004-03-15

    Alpha-actinin is a cell-adhesion and cytoskeletal protein that bundles actin microfilaments and links these filaments directly to integrin-adhesion receptors. Phosphoinositides bind to and regulate the interaction of a-actinin with actin filaments and integrin receptors. In the present study, we demonstrate that PtdIns(3,4,5)P3 inhibits and disrupts a-actinin-bundling activity, whereas PtdIns(4,5)P2 can only inhibit activity. In addition, a protease-sensitivity assay was developed to examine the flexibility of the linker region between the actin-binding domain and the spectrin repeats of a-actinin. Both phosphoinositides influenced the extent of proteolysis and the cleavage sites. PtdIns(4,5)P2 binding decreased the proteolysis of a-actinin, suggesting a role in stabilizing the structure of the protein. In contrast, PtdIns(3,4,5)P3 binding enhanced a-actinin proteolysis, indicating an increase in the flexibility of the protein. Furthermore, phosphoinositide binding influenced the proteolysis of the N- and C-terminal domains of a-actinin, indicating regulation of structure within both domains. These results support the hypothesis that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 differentially regulate a-actinin function by modulating the structure and flexibility of the protein.

  1. Phosphoinositides differentially regulate alpha-actinin flexibility and function.

    PubMed Central

    Corgan, Anne Marie; Singleton, CoreyAyne; Santoso, Cynthia B; Greenwood, Jeffrey A

    2004-01-01

    Alpha-actinin is a cell-adhesion and cytoskeletal protein that bundles actin microfilaments and links these filaments directly to integrin-adhesion receptors. Phosphoinositides bind to and regulate the interaction of a-actinin with actin filaments and integrin receptors. In the present study, we demonstrate that PtdIns(3,4,5)P3 inhibits and disrupts a-actinin-bundling activity, whereas PtdIns(4,5)P2 can only inhibit activity. In addition, a protease-sensitivity assay was developed to examine the flexibility of the linker region between the actin-binding domain and the spectrin repeats of a-actinin. Both phosphoinositides influenced the extent of proteolysis and the cleavage sites. PtdIns(4,5)P2 binding decreased the proteolysis of a-actinin, suggesting a role in stabilizing the structure of the protein. In contrast, PtdIns(3,4,5)P3 binding enhanced a-actinin proteolysis, indicating an increase in the flexibility of the protein. Furthermore, phosphoinositide binding influenced the proteolysis of the N- and C-terminal domains of a-actinin, indicating regulation of structure within both domains. These results support the hypothesis that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 differentially regulate a-actinin function by modulating the structure and flexibility of the protein. PMID:14670080

  2. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    PubMed

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein. PMID:27182737

  3. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    PubMed

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein.

  4. Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells.

    PubMed

    Zia, S; Ndoye, A; Nguyen, V T; Grando, S A

    1997-09-01

    The purpose of this study was to investigate the possibility of direct toxic effects of nicotine (Nic) on human bronchial epithelial cells (BEC) suggested by our previous findings of functional nicotinic acetylcholine receptors (nAChRs) in the epithelial cells lining mucocutaneous membranes. We now demonstrate for the first time that human and murine BEC both in vivo and in vitro express functional nAChRs, and that classic alpha 3, alpha 4, alpha 5 and alpha 7 subunits can contribute to formation of these acetylcholine-gated ion channels. In human bronchial and mouse lung tissues, and in cultures of human BEC, the nAChRs were visualized by subunit-specific antibodies on the cell membranes, particularly at the sites of cell-to-cell contacts. The epithelial cells of submucosal glands abundantly expressed alpha 7 nAChRs. Smoking significantly (p < 0.05) increased the relative numbers of nAChRs, and this effects could be reproduced in cultures of BEC exposed to 10 microM Nic. At a higher dose, Nic decreased the relative numbers of alpha 5-containing nAChRs, suggesting a role for receptor desensitization. The function of the nAChR channels expressed by BEC was demonstrated by biphasic increase in the concentrations of intracellular calcium ([Ca++]i) in response to activation of the channel by Nic and fluctuations of [Ca++]i due to channel blockade by mecamylamine (Mec). Long-term exposure to milimolar concentrations of Nic resulted in a steady increase of [Ca++]i, which may lead to cell damage. The biological roles of epithelial nAChRs apparently involve regulation of cell-to-cell communications, adhesion and motility, because Mec caused rapid and profound changes in these cell functions which were reversible by Nic. An over exposure of BEC to Nic, however, produced an antagonist-like effect, suggesting that the pathobiological effects of Nic toxicity might result from both activation of nAChR channels and nAChR desensitization. We conclude that medical consequences of

  5. Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif.

    PubMed

    Serrano, Pedro; Aubol, Brandon E; Keshwani, Malik M; Forli, Stefano; Ma, Chen-Ting; Dutta, Samit K; Geralt, Michael; Wüthrich, Kurt; Adams, Joseph A

    2016-06-01

    Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2-RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function. PMID:27091468

  6. G alpha 12/13 basally regulates p53 through Mdm4 expression.

    PubMed

    Kim, Mi-Sung; Lee, Sang Min; Kim, Won Dong; Ki, Sung Hwan; Moon, Aree; Lee, Chang Ho; Kim, Sang Geon

    2007-05-01

    G alpha(12/13), which belongs to the G alpha(12) family, participates in the regulation of diverse physiologic processes. In view of the control of G alpha(12/13) in cell proliferation, this study investigated the role of G alpha(12/13) in the regulation of p53 and mdm4. Immunoblotting and immunocytochemistry revealed that p53 was expressed in control embryonic fibroblasts and was largely localized in the nuclei. G alpha(12) deficiency decreased p53 levels and its DNA binding activity, accompanying p21 repression with Bcl(2) induction, whereas G alpha(13) deficiency exerted weak effects. G alpha(12) or G alpha(13) deficiency did not change p53 mRNA expression. ERK1/2 or Akt was not responsible for p53 repression due to G alpha(12) deficiency. Mdm4, a p53-stabilizing protein, was repressed by G alpha(12) deficiency and to a lesser extent by G alpha(13) deficiency, whereas mdm2, PTEN, beta-catenin, ATM, and Chk2 were unaffected. p53 accumulation by proteasomal inhibition during G alpha(12) deficiency suggested the role of G alpha(12) in p53 stabilization. Constitutively active G alpha(12) (G alpha(12)QL) or G alpha(13) (G alpha(13)QL) promoted p53 accumulation with mdm4 induction in MCF10A cells. p53 accumulation by mdm4 overexpression, but no mdm4 induction by p53 overexpression, and small interfering RNA knockdown verified the regulatory role of mdm4 for p53 downstream of G alpha(12/13). In control or G alpha(12)/G alpha(13)-deficient cells, genotoxic stress led to p53 accumulation. At concentrations increasing the flow cytometric pre-G(1) phase, doxorubicin or etoposide treatment caused serine phosphorylations in G alpha(12)-/- or G alpha(12/13)-/- cells, but did not induce mdm4. G alpha(12/13)QL transfection failed to phosphorylate p53 at serines. Our results indicate that G alpha(12/13) regulate basal p53 levels via mdm4, which constitutes a cell signaling pathway distinct from p53 phosphorylations elicited by genotoxic stress.

  7. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

    SciTech Connect

    Tsukasaki, Masayuki; Yamada, Atsushi; Suzuki, Dai; Aizawa, Ryo; Miyazono, Agasa; Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro; Morimura, Naoko; Yamamoto, Matsuo; Kamijo, Ryutaro

    2011-07-15

    Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.

  8. Functional analysis of developmentally regulated chromatin-hypersensitive domains carrying the alpha 1-fetoprotein gene promoter and the albumin/alpha 1-fetoprotein intergenic enhancer.

    PubMed Central

    Bernier, D; Thomassin, H; Allard, D; Guertin, M; Hamel, D; Blaquière, M; Beauchemin, M; LaRue, H; Estable-Puig, M; Bélanger, L

    1993-01-01

    During liver development, the tandem alpha 1-fetoprotein (AFP)/albumin locus is triggered at the AFP end and then asymmetrically enhanced; this is followed by autonomous repression of the AFP-encoding gene. To understand this regulation better, we characterized the two early developmental stage-specific DNase I-hypersensitive (DH) sites so far identified in rat liver AFP/albumin chromatin: an intergenic DH-enhancer site and the AFP DH-promoter site. Mutation-transfection analyses circumscribed the DH-enhancer domain to a 200-bp DNA segment stringently conserved among species. Targeted mutations, DNA-protein-binding assays, and coexpression experiments pinpointed C/EBP as the major activatory component of the intergenic enhancer. Structure-function relationships at the AFP DH-promoter site defined a discrete glucocorticoid-regulated domain activated cooperatively by HNF1 and a highly specific AFP transcription factor, FTF, which binds to a steroid receptor recognition motif. The HNF1/FTF/DNA complex is deactivated by glucocorticoid receptors or by the ubiquitous factor NF1, which eliminates HNF1 by competition at an overlapping, high-affinity binding site. We propose that the HNF1-NF1 site might serve as a developmental switch to direct autonomous AFP gene repression in late liver development. We also conclude that the intergenic enhancer is driven by C/EBP alpha primarily to fulfill albumin gene activation functions at early developmental stages. Factor FTF seems to be the key regulator of AFP gene-specific functions in carcinoembryonic states. Images PMID:7680097

  9. Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

    PubMed

    Boudjelal, M; Wang, Z; Voorhees, J J; Fisher, G J

    2000-04-15

    Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of

  10. An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis.

    PubMed

    Oliver, Stefan L; Brady, Jennifer J; Sommer, Marvin H; Reichelt, Mike; Sung, Phillip; Blau, Helen M; Arvin, Ann M

    2013-01-29

    Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.

  11. Alpha-Synuclein Regulates Neuronal Levels of Manganese and Calcium.

    PubMed

    Dučić, Tanja; Carboni, Eleonora; Lai, Barry; Chen, Si; Michalke, Bernhard; Lázaro, Diana F; Outeiro, Tiago F; Bähr, Mathias; Barski, Elisabeth; Lingor, Paul

    2015-10-21

    Manganese (Mn) may foster aggregation of alpha-synuclein (αSyn) contributing to the pathogenesis of PD. Here, we examined the influence of αSyn overexpression on distribution and oxidation states of Mn in frozen-hydrated primary midbrain neurons (PMNs) by synchrotron-based X-ray fluorescence (XRF) and X-ray absorption near edge structure spectroscopy (XANES). Overexpression of αSyn increased intracellular Mn levels, whereas levels of Ca, Zn, K, P, and S were significantly decreased. Mn oxidation states were not altered. A strong correlation between Cu-/Mn-levels as well as Fe-/Mn-levels was observed in αSyn-overexpressing cells. Subcellular resolution revealed a punctate or filament-like perinuclear and neuritic distribution of Mn, which resembled the expression of DMT1 and MnSOD. While overexpression of αSyn did not significantly alter the expression patterns of the most-expressed Mn transport proteins (DMT1, VGCC, Fpn1), it attenuated the Mn release from Mn-treated neurons. Thus, these data suggest that αSyn may act as an intracellular Mn store. In total, neurotoxicity in PD could be mediated via regulation of transition metal levels and the metal-binding capacity of αSyn, which could represent a promising therapeutic target for this neurodegenerative disorder.

  12. The inter-alpha-inhibitor family: from structure to regulation.

    PubMed Central

    Salier, J P; Rouet, P; Raguenez, G; Daveau, M

    1996-01-01

    Inter-alpha-inhibitor (IalphaI) and related molecules, collectively referred to as the IalphaI family, are a group of plasma protease inhibitors. They display attractive features such as precursor polypeptides that give rise to mature chains with quite distinct fates and functions, and inter-chain glycosaminoglycan bonds within the various molecules. The discovery of an ever growing number of such molecules has raised pertinent questions about their pathophysiological functions. The knowledge of this family has long been structure-oriented, whereas the structure/function and structure/regulation relationships of the family members and their genes have been largely ignored. These relationships are now being elucidated in events such as gene transcription, precursor processing, changes in plasma protein levels in health and disease and binding capacities that involve hyaluronan as well as other plasma proteins as ligands. This review presents some recent progress made in these fields that paves the way for an understanding of the functions of IalphaI family members in vivo. Finally, given the wealth of heterogeneous, complicated and sometimes contradictory nomenclatures and acronyms currently in use for this family, a new, uniform, nomenclature is proposed for IalphaI family genes, precursor polypeptides and assembled proteins. PMID:8670091

  13. Alpha-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase.

    PubMed

    Manna, Sunil K; Sarkar, Abira; Sreenivasan, Yashin

    2006-03-01

    Considering the role of interleukin-8 (IL-8) in a large number of acute and chronic inflammatory diseases, the regulation of IL-8-mediated biological responses is important. Alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that alpha-MSH interacts predominantly with melanocortin-1 receptors and inhibits several IL-8-induced biological responses in macrophages and neutrophils. It down-regulated receptors for IL-8 but not for TNF, IL-4, IL-13 or TNF-related apoptosis-inducing ligand (TRAIL) in neutrophils. It down-regulated CXCR type 1 and 2 but not mRNA levels. alpha-MSH did not inhibit IL-8 binding in purified cell membrane or affinity-purified CXCR. IL-8 or anti-CXCR Ab protected against alpha-MSH-mediated inhibition of IL-8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in alpha-MSH-treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in alpha-MSH-induced down-regulation of CXCR. These studies suggest that alpha-MSH inhibits IL-8-mediated biological responses by down-regulating CXCR through induction of serine protease and that alpha-MSH acts as a potent immunomodulator in neutrophil-driven inflammatory distress. PMID:16479540

  14. CCAAT/enhancer binding protein alpha regulates p21 protein and hepatocyte proliferation in newborn mice.

    PubMed Central

    Timchenko, N A; Harris, T E; Wilde, M; Bilyeu, T A; Burgess-Beusse, B L; Finegold, M J; Darlington, G J

    1997-01-01

    CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP alpha suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP alpha blocks proteolytic degradation of p21. Our data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels. PMID:9372966

  15. A G-Box-Like Motif Is Necessary for Transcriptional Regulation by Circadian Pseudo-Response Regulators in Arabidopsis1[OPEN

    PubMed Central

    Newton, Linsey; Liu, Ming-Jung

    2016-01-01

    PSEUDO-RESPONSE REGULATORs (PRRs) play overlapping and distinct roles in maintaining circadian rhythms and regulating diverse biological processes, including the photoperiodic control of flowering, growth, and abiotic stress responses. PRRs act as transcriptional repressors and associate with chromatin via their conserved C-terminal CCT (CONSTANS, CONSTANS-like, and TIMING OF CAB EXPRESSION 1 [TOC1/PRR1]) domains by a still-poorly understood mechanism. Here, we identified genome-wide targets of PRR9 using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and compared them with PRR7, PRR5, and TOC1/PRR1 ChIP-seq data. We found that PRR binding sites are located within genomic regions of low nucleosome occupancy and high DNase I hypersensitivity. Moreover, conserved noncoding regions among Brassicaceae species are enriched around PRR binding sites, indicating that PRRs associate with functionally relevant cis-regulatory regions. The PRRs shared a significant number of binding regions, and our results indicate that they coordinately restrict the expression of target genes to around dawn. A G-box-like motif was overrepresented at PRR binding regions, and we showed that this motif is necessary for mediating transcriptional regulation of CIRCADIAN CLOCK ASSOCIATED 1 and PRR9 by the PRRs. Our results further our understanding of how PRRs target specific promoters and provide an extensive resource for studying circadian regulatory networks in plants. PMID:26586835

  16. Protein kinase A regulation of P2X(4) receptors: requirement for a specific motif in the C-terminus.

    PubMed

    Brown, David A; Yule, David I

    2010-02-01

    The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X(4)R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X(4)R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X(4)R expressing HEK-293 cells. To define which region of the P2X(4)R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X(4)R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X(4)R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\\ P2X(4)-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca(2+) signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X(4)R, markedly enhances ATP-stimulated P2X(4)R currents and hence cytosolic Ca(2+) signals. This may occur at least in part, by altering the trafficking of a population of

  17. A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens

    PubMed Central

    El Sahili, Abbas; Li, Si-Zhe; Lang, Julien; Virus, Cornelia; Planamente, Sara; Ahmar, Mohammed; Guimaraes, Beatriz G.; Aumont-Nicaise, Magali; Vigouroux, Armelle; Soulère, Laurent; Reader, John; Queneau, Yves; Faure, Denis; Moréra, Solange

    2015-01-01

    Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3’-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of

  18. A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

    PubMed

    El Sahili, Abbas; Li, Si-Zhe; Lang, Julien; Virus, Cornelia; Planamente, Sara; Ahmar, Mohammed; Guimaraes, Beatriz G; Aumont-Nicaise, Magali; Vigouroux, Armelle; Soulère, Laurent; Reader, John; Queneau, Yves; Faure, Denis; Moréra, Solange

    2015-08-01

    Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of

  19. Selective down-regulation of the alpha6-integrin subunit in melanocytes by UVB light.

    PubMed

    Krengel, Sven; Stark, Imke; Geuchen, Christian; Knoppe, Bettina; Scheel, Gabriele; Schlenke, Peter; Gebert, Andreas; Wünsch, Lutz; Brinckmann, Jürgen; Tronnier, Michael

    2005-06-01

    In vivo, melanocytes bind to laminin (LM) molecules of the basement membrane (BM) via the integrins alpha3beta1 and alpha6beta1, and they adhere to neighbouring keratinocytes via E-cadherin. Only few studies have addressed the impact of ultraviolet (UV) light on the interaction of melanocytes with their microenvironment. In this report, we examined the influence of UVB irradiation on the expression of the most important melanocyte-adhesion molecules (E-, N-cadherin, alpha2-, alpha3-, alpha5-, alpha6-, alphaV-, beta1-, beta3-integrins and ICAM-1) in vitro by flow cytometry. We were able to demonstrate that the alpha6-integrin subunit is selectively and reversibly down-regulated by UVB in a dwzm 150ose-dependent manner. In comparison, keratinocytes lacked UVB-inducible alterations in the expression of alpha6-integrin. In the presence of LM-1, the UVB-induced down-regulation of alpha6-integrin in melanocytes was significantly reduced. Moreover, LM-1 increased the resistance of melanocytes to UVB-induced cell death, as measured by annexinV-binding analysis. This effect was reversed by preincubation with an alpha6-integrin-blocking antibody. By immunofluorescence, we could demonstrate that UVB leads to a dose-dependent internalization of alpha6-integrin, providing an obvious explanation for the down-regulation on the outer cell surface observed by flow cytometry. We suggest that adhesion to LM-1 through alpha6-integrin represents a protective mechanism for melanocytes to withstand UVB damage. Through alpha6-integrin internalization, sunburns might alter the interaction between melanocytes and the BM, resulting in apoptosis induced by loss of anchorage (anoikis). Repeated sunburns may then lead to the selection of a population of melanocytes which are capable of anchorage-independent survival, culminating in solar nevogenesis and melanoma development.

  20. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface. PMID:23580642

  1. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.

  2. Genome-wide study predicts promoter-G4 DNA motifs regulate selective functions in bacteria: radioresistance of D. radiodurans involves G4 DNA-mediated regulation.

    PubMed

    Beaume, Nicolas; Pathak, Rajiv; Yadav, Vinod Kumar; Kota, Swathi; Misra, Hari S; Gautam, Hemant K; Chowdhury, Shantanu

    2013-01-01

    A remarkable number of guanine-rich sequences with potential to adopt non-canonical secondary structures called G-quadruplexes (or G4 DNA) are found within gene promoters. Despite growing interest, regulatory role of quadruplex DNA motifs in intrinsic cellular function remains poorly understood. Herein, we asked whether occurrence of potential G4 (PG4) DNA in promoters is associated with specific function(s) in bacteria. Using a normalized promoter-PG4-content (PG4(P)) index we analysed >60,000 promoters in 19 well-annotated species for (a) function class(es) and (b) gene(s) with enriched PG4(P). Unexpectedly, PG4-associated functional classes were organism specific, suggesting that PG4 motifs may impart specific function to organisms. As a case study, we analysed radioresistance. Interestingly, unsupervised clustering using PG4(P) of 21 genes, crucial for radioresistance, grouped three radioresistant microorganisms including Deinococcus radiodurans. Based on these predictions we tested and found that in presence of nanomolar amounts of the intracellular quadruplex-binding ligand N-methyl mesoporphyrin (NMM), radioresistance of D. radiodurans was attenuated by ~60%. In addition, important components of the RecF recombinational repair pathway recA, recF, recO, recR and recQ genes were found to harbour promoter-PG4 motifs and were also down-regulated in presence of NMM. Together these results provide first evidence that radioresistance may involve G4 DNA-mediated regulation and support the rationale that promoter-PG4s influence selective functions. PMID:23161683

  3. HMGI(Y) and Sp1 in addition to NF-kappa B regulate transcription of the MGSA/GRO alpha gene.

    PubMed Central

    Wood, L D; Farmer, A A; Richmond, A

    1995-01-01

    Expression of the chemokine MGSA/GRO is upregulated as melanocytes progress to melanoma cells. We demonstrate that constitutive and cytokine induced MGSA/GRO alpha expression requires multiple DNA regulatory regions between positions -143 to -62. We have previously shown that the NF-kappa B element at -83 to -65 is essential for basal and cytokine induced MGSA/GRO alpha promoter activity in the Hs294T melanoma and normal retinal pigment epithelial (RPE) cells, respectively. Here, we have determined that the Sp1 binding element located approximately 42 base pairs upstream from the NF-kappa B element binds Sp1 and Sp3 constitutively and this element is necessary for basal MGSA/GRO alpha promoter activity. We demonstrate that the high mobility group proteins HMGI(Y) recognize the AT-rich motif nested within the NF-kappa B element in the MGSA/GRO alpha promoter. Loss of either NF-kappa B or HMGI(Y) complex binding by selected point mutations in the NF-kappa B element results in decreased basal and cytokine induced MGSA/GRO alpha promoter activity. Thus, these results indicate that transcriptional regulation of the chemokine MGSA/GRO alpha requires at least three transcription factors: Sp1, NF-kappa B and HMGI(Y). Images PMID:7479086

  4. A lymphokine regulates expression of alpha-1-proteinase inhibitor in human monocytes and macrophages.

    PubMed Central

    Takemura, S; Rossing, T H; Perlmutter, D H

    1986-01-01

    Biosynthesis and secretion of alpha-1-proteinase inhibitor (alpha 1 PI) has been demonstrated in primary cultures of human mononuclear phagocytes, making it possible to study regulation of alpha 1 PI in normal (PiMM) and homozygous-deficient (PiZZ) individuals. In this study, expression of alpha 1 PI by blood monocytes, bronchoalveolar, and breast milk macrophages decreased during 1 wk in culture whereas expression of other secreted proteins increased. The addition of crude supernatants from mitogen-stimulated peripheral blood mononuclear cells to confluent monolayers of mononuclear phagocytes after 1 wk in culture resulted in a 2- to 2.5-fold increase in alpha 1 PI expression. The increase in alpha 1 PI expression was dose- and time-dependent, and involved a mechanism acting at a pretranslational level as shown by an increase in specific messenger RNA content corresponding to the increase in synthesis and secretion of alpha 1 PI. Although alpha 1 PI was expressed in native form and in forms complexed with serine protease by monocytes early in culture, it was expressed in its native form alone when monocytes were incubated with the lymphokine after 1 wk in culture. The regulating factor had the characteristics of a polypeptide and was derived from T lymphocytes, but it was not interferon-alpha, -beta, -gamma, or interleukin 2. This lymphokine also stimulated synthesis of alpha 1 PI in monocytes of homozygous-deficient PiZZ individuals, but had minimal effect on secretion, thereby increasing the intracellular accumulation of the inhibitor and exaggerating the defect in secretion of alpha 1 PI in these individuals. Regulation of mononuclear phagocyte alpha 1 PI expression by a lymphokine provides a model for further analysis of the effect of enhanced synthesis on a defect in posttranslational processing/secretion and for analysis of differential regulation of protease and inhibitor expressed in the same cells. Images PMID:3485658

  5. Condensin II Regulates Interphase Chromatin Organization Through the Mrg-Binding Motif of Cap-H2

    PubMed Central

    Wallace, Heather A.; Klebba, Joseph E.; Kusch, Thomas; Rogers, Gregory C.; Bosco, Giovanni

    2015-01-01

    The spatial organization of the genome within the eukaryotic nucleus is a dynamic process that plays a central role in cellular processes such as gene expression, DNA replication, and chromosome segregation. Condensins are conserved multi-subunit protein complexes that contribute to chromosome organization by regulating chromosome compaction and homolog pairing. Previous work in our laboratory has shown that the Cap-H2 subunit of condensin II physically and genetically interacts with the Drosophila homolog of human MORF4-related gene on chromosome 15 (MRG15). Like Cap-H2, Mrg15 is required for interphase chromosome compaction and homolog pairing. However, the mechanism by which Mrg15 and Cap-H2 cooperate to maintain interphase chromatin organization remains unclear. Here, we show that Cap-H2 localizes to interband regions on polytene chromosomes and co-localizes with Mrg15 at regions of active transcription across the genome. We show that co-localization of Cap-H2 on polytene chromosomes is partially dependent on Mrg15. We have identified a binding motif within Cap-H2 that is essential for its interaction with Mrg15, and have found that mutation of this motif results in loss of localization of Cap-H2 on polytene chromosomes and results in partial suppression of Cap-H2-mediated compaction and homolog unpairing. Our data are consistent with a model in which Mrg15 acts as a loading factor to facilitate Cap-H2 binding to chromatin and mediate changes in chromatin organization. PMID:25758823

  6. Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha

    PubMed Central

    Chorley, Brian N.; Campbell, Michelle R.; Wang, Xuting; Karaca, Mehmet; Sambandan, Deepa; Bangura, Fatu; Xue, Peng; Pi, Jingbo; Kleeberger, Steven R.; Bell, Douglas A.

    2012-01-01

    Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells’ SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis. PMID:22581777

  7. DNA Repair, Redox Regulation and Modulation of Estrogen Receptor Alpha Mediated Transcription

    ERIC Educational Resources Information Center

    Curtis-Ducey, Carol Dianne

    2009-01-01

    Interaction of estrogen receptor [alpha] (ER[alpha]) with 17[beta]-estradiol (E[subscript 2]) facilitates binding of the receptor to estrogen response elements (EREs) in target genes, which in turn leads to recruitment of coregulatory proteins. To better understand how estrogen-responsive genes are regulated, our laboratory identified a number of…

  8. Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

    2003-04-01

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  9. Cutting edge: the UNC93B1 tyrosine-based motif regulates trafficking and TLR responses via separate mechanisms.

    PubMed

    Pelka, Karin; Phulphagar, Kshiti; Zimmermann, Jana; Stahl, Rainer; Schmid-Burgk, Jonathan L; Schmidt, Tobias; Spille, Jan-Hendrik; Labzin, Larisa I; Agrawal, Sudhir; Kandimalla, Ekambar R; Casanova, Jean-Laurent; Hornung, Veit; Marshak-Rothstein, Ann; Höning, Stefan; Latz, Eicke

    2014-10-01

    Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.

  10. A Novel SUMO1-specific Interacting Motif in Dipeptidyl Peptidase 9 (DPP9) That Is Important for Enzymatic Regulation*

    PubMed Central

    Pilla, Esther; Möller, Ulrike; Sauer, Guido; Mattiroli, Francesca; Melchior, Frauke; Geiss-Friedlander, Ruth

    2012-01-01

    Sumoylation affects many cellular processes by regulating the interactions of modified targets with downstream effectors. Here we identified the cytosolic dipeptidyl peptidase 9 (DPP9) as a SUMO1 interacting protein. Surprisingly, DPP9 binds to SUMO1 independent of the well known SUMO interacting motif, but instead interacts with a loop involving Glu67 of SUMO1. Intriguingly, DPP9 selectively associates with SUMO1 and not SUMO2, due to a more positive charge in the SUMO1-loop. We mapped the SUMO-binding site of DPP9 to an extended arm structure, predicted to directly flank the substrate entry site. Importantly, whereas mutants in the SUMO1-binding arm are less active compared with wild-type DPP9, SUMO1 stimulates DPP9 activity. Consistent with this, silencing of SUMO1 leads to a reduced cytosolic prolyl-peptidase activity. Taken together, these results suggest that SUMO1, or more likely, a sumoylated protein, acts as an allosteric regulator of DPP9. PMID:23152501

  11. Transcriptional regulation of human retinoic acid receptor-alpha (RAR-{alpha}) by Wilms` tumour gene product

    SciTech Connect

    Goodyer, P.R.; Torban, E.; Dehbi, M.

    1994-09-01

    The Wilms` tumor gene encodes a 47-49 kDa transcription factor expressed in kidney, gonads and mesothelium during embryogenesis. Inherited mutations of WT1 lead to aberrant urogenital development and Wilms` tumor, but the role of WT1 in development is not fully understood. Since the human RAR-{alpha} gene contains a potential WT1 binding site at its 5{prime} end, we studied the effect of WT1 co-transfection on expression of an RAR-{alpha} promoter/CAT reporter construct in COS cells. COS cells were plated at 5X10{sup 5} cells/dish in DMEM with 10% FBS and transfected by the Ca/PO4 method with an expression plasmid containing the full-length WT1 (-/-) cDNA under the control of the CMV promoter, plasmid containing the RAR-{alpha} promoter (-519 to +36)/CAT reporter and TK/growth hormone plasmid to control for efficiency of transfection. CAT/GH activity at 48 hours was inhibited by co-transfection with increasing amounts of WT1 (-/-); maximum inhibition = 5% of control. WT1 co-transfection did not affect expression of TKGH, nor of a CMV-CAT vector. Expression of WT1 protein in tranfected COS cells was demonstrated by Western blotting. Minimal inhibiton of RAR-{alpha}/CAT activity was seen when cells were co-transfected with vectors containing WT1 deletion mutants, alternate WT1 splicing variants, or WT1 (-/-) cDNA bearing a mutation identified in a patient with Drash syndrome. Gel shift assays indicated binding of WT1 to RAR-{alpha} cDNA but not to an RAR-{alpha} deletion mutant lacking the GCGGGGGGCG site. These observations suggest that WT1 may function to regulate RAR-{alpha} expression during normal development.

  12. Role of PDZ Proteins in Regulating Trafficking, Signaling, and Function of GPCRs: Means, Motif, and Opportunity

    PubMed Central

    Romero, Guillermo; von Zastrow, Mark; Friedman, Peter A.

    2016-01-01

    PDZ proteins, named for the common structural domain shared by the postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (ZO-1), constitute a family of 200–300 recognized members. These cytoplasmic adapter proteins are capable of assembling a variety of membrane-associated proteins and signaling molecules in short-lived functional units. Here, we review PDZ proteins that participate in the regulation of signaling, trafficking, and function of G protein-coupled receptors. Salient structural features of PDZ proteins that allow them to recognize targeted GPCRs are considered. Scaffolding proteins harboring PDZ domains may contain single or multiple PDZ modules and may also include other protein–protein interaction modules. PDZ proteins may impact receptor signaling by diverse mechanisms that include retaining the receptor at the cell membrane, thereby increasing the duration of ligand binding, as well as importantly influencing GPCR internalization, trafficking, recycling, and intracellular sorting. PDZ proteins are also capable of modifying the assembled complex of accessory proteins such as β-arrestins that themselves regulate GPCR signaling. Additionally, PDZ proteins may modulate GPCR signaling by altering the G protein to which the receptor binds, or affect other regulatory proteins that impact GTPase activity, protein kinase A, phospholipase C, or modify downstream signaling events. Small molecules targeting the PDZ protein-GPCR interaction are being developed and may become important and selective drug candidates. PMID:21907913

  13. Plasma membrane CFTR regulates RANTES expression via its C-terminal PDZ-interacting motif.

    PubMed

    Estell, Kim; Braunstein, Gavin; Tucker, Torry; Varga, Karoly; Collawn, James F; Schwiebert, Lisa M

    2003-01-01

    Despite the identification of 1,000 mutations in the cystic fibrosis gene product CFTR, there remains discordance between CFTR genotype and lung disease phenotype. The study of CFTR, therefore, has expanded beyond its chloride channel activity into other possible functions, such as its role as a regulator of gene expression. Findings indicate that CFTR plays a role in the expression of RANTES in airway epithelia. RANTES is a chemokine that has been implicated in the regulation of mucosal immunity and the pathogenesis of airway inflammatory diseases. Results demonstrate that CFTR triggers RANTES expression via a mechanism that is independent of CFTR's chloride channel activity. Neither pharmacological inhibition of CFTR nor activation of alternative chloride channels, including hClC-2, modulated RANTES expression. Through the use of CFTR disease-associated and truncation mutants, experiments suggest that CFTR-mediated transcription factor activation and RANTES expression require (i) insertion of CFTR into the plasma membrane and (ii) an intact CFTR C-terminal PDZ-interacting domain. Expression of constructs encoding wild-type or dominant-negative forms of the PDZ-binding protein EBP50 suggests that EBP50 may be involved in CFTR-dependent RANTES expression. Together, these data suggest that CFTR modulates gene expression in airway epithelial cells while located in a macromolecular signaling complex at the plasma membrane. PMID:12509457

  14. Top-level dynamics and the regulated gene response of feed-forward loop transcriptional motifs

    NASA Astrophysics Data System (ADS)

    Mayo, Michael; Abdelzaher, Ahmed; Perkins, Edward J.; Ghosh, Preetam

    2014-09-01

    Feed-forward loops are hierarchical three-node transcriptional subnetworks, wherein a top-level protein regulates the activity of a target gene via two paths: a direct-regulatory path, and an indirect route, whereby the top-level proteins act implicitly through an intermediate transcription factor. Using a transcriptional network of the model bacterium Escherichia coli, we confirmed that nearly all types of feed-forward loop were significantly overrepresented in the bacterial network. We then used mathematical modeling to study their dynamics by manipulating the rise times of the top-level protein concentration, termed the induction time, through alteration of the protein destruction rates. Rise times of the regulated proteins exhibited two qualitatively different regimes, depending on whether top-level inductions were "fast" or "slow." In the fast regime, rise times were nearly independent of rapid top-level inductions, indicative of biological robustness, and occurred when RNA production rate-limits the protein yield. Alternatively, the protein rise times were dependent upon slower top-level inductions, greater than approximately one bacterial cell cycle. An equation is given for this crossover, which depends upon three parameters of the direct-regulatory path: transcriptional cooperation at the DNA-binding site, a protein-DNA dissociation constant, and the relative magnitude of the top-level protien concentration.

  15. Top-level dynamics and the regulated gene response of feed-forward loop transcriptional motifs.

    PubMed

    Mayo, Michael; Abdelzaher, Ahmed; Perkins, Edward J; Ghosh, Preetam

    2014-09-01

    Feed-forward loops are hierarchical three-node transcriptional subnetworks, wherein a top-level protein regulates the activity of a target gene via two paths: a direct-regulatory path, and an indirect route, whereby the top-level proteins act implicitly through an intermediate transcription factor. Using a transcriptional network of the model bacterium Escherichia coli, we confirmed that nearly all types of feed-forward loop were significantly overrepresented in the bacterial network. We then used mathematical modeling to study their dynamics by manipulating the rise times of the top-level protein concentration, termed the induction time, through alteration of the protein destruction rates. Rise times of the regulated proteins exhibited two qualitatively different regimes, depending on whether top-level inductions were "fast" or "slow." In the fast regime, rise times were nearly independent of rapid top-level inductions, indicative of biological robustness, and occurred when RNA production rate-limits the protein yield. Alternatively, the protein rise times were dependent upon slower top-level inductions, greater than approximately one bacterial cell cycle. An equation is given for this crossover, which depends upon three parameters of the direct-regulatory path: transcriptional cooperation at the DNA-binding site, a protein-DNA dissociation constant, and the relative magnitude of the top-level protien concentration.

  16. PPAR{alpha} is a key regulator of hepatic FGF21

    SciTech Connect

    Lundasen, Thomas; Hunt, Mary C.; Nilsson, Lisa-Mari; Sanyal, Sabyasachi; Angelin, Bo; Alexson, Stefan E.H.; Rudling, Mats . E-mail: mats.rudling@cnt.ki.se

    2007-08-24

    The metabolic regulator fibroblast growth factor 21 (FGF21) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF21 is regulated by the peroxisome proliferator-activated receptor alpha (PPAR{alpha}). Fasting or treatment of mice with the PPAR{alpha} agonist Wy-14,643 induced FGF21 mRNA by 10-fold and 8-fold, respectively. In contrast, FGF21 mRNA was low in PPAR{alpha} deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF21. Obese ob/ob mice, known to have increased PPAR{alpha} levels, displayed 12-fold increased hepatic FGF21 mRNA levels. The potential importance of PPAR{alpha} for FGF21 expression also in human liver was shown by Wy-14,643 induction of FGF21 mRNA in human primary hepatocytes, and PPAR{alpha} response elements were identified in both the human and mouse FGF21 promoters. Further studies on the mechanisms of regulation of FGF21 by PPAR{alpha} in humans will be of great interest.

  17. Integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

    SciTech Connect

    Sansing, Hope A.; Sarkeshik, Ali; Yates, John R.; Patel, Vyomesh; Gutkind, J. Silvio; Yamada, Kenneth M.; Berrier, Allison L.

    2011-03-11

    Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.

  18. c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells.

    PubMed Central

    Nakao, K; Lawless, D; Ohe, Y; Miyao, Y; Nakabayashi, H; Kamiya, H; Miura, K; Ohtsuka, E; Tamaoki, T

    1990-01-01

    We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif). In contrast, rasVal-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-S1 genes even though these promoters contain homologous A + T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-S1 promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli. Images PMID:1690841

  19. Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1

    PubMed Central

    Choi, Young Jun

    2016-01-01

    AU-rich element binding/degradation factor 1 (AUF1) plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE) in the 3′-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM) and a Gln- (Q-) rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1) was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding. PMID:27437398

  20. Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1.

    PubMed

    Choi, Young Jun; Yoon, Je-Hyun; Chang, Jeong Ho

    2016-01-01

    AU-rich element binding/degradation factor 1 (AUF1) plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE) in the 3'-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM) and a Gln- (Q-) rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1) was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding. PMID:27437398

  1. IQ Motif-Containing GTPase-Activating Protein 2 (IQGAP2) Is a Novel Regulator of Colonic Inflammation in Mice

    PubMed Central

    Ghaleb, Amr M.; Bialkowska, Agnieszka B.; Snider, Ashley J.; Gnatenko, Dmitri V.; Hannun, Yusuf A.; Yang, Vincent W.; Schmidt, Valentina A.

    2015-01-01

    IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca2+/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer. PMID:26047140

  2. The quorum-sensing regulator ComA from Bacillus subtilis activates transcription using topologically distinct DNA motifs

    PubMed Central

    Wolf, Diana; Rippa, Valentina; Mobarec, Juan Carlos; Sauer, Patricia; Adlung, Lorenz; Kolb, Peter; Bischofs, Ilka B.

    2016-01-01

    ComA-like transcription factors regulate the quorum response in numerous Gram-positive bacteria. ComA proteins belong to the tetrahelical helix-turn-helix superfamily of transcriptional activators, which bind as homodimers to inverted sequence repeats in the DNA. Here, we report that ComA from Bacillus subtilis recognizes a topologically distinct motif, in which the binding elements form a direct repeat. We provide in vitro and in vivo evidence that the canonical and non-canonical site play an important role in facilitating type I and type II promoter activation, respectively, by interacting with different subunits of RNA polymerase. We furthermore show that there is a variety of contexts in which the non-canonical site can occur and identify new direct target genes that are located within the integrative and conjugative element ICEBs1. We therefore suggest that ComA acts as a multifunctional transcriptional activator and provides a striking example for complexity in protein–DNA interactions that evolved in the context of quorum sensing. PMID:26582911

  3. Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function

    PubMed Central

    Bach, Harold H; Wong, Yee M; Tripathi, Abhishek; Nevins, Amanda M; Gamelli, Richard L; Volkman, Brian F; Byron, Kenneth L; Majetschak, Matthias

    2014-01-01

    Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α1-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α1-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α1-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca2+ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α1-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α1-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure. PMID:25032954

  4. Ginsenoside Rf, a component of ginseng, regulates lipoprotein metabolism through peroxisome proliferator-activated receptor {alpha}

    SciTech Connect

    Lee, Hyunghee; Gonzalez, Frank J.; Yoon, Michung . E-mail: yoon60@mokwon.ac.kr

    2006-01-06

    We investigated whether ginseng regulates lipoprotein metabolism by altering peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})-mediated pathways, using a PPAR{alpha}-null mouse model. Administration of ginseng extract, ginsenosides, and ginsenoside Rf (Rf) to wild-type mice not only significantly increased basal levels of hepatic apolipoprotein (apo) A-I and C-III mRNA compared with wild-type controls, but also substantially reversed the reductions in mRNA levels of apo A-I and C-III expected following treatment with the potent PPAR{alpha} ligand Wy14,643. In contrast, no effect was detected in the PPAR{alpha}-null mice. Testing of eight main ginsenosides on PPAR{alpha} reporter gene expression indicated that Rf was responsible for the effects of ginseng on lipoprotein metabolism. Furthermore, the inhibition of PPAR{alpha}-dependent transactivation by Rf seems to occur at the level of DNA binding. These results demonstrate that ginseng component Rf regulates apo A-I and C-III mRNA and the actions of Rf on lipoprotein metabolism are mediated via interactions with PPAR{alpha}.

  5. Transcriptional regulation of collagenase-3 by interleukin-1 alpha in osteoblasts.

    PubMed

    Varghese, Samuel; Canalis, Ernesto

    2003-12-01

    Interleukin-1 (IL-1)alpha is an autocrine/paracrine agent of the skeletal tissue and it regulates bone remodeling. Collagenase-3 or matrix metalloproteinase (MMP)-13 is expressed in osteoblasts and its expression is modulated by several cytokines including IL-1alpha. Because the molecular mechanism of increased synthesis of collagenase-3 in bone cells by IL-1alpha is not known, we investigated if collagenase-3 expression by IL-1alpha in osteoblasts is mediated by transcriptional or post-transcriptional mechanisms. Exposure of rat osteoblastic cultures (Ob cells) to IL-1alpha at concentrations higher than 0.5 nM increased the synthesis of collagenase-3 mRNA up to eightfold and the secretion of immunoreactive protein up to 21-fold. The effects of IL-1alpha on collagenase-3 were time- and dose-dependent. Although prostaglandins stimulate collagenase-3 expression, stimulation of collagenase-3 in Ob cells by IL-1alpha was not mediated through increased biosynthesis of prostaglandins. The half-life of collagenase-3 mRNA from control and IL-1alpha-treated Ob cells was similar suggesting that the stabilization of collagenase-3 mRNA did not contribute to the increase in collagenase-3. However, IL-1alpha stimulated the rate of transcription of the collagenase-3 gene by twofold to fourfold indicating regulation of collagenase-3 expression in Ob cells at the transcriptional level. Stimulation of collagenase-3 by IL-1alpha in osteoblasts may in part mediate the effects of IL-1alpha in bone metabolism.

  6. Tumor necrosis factor alpha regulates in vivo intrapulmonary expression of ICAM-1.

    PubMed Central

    Mulligan, M. S.; Vaporciyan, A. A.; Miyasaka, M.; Tamatani, T.; Ward, P. A.

    1993-01-01

    Lung injury following deposition of IgG immune complexes is neutrophil-dependent and requires both tumor necrosis factor alpha (TNF alpha) and CD18. In the current studies, we have evaluated the relationship between TNF alpha and expression of intracellular adhesion molecule-1 (ICAM-1) in vitro and in vivo. In both rat pulmonary artery endothelial cells and human umbilical vein endothelial cells, TNF alpha induced an early (within 60 minutes) increase in ICAM-1 expression, followed by a peak at 6 to 8 hours, with relatively stable expression at 24 hours. Expression of E-selectin did not show the early phase (within 60 minutes) of up-regulation, peaked at 4 hours, and then declined thereafter. Using a radioimmunochemical assay in vivo, it was demonstrated that intrapulmonary deposition of IgG immune complexes caused a progressive increase in ICAM-1 expression in lung over an 8-hour period. In animals pretreated with antibody to TNF alpha, the intrapulmonary expression of ICAM-1 was significantly reduced. These results were confirmed by immunoperoxidase analysis of lung tissue. It was also shown that airway instillation of TNF alpha caused up-regulation of ICAM-1 in lung. These data support the concept that deposition of IgG immune complexes in lung induces intrapulmonary up-regulation of ICAM-1 in a manner that is TNF alpha-dependent. Images Figure 2 Figure 7 PMID:7685152

  7. A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression

    PubMed Central

    Venot, Quitterie; Delaunay, Jean-Louis; Fouassier, Laura; Delautier, Danièle; Falguières, Thomas; Housset, Chantal; Maurice, Michèle; Aït-Slimane, Tounsia

    2016-01-01

    ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1) domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL), which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane. PMID:26789121

  8. Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

    PubMed Central

    Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

    1995-01-01

    The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7542585

  9. Integrin engagement by the helical RGD motif of the Helicobacter pylori CagL protein is regulated by pH-induced displacement of a neighboring helix.

    PubMed

    Bonsor, Daniel A; Pham, Kieu T; Beadenkopf, Robert; Diederichs, Kay; Haas, Rainer; Beckett, Dorothy; Fischer, Wolfgang; Sundberg, Eric J

    2015-05-15

    Arginine-aspartate-glycine (RGD) motifs are recognized by integrins to bridge cells to one another and the extracellular matrix. RGD motifs typically reside in exposed loop conformations. X-ray crystal structures of the Helicobacter pylori protein CagL revealed that RGD motifs can also exist in helical regions of proteins. Interactions between CagL and host gastric epithelial cell via integrins are required for the translocation of the bacterial oncoprotein CagA. Here, we have investigated the molecular basis of the CagL-host cell interactions using structural, biophysical, and functional analyses. We solved an x-ray crystal structure of CagL that revealed conformational changes induced by low pH not present in previous structures. Using analytical ultracentrifugation, we found that pH-induced conformational changes in CagL occur in solution and not just in the crystalline environment. By designing numerous CagL mutants based on all available crystal structures, we probed the functional roles of CagL conformational changes on cell surface integrin engagement. Together, our data indicate that the helical RGD motif in CagL is buried by a neighboring helix at low pH to inhibit CagL binding to integrin, whereas at neutral pH the neighboring helix is displaced to allow integrin access to the CagL RGD motif. This novel molecular mechanism of regulating integrin-RGD motif interactions by changes in the chemical environment provides new insight to H. pylori-mediated oncogenesis.

  10. Regulation of Class A scavenger receptor-mediated cell adhesion and surface localization by PI3K: identification of a regulatory cytoplasmic motif

    PubMed Central

    Cholewa, Jill; Nikolic, Dejan; Post, Steven R.

    2010-01-01

    The importance of cytoplasmic motifs in differentially regulating SR-A function was demonstrated by deleting the first 49 cytoplasmic aa (SR-AΔ1–49), which abolished SR-A-mediated ligand internalization without reducing cell adhesion. To identify additional cytoplasmic motifs within the first 49 aa that regulate SR-A function, the acidic residues in a conserved motif (EDAD) were changed to their amide derivatives (SR-AQNAN). The function and regulation of SR-AQNAN were compared with that of SR-AΔ1–49 and SR-A in transfected HEK-293 cells. Blocking PI3K activation inhibited SR-A, but not SR-AΔ1–49- or SR-AQNAN-mediated cell adhesion. Although deleting (SR-AΔ1–49) or mutating (SR-AQNAN) the EDAD motif abolished the PI3K sensitivity of SR-A-mediated cell adhesion, these mutations did not affect ligand internalization or PI3K activation during cell adhesion. To define the mechanism by which PI3K regulates SR-A-mediated cell adhesion, the cellular localization of wild-type and mutant SR-A was examined. PI3K inhibition reduced surface localization of SR-A but not of SR-AΔ1–49 or SR-AQNAN. The regulation of SR-A surface localization by PI3K was confirmed in peritoneal macrophages, which endogenously express SR-A. Together, these results suggest a pathway in which SR-A binding to an immobilized ligand activates PI3K to recruit more receptor to the plasma membrane and enhances cell adhesion. PMID:19952357

  11. Fibronectin-bound TNF-alpha stimulates monocyte matrix metalloproteinase-9 expression and regulates chemotaxis.

    PubMed

    Vaday, G G; Hershkoviz, R; Rahat, M A; Lahat, N; Cahalon, L; Lider, O

    2000-11-01

    Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF-alpha avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF-alpha complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM-bound TNF-alpha by examining its effects on MMP-9 secretion by monocytes. Immunofluorescent staining indicated that LPS-activated monocytes deposited newly synthesized TNF-alpha into ECM-FN. FN-bound TNF-alpha (FN/TNF-alpha) significantly up-regulated MMP-9 expression and secretion by the human monocytic cell line MonoMac-6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF-alpha activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Cheniotaxis through ECM gels in the presence of soluble or bound TNF-alpha was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac-6 cells to FN/TNF-alpha required functional activated beta1 integrins, FN/TNF-alpha-induced MMP-9 secretion was independent of binding to beta1 integrins, since MMP-9 secretion was unaffected by: (1) neutralizing nAb to alpha4, alpha5, and beta1 subunits, which blocked cell adhesion; (2) a mAb that stimulated beta1 integrin-mediated adhesion; and (3) binding TNF-alpha to the 30-kDa amino-terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell-adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM-degrading enzymes. These TNF-alpha

  12. [Prediction of Promoter Motifs in Virophages].

    PubMed

    Gong, Chaowen; Zhou, Xuewen; Pan, Yingjie; Wang, Yongjie

    2015-07-01

    Virophages have crucial roles in ecosystems and are the transport vectors of genetic materials. To shed light on regulation and control mechanisms in virophage--host systems as well as evolution between virophages and their hosts, the promoter motifs of virophages were predicted on the upstream regions of start codons using an analytical tool for prediction of promoter motifs: Multiple EM for Motif Elicitation. Seventeen potential promoter motifs were identified based on the E-value, location, number and length of promoters in genomes. Sputnik and zamilon motif 2 with AT-rich regions were distributed widely on genomes, suggesting that these motifs may be associated with regulation of the expression of various genes. Motifs containing the TCTA box were predicted to be late promoter motif in mavirus; motifs containing the ATCT box were the potential late promoter motif in the Ace Lake mavirus . AT-rich regions were identified on motif 2 in the Organic Lake virophage, motif 3 in Yellowstone Lake virophage (YSLV)1 and 2, motif 1 in YSLV3, and motif 1 and 2 in YSLV4, respectively. AT-rich regions were distributed widely on the genomes of virophages. All of these motifs may be promoter motifs of virophages. Our results provide insights into further exploration of temporal expression of genes in virophages as well as associations between virophages and giant viruses. PMID:26524912

  13. PKC{alpha} expression regulated by Elk-1 and MZF-1 in human HCC cells

    SciTech Connect

    Hsieh, Y.-H.; Wu, T.-T.; Tsai, J.-H.; Huang, C.-Y.; Hsieh, Y.-S.; Liu, J.-Y. . E-mail: jyl@csmu.edu.tw

    2006-01-06

    Our previous study found that PKC{alpha} was highly expressed in the poor-differentiated human HCC cells and associated with cell migration and invasion. In this study, we further investigated the gene regulation of this enzyme. We showed that PKC{alpha} expression enhancement in the poor-differentiated human HCC cells was found neither by DNA amplification nor by increasing mRNA stability using differential PCR and mRNA decay assays. After screening seven transcription factors in the putative cis-acting regulatory elements of human PKC{alpha} promoters, only Elk-1 and MZF-1 antisense oligonucleotide showed a significant reduction in the PKC{alpha} mRNA level. They also reduced cell proliferation, cell migratory and invasive capabilities, and DNA binding activities in the PKC{alpha} promoter region. Over-expression assay confirmed that the PKC{alpha} expression may be modulated by these two factors at the transcriptional level. Therefore, these results may provide a novel mechanism for PKC{alpha} expression regulation in human HCC cells.

  14. Androgen regulated expression of the alpha 2u-globulin gene in pancreatic hepatocytes of rat

    PubMed Central

    1990-01-01

    Under a copper-deficient regimen, pancreatic cells in the adult rat can be found to undergo differentiation into hepatocytes. Pancreatic hepatocytes induced in male and female rats were examined for the expression of the androgen-inducible hepatic protein, alpha 2u- globulin. Alpha 2u-Globulin protein was demonstrable by immunoperoxidase method in all the pancreatic hepatocytes of male rats. Northern blot analysis confirmed the presence of 1.3 kb alpha 2u- globulin mRNA transcript in the pancreas of male rats with hepatocytes. Orchiectomy resulted in marked decrease of alpha 2u-globulin protein and its mRNA. Administration of dihydrotestosterone to castrated rats resulted in increased levels of alpha 2u-globulin mRNA and the amount of alpha 2u-globulin protein in the pancreatic hepatocytes. Unlike normal males, in intact and ovariectomized females alpha 2u-globulin was not detectable in pancreatic hepatocytes. These results indicate that similar to hepatic parenchymal cells pancreatic hepatocytes synthesize alpha 2u-globulin under androgenic regulation. Furthermore, unlike in liver where it is expressed predominantly in perivenular and midlobular hepatocytes, there is no localized difference in the expression of this gene in the transdifferentiated pancreatic hepatocytes. PMID:1688854

  15. Consensus PP1 binding motifs regulate transcriptional corepression and alternative RNA splicing activities of the steroid receptor coregulators, p54nrb and PSF.

    PubMed

    Liu, Liangliang; Xie, Ning; Rennie, Paul; Challis, John R G; Gleave, Martin; Lye, Stephen J; Dong, Xuesen

    2011-07-01

    Originally identified as essential pre-mRNA splicing factors, non-POU-domain-containing, octamer binding protein (p54nrb) and PTB-associated RNA splicing factor (PSF) are also steroid receptor corepressors. The mechanisms by which p54nrb and PSF regulate gene transcription remain unclear. Both p54nrb and PSF contain protein phosphatase 1 (PP1) consensus binding RVxF motifs, suggesting that PP1 may regulate phosphorylation status of p54nrb and PSF and thus their function in gene transcription. In this report, we demonstrated that PP1 forms a protein complex with both p54nrb and PSF. PP1 interacts directly with the RVxF motif only in p54nrb, but not in PSF. Association with PP1 results in dephosphorylation of both p54nrb and PSF in vivo and the loss of their transcriptional corepressor activities. Using the CD44 minigene as a reporter, we showed that PP1 regulates p54nrb and PSF alternative splicing activities that determine exon skipping vs. inclusion in the final mature RNA for translation. In addition, changes in transcriptional corepression and RNA splicing activities of p54nrb and PSF are correlated with alterations in protein interactions of p54nrb and PSF with transcriptional corepressors such as Sin3A and histone deacetylase 1, and RNA splicing factors such as U1A and U2AF. Furthermore, we demonstrated a novel function of the RVxF motif within PSF that enhances its corepression and RNA splicing activities independent of PP1. We conclude that the RVxF motifs play an important role in controlling the multifunctional properties of p54nrb and PSF in the regulation of gene transcription.

  16. The orphan nuclear receptor SHP regulates PGC-1alpha expression and energy production in brown adipocytes.

    PubMed

    Wang, Li; Liu, Jun; Saha, Pradip; Huang, Jiansheng; Chan, Lawrence; Spiegelman, Bruce; Moore, David D

    2005-10-01

    Brown adipocytes increase energy production in response to induction of PGC-1alpha, a dominant regulator of energy metabolism. We have found that the orphan nuclear receptor SHP (NR0B2) is a negative regulator of PGC-1alpha expression in brown adipocytes. Mice lacking SHP show increased basal expression of PGC-1alpha, increased energy expenditure, and resistance to diet-induced obesity. Increased PGC-1alpha expression in SHP null brown adipose tissue is not due to beta-adrenergic activation, since it is also observed in primary cultures of SHP(-/-) brown adipocytes that are not exposed to such stimuli. In addition, acute inhibition of SHP expression in cultured wild-type brown adipocytes increases basal PGC-1alpha expression, and SHP overexpression in SHP null brown adipocytes decreases it. The orphan nuclear receptor ERRgamma is expressed in BAT and its transactivation of the PGC-1alpha promoter is potently inhibited by SHP. We conclude that SHP functions as a negative regulator of energy production in BAT.

  17. Down-regulation of the G-proteins Gq alpha and G11 alpha by transfected human M3 muscarinic acetylcholine receptors in Chinese hamster ovary cells is independent of receptor down-regulation.

    PubMed Central

    van de Westerlo, E; Yang, J; Logsdon, C; Williams, J A

    1995-01-01

    Chinese hamster ovary cells stably transfected with human M3 muscarinic acetylcholine receptors show a 40-50% reduction in the immunoreactive G-proteins Gq alpha and G11 alpha when stimulated with the cholinergic agonist carbachol. This effect is seen after 9 h, is maximal after 24 h, and occurs over a range of carbachol concentrations that activate phosphoinositide hydrolysis in these cells. The effect is specific for Gq alpha family proteins as Gs alpha was slightly increased after carbachol treatment and G13 alpha was unchanged. Using a urea gel system, we were able to resolve Gq alpha and G11 alpha, both of which were down-regulated by carbachol. An M3 receptor mutant, with C-terminal threonines changed to alanines as described previously, binds ligand and activates phosphoinositide hydrolysis normally but is not down-regulated in response to carbachol. This receptor, however, induces Gq alpha/G11 alpha down-regulation similarly to wild-type M3 receptors, indicating that G-protein down-regulation is not directly coupled to receptor down-regulation. Thus down-regulation of Gq alpha and G11 alpha may contribute to heterologous desensitization particularly at longer times of agonist exposure. Images Figure 1 Figure 4 PMID:7654194

  18. Leucine zipper motif in RRS1 is crucial for the regulation of Arabidopsis dual resistance protein complex RPS4/RRS1.

    PubMed

    Narusaka, Mari; Toyoda, Kazuhiro; Shiraishi, Tomonori; Iuchi, Satoshi; Takano, Yoshitaka; Shirasu, Ken; Narusaka, Yoshihiro

    2016-01-01

    Arabidopsis thaliana leucine-rich repeat-containing (NLR) proteins RPS4 and RRS1, known as dual resistance proteins, confer resistance to multiple pathogen isolates, such as the bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum and the fungal pathogen Colletotrichum higginsianum. RPS4 is a typical Toll/interleukin 1 Receptor (TIR)-type NLR, whereas RRS1 is an atypical TIR-NLR that contains a leucine zipper (LZ) motif and a C-terminal WRKY domain. RPS4 and RRS1 are localised near each other in a head-to-head orientation. In this study, direct mutagenesis of the C-terminal LZ motif in RRS1 caused an autoimmune response and stunting in the mutant. Co-immunoprecipitation analysis indicated that full-length RPS4 and RRS1 are physically associated with one another. Furthermore, virus-induced gene silencing experiments showed that hypersensitive-like cell death triggered by RPS4/LZ motif-mutated RRS1 depends on EDS1. In conclusion, we suggest that the RRS1-LZ motif is crucial for the regulation of the RPS4/RRS1 complex. PMID:26750751

  19. Leucine zipper motif in RRS1 is crucial for the regulation of Arabidopsis dual resistance protein complex RPS4/RRS1

    PubMed Central

    Narusaka, Mari; Toyoda, Kazuhiro; Shiraishi, Tomonori; Iuchi, Satoshi; Takano, Yoshitaka; Shirasu, Ken; Narusaka, Yoshihiro

    2016-01-01

    Arabidopsis thaliana leucine-rich repeat-containing (NLR) proteins RPS4 and RRS1, known as dual resistance proteins, confer resistance to multiple pathogen isolates, such as the bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum and the fungal pathogen Colletotrichum higginsianum. RPS4 is a typical Toll/interleukin 1 Receptor (TIR)-type NLR, whereas RRS1 is an atypical TIR-NLR that contains a leucine zipper (LZ) motif and a C-terminal WRKY domain. RPS4 and RRS1 are localised near each other in a head-to-head orientation. In this study, direct mutagenesis of the C-terminal LZ motif in RRS1 caused an autoimmune response and stunting in the mutant. Co-immunoprecipitation analysis indicated that full-length RPS4 and RRS1 are physically associated with one another. Furthermore, virus-induced gene silencing experiments showed that hypersensitive-like cell death triggered by RPS4/LZ motif-mutated RRS1 depends on EDS1. In conclusion, we suggest that the RRS1-LZ motif is crucial for the regulation of the RPS4/RRS1 complex. PMID:26750751

  20. Leucine zipper motif in RRS1 is crucial for the regulation of Arabidopsis dual resistance protein complex RPS4/RRS1.

    PubMed

    Narusaka, Mari; Toyoda, Kazuhiro; Shiraishi, Tomonori; Iuchi, Satoshi; Takano, Yoshitaka; Shirasu, Ken; Narusaka, Yoshihiro

    2016-01-11

    Arabidopsis thaliana leucine-rich repeat-containing (NLR) proteins RPS4 and RRS1, known as dual resistance proteins, confer resistance to multiple pathogen isolates, such as the bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum and the fungal pathogen Colletotrichum higginsianum. RPS4 is a typical Toll/interleukin 1 Receptor (TIR)-type NLR, whereas RRS1 is an atypical TIR-NLR that contains a leucine zipper (LZ) motif and a C-terminal WRKY domain. RPS4 and RRS1 are localised near each other in a head-to-head orientation. In this study, direct mutagenesis of the C-terminal LZ motif in RRS1 caused an autoimmune response and stunting in the mutant. Co-immunoprecipitation analysis indicated that full-length RPS4 and RRS1 are physically associated with one another. Furthermore, virus-induced gene silencing experiments showed that hypersensitive-like cell death triggered by RPS4/LZ motif-mutated RRS1 depends on EDS1. In conclusion, we suggest that the RRS1-LZ motif is crucial for the regulation of the RPS4/RRS1 complex.

  1. Regulation of 5alpha-reductase isoforms by oxytocin in the rat ventral prostate.

    PubMed

    Assinder, S J; Johnson, C; King, K; Nicholson, H D

    2004-12-01

    Oxytocin (OT) is present in the male reproductive tract, where it is known to modulate contractility, cell growth, and steroidogenesis. Little is known about how OT regulates these processes. This study describes the localization of OT receptor in the rat ventral prostate and investigates if OT regulates gene expression and/or activity of 5alpha-reductase isoforms I and II. The ventral prostates of adult male Wistar rats were collected following daily sc administration of saline (control), OT, a specific OT antagonist or both OT plus antagonist for 3 d. Expression of the OT receptor was identified in the ventral prostate by RT-PCR and Western blot, and confirmed to be a single active binding site by radioreceptor assay. Immunohistochemistry localized the receptor to the epithelium of prostatic acini and to the stromal tissue. Real-time RT-PCR determined that OT treatment significantly reduced expression of 5alpha-reductase I but significantly increased 5alpha-reductase II expression in the ventral prostate. Activity of both isoforms of 5alpha-reductase was significantly increased by OT, resulting in increased concentration of prostatic dihydrotestosterone. In conclusion, OT is involved in regulating conversion of testosterone to the biologically active dihydrotestosterone in the rat ventral prostate. It does so by differential regulation of 5alpha-reductase isoforms I and II.

  2. Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine.

    PubMed Central

    Tomsig, Jose Luis; Sohma, Hitoshi; Creutz, Carl E

    2004-01-01

    The role of copines in regulating signalling from the TNF-alpha (tumour necrosis factor-alpha) receptor was probed by the expression of a copine dominant-negative construct in HEK293 (human embryonic kidney 293) cells. The construct was found to reduce activation of the transcription factor NF-kappaB (nuclear factor-kappaB) by TNF-alpha. The introduction of calcium into HEK293 cells either through the activation of muscarinic cholinergic receptors or through the application of the ionophore A23187 was found to enhance TNF-alpha-dependent activation of NF-kappaB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-alpha was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-alpha signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-alpha-dependent degradation of IkappaB, a regulator of NF-kappaB. All of the effects of the dominant-negative construct could be reversed by overexpression of full-length copine I, suggesting that the construct acts specifically through competitive inhibition of copine. One of the identified targets of copine I is the NEDD8-conjugating enzyme UBC12 (ubiquitin C12), that promotes the degradation of IkappaB through the ubiquitin ligase enzyme complex SCF(betaTrCP). Therefore the copine dominant-negative construct might inhibit TNF-alpha signalling by dysregulation or mislocalization of UBC12. Based on these results, a hypothesis is presented for possible roles of copines in regulating other signalling pathways in animals, plants and protozoa. PMID:14674885

  3. Nicotinic acetylcholine receptor alpha 7 regulates cAMP signal within lipid rafts.

    PubMed

    Oshikawa, Jin; Toya, Yoshiyuki; Fujita, Takayuki; Egawa, Masato; Kawabe, Junichi; Umemura, Satoshi; Ishikawa, Yoshihiro

    2003-09-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR alpha 7-subunit has a property of high Ca2+ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChR alpha 7 existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR alpha 5- and beta2-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChR alpha 7. Cholesterol depletion from plasma membranes with methyl-beta-cyclodextrin redistributed nAChR alpha 7 and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with alpha-bungarotoxin, a specific antagonist of nAChR alpha 7, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChR alpha 7 has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChR alpha 7 with AC within plasma membranes. In addition, nAChR alpha 7 may regulate the AC activity via Ca2+ within lipid rafts.

  4. Down-regulating alpha-galactosidase enhances freezing tolerance in transgenic petunia.

    PubMed

    Pennycooke, Joyce C; Jones, Michelle L; Stushnoff, Cecil

    2003-10-01

    Alpha-galactosidase (alpha-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the alpha-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of alpha-Gal was modified in transgenic petunia (Petunia x hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that alpha-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of alpha-Gal mRNAs. alpha-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from -4 degrees C for cold-acclimated wild-type plants to -8 degrees C for the most tolerant antisense line. Down-regulating alpha-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the alpha-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with alpha-Gal provides an additional method for improving the freezing tolerance of plants.

  5. P70S6K 1 regulation of angiogenesis through VEGF and HIF-1{alpha} expression

    SciTech Connect

    Bian, Chuan-Xiu; Shi, Zhumei; Meng, Qiao; Jiang, Yue; Liu, Ling-Zhi; Jiang, Bing-Hua

    2010-07-30

    Research highlights: {yields} P70S6K1 regulates VEGF expression; {yields} P70S6K1 induces transcriptional activation through HIF-1{alpha} binding site; {yields} P70S6K1 regulates HIF-1{alpha}, but not HIF-1{beta} protein expression; {yields} P70S6K1 mediates tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression. -- Abstract: The 70 kDa ribosomal S6 kinase 1 (p70S6K1), a downstream target of phosphoinositide 3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK), is an important regulator of cell cycle progression, and cell proliferation. Recent studies indicated an important role of p70S6K1 in PTEN-negative and AKT-overexpressing tumors. However, the mechanism of p70S6K1 in tumor angiogenesis remains to be elucidated. In this study, we specifically inhibited p70S6K1 activity in ovarian cancer cells using vector-based small interfering RNA (siRNA) against p70S6K1. We found that knockdown of p70S6K1 significantly decreased VEGF protein expression and VEGF transcriptional activation through the HIF-1{alpha} binding site at its enhancer region. The expression of p70S6K1 siRNA specifically inhibited HIF-1{alpha}, but not HIF-1{beta} protein expression. We also found that p70S6K1 down-regulation inhibited ovarian tumor growth and angiogenesis, and decreased cell proliferation and levels of VEGF and HIF-1{alpha} expression in tumor tissues. Our results suggest that p70S6K1 is required for tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression, providing a molecular mechanism of human ovarian cancer mediated by p70S6K1 signaling.

  6. Phytol directly activates peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and regulates gene expression involved in lipid metabolism in PPAR{alpha}-expressing HepG2 hepatocytes

    SciTech Connect

    Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo . E-mail: fat@kais.kyoto-u.ac.jp

    2005-11-18

    The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPAR{alpha}-specific activator. Phytol induced the increase in PPAR{alpha}-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPAR{alpha}. Moreover, the addition of phytol upregulated the expression of PPAR{alpha}-target genes at both mRNA and protein levels in PPAR{alpha}-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPAR{alpha} ligand and that it stimulates the expression of PPAR{alpha}-target genes in intact cells. Because PPAR{alpha} activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism.

  7. AMPK activation regulates apoptosis, adipogenesis, and lipolysis by eIF2{alpha} in adipocytes

    SciTech Connect

    Dagon, Yossi; Avraham, Yosefa; Berry, Elliot M. . E-mail: Berry@md.huji.ac.il

    2006-02-03

    AMP-activated protein kinase (AMPK) is a metabolic master switch regulating glucose and lipid metabolism. Recently, AMPK has been implicated in the control of adipose tissue content. Yet, the nature of this action is controversial. We examined the effect on F442a adipocytes of the AMPK activator-AICAR. Activation of AMPK induced dose-dependent apoptotic cell death, inhibition of lipolysis, and downregulatation key adipogenic genes, such as peroxisome proliferator-activated receptor (PPAR{gamma}) and CCAAT/enhancer-binding protein alpha (C/EBP{alpha}). We have identified the {alpha}-subunit of the eukaryotic initiation factor-2 (eIF2{alpha}) as a target gene which is phosphorylated following AICAR treatment. Such phosphorylation is one of the best-characterized mechanisms for downregulating protein synthesis. 2-Aminopurine (2-AP), an inhibitor of eIF2{alpha} kinases, could overcome the apoptotic effect of AICAR, abolishing the reduction of PPAR{gamma} and C/EBP{alpha} and the lipolytic properties of AMPK. Thus, AMPK may diminish adiposity via reduction of fat cell number through eIF2{alpha}-dependent translation shutdown.

  8. Role of upstream DNase I hypersensitive sites in the regulation of human alpha globin gene expression.

    PubMed

    Sharpe, J A; Summerhill, R J; Vyas, P; Gourdon, G; Higgs, D R; Wood, W G

    1993-09-01

    Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells. PMID:7689876

  9. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    EPA Science Inventory

    Peroxisome proliferator-activated receptor alpha (PPARα) regulates transcription of genes involved both in lipid and glucose metabolism as well as inflammation. Fibrates are PPARα ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. Fibrates...

  10. Regulation of homocysteine homeostasis through the transcriptional coactivator PGC-1alpha.

    PubMed

    Li, Siming; Arning, Erland; Liu, Chang; Vitvitsky, Victor; Hernandez, Carlos; Banerjee, Ruma; Bottiglieri, Teodoro; Lin, Jiandie D

    2009-03-01

    Plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. Hcy is a nonprotein amino acid derivative that is generated from the methionine cycle, which provides the methyl group for essentially all biological methylation reactions. Although plasma Hcy levels are elevated in patients with cardiovascular disease, the mechanisms that regulate Hcy homeostasis remain poorly defined. In this study, we found that the expression of key enzymes involved in Hcy metabolism is induced in the liver in response to fasting. This induction coincides with increased expression of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha, a transcriptional coactivator that regulates hepatic gluconeogenesis and mitochondrial function. PGC-1alpha stimulates the expression of genes involved in Hcy metabolism in cultured primary hepatocytes as well as in the liver. Adenoviral-mediated expression of PGC-1alpha in vivo leads to elevated plasma Hcy levels. In contrast, mice deficient in PGC-1alpha have lower plasma Hcy concentrations. These results define a novel role for the PGC-1alpha coactivator pathway in the regulation of Hcy homeostasis and suggest a potential pathogenic mechanism that contributes to hyperhomocysteinemia.

  11. A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus.

    PubMed Central

    Görlich, D; Henklein, P; Laskey, R A; Hartmann, E

    1996-01-01

    The complex of importin-alpha and -beta is essential for nuclear protein import. It binds the import substrate in the cytosol, and the resulting trimeric complex moves through the nuclear pores, probably as a single entity. Importin-alpha provides the nuclear localization signal binding site, importin-beta the site of initial docking to the pore. Here we show that the conserved, basic N-terminus of importin-alpha is sufficient for importin-beta binding and essential for protein import. The fusion product of this 41 amino acid domain to a heterologous protein if transported into the nucleus in the same way as full-length importin-alpha itself. Transport is dependent on importin-beta but competed by importin-alpha. As no additional part of importin-alpha is needed for translocation, the movement which drives the import substrate complex into the nucleus appears to be generated between importin-beta and structures of the nuclear pore. The domain that binds to importin-beta appears to confer import only, but not re-export out of the nucleus, suggesting that the return of importin-alpha into the cytoplasm is not a simple reversal of its entry. Images PMID:8617226

  12. Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

    PubMed Central

    Thomas, Maria; Winter, Stefan; Klumpp, Britta; Turpeinen, Miia; Klein, Kathrin; Schwab, Matthias; Zanger, Ulrich M.

    2015-01-01

    The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions –2762/–2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype. PMID:26582990

  13. Role of hypoxia-inducible transcription factors 1alpha and 2alpha in the regulation of plasminogen activator inhibitor-1 expression in a human trophoblast cell line.

    PubMed

    Meade, E S; Ma, Y Y; Guller, S

    2007-10-01

    The plasminogen activator inhibitors (PAIs) play critical roles in regulating hemostatic and invasive functions of trophoblasts through suppression of plasmin-dependent fibrinolysis and extracellular matrix degradation. The expression of PAI-1 is increased under hypoxic conditions, although the mechanism remains incompletely understood. In the current study we used HTR-8/SVneo cells, a first trimester extravillous trophoblast cell line, and siRNA technology to examine the role of hypoxia-inducible transcription factors (HIFs)-1alpha and -2alpha in the regulation of PAI-1 expression. Using serum-containing and serum-free media culture media it was initially noted that levels of PAI-1, but not PAI-2 protein, were markedly induced by hypoxic (2-3% oxygen) treatment. Under hypoxic conditions, Western blotting revealed that the presence of siRNAs to HIF-1alpha and HIF-2alpha suppressed expression of their respective proteins, whereas treatment with non-targeting and cyclophilin B siRNAs did not. Importantly, incubation with siRNA to HIF-1alpha or HIF-2alpha alone reduced PAI-1 protein levels to a similar extent, with the combined treatment inducing a more profound effect. The presence of HIF siRNAs reduced levels of PAI-1 mRNA as measured by quantitative real-time PCR, indicating that HIF-1alpha and HIF-2 alpha regulate PAI-1 expression at a transcriptional level. These results indicate that both HIF-1alpha and HIF-2alpha play important and similar roles in hypoxia-mediated stimulation of PAI-1 expression in HTR-8/SVneo cells. Our findings provide insight into the physiological regulation of trophoblast PAI-1 expression in early pregnancy when placental oxygen levels are low, as well as a mechanism for over-expression of placental PAI-1 noted in pregnancies with preeclampsia.

  14. Regulation of lipid peroxidation by nitric oxide and PGF2alpha during luteal regression in rats.

    PubMed

    Motta, A B; Estevez, A; Franchi, A; Perez-Martinez, S; Farina, M; Ribeiro, M L; Lasserre, A; Gimeno, M F

    2001-04-01

    Corpus luteum regression is related to an increased generation of reactive oxygen species. Although several studies indicate that PGF(2alpha) is involved in regression of the corpus luteum in mammalian species through an increase in reactive oxygen species, the exact mechanism remains unknown. In the present study, the relationship between nitric oxide and PGF(2alpha) in regulation of lipid peroxidation was studied. Ovarian tissue from pseudopregnant rats at mid- (day 5) or late phase or at the time of regression (day 9 of pseudopregnancy) of corpus luteum development was used. Thiobarbituric acid reactants, used as a lipid peroxidation index, were higher on day 9 of pseudopregnancy than on day 5. In contrast, glutathione content (an antioxidant metabolite) was lower on day 9 than on day 5 of pseudopregnancy. These results indicate that there was an enhanced oxidative status in ovarian tissue during luteolysis. Administration of N(omega)-nitro-L-arginine methyl ester (L-NAME: 600 micromol l(-1)), a competitive nitric oxide synthase (NOS) inhibitor, led to a decrease in basal thiobarbituric acid reactant content in ovarian tissue from rats on day 9 of pseudopregnancy only, indicating that during regression of the corpus luteum, NO could act as intermediary in ovarian lipid peroxidation. Administration of a luteolytic dose (3 microg kg(-1) body weight i.p.) of a synthetic PGF(2alpha) increased thiobarbituric acid reactant content in ovaries from rats on day 9 of pseudopregnancy. As this effect was reversed partially by L-NAME, it is proposed that during regression of corpora lutea, PGF(2alpha) and NO are involved in regulation of lipid peroxidation. As this effect was only reversed partially, it is possible that there is another mechanism involving PGF(2alpha) (but not the NO-NOS pathway) in regulation of ovarian lipid peroxidation. Furthermore, the administration of PGF(2alpha) enhanced ovarian NOS activity, whereas cyclooxygenase inhibition (by indomethacin

  15. Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    PubMed Central

    Tammimies, Kristiina; Bieder, Andrea; Lauter, Gilbert; Sugiaman-Trapman, Debora; Torchet, Rachel; Hokkanen, Marie-Estelle; Burghoorn, Jan; Castrén, Eero; Kere, Juha; Tapia-Páez, Isabel; Swoboda, Peter

    2016-01-01

    DYX1C1, DCDC2, and KIAA0319 are three of the most replicated dyslexia candidate genes (DCGs). Recently, these DCGs were implicated in functions at the cilium. Here, we investigate the regulation of these DCGs by Regulatory Factor X transcription factors (RFX TFs), a gene family known for transcriptionally regulating ciliary genes. We identify conserved X-box motifs in the promoter regions of DYX1C1, DCDC2, and KIAA0319 and demonstrate their functionality, as well as the ability to recruit RFX TFs using reporter gene and electrophoretic mobility shift assays. Furthermore, we uncover a complex regulation pattern between RFX1, RFX2, and RFX3 and their significant effect on modifying the endogenous expression of DYX1C1 and DCDC2 in a human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1). In addition, induction of ciliogenesis increases the expression of RFX TFs and DCGs. At the protein level, we show that endogenous DYX1C1 localizes to the base of the cilium, whereas DCDC2 localizes along the entire axoneme of the cilium, thereby validating earlier localization studies using overexpression models. Our results corroborate the emerging role of DCGs in ciliary function and characterize functional noncoding elements, X-box promoter motifs, in DCG promoter regions, which thus can be targeted for mutation screening in dyslexia and ciliopathies associated with these genes.—Tammimies, K., Bieder, A., Lauter, G., Sugiaman-Trapman, D., Torchet, R., Hokkanen, M.-E., Burghoorn, J., Castrén, E., Kere, J., Tapia-Páez, I., Swoboda, P. Ciliary dyslexia candidate genes DYX1C1 and DCDC2 are regulated by Regulatory Factor (RF) X transcription factors through X-box promoter motifs. PMID:27451412

  16. Complex regulation and diverse functions of alpha-klotho.

    PubMed

    Maeda, Ryota; Imura, Akihiro; Nabeshima, Yo-ichi

    2013-01-01

    It has been understood that the α-klotho gene, first identified as an aging-related gene, is actually necessary for regulating mineral homeostasis in vertebrates. All vertebrates, including humans, actively maintain calcium and phosphate ions in bones, circulating blood, and cerebrospinal fluid. Therefore, disruptions in homeostasis cause osteoporosis, ectopic calcification, and epilepsy. Vitamin D and parathyroid hormone are well-known hormones for maintaining calcium and phosphate balance. Synthesis of vitamin D, secretion of parathyroid hormone, and absorption of calcium and phosphate ions are all finely controlled by α-Klotho-dependent machineries. However, the precise molecular mechanisms and functions of α-Klotho are still unclear. In this article, we present an overview of recent progress in α-klotho research, with a main focus on molecular aspects. PMID:23652548

  17. Substitution of a conserved cysteine-996 in a cysteine-rich motif of the laminin {alpha}2-chain in congenital muscular dystrophy with partial deficiency of the protein

    SciTech Connect

    Nissinen, M.; Xu Zhang; Tryggvason, K.

    1996-06-01

    Congenital muscular dystrophies (CMDs) are autosomal recessive muscle disorders of early onset. Approximately half of CMD patients present laminin {alpha}2-chain (merosin) deficiency in muscle biopsies, and the disease locus has been mapped to the region of the LAMA2 gene (6q22-23) in several families. Recently, two nonsense mutations in the laminin {alpha}2-chain gene were identified in CMD patients exhibiting complete deficiency of the laminin {alpha}2-chain in muscle biopsies. However, a subset of CMD patients with linkage to LAMA2 show only partial absence of the laminin {alpha}2-chain around muscle fibers, by immunocytochemical analysis. In the present study we have identified a homozygous missense mutation in the {alpha}2-chain gene of a consanguineous Turkish family with partial laminin {alpha}2-chain deficiency. The T{r_arrow}C transition at position 3035 in the cDNA sequence results in a Cys996{r_arrow}Arg substitution. The mutation that affects one of the conserved cysteine-rich repeats in the short arm of the laminin {alpha}2-chain should result in normal synthesis of the chain and in formation and secretion of a heterotrimeric laminin molecule. Muscular dysfunction is possibly caused either by abnormal disulfide cross-links and folding of the laminin repeat, leading to the disturbance of an as yet unknown binding function of the laminin {alpha}2-chain and to shorter half-life of the muscle-specific laminin-2 and laminin-4 isoforms, or by increased proteolytic sensitivity, leading to truncation of the short arm. 42 refs., 7 figs.

  18. Nef-induced CD4 and major histocompatibility complex class I (MHC-I) down-regulation are governed by distinct determinants: N-terminal alpha helix and proline repeat of Nef selectively regulate MHC-I trafficking.

    PubMed

    Mangasarian, A; Piguet, V; Wang, J K; Chen, Y L; Trono, D

    1999-03-01

    The Nef protein of primate lentiviruses triggers the accelerated endocytosis of CD4 and of class I major histocompatibility complex (MHC-I), thereby down-modulating the cell surface expression of these receptors. Nef acts as a connector between the CD4 cytoplasmic tail and intracellular sorting pathways both in the Golgi and at the plasma membrane, triggering the de novo formation of CD4-specific clathrin-coated pits (CCP). The downstream partners of Nef in this event are the adapter protein complex (AP) of CCP and possibly a subunit of the vacuolar ATPase. Whether Nef-induced MHC-I down-regulation stems from a similar mechanism is unknown. By comparing human immunodeficiency virus type 1 (HIV-1) Nef mutants for their ability to affect either CD4 or MHC-I expression, both in transient-transfection assays and in the context of HIV-1 infection, it was determined that Nef-induced CD4 and MHC-I down-regulation constitute genetically and functionally separate properties. Mutations affecting only CD4 regulation mapped to residues previously shown to mediate the binding of Nef to this receptor, such as W57 and L58, as well as to an AP-recruiting dileucine motif and to an acidic dipeptide in the C-terminal region of the protein. In contrast, mutation of residues in an alpha-helical region in the proximal portion of Nef and amino acid substitutions in a proline-based SH3 domain-binding motif selectively affected MHC-I down-modulation. Although both the N-terminal alpha-helix and the proline-rich region of Nef have been implicated in recruiting Src family protein kinases, the inhibitor herbimycin A did not block MHC-I down-regulation, suggesting that the latter process is not mediated through an activation of this family of tyrosine kinases. PMID:9971776

  19. Agrin regulation of alpha3 sodium-potassium ATPase activity modulates cardiac myocyte contraction.

    PubMed

    Hilgenberg, Lutz G W; Pham, Bryan; Ortega, Maria; Walid, Saif; Kemmerly, Thomas; O'Dowd, Diane K; Smith, Martin A

    2009-06-19

    Drugs that inhibit Na,K-ATPases, such as digoxin and ouabain, alter cardiac myocyte contractility. We recently demonstrated that agrin, a protein first identified at the vertebrate neuromuscular junction, binds to and regulates the activity of alpha3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the alpha3 Na,K-ATPase are expressed in heart, but their potential for interaction and effect on cardiac myocyte function was unknown. Here we show that agrin binds to the alpha3 subunit of the Na,K-ATPase in cardiac myocyte membranes, inducing tyrosine phosphorylation and inhibiting activity of the pump. Agrin also triggers a rapid increase in cytoplasmic Na(+) in cardiac myocytes, suggesting a role in cardiac myocyte function. Consistent with this hypothesis, spontaneous contraction frequencies of cultured cardiac myocytes prepared from mice in which agrin expression is blocked by mutation of the Agrn gene are significantly higher than in the wild type. The Agrn mutant phenotype is rescued by acute treatment with recombinant agrin. Furthermore, exposure of wild type myocytes to an agrin antagonist phenocopies the Agrn mutation. These data demonstrate that the basal frequency of myocyte contraction depends on endogenous agrin-alpha3 Na,K-ATPase interaction and suggest that agrin modulation of the alpha3 Na,K-ATPase is important in regulating heart function.

  20. Osmotic pressure regulates alpha beta gamma-rENaC expressed in Xenopus oocytes.

    PubMed

    Ji, H L; Fuller, C M; Benos, D J

    1998-11-01

    The hypothesis that amiloride-sensitive Na+ channels (ENaC) are involved in cell volume regulation was tested. Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used to superfuse Xenopus oocytes expressing alpha beta gamma-rat ENaC (alpha beta gamma-rENaC). Whole cell currents were reversibly dependent on external osmolarity. Under conditions of swelling (70 mosM) or shrinkage (450 mosM), current amplitude decreased and increased, respectively. In contrast, there was no change in current amplitude of H2O-injected oocytes to the above osmotic insults. Currents recorded from alpha beta gamma-rENaC-injected oocytes were not sensitive to external Cl- concentration or to the K+ channel inhibitor BaCl2. They were sensitive to amiloride. The concentration of amiloride necessary to inhibit one-half of the maximal rENaC current expressed in oocytes (Ki; apparent dissociation constant) decreased in swollen cells and increased in shrunken oocytes. The osmotic pressure-induced Na+ currents showed properties similar to those of stretch-activated channels, including inhibition by Gd3+ and La3+, and decreased selectivity for Na+. alpha beta gamma-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the first demonstration that alpha beta gamma-rENaC heterologously expressed in Xenopus oocytes may contribute to oocyte volume regulation following shrinkage. PMID:9814964

  1. Filamin A negatively regulates the transcriptional activity of p73{alpha} in the cytoplasm

    SciTech Connect

    Kim, Eun-Joo; Park, Jong-Sup; Um, Soo-Jong

    2007-11-03

    The transcription regulator p73{alpha} is structurally different from p53 in that it possesses a unique C-terminal domain, which has been implicated in transcriptional repression. To dissect the mechanism of repression by this domain, we performed a yeast two-hybrid screen of a HeLa cDNA library using residues 487-636 of p73{alpha} as bait and isolated a cDNA clone encoding the C-terminal portion (residues 2210-2647) of filamin A, a 280-kDa actin-binding protein. Additional yeast two-hybrid assays indicated that filamin A specifically interacts with the p73{alpha} C-terminus, which is lacking in p53 and p73{beta}. The interaction was confirmed by GST pull-down assays in vitro and by immunoprecipitation analysis in vivo. Immunofluorescence microscopy revealed that p73{alpha} remained in the cytoplasm in A7 melanoma cells stably expressing filamin A, whereas it was localized in the nucleus of filamin A-deficient M2 cells. Deletion of the C-terminus of p73{alpha} (residues 487-636) resulted in nuclear localization in both cell types. Consistent with our interaction data, transient co-expression of filamin A resulted in the down-regulation of p73{alpha}, but not of p53, transcriptional activity on various p53-responsive promoters. Taken together, our data suggest that p73{alpha} is sequestered in the cytoplasm by filamin A, thereby inhibiting its transcriptional activity.

  2. The VTLISFG motif in the BH1 domain plays a significant role in regulating the degradation of Mcl-1.

    PubMed

    Xiao, Kang; Chen, Pengxuan; Chang, Donald Choy

    2014-01-01

    Mcl-1 is a member of the Bcl-2 family protein; its degradation is required for the initiation of apoptosis. The mechanism, however, is not yet clearly known. Previously, it was reported that Mcl-1 is degraded through the ubiquitination-mediated pathway and the PEST domain is the motif responsible for promoting this degradation. We found evidence that this may not be true. We generated several Mcl-1 deletion mutants and examined their effects on protein stability. Deletion of the PEST domain did not prevent the degradation of Mcl-1 during apoptosis. The BH1 domain, but not the PEST, BH3 or BH2 domain, exhibited a short half-life. A peptide named "F3" (VTLISFG) in the C-terminus of the BH1 domain appears to be critical for the rapid turnover of Mcl-1. Deletion of F3 from GFP-Mcl-1-ΔPEST retarded the degradation of this mutant. F3 appeared to be the minimum functional sequence of the degradation motif, since deletion of a single residue was sufficient to abrogate its short half-life. Fusion of F3 with p32 resulted in the degradation of p32 during UV-induced apoptosis, while wild type p32 was not affected. Taken together, these findings suggest that F3 (VTLISFG), instead of PEST, is the major motif responsible for the degradation of Mcl-1 during apoptosis.

  3. Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

    PubMed

    Paulhe, F; Racaud-Sultan, C; Ragab, A; Albiges-Rizo, C; Chap, H; Iberg, N; Morand, O; Perret, B

    2001-11-01

    Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

  4. Genome-wide Prediction and Functional Validation of Promoter Motifs Regulating Gene Expression in Spore and Infection Stages of Phytophthora infestans

    PubMed Central

    Roy, Sourav; Kagda, Meenakshi; Judelson, Howard S.

    2013-01-01

    Most eukaryotic pathogens have complex life cycles in which gene expression networks orchestrate the formation of cells specialized for dissemination or host colonization. In the oomycete Phytophthora infestans, the potato late blight pathogen, major shifts in mRNA profiles during developmental transitions were identified using microarrays. We used those data with search algorithms to discover about 100 motifs that are over-represented in promoters of genes up-regulated in hyphae, sporangia, sporangia undergoing zoosporogenesis, swimming zoospores, or germinated cysts forming appressoria (infection structures). Most of the putative stage-specific transcription factor binding sites (TFBSs) thus identified had features typical of TFBSs such as position or orientation bias, palindromy, and conservation in related species. Each of six motifs tested in P. infestans transformants using the GUS reporter gene conferred the expected stage-specific expression pattern, and several were shown to bind nuclear proteins in gel-shift assays. Motifs linked to the appressoria-forming stage, including a functionally validated TFBS, were over-represented in promoters of genes encoding effectors and other pathogenesis-related proteins. To understand how promoter and genome architecture influence expression, we also mapped transcription patterns to the P. infestans genome assembly. Adjacent genes were not typically induced in the same stage, including genes transcribed in opposite directions from small intergenic regions, but co-regulated gene pairs occurred more than expected by random chance. These data help illuminate the processes regulating development and pathogenesis, and will enable future attempts to purify the cognate transcription factors. PMID:23516354

  5. Alpha2 adrenoceptors regulate proliferation of human intestinal epithelial cells

    PubMed Central

    Schaak, S; Cussac, D; Cayla, C; Devedjian, J; Guyot, R; Paris, H; Denis, C

    2000-01-01

    BACKGROUND AND AIMS—Previous studies on rodents have suggested that catecholamines stimulate proliferation of the intestinal epithelium through activation of α2 adrenoceptors located on crypt cells. The occurrence of this effect awaits demonstration in humans and the molecular mechanisms involved have not yet been elucidated. Here, we examined the effect of α2 agonists on a clone of Caco2 cells expressing the human α2A adrenoceptor.
METHODS—Cells were transfected with a bicistronic plasmid containing the α2C10 and neomycin phosphotransferase genes. G418 resistant clones were assayed for receptor expression using radioligand binding. Receptor functionality was assessed by testing its ability to couple Gi proteins and to inhibit cAMP production. Mitogen activated protein kinase (MAPK) phosphorylation was followed by western blot, and cell proliferation was estimated by measuring protein and DNA content.
RESULTS—Permanent transfection of Caco2 cells allowed us to obtain a clone (Caco2-3B) expressing α2A adrenoceptors at a density similar to that found in normal human intestinal epithelium. Caco2-3B retained morphological features and brush border enzyme expression characteristic of enterocytic differentiation. The receptor was coupled to Gi2/Gi3 proteins and its stimulation caused marked diminution of forskolin induced cAMP production. Treatment of Caco2-3B with UK14304 (α2 agonist) induced a rapid increase in the phosphorylation state of MAPK, extracellular regulated protein kinase 1 (Erk1), and 2 (Erk2). This event was totally abolished in pertussis toxin treated cells and in the presence of kinase inhibitors (genistein or PD98059). It was unaffected by protein kinase C downregulation but correlated with a transient increase in Shc tyrosine phosphorylation. Finally, sustained exposure of Caco2-3B to UK14304 resulted in modest but significant acceleration of cell proliferation. None of these effects was observed in the parental cell line Caco2.

  6. Potential Role of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs (RECK) in Regulation of Matrix Metalloproteinases (MMPs) Expression in Periodontal Diseases

    PubMed Central

    Liu, Nian; Zhou, Bin; Zhu, Guangxun

    2016-01-01

    Periodontal diseases are characterized by pathological destruction of extracellular matrix (ECM) of periodontal tissues. Matrix metalloproteinases (MMPs) are a significant part of the degradation of ECM. However, the regulation of MMPs expression level in periodontal diseases is as yet undetermined. RECK (reversion-inducing cysteine-rich protein with Kazal motifs), a novel membrane-anchored inhibitor of MMPs, could regulate the expressions of MMP-2, 9 and MT1-MMP as a cell surface-signaling molecule. Thus, we propose that RECK may play an important role in regulating MMPs in the ECM degradation of periodontal diseases. The RECK/MMPs signaling pathway could provide a new approach for prevention and treatment of RECK in periodontal diseases by blocking MMPs. PMID:27272560

  7. The endocytosis and signaling of the γδ T cell coreceptor WC1 are regulated by a dileucine motif.

    PubMed

    Hsu, Haoting; Baldwin, Cynthia L; Telfer, Janice C

    2015-03-01

    WC1 proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WC1 cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens.

  8. Alpha-adrenergic regulation of angiotensin II receptors in neuronal cultures from rat brain

    SciTech Connect

    Sumners, C.; Watkins, L.L.; Raizada, M.K.

    1986-03-05

    Our previous studies have suggested that endogenous catecholamine (CA) levels are important regulators of angiotensin II receptors (AngII-R) in neuronal cultures. The present study was undertaken to determine the possible mechanisms by which CA exert their effects on AngII-R. Incubation of neuronal cultures with norepinephrine (NE) resulted in time and dose dependent decreases in (/sup 125/I)-AngII specific binding. A maximal effect of 60-70% was observed between 4-8 hours at 1..mu..M NE, which was the result of a decrease in Bmax (104 +/- 16.3 fmol/mg protein in controls vs 41.4 +/- 9.3 fmol/mg protein in NE treated) and little change in Kd. Similar effects were observed when neuronal cultures were incubated with dopamine (DA) or phenylephrine, but serotonin, epinephrine and isoproterenol had no effect. The NE and DA stimulated decrease in AngII-R appears to be an ..cap alpha../sub 1/=adrenergic receptor mediated phenomenon because it is abolished by coincubation with prazosin (..cap alpha../sub 1/-antagonist) and not by ..cap alpha../sub 2/- or ..beta..-antagonists. Similar incubations of neuronal cultures with NE also caused a time and dose dependent downregulation of ..cap alpha../sub 1/-adrenergic receptors. Taken together these observations indicate that the NE induced downregulation of AngII-R is mediated by ..cap alpha../sub 1/-receptors, and suggest that one possible mechanism may be the cointernalization of both ..cap alpha../sub 1/- and AngII-R stimulated by ..cap alpha../sub 1/-adrenergic agonists.

  9. The actin cytoskeleton differentially regulates platelet alpha-granule and dense-granule secretion.

    PubMed

    Flaumenhaft, Robert; Dilks, James R; Rozenvayn, Nataliya; Monahan-Earley, Rita A; Feng, Dian; Dvorak, Ann M

    2005-05-15

    Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced alpha-granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced alpha-granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca(2+)- or guanosine triphosphate (GTP)-gamma-S-induced alpha-granule secretion. Latrunculin A-dependent alpha-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates alpha-granule and dense granule secretion. PMID:15671445

  10. Venom gland EST analysis of the saw-scaled viper, Echis ocellatus, reveals novel alpha9beta1 integrin-binding motifs in venom metalloproteinases and a new group of putative toxins, renin-like aspartic proteases.

    PubMed

    Wagstaff, Simon C; Harrison, Robert A

    2006-08-01

    Echis ocellatus is the most medically important snake in West Africa. However, the composition of its venom and the differential contribution of these venom components to the severe haemorrhagic and coagulopathic pathology of envenoming are poorly understood. To address this situation we assembled a toxin transcriptome based upon 1000 expressed sequence tags (EST) from a cDNA library constructed from pooled venom glands of 10 individual E. ocellatus. We used a variety of bioinformatic tools to construct a fully annotated venom-toxin transcriptome that was interrogated with a combination of BLAST annotation, gene ontology cataloguing and disintegrin-motif searching. The results of these analyses revealed an unusually abundant and diverse expression of snake venom metalloproteinases (SVMP) and a broad toxin-expression profile including several distinct isoforms of bradykinin-potentiating peptides, phospholipase A(2), C-type lectins, serine proteinases and l-amino oxidases. Most significantly, we identified for the first time a conserved alpha(9)beta(1) integrin-binding motif in several SVMPs, and a new group of putative venom toxins, renin-like aspartic proteases. PMID:16713134

  11. [Regulation in the expression of alpha-galactosidase gene in raf operon in Escherichia coli].

    PubMed

    Su, T Z; Qi, S; Yun, W H; Xiu, L

    1989-06-01

    The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2551100

  12. P70S6K 1 regulation of angiogenesis through VEGF and HIF-1alpha expression.

    PubMed

    Bian, Chuan-Xiu; Shi, Zhumei; Meng, Qiao; Jiang, Yue; Liu, Ling-Zhi; Jiang, Bing-Hua

    2010-07-30

    The 70kDa ribosomal S6 kinase 1 (p70S6K1), a downstream target of phosphoinositide 3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK), is an important regulator of cell cycle progression, and cell proliferation. Recent studies indicated an important role of p70S6K1 in PTEN-negative and AKT-overexpressing tumors. However, the mechanism of p70S6K1 in tumor angiogenesis remains to be elucidated. In this study, we specifically inhibited p70S6K1 activity in ovarian cancer cells using vector-based small interfering RNA (siRNA) against p70S6K1. We found that knockdown of p70S6K1 significantly decreased VEGF protein expression and VEGF transcriptional activation through the HIF-1alpha binding site at its enhancer region. The expression of p70S6K1 siRNA specifically inhibited HIF-1alpha, but not HIF-1beta protein expression. We also found that p70S6K1 down-regulation inhibited ovarian tumor growth and angiogenesis, and decreased cell proliferation and levels of VEGF and HIF-1alpha expression in tumor tissues. Our results suggest that p70S6K1 is required for tumor growth and angiogenesis through HIF-1alpha and VEGF expression, providing a molecular mechanism of human ovarian cancer mediated by p70S6K1 signaling.

  13. Uncoupling protein-2 up-regulation and enhanced cyanide toxicity are mediated by PPAR{alpha} activation and oxidative stress

    SciTech Connect

    Zhang, X.; Li, L.; Prabhakaran, K.; Zhang, L.; Leavesley, H.B.; Borowitz, J.L.; Isom, G.E.

    2007-08-15

    Uncoupling protein 2 (UCP-2) is an inner mitochondrial membrane proton carrier that modulates mitochondrial membrane potential ({delta}{psi}{sub m}) and uncouples oxidative phosphorylation. We have shown that up-regulation of UCP-2 by Wy14,643, a selective peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) agonist, enhances cyanide cytotoxicity. The pathway by which Wy14,643 up-regulates UCP-2 was determined in a dopaminergic cell line (N27 cells). Since dopaminergic mesencephalic cells are a primary brain target of cyanide, the N27 immortalized mesencephalic cell was used in this study. Wy14,643 produced a concentration- and time-dependent up-regulation of UCP-2 that was linked to enhanced cyanide-induced cell death. MK886 (PPAR{alpha} antagonist) or PPAR{alpha} knock-down by RNA interference (RNAi) inhibited PPAR{alpha} activity as shown by the peroxisome proliferator response element-luciferase reporter assay, but only partially decreased up-regulation of UCP-2. The role of oxidative stress as an alternative pathway to UCP-2 up-regulation was determined. Wy14,643 induced a rapid surge of ROS generation and loading cells with glutathione ethyl ester (GSH-EE) or pre-treatment with vitamin E attenuated up-regulation of UCP-2. On the other hand, RNAi knockdown of PPAR{alpha} did not alter ROS generation, suggesting a PPAR{alpha}-independent component to the response. Co-treatment with PPAR{alpha}-RNAi and GSH-EE blocked both the up-regulation of UCP-2 by Wy14,643 and the cyanide-induced cell death. It was concluded that a PPAR{alpha}-mediated pathway and an oxidative stress pathway independent of PPAR{alpha} mediate the up-regulation of UCP-2 and subsequent increased vulnerability to cyanide-induced cytotoxicity.

  14. SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms.

    PubMed

    Liang, Yueyang; Ward, Sally; Li, Ping; Bennett, Tom; Leyser, Ottoline

    2016-07-01

    Strigolactones (SLs) are hormonal signals that regulate multiple aspects of shoot architecture, including shoot branching. Like many plant hormonal signaling systems, SLs act by promoting ubiquitination of target proteins and their subsequent proteasome-mediated degradation. Recently, SMXL6, SMXL7, and SMXL8, members of the SMAX1-LIKE (SMXL) family of chaperonin-like proteins, have been identified as proteolytic targets of SL signaling in Arabidopsis thaliana However, the mechanisms by which these proteins regulate downstream events remain largely unclear. Here, we show that SMXL7 functions in the nucleus, as does the SL receptor, DWARF14 (D14). We show that nucleus-localized D14 can physically interact with both SMXL7 and the MAX2 F-box protein in a SL-dependent manner and that disruption of specific conserved domains in SMXL7 affects its localization, SL-induced degradation, and activity. By expressing and overexpressing these SMXL7 protein variants, we show that shoot tissues are broadly sensitive to SMXL7 activity, but degradation normally buffers the effect of increasing SMXL7 expression. SMXL7 contains a well-conserved EAR (ETHYLENE-RESPONSE FACTOR Amphiphilic Repression) motif, which contributes to, but is not essential for, SMXL7 functionality. Intriguingly, different developmental processes show differential sensitivity to the loss of the EAR motif, raising the possibility that there may be several distinct mechanisms at play downstream of SMXL7.

  15. SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms.

    PubMed

    Liang, Yueyang; Ward, Sally; Li, Ping; Bennett, Tom; Leyser, Ottoline

    2016-07-01

    Strigolactones (SLs) are hormonal signals that regulate multiple aspects of shoot architecture, including shoot branching. Like many plant hormonal signaling systems, SLs act by promoting ubiquitination of target proteins and their subsequent proteasome-mediated degradation. Recently, SMXL6, SMXL7, and SMXL8, members of the SMAX1-LIKE (SMXL) family of chaperonin-like proteins, have been identified as proteolytic targets of SL signaling in Arabidopsis thaliana However, the mechanisms by which these proteins regulate downstream events remain largely unclear. Here, we show that SMXL7 functions in the nucleus, as does the SL receptor, DWARF14 (D14). We show that nucleus-localized D14 can physically interact with both SMXL7 and the MAX2 F-box protein in a SL-dependent manner and that disruption of specific conserved domains in SMXL7 affects its localization, SL-induced degradation, and activity. By expressing and overexpressing these SMXL7 protein variants, we show that shoot tissues are broadly sensitive to SMXL7 activity, but degradation normally buffers the effect of increasing SMXL7 expression. SMXL7 contains a well-conserved EAR (ETHYLENE-RESPONSE FACTOR Amphiphilic Repression) motif, which contributes to, but is not essential for, SMXL7 functionality. Intriguingly, different developmental processes show differential sensitivity to the loss of the EAR motif, raising the possibility that there may be several distinct mechanisms at play downstream of SMXL7. PMID:27317673

  16. SMAX1-LIKE7 Signals from the Nucleus to Regulate Shoot Development in Arabidopsis via Partially EAR Motif-Independent Mechanisms[OPEN

    PubMed Central

    Li, Ping

    2016-01-01

    Strigolactones (SLs) are hormonal signals that regulate multiple aspects of shoot architecture, including shoot branching. Like many plant hormonal signaling systems, SLs act by promoting ubiquitination of target proteins and their subsequent proteasome-mediated degradation. Recently, SMXL6, SMXL7, and SMXL8, members of the SMAX1-LIKE (SMXL) family of chaperonin-like proteins, have been identified as proteolytic targets of SL signaling in Arabidopsis thaliana. However, the mechanisms by which these proteins regulate downstream events remain largely unclear. Here, we show that SMXL7 functions in the nucleus, as does the SL receptor, DWARF14 (D14). We show that nucleus-localized D14 can physically interact with both SMXL7 and the MAX2 F-box protein in a SL-dependent manner and that disruption of specific conserved domains in SMXL7 affects its localization, SL-induced degradation, and activity. By expressing and overexpressing these SMXL7 protein variants, we show that shoot tissues are broadly sensitive to SMXL7 activity, but degradation normally buffers the effect of increasing SMXL7 expression. SMXL7 contains a well-conserved EAR (ETHYLENE-RESPONSE FACTOR Amphiphilic Repression) motif, which contributes to, but is not essential for, SMXL7 functionality. Intriguingly, different developmental processes show differential sensitivity to the loss of the EAR motif, raising the possibility that there may be several distinct mechanisms at play downstream of SMXL7. PMID:27317673

  17. Rapid regulation of pancreatic alpha- and beta- cell signalling systems by estrogens.

    PubMed

    Ripoll, Cristina; Ropero, Ana B; Alonso-Magdalena, Paloma; Quesada, Ivan; Fuentes, Esther; Nadal, Angel

    2008-03-01

    Rapid estrogen actions are triggered after estrogens are bound to a variety of proteins in organelles other than the nucleus. Those include classic estrogen receptors ERalpha and ERbeta, novel membrane proteins that behave as estrogen receptors such as GPR30, ion channels, and other ligand receptors. In pancreatic alpha and beta-cells, estrogens binding to a non-classical membrane estrogen receptors at physiological concentrations regulate ion channels and [Ca(2+)](i) signals, provoking important physiological responses. In beta-cells, 17beta-estradiol regulates K(ATP) channel activity and glucose-induced [Ca(2+)](i) oscillations, eliciting changes in insulin release and the activation of Ca(2+)-dependent transcription factors. In alpha-cells, 17beta-estradiol abolishes low glucose-induced [Ca(2+)](i) oscillations.

  18. alpha-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with alpha 7 subunits and up-regulation by chronic nicotine treatment.

    PubMed

    Barrantes, G E; Rogers, A T; Lindstrom, J; Wonnacott, S

    1995-02-20

    High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [3H]nicotine or [3H]cytisine to live cells in situ was detected although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [3H]QNB were present at a density of 0.75 pmol/mg protein. [125I]alpha-Bungarotoxin (alpha Bgt) bound to hippocampal cultures with a Bmax of 128 fmol/mg protein and a Kd of 0.6 nM; cortical cultures expressed five times fewer [125I]alpha-Bgt binding sites. Fluorescence cytochemistry with rhodamine-alpha-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 x 10(4) and 2 x 10(4) binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat alpha 7 nicotinic receptor subunit) and rhodamine-alpha-Bgt gave coincident labelling patterns, supporting the correlation between the alpha 7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 microM nicotine for 14 days elicited a 40% increase in the numbers of [125I]alpha-Bgt binding sites, mimicking the up-regulation observed in in vivo studies. Primary cultures offer a useful in vitro system for investigating the expression and regulation of brain alpha-Bgt-sensitive receptors.

  19. Tumour-derived interleukin 1alpha (IL-1alpha) up-regulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) by endothelial cells.

    PubMed Central

    Fonsatti, E.; Altomonte, M.; Coral, S.; Cattarossi, I.; Nicotra, M. R.; Gasparollo, A.; Natali, P. G.; Maio, M.

    1997-01-01

    Levels of circulating soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in patients affected by solid malignancies; however, the cellular sources generating high levels of sICAM-1 remain to be characterized. Using conditioned media (CM) from seven ICAM-1-positive or -negative neoplastic cells, we demonstrate that tumour-derived interleukin 1alpha (IL-1alpha) significantly (P < 0.05) up-regulates the release of sICAM-1 by human umbilical vein endothelial cells. The intensity of the effect correlated with the amounts of IL-1alpha detectable in CM. Levels of ICAM-1 mRNA were also up-regulated by tumour-secreted IL-1alpha. The up-regulation of the shedding of sICAM-1 and of its expression at protein and mRNA level were completely reversed by the addition of anti-IL-1alpha neutralizing antibodies. Consistent with the in vitro data, tumour endothelia were strongly stained for ICAM-1 compared with autologous normal tissue endothelia. Taken altogether, our observations reveal an IL-1alpha-mediated tumour-endothelium relationship sustaining the shedding of sICAM-1 by endothelial cells. This is a general phenomenon in solid malignancies that correlates with the ability of neoplastic cells to secrete IL-1alpha rather than with their expression of ICAM-1 and/or histological origin. sICAM-1 has been previously shown to inhibit LFA-1/ICAM-1-mediated cell-cell interactions; therefore, the ability of neoplastic cells to secrete IL-1alpha is likely to represent a mechanism for their escape from immune interaction. Images Figure 5 Figure 6 PMID:9374368

  20. Adenovirus E4-ORF3-dependent relocalization of TIF1{alpha} and TIF1{gamma} relies on access to the Coiled-Coil motif

    SciTech Connect

    Vink, Elizabeth I.; Yondola, Mark A.; Wu, Kai; Hearing, Patrick

    2012-01-20

    The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1{alpha}), but not homologous TIF1{beta}. Here, we introduce TIF1{gamma} as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1{gamma} in virus-infected cells in vivo and binds to TIF1{gamma} in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1{alpha}, TIF1{beta}, and TIF1{gamma}, and that the C-terminal half of TIF1{beta} interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.

  1. Retinoid X receptor alpha controls innate inflammatory responses through the up-regulation of chemokine expression.

    PubMed

    Núñez, Vanessa; Alameda, Daniel; Rico, Daniel; Mota, Rubén; Gonzalo, Pilar; Cedenilla, Marta; Fischer, Thierry; Boscá, Lisardo; Glass, Christopher K; Arroyo, Alicia G; Ricote, Mercedes

    2010-06-01

    The retinoid X receptor alpha (RXRalpha) plays a central role in the regulation of many intracellular receptor signaling pathways and can mediate ligand-dependent transcription by forming homodimers or heterodimers with other nuclear receptors. Although several members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression, the existence in vivo of an RXR signaling pathway in macrophages has not been established. Here, we provide evidence that RXRalpha regulates the transcription of the chemokines Ccl6 and Ccl9 in macrophages independently of heterodimeric partners. Mice lacking RXRalpha in myeloid cells exhibit reduced levels of CCL6 and CCL9, impaired recruitment of leukocytes to sites of inflammation, and lower susceptibility to sepsis. These studies demonstrate that macrophage RXRalpha plays key roles in the regulation of innate immunity and represents a potential target for immunotherapy of sepsis.

  2. Regulation of hepatic branched-chain alpha-keto acid dehydrogenase complex in rats fed a high-fat diet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Branched-chain alpha-keto acid dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of branched chain alpha-ketoacid (BCKA) catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal...

  3. Central tolerance regulates B cells reactive with Goodpasture antigen alpha3(IV)NC1 collagen

    PubMed Central

    Zhang, Ying; Su, Susan C.; Hecox, Douglas B.; Brady, Graham F.; Mackin, Katherine M.; Clark, Amy G.; Foster, Mary H.

    2008-01-01

    Patients and rodents with Goodpasture’s syndrome (GPS) develop severe autoimmune crescentic glomerulonephritis, kidney failure, and lung hemorrhage due to binding of pathogenic autoantibodies to the NC1 domain of the alpha3 chain of type IV collagen. Target epitopes are cryptic, normally hidden from circulating antibodies by protein-protein interactions and the highly tissue-restricted expression of the alpha3(IV) collagen chain. Based on this limited antigen exposure, it has been suggested that target epitopes are not available as B cell tolerogens. To determine how pathogenic anti-GPS autoantibody responses are regulated, we generated an immunoglobulin (Ig) transgenic (Tg) mouse model that expresses an Ig that binds alpha3(IV)NC1 collagen epitopes recognized by serum IgG of patients with GPS. Phenotypic analysis reveals B cell depletion and light chain editing in Tg mice. To determine the default tolerance phenotype in the absence of receptor editing and endogenous lymphocyte populations, we crossed Tg mice two generations with mice deficient in recombinase activating gene (Rag). Resulting Tg Rag-deficient mice have central B cell deletion. Thus development of Tg anti-alpha3(IV)NC1 collagen B cells is halted in the bone marrow, at which point the cells are deleted unless rescued by a Rag enzyme-dependent process, such as editing. The central tolerance phenotype implies that tolerizing self antigen is expressed in bone marrow. PMID:18941198

  4. Glucocorticoid regulation of branched-chain alpha-ketoacid dehydrogenase E2 subunit gene expression.

    PubMed Central

    Costeas, P A; Chinsky, J M

    2000-01-01

    Regulation of the mammalian branched-chain alpha-ketoacid dehydrogenase complex (BCKAD) occurs under a variety of stressful conditions associated with changes in circulating glucocorticoids. Multiple levels of regulation in hepatocytes, including alteration of the levels of the structural subunits available for assembly (E1, alpha-ketoacid decarboxylase; E2, dihydrolipoamide acyltransferase; and E3, dihydrolipoamide dehydrogenase), as well as BCKAD kinase, which serves to phosphorylate the E1alpha subunit and inactivate complex activity, have been proposed. The direct role of glucocorticoids in regulating the expression of the murine gene encoding the major BCKAD subunit E2, upon which the other BCKAD subunits assemble, was therefore examined. Deletion analysis of the 5' proximal 7.0 kb of the murine E2 promoter sequence, using E2 promoter/luciferase expression minigene plasmids introduced into the hepatic H4IIEC3 cell line, suggested a promoter proximal region responsive to glucocorticoid regulation. Linker-scanning mutagenesis combined with deletion analysis established this functional glucocorticoid-responsive unit (GRU) to be located near the murine E2 proximal promoter site at -140 to -70 bp upstream from the transcription initiation site. The presence of this region in plasmid minigenes, containing varying amounts of the murine genomic sequence 5' upstream from proximal E2 promoter sequences, conferred 2-10 fold increases in luciferase reporter gene expression in H4IIEC3 cells, whether introduced by transient transfection or following co-selection for stable transfectants. The GRU region itself appeared to contain multiple interacting elements that combine to regulate overall E2 promoter activity in response to changing physiological conditions associated with varying concentrations of glucocorticoids and likely other hormonal effectors. PMID:10749674

  5. The 4-Cysteine Zinc-Finger Motif of the RNA Polymerase Regulator DksA serves as a Thiol Switch for Sensing Oxidative and Nitrosative Stress

    PubMed Central

    Henard, Calvin A.; Tapscott, Timothy; Crawford, Matthew A.; Husain, Maroof; Doulias, Paschalis-Thomas; Porwollik, Steffen; Liu, Lin; McClelland, Michael; Ischiropoulos, Harry; Vázquez-Torres, Andrés

    2014-01-01

    We show that thiols in the 4-cysteine zinc-finger motif of DksA, an RNA polymerase accessory protein known to regulate the stringent response, sense oxidative and nitrosative stress. Hydrogen peroxide- or nitric oxide (NO)-mediated modifications of thiols in the DksA 4-cysteine zinc-finger motif release the metal cofactor and drive reversible changes in the α-helicity of the protein. Wild-type and relA spoT mutant Salmonella, but not isogenic dksA-deficient bacteria, experience the downregulation of r-protein and amino acid transport expression after NO treatment, suggesting that DksA can regulate gene expression in response to NO congeners independently of the ppGpp alarmone. Oxidative stress enhances the DksA-dependent repression of rpsM, while preventing the activation of livJ and hisG gene transcription that is supported by reduced, zinc-bound DksA. The inhibitory effects of oxidized DksA on transcription are reversible with dithiothreitol. Our investigations indicate that sensing of reactive species by DksA redox active thiols fine-tunes the expression of translational machinery and amino acid assimilation and biosynthesis in accord with the metabolic stress imposed by oxidative and nitrosative stress. Given the conservation of Cys114, and neighboring hydrophobic and charged amino acids in DksA orthologues, phylogenetically diverse microorganisms may use the DksA thiol switch to regulate transcriptional responses to oxidative and nitrosative stress. PMID:24354846

  6. Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

    PubMed

    de Vries, R P; van de Vondervoort, P J I; Hendriks, L; van de Belt, M; Visser, J

    2002-09-01

    The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

  7. Alpha 7 subunit of nAChR regulates migration of human mesenchymal stem cells.

    PubMed

    Schraufstatter, Ingrid U; DiScipio, Richard G; Khaldoyanidi, Sophia K

    2009-01-01

    The efficient migration of mesenchymal stem cells (MSCs) to diseased tissues is required for the fulfillment of their regenerative potential. Recruitment of circulating cells into the damaged tissues is regulated by a complex network, which includes the non-neural cholinergic system. We found that human MSCs (hMSCs) express nicotinic acetylcholine receptor subunits alpha 7, beta 2 and beta 4. The receptor agonist nicotine caused calcium (Ca(++)) influx into hMSCs suggesting that the calcium ion channel alpha 7 homopolymer mediated this response. While high concentrations of nicotine (10(5)M) induced hMSC apoptosis, physiological concentrations (10(7)M) did not interfere with cell survival. At non-toxic concentrations, nicotine increased spontaneous migration of hMSCs, whereas chemotaxis of hMSCs toward C3a and bFGF in vitro and migration of intravenously infusion hMSCs into bone marrow and spleen in vivo were inhibited. The antagonist for the alpha 7 homopolymer, bungarotoxin, blocked the inhibitory effect of nicotine on chemotactic factor-induced migration of hMSCs. These findings reveal an involvement of the non-neural cholinergic system in regulation of hMSC migration.

  8. Inverse regulation of human ERBB2 and epidermal growth factor receptors by tumor necrosis factor alpha.

    PubMed

    Kalthoff, H; Roeder, C; Gieseking, J; Humburg, I; Schmiegel, W

    1993-10-01

    Recombinant human tumor necrosis factor (TNF) alpha decreased the expression of ERBB2 mRNA by stimulating p55 TNF receptors of pancreatic tumor cells. This decrease contrasts with an increase in epidermal growth factor receptor (EGFR) mRNA. Both effects were selectively achieved by TNF-alpha or -beta, whereas interferon alpha or gamma or transforming growth factor beta showed no such effects. The inverse regulatory effects of TNF on ERBB2 and EGFR mRNA levels were evoked by different signaling pathways of p55 TNF receptors. The TNF-mediated ERBB2 mRNA decrease was followed by a reduction in protein. Four of five pancreatic tumor cell lines exhibited this down-regulation. This decrease of ERBB2 is a singular example of a modulation of this growth factor receptor by TNF. Overexpression of ERBB2 has been reported to cause resistance to TNF and other cytotoxic cytokines. In our study we show that the TNF-mediated down-regulation of ERBB2 in pancreatic tumor cells is accompanied by an increase in growth inhibition at low doses of TNF. The simultaneous alteration of the ERBB2/EGFR balance by TNF represents a striking model of cytokine receptor transregulation in the growth control of malignant pancreatic epithelial cells.

  9. Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.

    PubMed Central

    O'Toole, J J; Deyst, K A; Bowe, M A; Nastuk, M A; McKechnie, B A; Fallon, J R

    1996-01-01

    Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8693000

  10. Inverse regulation of human ERBB2 and epidermal growth factor receptors by tumor necrosis factor alpha.

    PubMed Central

    Kalthoff, H; Roeder, C; Gieseking, J; Humburg, I; Schmiegel, W

    1993-01-01

    Recombinant human tumor necrosis factor (TNF) alpha decreased the expression of ERBB2 mRNA by stimulating p55 TNF receptors of pancreatic tumor cells. This decrease contrasts with an increase in epidermal growth factor receptor (EGFR) mRNA. Both effects were selectively achieved by TNF-alpha or -beta, whereas interferon alpha or gamma or transforming growth factor beta showed no such effects. The inverse regulatory effects of TNF on ERBB2 and EGFR mRNA levels were evoked by different signaling pathways of p55 TNF receptors. The TNF-mediated ERBB2 mRNA decrease was followed by a reduction in protein. Four of five pancreatic tumor cell lines exhibited this down-regulation. This decrease of ERBB2 is a singular example of a modulation of this growth factor receptor by TNF. Overexpression of ERBB2 has been reported to cause resistance to TNF and other cytotoxic cytokines. In our study we show that the TNF-mediated down-regulation of ERBB2 in pancreatic tumor cells is accompanied by an increase in growth inhibition at low doses of TNF. The simultaneous alteration of the ERBB2/EGFR balance by TNF represents a striking model of cytokine receptor transregulation in the growth control of malignant pancreatic epithelial cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8105469

  11. Alternative Splicing in CaV2.2 Regulates Neuronal Trafficking via Adaptor Protein Complex-1 Adaptor Protein Motifs

    PubMed Central

    Macabuag, Natsuko

    2015-01-01

    N-type voltage-gated calcium (CaV2.2) channels are expressed in neurons and targeted to the plasma membrane of presynaptic terminals, facilitating neurotransmitter release. Here, we find that the adaptor protein complex-1 (AP-1) mediates trafficking of CaV2.2 from the trans-Golgi network to the cell surface. Examination of splice variants of CaV2.2, containing either exon 37a (selectively expressed in nociceptors) or 37b in the proximal C terminus, reveal that canonical AP-1 binding motifs, YxxΦ and [DE]xxxL[LI], present only in exon 37a, enhance intracellular trafficking of exon 37a-containing CaV2.2 to the axons and plasma membrane of rat DRG neurons. Finally, we identify differential effects of dopamine-2 receptor (D2R) and its agonist-induced activation on trafficking of CaV2.2 isoforms. D2R slowed the endocytosis of CaV2.2 containing exon 37b, but not exon 37a, and activation by the agonist quinpirole reversed the effect of the D2R. Our work thus reveals key mechanisms involved in the trafficking of N-type calcium channels. SIGNIFICANCE STATEMENT CaV2.2 channels are important for neurotransmitter release, but how they are trafficked is still poorly understood. Here, we describe a novel mechanism for trafficking of CaV2.2 from the trans-Golgi network to the cell surface which is mediated by the adaptor protein AP-1. Alternative splicing of exon 37 produces CaV2.2-exon 37a, selectively expressed in nociceptors, or CaV2.2-exon 37b, which is the major splice isoform. Our study reveals that canonical AP-1 binding motifs (YxxΦ and [DE]xxxL[LI]), present in exon 37a, but not 37b, enhance intracellular trafficking of exon 37a-containing CaV2.2 to axons and plasma membrane of DRG neurons. Interaction of APs with CaV2.2 channels may also be key underlying mechanisms for differential effects of the dopamine D2 receptor on trafficking of CaV2.2 splice variants. PMID:26511252

  12. Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells.

    PubMed

    Miller, K A; Eklund, E A; Peddinghaus, M L; Cao, Z; Fernandes, N; Turk, P W; Thimmapaya, B; Weitzman, S A

    2001-11-16

    Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha3A, beta3, and gamma2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin alpha3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.

  13. Interlocked positive and negative feedback network motifs regulate β-catenin activity in the adherens junction pathway

    PubMed Central

    Klinke, David J.; Horvath, Nicholas; Cuppett, Vanessa; Wu, Yueting; Deng, Wentao; Kanj, Rania

    2015-01-01

    The integrity of epithelial tissue architecture is maintained through adherens junctions that are created through extracellular homotypic protein–protein interactions between cadherin molecules. Cadherins also provide an intracellular scaffold for the formation of a multiprotein complex that contains signaling proteins, including β-catenin. Environmental factors and controlled tissue reorganization disrupt adherens junctions by cleaving the extracellular binding domain and initiating a series of transcriptional events that aim to restore tissue homeostasis. However, it remains unclear how alterations in cell adhesion coordinate transcriptional events, including those mediated by β-catenin in this pathway. Here were used quantitative single-cell and population-level in vitro assays to quantify the endogenous pathway dynamics after the proteolytic disruption of the adherens junctions. Using prior knowledge of isolated elements of the overall network, we interpreted these data using in silico model-based inference to identify the topology of the regulatory network. Collectively the data suggest that the regulatory network contains interlocked network motifs consisting of a positive feedback loop, which is used to restore the integrity of adherens junctions, and a negative feedback loop, which is used to limit β-catenin–induced gene expression. PMID:26224311

  14. An Algorithm for Motif Discovery with Iteration on Lengths of Motifs.

    PubMed

    Fan, Yetian; Wu, Wei; Yang, Jie; Yang, Wenyu; Liu, Rongrong

    2015-01-01

    Analysis of DNA sequence motifs is becoming increasingly important in the study of gene regulation, and the identification of motif in DNA sequences is a complex problem in computational biology. Motif discovery has attracted the attention of more and more researchers, and varieties of algorithms have been proposed. Most existing motif discovery algorithms fix the motif's length as one of the input parameters. In this paper, a novel method is proposed to identify the optimal length of the motif and the optimal motif with that length, through an iteration process on increasing length numbers. For each fixed length, a modified genetic algorithm (GA) is used for finding the optimal motif with that length. Three operators are used in the modified GA: Mutation that is similar to the one used in usual GA but is modified to avoid local optimum in our case, and Addition and Deletion that are proposed by us for the problem. A criterion is given for singling out the optimal length in the increasing motif's lengths. We call this method AMDILM (an algorithm for motif discovery with iteration on lengths of motifs). The experiments on simulated data and real biological data show that AMDILM can accurately identify the optimal motif length. Meanwhile, the optimal motifs discovered by AMDILM are consistent with the real ones and are similar with the motifs obtained by the three well-known methods: Gibbs Sampler, MEME and Weeder. PMID:26357084

  15. Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene.

    PubMed Central

    Ho, I C; Vorhees, P; Marin, N; Oakley, B K; Tsai, S F; Orkin, S H; Leiden, J M

    1991-01-01

    In addition to its role in the recognition of foreign antigens, the T cell receptor (TCR) alpha gene serves as a model system for studies of developmentally-regulated, lineage-specific gene expression in T cells. TCR alpha gene expression is restricted to cells of the TCR alpha/beta+ lineage, and is controlled by a T cell-specific transcriptional enhancer located 4.5 kb 3' to the C alpha gene segment. The TCR alpha enhancer contains four nuclear protein binding sites called T alpha 1-T alpha 4. In this report we describe the identification and characterization of a novel human cDNA, hGATA-3 that binds to the T alpha 3 element of the human TCR alpha enhancer. hGATA-3 contains a zinc finger domain that is highly related to the DNA-binding domain of the erythroid-specific transcription factor, GATA-1, and binds to a region of T alpha 3 that contains a consensus GATA binding site (AGATAG). Northern blot analyses of hematopoietic cell lines demonstrate that hGATA-3 is expressed exclusively in T cells. Overexpression of hGATA-3 in HeLa cells or human B cells specifically activated transcription from a co-transfected reporter plasmid containing two copies of the T alpha 3 binding site located upstream of the minimal SV40 promoter. Taken together these results demonstrate that hGATA-3 is a novel lineage-specific hematopoietic transcription factor that appears to play an important role in regulating the T cell-specific expression of the TCR alpha gene. Images PMID:1827068

  16. Effects of PGF{sub 2{alpha}} on human melanocytes and regulation of the FP receptor by ultraviolet radiation

    SciTech Connect

    Scott, Glynis . E-mail: Glynis_Scott@urmc.rochester.edu; Jacobs, Stacey; Leopardi, Sonya; Anthony, Frank A.; Learn, Doug; Malaviya, Rama; Pentland, Alice

    2005-04-01

    Prostaglandins are potent lipid hormones that activate multiple signaling pathways resulting in regulation of cellular growth, differentiation, and apoptosis. In the skin, prostaglandins are rapidly released by keratinocytes following ultraviolet radiation and are chronically present in inflammatory skin lesions. We have shown previously that melanocytes, which provide photoprotection to keratinocytes through the production of melanin, express several receptors for prostaglandins, including the PGE{sub 2} receptors EP{sub 1} and EP{sub 3} and the PGF{sub 2{alpha}} receptor FP, and that PGF{sub 2{alpha}} stimulates melanocyte dendricity. We now show that PGF{sub 2{alpha}} stimulates the activity and expression of tyrosinase, the rate-limiting enzyme in melanin synthesis. Analysis of FP receptor regulation showed that the FP receptor is regulated by ultraviolet radiation in melanocytes in vitro and in human skin in vivo. We also show that ultraviolet irradiation stimulates production of PGF{sub 2{alpha}} by melanocytes. These results show that PGF{sub 2{alpha}} binding to the FP receptor activates signals that stimulate a differentiated phenotype (dendricity and pigmentation) in melanocytes. The regulation of the FP receptor and the stimulation of production of PGF{sub 2{alpha}} in melanocytes in response to ultraviolet radiation suggest that PGF{sub 2{alpha}} could act as an autocrine factor for melanocyte differentiation.

  17. MIR@NT@N: a framework integrating transcription factors, microRNAs and their targets to identify sub-network motifs in a meta-regulation network model

    PubMed Central

    2011-01-01

    Background To understand biological processes and diseases, it is crucial to unravel the concerted interplay of transcription factors (TFs), microRNAs (miRNAs) and their targets within regulatory networks and fundamental sub-networks. An integrative computational resource generating a comprehensive view of these regulatory molecular interactions at a genome-wide scale would be of great interest to biologists, but is not available to date. Results To identify and analyze molecular interaction networks, we developed MIR@NT@N, an integrative approach based on a meta-regulation network model and a large-scale database. MIR@NT@N uses a graph-based approach to predict novel molecular actors across multiple regulatory processes (i.e. TFs acting on protein-coding or miRNA genes, or miRNAs acting on messenger RNAs). Exploiting these predictions, the user can generate networks and further analyze them to identify sub-networks, including motifs such as feedback and feedforward loops (FBL and FFL). In addition, networks can be built from lists of molecular actors with an a priori role in a given biological process to predict novel and unanticipated interactions. Analyses can be contextualized and filtered by integrating additional information such as microarray expression data. All results, including generated graphs, can be visualized, saved and exported into various formats. MIR@NT@N performances have been evaluated using published data and then applied to the regulatory program underlying epithelium to mesenchyme transition (EMT), an evolutionary-conserved process which is implicated in embryonic development and disease. Conclusions MIR@NT@N is an effective computational approach to identify novel molecular regulations and to predict gene regulatory networks and sub-networks including conserved motifs within a given biological context. Taking advantage of the M@IA environment, MIR@NT@N is a user-friendly web resource freely available at http://mironton.uni.lu which will be

  18. Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with alpha9beta1 and alpha4beta1 integrins.

    PubMed

    Grassinger, Jochen; Haylock, David N; Storan, Melonie J; Haines, Gemma O; Williams, Brenda; Whitty, Genevieve A; Vinson, Andrew R; Be, Cheang Ly; Li, Songhui; Sørensen, Esben S; Tam, Patrick P L; Denhardt, David T; Sheppard, Dean; Choong, Peter F; Nilsson, Susan K

    2009-07-01

    Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express alpha(9)beta(1) integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the alpha(9)beta(1) and alpha(4)beta(1) integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with alpha(9)beta(1) and alpha(4)beta(1) integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.

  19. Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases*

    PubMed Central

    Gentry, Leanna R.; Nishimura, Akiyuki; Cox, Adrienne D.; Martin, Timothy D.; Tsygankov, Denis; Nishida, Motohiro; Elston, Timothy C.; Der, Channing J.

    2015-01-01

    The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB. PMID:26216878

  20. Regulation of the alpha-galactosidase activity in Streptococcus pneumoniae: characterization of the raffinose utilization system.

    PubMed

    Rosenow, C; Maniar, M; Trias, J

    1999-12-01

    A 10.2-kb gene region was identified in the Streptococcus pneumoniae genome sequence that contains eight genes involved in regulation and metabolism of raffinose. The genes rafR and rafS are transcribed as one operon, and their gene products regulate the raffinose-dependent stimulation of a divergently transcribed second promoter (P(A)) directing the expression of aga, the structural gene for alpha-galactosidase. Raffinose-mediated transcription from P(A) results in a 500-fold increase in alpha-galactosidase activity in the cell. A third promoter within the cluster is responsible for the transcription of the remaining five genes (rafE, rafF, rafG, gtfA, and rafX), whose gene products might be involved in transport and metabolism of raffinose. The presence of additional internal promoters cannot be excluded. The aga promoter P(A) is negatively regulated by the presence of sucrose in the growth medium. Consistent with catabolite repression (CR), a DNA sequence with high homology to the CRE (cis-active element) was identified upstream of the aga promoter. Sucrose-mediated CR depends on the phosphoenolpyruvate: sucrose phosphotransferase system (PTS) but is unaffected by a mutation in a gene encoding a homolog of the CRE regulatory protein CcpA.

  1. Detecting correlations among functional-sequence motifs

    NASA Astrophysics Data System (ADS)

    Pirino, Davide; Rigosa, Jacopo; Ledda, Alice; Ferretti, Luca

    2012-06-01

    Sequence motifs are words of nucleotides in DNA with biological functions, e.g., gene regulation. Identification of such words proceeds through rejection of Markov models on the expected motif frequency along the genome. Additional biological information can be extracted from the correlation structure among patterns of motif occurrences. In this paper a log-linear multivariate intensity Poisson model is estimated via expectation maximization on a set of motifs along the genome of E. coli K12. The proposed approach allows for excitatory as well as inhibitory interactions among motifs and between motifs and other genomic features like gene occurrences. Our findings confirm previous stylized facts about such types of interactions and shed new light on genome-maintenance functions of some particular motifs. We expect these methods to be applicable to a wider set of genomic features.

  2. The CcpA regulon of Streptococcus suis reveals novel insights into the regulation of the streptococcal central carbon metabolism by binding of CcpA to two distinct binding motifs.

    PubMed

    Willenborg, Jörg; de Greeff, Astrid; Jarek, Michael; Valentin-Weigand, Peter; Goethe, Ralph

    2014-04-01

    Streptococcus suis (S. suis) is a neglected zoonotic streptococcus causing fatal diseases in humans and in pigs. The transcriptional regulator CcpA (catabolite control protein A) is involved in the metabolic adaptation to different carbohydrate sources and virulence of S. suis and other pathogenic streptococci. In this study, we determined the DNA binding characteristics of CcpA and identified the CcpA regulon during growth of S. suis. Electrophoretic mobility shift analyses showed promiscuous DNA binding of CcpA to cognate cre sites in vitro. In contrast, sequencing of immunoprecipitated chromatin revealed two specific consensus motifs, a pseudo-palindromic cre motif (WWGAAARCGYTTTCWW) and a novel cre2 motif (TTTTYHWDHHWWTTTY), within the regulatory elements of the genes directly controlled by CcpA. Via these elements CcpA regulates expression of genes involved in carbohydrate uptake and conversion, and in addition in important metabolic pathways of the central carbon metabolism, like glycolysis, mixed-acid fermentation, and the fragmentary TCA cycle. Furthermore, our analyses provide evidence that CcpA regulates the genes of the central carbon metabolism by binding either the pseudo-palindromic cre motif or the cre2 motif in a HPr(Ser)∼P independent conformation.

  3. Alpha-inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin.

    PubMed

    Crawford, R J; Hammond, V E; Evans, B A; Coghlan, J P; Haralambidis, J; Hudson, B; Penschow, J D; Richards, R I; Tregear, G W

    1987-10-01

    Inhibin is a glycoprotein hormone composed of two nonidentical subunits. It is produced by the ovary and testis and plays a vital role in gonadal function by inhibiting the secretion of FSH. More recently, additional activities associated with inhibin peptides have been identified. Inhibin heterodimers (alpha-beta) are reported to act directly on ovarian granulosa cells and inhibit estrogen production induced by FSH. Furthermore, homodimers of beta-inhibin subunits stimulate the secretion of FSH, an activity that is directly opposite to that of inhibin. Each of these inhibin-related activities are concerned with the hypothalamic-pituitary-gonadal axis. We have investigated further the complexity of inhibin activity by determining whether inhibin genes are expressed in nongonadal tissue. RNA hybridization experiments demonstrate that the alpha-inhibin gene is expressed in the sheep adrenal cortex and hybridization histochemistry shows that this gene is expressed in each of the functional zones within the cortex. Dot blot analysis showed that the level of alpha mRNA within the adrenal is influenced by ACTH, one of the major regulators of adrenal cortex function. These observations imply that there are inhibin-related peptides not directly associated with the gonads. beta-inhibin gene expression was not clearly detected in the adrenal and we conclude that if expression occurs then it does so at extremely low levels.

  4. Banana fruit VQ motif-containing protein5 represses cold-responsive transcription factor MaWRKY26 involved in the regulation of JA biosynthetic genes

    PubMed Central

    Ye, Yu-Jie; Xiao, Yun-Yi; Han, Yan-Chao; Shan, Wei; Fan, Zhong-Qi; Xu, Qun-Gang; Kuang, Jian-Fei; Lu, Wang-Jin; Lakshmanan, Prakash; Chen, Jian-Ye

    2016-01-01

    Most harvested fruits and vegetables are stored at low temperature but many of them are highly sensitive to chilling injury. Jasmonic acid (JA), a plant hormone associated with various stress responses, is known to reduce chilling injury in fruits. However, little is known about the transcriptional regulation of JA biosynthesis in relation to cold response of fruits. Here, we show the involvement of a Group I WRKY transcription factor (TF) from banana fruit, MaWRKY26, in regulating JA biosynthesis. MaWRKY26 was found to be nuclear-localized with transcriptional activation property. MaWRKY26 was induced by cold stress or by methyl jasmonate (MeJA), which enhances cold tolerance in banana fruit. More importantly, MaWRKY26 transactivated JA biosynthetic genes MaLOX2, MaAOS3 and MaOPR3 via binding to their promoters. Further, MaWRKY26 physically interacted with a VQ motif-containing protein MaVQ5, and the interaction attenuated MaWRKY26-induced transactivation of JA biosynthetic genes. These results strongly suggest that MaVQ5 might act as a repressor of MaWRKY26 in activating JA biosynthesis. Taken together, our findings provide new insights into the transcriptional regulation of JA biosynthesis in response to cold stress and a better understanding of the molecular aspects of chilling injury in banana fruit. PMID:27004441

  5. Up-regulation of alpha1-microglobulin by hemoglobin and reactive oxygen species in hepatoma and blood cell lines.

    PubMed

    Olsson, Magnus G; Allhorn, Maria; Olofsson, Tor; Akerström, Bo

    2007-03-15

    alpha(1)-Microglobulin is a 26-kDa glycoprotein synthesized in the liver, secreted to the blood, and rapidly distributed to the extravascular compartment of all tissues. Recent results show that alpha(1)-microglobulin has heme-binding and heme-degrading properties and it has been suggested that the protein is involved in the defense against oxidation by heme and reactive oxygen species. In the present study the influence of hemoglobin and reactive oxygen species (ROS) on the cellular expression of alpha(1)-microglobulin was investigated. Oxy- and methemoglobin, free heme, and Fenton reaction-induced hydroxyl radicals induced a dose-dependent up-regulation of alpha(1)-microglobulin on both mRNA and protein levels in hepatoma cells and an increased secretion of alpha(1)-microglobulin. The up-regulation was reversed by the addition of catalase and ascorbate, and by reacting hemoglobin with cyanide which prevents redox reactions. Furthermore, the blood cell lines U937 and K562 expressed alpha(1)-microglobulin at low levels, and this expression increased up to 11-fold by the addition of hemoglobin. These results suggest that alpha(1)-microglobulin expression is induced by ROS, arising from redox reactions of hemoglobin or from other sources and are consistent with the hypothesis that alpha(1)-microglobulin participates in the defense against oxidation by hemoglobin, heme, and reactive oxygen species.

  6. Coactivator PGC-1{alpha} regulates the fasting inducible xenobiotic-metabolizing enzyme CYP2A5 in mouse primary hepatocytes

    SciTech Connect

    Arpiainen, Satu; Jaervenpaeae, Sanna-Mari; Manninen, Aki; Viitala, Pirkko; Lang, Matti A.; Pelkonen, Olavi; Hakkola, Jukka

    2008-10-01

    The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1{alpha} and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1{alpha} expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1{alpha} expression vector demonstrated that PGC-1{alpha} is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4{alpha} response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1{alpha} binds, together with HNF-4{alpha}, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1{alpha} mediates the expression of CYP2A5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4{alpha}. This strongly suggests that PGC-1{alpha} is the major factor mediating the fasting response of CYP2A5.

  7. A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.

    PubMed

    Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J

    2015-02-01

    Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

  8. A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.

    PubMed

    Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J

    2015-02-01

    Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk. PMID:25646814

  9. A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    PubMed Central

    Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J.

    2015-01-01

    Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk. PMID:25646814

  10. Immunoreceptor tyrosine-based inhibitory motif (ITIM)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases: a case study involving PECAM-1.

    PubMed

    Tourdot, Benjamin E; Brenner, Michelle K; Keough, Kathleen C; Holyst, Trudy; Newman, Peter J; Newman, Debra K

    2013-04-16

    The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing nonreceptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton's tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals. PMID:23418871

  11. Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs.

    PubMed

    Liu, Yujia; Ji, Xiaoyu; Nie, Xianguang; Qu, Min; Zheng, Lei; Tan, Zilong; Zhao, Huimin; Huo, Lin; Liu, Shengnan; Zhang, Bing; Wang, Yucheng

    2015-08-01

    Plant basic helix-loop-helix (bHLH) transcription factors play essential roles in abiotic stress tolerance. However, most bHLHs have not been functionally characterized. Here, we characterized the functional role of a bHLH transcription factor from Arabidopsis, AtbHLH112, in response to abiotic stress. AtbHLH112 is a nuclear-localized protein, and its nuclear localization is induced by salt, drought and abscisic acid (ABA). In addition, AtbHLH112 serves as a transcriptional activator, with the activation domain located at its N-terminus. In addition to binding to the E-box motifs of stress-responsive genes, AtbHLH112 binds to a novel motif with the sequence 'GG[GT]CC[GT][GA][TA]C' (GCG-box). Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by increasing the expression of P5CS genes and reducing the expression of P5CDH and ProDH genes to increase proline levels. AtbHLH112 also increases the expression of POD and SOD genes to improve reactive oxygen species (ROS) scavenging ability. We present a model suggesting that AtbHLH112 is a transcriptional activator that regulates the expression of genes via binding to their GCG- or E-boxes to mediate physiological responses, including proline biosynthesis and ROS scavenging pathways, to enhance stress tolerance.

  12. PGC-1{beta} regulates mouse carnitine-acylcarnitine translocase through estrogen-related receptor {alpha}

    SciTech Connect

    Gacias, Mar; Perez-Marti, Albert; Pujol-Vidal, Magdalena; Marrero, Pedro F.; Haro, Diego; Relat, Joana

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer The Cact gene is induced in mouse skeletal muscle after 24 h of fasting. Black-Right-Pointing-Pointer The Cact gene contains a functional consensus sequence for ERR. Black-Right-Pointing-Pointer This sequence binds ERR{alpha} both in vivo and in vitro. Black-Right-Pointing-Pointer This ERRE is required for the activation of Cact expression by the PGC-1/ERR axis. Black-Right-Pointing-Pointer Our results add Cact as a genuine gene target of these transcriptional regulators. -- Abstract: Carnitine/acylcarnitine translocase (CACT) is a mitochondrial-membrane carrier proteins that mediates the transport of acylcarnitines into the mitochondrial matrix for their oxidation by the mitochondrial fatty acid-oxidation pathway. CACT deficiency causes a variety of pathological conditions, such as hypoketotic hypoglycemia, cardiac arrest, hepatomegaly, hepatic dysfunction and muscle weakness, and it can be fatal in newborns and infants. Here we report that expression of the Cact gene is induced in mouse skeletal muscle after 24 h of fasting. To gain insight into the control of Cact gene expression, we examine the transcriptional regulation of the mouse Cact gene. We show that the 5 Prime -flanking region of this gene is transcriptionally active and contains a consensus sequence for the estrogen-related receptor (ERR), a member of the nuclear receptor family of transcription factors. This sequence binds ERR{alpha}in vivo and in vitro and is required for the activation of Cact expression by the peroxisome proliferator-activated receptor gamma coactivator (PGC)-1/ERR axis. We also demonstrate that XTC790, the inverse agonist of ERR{alpha}, specifically blocks Cact activation by PGC-1{beta} in C2C12 cells.

  13. Regulation of adherence and virulence by the Entamoeba histolytica lectin cytoplasmic domain, which contains a beta2 integrin motif.

    PubMed

    Vines, R R; Ramakrishnan, G; Rogers, J B; Lockhart, L A; Mann, B J; Petri, W A

    1998-08-01

    Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the beta2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin's role in virulence.

  14. A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles.

    PubMed

    Ziegler, Christopher M; Eisenhauer, Philip; Bruce, Emily A; Beganovic, Vedran; King, Benjamin R; Weir, Marion E; Ballif, Bryan A; Botten, Jason

    2016-09-01

    We report that the lymphocytic choriomeningitis virus (LCMV) matrix protein, which drives viral budding, is phosphorylated at serine 41 (S41). A recombinant (r)LCMV bearing a phosphomimetic mutation (S41D) was impaired in infectious and defective interfering (DI) particle release, while a non-phosphorylatable mutant (S41A) was not. The S41D mutant was disproportionately impaired in its ability to release DI particles relative to infectious particles. Thus, DI particle production by LCMV may be dynamically regulated via phosphorylation of S41.

  15. PPAR{alpha} gene expression is up-regulated by LXR and PXR activators in the small intestine

    SciTech Connect

    Inoue, Jun; Satoh, Shin-ichi; Kita, Mariko; Nakahara, Mayuko; Hachimura, Satoshi; Miyata, Masaaki; Nishimaki-Mogami, Tomoko; Sato, Ryuichiro

    2008-07-11

    LXR, PXR, and PPAR{alpha} are members of a nuclear receptor family which regulate the expression of genes involved in lipid metabolism. Here, we show the administration of T0901317 stimulates PPAR{alpha} gene expression in the small intestine but not in the liver of both normal and FXR-null mice. The administration of LXR specific ligand GW3965, or PXR specific ligand PCN has the same effect, indicating that ligand-dependent activation of LXR and PXR, but not FXR, is responsible for the increased gene expression of PPAR{alpha} in the mouse small intestine.

  16. Dietary phosphorus transcriptionally regulates 25-hydroxyvitamin D-1alpha-hydroxylase gene expression in the proximal renal tubule.

    PubMed

    Zhang, Martin Y H; Wang, Xuemei; Wang, Jonathan T; Compagnone, Nathalie A; Mellon, Synthia H; Olson, Jean L; Tenenhouse, Harriet S; Miller, Walter L; Portale, Anthony A

    2002-02-01

    Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1alpha-hydroxylase activity and the renal expression of P450c1alpha and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1alpha-hydroxylase activity and renal P450c1alpha mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1alpha-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowman's capsule, thick ascending limb of Henle's loop, distal tubule, and collecting duct. In mice fed a phosphorus-restricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowman's capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1alpha gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1alpha mRNA and protein. The increase in P450c1alpha gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1alpha gene.

  17. Mechanisms regulating male sexual behavior in the rat: role of 3 alpha- and 3 beta-androstanediols.

    PubMed

    Morali, G; Oropeza, M V; Lemus, A E; Perez-Palacios, G

    1994-09-01

    To assess whether naturally occurring 5 alpha-androstanediols (5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol) play a role in the regulation of male sexual behavior in the rat, their capability to restore copulatory behavior in castrated animals was evaluated. Androstanediols were chronically administered either alone or in combination with 5 alpha-dihydrotestosterone (DHT) or with estradiol-17 beta (E2). Animals treated with testosterone (T), DHT, E2, and vehicle, either alone or in different combinations, served as controls. The occurrence of mounting, intromission, and ejaculation as well as detailed parameters of copulatory behavior were recorded twice per week for 3 weeks. At the end of treatments, the weights of sex accessory organs were also recorded. When 3 beta, 5 alpha-androstanediol (3 beta-diol; 500 micrograms/day) was administered in combination with DHT (300 micrograms/day), full copulatory behavior was restored in all subjects in a manner similar to that obtained with E2 plus DHT or T plus DHT combinations, thus indicating an estrogen-like behavioral effect of 3 beta-diol. Administration of 3 alpha, 5 alpha-androstanediol (3 alpha-diol; 500 micrograms/day) combined with DHT also restored sexual behavior, though to a lesser extent. When 3 alpha-diol (500 micrograms/day) was simultaneously administered with E2 (5 micrograms/day), the copulatory behavior of castrated animals was fully restored in a fashion similar to that observed after administration of DHT plus E2 and T plus E2 combinations, indicating a potent androgen-like effect of 3 alpha-diol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7803627

  18. RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond.

    PubMed

    Gama-Castro, Socorro; Salgado, Heladia; Santos-Zavaleta, Alberto; Ledezma-Tejeida, Daniela; Muñiz-Rascado, Luis; García-Sotelo, Jair Santiago; Alquicira-Hernández, Kevin; Martínez-Flores, Irma; Pannier, Lucia; Castro-Mondragón, Jaime Abraham; Medina-Rivera, Alejandra; Solano-Lira, Hilda; Bonavides-Martínez, César; Pérez-Rueda, Ernesto; Alquicira-Hernández, Shirley; Porrón-Sotelo, Liliana; López-Fuentes, Alejandra; Hernández-Koutoucheva, Anastasia; Del Moral-Chávez, Víctor; Rinaldi, Fabio; Collado-Vides, Julio

    2016-01-01

    RegulonDB (http://regulondb.ccg.unam.mx) is one of the most useful and important resources on bacterial gene regulation,as it integrates the scattered scientific knowledge of the best-characterized organism, Escherichia coli K-12, in a database that organizes large amounts of data. Its electronic format enables researchers to compare their results with the legacy of previous knowledge and supports bioinformatics tools and model building. Here, we summarize our progress with RegulonDB since our last Nucleic Acids Research publication describing RegulonDB, in 2013. In addition to maintaining curation up-to-date, we report a collection of 232 interactions with small RNAs affecting 192 genes, and the complete repertoire of 189 Elementary Genetic Sensory-Response units (GENSOR units), integrating the signal, regulatory interactions, and metabolic pathways they govern. These additions represent major progress to a higher level of understanding of regulated processes. We have updated the computationally predicted transcription factors, which total 304 (184 with experimental evidence and 120 from computational predictions); we updated our position-weight matrices and have included tools for clustering them in evolutionary families. We describe our semiautomatic strategy to accelerate curation, including datasets from high-throughput experiments, a novel coexpression distance to search for 'neighborhood' genes to known operons and regulons, and computational developments. PMID:26527724

  19. RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond

    PubMed Central

    Gama-Castro, Socorro; Salgado, Heladia; Santos-Zavaleta, Alberto; Ledezma-Tejeida, Daniela; Muñiz-Rascado, Luis; García-Sotelo, Jair Santiago; Alquicira-Hernández, Kevin; Martínez-Flores, Irma; Pannier, Lucia; Castro-Mondragón, Jaime Abraham; Medina-Rivera, Alejandra; Solano-Lira, Hilda; Bonavides-Martínez, César; Pérez-Rueda, Ernesto; Alquicira-Hernández, Shirley; Porrón-Sotelo, Liliana; López-Fuentes, Alejandra; Hernández-Koutoucheva, Anastasia; Moral-Chávez, Víctor Del; Rinaldi, Fabio; Collado-Vides, Julio

    2016-01-01

    RegulonDB (http://regulondb.ccg.unam.mx) is one of the most useful and important resources on bacterial gene regulation,as it integrates the scattered scientific knowledge of the best-characterized organism, Escherichia coli K-12, in a database that organizes large amounts of data. Its electronic format enables researchers to compare their results with the legacy of previous knowledge and supports bioinformatics tools and model building. Here, we summarize our progress with RegulonDB since our last Nucleic Acids Research publication describing RegulonDB, in 2013. In addition to maintaining curation up-to-date, we report a collection of 232 interactions with small RNAs affecting 192 genes, and the complete repertoire of 189 Elementary Genetic Sensory-Response units (GENSOR units), integrating the signal, regulatory interactions, and metabolic pathways they govern. These additions represent major progress to a higher level of understanding of regulated processes. We have updated the computationally predicted transcription factors, which total 304 (184 with experimental evidence and 120 from computational predictions); we updated our position-weight matrices and have included tools for clustering them in evolutionary families. We describe our semiautomatic strategy to accelerate curation, including datasets from high-throughput experiments, a novel coexpression distance to search for ‘neighborhood’ genes to known operons and regulons, and computational developments. PMID:26527724

  20. RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond.

    PubMed

    Gama-Castro, Socorro; Salgado, Heladia; Santos-Zavaleta, Alberto; Ledezma-Tejeida, Daniela; Muñiz-Rascado, Luis; García-Sotelo, Jair Santiago; Alquicira-Hernández, Kevin; Martínez-Flores, Irma; Pannier, Lucia; Castro-Mondragón, Jaime Abraham; Medina-Rivera, Alejandra; Solano-Lira, Hilda; Bonavides-Martínez, César; Pérez-Rueda, Ernesto; Alquicira-Hernández, Shirley; Porrón-Sotelo, Liliana; López-Fuentes, Alejandra; Hernández-Koutoucheva, Anastasia; Del Moral-Chávez, Víctor; Rinaldi, Fabio; Collado-Vides, Julio

    2016-01-01

    RegulonDB (http://regulondb.ccg.unam.mx) is one of the most useful and important resources on bacterial gene regulation,as it integrates the scattered scientific knowledge of the best-characterized organism, Escherichia coli K-12, in a database that organizes large amounts of data. Its electronic format enables researchers to compare their results with the legacy of previous knowledge and supports bioinformatics tools and model building. Here, we summarize our progress with RegulonDB since our last Nucleic Acids Research publication describing RegulonDB, in 2013. In addition to maintaining curation up-to-date, we report a collection of 232 interactions with small RNAs affecting 192 genes, and the complete repertoire of 189 Elementary Genetic Sensory-Response units (GENSOR units), integrating the signal, regulatory interactions, and metabolic pathways they govern. These additions represent major progress to a higher level of understanding of regulated processes. We have updated the computationally predicted transcription factors, which total 304 (184 with experimental evidence and 120 from computational predictions); we updated our position-weight matrices and have included tools for clustering them in evolutionary families. We describe our semiautomatic strategy to accelerate curation, including datasets from high-throughput experiments, a novel coexpression distance to search for 'neighborhood' genes to known operons and regulons, and computational developments.

  1. G protein-coupled receptor 30 expression is up-regulated by EGF and TGF alpha in estrogen receptor alpha-positive cancer cells.

    PubMed

    Vivacqua, Adele; Lappano, Rosamaria; De Marco, Paola; Sisci, Diego; Aquila, Saveria; De Amicis, Francesca; Fuqua, Suzanne A W; Andò, Sebastiano; Maggiolini, Marcello

    2009-11-01

    In the present study, we evaluated the regulation of G protein-coupled receptor (GPR)30 expression in estrogen receptor (ER)-positive endometrial, ovarian, and estrogen-sensitive, as well as tamoxifen-resistant breast cancer cells. We demonstrate that epidermal growth factor (EGF) and TGF alpha transactivate the GPR30 promoter and accordingly up-regulate GPR30 mRNA and protein levels only in endometrial and tamoxifen-resistant breast cancer cells. These effects exerted by EGF and TGF alpha were dependent on EGF receptor (EGFR) expression and activation and involved phosphorylation of the Tyr(1045) and Tyr(1173) EGFR sites. Using gene-silencing experiments and specific pharmacological inhibitors, we have ascertained that EGF and TGF alpha induce GPR30 expression through the EGFR/ERK transduction pathway, and the recruitment of c-fos to the activator protein-1 site located within GPR30 promoter sequence. Interestingly, we show that functional cross talk of GPR30 with both activated EGFR and ER alpha relies on a physical interaction among these receptors, further extending the potential of estrogen to trigger a complex stimulatory signaling network in hormone-sensitive tumors. Given that EGFR/HER2 overexpression is associated with tamoxifen resistance, our data may suggest that ligand-activated EGFR could contribute to the failure of tamoxifen therapy also by up-regulating GPR30, which in turn could facilitates the action of estrogen. In addition, important for resistance is the ability of tamoxifen to bind to and activate GPR30, the expression of which is up-regulated by EGFR activation. Our results emphasize the need for new endocrine agents able to block widespread actions of estrogen without exerting any stimulatory activity on transduction pathways shared by the steroid and growth factor-signaling networks.

  2. The amiloride-sensitive epithelial sodium channel alpha-subunit is transcriptionally down-regulated in rat parotid cells by the extracellular signal-regulated protein kinase pathway.

    PubMed

    Zentner, M D; Lin, H H; Wen, X; Kim, K J; Ann, D K

    1998-11-13

    Previous studies have shown that an inducible Raf-1 kinase protein, DeltaRaf-1:ER, activates the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK)-signaling pathway, which is required for the transformation of the rat salivary epithelial cell line, Pa-4. Differential display polymerase chain reaction was employed to search for mRNAs repressed by DeltaRaf-1:ER activation. Through this approach, the gene encoding the alpha-subunit of the amiloride-sensitive epithelial sodium channel (alpha-ENaC) was identified as a target of activated Raf-1 kinases. alpha-ENaC down-regulation could also be seen in cells treated with 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA), indicating that the repression of steady-state alpha-ENaC mRNA level was dependent upon the activity of protein kinase C, the target of TPA, as well. Pretreatment of cells with a specific inhibitor of the ERK kinase pathway, PD 98059, markedly abolished the down-regulation of alpha-ENaC expression, consistent with the hypothesis that the ERK kinase-signaling pathway is involved in TPA-mediated repression. Moreover, through the use of transient transfection assays with alpha-ENaC-reporter and activated Raf expression construct(s), we provide the first evidence that activation of the ERK pathway down-regulates alpha-ENaC expression at the transcriptional level. Elucidating the molecular programming that modulates the expression of the alpha-subunit may provide new insights into the modulation of sodium reabsorption across epithelia. PMID:9804854

  3. Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

    SciTech Connect

    Zhang, Yu; Cheng, Jung-Chien; Huang, He-Feng; Leung, Peter C.K.

    2013-11-01

    Highlights: •HOXA7 regulates MCF7 cell proliferation. •HOXA7 up-regulates ERα expression. •HOXA7 mediates estrogen-induced MCF7 cell proliferation. -- Abstract: Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited. In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.

  4. The pyruvate dehydrogenase E1 alpha gene is testosterone and prolactin regulated in prostate epithelial cells.

    PubMed

    Costello, L C; Liu, Y; Zou, J; Franklin, R B

    2000-02-01

    The prostate gland of humans and other animals has the unique function of accumulating and secreting extraordinarily high levels of citrate. The prostate secretory epithelial cells synthesize citrate which, due to a limiting mitochondrial (m-) aconitase, accumulates rather than being oxidized. Thus citrate is essentially an end product of metabolism in prostate. For continued net citrate production, a continual source of oxaloacetate (OAA) and acetyl CoA is required. Glucose via pyruvate oxidation provides the source of Acetyl CoA. In prostate cells, citrate production is regulated by testosterone and/or by prolactin. Both hormones selectively regulate the level and activity of pyruvate dehydrogenase E1 alpha (E1a) in animal prostate cells; thereby regulating the availability of acetyl CoA for citrate synthesis. Studies were conducted to determine if testosterone and prolactin might regulate the expression of the E1a gene in prostate epithelial cells. Prolactin treatment of rat ventral and lateral prostate cells and human PC3 cells increased the levels of E1a mRNA and the rates of transcription of the E1a gene. Testosterone also increased the mRNA level and transcription of E1a in rat ventral prostate cells, and in PC3 cells transfected with androgen receptor. However, testosterone treatment resulted in a repression of E1a gene expression in lateral prostate cells. Evidence is presented which supports the view that prolactin regulation of E1a is mediated via PKC. The rapidity of the effects of both hormones is representative of an immediate-early gene response. To our knowledge this represents the first report in any mammalian cells that, in addition to its constitutive expression in all mammalian cells, the E1a gene is a hormonally-regulated gene in specifically targeted prostate epithelial cells. PMID:10711720

  5. Mining Conditional Phosphorylation Motifs.

    PubMed

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou

    2014-01-01

    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/. PMID:26356863

  6. Alpha actinin-1 regulates cell-matrix adhesion organization in keratinocytes: consequences for skin cell motility.

    PubMed

    Hamill, Kevin J; Hiroyasu, Sho; Colburn, Zachary T; Ventrella, Rosa V; Hopkinson, Susan B; Skalli, Omar; Jones, Jonathan C R

    2015-04-01

    The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton, and matrix adhesions. In this study, we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via a small hairpin RNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower compared with their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion, and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics, and directed migration. PMID:25431851

  7. Down-regulation of. alpha. sub 2 adrenoceptors in ventrolateral medulla of spontaneously hypertensive rats

    SciTech Connect

    Gulati, A. )

    1991-01-01

    The binding of ({sup 3}H)idaxazon to imidazole sites and ({sup 3}H)rauwolscine to {alpha}{sub 2} adrenoceptors of neuronal membranes prepared from cerebral cortex and ventrolateral medulla of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. ({sup 3}H)idaxazon bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of ({sup 3}H)idaxazon in ventrolateral medulla and cerebral cortex was found to be similar in SHR and WKY rats. ({sup 3}H)Rauwolscine bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of ({sup 3}H)rauwolscine in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla ({sup 3}H)rauwolscine binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due a decrease (32%) in the B{sub max} value in SHR rats as compared to WKY rats. The K{sub d} values were similar in SHR and WKY rats. It is concluded that imidazole binding sites are not affected while, {alpha}{sub 2} adrenergic binding sites are decreased in the ventrolateral medulla of SHR rats and may be contributing to the regulation of blood pressure.

  8. PPAR{alpha} regulates the hepatotoxic biomarker alanine aminotransferase (ALT1) gene expression in human hepatocytes

    SciTech Connect

    Thulin, Petra; Rafter, Ingalill; Stockling, Kenneth; Tomkiewicz, Celine; Norjavaara, Ensio; Aggerbeck, Martine; Hellmold, Heike; Ehrenborg, Ewa; Andersson, Ulf; Cotgreave, Ian; Glinghammar, Bjoern

    2008-08-15

    In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) {alpha} agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPAR{alpha} agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at - 574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum.

  9. Poly (ADP-ribose) polymerase 1 transcriptional regulation: A novel crosstalk between histone modification H3K9ac and ETS1 motif hypomethylation in BRCA1-mutated ovarian cancer

    PubMed Central

    Li, Da; Bi, Fang-Fang; Cao, Ji-Min; Cao, Chen; Li, Chun-Yan; Liu, Bo; Yang, Qing

    2014-01-01

    Poly (ADP-ribose) polymerase 1 (PARP1) plays a critical role in ovarian cancer progression. However, the epigenetic mechanism regulating PARP1 transcription remains largely unknown. Here, we show that the hypomethylated ETS1 motif is a key regulatory element for the PARP1 gene in BRCA1-mutated ovarian cancer. Mechanistically, the ETS1 motif hypomethylation-mediated increase of active histone marker H3K9ac and transcription factor ETS1 enrichment synergistically activates PARP1 transcription. Clinicopathological data indicate that a hypomethylated ETS1 motif was associated with high-grade tumors (P = 0.026) and pN1 (P = 0.002). Univariate survival analysis demonstrated an association between the hypomethylated ETS1 motif and an increased risk of death in BRCA1-mutated ovarian cancer patients. Our findings imply that the genetic (such as BRCA1 mutation) and epigenetic mechanisms (such as hypomethylated ETS1 motif, and histone modification H3K9ac and transcription factor ETS1 binding) are jointly involved in the malignant progression of PARP1-related ovarian cancer. PMID:24448423

  10. Identification of common motifs in the regulation of light harvesting: The case of cyanobacteria IsiA.

    PubMed

    Wahadoszamen, Md; D'Haene, Sandrine; Ara, Anjue Mane; Romero, Elisabet; Dekker, Jan P; Grondelle, Rienk van; Berera, Rudi

    2015-01-01

    When cyanobacteria are grown under iron-limited or other oxidative stress conditions the iron stress inducible pigment-protein IsiA is synthesized in variable amounts. IsiA accumulates in aggregates inside the photosynthetic membrane that strongly dissipate chlorophyll excited state energy. In this paper we applied Stark fluorescence (SF) spectroscopy at 77K to IsiA aggregates to gain insight into the nature of the emitting and energy dissipating state(s). Our study shows that two emitting states are present in the system, one emitting at 684 nm and the other emitting at about 730 nm. The new 730 nm state exhibits strongly reduced fluorescence (F) together with a large charge transfer character. We discuss these findings in the light of the energy dissipation mechanisms involved in the regulation of photosynthesis in plants, cyanobacteria and diatoms. Our results suggest that photosynthetic organisms have adopted common mechanisms to cope with the deleterious effects of excess light under unfavorable growth conditions.

  11. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis

    PubMed Central

    Weigel, Christoph; Veldwijk, Marlon R.; Oakes, Christopher C.; Seibold, Petra; Slynko, Alla; Liesenfeld, David B.; Rabionet, Mariona; Hanke, Sabrina A.; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-01-01

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy. PMID:26964756

  12. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis.

    PubMed

    Weigel, Christoph; Veldwijk, Marlon R; Oakes, Christopher C; Seibold, Petra; Slynko, Alla; Liesenfeld, David B; Rabionet, Mariona; Hanke, Sabrina A; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-03-11

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy.

  13. SIRT1 associates with eIF2-alpha and regulates the cellular stress response

    PubMed Central

    Ghosh, Hiyaa Singhee; Reizis, Boris; Robbins, Paul D.

    2011-01-01

    SIRT1 is a NAD+ dependent protein deacetylase known to increase longevity in model organisms. SIRT1 regulates cellular response to oxidative and/or genotoxic stress by regulating proteins such as p53 and FOXO. The eukaryotic initiation factor-2, eIF2, plays a critical role in the integrated stress response pathway. Under cellular stress, phosphorylation of the alpha subunit of eIF2 is essential for immediate shut-off of translation and activation of stress response genes. Here we demonstrate that SIRT1 interacts with eIF2α. Loss of SIRT1 results in increased phosphorylation of eIF2α. However, the downstream stress induced signaling pathway is compromised in SIRT1-deficient cells, indicated by delayed expression of the downstream target genes CHOP and GADD34 and a slower post-stress translation recovery. Finally, SIRT1 co-immunoprecipitates with mediators of eIF2α dephosphorylation, GADD34 and CreP, suggesting a role for SIRT1 in the negative feedback regulation of eIF2α phosphorylation. PMID:22355666

  14. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 Regulates the Cross Talk between Immunity and Abscisic Acid Responses1[W][OPEN

    PubMed Central

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-01-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens. PMID:24639336

  15. Structure of GrlR and the Implication of its EDED Motif in Mediating the Regulation of Type III Secretion System in EHEC

    SciTech Connect

    Jobichen,C.; Li, M.; Yerushalmi, G.; Tan, Y.; Mok, Y.; Rosenshine, I.; Leung, K.; Sivaraman, J.

    2007-01-01

    Enterohemorrhagic Escherichia coli (EHEC) is a common cause of severe hemorrhagic colitis. EHEC's virulence is dependent upon a type III secretion system (TTSS) encoded by 41 genes. These genes are organized in several operons clustered in the locus of enterocyte effacement. Most of the locus of enterocyte effacement genes, including grlA and grlR, are positively regulated by Ler, and Ler expression is positively and negatively modulated by GrlA and GrlR, respectively. However, the molecular basis for the GrlA and GrlR activity is still elusive. We have determined the crystal structure of GrlR at 1.9 Angstroms resolution. It consists of a typical {beta}-barrel fold with eight {beta}-strands containing an internal hydrophobic cavity and a plug-like loop on one side of the barrel. Strong hydrophobic interactions between the two {beta}-barrels maintain the dimeric architecture of GrlR. Furthermore, a unique surface-exposed EDED (Glu-Asp-Glu-Asp) motif is identified to be critical for GrlA-GrlR interaction and for the repressive activity of GrlR. This study contributes a novel molecular insight into the mechanism of GrlR function.

  16. Role of the SUMO-interacting motif in HIPK2 targeting to the PML nuclear bodies and regulation of p53

    SciTech Connect

    Sung, Ki Sa; Lee, Yun-Ah; Kim, Eui Tae; Lee, Seung-Rock; Ahn, Jin-Hyun; Choi, Cheol Yong

    2011-04-15

    Homeodomain-interacting protein kinase 2 (HIPK2) is a key regulator of various transcription factors including p53 and CtBP in the DNA damage signaling pathway. PML-nuclear body (NB) is required for HIPK2-mediated p53 phosphorylation at Ser46 and induction of apoptosis. Although PML-NB targeting of HIPK2 has been shown, much is not clear about the molecular mechanism of HIPK2 recruitment to PML-NBs. Here we show that HIPK2 colocalizes specifically with PML-I and PML-IV. Mutational analysis showed that HIPK2 recruitment to PML-IV-NBs is mediated by the SUMO-interaction motifs (SIMs) of both PML-IV and HIPK2. Wild-type HIPK2 associated with SUMO-conjugated PML-IV at a higher affinity than with un-conjugated PML-IV, while the association of a HIPK2 SIM mutant with SUMO-modified PML-IV was impaired. In colony formation assays, HIPK2 strongly suppressed cell proliferation, but HIPK2 SIM mutants did not. In addition, activation and phosphorylation of p53 at the Ser46 residue were impaired by HIPK2 SIM mutants. These results suggest that SIM-mediated HIPK2 targeting to PML-NBs is crucial for HIPK2-mediated p53 activation and induction of apoptosis.

  17. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 regulates the cross talk between immunity and abscisic acid responses.

    PubMed

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-05-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens.

  18. Regulation of glycoprotein D synthesis: does alpha 4, the major regulatory protein of herpes simplex virus 1, regulate late genes both positively and negatively?

    PubMed Central

    Arsenakis, M; Campadelli-Fiume, G; Roizman, B

    1988-01-01

    Earlier studies have described the alpha 4/c113 baby hamster kidney cell line which constitutively expresses the alpha 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1). Introduction of the HSV-1 glycoprotein B (gB) gene, regulated as a gamma 1 gene, into these cells yielded a cell line which constitutively expressed both the alpha 4 and gamma 1 gB genes. The expression of the gB gene was dependent on the presence of functional alpha 4 protein. In this article we report that we introduced into the alpha 4/c113 and into the parental BHK cells, the HSV-1 BamHI J fragment, which encodes the domains of four genes, including those of glycoproteins D, G, and I (gD, gG, and gI), and most of the coding sequences of the glycoprotein E (gE) gene. In contrast to the earlier studies, we obtained significant constitutive expression of gD (also a gamma 1 gene) in a cell line (BJ) derived from parental BHK cells, but not in a cell line (alpha 4/BJ) which expresses functional alpha 4 protein. RNA homologous to the gD gene was present in significant amounts in the BJ cell line; smaller amounts of this RNA were detected in the alpha 4/BJ cell line. RNA homologous to gE, presumed to be polyadenylated from signals in the vector sequences, was present in the BJ cells but not in the alpha 4/BJ cells. The expression of the HSV-1 gD and gE genes was readily induced in the alpha 4/BJ cells by superinfection with HSV-2. The BJ cell line was, in contrast, resistant to expression of HSV-1 and HSV-2 genes. The BamHI J DNA fragment copy number was approximately 1 per BJ cell genome equivalent and 30 to 50 per alpha 4/BJ cell genome equivalent. We conclude that (i) the genes specifying gD and gB belong to different viral regulatory gene subsets, (ii) the gD gene is subject to both positive and negative regulation, (iii) both gD and gE mRNAs are subject to translational controls although they may be different, and (iv) the absence of expression of gD in the alpha 4/BJ

  19. Peroxisome proliferator-activated receptor {alpha} agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

    SciTech Connect

    Chen Xiang; Li Ming; Sun Weiping; Bi Yan; Cai Mengyin; Liang Hua; Yu Qiuqiong; He Xiaoying; Weng Jianping

    2008-04-18

    It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11{beta}-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPAR{alpha}), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPAR{alpha} activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosterone level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPAR{alpha} inhibitor MK886, suggesting that fenofibrate activated through PPAR{alpha}. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPAR{alpha}.

  20. Primer extension studies on alpha-amylase mRNAs in barley aleurone. II. Hormonal regulation of expression.

    PubMed

    Chandler, P M; Jacobsen, J V

    1991-04-01

    Relative levels of different alpha-amylase mRNAs were assessed by primer extension experiments using RNA prepared from aleurone of barley (Hordeum vulgare L. cv. Himalaya). Three different aleurone systems were studied: protoplasts prepared from aleurone layers, isolated aleurone layers, and aleurone from germinated grain. Oligonucleotide primers specific for the low-pI and high-pI alpha-amylase groups allowed the levels of different alpha-amylase mRNAs to be assessed both within and between the two groups. In all aleurone systems the same set of alpha-amylase mRNAs was produced in response to either applied gibberellic acid (aleurone protoplasts, isolated aleurone layers) or, presumably, native gibberellin(s) (germinated grain). This result indicates that the same set of genes is being expressed in each case. Differences were observed between the different aleurone systems in regulation of levels of alpha-amylase mRNAs. In particular, the regulation of alpha-amylase mRNA levels in aleurone of germinated grain has unique features which are not adequately explained by the response of isolated aleurone layers to gibberellic acid.

  1. Nucleotide sequences required for the regulation of a rat alpha 2u-globulin gene by glucocorticoids.

    PubMed Central

    Addison, W R; Kurtz, D T

    1986-01-01

    alpha 2u-Globulin is a rat protein of as yet unknown function whose synthesis can be induced by glucocorticoids and several other hormones. Induction by glucocorticoids is a secondary response to the hormone: protein synthesis is required before the hormone can exert its stimulatory effect on alpha 2u-globulin transcription. We have used the linker-scanning mutagenesis procedure, followed by transfer of the mutant genes into mouse L-cells for analysis of their phenotype, to determine sequences within a cloned alpha 2u-globulin promoter that are required for its regulation by glucocorticoids. Mutations between positions -115 and -160 abolish or greatly reduce the inducibility of alpha 2u-globulin by the hormone. Mutations just upstream from this region, between positions -177 and -220, have an opposite effect; they increase induction two- to fourfold. Images PMID:2431290

  2. Tripartite motif-containing protein 38 negatively regulates TLR3/4- and RIG-I-mediated IFN-β production and antiviral response by targeting NAP1.

    PubMed

    Zhao, Wei; Wang, Lijuan; Zhang, Meng; Wang, Peng; Yuan, Chao; Qi, Jianni; Meng, Hong; Gao, Chengjiang

    2012-06-01

    Recognition of RNA virus through TLR and RIG-I-like receptor results in rapid expression of type I IFNs, which play an essential role in host antiviral responses. However, the mechanisms to terminate the production of type I IFNs are not well defined. In the current study, we identified a member of the tripartite motif (TRIM) family, TRIM38, as a negative regulator in TLR3/4- and RIG-I-mediated IFN-β signaling. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of IFN regulatory factor 3 and enhanced expression of IFN-β, whereas overexpression of TRIM38 had opposite effects. Coimmunoprecipitation and colocalization experiments demonstrated that TRIM38 interacted with NF-κB-activating kinase-associated protein 1 (NAP1), which is required for TLR-induced IFN regulatory factor 3 activation and IFN-β production. As an E3 ligase, TRIM38 promoted K48-linked polyubiquitination and proteasomal degradation of NAP1. Thus, knockdown of TRIM38 expression resulted in higher protein level of NAP1 in primary macrophages. Consistent with the inhibitory roles in TLR3/4- and RIG-I-mediated IFN-β signaling, knockdown of TRIM38 significantly inhibited the replication of vesicular stomatitis virus. Overexpression of TRIM38 resulted in enhanced replication of vesicular stomatitis virus. Therefore, our results demonstrate that TRIM38 is a negative regulator for TLR and RIG-I-mediated IFN-β production by targeting NAP1 for ubiquitination and subsequent proteasome-mediated degradation. PMID:22539786

  3. Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site.

    PubMed

    Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

    2015-11-15

    Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD.

  4. ERR{alpha} regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

    SciTech Connect

    Rajalin, Ann-Marie; Pollock, Hanna; Aarnisalo, Piia

    2010-05-28

    The orphan nuclear receptor estrogen-related receptor-{alpha} (ERR{alpha}) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERR{alpha} in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERR{alpha} deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERR{alpha} deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERR{alpha} in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERR{alpha} deficient MSCs and enhanced upon ERR{alpha} overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERR{alpha}. Under adipogenic conditions, ERR{alpha} deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERR{alpha} in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERR{alpha} may play different roles in bone under different physiological conditions.

  5. Perilipin, a critical regulator of fat storage and breakdown, is a target gene of estrogen receptor-related receptor {alpha}

    SciTech Connect

    Akter, Mst. Hasina; Yamaguchi, Tomohiro; Hirose, Fumiko; Osumi, Takashi

    2008-04-11

    Perilipin is a protein localized on lipid droplet surfaces in adipocytes and steroidogenic cells, playing a central role in regulated lipolysis. Expression of the perilipin gene is markedly induced during adipogenesis. We found that transcription from the perilipin gene promoter is activated by an orphan nuclear receptor, estrogen receptor-related receptor (ERR){alpha}. A response element to this receptor was identified in the promoter region by a gene reporter assay, the electrophoretic-gel mobility-shift assay and the chromatin immunoprecipitation assay. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} enhanced, whereas small heterodimer partner (SHP) repressed, the transactivating function of ERR{alpha} on the promoter. Thus, the perilipin gene expression is regulated by a transcriptional network controlling energy metabolism, substantiating the functional importance of perilipin in the maintenance of body energy balance.

  6. Cell type-specific transcriptional regulation of the gene encoding importin-{alpha}1

    SciTech Connect

    Kamikawa, Yasunao; Yasuhara, Noriko; Yoneda, Yoshihiro

    2011-08-15

    Importin-{alpha}1 belongs to a receptor family that recognizes classical nuclear localization signals. Encoded by Kpna2, this receptor subtype is highly expressed in mouse embryonic stem (ES) cells. In this study, we identified a critical promoter region in Kpna2 and showed that the expression of this gene is differentially regulated in ES cells and NIH3T3 cells. Conserved CCAAT boxes are required for Kpna2 promoter activity in both ES and NIH3T3 cells. Interestingly, deletion of the region from nucleotide position - 251 to - 179 bp resulted in a drastic reduction in Kpna2 transcriptional activity only in ES cells. This region contains Krueppel-like factor (Klf) binding sequences and is responsible for transactivation of the gene by Klf2 and Klf4. Accordingly, endogenous Kpna2 mRNA levels decreased in response to depletion of Klf2 and Klf4 in ES cells. Our results suggest that Klf2 and Klf4 function redundantly to drive high level of Kpna2 expression in ES cells. -- Research Highlights: {yields} We showed the cell type-specific transcriptional regulation of Kpna2 encoding importin-al. {yields} NF-Y binds the CCAAT boxes to activate Kpna2 transcription in NIH3T3 cells. {yields} Klf2 and Klf4 redundantly activate the expression of Kpna2 in ES cells.

  7. LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

    PubMed Central

    Mattijssen, Sandy

    2015-01-01

    LARP4 is a protein with unknown function that independently binds to poly(A) RNA, RACK1, and the poly(A)-binding protein (PABPC1). Here, we report on its regulation. We found a conserved AU-rich element (ARE) in the human LARP4 mRNA 3′ untranslated region (UTR). This ARE, but not its antisense version or a point-mutated version, significantly decreased the stability of β-globin reporter mRNA. We found that overexpression of tristetraprolin (TTP), but not its RNA binding mutant or the other ARE-binding proteins tested, decreased cellular LARP4 levels. RNA coimmunoprecipitation showed that TTP specifically associated with LARP4 mRNA in vivo. Consistent with this, mouse LARP4 accumulated to higher levels in TTP gene knockout (KO) cells than in control cells. Stimulation of WT cells with tumor necrosis factor alpha (TNF-α), which rapidly induces TTP, robustly decreased LARP4 with a coincident time course but had no such effect on LARP4B or La protein or on LARP4 in the TTP KO cells. The TNF-α-induced TTP pulse was followed by a transient decrease in LARP4 mRNA that was quickly followed by a subsequent transient decrease in LARP4 protein. Involvement of LARP4 as a target of TNF-α–TTP regulation provides a clue as to how its functional activity may be used in a physiologic pathway. PMID:26644407

  8. Hepatic triacylglycerol hydrolysis regulates peroxisome proliferator-activated receptor alpha activity.

    PubMed

    Sapiro, Jessica M; Mashek, Mara T; Greenberg, Andrew S; Mashek, Douglas G

    2009-08-01

    Recent evidence suggests that fatty acids generated from intracellular triacylglycerol (TAG) hydrolysis may have important roles in intracellular signaling. This study was conducted to determine if fatty acids liberated from TAG hydrolysis regulate peroxisome proliferator-activated receptor alpha (PPARalpha). Primary rat hepatocyte cultures were treated with adenoviruses overexpressing adipose differentiation-related protein (ADRP) or adipose triacylglycerol lipase (ATGL) or treated with short interfering RNA (siRNA) targeted against ADRP. Subsequent effects on TAG metabolism and PPARalpha activity and target gene expression were determined. Overexpressing ADRP attenuated TAG hydrolysis, whereas siRNA-mediated knockdown of ADRP or ATGL overexpression resulted in enhanced TAG hydrolysis. Results from PPARalpha reporter activity assays demonstrated that decreasing TAG hydrolysis by ADRP overexpression resulted in a 35-60% reduction in reporter activity under basal conditions or in the presence of fatty acids. As expected, PPARalpha target genes were also decreased in response to ADRP overexpression. However, the PPARalpha ligand, WY-14643, was able to restore PPARalpha activity following ADRP overexpression. Despite its effects on PPARalpha, overexpressing ADRP did not affect PPARgamma activity. Enhancing TAG hydrolysis through ADRP knockdown or ATGL overexpression increased PPARalpha activity. These results indicate that TAG hydrolysis and the consequential release of fatty acids regulate PPARalpha activity.

  9. Thyroid hormone regulates vitellogenin by inducing estrogen receptor alpha in the goldfish liver.

    PubMed

    Nelson, Erik R; Habibi, Hamid R

    2016-11-15

    Vitellogenin (Vtg) is an egg-yolk precursor protein that is synthesized in the liver of oviparous species and taken up from the circulation by the ovary. It is well known that Vtg is induced by circulating estrogens. However, other endocrine factors that regulate the expression of Vtg are less well characterized; factors that might play significant roles, especially in seasonal spawners such as the goldfish which require increased quantities of Vtg for the development of hundreds of follicles. In this regard, thyroid hormones have been shown to cycle with the reproductive season. Therefore, we hypothesized that the thyroid hormones might influence the synthesis of Vtg. Treatment of female goldfish with triiodothyronine (T3) resulted in increased Vtg, an observation that was absent in males. Furthermore, T3 failed to induce Vtg in cultured hepatocytes of either sex. Interestingly however, T3 consistently up-regulated the expression of the estrogen receptor alpha (ERα). The T3 mediated upregulation of ERα requires the presence of both thyroid receptor (TR) α-1 and TRβ. When goldfish or cultured hepatocytes were treated with T3 followed by estradiol, there was a synergistic increase in Vtg, a response which is dependent on the presence of ERα. Therefore, by upregulating ERα, T3 serves to prime the liver to subsequent stimuli from estradiol. This cross-talk likely reveals an important physiologic mechanism by which thyroid hormones, whose circulating levels are high during early gonadal recrudescence, facilitate the production of large amounts of Vtg required for egg development. PMID:27585488

  10. Discovery of the first known small-molecule inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase.

    PubMed

    Rosen, Mark D; Woods, Craig R; Goldberg, Steven D; Hack, Michael D; Bounds, A Dawn; Yang, Young; Wagaman, Pamela C; Phuong, Victor K; Ameriks, Angela P; Barrett, Terrance D; Kanelakis, Kimon C; Chuang, Jui Chang; Chang, Jui; Shankley, Nigel P; Rabinowitz, Michael H

    2009-12-01

    A series of indeno[1,2-c]pyrazoles were discovered to be the first known inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase. The synthesis, structure-activity relationship profile, and in-vitro pharmacological characterization of this inaugural series of HRI kinase inhibitors are detailed.

  11. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    PubMed

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  12. A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha

    PubMed Central

    Gesson, Kevin; Rescheneder, Philipp; Skoruppa, Michael P.; von Haeseler, Arndt; Dechat, Thomas

    2016-01-01

    Lamins are components of the peripheral nuclear lamina and interact with heterochromatic genomic regions, termed lamina-associated domains (LADs). In contrast to lamin B1 being primarily present at the nuclear periphery, lamin A/C also localizes throughout the nucleus, where it associates with the chromatin-binding protein lamina-associated polypeptide (LAP) 2 alpha. Here, we show that lamin A/C also interacts with euchromatin, as determined by chromatin immunoprecipitation of euchromatin- and heterochromatin-enriched samples. By way of contrast, lamin B1 was only found associated with heterochromatin. Euchromatic regions occupied by lamin A/C overlap with those bound by LAP2alpha, and lack of LAP2alpha in LAP2alpha-deficient cells shifts binding of lamin A/C toward more heterochromatic regions. These alterations in lamin A/C-chromatin interactions correlate with changes in epigenetic histone marks in euchromatin but do not significantly affect gene expression. Loss of lamin A/C in heterochromatic regions in LAP2alpha-deficient cells, however, correlated with increased gene expression. Our data show a novel role of nucleoplasmic lamin A/C and LAP2alpha in regulating euchromatin. PMID:26798136

  13. Recombinant human tumor necrosis factor-alpha. Regulation of N-formylmethionylleucylphenylalanine receptor affinity and function on human neutrophils.

    PubMed Central

    Atkinson, Y H; Marasco, W A; Lopez, A F; Vadas, M A

    1988-01-01

    Preincubation of neutrophils with recombinant human tumor necrosis factor-alpha (rH TNF-alpha) enhanced the subsequent release of superoxide anion in response to various concentrations of N-formylmethionylleucylphenylalanine (FMLP). Enhanced superoxide anion production was evident by 5 min and had reached a plateau by 15 min. Not only was the total amount of superoxide anion released greater, but the rate of release was also enhanced threefold by rH TNF-alpha. In contrast, rH TNF-alpha reduced or abolished neutrophil locomotion under agarose in response to a gradient of FMLP. Binding studies of f-Met-Leu-[3H]Phe to purified human neutrophils revealed a heterogeneous binding to unstimulated cells. The high affinity component consisted of approximately 2,000 sites per cell and had an average Kd of 2 +/- 0.7 nM (n = 4). The low affinity component consisted of approximately 40,000 sites per cell and had an average Kd of 180 +/- 50 nM (n = 4). rH TNF-alpha caused conversion to a linear Scatchard plot showing no significant change in total binding sites but a single Kd of 40 +/- 10 nM (n = 4). These data indicate that rH TNF-alpha may influence neutrophil responses to FMLP by regulating the affinity of FMLP receptors. PMID:2830314

  14. The liver-enriched transcription factor CREBH is nutritionally regulated and activated by fatty acids and PPAR{alpha}

    SciTech Connect

    Danno, Hirosuke; Ishii, Kiyo-aki; Nakagawa, Yoshimi; Mikami, Motoki; Yamamoto, Takashi; Yabe, Sachiko; Furusawa, Mika; Kumadaki, Shin; Watanabe, Kazuhisa; Shimizu, Hidehisa; Matsuzaka, Takashi; Kobayashi, Kazuto; Takahashi, Akimitsu; Yatoh, Shigeru; Suzuki, Hiroaki; Yamada, Nobuhiro; Shimano, Hitoshi

    2010-01-08

    To elucidate the physiological role of CREBH, the hepatic mRNA and protein levels of CREBH were estimated in various feeding states of wild and obesity mice. In the fast state, the expression of CREBH mRNA and nuclear protein were high and profoundly suppressed by refeeding in the wild-type mice. In ob/ob mice, the refeeding suppression was impaired. The diet studies suggested that CREBH expression was activated by fatty acids. CREBH mRNA levels in the mouse primary hepatocytes were elevated by addition of the palmitate, oleate and eicosapenonate. It was also induced by PPAR{alpha} agonist and repressed by PPAR{alpha} antagonist. Luciferase reporter gene assays indicated that the CREBH promoter activity was induced by fatty acids and co-expression of PPAR{alpha}. Deletion studies identified the PPRE for PPAR{alpha} activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay confirmed that PPAR{alpha} directly binds to the PPRE. Activation of CREBH at fasting through fatty acids and PPAR{alpha} suggest that CREBH is involved in nutritional regulation.

  15. The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

    SciTech Connect

    Klapper, Maja . E-mail: klapper@molnut.uni-kiel.de; Boehme, Mike; Nitz, Inke; Doering, Frank

    2007-04-27

    The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

  16. OsCCD1, a novel small calcium-binding protein with one EF-hand motif, positively regulates osmotic and salt tolerance in rice.

    PubMed

    Jing, Pei; Zou, Juanzi; Kong, Lin; Hu, Shiqi; Wang, Biying; Yang, Jun; Xie, Guosheng

    2016-06-01

    Calcium-binding proteins play key roles in the signal transduction in the growth and stress response in eukaryotes. However, a subfamily of proteins with one EF-hand motif has not been fully studied in higher plants. Here, a novel small calcium-binding protein with a C-terminal centrin-like domain (CCD1) in rice, OsCCD1, was characterized to show high similarity with a TaCCD1 in wheat. As a result, OsCCD1 can bind Ca(2+) in the in vitro EMSA and the fluorescence staining calcium-binding assays. Transient expression of green fluorescent protein (GFP)-tagged OsCCD1 in rice protoplasts showed that OsCCD1 was localized in the nucleus and cytosol of rice cells. OsCCD1 transcript levels were transiently induced by osmotic stress and salt stress through the calcium-mediated ABA signal. The rice seedlings of T-DNA mutant lines showed significantly less tolerance to osmotic and salt stresses than wild type plants (p<0.01). Conversely, its overexpressors can significantly enhance the tolerance to osmotic and salt stresses than wild type plants (p<0.05). Semi-quantitative RT-PCR analysis revealed that, OsDREB2B, OsAPX1 and OsP5CS genes are involved in the rice tolerance to osmotic and salt stresses. In sum, OsCCD1 gene probably affects the DREB2B and its downstream genes to positively regulate osmotic and salt tolerance in rice seedlings. PMID:27095404

  17. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1.

    PubMed

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-03-18

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1.

  18. The GABA(A) receptor alpha1 subtype in the ventral pallidum regulates alcohol-seeking behaviors.

    PubMed

    Harvey, Scott C; Foster, Katrina L; McKay, Pete F; Carroll, Michelle R; Seyoum, Regat; Woods, James E; Grey, Collette; Jones, Cecily M; McCane, Shannan; Cummings, Rancia; Mason, Dynesha; Ma, Chunrong; Cook, James M; June, Harry L

    2002-05-01

    We investigated the potential role of the alpha1-containing GABA(A) receptor in regulating the reinforcing properties of alcohol. To accomplish this, we developed 3-propoxy-beta-carboline hydrochloride (3-PBC), a mixed agonist-antagonist benzodiazepine site ligand with binding selectivity at the alpha1 receptor. We then tested the capacity of 3-PBC to block alcohol-maintained responding in the ventral pallidum (VP), a novel alcohol reward substrate, which primarily expresses the alpha1-receptor isoform. Our results demonstrated that bilateral microinfusion of 3-PBC (0.5-40 microg) in the anterior and medial VP produced marked reductions in alcohol-maintained responding in a genetically selected rodent model of alcohol drinking. The VP infusions showed both neuroanatomical and reinforcer specificity because no effects were seen in sites dorsal to the VP (e.g., nucleus accumbens, caudate putamen). The saccharin-maintained responding was reduced only with the highest dose (40 microg). Parenteral injections of 3-PBC (1-20 mg/kg) also showed a similar selectivity on alcohol-maintained responding. Complementary in vitro studies revealed that 3-PBC exhibited a low partial agonist efficacy profile at recombinant diazepam-sensitive receptors (e.g., alpha1beta3gamma2, alpha2beta3gamma, and alpha3beta3gamma2). The selective suppression of 3-PBC on alcohol-maintained responding after central and parenteral administrations, together with its low-efficacy agonist profile, suggest that the reduction in alcohol-maintained behaviors was not attributable to a general suppression on consummatory behaviors. These results demonstrate that the alpha1-containing GABA(A) receptors in both the anterior and medial VP are important in regulating the reinforcing properties of alcohol. These receptors represent novel targets in the design and development of pharmacotherapies for alcohol-dependent subjects. PMID:11978852

  19. Induced heteroduplex genotyping of TNF-alpha, IL-1beta, IL-6 and IL-10 polymorphisms associated with transcriptional regulation.

    PubMed

    Morse, H R; Olomolaiye, O O; Wood, N A; Keen, L J; Bidwell, J L

    1999-10-01

    We describe the construction and use of 7 induced heteroduplex generators, reagents for the rapid and unequivocal genotyping of nucleotide sequence polymorphism in TNF-alpha, IL-1beta, IL-6 and IL-10. Polymorphisms detected are those previously associated with regulation of gene transcription: TNF-alpha positions -308 and -238; IL-1beta position +3953; IL-6 position -174; and IL-10 positions -1082, -819 and -592. The reagents were used for analysis of allele and haplotype frequencies in a population of healthy Caucasian volunteer blood donors.

  20. Regulation of the murine alpha B-crystallin/small heat shock protein gene in cardiac muscle.

    PubMed Central

    Gopal-Srivastava, R; Haynes, J I; Piatigorsky, J

    1995-01-01

    The murine alpha B-crystallin/small heat shock protein gene is expressed at high levels in the lens and at lower levels in the heart, skeletal muscle, and numerous other tissues. Previously we have found a skeletal-muscle-preferred enhancer at positions -427 to -259 of the alpha B-crystallin gene containing at least four cis-acting regulatory elements (alpha BE-1, alpha BE-2, alpha BE-3, and MRF, which has an E box). Here we show that in transgenic mice, the alpha B-crystallin enhancer directs the chloramphenicol acetyltransferase reporter gene driven by the alpha B-crystallin promoter specifically to myocardiocytes of the heart. The alpha B-crystallin enhancer was active in conjugation with the herpes simplex virus thymidine kinase promoter/human growth hormone reporter gene in transfected rat myocardiocytes. DNase I footprinting and site-specific mutagenesis experiments showed that alpha BE-1, alpha BE-2, alpha BE-3, MRF, and a novel, heart-specific element called alpha BE-4 are required for alpha B-crystallin enhancer activity in transfected myocardiocytes. By contrast, alpha BE-4 is not utilized for enhancer activity in transfected lens or skeletal muscle cell lines. Alpha BE-4 contains an overlapping heat shock sequence and a reverse CArG box [5'-GG(A/T)6CC-3']. Electrophoretic mobility shift assays with an antibody to serum response factor and a CArG-box-competing sequence from the c-fos promoter indicated that a cardiac-specific protein with DNA-binding and antigenic similarities to serum response factor binds to alpha BE-4 via the reverse CArG box; electrophoretic mobility shift assays and antibody experiments with anti-USF antiserum and heart nuclear extract also raised the possibility that the MRF E box utilizes USF or an antigenically related protein. We conclude that the activity of the alpha B-crystallin enhancer in the heart utilizes a reverse CArG box and an E-box-dependent pathway. PMID:8524275

  1. A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.

    PubMed

    Couturier, S; Bertrand, D; Matter, J M; Hernandez, M C; Bertrand, S; Millar, N; Valera, S; Barkas, T; Ballivet, M

    1990-12-01

    cDNA and genomic clones encoding alpha 7, a novel neuronal nicotinic acetylcholine receptor (nAChR) alpha subunit, were isolated and sequenced. The mature alpha 7 protein (479 residues) has moderate homology with all other alpha and non-alpha nAChR subunits and probably assumes the same transmembrane topology. alpha 7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of E12 tectum. In Xenopus oocytes, the alpha 7 protein assembles into a homo-oligomeric channel responding to acetylcholine and nicotine. The alpha 7 channel desensitizes very rapidly, rectifies strongly above -20 mV, and is blocked by alpha-bungarotoxin. A bacterial fusion protein encompassing residues 124-239 of alpha 7 binds labeled alpha-bungarotoxin. We conclude that alpha-bungarotoxin binding proteins in the vertebrate nervous system can function as nAChRs.

  2. Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-6-dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains.

    PubMed Central

    Schwabe, M; Brini, A T; Bosco, M C; Rubboli, F; Egawa, M; Zhao, J; Princler, G L; Kung, H F

    1994-01-01

    IL-6 is an autocrine growth factor for U266 myeloma cells and their growth is inhibited by IFN-alpha or IL-6 mAb. We asked, therefore, whether IFN-alpha-induced growth inhibition involved IL-6. IFN-alpha and mAb against IL-6, the IL-6R alpha-(gp80) or beta-chain (gp130) potently inhibited U266 cells. Remarkably, this effect occurred despite IFN-alpha-augmented secretion of endogenous IL-6. However, examining the IL-6R revealed that IFN-alpha drastically curtailed expression of the IL-6R alpha- and beta-chain. This effect occurred on two different levels (protein and mRNA) and by two different mechanisms (directly and indirectly through IL-6). First, IFN-alpha, but not IL-6, greatly decreased gp80 and, to a lesser extent, gp130 mRNA levels which resulted in a loss of IL-6 binding sites. Second, IFN-alpha-induced IL-6 predominantly down-regulated membrane-bound gp130. IFN-alpha-mediated decrease of gp80 levels was not detected on IL-6-independent myeloma (RPMI 8226) or myeloid cells (U937). We conclude that IFN-alpha inhibited IL-6-dependent myeloma cell growth by depriving U266 cells of an essential component of their autocrine growth loop, a functional IL-6R. Images PMID:7989587

  3. Network Analysis Implicates Alpha-Synuclein (Snca) in the Regulation of Ovariectomy-Induced Bone Loss

    PubMed Central

    Calabrese, Gina; Mesner, Larry D.; Foley, Patricia L.; Rosen, Clifford J.; Farber, Charles R.

    2016-01-01

    The postmenopausal period in women is associated with decreased circulating estrogen levels, which accelerate bone loss and increase the risk of fracture. Here, we gained novel insight into the molecular mechanisms mediating bone loss in ovariectomized (OVX) mice, a model of human menopause, using co-expression network analysis. Specifically, we generated a co-expression network consisting of 53 gene modules using expression profiles from intact and OVX mice from a panel of inbred strains. The expression of four modules was altered by OVX, including module 23 whose expression was decreased by OVX across all strains. Module 23 was enriched for genes involved in the response to oxidative stress, a process known to be involved in OVX-induced bone loss. Additionally, module 23 homologs were co-expressed in human bone marrow. Alpha synuclein (Snca) was one of the most highly connected “hub” genes in module 23. We characterized mice deficient in Snca and observed a 40% reduction in OVX-induced bone loss. Furthermore, protection was associated with the altered expression of specific network modules, including module 23. In summary, the results of this study suggest that Snca regulates bone network homeostasis and ovariectomy-induced bone loss. PMID:27378017

  4. Interferon alpha-inducible protein 6 regulates NRASQ61K-induced melanomagenesis and growth

    PubMed Central

    Gupta, Romi; Forloni, Matteo; Bisserier, Malik; Dogra, Shaillay Kumar; Yang, Qiaohong; Wajapeyee, Narendra

    2016-01-01

    Mutations in the NRAS oncogene are present in up to 20% of melanoma. Here, we show that interferon alpha-inducible protein 6 (IFI6) is necessary for NRASQ61K-induced transformation and melanoma growth. IFI6 was transcriptionally upregulated by NRASQ61K, and knockdown of IFI6 resulted in DNA replication stress due to dysregulated DNA replication via E2F2. This stress consequentially inhibited cellular transformation and melanoma growth via senescence or apoptosis induction depending on the RB and p53 pathway status of the cells. NRAS-mutant melanoma were significantly more resistant to the cytotoxic effects of DNA replication stress-inducing drugs, and knockdown of IFI6 increased sensitivity to these drugs. Pharmacological inhibition of IFI6 expression by the MEK inhibitor trametinib, when combined with DNA replication stress-inducing drugs, blocked NRAS-mutant melanoma growth. Collectively, we demonstrate that IFI6, via E2F2 regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS-mutant melanoma. DOI: http://dx.doi.org/10.7554/eLife.16432.001 PMID:27608486

  5. Drosophila glucome screening identifies Ck1alpha as a regulator of mammalian glucose metabolism

    PubMed Central

    Ugrankar, Rupali; Berglund, Eric; Akdemir, Fatih; Tran, Christopher; Kim, Min Soo; Noh, Jungsik; Schneider, Rebekka; Ebert, Benjamin; Graff, Jonathan M.

    2015-01-01

    Circulating carbohydrates are an essential energy source, perturbations in which are pathognomonic of various diseases, diabetes being the most prevalent. Yet many of the genes underlying diabetes and its characteristic hyperglycaemia remain elusive. Here we use physiological and genetic interrogations in D. melanogaster to uncover the ‘glucome', the complete set of genes involved in glucose regulation in flies. Partial genomic screens of ∼1,000 genes yield ∼160 hyperglycaemia ‘flyabetes' candidates that we classify using fat body- and muscle-specific knockdown and biochemical assays. The results highlight the minor glucose fraction as a physiological indicator of metabolism in Drosophila. The hits uncovered in our screen may have conserved functions in mammalian glucose homeostasis, as heterozygous and homozygous mutants of Ck1alpha in the murine adipose lineage, develop diabetes. Our findings demonstrate that glucose has a role in fly biology and that genetic screenings carried out in flies may increase our understanding of mammalian pathophysiology. PMID:25994086

  6. MISAE: a new approach for regulatory motif extraction.

    PubMed

    Sun, Zhaohui; Yang, Jingyi; Deogun, Jitender S

    2004-01-01

    The recognition of regulatory motifs of co-regulated genes is essential for understanding the regulatory mechanisms. However, the automatic extraction of regulatory motifs from a given data set of the upstream non-coding DNA sequences of a family of co-regulated genes is difficult because regulatory motifs are often subtle and inexact. This problem is further complicated by the corruption of the data sets. In this paper, a new approach called Mismatch-allowed Probabilistic Suffix Tree Motif Extraction (MISAE) is proposed. It combines the mismatch-allowed probabilistic suffix tree that is a probabilistic model and local prediction for the extraction of regulatory motifs. The proposed approach is tested on 15 co-regulated gene families and compares favorably with other state-of-the-art approaches. Moreover, MISAE performs well on "corrupted" data sets. It is able to extract the motif from a "corrupted" data set with less than one fourth of the sequences containing the real motif.

  7. A novel alpha kinase EhAK1 phosphorylates actin and regulates phagocytosis in Entamoeba histolytica.

    PubMed

    Mansuri, M Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-10-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.

  8. BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells.

    PubMed

    Wakle-Prabagaran, Monali; Lorca, Ramón A; Ma, Xiaofeng; Stamnes, Susan J; Amazu, Chinwendu; Hsiao, Jordy J; Karch, Celeste M; Hyrc, Krzysztof L; Wright, Michael E; England, Sarah K

    2016-04-19

    The large-conductance, voltage-gated, calcium (Ca(2+))-activated potassium channel (BKCa) plays an important role in regulating Ca(2+)signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCain myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCaand both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCain an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca(2+)to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca(2+)-free medium and inhibitors of various Ca(2+)signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca(2+)through store-operated Ca(2+)channels. Finally, we found that the specific BKCablocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCachannel in human myometrium and that BKCaand its immunomodulatory interacting partners regulate Ca(2+)dynamics in hMSMCs during pregnancy. PMID:27044074

  9. Human Lineage-Specific Transcriptional Regulation through GA-Binding Protein Transcription Factor Alpha (GABPa)

    PubMed Central

    Perdomo-Sabogal, Alvaro; Nowick, Katja; Piccini, Ilaria; Sudbrak, Ralf; Lehrach, Hans; Yaspo, Marie-Laure; Warnatz, Hans-Jörg; Querfurth, Robert

    2016-01-01

    A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes. PMID:26814189

  10. Hepatitis C virus suppresses Hepatocyte Nuclear Factor 4 alpha, a key regulator of hepatocellular carcinoma.

    PubMed

    Vallianou, Ioanna; Dafou, Dimitra; Vassilaki, Niki; Mavromara, Penelope; Hadzopoulou-Cladaras, Margarita

    2016-09-01

    Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC. PMID:27477312

  11. A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    PubMed Central

    Mansuri, M. Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-01-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics. PMID:25299184

  12. Effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

    SciTech Connect

    Fisher, R.A.; Tanabe, S.; Buxton, D.B.; Olson, M.S.

    1985-08-05

    The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of UCO2 from infused (1- UC) pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Results show that alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.

  13. Regulation of androgen receptor and 5 alpha-reductase in the skin of normal and hirsute women.

    PubMed

    Mauvais-Jarvis, P

    1986-05-01

    The hormonal activity of androgens is mediated in target cells, particularly in human skin, by two kinds of proteins: the androgen receptor and the enzyme 5 alpha-reductase. In well differentiated androgen target cells, 5 alpha-reductase achieves the transformation of testosterone (T) into dihydrotestosterone (DHT), a more active androgen than T, because of its higher affinity for the receptor. In other words, 5 alpha-reductase acts as an amplifier of the androgen signal but is not absolutely required for androgen action. Regarding the regulation of the androgen receptor, minimal information is available. However, in genital skin, the receptor seems to be predominantly localized in the cytosolic compartment before puberty in males and in the nuclear compartment after puberty. In hirsute patients, recent data on genital skin fibroblasts do not show significant differences between the binding capacity of fibroblasts from normal and hirsute women whereas there is no difference between normal men and women. 5 alpha-Reductase activity seems to be a very important step in the processes involved in androgen action. While 5 alpha-reductase activity present in the skin of external genitalia does not seem to be androgen dependent, this is not the case for the enzyme located in pubic skin. In this area, a sex difference between males and females may be observed both in skin homogenates and in cultured fibroblasts. In addition DHT added to a medium of pubic skin fibroblasts is capable of increasing 5 alpha-reductase activity. This increase is not observed when cyproterone acetate is added to the medium and in patients with testicular feminization syndrome without receptors. Pubic 5 alpha-reductase activity is an androgen receptor mediated phenomenon. In patients with hirsutism, and particularly idiopathic hirsutism, 5 alpha-reductase activity is high without an increase in circulating androgens. This may be observed both in pubic skin homogenates and in cultured fibroblasts

  14. [Regulation of lipid metabolism in blood of weaving shop workers (noise level of about 90 dB A) by alpha-tocopherol acetate].

    PubMed

    Melkonian, M M

    1993-01-01

    alpha-Tocopherol acetate, used for repeated treatment within a year of weavers working under highly uncomfortable conditions (the level of noise up to 90 dB), was shown to exhibit the regulating effect on the rate of lipid peroxidation and on content of alpha-tocopherol and cholesterol in blood plasma and erythrocyte membranes as well as on the ratios alpha-tocopherol acetate/total lipids, alpha-tocopherol acetate/cholesterol in blood plasma and cholesterol/alpha-tocopherol acetate in erythrocyte membranes. The vitamin decreased distinctly the atherogenicity coefficient; these data suggest that treatment with tocopherol is essential for prophylaxis of noise-produced impairments.

  15. In vivo regulation of gene transcription by alpha- and gamma-Tocopherol in murine T lymphocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Of the 8 different analogues (alpha-, beta-, gamma-, delta-tocopherols and tocotrienols) designated as vitamin E, alpha-tocopherol (a-T) has been mostly studied, together with gamma-tocopherol (g-T) which is abundant in the US diet. We compared the effect of dietary supplementation with adequate or ...

  16. Regulation of miR-200c by nuclear receptors PPAR{alpha}, LRH-1 and SHP

    SciTech Connect

    Zhang, Yuxia; Yang, Zhihong; Whitby, Richard; Wang, Li

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Knockdown of PPAR{alpha} and LRH-1 abolishes miR-200c inhibition of HCC cell migration. Black-Right-Pointing-Pointer SHP represses miR-200c expression via inhibition of the activity of PPAR{alpha} and LRH-1. Black-Right-Pointing-Pointer RJW100 exhibits strong ability to downregulate ZEB1 and ZEB2 proteins. -- Abstract: We investigated regulation of miR-200c expression by nuclear receptors. Ectopic expression of miR-200c inhibited MHCC97H cell migration, which was abrogated by the synergistic effects of PPAR{alpha} and LRH-1 siRNAs. The expression of miR-200c was decreased by PPAR{alpha}/LRH-1 siRNAs and increased by SHP siRNAs, and overexpression of the receptors reversed the effects of their respective siRNAs. SHP siRNAs also drastically enhanced the ability of the LRH-1 agonist RJW100 to induce miR-200c and downregulate ZEB1 and ZEB2 proteins. Co-expression of PPAR{alpha} and LRH-1 moderately transactivated the miR-200c promoter, which was repressed by SHP co-expression. RJW100 caused strong activation of the miR-200c promoter. This is the first report to demonstrate that miR-200c expression is controlled by nuclear receptors.

  17. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells.

    PubMed Central

    Stefanovic, B; Hellerbrand, C; Holcik, M; Briendl, M; Aliebhaber, S; Brenner, D A

    1997-01-01

    The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR. PMID:9271398

  18. Motifs in brain networks.

    PubMed

    Sporns, Olaf; Kötter, Rolf

    2004-11-01

    Complex brains have evolved a highly efficient network architecture whose structural connectivity is capable of generating a large repertoire of functional states. We detect characteristic network building blocks (structural and functional motifs) in neuroanatomical data sets and identify a small set of structural motifs that occur in significantly increased numbers. Our analysis suggests the hypothesis that brain networks maximize both the number and the diversity of functional motifs, while the repertoire of structural motifs remains small. Using functional motif number as a cost function in an optimization algorithm, we obtain network topologies that resemble real brain networks across a broad spectrum of structural measures, including small-world attributes. These results are consistent with the hypothesis that highly evolved neural architectures are organized to maximize functional repertoires and to support highly efficient integration of information.

  19. Motifs in Brain Networks

    PubMed Central

    2004-01-01

    Complex brains have evolved a highly efficient network architecture whose structural connectivity is capable of generating a large repertoire of functional states. We detect characteristic network building blocks (structural and functional motifs) in neuroanatomical data sets and identify a small set of structural motifs that occur in significantly increased numbers. Our analysis suggests the hypothesis that brain networks maximize both the number and the diversity of functional motifs, while the repertoire of structural motifs remains small. Using functional motif number as a cost function in an optimization algorithm, we obtain network topologies that resemble real brain networks across a broad spectrum of structural measures, including small-world attributes. These results are consistent with the hypothesis that highly evolved neural architectures are organized to maximize functional repertoires and to support highly efficient integration of information. PMID:15510229

  20. Hormonal Regulation of alpha-Amylase Gene Transcription in Wild Oat (Avena fatua L.) Aleurone Protoplasts.

    PubMed

    Zwar, J A; Hooley, R

    1986-02-01

    The time of appearance and relative amounts of alpha-amylase mRNA in wild oat (Avena fatua L.) aleurone protoplasts incubated with 1 micromolar gibberellin A(4) (GA(4)) were closely correlated with the amounts of alpha-amylase enzyme secreted by the protoplasts. In the absence of GA(4), or when protoplasts were incubated with 25 micromolar abscisic acid (ABA) together with 1 micromolar GA(4) no alpha-amylase mRNA was detected and only very low levels of alpha-amylase were secreted. Nuclei were isolated in high yields (65-71%) from aleurone protoplasts and in an in vitro transcription system displayed characteristics of a faithful DNA-dependent RNA synthesizing system. The time course of incorporation of [(3)H]-UTP suggested that the RNA synthesized was mainly ;run off' transcription and therefore that the transcripts produced in vitro were those being synthesized in the protoplasts at the times when the nuclei were isolated. By hybridizing in vitro synthesized [(32)P]RNA to barley alpha-amylase cDNA and control filters we have estimated that 90 +/- 10 ppm of the transcripts synthesized by nuclei isolated from GA(4) treated protoplasts can be attributed to alpha-amylase sequences and that statistically insignificant amounts of these transcripts are obtained from control and GA(4) plus ABA treatments. The results suggest that GA(4) and ABA influence the transcription of alpha-amylase genes in aleurone protoplasts of wild oat.

  1. Presynaptic targeting of alpha4beta 2 nicotinic acetylcholine receptors is regulated by neurexin-1beta.

    PubMed

    Cheng, Shi-Bin; Amici, Stephanie A; Ren, Xiao-Qin; McKay, Susan B; Treuil, Magdalen W; Lindstrom, Jon M; Rao, Jayaraman; Anand, Rene

    2009-08-28

    The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between alpha4beta2 AChRs and the presynaptic cell adhesion molecule, neurexin-1beta. In non-neuronal tsA 201 cells, recombinant neurexin-1beta and mature alpha4beta2 AChRs form complexes. alpha4beta2 AChRs and neurexin-1beta also coimmunoprecipitate from rat brain lysates. When exogenous alpha4beta2 AChRs and neurexin-1beta are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1beta. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1beta lacking the extracellular domain. Additionally, when alpha4beta2 AChRs, alpha7 AChRs, and neurexin-1beta are coexpressed in the same neuron, only the alpha4beta2 AChR colocalizes with neurexin-1beta at presynaptic terminals. Collectively, these data suggest that neurexin-1beta targets alpha4beta2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on alpha4beta2 AChRs, may contribute to the etiology of these neurological disorders.

  2. Developmental regulation of {beta}-hexosaminidase {alpha}- and {beta}-subunit gene expression in the rat reproductive system

    SciTech Connect

    Trasler, J.M.; Wakamatsu, N.; Gravel, R.A.; Benoit, G.

    1994-09-01

    {beta}-Hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G{sub M2} gangliosidoses. Enzyme activity for {beta}-hexosaminidase is many fold higher in the epididymis than in other tissues, is present in sperm and is postulated to be required for mammalian fertilization. To better understand how {beta}-hexosaminidase is regulated in the reproductive system, we quantitated the mRNA expression of the {alpha}- and {beta}-subunits (Hex {alpha} and Hex {beta}) of the enzyme in the developing rat testis and epididymis. Hex {alpha} mRNA was differentially expressed and abundant in adult rat testis and epididymis, 13- and 2-fold brain levels, respectively. In contrast, Hex {beta} mRNA levels in the testis and epididymis were .3- and 5-fold brain levels. Within the epididymis both Hex {alpha} and Hex {beta} mRNA concentrations were highest in the corpus, 1.5-fold and 9-fold initial segment values, respectively. During testis development from 7-91 days of age, testis levels of Hex {alpha} mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium. In isolated male germ cells, Hex {alpha} expression was most abundant in haploid round spermatids. Hex {alpha} mRNA was undetectable after hypophysectomy and returned to normal after testosterone administration and the return of advanced germ cells to the testis. Hex {beta} mRNA was expressed at constant low levels throughout testis development. In the caput-corpus and cauda regions of the epididymis Hex {alpha} mRNA levels increased 2-fold between 14 and 91 days; during the same developmental period epididymal Hex {beta} mRNA levels increased dramatically, by 10-20 fold. In summary, Hex {alpha} and Hex {beta} mRNAs are differentially and developmentally expressed at high levels in the rat testis and epididymis and augur for an important role for {beta}-hexosaminidase in normal male reproductive function.

  3. Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

    NASA Technical Reports Server (NTRS)

    Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

    2003-01-01

    The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

  4. The role and regulation of hypoxia-inducible factor-1alpha expression in brain development and neonatal hypoxic-ischemic brain injury.

    PubMed

    Fan, Xiyong; Heijnen, Cobi J; van der Kooij, Michael A; Groenendaal, Floris; van Bel, Frank

    2009-12-11

    During neonatal hypoxic-ischemic brain injury, activation of transcription of a series of genes is induced to stimulate erythropoiesis, anti-apoptosis, apoptosis, necrosis and angiogenesis. A key factor mediating these gene transcriptions is hypoxia-inducible factor-1alpha (HIF-1alpha). During hypoxia, HIF-1alpha protein is stabilized and heterodimerizes with HIF-1beta to form HIF-1, subsequently regulating the expression of target genes. HIF-1alpha participates in early brain development and proliferation of neuronal precursor cells. Under pathological conditions, HIF-1alpha is known to play an important role in neonatal hypoxic-ischemic brain injury: on the one hand, HIF-1alpha has neuroprotective effects whereas it can also have neurotoxic effects. HIF-1alpha regulates the transcription of erythropoietin (EPO), which induces several pathways associated with neuroprotection. HIF-1alpha also promotes the expression of vascular endothelial cell growth factor (VEGF), which is related to neovascularization in hypoxic-ischemic brain areas. In addition, HIF-1alpha has an anti-apoptotic effect by increasing the expression of anti-apoptotic factors such as EPO during mild hypoxia. The neurotoxic effects of HIF-1alpha are represented by its participation in the apoptotic process by increasing the stability of the tumor suppressor protein p53 during severe hypoxia. Moreover, HIF-1alpha plays a role in cell necrosis, by interacting with calcium and calpain. HIF-1alpha can also exacerbate brain edema via increasing the permeability of the blood-brain barrier (BBB). Given these properties, HIF-1alpha has both neuroprotective and neurotoxic effects after hypoxia-ischemia. These events are cell type specific and related to the severity of hypoxia. Unravelling of the complex functions of HIF-1alpha may be important when designing neuroprotective therapies for hypoxic-ischemic brain injury.

  5. Regulation of valine and. alpha. -ketoisocaproate metabolism in rat kidney mitochondria

    SciTech Connect

    Miller, R.H.; Harper, A.E. )

    1988-10-01

    Activities of branched-chain amino acid (BCAA) aminotransferase (BCAT) and {alpha}-keto acid dehydrogenase (BCKD) were assayed in mitochondria isolated from kidneys of rats. Rates of transamination of valine and oxidation of keto acids {alpha}-ketoisocaproate (KIC) or {alpha}-ketoisovalerate (KIV) were estimated using radioactive tracers of the appropriate substrate from amounts of {sup 14}C-labeled products formed. Because of the high mitochondrial BCAT activity, an amino acceptor for BCAT, {alpha}-ketoglutarate ({alpha}-KG) or KIC, was added to the assay medium when valine was the substrate. Rates of valine transamination and subsequent oxidation of the KIV formed were determined with 0.5 mM {alpha}-KG as the amino acceptor; these rates were 5- to 50-fold those without added {alpha}-KG. Rates of CO{sub 2} evolution from valine also increased when KIC was present; however, with KIC concentrations above 0.2 mM, rates of CO{sub 2} evolution from valine declined although rates of transamination continued to rise. When 0.05 mM KIC was added to the assay medium, oxidation of KIC was suppressed by inclusion of valine or glutamate in the medium. When valine was present KIC was not oxidized preferentially, presumably because it was also serving as an amino acceptor for BCAT. These results indicate that as the supply of amino acceptor, {alpha}-KG or KIC, is increased in mitochondria not only is the rate of valine transamination stimulated but also the rate of oxidation of the KIV formed from valine. Thus the rate of oxidation of BCAA can be controlled by factors that influence the rate and direction of BCAA transamination and, thereby, the supply of substrate for BCKD.

  6. Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice.

    PubMed Central

    Fox, N; Solter, D

    1988-01-01

    Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta. Images PMID:2468998

  7. Regulation and function of the alpha/sub 2/ adrenergic autoreceptor in the central nervous system

    SciTech Connect

    Spengler, R.N.

    1987-01-01

    The purpose of this investigation was to determine whether changes observed in the number of alpha/sub 2/ adrenergic receptors in the brain as measured by radioligand binding experiments reflect changes in the function of alpha/sub 2/ autoregulatory receptors which are located on noradrenergic nerve terminals. Inhibition by clonidine of field stimulated /sup 3/H-norepinephrine (/sup 3/H-NE) release from rat hippocampal slices before and after several drug treatments was analyzed to investigate changes in alpha/sub 2/ adrenergic receptor function. Clonidine in a concentration-dependent manner inhibited /sup 3/H-NE release. The effect of clonidine was blocked by the specific alpha/sub 2/ adrenergic receptor antagonist, idazoxan. The cumulative administration of clonidine generated a smooth and well-fitted log-concentration-effect curve. Results are presented which demonstrate that this technique can be employed to investigate the role of changes in the function of the alpha/sub 2/ autoregulatory receptor. The present investigation also examined representatives of four drug classes which have been shown to alter the specific binding of /sup 3/H-clonidine to neural membranes to determine whether changes in the alpha/sub 2/ autoregulatory receptor function also occur.

  8. The C-Terminal Sequence and PI motif of the Orchid (Oncidium Gower Ramsey) PISTILLATA (PI) Ortholog Determine its Ability to Bind AP3 Orthologs and Enter the Nucleus to Regulate Downstream Genes Controlling Petal and Stamen Formation.

    PubMed

    Mao, Wan-Ting; Hsu, Hsing-Fun; Hsu, Wei-Han; Li, Jen-Ying; Lee, Yung-I; Yang, Chang-Hsien

    2015-11-01

    This study focused on the investigation of the effects of the PI motif and C-terminus of the Oncidium Gower Ramsey MADS box gene 8 (OMADS8), a PISTILLATA (PI) ortholog, on floral organ formation. 35S::OMADS8 completely rescued and 35S::OMADS8-PI (with the PI motif deleted) partially rescued petal/stamen formation, whereas these deficiencies were not rescued by 35S::OMADS8-C (C-terminal 29 amino acids deleted) in pi-1 mutants. OMADS8 could interact with Arabidopsis APETALA3 (AP3) and enter the nucleus. The nuclear entry efficiency was reduced for OMADS8-PI/AP3 and OMADS8-C/AP3. OMADS8 could also interact with OMADS5/OMADS9 (the Oncidium AP3 ortholog) and enter the nucleus with an efficiency only slightly affected by the deletion of the C-terminal sequence or PI motif. However, the stability of the OMADS8/OMADS5 and OMADS8/OMADS9 complexes was significantly reduced by deletion of the C-terminal sequence or PI motif. Further analysis indicated that the expression of genes downstream of AP3/PI (BNQ1/BNQ2/GNC/At4g30270) was compensated by 35S::OMADS8 and 35S::OMADS8-PI to a level similar to wild-type plants but was not affected by 35S::OMADS8-C in the pi-1 mutants. A similar FRET (fluorescence resonance energy transfer) efficiency was observed for Arabidopsis AGAMOUS (AG) and the Oncidium AG ortholog OMADS4 for OMADS8, OMADS8-PI and OMADS8-C. These results indicated that the OMADS8 PI motif and C-terminus were valuable for the interaction of OMADS8 with the AP3 orthologs to form higher order heterotetrameric complexes that regulated petal/stamen formation in both Oncidium orchids and transgenic Arabidopsis. However, the C-terminal sequence and PI motif were dispensable for the interaction of OMADS8 with the AG orthologs.

  9. Regulation of the synthesis of barley aleurone. cap alpha. -amylase by gibberellic acid and calcium ions

    SciTech Connect

    Jones, R.L.; Carbonell, J.

    1984-09-01

    The effects of gibberellic acid (GA/sub 3/) and calcium ions on the production of ..cap alpha..-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA/sub 3/ or CA/sup 2 +/ show qualitative and quantitative changes in hydrolase production following incubation in either GA/sub 3/ or CA/sup 2 +/ or both. In cubation in H/sub 2/O or CA/sup 2 +/ results in the production of low levels of ..cap alpha..-amylase or acid phosphatase. The addition of GA/sub 3/ to the incubation medium causes 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of CA/sup 2 +/ at 10 millimolar causes a further 8- to 9-fold increase in ..cap alpha..-amylase release and a 75% increase in phosphatase release. Production of ..cap alpha..-amylase isoenzymes is also modified by the levels of GA/sub 3/ and CA/sup 2 +/ in the incubation medium. ..cap alpha..-amylase 2 is produced under all conditions of incubation, while ..cap alpha..-amylase 1 appears only when layers are incubated in GA/sub 3/ or GA/sub 3/ plus CA/sup 2 +/. The synthesis of ..cap alpha..-amylases 3 and 4 requires the presence of both GA/sub 3/ and CA/sup 2 +/ in the incubation medium. Laurell rocket immunoelectrophoresis shows that two distinct groups of ..cap alpha..-amylase antigens are present in incubation media of aleurone layers incubated with both GA/sub 3/ and CA/sup 2 +/, while only one group of antigens is found in media of layers incubated in GA/sub 3/ alone. Strontium ions can be substituted for CA/sup 2 +/ in increasing hydrolase production, although higher concentrations of Sr/sup 2 +/ are requried for maximal response. We conclude that GA/sub 3/ is required for the production of ..cap alpha..-amylase 1 and that both GA/sub 3/ and either CA/sup 2 +/ or Sr/sup 2 +/ are required for the production of isoenzymes 3 and 4 of barley aleurone ..cap alpha..-amylase. 22 references, 8

  10. Redundant ERF-VII Transcription Factors Bind to an Evolutionarily Conserved cis-Motif to Regulate Hypoxia-Responsive Gene Expression in Arabidopsis.

    PubMed

    Gasch, Philipp; Fundinger, Moritz; Müller, Jana T; Lee, Travis; Bailey-Serres, Julia; Mustroph, Angelika

    2016-01-01

    The response of Arabidopsis thaliana to low-oxygen stress (hypoxia), such as during shoot submergence or root waterlogging, includes increasing the levels of ∼50 hypoxia-responsive gene transcripts, many of which encode enzymes associated with anaerobic metabolism. Upregulation of over half of these mRNAs involves stabilization of five group VII ethylene response factor (ERF-VII) transcription factors, which are routinely degraded via the N-end rule pathway of proteolysis in an oxygen- and nitric oxide-dependent manner. Despite their importance, neither the quantitative contribution of individual ERF-VIIs nor the cis-regulatory elements they govern are well understood. Here, using single- and double-null mutants, the constitutively synthesized ERF-VIIs RELATED TO APETALA2.2 (RAP2.2) and RAP2.12 are shown to act redundantly as principle activators of hypoxia-responsive genes; constitutively expressed RAP2.3 contributes to this redundancy, whereas the hypoxia-induced HYPOXIA RESPONSIVE ERF1 (HRE1) and HRE2 play minor roles. An evolutionarily conserved 12-bp cis-regulatory motif that binds to and is sufficient for activation by RAP2.2 and RAP2.12 is identified through a comparative phylogenetic motif search, promoter dissection, yeast one-hybrid assays, and chromatin immunopurification. This motif, designated the hypoxia-responsive promoter element, is enriched in promoters of hypoxia-responsive genes in multiple species. PMID:26668304

  11. DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

    PubMed

    Periz, G; Keller, L R

    1997-07-01

    Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements.

  12. DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

    PubMed Central

    Periz, G; Keller, L R

    1997-01-01

    Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements. PMID:9199320

  13. Small heat shock protein alphaA-crystallin regulates epithelial sodium channel expression.

    PubMed

    Kashlan, Ossama B; Mueller, Gunhild M; Qamar, Mohammad Z; Poland, Paul A; Ahner, Annette; Rubenstein, Ronald C; Hughey, Rebecca P; Brodsky, Jeffrey L; Kleyman, Thomas R

    2007-09-21

    Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo post-translational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which misfolded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular chaperones, including the small heat shock protein alphaA-crystallin, have recently been shown to play a role in this process. We have now found that alphaA-crystallin is expressed in cultured mouse collecting duct cells, where apical Na(+) transport is mediated by epithelial Na(+) channels (ENaC). ENaC-mediated Na(+) currents in Xenopus oocytes were reduced by co-expression of alphaA-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of alphaA-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that alphaA-crystallin and ENaC co-immunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ER-associated degradation. PMID:17664274

  14. Concerted regulation of inhibitory activity of alpha 1-antitrypsin by the native strain distributed throughout the molecule.

    PubMed

    Seo, Eun Joo; Lee, Cheolju; Yu, Myeong-Hee

    2002-04-19

    The native forms of common globular proteins are in their most stable state but the native forms of plasma serpins (serine protease inhibitors) show high energy state interactions. The high energy state strain of alpha(1)-antitrypsin, a prototype serpin, is distributed throughout the whole molecule, but the strain that regulates the function directly appears to be localized in the region where the reactive site loop is inserted during complex formation with a target protease. To examine the functional role of the strain at other regions of alpha(1)-antitrypsin, we increased the stability of the molecule greatly via combining various stabilizing single amino acid substitutions that did not affect the activity individually. The results showed that a substantial increase of stability, over 13 kcal mol(-1), affected the inhibitory activity with a correlation of 11% activity loss per kcal mol(-1). Addition of an activity affecting single residue substitution in the loop insertion region to these very stable substitutions caused a further activity decrease. The results suggest that the native strain of alpha(1)-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner. PMID:11834734

  15. Using SCOPE to identify potential regulatory motifs in coregulated genes.

    PubMed

    Martyanov, Viktor; Gross, Robert H

    2011-05-31

    SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data. In this article, we utilize a web version of SCOPE to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs and has been used in other studies. The three algorithms that comprise SCOPE are BEAM, which finds non-degenerate motifs (ACCGGT), PRISM, which finds degenerate motifs (ASCGWT), and SPACER, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well. Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor. Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from

  16. PPAR{alpha} agonists up-regulate organic cation transporters in rat liver cells

    SciTech Connect

    Luci, Sebastian; Geissler, Stefanie; Koenig, Bettina; Koch, Alexander; Stangl, Gabriele I.; Hirche, Frank; Eder, Klaus . E-mail: klaus.eder@landw.uni-halle.de

    2006-11-24

    It has been shown that clofibrate treatment increases the carnitine concentration in the liver of rats. However, the molecular mechanism is still unknown. In this study, we observed for the first time that treatment of rats with the peroxisome proliferator activated receptor (PPAR)-{alpha} agonist clofibrate increases hepatic mRNA concentrations of organic cation transporters (OCTNs)-1 and -2 which act as transporters of carnitine into the cell. In rat hepatoma (Fao) cells, treatment with WY-14,643 also increased the mRNA concentration of OCTN-2. mRNA concentrations of enzymes involved in carnitine biosynthesis were not altered by treatment with the PPAR{alpha} agonists in livers of rats and in Fao cells. We conclude that PPAR{alpha} agonists increase carnitine concentrations in livers of rats and cells by an increased uptake of carnitine into the cell but not by an increased carnitine biosynthesis.

  17. Two RGD-independent alpha vbeta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties.

    PubMed

    Maeshima, Y; Colorado, P C; Kalluri, R

    2000-08-01

    Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (alpha3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54-132) anti-angiogenic and a C-terminal (amino acids 185-203) anti-tumor cell activity (Maeshima, Y., et al. 2000) J. Biol. Chem. 275, 21340-21348). Previous studies have identified the 185-203 amino acid sequence as a ligand for alpha(v)beta(3) integrin (Shahan, T. A., et al. (1999) Cancer Res. 59, 4584-4590). In the present study, we found distinct additional RGD-independent alpha(v)beta(3) integrin binding site within 54-132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54-132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185-203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the alpha(v)beta(3) integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique alpha(v)beta(3) integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin. PMID:10837460

  18. A T-to-G transversion at nucleotide -567 upstream of HBG2 in a GATA-1 binding motif is associated with elevated hemoglobin F.

    PubMed

    Chen, Zhiyi; Luo, Hong-Yuan; Basran, Raveen K; Hsu, Tien-Huei; Mang, Daniel W H; Nuntakarn, Lalana; Rosenfield, Cathy G; Patrinos, George P; Hardison, Ross C; Steinberg, Martin H; Chui, David H K

    2008-07-01

    Increased fetal hemoglobin (Hb F; alpha(2)gamma(2)) production in adults can ameliorate the clinical severity of sickle cell disease and beta-thalassemia major. Thus, understanding the regulation of gamma-globin gene expression and its silencing in adults has potential therapeutic implications. We studied a father and son in an Iranian-American family who had elevated Hb F levels and found a novel T-to-G transversion at nucleotide (nt) -567 of the HBG2 promoter. This mutation alters a GATA-1 binding motif to a GAGA sequence located within a previously identified silencing element. DNA-protein binding assays showed that the GATA motif of interest is capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of the HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between nt -675 and -526. In addition, the T-to-G mutation at the GATA motif increased the promoter activity by two- to threefold in transiently transfected erythroid cell lines. The binding motif is uniquely conserved in simian primates with a fetal pattern of gamma-globin gene expression. These results suggest that the GATA motif under study has a functional role in silencing gamma-globin gene expression in adults. The T-to-G mutation in this motif disrupts GATA-1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up-regulation of gamma-globin gene expression in adults.

  19. Nicotinic acetylcholine receptors containing alpha 7 subunits on rat cortical neurons do not undergo long-lasting inactivation even when up-regulated by chronic nicotine exposure.

    PubMed

    Kawai, H; Berg, D K

    2001-09-01

    Chronic exposure to (-)nicotine has been widely reported to up-regulate nicotinic acetylcholine receptors on neurons and induce long-term inactivation as a possible cause. Nicotinic receptors containing alpha 7 subunits are among the most abundant in brain and influence diverse cellular events. Whole-cell patch clamp recording from embryonic rat cortical neurons in culture was used to identify responses from alpha 7-containing receptors. Immunochemical staining for glutamic acid decarboxylase (GAD) indicated that both GABAergic and non-GABAergic neurons expressed the receptors. Exposure to micromolar concentrations of nicotine for 1-4 days caused up-regulation of the receptors as measured by [alpha-(125)I]-bungarotoxin binding. Carbachol produced the same up-regulation, and cell counts demonstrated that neuronal survival was unchanged. The up-regulation was accompanied by an increased whole-cell response; no evidence was found for long-lasting inactivation. Autonomic alpha 7-containing receptors also avoided long-lasting inactivation, even though the receptors were down-regulated by nicotine. Blocking protein synthesis or protein glycosylation prevented receptor up-regulation on cortical neurons, suggesting that new synthesis was required. No evidence was found for a pre-existing intracellular pool that supplied receptors to the surface. The results indicate that alpha 7-containing receptors differ from other receptor subtypes in their regulation by nicotine and demonstrate further that long-lasting inactivation is not an obligatory requirement for up-regulation in this case.

  20. Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha 1-acid glycoprotein (AGP, ORM-1) is a highly glycosylated mammalian acute phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute phase protein in this ...

  1. DeltaNp73alpha regulates MDR1 expression by inhibiting p53 function.

    PubMed

    Vilgelm, A; Wei, J X; Piazuelo, M B; Washington, M K; Prassolov, V; El-Rifai, W; Zaika, A

    2008-04-01

    The p73 protein is a transcription factor and member of the p53 protein family that expresses as a complex variety of isoforms. DeltaNp73alpha is an N-terminally truncated isoform of p73. We found that DeltaNp73 protein is upregulated in human gastric carcinoma suggesting that DeltaNp73 may play an oncogenic role in these tumors. Although it has been shown that DeltaNp73alpha inhibits apoptosis and counteracts the effect of chemotherapeutic drugs, the underlying mechanism by which this p73 isoform contributes to chemotherapeutic drug response remains to be explored. We found that DeltaNp73alpha upregulates MDR1 mRNA and p-glycoprotein (p-gp), which is involved in chemotherapeutic drug transport. This p-gp upregulation was accompanied by increased p-gp functional activity in gastric cancer cells. Our data suggest that upregulation of MDR1 by DeltaNp73alpha is mediated by interaction with p53 at the MDR1 promoter.

  2. Thyroid hormone regulation of transmembrane signalling in neonatal rat ventricular myocytes by selective alteration of the expression and coupling of G-protein alpha-subunits.

    PubMed Central

    Bahouth, S W

    1995-01-01

    Thyroid hormone exerts profound effects on the activity of the hormone-sensitive adenylate cyclase system in the heart. Distinct guanine nucleotide-binding regulatory proteins (G-proteins) mediate stimulatory and inhibitory influences on adenylate cyclase activity. To examine whether the effects of thyroid hormone on adenylate cyclase involve specific changes in G-protein subunit expression, the influence of tri-iodothyronine (T3) on the biosynthesis and activity of G-proteins in neonatal rat ventricular myocytes was determined. In myocytes challenged with T3 for 5 days, Gs alpha levels increased by 4 +/- 0.5-fold, whereas Gi2 alpha levels declined by more than 80%. T3 down-regulated Gi2 alpha mRNA by 60% within 3 days, but had no effect on Gs alpha mRNA. The basis for the decline in Gi2 alpha mRNA was the T3-mediated suppression of Gi2 alpha gene transcription by 80 +/- 9% within 4 h. The decline in Gi2 alpha mRNA in response to T3 produced a 2-fold decrease in relative rate of synthesis of Gi2 alpha but not in its half-life (46 +/- 7 h). Gs alpha synthesis was not altered by T3, but the half-life of Gs alpha increased from 50 +/- 6 h in control cells to 72 +/- 8 h in T3-treated cells. In addition, T3 provoked the translocation of Gs alpha from the cytoplasmic to the membranous compartment. Membranous Gs alpha increased from 30 +/- 6% to 61 +/- 7% of total cellular Gs alpha, whereas cytoplasmic Gs alpha declined from 68 +/- 6% to 33 +/- 8% within 1 day of exposure to T3. T3-mediated up-regulation of Gs alpha enhanced the activation of myocardial adenylate cyclase by the stimulatory pathway whereas the down-regulation of Gi2 alpha attenuated the deactivation of myocardial adenylate cyclase by the inhibitory pathway. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7741715

  3. Opposite effects of the acute promyelocytic leukemia PML-retinoic acid receptor alpha (RAR alpha) and PLZF-RAR alpha fusion proteins on retinoic acid signalling.

    PubMed Central

    Ruthardt, M; Testa, U; Nervi, C; Ferrucci, P F; Grignani, F; Puccetti, E; Grignani, F; Peschle, C; Pelicci, P G

    1997-01-01

    Fusion proteins involving the retinoic acid receptor alpha (RAR alpha) and the PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemias (APLs). APLs with the PML-RAR alpha or the PLZF-RAR alpha fusion protein are phenotypically indistinguishable except that they differ in their sensitivity to retinoic acid (RA)-induced differentiation: PML-RAR alpha blasts are sensitive to RA and patients enter disease remission after RA treatment, while patients with PLZF-RAR alpha do not. We here report that (i) like PML-RAR alpha expression, PLZF-RAR alpha expression blocks terminal differentiation of hematopoietic precursor cell lines (U937 and HL-60) in response to different stimuli (vitamin D3, transforming growth factor beta1, and dimethyl sulfoxide); (ii) PML-RAR alpha, but not PLZF-RAR alpha, increases RA sensitivity of hematopoietic precursor cells and restores RA sensitivity of RA-resistant hematopoietic cells; (iii) PML-RAR alpha and PLZF-RAR alpha have similar RA binding affinities; and (iv) PML-RAR alpha enhances the RA response of RA target genes (those for RAR beta, RAR gamma, and transglutaminase type II [TGase]) in vivo, while PLZF-RAR alpha expression has either no effect (RAR beta) or an inhibitory activity (RAR gamma and type II TGase). These data demonstrate that PML-RAR alpha and PLZF-RAR alpha have similar (inhibitory) effects on RA-independent differentiation and opposite (stimulatory or inhibitory) effects on RA-dependent differentiation and that they behave in vivo as RA-dependent enhancers or inhibitors of RA-responsive genes, respectively. Their different activities on the RA signalling pathway might underlie the different responses of PML-RAR alpha and PLZF-RAR alpha APLs to RA treatment. The PLZF-RAR alpha fusion protein contains an approximately 120-amino-acid N-terminal motif (called the POZ domain), which is also found in a variety of zinc finger proteins and a group of poxvirus proteins and which mediates protein

  4. Hydrogen-bond motifs in the crystals of hydrophobic amino acids.

    PubMed

    Fábián, László; Chisholm, James A; Galek, Peter T A; Motherwell, W D Samuel; Feeder, Neil

    2008-08-01

    A computer program has been developed to survey a set of crystal structures for hydrogen-bond motifs. Possible ring and chain motifs are generated automatically from a user-defined list of interacting molecular fragments and intermolecular interactions. The new program was used to analyse the hydrogen-bond networks in the crystals of 52 zwitterionic alpha-amino acids. All the possible chain motifs (repeating 1-4 molecules) are frequent, while the frequency of ring motifs (2-6 molecules) ranges from 0 to 85% of the structures. The list of motifs displayed by each structure reveals structural similarities and it can be used to compare polymorphs. The motifs formed in cocrystals of alpha-amino acids and in crystals of beta- and gamma-amino acids are similar to those of alpha-amino acids. PMID:18641453

  5. The microRNA-124-iGluR2/3 pathway regulates glucagon release from alpha cells

    PubMed Central

    Zhang, Haiyang; Liu, Rui; Deng, Ting; Wang, Xia; Lang, Hongmei; Qu, Yanjun; Duan, Jingjing; Huang, Dingzhi; Ying, Guoguang; Ba, Yi

    2016-01-01

    Glucagon, secreted from islet alpha cells, plays an important role in regulating glucose homeostasis; however, the molecular mechanism underlying this process is not fully understood. Previous studies have demonstrated that miRNAs are involved in the function of alpha cells. Glutamate promotes glucagon secretion by mediating the opening of Ca2+ channels. In this present, iGluR2 and iGluR3 levels were significantly increased in fasting-treated mouse islets. Additional studies showed that miR-124-3p simultaneously regulates the expression of iGluR2 and iGluR3 through the direct targeting of mRNA 3’UTR of these two genes. The miR-124-iGluRs pathway also contributed to the high level of glucagon secretion through long-term high glucose levels. Thus, a novel pathway comprising miRNA, glutamate and iGluRs has been demonstrated to regulate the biological process of glucagon release. PMID:27013590

  6. Comparison of alpha-2 adrenergic receptors and their regulation in rodent and porcine species

    SciTech Connect

    Feller, D.J.; Bylund, D.B.

    1984-02-01

    The alpha-2 adrenergic antagonist (/sup 3/H)yohimbine (YOH) and the alpha-2 agonist (/sup 3/H)p-aminoclonidine (PAC) saturably label high-affinity binding sites in the submandibular gland from 3-week-old rats and 5-week-old pigs and in the lung from neonatal rats and 5-week-old pigs. (/sup 3/H)YOH had KD values of 5.5, 1.8, 0.45 and 0.22 nM in the rat gland and lung and porcine gland and lung, respectively. KD values of 2.4, 5.3 and 1.3 nM were found for (/sup 3/H)PAC in rodent and pig submandibular gland and pig lung, respectively. Both /sup 3/H-ligands labeled approximately the same density of sites within each tissue except in the rat lung in which (/sup 3/H)PAC binding was too low to reliably estimate. In all cases the pharmacologic profile was indicative of an alpha-2 adrenergic receptor site. However, the Ki of yohimbine vs. (/sup 3/H)PAC was 30- to 140-fold higher for the rodent relative to the porcine species. GTP decreased the affinity of (-)-epinephrine and PAC at (/sup 3/H)YOH-labeled sites in the pig gland and lung, but did not shift the affinity of epinephrine in the rat gland. These results suggest the possibility of subtype or species differences for the alpha-2 receptor. The Ki values of the antagonists YOH and phentolamine were different at (/sup 3/H)PAC and (/sup 3/H)YOH sites. GTP caused a dose-dependent reduction in (/sup 3/H)PAC binding in the porcine submandibular gland and lung. At 10 microM GTP, this loss was due to a decrease in /sup 3/H-agonist affinity, but not density.

  7. Synthetic biology with RNA motifs.

    PubMed

    Saito, Hirohide; Inoue, Tan

    2009-02-01

    Structural motifs in naturally occurring RNAs and RNPs can be employed as new molecular parts for synthetic biology to facilitate the development of novel devices and systems that modulate cellular functions. In this review, we focus on the following: (i) experimental evolution techniques of RNA molecules in vitro and (ii) their applications for regulating gene expression systems in vivo. For experimental evolution, new artificial RNA aptamers and RNA enzymes (ribozymes) have been selected in vitro. These functional RNA molecules are likely to be applicable in the reprogramming of existing gene regulatory systems. Furthermore, they may be used for designing hypothetical RNA-based living systems in the so-called RNA world. For the regulation of gene expressions in living cells, the development of new riboswitches allows us to modulate the target gene expression in a tailor-made manner. Moreover, recently RNA-based synthetic genetic circuits have been reported by employing functional RNA molecules, expanding the repertory of synthetic biology with RNA motifs. PMID:18775792

  8. Differential splicing creates a diversity of transcripts from a neurospecific developmentally regulated gene encoding a protein with new zinc-finger motifs.

    PubMed Central

    Buchman, V L; Ninkina, N N; Bogdanov, Y D; Bortvin, A L; Akopian, H N; Kiselev, S L; Krylova OYu; Anokhin, K V; Georgiev, G P

    1992-01-01

    We have cloned a novel neurospecific gene, named neuro-d4, by differential screening a rat cerebral cortex cDNA library. Northern blot hybridization showed that neuro-d4 expression is restricted to neuronal tissues both in newborn and adult animals. The level of neuro-d4 mRNA in the rat central nervous system is high during the later stages of embryonic development and gradually decreases during the postnatal period. In situ hybridization suggests that the gene transcripts are localized in neuronal cell bodies. Nucleotide sequences of overlapped cDNA clones and all 12 exons in genomic clone were determined. The deduced protein has consensus sequences for a nuclear localization signal, a Krüppel-type zinc-finger and a new type of cysteine/histidine-rich motif resembling zinc-fingers. Several differential splicing variants were found, each of which influences the structure of the encoded protein. Images PMID:1454523

  9. Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

    PubMed Central

    Moon, Sung-Kyun; Lee, Haa-Yung; Pan, Huiqi; Takeshita, Tamotsu; Park, Raekil; Cha, Kiweon; Andalibi, Ali; Lim, David J

    2006-01-01

    Background We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. Methods The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. Results Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. Conclusion We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway. PMID:16433908

  10. [Personal motif in art].

    PubMed

    Gerevich, József

    2015-01-01

    One of the basic questions of the art psychology is whether a personal motif is to be found behind works of art and if so, how openly or indirectly it appears in the work itself. Analysis of examples and documents from the fine arts and literature allow us to conclude that the personal motif that can be identified by the viewer through symbols, at times easily at others with more difficulty, gives an emotional plus to the artistic product. The personal motif may be found in traumatic experiences, in communication to the model or with other emotionally important persons (mourning, disappointment, revenge, hatred, rivalry, revolt etc.), in self-searching, or self-analysis. The emotions are expressed in artistic activity either directly or indirectly. The intention nourished by the artist's identity (Kunstwollen) may stand in the way of spontaneous self-expression, channelling it into hidden paths. Under the influence of certain circumstances, the artist may arouse in the viewer, consciously or unconsciously, an illusionary, misleading image of himself. An examination of the personal motif is one of the important research areas of art therapy.

  11. [Personal motif in art].

    PubMed

    Gerevich, József

    2015-01-01

    One of the basic questions of the art psychology is whether a personal motif is to be found behind works of art and if so, how openly or indirectly it appears in the work itself. Analysis of examples and documents from the fine arts and literature allow us to conclude that the personal motif that can be identified by the viewer through symbols, at times easily at others with more difficulty, gives an emotional plus to the artistic product. The personal motif may be found in traumatic experiences, in communication to the model or with other emotionally important persons (mourning, disappointment, revenge, hatred, rivalry, revolt etc.), in self-searching, or self-analysis. The emotions are expressed in artistic activity either directly or indirectly. The intention nourished by the artist's identity (Kunstwollen) may stand in the way of spontaneous self-expression, channelling it into hidden paths. Under the influence of certain circumstances, the artist may arouse in the viewer, consciously or unconsciously, an illusionary, misleading image of himself. An examination of the personal motif is one of the important research areas of art therapy. PMID:26202617

  12. Novel Epigenetic Regulation of Alpha-Synuclein Expression in Down Syndrome.

    PubMed

    Ramakrishna, Narayan; Meeker, Harry C; Brown, W Ted

    2016-01-01

    Alpha-synuclein (SNCA), a presynaptic protein, is significantly reduced in individuals with Down syndrome (DS) and Ts65Dn mice, a mouse model of DS. Methylation analyses of promoter proximal CpG sites indicate similar reduction in Ts65Dn mice compared to control mice. Epigallocatechin-3-gallate (EGCG), a polyphenolic catechin present in green tea extract, increases methylation of SNCA promoter proximal CpG sites and expression in Ts65Dn mice. These results suggest a positive link between CpG methylation and SNCA expression in Down syndrome. PMID:25416858

  13. Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

    SciTech Connect

    Chung, Eric; Jakinovich, Paul; Bae, Aekyung; Rebecchi, Mario

    2012-10-01

    Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

  14. Regulatory motifs in Chk1

    PubMed Central

    Caparelli, Michael L.; O’Connell, Matthew J.

    2013-01-01

    Chk1 is the effector kinase of the G2 DNA damage checkpoint. Chk1 homologs possess a highly conserved N-terminal kinase domain and a less conserved C-terminal regulatory domain. In response to DNA damage, Chk1 is recruited to mediator proteins assembled at lesions on replication protein A (RPA)-coated single-stranded DNA (ssDNA). Chk1 is then activated by phosphorylation on S345 in the C-terminal regulatory domain by the PI3 kinase-related kinases ATM and ATR to enforce a G2 cell cycle arrest to allow time for DNA repair. Models have emerged in which this C-terminal phosphorylation relieves auto-inhibitory regulation of the kinase domain by the regulatory domain. However, experiments in fission yeast have shown that deletion of this putative auto-inhibitory domain actually inactivates Chk1 function. We show here that Chk1 homologs possess a kinase-associated 1 (KA1) domain that possesses residues previously implicated in Chk1 auto-inhibition. In addition, all Chk1 homologs have a small and highly conserved C-terminal extension (CTE domain). In fission yeast, both of these motifs are essential for Chk1 activation through interaction with the mediator protein Crb2, the homolog of human 53BP1. Thus, through different intra- and intermolecular interactions, these motifs explain why the regulatory domain exerts both positive and negative control over Chk1 activation. Such motifs may provide alternative targets to the ATP-binding pocket on which to dock Chk1 inhibitors as anticancer therapeutics. PMID:23422000

  15. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    PubMed Central

    Brown, M. J.; Bristow, D. R.

    1996-01-01

    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect

  16. Transcriptional Regulation of Apolipoprotein A5 Gene Expression by the Nuclear Receptor ROR alpha

    SciTech Connect

    Genoux, Annelise; Dehondt, Helene; Helleboid-Chapman, Audrey; Duhem, Christian; Hum, Dean W.; Martin, Genevieve; Pennacchio, Len; Staels, Bart; Fruchart-Najib, Jamila; Fruchart, Jean-Charles

    2004-10-01

    Apolipoprotein A5 has recently been identified as a crucial determinant of plasma triglyceride levels. Our results showed that RORa up-regulates human APOA5 but has no effect on mouse apoa5 promoter. These data suggest an additional important physiological role for RORa in the regulation of genes involved in plasma triglyceride homeostasis in human and probably in the development of atherosclerosis

  17. Phosphorylation of the protein kinase C-theta activation loop and hydrophobic motif regulates its kinase activity, but only activation loop phosphorylation is critical to in vivo nuclear-factor-kappaB induction.

    PubMed Central

    Liu, Yin; Graham, Caroline; Li, Aiqun; Fisher, Robert J; Shaw, Stephen

    2002-01-01

    Protein kinase C (PKC)-theta, a member of the 'novel' subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for 'classical' PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr(538)), turn motif (Ser(676)) and hydrophobic motif (Ser(695)). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (>100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical in vivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor kappaB (NF-kappaB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-kappaB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-kappaB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive

  18. FXR regulates organic solute transporters alpha and beta in the adrenal gland, kidney, and intestine.

    PubMed

    Lee, Hans; Zhang, Yanqiao; Lee, Florence Y; Nelson, Stanley F; Gonzalez, Frank J; Edwards, Peter A

    2006-01-01

    Expression of the farnesoid X receptor (FXR; NR1H4) is limited to the liver, intestine, kidney, and adrenal gland. However, the role of FXR in the latter two organs is unknown. In the current study, we performed microarray analysis using RNA from H295R cells infected with constitutively active FXR. Several putative FXR target genes were identified, including the organic solute transporters alpha and beta (OSTalpha and OSTbeta). Electromobility shift assays and promoter-reporter studies identified functional farnesoid X receptor response elements (FXREs) in the promoters of both human genes. These FXREs are conserved in both mouse genes. Treatment of wild-type mice with 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic FXR agonist, induced OSTalpha and OSTbeta mRNAs in the intestine and kidney. Both mRNAs were also induced when wild-type, but not FXR-deficient (FXR-/-), adrenals were cultured in the presence of GW4064. OSTalpha and OSTbeta mRNA levels were also induced in the adrenals and kidneys of wild-type, but not FXR-/-, mice after the increase of plasma bile acids in response to the hepatotoxin alpha-naphthylisothiocyanate. Finally, overexpression of human OSTalpha and OSTbeta facilitated the uptake of conjugated chenodeoxycholate and the activation of FXR target genes. These results demonstrate that OSTalpha and OSTbeta are novel FXR target genes that are expressed in the adrenal gland, kidney, and intestine.

  19. Cloning, sequencing, and oxygen regulation of the Rhodobacter capsulatus alpha-ketoglutarate dehydrogenase operon.

    PubMed Central

    Dastoor, F P; Forrest, M E; Beatty, J T

    1997-01-01

    The Rhodobacter capsulatus sucA, sucB, and lpd genes, which encode the alpha-ketoglutarate dehydrogenase (E1o), the dihydrolipoamide succinyltransferase (E2o), and the dihydrolipoamide dehydrogenase (E3) components of the alpha-ketoglutarate dehydrogenase complex (KGD), respectively, were cloned, sequenced, and used for regulatory analyses. The KGD enzymatic activity was greater in cells grown under aerobic, respiratory growth conditions than under anaerobic, photosynthetic conditions. Similarly, the sucA gene was transcribed differentially, leading to a greater accumulation of sucA mRNAs under respiratory growth conditions than under photosynthetic conditions, although differential rates of mRNA decay could also contribute to the different amounts of sucA mRNAs under these two growth conditions. The sucA promoter was located about 4 kb upstream of the 5' end of the sucA gene, and transcripts greater than 9.5 kb hybridized to a sucA probe, suggesting the presence of an operon that produces a polycistronic mRNA. Thus, these genes seem to be expressed as an unstable primary transcript, and we speculate that posttranscriptional processes control the stoichiometry of KGD proteins. PMID:9226266

  20. Disordered regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase gene expression by phosphorus in X-linked hypophosphatemic (hyp) mice.

    PubMed

    Azam, Nasreen; Zhang, Martin Y H; Wang, Xuemei; Tenenhouse, Harriet S; Portale, Anthony A

    2003-08-01

    X-linked hypophosphatemic (Hyp) mice exhibit hypophosphatemia, impaired renal phosphate reabsorption, defective skeletal mineralization, and disordered regulation of vitamin D metabolism: In Hyp mice, restriction of dietary phosphorus induces a decrease in serum concentration of 1,25-dihydroxyvitamin D and renal activity of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase), and induces an increase in renal activity of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase). In contrast, in wild-type mice, phosphorus restriction stimulates renal 1alpha-hydroxylase gene expression and suppresses that of 24-hydroxylase. To determine the molecular basis for the disordered regulation of vitamin D metabolism in Hyp mice, we determined renal mitochondrial 1alpha-hydroxylase activity and the renal abundance of p450c1alpha and p450c24 mRNA in wild-type and Hyp mice fed either control, low-, or high-phosphorus diets for 5 d. In wild-type mice, phosphorus restriction increased 1alpha-hydroxylase activity and p450c1alpha mRNA expression by 6-fold and 3-fold, respectively, whereas in the Hyp strain the same diet induced changes of similar magnitude but opposite in direction. Phosphorus supplementation was without effect in wild-type mice, whereas in Hyp mice the same diet induced 3-fold and 2-fold increases, respectively, in enzyme activity and p450c1alpha mRNA abundance. In wild-type mice, both renal 1alpha-hydroxylase activity and p450c1alpha mRNA abundance varied inversely and significantly with serum phosphorus concentrations, whereas in Hyp mice the relationship between both renal parameters and serum phosphorus concentration was direct. In Hyp mice, phosphorus restriction induced a significant increase in renal p450c24 mRNA abundance, in contrast to the lack of effect observed in wild-type mice. The present findings demonstrate that regulation of both the p450c1alpha and p45024 genes by phosphorus is disordered in Hyp mice at the level of renal 1alpha

  1. Transport of prostaglandin F(2alpha) pulses from the uterus to the ovary at the time of luteolysis in ruminants is regulated by prostaglandin transporter-mediated mechanisms.

    PubMed

    Lee, JeHoon; McCracken, John A; Banu, Sakhila K; Rodriguez, Royce; Nithy, Thamizh K; Arosh, Joe A

    2010-07-01

    In ruminants, prostaglandin F2alpha (PGF(2alpha)) is the uterine luteolytic hormone. During luteolysis, PGF(2alpha) is synthesized and released from the endometrium in a pulsatile pattern. The unique structure of the vascular utero-ovarian plexus (UOP) allows transport of luteolytic PGF(2alpha) pulses directly from the uterus to the ovary, thus bypassing the systemic circulation. However, the underlying molecular mechanism is not known. The objective of the present study was to determine a role for PG transporter protein (PGT) in the compartmental transport of PGF(2alpha) from uterus to ovary through the UOP at the time of luteolysis using the sheep as a ruminant model. [(3)H]PGF(2alpha), with or without a PGT inhibitor, was infused into UOP, and PGF(2alpha) transport and PGT protein expression were determined. Results indicate that PGT protein is expressed in tunica intima, tunica media, and tunica adventitia of the utero-ovarian vein and the ovarian artery of the UOP, and the expression levels are higher on d 10-15 compared with d 3-6 of the estrous cycle. Pharmacological inhibition of PGT prevented transport of exogenous [(3)H]PGF(2alpha) as well as oxytocin-induced endogenous luteolytic PGF(2alpha) pulse up to 80% from uterine venous blood into ovarian arterial blood through the UOP at the time of luteolysis in sheep. Taken together, these results indicate that at the time of luteolysis, transport of PGF(2alpha) from uterus to ovary through the UOP is regulated by PGT-mediated mechanisms. These findings also suggest that impaired PGT-mediated transport of PGF(2alpha) from the utero-ovarian vein into the ovarian artery could adversely influence luteolysis and thus affect fertility in ruminants.

  2. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    PubMed

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. A Gibbs sampler for motif detection in phylogenetically close sequences

    NASA Astrophysics Data System (ADS)

    Siddharthan, Rahul; van Nimwegen, Erik; Siggia, Eric

    2004-03-01

    Genes are regulated by transcription factors that bind to DNA upstream of genes and recognize short conserved ``motifs'' in a random intergenic ``background''. Motif-finders such as the Gibbs sampler compare the probability of these short sequences being represented by ``weight matrices'' to the probability of their arising from the background ``null model'', and explore this space (analogous to a free-energy landscape). But closely related species may show conservation not because of functional sites but simply because they have not had sufficient time to diverge, so conventional methods will fail. We introduce a new Gibbs sampler algorithm that accounts for common ancestry when searching for motifs, while requiring minimal ``prior'' assumptions on the number and types of motifs, assessing the significance of detected motifs by ``tracking'' clusters that stay together. We apply this scheme to motif detection in sporulation-cycle genes in the yeast S. cerevisiae, using recent sequences of other closely-related Saccharomyces species.

  4. Occurrence of poly(alpha2,8-deaminoneuraminic acid) in mammalian tissues: widespread and developmentally regulated but highly selective expression on glycoproteins.

    PubMed Central

    Ziak, M; Qu, B; Zuo, X; Zuber, C; Kanamori, A; Kitajima, K; Inoue, S; Inoue, Y; Roth, J

    1996-01-01

    In tissues of higher organisms homopolymers of alpha2,8-linked N-acetylneuraminic acid can be found as a posttranslational modification on selected proteins. We report here the discovery of homopolymers of alpha2,8-linked deaminoneuraminic acid [poly(alpha2,8-KDN)] in various tissues derived from all three germ layers in vertebrates including mammals. The monoclonal antibody kdn8kdn in conjunction with a bacterial KDNase permitted the detection of poly(alpha2,8-KDN) by immunohistochemistry and immunoblotting. Further evidence for the existence of poly(alpha2,8-KDN) was obtained by gas/liquid chromatography. The poly(alpha2,8-KDN) glycan was detectable in all tissues studied with the exception of mucus-producing cells present in various organs, the extracellular matrix, and basement membranes. However, in certain organs such as muscle, kidney, lung, and brain its expression was developmentally regulated. Despite its widespread tissue distribution, the poly(alpha2,8-KDN) glycan was detected on a single 150-kDa glycoprotein except for a single >350-kDa glycoprotein in kidney, which makes it most distinctive among polysialic acids. The ubiquitous yet selective expression may be indicative of a general function of the poly(alpha2,8-KDN)-bearing glycoproteins. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8610115

  5. Gene expression profiling reveals novel regulation by bisphenol-A in estrogen receptor-{alpha}-positive human cells

    SciTech Connect

    Singleton, David W.; Feng, Yuxin; Yang, Jun; Puga, Alvaro; Lee, Adrian V.; Khan, Sohaib A. . E-mail: sohaib.khan@uc.edu

    2006-01-15

    Bisphenol-A (BPA) shows proliferative actions in uterus and mammary glands and may influence the development of male and female reproductive tracts in utero or during early postnatal life. Because of its ability to function as an estrogen receptor (ER) agonist, BPA has the potential to disrupt normal endocrine signaling through regulation of ER target genes. Some genes are regulated by both estradiol (E2) and BPA, but those exclusive to either agent have not been described. Using a yeast strain incorporating a vitellogenin A2 ERE-LacZ reporter gene into the genome, we found that BPA induced expression of the reporter in colonies transformed with the ER{alpha} expression plasmid, illustrating BPA-mediated regulation within a chromatin context. Additionally, a reporter gene transiently transfected into the endometrial cancer (Ishikawa) cell line also showed BPA activity, although at 100-fold less potency than E2. To compare global gene expression in response to BPA and E2, we used a variant of the MCF-7 breast cancer cell line stably expressing HA-tagged ER{alpha}. Cultures were treated for 3 h with an ethanol vehicle, E2 (10{sup -8} M), or BPA (10{sup -6} M), followed by isolation of RNA and microarray analysis with the human U95A probe array (Affymetrix, Santa Clara, CA, USA). More than 300 genes were changed 2-fold or more by either or both agents, with roughly half being up-regulated and half down-regulated. A number of growth- and development-related genes, such as HOXC1 and C6, Wnt5A, Frizzled, TGF{beta}-2, and STAT inhibitor 2, were found to be affected exclusively by BPA. We used quantitative real-time PCR to verify regulation of the HOXC6 gene, which showed decreased expression of approximately 2.5-fold by BPA. These results reveal novel effects by BPA and E2, raising interesting possibilities regarding the role of endocrine disruptors in sexual development.

  6. 1-alpha-Hydroxyvitamin D3 treatment decreases bone turnover and modulates calcium-regulating hormones in early postmenopausal women.

    PubMed

    Chen, J T; Shiraki, M; Hasumi, K; Tanaka, N; Katase, K; Kato, T; Hirai, Y; Nakamura, T; Ogata, E

    1997-06-01

    50 Japanese women within 10 years after menopause (mean age 52.5 years) were studied to determine the effects of 0.75 microgram of 1-alpha-hydroxyvitamin D3 [1-alpha-(OH)D3] with calcium (150 mg/day) (treated group: N = 25) and calcium only (control group: N = 25) for 12 months on bone mass and metabolism. Their L2-4 BMD measurements were 1.5 SD below the mean value of Japanese young, normal women. L2-4 BMDs increased significantly in the treated group (+2.1%; p < 0.01), but decreased significantly in controls (-2.1%; p < 0.01). Although serum calcium and creatinine remained unchanged in both groups, phosphorus levels increased significantly in the treated group (p < 0.01). Urinary calcium/creatinine (Cr) increased in both groups. Urinary pyridinoline/Cr and deoxypyridinoline/Cr decreased significantly in the treated group (p < 0.05), but not in the control group. Serum osteocalcin levels remained unchanged in both groups. Intact parathyroid hormone levels decreased significantly (p < 0.05) and calcitonin levels significantly increased in the treated group (p < 0.05), but these changes were not observed in the control group. These data clearly demonstrate that 0.75 microgram of 1-alpha-(OH)D3 maintained bone mass by reducing bone resorption by modulation of calcium-regulating hormones. Temporarily increased urinary calcium excretion was observed in control group, but did not appear to be effective in modulating bone turnover. PMID:9177871

  7. Effects of adrenalectomy on the alpha-adrenergic regulation of cytosolic free calcium in hepatocytes

    SciTech Connect

    Freudenrich, C.C.; Borle, A.B.

    1988-06-25

    We have previously published that bilateral adrenalectomy in the rat reduces the Ca2+-mediated alpha-adrenergic activation of hepatic glycogenolysis, while it increases the cellular calcium content of hepatocytes. In the experiments presented here, the concentration of cytosolic free calcium (Ca2+i) at rest and in response to epinephrine was measured in aequorin-loaded hepatocytes isolated from sham and adrenalectomized male rats. We found that in adrenalectomized rats the resting Ca2+i was elevated, the rise in Ca2+i evoked by epinephrine was reduced, and the rise in /sup 45/Ca efflux that follows such stimulation was depressed. Furthermore, the slope of the relationship between Ca2+i and calcium efflux was decreased 60% in adrenalectomized. Adrenalectomy did not change Ca2+ release from intracellular calcium pools in response to IP3 in saponin-permeabilized hepatocytes. The EC50 for inositol 1,4,5-triphosphate and the maximal Ca2+ released were similar in both sham and adrenalectomized animals. Finally, the liver calmodulin content determined by radioimmunoassay was not significantly different between sham and adrenalectomized rats. These results suggest that 1) adrenalectomy reduces calcium efflux from the hepatocyte, probably by an effect on the plasma membrane (Ca2+-Mg2+)-ATPase-dependent Ca2+ pump and thus alters cellular calcium homeostasis; 2) adrenalectomy decreases the rise in Ca2+i in response to epinephrine; 3) this decreased rise in Ca2+i is not due to defects in the intracellular Ca2+ storage and mobilization processes; and 4) the effects of adrenalectomy on cellular calcium metabolism and on alpha-adrenergic activation of glycogenolysis are not caused by a reduction in soluble calmodulin.

  8. Plasminogen activator inhibitor-I-related regulation of procollagen I ({alpha}{sub 1} and {alpha}{sub 2}) by antitransforming growth factor-{beta}{sub 1} treatment during radiation-impaired wound healing

    SciTech Connect

    Schultze-Mosgau, Stefan; Thorwarth, Michael; Roedel, Franz; Melnychenko, Ivan; Grabenbauer, Gerhard G.; Amann, Kerstin; Wehrhan, Falk

    2006-01-01

    Purpose: Plasminogen activator inhibitor (PAI)-1 mediates transforming growth factor-{beta}{sub 1} (TGF-{beta}{sub 1})-related signaling by stimulating collagen Type I synthesis in radiation-impaired wound healing. The regulation of {alpha}(I)-procollagen is contradictory in fibroblasts of different fibrotic lesions. It is not known whether anti-TGF-{beta}{sub 1} treatment specifically inhibits {alpha}(I)-procollagen synthesis. We used an experimental wound healing study to address anti-TGF-{beta}{sub 1}-associated influence on {alpha}(I)-procollagen synthesis. Methods and Materials: A free flap was transplanted into the preirradiated (40 Gy) or nonirradiated neck region of Wistar rats: Group 1 (n = 8) surgery alone; Group 2 (n = 14) irradiation and surgery; Group 3 (n = 8) irradiation and surgery and anti-TGF-{beta}{sub 1} treatment. On the 14th postoperative day, skin samples were processed for fibroblast culture, in situ hybridization for TGF-{beta}{sub 1}, immunohistochemistry, and immunoblotting for PAI-1, {alpha}{sub 1}/{alpha}{sub 2}(I)-procollagen. Results: Anti-TGF-{beta}{sub 1} significantly reduced TGF-{beta}{sub 1} mRNA (p < 0.05) and PAI-1 expression (p < 0.05). Anti-TGF-{beta}{sub 1} treatment in vivo significantly reduced {alpha}{sub 1}(I)-procollagen protein (p < 0.05) and the number of expressing cells (p < 0.05) in contrast to significantly increased (p < 0.05) {alpha}{sub 2}(I)-procollagen expression. Conclusion: These results emphasize anti-TGF-{beta}{sub 1} treatment to reduce radiation-induced fibrosis by decreasing {alpha}{sub 1}(I)-procollagen synthesis in vivo. {alpha}{sub 1}(I)-procollagen and {alpha}{sub 2}(I)-procollagen might be differentially regulated by anti-TGF-{beta}{sub 1} treatment. Increased TGF-{beta} signaling in irradiated skin fibroblasts seemed to be reversible, as shown by a reduction in PAI-1 expression after anti-TGF-{beta}{sub 1} treatment.

  9. Role of SIRT1 in regulation of LPS- or two ethanol metabolites-induced TNF-alpha production in cultured macrophage cell lines.

    PubMed

    Shen, Zheng; Ajmo, Joanne M; Rogers, Christopher Q; Liang, Xiaomei; Le, Lisa; Murr, Michel M; Peng, Yanhua; You, Min

    2009-05-01

    Dysregulation of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of alcoholic liver injury. Sirtuin 1 (SIRT1) is an NAD(+)-dependent class III protein deacetylase that is known to be involved in regulating production of proinflammatory cytokines including TNF-alpha. In the present study, we examined the role of SIRT1 signaling in TNF-alpha generation stimulated by either lipopolysaccharide (LPS), acetaldehyde (AcH), or acetate (two major metabolites of ethanol) in two cultured macrophage cell lines. In both rat Kupffer cell line 1 (RKC1) and murine RAW 264.7 macrophages, treatment with either LPS, AcH, or acetate caused significant decreases in SIRT1 transcription, translation, and activation, which essentially demonstrated an inverse relationship with TNF-alpha levels. LPS, AcH, and acetate each provoked the release of TNF-alpha from RKC1 cells, whereas coincubation with resveratrol (a potent SIRT1 agonist) inhibited this effect. Conversely, addition of sirtinol (a known SIRT1 inhibitor) or knocking down SIRT1 by the small silencing SIRT1 plasmid (SIRT1shRNA) augmented TNF-alpha release, suggesting that impairment of SIRT1 may contribute to TNF-alpha secretion. Further mechanistic studies revealed that inhibition of SIRT1 by LPS, AcH, or acetate was associated with a marked increase in the acetylation of the RelA/p65 subunit of nuclear transcription factor (NF-kappaB) and promotion of NF-kappaB transcriptional activity. Taken together, our findings suggest that SIRT1-NF-kappaB signaling is involved in regulating LPS- and metabolites-of-ethanol-mediated TNF-alpha production in rat Kupffer cells and in murine macrophages. Our study provides new insights into understanding the molecular mechanisms underlying the development of alcoholic steatohepatitis. PMID:19299582

  10. Hypoxia-Inducible Factor 2 Alpha Is Essential for Hepatic Outgrowth and Functions via the Regulation of leg1 Transcription in the Zebrafish Embryo

    PubMed Central

    Lin, Tzung-Yi; Chou, Chi-Fu; Chung, Hsin-Yu; Chiang, Chia-Yin; Li, Chung-Hao; Wu, Jen-Leih; Lin, Han-Jia; Pai, Tun-Wen; Hu, Chin-Hwa; Tzou, Wen-Shyong

    2014-01-01

    The liver plays a vital role in metabolism, detoxification, digestion, and the maintenance of homeostasis. During development, the vertebrate embryonic liver undergoes a series of morphogenic processes known as hepatogenesis. Hepatogenesis can be separated into three interrelated processes: endoderm specification, hepatoblast differentiation, and hepatic outgrowth. Throughout this process, signaling molecules and transcription factors initiate and regulate the coordination of cell proliferation, apoptosis, differentiation, intercellular adhesion, and cell migration. Hifs are already recognized to be essential in embryonic development, but their role in hepatogenesis remains unknown. Using the zebrafish embryo as a model organism, we report that the lack of Hif2-alpha but not Hif1-alpha blocks hepatic outgrowth. While Hif2-alpha is not involved in hepatoblast specification, this transcription factor regulates hepatocyte cell proliferation during hepatic outgrowth. Furthermore, we demonstrated that the lack of Hif2-alpha can reduce the expression of liver-enriched gene 1 (leg1), which encodes a secretory protein essential for hepatic outgrowth. Additionally, exogenous mRNA expression of leg1 can rescue the small liver phenotype of hif2-alpha morphants. We also showed that Hif2-alpha directly binds to the promoter region of leg1 to control leg1 expression. Interestingly, we discovered overrepresented, high-density Hif-binding sites in the potential upstream regulatory sequences of leg1 in teleosts but not in terrestrial mammals. We concluded that hif2-alpha is a key factor required for hepatic outgrowth and regulates leg1 expression in zebrafish embryos. We also proposed that the hif2-alpha-leg1 axis in liver development may have resulted from the adaptation of teleosts to their environment. PMID:25000307

  11. The N-terminus region of the putative C2H2 transcription factor Ada1 harbors a species-specific activation motif that regulates asexual reproduction in Fusarium verticillioides.

    PubMed

    Malapi-Wight, Martha; Kim, Jung-Eun; Shim, Won-Bo

    2014-01-01

    Fusarium verticillioides is an important plant pathogenic fungus causing maize ear and stalk rots. In addition, the fungus is directly associated with fumonisin contamination of food and feeds. Here, we report the functional characterization of Ada1, a putative Cys2-His2 zinc finger transcription factor with a high level of similarity to Aspergillus nidulans FlbC, which is required for the activation of the key regulator of conidiation brlA. ADA1 is predicted to encode a protein with two DNA binding motifs at the C terminus and a putative activator domain at the N terminus region. Deletion of the flbC gene in A. nidulans results in "fluffy" cotton-like colonies, with a defect in transition from vegetative growth to asexual development. In this study we show that Ada1 plays a key role in asexual development in F. verticillioides. Conidia production was significantly reduced in the knockout mutant (Δada1), in which aberrant conidia and conidiophores were also observed. We identified genes that are predicted to be downstream of ADA1, based on A. nidulans conidiation signaling pathway. Among them, the deletion of stuA homologue, FvSTUA, resulted in near absence of conidia production. To further investigate the functional conservation of this transcription factor, we complemented the Δada1 strain with A. nidulans flbC, F. verticillioides ADA1, and chimeric constructs. A. nidulans flbC failed to restore conidia production similar to the wild-type level. However, the Ada1N-terminal domain, which contains a putative activator, fused to A. nidulans FlbC C-terminal motif successfully complemented the Δada1 mutant. Taken together, Ada1 is an important transcriptional regulator of asexual development in F. verticillioides and that the N-terminus domain is critical for proper function of this transcription factor.

  12. Regulation of ionizing radiation-induced adhesion of breast cancer cells to fibronectin by alpha5beta1 integrin.

    PubMed

    Lee, Shin Hee; Cheng, Huiwen; Yuan, Ye; Wu, Shiyong

    2014-06-01

    Ionizing radiation (IR) is commonly used for cancer therapy, however, its potential influence on cancer metastatic potential remains controversial. In this study, we elucidated the role of integrins in regulation of IR-altered adhesion between breast cancer cells and extracellular matrix (ECM) proteins, which is a key step in the initial phase of metastasis. Our data suggest that the extent of effect that ionizing radiation had on cell adhesion depended on the genetic background of the breast cancer cells. Ionizing radiation was a better adhesion inducer for p53-mutated cells, such as MDA-MB-231 cells, than for p53 wild-type cells, such as MCF-7 cells. While IR-induced adhesions between MDA-MB-231 cells to fibronectin, laminin, collagen I and collagen IV, only blocking of the adhesion between α5β1 integrin and fibronectin using anti-α5β1 integrin antibody could completely inhibit the radiation-induced adhesion of the cells. A soluble Arg-Gly-Asp peptide, the binding motif for fibronectin binding integrins, could also reduce the adhesion of the cells to fibronectin with or without ionizing radiation exposure. The inhibition of the cell-fibronectin interaction also affected, but did not always correlate with, transwell migration of the cancer cells. In addition, our data showed that the total expression of α5 integrin and surface expression of α5β1 integrin were increased in the cells treated with ionizing radiation. The increased surface expression of α5β1 integrin, along with the adhesion between the cells and fibronectin, could be inhibited by both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) kinase inhibitors. These results suggested that ATM/ATR-mediated surface expression of α5β1 integrin might play a central role in regulation of ionizing radiation-altered adhesion. PMID:24785587

  13. Regulation of vitamin D receptor expression by retinoic acid receptor alpha in acute myeloid leukemia cells.

    PubMed

    Marchwicka, Aleksandra; Cebrat, Małgorzata; Łaszkiewicz, Agnieszka; Śnieżewski, Łukasz; Brown, Geoffrey; Marcinkowska, Ewa

    2016-05-01

    Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D. PMID:26969398

  14. The functional role of the alpha-1 adrenergic receptors in cerebral blood flow regulation.

    PubMed

    Purkayastha, Sushmita; Raven, Peter B

    2011-09-01

    Cerebral vasculature is richly innervated by the α-1 adrenergic receptors similar to that of the peripheral vasculature. However, the functional role of the α-1adrenergic receptors in cerebral blood flow (CBF) regulation is yet to be established. The traditional thinking being that during normotension and normocapnia sympathetic neural activity does not play a significant role in CBF regulation. Reports in the past have stated that catecholamines do not penetrate the blood brain barrier (BBB) and therefore only influence cerebral vessels from outside the BBB and hence, have a limited role in CBF regulation. However, with the advent of dynamic measurement techniques, beat-to-beat CBF assessment can be done during dynamic changes in arterial blood pressure. Several studies in the recent years have reported a functional role of the α-1adrenergic receptors in CBF regulation. This review focuses on the recent developments on the role of the sympathetic nervous system, specifically that of the α-1 adrenergic receptors in CBF regulation.

  15. Regulation of PGE2 signaling pathways and TNF-alpha signaling pathways on the function of bone marrow-derived dendritic cells and the effects of CP-25.

    PubMed

    Li, Ying; Sheng, Kangliang; Chen, Jingyu; Wu, Yujing; Zhang, Feng; Chang, Yan; Wu, Huaxun; Fu, Jingjing; Zhang, Lingling; Wei, Wei

    2015-12-15

    This study was to investigate PGE2 and TNF-alpha signaling pathway involving in the maturation and activation of bone marrow dendritic cells (DCs) and the effect of CP-25. Bone marrow DCs were isolated and stimulated by PGE2 and TNF-alpha respectively. The markers of maturation and activation expressed on DCs, such as CD40, CD80, CD83, CD86, MHC-II, and the ability of antigen uptake of DCs were analyzed by flow cytometry. The proliferation of T cells co-cultured with DCs, the signaling pathways of PGE2-EP4-cAMP and TNF-alpha-TRADD-TRAF2-NF-κB in DCs were analyzed. The results showed that both PGE2 and TNF-alpha up-regulated the expressions of CD40, CD80, CD83, CD86, and MHC-II, decreased the antigen uptake of DCs, and DCs stimulated by PGE2 or TNF-alpha could increase T cell proliferation. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) decreased significantly the expressions of CD40, CD80, CD83, CD86 and MHC-II, increased the antigen uptake of DCs, and suppressed T cell proliferation induced by DCs. PGE2 increased the expressions of EP4, NF-κB and down-regulated cAMP level of DCs. TNF-alpha could also up-regulate TNFR1, TRADD, TRAF2, and NF-κB expression of DCs. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) decreased the expressions of EP4 and NF-κB, increased cAMP level in DCs stimulated by PGE2. CP-25 (10(-5), 10(-6), and 10(-7)mol/l) also could down-regulate significantly TNFR1, TRADD, TRAF2, and NF-κB expression in DCs stimulated by TNF-alpha. These results demonstrate that PGE2 and TNF-alpha could enhance DCs functions by mediating PGE2-EP4-cAMP pathway, TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB pathway respectively. CP-25 might inhibit the function of DCs through regulating PGE2-EP4-cAMP and TNF-alpha-TNFR1-TRADD-TRAF2-NF-κB pathways.

  16. 1. alpha. ,25-dihydroxyvitamin D sub 3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells

    SciTech Connect

    Billecocq, A.; Emanuel, J.R.; Levenson, R.; Baron, R. )

    1990-08-01

    1{alpha},25-Dihydroxyvitamin D{sub 3} (1,25(OH){sub 2}D{sub 3}), the active metabolite of the steroid hormone vitamin D, is a potent regulator of macrophage and osteoclast differentiation. The mature osteoclast, unlike the circulating monocyte or the tissue macrophage, expresses high levels of carbonic anhydrase II (CAII). This enzyme generates protons and bicarbonate from water and carbon dioxide and is involved in bone resorption and acid-base regulation. To test whether 1,25(OH){sub 2}D{sub 3} could induce the differentiation of myelomonocytic precursors toward osteoclasts rather than macrophages, analyzed its effects on the expression of CAII in bone marrow cultures containing precursors common to both cell types. The expression of CAII was markedly increased by 1,25(OH){sub 2}D{sub 3} in a dose-and time-dependent manner. In bone marrow, this increase occurred at the mRNA and protein levels and was detectable as early as 24 hr after stimulation. 1,25(OH){sub 2}D{sub 3} was also found to induce CAII expression in a transformed myelomonocytic avian cell line. These results suggest that 1,25(OH){sub 2}D{sub 3} regulates the level at which myelomonocytic precursors express CAII, an enzyme that is involved in the function of the mature osteoclast.

  17. Cartilage development requires the function of Estrogen-related receptor alpha that directly regulates sox9 expression in zebrafish.

    PubMed

    Kim, Yong-Il; No Lee, Joon; Bhandari, Sushil; Nam, In-Koo; Yoo, Kyeong-Won; Kim, Se-Jin; Oh, Gi-Su; Kim, Hyung-Jin; So, Hong-Seob; Choe, Seong-Kyu; Park, Raekil

    2015-12-10

    Estrogen-related receptor alpha (ESRRa) regulates a number of cellular processes including development of bone and muscles. However, direct evidence regarding its involvement in cartilage development remains elusive. In this report, we establish an in vivo role of Esrra in cartilage development during embryogenesis in zebrafish. Gene expression analysis indicates that esrra is expressed in developing pharyngeal arches where genes necessary for cartilage development are also expressed. Loss of function analysis shows that knockdown of esrra impairs expression of genes including sox9, col2a1, sox5, sox6, runx2 and col10a1 thus induces abnormally formed cartilage in pharyngeal arches. Importantly, we identify putative ESRRa binding elements in upstream regions of sox9 to which ESRRa can directly bind, indicating that Esrra may directly regulate sox9 expression. Accordingly, ectopic expression of sox9 rescues defective formation of cartilage induced by the knockdown of esrra. Taken together, our results indicate for the first time that ESRRa is essential for cartilage development by regulating sox9 expression during vertebrate development.

  18. Cartilage development requires the function of Estrogen-related receptor alpha that directly regulates sox9 expression in zebrafish

    PubMed Central

    Kim, Yong-Il; No Lee, Joon; Bhandari, Sushil; Nam, In-Koo; Yoo, Kyeong-Won; Kim, Se-Jin; Oh, Gi-Su; Kim, Hyung-Jin; So, Hong-Seob; Choe, Seong-Kyu; Park, Raekil

    2015-01-01

    Estrogen-related receptor alpha (ESRRa) regulates a number of cellular processes including development of bone and muscles. However, direct evidence regarding its involvement in cartilage development remains elusive. In this report, we establish an in vivo role of Esrra in cartilage development during embryogenesis in zebrafish. Gene expression analysis indicates that esrra is expressed in developing pharyngeal arches where genes necessary for cartilage development are also expressed. Loss of function analysis shows that knockdown of esrra impairs expression of genes including sox9, col2a1, sox5, sox6, runx2 and col10a1 thus induces abnormally formed cartilage in pharyngeal arches. Importantly, we identify putative ESRRa binding elements in upstream regions of sox9 to which ESRRa can directly bind, indicating that Esrra may directly regulate sox9 expression. Accordingly, ectopic expression of sox9 rescues defective formation of cartilage induced by the knockdown of esrra. Taken together, our results indicate for the first time that ESRRa is essential for cartilage development by regulating sox9 expression during vertebrate development. PMID:26657540

  19. Nucleotide sequence analysis of HLA-B*1523 and B*8101. Dominant alpha-helical motifs produce complex serologic recognition patterns for the HLA-B"DT" and HLA-B"NM5" antigens.

    PubMed

    Ellexson, M E; Zhang, G; Stewart, D; Lau, M; Teresi, G; Terasaki, P; Roe, B; Hildebrand, W

    1995-10-01

    Assigning a precise serologic specificity to the class I HLA-B"NM5" and HLA-B"DT" molecules has proven difficult, with patterns of serologic cross-reactivity suggesting that NM5 is most like antigens in the B5 CREG and that DT is either B7 or B40 like. To better understand the relationship these antigens share with other HLA-B molecules we determined the nucleotide sequence of the alleles encoding HLA-B"NM5" and HLA-B"DT". Sequencing results show that NM5 shares the most overall sequence homology with the B70 antigens and that differences at the alpha-helical Bw4/Bw6 epitope preclude serologic cross-reactivity between NM5 and the B70 antigens. Accordingly, NM5 has been assigned the name B*1523. The strong serologic impact of helical sequence conservations and variations is reiterated for the class I HLA-B"DT" molecule. Comparative analysis demonstrates that sequence conservations in the first domain's alpha-helix stimulate cross-reactivity between HLA-B"DT" and HLA-B7, whereas epitopes conserved in the second domain's alpha-helix impel cross-reactivity between HLA-B"DT" and HLA-B48. To convey the unique lineage of this hybrid B7/B48 molecule the name HLA-B*8101 has been assigned to HLA-B"DT".

  20. De Novo Regulatory Motif Discovery Identifies Significant Motifs in Promoters of Five Classes of Plant Dehydrin Genes

    PubMed Central

    Zolotarov, Yevgen; Strömvik, Martina

    2015-01-01

    Plants accumulate dehydrins in response to osmotic stresses. Dehydrins are divided into five different classes, which are thought to be regulated in different manners. To better understand differences in transcriptional regulation of the five dehydrin classes, de novo motif discovery was performed on 350 dehydrin promoter sequences from a total of 51 plant genomes. Overrepresented motifs were identified in the promoters of five dehydrin classes. The Kn dehydrin promoters contain motifs linked with meristem specific expression, as well as motifs linked with cold/dehydration and abscisic acid response. KS dehydrin promoters contain a motif with a GATA core. SKn and YnSKn dehydrin promoters contain motifs that match elements connected with cold/dehydration, abscisic acid and light response. YnKn dehydrin promoters contain motifs that match abscisic acid and light response elements, but not cold/dehydration response elements. Conserved promoter motifs are present in the dehydrin classes and across different plant lineages, indicating that dehydrin gene regulation is likely also conserved. PMID:26114291

  1. TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k

    NASA Technical Reports Server (NTRS)

    Li, Yi-Ping; Lecker, Stewart H.; Chen, Yuling; Waddell, Ian D.; Goldberg, Alfred L.; Reid, Michael B.

    2003-01-01

    In some inflammatory diseases, TNF-alpha is thought to stimulate muscle catabolism via an NF-kappaB-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF-alpha stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-kappaB binding site; NF-kappaB binding to this sequence is increased by TNF-alpha stimulation. A dominant negative inhibitor of NF-kappaB activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-alpha. In extracts from TNF-alpha-treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-alpha/NF-kappaB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.

  2. Estrogen Receptor Alpha (ESR1)-Dependent Regulation of the Mouse Oviductal Transcriptome.

    PubMed

    Cerny, Katheryn L; Ribeiro, Rosanne A C; Jeoung, Myoungkun; Ko, CheMyong; Bridges, Phillip J

    2016-01-01

    Estrogen receptor-α (ESR1) is an important transcriptional regulator in the mammalian oviduct, however ESR1-dependent regulation of the transcriptome of this organ is not well defined, especially at the genomic level. The objective of this study was therefore to investigate estradiol- and ESR1-dependent regulation of the transcriptome of the oviduct using transgenic mice, both with (ESR1KO) and without (wild-type, WT) a global deletion of ESR1. Oviducts were collected from ESR1KO and WT littermates at 23 days of age, or ESR1KO and WT mice were treated with 5 IU PMSG to stimulate follicular development and the production of ovarian estradiol, and the oviducts collected 48 h later. RNA extracted from whole oviducts was hybridized to Affymetrix Genechip Mouse Genome 430-2.0 arrays (n = 3 arrays per genotype and treatment) or reverse transcribed to cDNA for analysis of the expression of selected mRNAs by real-time PCR. Following microarray analysis, a statistical two-way ANOVA and pairwise comparison (LSD test) revealed 2428 differentially expressed transcripts (DEG's, P < 0.01). Genotype affected the expression of 2215 genes, treatment (PMSG) affected the expression of 465 genes, and genotype x treatment affected the expression of 438 genes. With the goal of determining estradiol/ESR1-regulated function, gene ontology (GO) and bioinformatic pathway analyses were performed on DEG's in the oviducts of PMSG-treated ESR1KO versus PMSG-treated WT mice. Significantly enriched GO molecular function categories included binding and catalytic activity. Significantly enriched GO cellular component categories indicated the extracellular region. Significantly enriched GO biological process categories involved a single organism, modulation of a measurable attribute and developmental processes. Bioinformatic analysis revealed ESR1-regulation of the immune response within the oviduct as the primary canonical pathway. In summary, a transcriptomal profile of estradiol- and ESR1

  3. Effect of some plant growth regulators on lindane and alpha-endosulfan toxicity to Brassica chinensis.

    PubMed

    Chouychai, Waraporn

    2012-07-01

    The effect of indolebutyric acid (IBA) and gibberellic acid (GA3), to alleviate the organochlorine phytotoxicity were studied in Brassica chinensis. Presence of organochlorine decreased Brassica chinensis seedlings growth in contaminated alkaline soil. One mg l(-1) IBA could enhance 14 and 26% shoot and root length of B. chinensis seedlings grown at 40 mg kg(-1) lindane contaminated soil, respectively. Ten mg l(-1) IBA also increased 80 and 40% root fresh weight of seedling grown in 40 mg kg(-1) lindane and alpha-endosulfan contaminated soils, respectively. However, IBAhad no effect on shoot and root length of seedlings grown in endosulfan contaminated soil. On the other hand, 10 mg l(-1) GA3 only increased 80% of shoot and root fresh weigh of B. chinensisin 40 mg kg(-1) endosulfan contaminated soil. External auxin addition could increase B. chinensis growth in lindane more than endosulfan contaminated soil. External gibberellin was less effective than external auxin to increase B. chinensis growth in organochlorine contaminated soil. There is possibility that auxin could decrease organochlorine phytotoxicity in plants and hence can be useful for organochlorine phytoremediation.

  4. The psmα locus regulates production of Staphylococcus aureus alpha-toxin during infection.

    PubMed

    Berube, Bryan J; Sampedro, Georgia R; Otto, Michael; Bubeck Wardenburg, Juliane

    2014-08-01

    Staphylococcus aureus is a leading cause of human bacterial infection, causing a wide spectrum of disease ranging from skin and soft tissue infections to life-threatening pneumonia and sepsis. S. aureus toxins play an essential role in disease pathogenesis, contributing to both immunomodulation and host tissue injury. Prominent among these toxins are the membrane-active pore-forming cytolysin alpha-toxin (Hla) and the amphipathic α-helical phenol-soluble modulin (PSM) peptides. As deletion of either the hla or psm locus leads to a phenotypically similar virulence defect in skin and soft tissue infection, we sought to determine the relative contribution of each locus to disease pathogenesis. Here we show that production of Hla can be modulated by PSM expression. An S. aureus mutant lacking PSM expression exhibits a transcriptional delay in hla mRNA production and therefore fails to secrete normal levels of Hla at early phases of growth. This leads to attenuation of virulence in vitro and in murine skin and lung models of infection, correlating with reduced recovery of Hla from host tissues. Production of Hla and restoration of staphylococcal virulence can be achieved in the psm mutant by plasmid-driven overexpression of hla. Our study suggests the coordinated action of Hla and PSMs in host tissue during early pathogenesis, confirming a major role for Hla in epithelial injury during S. aureus infection. These findings highlight the possibility that therapeutics targeting PSM production may simultaneously prevent Hla-mediated tissue injury.

  5. Effect of some plant growth regulators on lindane and alpha-endosulfan toxicity to Brassica chinensis.

    PubMed

    Chouychai, Waraporn

    2012-07-01

    The effect of indolebutyric acid (IBA) and gibberellic acid (GA3), to alleviate the organochlorine phytotoxicity were studied in Brassica chinensis. Presence of organochlorine decreased Brassica chinensis seedlings growth in contaminated alkaline soil. One mg l(-1) IBA could enhance 14 and 26% shoot and root length of B. chinensis seedlings grown at 40 mg kg(-1) lindane contaminated soil, respectively. Ten mg l(-1) IBA also increased 80 and 40% root fresh weight of seedling grown in 40 mg kg(-1) lindane and alpha-endosulfan contaminated soils, respectively. However, IBAhad no effect on shoot and root length of seedlings grown in endosulfan contaminated soil. On the other hand, 10 mg l(-1) GA3 only increased 80% of shoot and root fresh weigh of B. chinensisin 40 mg kg(-1) endosulfan contaminated soil. External auxin addition could increase B. chinensis growth in lindane more than endosulfan contaminated soil. External gibberellin was less effective than external auxin to increase B. chinensis growth in organochlorine contaminated soil. There is possibility that auxin could decrease organochlorine phytotoxicity in plants and hence can be useful for organochlorine phytoremediation. PMID:23360012

  6. Regulated alpha-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping.

    PubMed

    Rodgers, Nancy D; Wang, Zuoren; Kiledjian, Megerditch

    2002-12-01

    The alpha-globin mRNA contains a C-rich stability element (CRE) in its 3' untranslated region (3' UTR) which is critical for the stability of this long-lived mRNA. A protein complex, termed the alpha-complex, forms on the CRE and has been shown to contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-binding protein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribonuclease. In this report, we demonstrate that the alpha-globin 3' UTR can confer stability on a heterologous mRNA in cells, and this stability is dependent on the alpha-complex. Moreover, the stability was exclusively detected with cytoplasmic mRNA, suggesting that the regulation of alpha-globin mRNA stability is a cytoplasmic event. An additional mechanism by which the alpha-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3' to 5' exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow the demise of the alpha-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3'-to-5' decay carried out by the exosome and ultimately hydrolysis of the residual cap structure.

  7. Tumour necrosis factor-alpha up-regulates the expression of BMP-4 mRNA but inhibits chondrogenesis in mouse clonal chondrogenic EC cells, ATDC5.

    PubMed

    Horiguchi, M; Akiyama, H; Ito, H; Shigeno, C; Nakamura, T

    2000-05-01

    Tumour necrosis factor (TNF)-alpha causes the degradation of articular cartilage in arthritis via direct actions on chondrocytes. However, it remains unknown whether TNF-alpha affects chondrogenesis in chondroprogenitors. In the present study, we assessed the effects of TNF-alpha in vitro on chondrogenesis using mouse clonal chondrogenic EC cells, ATDC5. TNF-alpha (10 ng/ml) stimulated [3H] thymidine incorporation in undifferentiated ATDC5 cells, and suppressed cartilaginous nodule formation and the accumulation of cartilage-specific proteoglycan. We recently showed that undifferentiated ATDC5 cells express BMP-4 and that exogenously administered BMP-4 promotes chondrogenesis in these cells. Interestingly, TNF-alpha up-regulated the expression of BMP-4 mRNA in undifferentiated ATDC5 cells in time- and dose-dependent manners. However, exogenously administered BMP-4 was not capable of reversing the inhibitory action of TNF-alpha on chondrogenesis in ATDC5 cells. These results indicate that TNF-alpha stimulates both cell proliferation and BMP-4 expression but inhibits chondrogenesis in chondroprogenitor-like ATDC5 cells. PMID:10857772

  8. Evidence for a role of the (alpha)-tubulin C terminus in the regulation of cyclin B synthesis in developing oocytes.

    PubMed

    Vée, S; Lafanechère, L; Fisher, D; Wehland, J; Job, D; Picard, A

    2001-03-01

    Microinjected mAb YL1/2, an (alpha)-tubulin antibody specific for the tyrosinated form of the protein, blocks the cell cycle in developing oocytes. Here, we have investigated the mechanism involved in the mAb effect. Both developing starfish and Xenopus oocytes were injected with two different (alpha)-tubulin C terminus antibodies. The injected antibodies blocked cell entry into mitosis through specific inhibition of cyclin B synthesis. The antibody effect was independent of the presence or absence of polymerized microtubules and was mimicked by injected synthetic peptides corresponding to the tyrosinated (alpha)-tubulin C terminus, whereas peptides lacking the terminal tyrosine were ineffective. These results indicate that tyrosinated (alpha)-tubulin, or another protein sharing the same C-terminal epitope, is involved in specific regulation of cyclin B synthesis in developing oocytes.

  9. Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids.

    PubMed Central

    Larsen, M H; Figurski, D H

    1994-01-01

    The kil-kor regulon was first identified on the broad-host-range IncP alpha plasmid RK2 by the presence of multiple kil loci (kilA, kilB, kilC, and recently kilE) that are lethal to Escherichia coli host cells in the absence of regulation by kor functions in various combinations. Whereas the kilB operon is required for mating-pair formation during conjugation, the functions encoded by the other kil loci are not known. They are not essential for replication or conjugal transfer, but their coregulation with replication and transfer genes indicates that they are likely to be important for RK2. In this report, we describe molecular and genetic studies on kilC. We determined the nucleotide sequence of the kilC region, which is located between the origin of vegetative replication (oriV) and transposon Tn1 on RK2. Primer extension analysis identified the transcriptional start site and showed that a sequence corresponding to a strong sigma 70 promoter is functional. The abundance of RNA initiated from the kilC promoter is reduced in the presence of korA and korC, as predicted from genetic analysis of kilC regulation. The first gene of the kilC operon (klcA) is sufficient to express the host-lethal phenotype of the kilC determinant in the absence of korA and korC. By comparing RK2 to the related IncP alpha plasmids pUZ8 and R995, we determined that the Tn1 transposon in RK2 interrupts a gene (klcB) immediately downstream of klcA. Thus, the kilC determinant is normally part of an autoregulated operon of three genes: klcA, klcB, and korC. klcA is predicted to encode a 15,856-Da polypeptide that is related to the ArdB antirestriction protein of the IncN plasmid pKM101, suggesting a role for klcA in the broad host ranges of IncP alpha plasmids. The predicted product of the uninterrupted klcB gene is a polypeptide of 51,133 Da that contains a segment with significant similarity to the RK2 regulatory proteins KorA and TrbA. Located 145 bp upstream of the kilC promoter is a 10th

  10. Sympathetic alpha-adrenergic regulation of blood flow and volume in hamsters arousing from hibernation.

    PubMed

    Osborne, P G; Sato, J; Shuke, N; Hashimoto, M

    2005-08-01

    Mammals arousing from hibernation display pronounced regional heterothermy, where the thoracic and head regions warm faster than the abdominal and hindlimb regions. We used laser-Doppler flowmetry to measure peripheral hind foot blood flow during hibernation and arousal and gamma imaging of technetium-labeled albumin to measure whole blood volume distribution in hamsters arousing from hibernation. It was discovered that the hibernating hamster responds to physical but not to sound or hypercapnic stimulation with rapid, 73% reduction of hind foot blood flow. Hind foot blood flow vasoconstriction was maintained from the onset of arousal until late in arousal when rectal temperature was rapidly increased. alpha-Adrenergic blockade early in arousal increased hind foot blood flow by 700%, suggesting that vasoconstriction was mediated by activation of sympathetic tone. Gamma imaging revealed that, by the early phase of arousal from hibernation, the blood volume of the body below the liver is greatly reduced, whereas blood volumes of the thorax and head are much greater than corresponding volumes in anesthetized hamsters. As arousal progresses and cardiac activity increases and regional heterothermy develops, this regional blood volume distribution is largely maintained; however, blood volume slowly decreases in the thoracic region and slowly increases in the shoulder and head regions. The rapid increase in rectal temperature, characteristic of mid- to late- arousal phases, is probably mediated, in part, by reduction of adrenergic tone on abdominal and hindlimb vasculature. Warm blood then moves into the hind body, produces an increase in temperature, blood flow, and blood volume in the hind body and compensatory reductions of blood volume in the neck, head, and thoracic regions.

  11. Alpha-arrestins Aly1 and Aly2 regulate intracellular trafficking in response to nutrient signaling.

    PubMed

    O'Donnell, Allyson F; Apffel, Alex; Gardner, Richard G; Cyert, Martha S

    2010-10-15

    Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. β-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein-coupled receptors. In contrast, we show that yeast α-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of α-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the γ-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how α-arrestins coordinate signaling events with protein trafficking.

  12. Regulation of Rnd3 localization and function by protein kinase C alpha-mediated phosphorylation.

    PubMed

    Madigan, James P; Bodemann, Brian O; Brady, Donita C; Dewar, Brian J; Keller, Patricia J; Leitges, Michael; Philips, Mark R; Ridley, Anne J; Der, Channing J; Cox, Adrienne D

    2009-11-15

    The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by post-translational modifications such as prenylation and phosphorylation. We show in the present study that, upon PKC (protein kinase C) agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCalpha-null cells or to a non-phosphorylatable mutant of Rnd3. We further show that PKCalpha directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signalling from the Rho-ROCK (Rho-kinase) pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signalling to alter cytoskeletal organization.

  13. CD36 is essential for regulation of the host innate response to Staphylococcus aureus alpha-toxin-mediated dermonecrosis

    PubMed Central

    Castleman, Moriah J.; Febbraio, Maria; Hall, Pamela R.

    2015-01-01

    Staphylococcus aureus is the primary cause of skin and skin structure infections (SSSI) in the USA. Alpha-hemolysin (Hla), a pore-forming toxin secreted by S. aureus and a major contributor to tissue necrosis, prompts recruitment of neutrophils critical for host defense against S. aureus infections. However, the failure to clear apoptotic neutrophils can result in damage to host tissues, suggesting that mechanisms of neutrophil clearance are essential to limiting Hla-mediated dermonecrosis. We hypothesized that CD36, a scavenger receptor which facilitates recognition of apoptosing cells, would play a significant role in regulating Hla-mediated inflammation and tissue injury during S. aureus SSSI. Here we show that CD36 on macrophages negatively regulates dermonecrosis caused by Hla-producing S. aureus. This regulation is independent of bacterial burden, as CD36 also limits dermonecrosis caused by intoxication with sterile bacterial supernatant or purified Hla. Dermonecrotic lesions of supernatant intoxicated CD36−/− mice are significantly larger, with increased neutrophil accumulation and IL-1β expression, compared to CD36+/+ (wild-type) mice. Neutrophil depletion of CD36−/− mice prevents this phenotype, demonstrating the contribution of neutrophils to tissue injury in this model. Furthermore, administration of CD36+/+, but not CD36−/−, macrophages near the site of intoxication reduces dermonecrosis, IL-1β production and neutrophil accumulation to levels seen in wild-type mice. This therapeutic effect is reversed by inhibiting actin polymerization in the CD36+/+ macrophages, supporting a mechanism of action whereby CD36-dependent macrophage phagocytosis of apoptotic neutrophils regulates Hla-mediated dermonecrosis. Together, these data demonstrate that CD36 is essential for controlling the host innate response to S. aureus skin infection. PMID:26223653

  14. The β-lactamase gene regulator AmpR is a tetramer that recognizes and binds the D-Ala-D-Ala motif of its repressor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide.

    PubMed

    Vadlamani, Grishma; Thomas, Misty D; Patel, Trushar R; Donald, Lynda J; Reeve, Thomas M; Stetefeld, Jörg; Standing, Kenneth G; Vocadlo, David J; Mark, Brian L

    2015-01-30

    Inducible expression of chromosomal AmpC β-lactamase is a major cause of β-lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to β-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide. When exposed to β-lactams, however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAc-pentapeptide from AmpR and convert it into an activator of ampC transcription. Here we describe the molecular interactions between AmpR (from Citrobacter freundii), its DNA operator, and repressor UDP-MurNAc-pentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N11-A LTTR binding motif and revealed that it can bind four repressor molecules in an apparently stepwise manner. A crystal structure of the AmpR effector-binding domain bound to UDP-MurNAc-pentapeptide revealed that the terminal D-Ala-D-Ala motif of the repressor forms the primary contacts with the protein. This observation suggests that 1,6-anhydroMurNAc-pentapeptide may convert AmpR into an activator of ampC transcription more effectively than 1,6-anhydroMurNAc-tripeptide (which lacks the D-Ala-D-Ala motif). Finally, small angle x-ray scattering demonstrates that the AmpR·DNA complex adopts a flat conformation similar to the LTTR protein AphB and undergoes only a slight conformational change when binding UDP-MurNAc-pentapeptide. Modeling the AmpR·DNA tetramer bound to UDP-MurNAc-pentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function. PMID:25480792

  15. The β-Lactamase Gene Regulator AmpR Is a Tetramer That Recognizes and Binds the d-Ala-d-Ala Motif of Its Repressor UDP-N-acetylmuramic Acid (MurNAc)-pentapeptide*

    PubMed Central

    Vadlamani, Grishma; Thomas, Misty D.; Patel, Trushar R.; Donald, Lynda J.; Reeve, Thomas M.; Stetefeld, Jörg; Standing, Kenneth G.; Vocadlo, David J.; Mark, Brian L.

    2015-01-01

    Inducible expression of chromosomal AmpC β-lactamase is a major cause of β-lactam antibiotic resistance in the Gram-negative bacteria Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is induced by the LysR-type transcriptional regulator (LTTR) AmpR, which activates ampC expression in response to changes in peptidoglycan (PG) metabolite levels that occur during exposure to β-lactams. Under normal conditions, AmpR represses ampC transcription by binding the PG precursor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide. When exposed to β-lactams, however, PG catabolites (1,6-anhydroMurNAc-peptides) accumulate in the cytosol, which have been proposed to competitively displace UDP-MurNAc-pentapeptide from AmpR and convert it into an activator of ampC transcription. Here we describe the molecular interactions between AmpR (from Citrobacter freundii), its DNA operator, and repressor UDP-MurNAc-pentapeptide. Non-denaturing mass spectrometry revealed AmpR to be a homotetramer that is stabilized by DNA containing the T-N11-A LTTR binding motif and revealed that it can bind four repressor molecules in an apparently stepwise manner. A crystal structure of the AmpR effector-binding domain bound to UDP-MurNAc-pentapeptide revealed that the terminal d-Ala-d-Ala motif of the repressor forms the primary contacts with the protein. This observation suggests that 1,6-anhydroMurNAc-pentapeptide may convert AmpR into an activator of ampC transcription more effectively than 1,6-anhydroMurNAc-tripeptide (which lacks the d-Ala-d-Ala motif). Finally, small angle x-ray scattering demonstrates that the AmpR·DNA complex adopts a flat conformation similar to the LTTR protein AphB and undergoes only a slight conformational change when binding UDP-MurNAc-pentapeptide. Modeling the AmpR·DNA tetramer bound to UDP-MurNAc-pentapeptide predicts that the UDP-MurNAc moiety of the repressor participates in modulating AmpR function. PMID:25480792

  16. Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

    SciTech Connect

    Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  17. Characterization of the Promoter Motif Regulated by PSPTO_1209 a FecI-like ECF Sigma Factor of Pseudomonas syringae pv. tomato DC3000

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomanads are renowned for their capacity to adapt to diverse environments, a fact that is reflected in the fraction of their genomes dedicated to encoding transcription regulators. Members of the Pseudomonas genus include species that are adapted to pathogenic and symbiotic lifestyles in associ...

  18. microRNA-155 Regulates Alpha-Synuclein-Induced Inflammatory Responses in Models of Parkinson Disease

    PubMed Central

    Thome, Aaron D.; Harms, Ashley S.; Volpicelli-Daley, Laura A.

    2016-01-01

    Increasing evidence points to inflammation as a chief mediator of Parkinson's disease (PD), a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein α-synuclein (α-syn). Recently, microRNAs, small, noncoding RNAs involved in regulating gene expression at the posttranscriptional level, have been recognized as important regulators of the inflammatory environment. Using an array approach, we found significant upregulation of microRNA-155 (miR-155) in an in vivo model of PD produced by adeno-associated-virus-mediated expression of α-syn. Using a mouse with a complete deletion of miR-155, we found that loss of miR-155 reduced proinflammatory responses to α-syn and blocked α-syn-induced neurodegeneration. In primary microglia from miR-155−/− mice, we observed a markedly reduced inflammatory response to α-syn fibrils, with attenuation of major histocompatibility complex class II (MHCII) and proinflammatory inducible nitric oxide synthase expression. Treatment of these microglia with a synthetic mimic of miR-155 restored the inflammatory response to α-syn fibrils. Our results suggest that miR-155 has a central role in the inflammatory response to α-syn in the brain and in α-syn-related neurodegeneration. These effects are at least in part due to a direct role of miR-155 on the microglial response to α-syn. These data implicate miR-155 as a potential therapeutic target for regulating the inflammatory response in PD. SIGNIFICANCE STATEMENT The main feature associated with Parkinson's disease (PD) is the accumulation of α-synuclein in the brain accompanied by signs of inflammation and immune activation. Our studies suggest that microRNA-155 is a key inflammation-initiating molecule that could be a viable target for PD therapeutics. PMID:26911687

  19. microRNA-155 Regulates Alpha-Synuclein-Induced Inflammatory Responses in Models of Parkinson Disease.

    PubMed

    Thome, Aaron D; Harms, Ashley S; Volpicelli-Daley, Laura A; Standaert, David G

    2016-02-24

    Increasing evidence points to inflammation as a chief mediator of Parkinson's disease (PD), a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein α-synuclein (α-syn). Recently, microRNAs, small, noncoding RNAs involved in regulating gene expression at the posttranscriptional level, have been recognized as important regulators of the inflammatory environment. Using an array approach, we found significant upregulation of microRNA-155 (miR-155) in an in vivo model of PD produced by adeno-associated-virus-mediated expression of α-syn. Using a mouse with a complete deletion of miR-155, we found that loss of miR-155 reduced proinflammatory responses to α-syn and blocked α-syn-induced neurodegeneration. In primary microglia from miR-155(-/-) mice, we observed a markedly reduced inflammatory response to α-syn fibrils, with attenuation of major histocompatibility complex class II (MHCII) and proinflammatory inducible nitric oxide synthase expression. Treatment of these microglia with a synthetic mimic of miR-155 restored the inflammatory response to α-syn fibrils. Our results suggest that miR-155 has a central role in the inflammatory response to α-syn in the brain and in α-syn-related neurodegeneration. These effects are at least in part due to a direct role of miR-155 on the microglial response to α-syn. These data implicate miR-155 as a potential therapeutic target for regulating the inflammatory response in PD.

  20. 5'-AMP-activated protein kinase alpha regulates stress granule biogenesis.

    PubMed

    Mahboubi, Hicham; Barisé, Ramla; Stochaj, Ursula

    2015-07-01

    Stress granule (SG) assembly represents a conserved eukaryotic defense strategy against various insults. Although essential for the ability to cope with deleterious conditions, the signaling pathways controlling SG formation are not fully understood. The energy sensor AMP-activated protein kinase (AMPK) is critical for the cellular stress response. Human cells produce two AMPK catalytic α-subunits with not only partially overlapping, but also unique functions. Here, we provide direct support for structural and functional links between AMPK-α isoforms and SGs. As such, several stressors promote SG association of AMPK-α2, but not AMPK-α1. Multiple lines of evidence link AMPK activity to SG biogenesis. First, pharmacological kinase inhibition interfered with SG formation. Second, AMPK-α knockdown combined with in-depth quantitative SG analysis revealed isoform-specific changes of SG characteristics. Third, overexpression of mutant α-subunits further substantiated that AMPK regulates SG parameters. Finally, we identified the SG-nucleating protein G3BP1 as an AMPK-α2 binding partner. This interaction is stimulated by stress and notably occurs in SGs. Collectively, our data define the master metabolic regulator AMPK as a novel SG constituent that also controls their biogenesis.

  1. Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNF{alpha}-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

    SciTech Connect

    Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu; Potts, Jay D.; Tang, Dong-Qi; Li, Dong-Sheng; Cui, Taixing

    2010-01-01

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

  2. Transforming growth factor alpha may be a physiological regulator of liver regeneration by means of an autocrine mechanism.

    PubMed Central

    Mead, J E; Fausto, N

    1989-01-01

    We investigated whether transforming growth factor alpha (TGF-alpha) is involved in hepatocyte growth responses both in vivo and in culture. During liver regeneration after partial hepatectomy in rats, TGF-alpha mRNA increased; it reached a maximum (approximately 9-fold higher than normal) at the peak of DNA synthesis. The message and the peptide were localized in hepatocytes and found in higher amounts in hepatocytes obtained from regenerating liver. TGF-alpha caused a 13-fold elevation of DNA synthesis in hepatocytes in primary culture and was slightly more effective than epidermal growth factor. TGF-beta blocked TGF-alpha stimulation when added either simultaneously with TGF-alpha or a day later. TGF-alpha message increased in hepatocytes stimulated to undergo DNA synthesis by TGF-alpha or epidermal growth factor, and the peptide was detected in the culture medium by RIA. In the regenerating liver, the increase in TGF-alpha mRNA during the first day after partial hepatectomy coincided with an increase in epidermal growth factor/TGF-alpha receptor mRNA and a decrease (already reported) in the number of these receptors. We conclude that TGF-alpha may function as a physiological inducer of hepatocyte DNA synthesis during liver regeneration by means of an autocrine mechanism and that its stimulatory effects in this growth process are balanced by the inhibitory action of TGF-beta 1. Images PMID:2922399

  3. Identification of the human ApoAV gene as a novel ROR{alpha} target gene

    SciTech Connect

    Lind, Ulrika; Nilsson, Tina; McPheat, Jane; Stroemstedt, Per-Erik; Bamberg, Krister; Balendran, Clare; Kang, Daiwu . E-mail: Daiwu.Kang@astrazeneca.com

    2005-04-29

    Retinoic acid receptor-related orphan receptor-{alpha} (ROR{alpha}) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that ROR{alpha} regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that ROR{alpha} also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of ROR{alpha} increased the endogenous expression of ApoAV in HepG2 cells and ROR{alpha} also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate ROR{alpha} transactivation, one of which overlaps with a previously identified binding site for PPAR{alpha}. Together, these results suggest a novel mechanism whereby ROR{alpha} modulates lipid metabolism and implies ROR{alpha} as a potential target for the treatment of dyslipidemia and atherosclerosis.

  4. Regulation of the alpha-fetoprotein promoter: Ku binding and DNA spatial conformation.

    PubMed

    Liénard, P; De Mees, C; Drèze, P-L; Dieu, M; Dierick, J-F; Raes, M; Szpirer, J; Szpirer, C

    2006-10-01

    This work shows that the proximal promoter of the mouse Afp gene contains a Ku binding site and that Ku binding is associated with down-regulation of the transcriptional activity of the Afp promoter. The Ku binding site is located in a segment able to adopt a peculiar structured form, probably a hairpin structure. Interestingly, the structured form eliminates the binding sites of the positive transcription factor HNF1. Furthermore, a DNAse hypersensitive site is detected in footprinting experiments done with extracts of AFP non-expressing hepatoma cells. These observations suggest that the structured form is stabilised by Ku and is associated with extinction of the gene in AFP non-expressing hepatic cells.

  5. Self-regulating galaxy formation. Part 1: HII disk and Lyman alpha pressure

    NASA Technical Reports Server (NTRS)

    Cox, D. P.

    1983-01-01

    Assuming a simple but physically based prototype for behavior of interstellar material during formation of a disk galaxy, coupled with the lowest order description of infall, a scenario is developed for self-regulated disk galaxy formation. Radiation pressure, particularly that of Lyman depha (from fluorescence conversion Lyman continuum), is an essential component, maintaining an inflated disk and stopping infall when only a small fraction of the overall perturbation has joined the disk. The resulting galaxies consist of a two dimensional family whose typical scales and surface density are expressable in terms of fundamental constants. The model leads naturally to galaxies with a rich circumgalactic environment and flat rotation curves (but is weak in its analysis of the subsequent evolution of halo material).

  6. Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking

    PubMed Central

    Barthel, Steven R.; Wiese, Georg K.; Cho, Jaehyung; Opperman, Matthew J.; Hays, Danielle L.; Siddiqui, Javed; Pienta, Kenneth J.; Furie, Bruce; Dimitroff, Charles J.

    2009-01-01

    How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLeX) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLeX and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that α1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLeX and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that α1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism. PMID:19889975

  7. Down-regulation of malignant potential by alpha linolenic acid in human and mouse colon cancer cells.

    PubMed

    Chamberland, John P; Moon, Hyun-Seuk

    2015-03-01

    Omega-3 fatty acids (also called ω-3 fatty acis or n-3 fatty acid) are polyunsaturated fatty acids (PUFAs) with a double bond (C=C) at the third carbon atom from the end of the carbon chain. Numerous test tube and animal studies have shown that omega-3 fatty acids may prevent or inhibit the growth of cancers, suggesting that omega-3 fatty acids are important in cancer physiology. Alpha-linolenic acid (ALA) is one of an essential omega-3 fatty acid and organic compound found in seeds (chia and flaxseed), nuts (notably walnuts), and many common vegetable oils. ALA has also been shown to down-regulate cell proliferation of prostate, breast, and bladder cancer cells. However, direct evidence that ALA suppresses to the development of colon cancer has not been studied. Also, no previous studies have evaluated whether ALA may regulate malignant potential (adhesion, invasion and colony formation) in colon cancer cells. In order to address the questions above, we conducted in vitro studies and evaluated whether ALA may down-regulate malignant potential in human (HT29 and HCT116) and mouse (MCA38) colon cancer cell lines. We observed that treatment with 1-5 mM of ALA inhibits cell proliferation, adhesion and invasion in both human and mouse colon cancer cell lines. Interestingly, we observed that ALA did not decrease total colony numbers when compared to control. By contrast, we found that size of colony was significantly changed by ALA treatment when compared to control in all colon cancer cell lines. We suggest that our data enhance our current knowledge of ALA's mechanism and provide crucial information to further the development of new therapies for the management or chemoprevention of colon cancer.

  8. Dietary fish oil up-regulates cholesterol 7alpha-hydroxylase mRNA in mouse liver leading to an increase in bile acid and cholesterol excretion.

    PubMed

    Bérard, Annie M; Dumon, Marie-France; Darmon, Michel

    2004-02-13

    To investigate the molecular events controlling reverse cholesterol transport, we compared gene expression of normal mouse liver to that of mice fed a long chain (LC) omega-3 fatty acid-enriched diet. Using cDNA microarrays, we assessed expression levels of 1176 genes, and we found that D-site binding protein (DBP) was three-fold increased in mice on a LC omega-3 fatty acid-rich diet compared to controls. DBP is known to increase transcriptional level of cholesterol 7alpha-hydroxylase (C7alpha), the rate-limiting enzyme for bile acid production and cholesterol excretion, and we found that C7alpha mRNA was also up-regulated by LC omega-3 fatty acids. Moreover, liver X receptor-alpha, another transcription factor up-regulating C7alpha, was three- to four-fold increased in liver of treated mice. On the other hand, we demonstrated that bile acid and cholesterol excretion were two-fold increased. These results show that LC omega-3 fatty acids control cholesterol metabolism in mice at a new endpoint.

  9. Identification and characterization of cis elements in the STAT3 gene regulating STAT3 alpha and STAT3 beta messenger RNA splicing.

    PubMed

    Shao, H; Quintero, A J; Tweardy, D J

    2001-12-15

    Signal transducer and activator of transcription 3 (STAT3) is an oncogene and a critical regulator of multiple cell-fate decisions, including myeloid cell differentiation. Two isoforms of STAT3 have been identified: alpha (p92) and beta (p83). These differ structurally in their C-terminal transactivation domains, resulting in distinct functional activities. The cis genetic elements that regulate the ratio of alpha to beta messenger RNA (mRNA) are unknown. In this study, cloning, sequencing, and splicing analysis of the human and murine STAT3 genes revealed a highly conserved 5' donor site for generation of both alpha and beta mRNA and distinct branch-point sequences, polypyrimidine tracts, and 3' acceptor sites (ASs) for each. The beta 3' AS was found to be located 50 nucleotides downstream of the alpha 3' AS in exon 23. Two additional cryptic 3' ASs (delta and epsilon) were also identified. Thus, we identified for the first time the cis regulatory sequences responsible for generation of STAT3 alpha and STAT3 beta mRNA.

  10. TNF-alpha-mediates neuroprotection against glutamate-induced excitotoxicity via NF-kappaB-dependent up-regulation of K2.2 channels.

    PubMed

    Dolga, Amalia M; Granic, Ivica; Blank, Thomas; Knaus, Hans-Guenther; Spiess, Joachim; Luiten, Paul G M; Eisel, Ulrich L M; Nijholt, Ingrid M

    2008-11-01

    Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) induces neuroprotection against excitotoxic damage in primary cortical neurons via sustained nuclear factor-kappa B (NF-kappaB) activation. The transcription factor NF-kappaB can regulate the expression of small conductance calcium-activated potassium (K(Ca)) channels. These channels reduce neuronal excitability and as such may yield neuroprotection against neuronal overstimulation. In the present study we investigated whether TNF-alpha-mediated neuroprotective signaling is inducing changes in the expression of small conductance K(Ca) channels. Interestingly, the expression of K(Ca)2.2 channel was up-regulated by TNF-alpha treatment in a time-dependent manner whereas the expression of K(Ca)2.1 and K(Ca)2.3 channels was not altered. The increase in K(Ca)2.2 channel expression after TNF-alpha treatment was shown to be dependent on TNF-R2 and NF-kappaB activation. Furthermore, activation of small conductance K(Ca) channels by 6,7-dichloro-1H-indole-2,3-dione 3-oxime or cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine-induced neuroprotection against a glutamate challenge. Treatment with the small conductance K(Ca) channel blocker apamin or K(Ca)2.2 channel siRNA reverted the neuroprotective effect elicited by TNF-alpha. We conclude that treatment of primary cortical neurons with TNF-alpha leads to increased K(Ca)2.2 channel expression which renders neurons more resistant to excitotoxic cell death.

  11. The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism

    PubMed Central

    Freitas, Fernanda Zanolli; Virgilio, Stela; Cupertino, Fernanda Barbosa; Kowbel, David John; Fioramonte, Mariana; Gozzo, Fabio Cesar; Glass, N. Louise; Bertolini, Maria Célia

    2016-01-01

    When exposed to stress conditions, all cells induce mechanisms resulting in an attempt to adapt to stress that involve proteins which, once activated, trigger cell responses by modulating specific signaling pathways. In this work, using a combination of pulldown assays and mass spectrometry analyses, we identified the Neurospora crassa SEB-1 transcription factor that binds to the Stress Response Element (STRE) under heat stress. Orthologs of SEB-1 have been functionally characterized in a few filamentous fungi as being involved in stress responses; however, the molecular mechanisms mediated by this transcription factor may not be conserved. Here, we provide evidences for the involvement of N. crassa SEB-1 in multiple cellular processes, including response to heat, as well as osmotic and oxidative stress. The Δseb-1 strain displayed reduced growth under these conditions, and genes encoding stress-responsive proteins were differentially regulated in the Δseb-1 strain grown under the same conditions. In addition, the SEB-1-GFP protein translocated from the cytosol to the nucleus under heat, osmotic, and oxidative stress conditions. SEB-1 also regulates the metabolism of the reserve carbohydrates glycogen and trehalose under heat stress, suggesting an interconnection between metabolism control and this environmental condition. We demonstrated that SEB-1 binds in vivo to the promoters of genes encoding glycogen metabolism enzymes and regulates their expression. A genome-wide transcriptional profile of the Δseb-1 strain under heat stress was determined by RNA-seq, and a broad range of cellular processes was identified that suggests a role for SEB-1 as a protein interconnecting these mechanisms. PMID:26994287

  12. Interaction with extracellular matrix proteins influences Lsh/Ity/Bcg (candidate Nramp) gene regulation of macrophage priming/activation for tumour necrosis factor-alpha and nitrite release.

    PubMed

    Formica, S; Roach, T I; Blackwell, J M

    1994-05-01

    The murine resistance gene Lsh/Ity/Bcg regulates activation of macrophages for tumour necrosis factor-alpha (TNF-alpha)-dependent production of nitric oxide mediating antimicrobial activity against Leishmania, Salmonella and Mycobacterium. As Lsh is differentially expressed in macrophages from different tissue sites, experiments were performed to determine whether interaction with extracellular matrix (ECM) proteins would influence the macrophage TNF-alpha response. Plating of bone marrow-derived macrophages onto purified fibrinogen or fibronectin-rich L929 cell-derived matrices, but not onto mannan, was itself sufficient to stimulate TNF-alpha release, with significantly higher levels released from congenic B10.L-Lshr compared to C57BL/10ScSn (Lshs) macrophages. Only macrophages plated onto fibrinogen also released measurable levels of nitrites, again higher in Lshr compared to Lshs macrophages. Addition of interferon-gamma (IFN-gamma), but not bacterial lipopolysaccharide or mycobacterial lipoarabinomannan, as a second signal enhanced the TNF-alpha and nitrite responses of macrophages plated onto fibrinogen, particularly in the Lshr macrophages. Interaction with fibrinogen and fibronectin also primed macrophages for an enhanced TNF-alpha response to leishmanial parasites, but this was only translated into enhanced nitrite responses in the presence of IFN-gamma. In these experiments, Lshr macrophages remained superior in their TNF-alpha responses throughout, but to a degree which reflected the magnitude of the difference observed on ECM alone. Hence, the specificity for the enhanced TNF-alpha responses of Lshr macrophages lay in their interaction with fibrinogen and fibronectin ECM, while a differential nitrite response was only observed with fibrinogen and/or IFN-gamma. The results are discussed in relation to the possible function of the recently cloned candidate gene Nramp, which has structural identity to eukaryote transporters and an N-terminal cytoplasmic

  13. Soybean C2H2-Type Zinc Finger Protein GmZFP3 with Conserved QALGGH Motif Negatively Regulates Drought Responses in Transgenic Arabidopsis

    PubMed Central

    Zhang, Dayong; Tong, Jinfeng; Xu, Zhaolong; Wei, Peipei; Xu, Ling; Wan, Qun; Huang, Yihong; He, Xiaolan; Yang, Jiayin; Shao, Hongbo; Ma, Hongxiang

    2016-01-01

    Plant response to environmental stresses is regulated by a complicated network of regulatory and functional genes. In this study, we isolated the putative stress-associated gene GmZFP3 (a C2H2-type Zinc finger protein gene) based on the previous finding that it was one of two genes located in the QTL region between the Satt590 and Satt567 markers related to soybean tolerance to drought. Temporal and spatial expression analysis using quantitative real-time PCR indicated that GmZFP3 was primarily expressed in roots, stems and leaf organs and was expressed at low levels in flowers and soybean pods. Moreover, GmZFP3 expression increased in response to polyethylene glycol (PEG) and Abscisic acid (ABA) treatments. In addition, subcellular localization analysis indicated that GmZFP3 was ubiquitously distributed in plant cells. Transgenic experiments indicated that GmZFP3 played a negative role in plant tolerance to drought. Analysis of ABA-related marker gene expression in Arabidopsis suggested that GmZFP3 might be involved in the ABA-dependent pathway during the drought stress response. Taken together, these results suggest that soybean GmZFP3 negatively regulates the drought response. PMID:27047508

  14. Expression of AtWRKY33 encoding a pathogen- or PAMP-responsive WRKY transcription factor is regulated by a composite DNA motif containing W box elements.

    PubMed

    Lippok, Bernadette; Birkenbihl, Rainer P; Rivory, Gaelle; Brümmer, Janna; Schmelzer, Elmon; Logemann, Elke; Somssich, Imre E

    2007-04-01

    WRKY transcription factors regulate distinct parts of the plant defense transcriptome. Expression of many WRKY genes themselves is induced by pathogens or pathogen-mimicking molecules. Here, we demonstrate that Arabidopsis WRKY33 responds to various stimuli associated with plant defense as well as to different kinds of phytopathogens. Although rapid pathogen-induced AtWRKY33 expression does not require salicylic acid (SA) signaling, it is dependent on PAD4, a key regulator upstream of SA. Activation of AtWRKY33 is independent of de novo protein synthesis, suggesting that it is at least partly under negative regulatory control. We show that a set of three WRKY-specific cis-acting DNA elements (W boxes) within the AtWRKY33 promoter is required for efficient pathogen- or PAMP-triggered gene activation. This strongly indicates that WRKY transcription factors are major components of the regulatory machinery modulating immediate to early expression of this gene in response to pathogen attack.

  15. Scaling and alpha-helix regulation of protein relaxation in a lipid bilayer

    NASA Astrophysics Data System (ADS)

    Qiu, Liming; Buie, Creighton; Cheng, Kwan Hon; Vaughn, Mark W.

    2014-12-01

    Protein conformation and orientation in the lipid membrane plays a key role in many cellular processes. Here we use molecular dynamics simulation to investigate the relaxation and C-terminus diffusion of a model helical peptide: beta-amyloid (Aβ) in a lipid membrane. We observed that after the helical peptide was initially half-embedded in the extracelluar leaflet of phosphatidylcholine (PC) or PC/cholesterol (PC/CHOL) membrane, the C-terminus diffused across the membrane and anchored to PC headgroups of the cytofacial lipid leaflet. In some cases, the membrane insertion domain of the Aβ was observed to partially unfold. Applying a sigmoidal fit to the process, we found that the characteristic velocity of the C-terminus, as it moved to its anchor site, scaled with θu-4/3, where θu is the fraction of the original helix that was lost during a helix to coil transition. Comparing this scaling with that of bead-spring models of polymer relaxation suggests that the C-terminus velocity is highly regulated by the peptide helical content, but that it is independent of the amino acid type. The Aβ was stabilized by the attachment of the positive Lys28 side chain to the negative phosphate of PC or 3β oxygen of CHOL in the extracellular lipid leaflet and of the C-terminus to its anchor site in the cytofacial lipid leaflet.

  16. Effects of alpha1-adrenergic receptor blockade by doxazosin on renin-angiotensin system-regulating aminopeptidase and vasopressin-degrading activities in male and female rat thalamus.

    PubMed

    de la Chica-Rodríguez, S; Cortés-Denia, P; Ramírez-Expósito, M J; Martínez-Martos, J M

    2007-11-01

    The thalamus has connections with central autonomic centers involved in cardiovascular control and is enervated by noradrenergic fibers. The excitability of thalamic neurons is due to a reduction of ionic currents mediated by alpha(1)-adrenoceptors. The brain renin- angiotensin system (RAS) and the peptide hormone arginine-vasopressin (AVP) are also involved in the central control of blood pressure, and fluid and electrolyte homeostasis. It has been extensively reported that aminopeptidase A (APA), aminopeptidase B (APB), aminopeptidase N (APN), and vasopressin-degrading cystyl aminopeptidase activity (AVP-DA) play an important role in the regulation of the activity of angiotensins and AVP. We have analyzed the effect of alpha(1)-adrenoceptor blockade by doxazosin on RAS-regulating aminopeptidase activities and AVP-DA in soluble and membrane-bound fractions of male and female rat thalamus. Our results show that alpha(1)-adrenoceptors blockade by doxazosin does not modify the RAS through its degrading peptidases at thalamic level either in male or female rats. However, alpha(1)-adrenoceptors blockade shows gender differences in AVP-DA, increasing in males but not in females, supporting an increased capacity of males against females to degrade AVP and, therefore, to regulate cardiovascular homeostasis, under this pharmacological manipulation.

  17. Regulated tissue-specific alternative splicing of enhanced green fluorescent protein transgenes conferred by alpha-tropomyosin regulatory elements in transgenic mice.

    PubMed

    Ellis, Peter D; Smith, Christopher W J; Kemp, Paul

    2004-08-27

    The mutually exclusive exons 2 and 3 of alpha-tropomyosin (alphaTM) have been used as a model system for strictly regulated alternative splicing. Exon 2 inclusion is only observed at high levels in smooth muscle (SM) tissues, whereas striated muscle and non-muscle cells use predominantly exon 3. Experiments in cell culture have shown that exon 2 selection results from repression of exon 3 and that this repression is mediated by regulatory elements flanking exon 3. We have now tested the cell culture-derived model in transgenic mice. We show that by harnessing the intronic splicing regulatory elements, expression of an enhanced green fluorescent protein transgene with a constitutively active promoter can be restricted to SM cells. Splicing of both endogenous alphaTM and a series of transgenes carrying regulatory element mutations was analyzed by reverse transcriptasePCR. These studies indicated that although SM-rich tissues are equipped to regulate splicing of high levels of endogenous or transgene alphaTM RNA, other non-SM tissues such as spleen, which express lower amounts of alphaTM, also splice significant proportions of exon 2, and this splicing pattern can be recapitulated by transgenes expressed at low levels. We confirm the importance in vivo of the negatively acting regulatory elements for regulated skipping of exon 3. Moreover, we provide evidence that some of the regulatory factors responsible for exon 3 skipping appear to be titratable, with loss of regulated splicing sometimes being associated with high transgene expression levels. PMID:15194683

  18. Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-alpha. Role in regulating cell death and pancreatitis.

    PubMed Central

    Gukovskaya, A S; Gukovsky, I; Zaninovic, V; Song, M; Sandoval, D; Gukovsky, S; Pandol, S J

    1997-01-01

    The aim of this study was to determine whether tumor necrosis factor-alpha (TNFalpha) and receptors for TNFalpha are expressed in the exocrine pancreas, and whether pancreatic acinar cells release and respond to TNFalpha. Reverse transcription PCR, immunoprecipitation, and Western blot analysis demonstrated the presence of TNFalpha and 55- and 75-kD TNFalpha receptors in pancreas from control rats, rats with experimental pancreatitis induced by supramaximal doses of cerulein, and in isolated pancreatic acini. Immunohistochemistry showed TNFalpha presence in pancreatic acinar cells. ELISA and bioassay measurements of TNFalpha indicated its release from pancreatic acinar cells during incubation in primary culture. Acinar cells responded to TNFalpha. TNFalpha potentiated NF-kappaB translocation into the nucleus and stimulated apoptosis in isolated acini while not affecting LDH release. In vivo studies demonstrated that neutralization of TNFalpha with an antibody produced a mild improvement in the parameters of cerulein-induced pancreatitis. However, TNFalpha neutralization greatly inhibited apoptosis in a modification of the cerulein model of pancreatitis which is associated with a high percentage of apoptotic cell death. The results indicate that pancreatic acinar cells produce, release, and respond to TNFalpha. This cytokine regulates apoptosis in both isolated pancreatic acini and experimental pancreatitis. PMID:9312187

  19. Cell Cycle Checkpoint Proteins p21 and Hus1 Regulating Intercellular Signaling Induced By Alpha Particle Irradiation

    NASA Astrophysics Data System (ADS)

    Wu, Lijun; Zhao, Ye; Wang, Jun; Hang, Haiying

    In recent years, the attentions for radiation induced bystander effects (RIBE) have been paid on the intercellular signaling events connecting the irradiated and non-irradiated cells. p21 is a member of the Cip/Kip family and plays essential roles in cell cycle progression arrest after cellular irradiation. DNA damage checkpoint protein Hus1 is a member of the Rad9-Rad1-Hus1 complex and functions as scaffold at the damage sites to facilitate the activation of downstream effectors. Using the medium trasfer method and the cells of MEF, MEF (p21-/-), MEF (p21-/-Hus1-/-) as either medium donor or receptor cells, it was found that with 5cGy alpha particle irradiation, the bystander cells showed a significant induction of -H2AX for normal MEFs (p¡0.05). However, the absence of p21 resulted in deficiency in inducing bystander effects. Further results indicated p21 affected the intercellular DNA damage signaling mainly through disrupting the production or release of the damage signals from irradiated cells. When Hus1 and p21 were both knocked out, an obvious induction of -H2AX recurred in bystander cells and the induction of -H2AX was GJIC (gap junction-mediated intercellular communication) dependent, indicating the interrelationship between p21 and Hus1 regulated the production and relay of DNA damage signals from irradiated cells to non-irradiated bystander cells.

  20. TIP30 interacts with an estrogen receptor alpha-interacting coactivator CIA and regulates c-myc transcription.

    PubMed

    Jiang, Chao; Ito, Mitsuhiro; Piening, Valerie; Bruck, Kristy; Roeder, Robert G; Xiao, Hua

    2004-06-25

    Deregulation of c-myc expression is implicated in the pathogenesis of many neoplasias. Estrogen receptor alpha (ERalpha) can increase the rate of c-myc transcription through the recruitment of a variety of cofactors to the promoter, yet the precise roles of these cofactors in transcription and tumorigenesis are largely unknown. We show here that a putative tumor suppressor TIP30, also called CC3 or Htatip2, interacts with an ERalpha-interacting coactivator CIA. Using chromatin immunoprecipitation assays, we demonstrate that TIP30 and CIA are distinct cofactors that are dynamically associated with the promoter and downstream regions of the c-myc gene in response to estrogen. Both TIP30 and CIA are recruited to the c-myc gene promoter by liganded ERalpha in the second transcription cycle. TIP30 overexpression represses ERalpha-mediated c-myc transcription, whereas TIP30 deficiency enhances c-myc transcription in both the absence and presence of estrogen. Ectopic CIA cooperates with TIP30 to repress ERalpha-mediated c-myc transcription. Moreover, virgin TIP30 knockout mice exhibit increased c-myc expression in mammary glands. Together, these results reveal an important role for TIP30 in the regulation of ERalpha-mediated c-myc transcription and suggest a mechanism for tumorigenesis promoted by TIP30 deficiency.

  1. Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells

    PubMed Central

    Fajardo, Alexandra M.; MacKenzie, Debra A.; Olguin, Sarah L.; Scariano, John K.; Rabinowitz, Ian; Thompson, Todd A.

    2016-01-01

    Tocopherylquinone (TQ), the oxidation product of alpha-tocopherol (AT), is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells), whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells) was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells. PMID:26986969

  2. A putative role of micro RNA in regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes.

    PubMed

    Song, Kwang-Hoon; Li, Tiangang; Owsley, Erika; Chiang, John Y L

    2010-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A1) plays a critical role in regulation of bile acid synthesis in the liver. CYP7A1 mRNAs have very short half-lives, and bile acids destabilize CYP7A1 mRNA via the 3'-untranslated region (3'-UTR). However, the underlying mechanism of translational regulation of CYP7A1 mRNA remains unknown. Screening of a human micro RNA (miRNA) microarray has identified five differentially expressed miRNAs in human primary hepatocytes treated with chenodeoxycholic acid, GW4064, or fibroblast growth factor (FGF)19. These compounds also significantly induced the expression of miR-122a, a liver-specific and the predominant miRNA in human hepatocytes. The putative recognition sequences for miR-122a and miR-422a were localized in the 3'-UTR of human CYP7A1 mRNA. The miR-122a and miR-422a mimics inhibited, whereas their inhibitors stimulated CYP7A1 mRNA expression. These miRNAs specifically inhibited the activity of the CYP7A1-3'-UTR reporter plasmids, and mutations of miRNA binding sites in 3'-UTR abrogated miRNA inhibition of reporter activity. These results suggest that miR-122a and miR-422a may destabilize CYP7A1 mRNA to inhibit CYP7A1 expression. However, these miRNAs did not play a role in mediating FGF19 inhibition of CYP7A1 transcription. Under certain conditions, miRNA may reduce CYP7A1 mRNA stability to inhibit bile acid synthesis, and the miR-122a antagomirs may stimulate bile acid synthesis to reduce serum cholesterol and triglycerides.

  3. Astrocyte inositol triphosphate receptor type 2 and cytosolic phospholipase A2 alpha regulate arteriole responses in mouse neocortical brain slices.

    PubMed

    He, Lihua; Linden, David J; Sapirstein, Adam

    2012-01-01

    Functional hyperemia of the cerebral vascular system matches regional blood flow to the metabolic demands of the brain. One current model of neurovascular control holds that glutamate released by neurons activates group I metabotropic glutamate receptors (mGluRs) on astrocytes, resulting in the production of diffusible messengers that act to regulate smooth muscle cells surrounding cerebral arterioles. The acute mouse brain slice is an experimental system in which changes in arteriole diameter can precisely measured with light microscopy. Stimulation of the brain slice triggers specific cellular responses that can be correlated to changes in arteriole diameter. Here we used inositol trisphosphate receptor type 2 (IP(3)R2) and cytosolic phospholipase A(2) alpha (cPLA(2)α) deficient mice to determine if astrocyte mGluR activation coupled to IP(3)R2-mediated Ca(2+) release and subsequent cPLA(2)α activation is required for arteriole regulation. We measured changes in astrocyte cytosolic free Ca(2+) and arteriole diameters in response to mGluR agonist or electrical field stimulation in acute neocortical mouse brain slices maintained in 95% or 20% O(2). Astrocyte Ca(2+) and arteriole responses to mGluR activation were absent in IP(3)R2(-/-) slices. Astrocyte Ca(2+) responses to mGluR activation were unchanged by deletion of cPLA(2)α but arteriole responses to either mGluR agonist or electrical stimulation were ablated. The valence of changes in arteriole diameter (dilation/constriction) was dependent upon both stimulus and O(2) concentration. Neuron-derived NO and activation of the group I mGluRs are required for responses to electrical stimulation. These findings indicate that an mGluR/IP(3)R2/cPLA(2)α signaling cascade in astrocytes is required to transduce neuronal glutamate release into arteriole responses.

  4. Pamidronate Down-regulates Tumor Necrosis Factor-alpha Induced Matrix Metalloproteinases Expression in Human Intervertebral Disc Cells

    PubMed Central

    Kang, Young-Mi; Hong, Seong-Hwan; Yang, Jae-Ho; Oh, Jin-Cheol; Park, Jin-Oh; Lee, Byung Ho; Lee, Sang-Yoon; Kim, Hak-Sun; Lee, Hwan-Mo

    2016-01-01

    Background N-containing bisphosphonates (BPs), such as pamidronate and risedronate, can inhibit osteoclastic function and reduce osteoclast number by inducing apoptotic cell death in osteoclasts. The aim of this study is to demonstrate the effect of pamidronate, second generation nitrogen-containing BPs and to elucidate matrix metallo-proteinases (MMPs) mRNA expression under serum starvation and/or tumor necrosis factor alpha (TNF-α) stimulation on metabolism of intervertebral disc (IVD) cells in vitro. Methods Firstly, to test the effect of pamidronate on IVD cells in vitro, various concentrations (10-12, 10-10, 10-8, and 10-6 M) of pamidronate were administered to IVD cells. Then DNA and proteoglycan synthesis were measured and messenger RNA (mRNA) expressions of type I collagen, type II collagen, and aggrecan were analyzed. Secondly, to elucidate the expression of MMPs mRNA in human IVD cells under the lower serum status, IVD cells were cultivated in full serum or 1% serum. Thirdly, to elucidate the expression of MMPs mRNA in IVD cells under the stimulation of 1% serum and TNF-α (10 ng/mL) In this study, IVD cells were cultivated in three dimensional alginate bead. Results Under the lower serum culture, IVD cells in alginate beads showed upregulation of MMP 2, 3, 9, 13 mRNA. The cells in lower serum and TNF-α also demonstrated upregulation of MMP-2, 3, 9, and 13 mRNA. The cells with various doses of pamidronate and lower serum and TNF-α were reveled partial down-regulation of MMPs. Conclusions Pamidronate, N-containing second generation BPs, was safe in metabolism of IVD in vitro maintaining chondrogenic phenotype and matrix synthesis, and down-regulated TNF-α induced MMPs expression.

  5. Estrogen receptor alpha (ESR1)-signaling regulates the expression of the taxane-response biomarker PRP4K.

    PubMed

    Lahsaee, Sara; Corkery, Dale P; Anthes, Livia E; Holly, Alice; Dellaire, Graham

    2016-01-01

    The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), is an essential kinase that is a component of the U5 snRNP and functions in spliceosome assembly. We demonstrated that PRP4K is a novel biological marker for taxane response in ovarian cancer patients and reduced levels of PRP4K correlate with intrinsic and acquired taxane resistance in both breast and ovarian cancer. Breast cancer treatments are chosen based on hormone and growth factor receptor status, with HER2 (ERBB2) positive breast cancer patients receiving anti-HER2 agents and taxanes and estrogen receptor alpha (ESR1) positive (ER+) breast cancer patients receiving anti-estrogen therapies such as tamoxifen. Here we demonstrate that PRP4K is expressed in the normal mammary duct epithelial cells of the mouse, and that estrogen induces PRP4K gene and protein expression in ER+ human MCF7 breast cancer cells. Estrogen acts through ESR1 to regulate PRP4K expression, as over-expression of ESR1 in the ER-negative MDA-MB-231 breast cancer cell line increased the expression of this kinase, and knock-down of ESR1 in ER+ T47D breast cancer cells reduced PRP4K levels. Furthermore, treatment with 4-hydroxytamoxifen (4-OHT) resulted in a dose-dependent decrease in PRP4K protein expression in MCF7 cells. Consistent with our previous studies identifying PRP4K as a taxane-response biomarker, reduced PRP4K expression in 4-OHT-treated cells correlated with reduced sensitivity to paclitaxel. Thus, PRP4K is novel estrogen regulated kinase, and its levels can be reduced by 4-OHT in ER+ breast cancer cells altering their response to taxanes. PMID:26712520

  6. Pamidronate Down-regulates Tumor Necrosis Factor-alpha Induced Matrix Metalloproteinases Expression in Human Intervertebral Disc Cells

    PubMed Central

    Kang, Young-Mi; Hong, Seong-Hwan; Yang, Jae-Ho; Oh, Jin-Cheol; Park, Jin-Oh; Lee, Byung Ho; Lee, Sang-Yoon; Kim, Hak-Sun; Lee, Hwan-Mo

    2016-01-01

    Background N-containing bisphosphonates (BPs), such as pamidronate and risedronate, can inhibit osteoclastic function and reduce osteoclast number by inducing apoptotic cell death in osteoclasts. The aim of this study is to demonstrate the effect of pamidronate, second generation nitrogen-containing BPs and to elucidate matrix metallo-proteinases (MMPs) mRNA expression under serum starvation and/or tumor necrosis factor alpha (TNF-α) stimulation on metabolism of intervertebral disc (IVD) cells in vitro. Methods Firstly, to test the effect of pamidronate on IVD cells in vitro, various concentrations (10-12, 10-10, 10-8, and 10-6 M) of pamidronate were administered to IVD cells. Then DNA and proteoglycan synthesis were measured and messenger RNA (mRNA) expressions of type I collagen, type II collagen, and aggrecan were analyzed. Secondly, to elucidate the expression of MMPs mRNA in human IVD cells under the lower serum status, IVD cells were cultivated in full serum or 1% serum. Thirdly, to elucidate the expression of MMPs mRNA in IVD cells under the stimulation of 1% serum and TNF-α (10 ng/mL) In this study, IVD cells were cultivated in three dimensional alginate bead. Results Under the lower serum culture, IVD cells in alginate beads showed upregulation of MMP 2, 3, 9, 13 mRNA. The cells in lower serum and TNF-α also demonstrated upregulation of MMP-2, 3, 9, and 13 mRNA. The cells with various doses of pamidronate and lower serum and TNF-α were reveled partial down-regulation of MMPs. Conclusions Pamidronate, N-containing second generation BPs, was safe in metabolism of IVD in vitro maintaining chondrogenic phenotype and matrix synthesis, and down-regulated TNF-α induced MMPs expression. PMID:27622181

  7. An Amino-Terminal Polo Kinase Interaction Motif Acts in the Regulation of Centrosome Formation and Reveals a Novel Function for centrosomin (cnn) in Drosophila.

    PubMed

    Eisman, Robert C; Phelps, Melissa A S; Kaufman, Thomas

    2015-10-01

    The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila melanogaster embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. We show a Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis.

  8. Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

    SciTech Connect

    Yoshioka, Toyo; Inagaki, Kenjiro; Noguchi, Tetsuya; Sakai, Mashito; Ogawa, Wataru; Hosooka, Tetsuya; Iguchi, Haruhisa; Watanabe, Eijiro; Matsuki, Yasushi; Hiramatsu, Ryuji; Kasuga, Masato

    2009-04-17

    The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.

  9. Biosynthesis and regulation of rat alpha 1-inhibitor3, a negative acute-phase reactant of the macroglobulin family.

    PubMed Central

    Geiger, T; Lamri, Y; Tran-Thi, T A; Gauthier, F; Feldmann, G; Decker, K; Heinrich, P C

    1987-01-01

    The biosynthesis of rat alpha 1-inhibitor3, a negative acute-phase reactant specifically found in rodents, was studied in vitro in a cell-free translation system from rabbit reticulocytes, in rat hepatocyte primary cultures and in vivo by immunocytochemistry using normal and turpentine-injected rats. By sucrose-gradient centrifugation and subsequent translation of the fractionated RNA in vitro it was found that the mRNA coding for alpha 1-inhibitor3 exhibited a size of about 28S. For the alpha 1-inhibitor3 translated in vitro an apparent Mr of 155,000 was determined. A continuous decrease in the level of alpha 1-inhibitor3 in serum during experimental inflammation induced by turpentine injection was demonstrated by means of quantitative 'rocket' immunoelectrophoresis. This result agrees with the observation by immunocytochemistry of a drastic decrease in alpha 1-inhibitor3 levels in hepatocytes 24 h after turpentine injection. At that time alpha 1-inhibitor3 is mainly located in the Golgi apparatus, whereas it is also present in the membranes of the rough and smooth endoplasmic reticulum when normal liver is used. All hepatocytes, but no other hepatic cells, contain alpha 1-inhibitor3. When hepatocyte primary cultures were labelled with [35S]methionine and alpha 1-inhibitor3 was immunoprecipitated from the hepatocyte medium and the supernatant of homogenized cells, two different forms of alpha 1-inhibitor3 were found. The intracellular form of alpha 1-inhibitor3, with an apparent Mr of 173,000, is characterized by oligosaccharide side chains of the high-mannose type. The form of alpha 1-inhibitor3 in the medium exhibited an Mr of 186,000 and carried carbohydrate side chains of the complex type. After labelling hepatocytes with radioactive sugars, [3H]mannose was found in both forms of alpha 1-inhibitor3, whereas [3H]fucose and [3H]galactose were incorporated only into the form found in the medium. In the presence of tunicamycin an unglycosylated alpha 1-inhibitor3

  10. Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110 alpha and beta by protease-activated receptor 2 and beta-arrestins.

    PubMed

    Wang, Ping; Kumar, Puneet; Wang, Chang; Defea, Kathryn A

    2007-12-01

    PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Galphaq/Ca2+-dependent activation and beta-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of beta-arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110alpha and p110beta) associated with p85alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110alpha- and p110beta-associated lipid kinase activities, and both p110alpha and p110beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that beta-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110alpha and p110beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.

  11. Association of protein kinase FA/GSK-3alpha (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression.

    PubMed

    Yang, S D; Yu, J S; Lee, T T; Ni, M H; Yang, C C; Ho, Y S; Tsen, T Z

    1995-10-01

    Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.

  12. The roles of nitric oxide synthase and eIF2alpha kinases in regulation of cell cycle upon UVB-irradiation.

    PubMed

    Wang, Lei; Liu, Yan; Wu, Shiyong

    2010-01-01

    In response to ultraviolet light (UV)-induced damage, cells initiate cellular recovery mechanisms including activation of repair genes and redistribution of cell cycle phases. While most studies have focused on DNA damage-inducible transcriptional regulation of cell cycle checkpoints, translational regulation also plays an important role in control of cell cycle progression upon UV-irradiation. UV-irradiation activates two kinases, PERK and GCN2, which phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and subsequently inhibit protein synthesis. We recently identified an upstream regulator, nitric oxide synthase (NOS), which controls the activation of both PERK and GCN2 upon UVB-irradiation. Our data suggested that UVB induces NOS activation and NO(.) production, which reacts with superoxide (O(2)(*-)) to form peroxynitrite (ONOO(-)) and activate PERK. The NO(*) production also leads to L-Arg depletion and GCN2 activation. The elevation of nitric oxide and activation of PERK/GCN2 have been shown to play roles in regulation of cell cycle upon UVB irradiation. In the present study, we show that the cell cycle phases were redistributed by inhibition of NOS activation or reduction of oxidative stress upon UVB irradiation, indicating the roles of NO(*) and its oxidative products in regulation of cell cycle. We also demonstrate that both PERK and GCN2 were involved in regulation of cell cycle upon UVB-irradiation, but the regulation is independent of eIF2alpha phosphorylation. While the mechanism for UVB-induced cell cycle control is yet to be unraveled, we here discuss the differential roles of NOS, PERK and GCN2 in regulation of cell cycle upon UVB-irradiation. PMID:20016280

  13. TNF-alpha, but not IFN-gamma, regulates CCN2 (CTGF), collagen type I, and proliferation in mesangial cells: possible roles in the progression of renal fibrosis.

    PubMed

    Cooker, Laurinda A; Peterson, Darryl; Rambow, Joann; Riser, Melisa L; Riser, Rebecca E; Najmabadi, Feridoon; Brigstock, David; Riser, Bruce L

    2007-07-01

    Connective tissue growth factor (CCN2) is a profibrotic factor acting downstream and independently of TGF-beta to mediate renal fibrosis. Although inflammation is often involved in the initiation and/or progression of fibrosis, the role of inflammatory cytokines in regulation of glomerular CCN2 expression, cellular proliferation, and extracellular matrix accumulation is unknown. We studied two such cytokines, TNF-alpha and IFN-gamma, for their effects on cultured mesangial cells in the presence or absence of TGF-beta, as a model for progressive renal fibrosis. Short-term treatment with TNF-alpha, like TGF-beta, significantly increased secreted CCN2 per cell, but unlike TGF-beta inhibited cellular replication. TNF-alpha combined with TGF-beta further increased CCN2 secretion and mRNA levels and reduced proliferation. Surprisingly, however, TNF-alpha treatment decreased baseline collagen type I protein and mRNA levels and largely blocked their stimulation by TGF-beta. Long-term treatment with TGF-beta or TNF-alpha alone no longer increased CCN2 protein levels. However, the combination synergistically increased CCN2. IFN-gamma had no effect on either CCN2 or collagen activity and produced a mild inhibition of TGF-beta-induced collagen only at a high concentration (500 U/ml). In summary, we report a strong positive regulatory role for TNF-alpha, but not IFN-gamma, in CCN2 production and secretion, including that driven by TGF-beta. The stimulation of CCN2 release by TNF-alpha, unlike TGF-beta, is independent of cellular proliferation and not linked to increased collagen type I accumulation. This suggests that the paradigm of TGF-beta-driven CCN2 with subsequent collagen production may be overridden by an as yet undefined inhibitory mechanism acting either directly or indirectly on matrix metabolism. PMID:17376761

  14. Suppression of VLDL secretion by cultured hepatocytes incubated with chylomicron remnants enriched in n-3 polyunsaturated fatty acids is regulated by hepatic nuclear factor-4alpha.

    PubMed

    López-Soldado, Iliana; Avella, Michael; Botham, Kathleen M

    2009-12-01

    Dietary n-3 polyunsaturated fatty acids (PUFA) suppress the secretion of very low density lipoprotein (VLDL) directly when delivered to the liver in chylomicron remnants (CMR). The role of sterol regulatory element-binding proteins (SREBPs) and hepatic nuclear factor-4alpha (HNF-4alpha) in the regulation of this effect was investigated. Chylomicron remnant-like particles (CRLPs) containing triacylglycerol (TG) from palm (rich in saturated fatty acids (SFA)) or fish (rich in n-3 PUFA) oil were incubated with cultured rat hepatocytes (24h) and the expression of protein and mRNA for SREBP-1, SREBP-2 and HNF-4alpha, and levels of mRNA for their target genes were determined. SREBP-1 and -2 protein expression in the membrane and nuclear fractions was unaffected by either type of CRLPs. mRNA abundance for SREBP-1c and -2 was also unchanged by CRLP-treatment, as were levels of mRNA for target genes of SREBP-1, including steroyl CoA desaturase, acetyl CoA carboxylase, fatty acid synthase and ATP citrate lyase, and SREBP-2 (3-hydroxy-3-methylglutaryl CoA reductase). In contrast, HNF-4alpha protein and mRNA levels were significantly decreased by CRLPs enriched in n-3 PUFA, but not SFA, and the expression of mRNA for HNF-4alpha target genes, including HNF-1alpha, apolipoprotein B and the microsomal TG transfer protein, was also lowered by n-3 PUFA-, but not SFA-enriched CRLPs. These findings suggest that the direct suppression of VLDL secretion by dietary n-3 PUFA delivered to the liver in CMR is mediated via decreased expression of HNF-4alpha.

  15. Characterisation and differential regulation of MAFbx/Atrogin-1 alpha and beta transcripts in skeletal muscle of Atlantic salmon (Salmo salar).

    PubMed

    Bower, Neil I; de la Serrana, Daniel Garcia; Johnston, Ian A

    2010-05-28

    MAFbx is an E3 ubiquitin ligase which plays important roles in myogenesis and muscle atrophy. We characterised the Atlantic salmon MAFbx gene, identifying two alternatively spliced MAFbx isoforms. The mRNA sequence of Atlantic salmon MAFbx-alpha is 1698 nucleotides long including a 134 bp 5' UTR and 1065 bp coding sequence which encodes a 355 amino acid protein with a predicted mass of 41,657Da and pI 8.74. Two different 3' UTRs were identified of 495 and 314 bp in length. MAFbx-beta is produced by the removal of the 116 bp exon 2 from MAFbx-alpha, resulting in a frame shift mutation and introduction of a premature stop codon. In contrast to mammals, MAFbx-alpha and beta were ubiquitously expressed in all salmon tissues examined. In vivo, expression was 600-fold (MAFbx-alpha) and 200-fold (MAFbx-beta) higher in fasted individuals than following 21 days refeeding to satiation. In primary myogenic cell cultures, MAFbx-alpha mRNA was highest in differentiated myotubes while MAFbx-beta mRNA had peak expression in mono-nucleated cells. Starving cells of serum and amino acids resulted in a 6-fold increase in MAFbx-alpha, whereas MAFbx-beta remained similar to control levels. In starved cells, MAFbx-alpha mRNA levels declined in response to amino acid, IGF-I and IGF-II treatments whereas MAFbx-beta levels only decreased in response to IGF-I. Addition of amino acids and IGF or insulin to starved cells increased MAFbx-beta levels after 12 and 24h. These results indicate that regulation of MAFbx in Atlantic salmon occurs at both the transcriptional and post-transcriptional level through production of the alternatively spliced MAFbx-beta, which is a likely target for non-sense mediated decay. PMID:20399749

  16. TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species.

    PubMed

    Jin, Shi; Ray, Ramesh M; Johnson, Leonard R

    2008-04-01

    Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.

  17. Reactive oxygen species mediate the down-regulation of mitochondrial transcripts and proteins by tumour necrosis factor-alpha in L929 cells.

    PubMed Central

    Sánchez-Alcázar, José A; Schneider, Erasmus; Hernández-Muñoz, Inmaculada; Ruiz-Cabello, Jesús; Siles-Rivas, Eva; de la Torre, Paz; Bornstein, Belen; Brea, Gloria; Arenas, Joaquín; Garesse, Rafael; Solís-Herruzo, José A; Knox, Alan J; Navas, Plácido

    2003-01-01

    In this study, we show that reactive oxygen species production induced by tumour necrosis factor alpha (TNF-alpha) in L929 cells was associated with a decrease in the steady-state mRNA levels of the mitochondrial transcript ATPase 6-8. Simultaneously, the transcript levels of two nuclear-encoded glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase, were increased. These changes were associated with decreased protein levels of the ATPase subunit a (encoded by the mitochondrial ATPase 6 gene) and cytochrome c oxidase subunit II, and increased protein levels of phosphofructokinase. Since TNF-alpha had no effect on the amount of mitochondrial DNA, the results suggested that TNF-alpha acted at the transcriptional and/or post-transcriptional level. Reactive oxygen species scavengers, such as butylated hydroxianisole and butylated hydroxytoluene, blocked the production of free radicals, prevented the down-regulation of ATPase 6-8 transcripts, preserved the protein levels of ATPase subunit a and cytochrome c oxidase subunit II, and attenuated the cytotoxic response to TNF-alpha, indicating a direct link between these two phenomena. PMID:12470298

  18. Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.

    PubMed Central

    Chen, J J; Pal, J K; Petryshyn, R; Kuo, I; Yang, J M; Throop, M S; Gehrke, L; London, I M

    1991-01-01

    We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases. Images PMID:1671169

  19. Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells.

    PubMed Central

    Lambertini, Elisabetta; Penolazzi, Letizia; Giordano, Silvia; Del Senno, Laura; Piva, Roberta

    2003-01-01

    (O)estrogen receptor-alpha (ERalpha), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERalpha gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at -117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of gamma-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PF a and PF b, that do not present binding sites for known transcription factors and that are involved in a strong DNA-protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERalpha gene expression in bone. These data provide evidence for different promoter usage of the ERalpha gene through the recruitment of tissue-specific transcription

  20. Elongation factor-1 alpha is a selective regulator of growth factor withdrawal and ER stress-induced apoptosis.

    PubMed

    Talapatra, S; Wagner, J D O; Thompson, C B

    2002-08-01

    To identify genes that contribute to apoptotic resistance, IL-3 dependent hematopoietic cells were transfected with a cDNA expression library and subjected to growth factor withdrawal. Transfected cells were enriched for survivors over two successive rounds of IL-3 withdrawal and reconstitution, resulting in the identification of a full-length elongation factor 1 alpha (EF-1alpha) cDNA. Ectopic EF-1alpha expression conferred protection from growth factor withdrawal and agents that induce endoplasmic reticulum stress, but not from nuclear damage or death receptor signaling. Overexpression of EF-1alpha did not lead to growth factor independent cell proliferation or global alterations in protein levels or rates of synthesis. These findings suggest that overexpression of EF-1alpha results in selective resistance to apoptosis induced by growth factor withdrawal and ER stress. PMID:12107828

  1. Identification of 7alpha-hydroxypregnenolone, a novel bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion.

    PubMed

    Tsutsui, Kazuyoshi; Haraguchi, Shogo; Matsunaga, Masahiro; Koyama, Teppei; Do Rego, Jean-Luc; Vaudry, Hubert

    2010-09-01

    We now know that steroids can be synthesized de novo by the brain and the peripheral nervous system. Such steroids are called neurosteroids and de novo neurosteroidogenesis from cholesterol is a conserved property of vertebrate brains. Our studies over the past decade have demonstrated that the brain expresses several kinds of steroidogenic enzymes and produces a variety of neurosteroids in sub-mammalian species. However, neurosteroid biosynthetic pathways in amphibians, as well as other vertebrates may still not be fully mapped. We first found that the newt brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. We then demonstrated that 7alpha-hydroxypregnenolone acts as a novel bioactive neurosteroid to stimulate locomotor activity of newt by means of the dopaminergic system. Subsequently, we analyzed the physiological roles of 7alpha-hydroxypregnenolone in the regulation of locomotor activity of newt. This paper summarizes the advances made in our understanding of 7alpha-hydroxypregnenolone, a newly discovered bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion in newt.

  2. Multiple Weak Linear Motifs Enhance Recruitment and Processivity in SPOP-Mediated Substrate Ubiquitination.

    PubMed

    Pierce, Wendy K; Grace, Christy R; Lee, Jihun; Nourse, Amanda; Marzahn, Melissa R; Watson, Edmond R; High, Anthony A; Peng, Junmin; Schulman, Brenda A; Mittag, Tanja

    2016-03-27

    Primary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert physiological relevance stronger than that suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli3(1-90)) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3(SPOP) and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH (meprin and TRAF homology) domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli3(1-90), the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli3(1-90) ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers regulating post-translational modification. PMID:26475525

  3. Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties.

    PubMed

    Rubio-Villena, Carla; Sanz, Pascual; Garcia-Gimeno, Maria Adelaida

    2015-01-01

    Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

  4. The C-terminal motif of SiAGO1b is required for the regulation of growth, development and stress responses in foxtail millet (Setaria italica (L.) P. Beauv)

    PubMed Central

    Liu, Xiaotong; Tang, Sha; Jia, Guanqing; Schnable, James C.; Su, Haixia; Tang, Chanjuan; Zhi, Hui; Diao, Xianmin

    2016-01-01

    Foxtail millet (Setaria italica (L.) P. Beauv), which belongs to the Panicoideae tribe of the Poaceae, is an important grain crop widely grown in Northern China and India. It is currently developing into a novel model species for functional genomics of the Panicoideae as a result of its fully available reference genome sequence, small diploid genome (2n=18, ~510Mb), short life cycle, small stature and prolific seed production. Argonaute 1 (AGO1), belonging to the argonaute (AGO) protein family, recruits small RNAs and regulates plant growth and development. Here, we characterized an AGO1 mutant (siago1b) in foxtail millet, which was induced by ethyl methanesulfonate treatment. The mutant exhibited pleiotropic developmental defects, including dwarfing stem, narrow and rolled leaves, smaller panicles and lower rates of seed setting. Map-based cloning analysis demonstrated that these phenotypic variations were attributed to a C–A transversion, and a 7-bp deletion in the C-terminus of the SiAGO1b gene in siago1b. Yeast two-hybrid assays and BiFC experiments revealed that the mutated region was an essential functional motif for the interaction between SiAGO1b and SiHYL1. Furthermore, 1598 differentially expressed genes were detected via RNA-seq-based comparison of SiAGO1b and wild-type plants, which revealed that SiAGO1b mutation influenced multiple biological processes, including energy metabolism, cell growth, programmed death and abiotic stress responses in foxtail millet. This study may provide a better understanding of the mechanisms by which SiAGO1b regulates the growth and development of crops. PMID:27045099

  5. VAMP subfamilies identified by specific R-SNARE motifs.

    PubMed

    Rossi, Valeria; Picco, Raffaella; Vacca, Marcella; D'Esposito, Maurizio; D'Urso, Michele; Galli, Thierry; Filippini, Francesco

    2004-05-01

    In eukaryotes, interactions among the alpha-helical coiled-coil domains (CCDs) of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in mediating the fusion among vesicles and target membranes. Surface residues of such CCDs are major candidates to regulate the specificity of membrane fusion, as they may alter local charge at the interaction layers and surface of the fusion complex, possibly modulating its formation and/or the binding of non-SNARE regulatory factors. Based on alternate patterns in surface residues, we have identified two motifs which group vesicular SNAREs in two novel subfamilies: RG-SNAREs and RD-SNAREs. The RG-SNARE CCD is common to all members of the widely conserved family of long VAMPs or longins and to yeast and non-neuronal VAMPs, possibly mediating "basic" fusion mechanisms; instead, only synaptobrevins from Bilateria share an RD-SNARE CCD, which is likely to mediate interactions to specific, yet unknown, regulatory factors and/or be the landmark of rapid fusion reactions like that mediating the release of neurotransmitters.

  6. GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry.

    PubMed

    June, H L; Harvey, S C; Foster, K L; McKay, P F; Cummings, R; Garcia, M; Mason, D; Grey, C; McCane, S; Williams, L S; Johnson, T B; He, X; Rock, S; Cook, J M

    2001-03-15

    GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating Et

  7. Prostaglandin F{sub 2{alpha}} regulates cytokine responses of mast cells through the receptors for prostaglandin E

    SciTech Connect

    Kaneko, Izumi; Hishinuma, Takanori; Suzuki, Kaori; Owada, Yuji; Kitanaka, Noriko; Kondo, Hisatake; Goto, Junichi; Furukawa, Hiroshi; Ono, Masao

    2008-03-14

    There is an increasing body of evidence that prostanoids modulate mast cell functions and contribute to the development of allergic inflammation. The present study aimed to identify an undetermined function of prostaglandin (PG) F{sub 2{alpha}} in mast cell activation and the signaling mechanism involved in it. Simultaneous quantification of prostanoids by liquid chromatography/tandem mass spectrometry revealed the constitutive release of PGF{sub 2{alpha}}, thromboxane B{sub 2}, and 6-keto-PGF{sub 1{alpha}} from bone marrow-derived mast cells (BMMCs). Upon activation of BMMCs by lipopolysaccharide, the cytokine production in BMMCs was enhanced when the culture was supplemented with PGF{sub 2{alpha}}. However, F prostanoid receptor-a selective receptor for PGF{sub 2{alpha}}-was not detected in BMMCs. Further investigations performed using prostanoid receptor antagonists revealed an alternative mechanism wherein the receptors for PGE species-E prostanoid receptors-mediated the PGF{sub 2{alpha}} signal in BMMCs. The present study provides an insight into a novel function of PGF{sub 2{alpha}}, i.e., an autocrine accelerator for mast cell activation.

  8. Triptolide inhibits COX-2 expression by regulating mRNA stability in TNF-{alpha}-treated A549 cells

    SciTech Connect

    Sun, Lixin; Zhang, Shuang; Jiang, Zhenzhou; Huang, Xin; Wang, Tao; Huang, Xiao; Li, Han; Zhang, Luyong

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Triptolide inhibited COX-2 expression and the half-life of COX-2 mRNA is decreased. Black-Right-Pointing-Pointer The HuR protein shuttling from nucleus to cytoplasm is inhibited by triptolide. Black-Right-Pointing-Pointer Triptolide inhibited 3 Prime -UTR fluorescence reporter gene activity. Black-Right-Pointing-Pointer COX-2 mRNA binding to HuR is decreased by triptolide in pull-down experiments. -- Abstract: Cyclooxygenase-2 (COX-2) over-expression is frequently associated with human non-small-cell lung cancer (NSCLC) and involved in tumor proliferation, invasion, angiogenesis and resistance to apoptosis. In the present study, the effects of triptolide on COX-2 expression in A549 cells were investigated and triptolide was found to inhibit TNF-{alpha}-induced COX-2 expression. In our further studies, it was found that triptolide decreased the half-life of COX-2 mRNA dramatically and that it inhibited 3 Prime -untranslated region (3 Prime -UTR) fluorescence reporter gene activity. Meanwhile, triptolide inhibited the HuR shuttling from nucleus to cytoplasm. After triptolide treatment, decreased COX-2 mRNA in pull-down experiments with anti-HuR antibodies was observed, indicating that the decreased cytoplasmic HuR is responsible for the decreased COX-2 mRNA. Taken together, our results provided evidence for the first time that triptolide inhibited COX-2 expression by COX-2 mRNA stability modulation and post-transcriptional regulation. These results provide a novel mechanism of action for triptolide which may be important in the treatment of lung cancer.

  9. Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity.

    PubMed

    Ji, H L; Parker, S; Langloh, A L; Fuller, C M; Benos, D J

    2001-07-01

    We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself. PMID:11401828

  10. Alpha-adrenergic regulation of systemic peripheral resistance and blood flow distribution in the turtle Trachemys scripta during anoxic submergence at 5 degrees C and 21 degrees C.

    PubMed

    Stecyk, J A W; Overgaard, J; Farrell, A P; Wang, T

    2004-01-01

    metabolism were indicated by increases in %.Q(sys) to the muscle at 21 degrees C (1.3-fold) and liver at 5 degrees C (1.7-fold). As well, the crucial importance of the turtle shell as a buffer reserve during anoxic submergence was indicated by 40-50% of .Q(sys) being directed towards the shell during anoxia at both 5 degrees C and 21 degrees C. Alpha-adrenergic stimulation and blockade during anoxia caused few changes in %.Q(sys) and absolute tissue blood flow. However, there was evidence of alpha-adrenergic vasoactivity contributing to blood flow regulation to the liver and shell during anoxic submergence at 5 degrees C.

  11. Network motif-based method for identifying coronary artery disease

    PubMed Central

    LI, YIN; CONG, YAN; ZHAO, YUN

    2016-01-01

    The present study aimed to develop a more efficient method for identifying coronary artery disease (CAD) than the conventional method using individual differentially expressed genes (DEGs). GSE42148 gene microarray data were downloaded, preprocessed and screened for DEGs. Additionally, based on transcriptional regulation data obtained from ENCODE database and protein-protein interaction data from the HPRD, the common genes were downloaded and compared with genes annotated from gene microarrays to screen additional common genes in order to construct an integrated regulation network. FANMOD was then used to detect significant three-gene network motifs. Subsequently, GlobalAncova was used to screen differential three-gene network motifs between the CAD group and the normal control data from GSE42148. Genes involved in the differential network motifs were then subjected to functional annotation and pathway enrichment analysis. Finally, clustering analysis of the CAD and control samples was performed based on individual DEGs and the top 20 network motifs identified. In total, 9,008 significant three-node network motifs were detected from the integrated regulation network; these were categorized into 22 interaction modes, each containing a minimum of one transcription factor. Subsequently, 1,132 differential network motifs involving 697 genes were screened between the CAD and control group. The 697 genes were enriched in 154 gene ontology terms, including 119 biological processes, and 14 KEGG pathways. Identifying patients with CAD based on the top 20 network motifs provided increased accuracy compared with the conventional method based on individual DEGs. The results of the present study indicate that the network motif-based method is more efficient and accurate for identifying CAD patients than the conventional method based on individual DEGs. PMID:27347046

  12. Alpha2,6-sialic acid on platelet endothelial cell adhesion molecule (PECAM) regulates its homophilic interactions and downstream antiapoptotic signaling.

    PubMed

    Kitazume, Shinobu; Imamaki, Rie; Ogawa, Kazuko; Komi, Yusuke; Futakawa, Satoshi; Kojima, Soichi; Hashimoto, Yasuhiro; Marth, Jamey D; Paulson, James C; Taniguchi, Naoyuki

    2010-02-26

    Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that alpha2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on alpha2,6-sialic acid. We also found that the absence of alpha2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis. PMID:20048157

  13. A motif present in the main cytoplasmic loop of nicotinic acetylcholine receptors and catalases.

    PubMed

    Morgado-Valle, C; García-Colunga, J; Miledi, R; Díaz-Muñoz, M

    2001-05-01

    A motif containing five conserved amino acids (RXPXTH(X)14P) was detected in 111 proteins, including 82 nicotinic acetylcholine receptor (nAChR) subunits and 20 catalases. To explore possible functional roles of this motif in nAChRs two approaches were used: first, the motif sequences in nAChR subunits and catalases were analysed and compared; and, second, deletions in the rat alpha2 and beta4 nAChR subunits expressed in Xenopus oocytes were analysed. Compared to the three-dimensional structure of bovine hepatic catalase, structural coincidences were found in the motif of catalases and nAChRs. On the other hand, partial deletions of the motif in the alpha2 or beta4 subunits and injection of the mutants into oocytes was followed by a very weak expression of functional nAChRs; oocytes injected with alpha2 and beta4 subunits in which the entire motif had been deleted failed to elicit any acetylcholine currents. The results suggest that the motif may play a role in the activation of nAChRs. PMID:11370971

  14. DMINDA: an integrated web server for DNA motif identification and analyses

    PubMed Central

    Ma, Qin; Zhang, Hanyuan; Mao, Xizeng; Zhou, Chuan; Liu, Bingqiang; Chen, Xin; Xu, Ying

    2014-01-01

    DMINDA (DNA motif identification and analyses) is an integrated web server for DNA motif identification and analyses, which is accessible at http://csbl.bmb.uga.edu/DMINDA/. This web site is freely available to all users and there is no login requirement. This server provides a suite of cis-regulatory motif analysis functions on DNA sequences, which are important to elucidation of the mechanisms of transcriptional regulation: (i) de novo motif finding for a given set of promoter sequences along with statistical scores for the predicted motifs derived based on information extracted from a control set, (ii) scanning motif instances of a query motif in provided genomic sequences, (iii) motif comparison and clustering of identified motifs, and (iv) co-occurrence analyses of query motifs in given promoter sequences. The server is powered by a backend computer cluster with over 150 computing nodes, and is particularly useful for motif prediction and analyses in prokaryotic genomes. We believe that DMINDA, as a new and comprehensive web server for cis-regulatory motif finding and analyses, will benefit the genomic research community in general and prokaryotic genome researchers in particular. PMID:24753419

  15. Regulation of vascular cell adhesion molecule-1 expression by IL-4 and TNF-alpha in cultured endothelial cells.

    PubMed Central

    Iademarco, M F; Barks, J L; Dean, D C

    1995-01-01

    Interaction between vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells and alpha 4 integrins on leukocytes is thought to mediate the selective recruitment of eosinophils and lymphocytes that occurs in allergic diseases. IL-4 is associated with allergic conditions, and it has been shown to selectively increase expression of VCAM-1 on endothelial cells in vivo, suggesting that it could be responsible for VCAM-1 expression in allergic disease. Using a combination of immunofluorescence, flow cytometry, and Northern analysis, we compared the effect of TNF-alpha and IL-4 on VCAM-1 expression. TNF-alpha is also associated with allergic diseases, and it rapidly increases transcription of the VCAM-1 gene. The effect of IL-4 was relatively modest with prolonged kinetics: VCAM-1 was not detected until 72 h after treatment with IL-4. However, when TNF-alpha and IL-4 were combined, there was a synergistic increase in VCAM-1 expression and a dramatic prolongation of the appearance of VCAM-1 on the cell surface. This synergy results from a combination of transcriptional activation by TNF-alpha and the stabilization of resulting transcripts by IL-4. We propose that IL-4 allows subthreshold concentrations of TNF-alpha (concentrations that would not normally activate expression of adhesion molecules on the endothelium) to selectively increase VCAM-1 expression and to prolong its appearance on the surface of cells in allergic disease. Images PMID:7529260

  16. Local graph alignment and motif search in biological networks

    NASA Astrophysics Data System (ADS)

    Berg, Johannes; Lässig, Michael

    2004-10-01

    Interaction networks are of central importance in postgenomic molecular biology, with increasing amounts of data becoming available by high-throughput methods. Examples are gene regulatory networks or protein interaction maps. The main challenge in the analysis of these data is to read off biological functions from the topology of the network. Topological motifs, i.e., patterns occurring repeatedly at different positions in the network, have recently been identified as basic modules of molecular information processing. In this article, we discuss motifs derived from families of mutually similar but not necessarily identical patterns. We establish a statistical model for the occurrence of such motifs, from which we derive a scoring function for their statistical significance. Based on this scoring function, we develop a search algorithm for topological motifs called graph alignment, a procedure with some analogies to sequence alignment. The algorithm is applied to the gene regulation network of Escherichia coli.

  17. Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8

    SciTech Connect

    Ogura, Hirotsugu; Tsukumo, Yoshinori; Sugimoto, Hikaru; Igarashi, Masayuki; Nagai, Kazuo; Kataoka, Takao

    2008-04-01

    The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in a dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-{alpha}-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8.

  18. Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency

    PubMed Central

    Pastore, Nunzia; Blomenkamp, Keith; Annunziata, Fabio; Piccolo, Pasquale; Mithbaokar, Pratibha; Maria Sepe, Rosa; Vetrini, Francesco; Palmer, Donna; Ng, Philip; Polishchuk, Elena; Iacobacci, Simona; Polishchuk, Roman; Teckman, Jeffrey; Ballabio, Andrea; Brunetti-Pierri, Nicola

    2013-01-01

    Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins. PMID:23381957

  19. Zinc finger protein 131 inhibits estrogen signaling by suppressing estrogen receptor {alpha} homo-dimerization

    SciTech Connect

    Oh, Yohan; Chung, Kwang Chul

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer ZNF131 directly interacts with ER{alpha}. Black-Right-Pointing-Pointer The binding affinity of ZNF131 to ER{alpha} increases upon E2 stimulation. Black-Right-Pointing-Pointer ZNF131 inhibits ER{alpha}-mediated trans-activation by suppressing its homo-dimerization. Black-Right-Pointing-Pointer ZNF131 inhibits ER{alpha}-dimerization and E2-induced breast cancer cell proliferation. Black-Right-Pointing-Pointer ZNF131 inhibits estrogen signaling by acting as an ER{alpha}-co-repressor. -- Abstract: Steroid hormone estrogen elicits various physiological functions, many of which are mediated through two structurally and functionally distinct estrogen receptors, ER{alpha} and ER{beta}. The functional role of zinc finger protein 131 (ZNF131) is poorly understood, but it is assumed to possess transcriptional regulation activity due to the presence of a DNA binding motif. A few recent reports, including ours, revealed that ZNF131 acts as a negative regulator of ER{alpha} and that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling. However, its molecular mechanism for ER{alpha} inhibition has not been elucidated in detail. Here, we demonstrate that ZNF131 directly interacts with ER{alpha}, which consequently inhibits ER{alpha}-mediated trans-activation by suppressing its homo-dimerization. Moreover, we show that the C-terminal region of ZNF131 containing the SUMOylation site is necessary for its inhibition of estrogen signaling. Taken together, these data suggest that ZNF131 inhibits estrogen signaling by acting as an ER{alpha}-co-repressor.

  20. Extrahepatic synthesis of plasminogen in the human cornea is up-regulated by interleukins-1alpha and -1beta.

    PubMed Central

    Twining, S S; Wilson, P M; Ngamkitidechakul, C

    1999-01-01

    The avascular cornea has limited access to plasma proteins, including plasminogen, a protein that is synthesized by the liver and supplied to most tissues via the blood. Recent experiments by others using plasminogen-deficient mice revealed the importance of plasmin, the active form of plasminogen, for the maintenance of the normal cornea and for corneal wound healing [Kao, Kao, Bugge, Kaufman, Kombrinck, Converse, Good and Degan (1998) Invest. Ophthalmol. Vis. Sci. 39, 502-508; Drew, Kaufman, Kombrinck, Danton, Daugherty, Degen and Bugge (1998) Blood 91, 1616-1624]. In the present experiments, plasmin was identified as a major serine proteinase in the human cornea. The major plasminogen and plasmin forms on non-reducing zymograms and Western blots had Mr values of 76x10(3) and 85x10(3), with minor forms of Mr 200x10(3), 135x10(3), 68x10(3) and 45x10(3). Angiostatin-like peptides with Mrs of 48x10(3), 45x10(3) and 38x10(3) were observed which bound to lysine-Sepharose and reacted with anti-plasminogen monoclonal antibodies directed towards kringle domains 1-3 of plasminogen. The cornea contained 1.1+/-0.15 microgram of plasminogen+plasmin/cornea, or 0.54+/-0.05 microgram of plasminogen+plasmin/mg of protein. Cornea conditioned medium contained nine times the amount of plasminogen+plasmin that could be extracted from the cornea. These data suggested that corneal cells, unlike most extrahepatic cells, synthesize plasminogen. The synthesis of plasminogen by the cornea was confirmed by immunoprecipitation of metabolically labelled plasminogen, sequencing of its cDNA obtained by reverse transcriptase-PCR and inhibition of protein synthesis. Interleukins-1alpha and -1beta stimulated corneal plasminogen synthesis 2-3-fold; however, interleukin-6 decreased corneal plasminogen synthesis by approx. 40% at early times after addition of the cytokine. By 24 h of culture, no differences were noted in the presence and absence of interleukin-6. Thus the cornea can synthesize

  1. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase.

    PubMed Central

    Chen, J J; Throop, M S; Gehrke, L; Kuo, I; Pal, J K; Brodsky, M; London, I M

    1991-01-01

    We have cloned the cDNA of the heme-regulated eIF-2 alpha kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2 alpha kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of approximately 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2 alpha kinase. This observation suggests that GCN2 protein kinase may be an eIF-2 alpha kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle. Images PMID:1679235

  2. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and. alpha. 1-antitrypsin genes

    SciTech Connect

    Costa, R.H. ); Grayson, D.R. ); Darnell, J.E. Jr. )

    1989-04-01

    Transthyretin (TTR) and {alpha}1-antitrypsin ({alpha}1-AT) are expressed at high levels in the liver and also in at least one other cell type. The authors report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the {alpha}1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. The authors had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and {alpha}1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the results of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and {alpha}1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.

  3. Transcriptional regulation of the human acid alpha-glucosidase gene. Identification of a repressor element and its transcription factors Hes-1 and YY1.

    PubMed

    Yan, B; Heus, J; Lu, N; Nichols, R C; Raben, N; Plotz, P H

    2001-01-19

    Acid alpha-glucosidase, the product of a housekeeping gene, is a lysosomal enzyme that degrades glycogen. A deficiency of this enzyme is responsible for a recessively inherited myopathy and cardiomyopathy, glycogenesis type II. We have previously demonstrated that the human acid alpha-glucosidase gene expression is regulated by a silencer within intron 1, which is located in the 5'-untranslated region. In this study, we have used deletion analysis, electrophoretic mobility shift assay, and footprint analysis to further localize the silencer to a 25-base pair element. The repressive effect on the TK promoter was about 50% in both orientations in expression plasmid, and two transcriptional factors were identified with antibodies binding specifically to the element. Mutagenesis and functional analyses of the element demonstrated that the mammalian homologue 1 of Drosophila hairy and Enhancer of split (Hes-1) binding to an E box (CACGCG) and global transcription factor-YY1 binding to its core site function as a transcriptional repressor. Furthermore, the overexpression of Hes-1 significantly enhanced the repressive effect of the silencer element. The data should be helpful in understanding the expression and regulation of the human acid alpha-glucosidase gene as well as other lysosomal enzyme genes.

  4. Estren (4-estren-3alpha,17beta-diol) is a prohormone that regulates both androgenic and estrogenic transcriptional effects through the androgen receptor.

    PubMed

    Centrella, Michael; McCarthy, Thomas L; Chang, Wei-Zhong; Labaree, David C; Hochberg, Richard B

    2004-05-01

    Alternative mechanisms of steroid action, through both traditional nuclear receptors and indirect pathways of gene activation, are emerging. Recent studies suggest that the synthetic steroid, 4-estrene-3alpha,17beta-diol (estren), has nongenotropic as well as sex-nonspecific osteogenic effects in ovariectomized and orchidectomized mice. We found limited estrogen receptor-dependent effects by estren on gene expression in primary osteoblast cultures and showed that it binds poorly to estrogen and androgen receptors in vitro. However, estren potently regulated direct and indirect androgen receptor-dependent effects on gene expression by osteoblasts. Consistent with this, osteoblasts produced the potent androgen 19-nortestosterone from estren by way of a 3alpha-hydroxysteroid dehydrogenase-like activity. Moreover, recombinant 3alpha-hydroxysteroid dehydrogenase (AKR1C9) and osteoblast-derived cell lysate each effectively converted estren to 19-nortestosterone in vitro, and mRNA encoding this enzyme occurs in osteoblasts. In addition to its androgenic activity, estren potently stimulated androgen receptor-dependent effects on gene expression through conventional estrogen-sensitive transcriptional elements in osteoblasts. Therefore, through local metabolism, estren indirectly activates the androgen receptor to regulate both androgen- and estrogen-like transcriptional responses by bone-forming cells.

  5. EIYMNVPV Motif is Essential for A1CF Nucleus Localization and A1CF (-8aa) Promotes Proliferation of MDA-MB-231 Cells via Up-Regulation of IL-6

    PubMed Central

    Zhou, Li; Hao, Jin; Yuan, Yue; Peng, Rui; Wang, Honglian; Ni, Dongsheng; Gu, Yuping; Huang, Liyuan; Mao, Zhaomin; Lyu, Zhongshi; Du, Yao; Liu, Zhicheng; Li, Yiman; Ju, Pan; Long, Yaoshui; Liu, Jianing; Zhou, Qin

    2016-01-01

    Apobec-1 complementation factor (A1CF) is a heterogeneous nuclear ribonuceloprotein (hnRNP) and mediates apolipoprotein-B mRNA editing. A1CF can promote the regeneration of the liver by post-transcriptionally stabilizing Interleukin-6 (IL-6) mRNA. It also contains two transcriptional variants-A1CF64 and A1CF65, distinguished by the appearance of a 24-nucleotide motif which contributes to the corresponding eight-amino acid motif of EIYMNVPV. For the first time, we demonstrated that the EIYMNVPV motif was essential for A1CF nucleus localization, A1CF deficient of the EIYMNVPV motif, A1CF (-8aa) showed cytoplasm distribution. More importantly, we found that A1CF (-8aa), but not its full-length counterpart, can promote proliferation of MDA-MB-231 cells accompanied with increased level of IL-6 mRNA. Furthermore, silencing of IL-6 attenuated A1CF (-8aa)-induced proliferation in MDA-MB-231 cells. In conclusion, notably, these findings suggest that A1CF (-8aa) promoted proliferation of MDA-MB-231 cells in vitro viewing IL-6 as a target. Thus, the EIYMNVPV motif could be developed as a potential target for basal-like breast cancer therapy. PMID:27231908

  6. Expression of alpha-expansin and expansin-like genes in deepwater rice.

    PubMed

    Lee, Yi; Kende, Hans

    2002-11-01

    Previously, we have studied the expression and regulation of four alpha- and 14 beta-expansin genes in deepwater rice (Oryza sativa). We now report on the structure, expression, and regulation of 22 additional alpha-expansin (Os-EXP) genes, four expansin-like (Os-EXPL) genes, and one expansin-related (Os-EXPR) gene, which have recently been identified in the expressed sequence tag and genomic databases of rice. Alpha-expansins are characterized by a series of conserved Cys residues in the N-terminal half of the protein, a histidine-phenylalanine-aspartate (HFD) motif in the central region, and a series of tryptophan residues near the carboxyl terminus. Of the 22 additional alpha-expansin genes, five are expressed in internodes and leaves, three in coleoptiles, and nine in roots, with high transcript levels in the growing regions of these organs. Transcripts of five alpha-expansin genes were found in roots only. Expression of five alpha-expansin genes was induced in the internode by treatment with gibberellin (GA) and by wounding. The wound response resulted from excising stem sections or from piercing pinholes into the stem of intact plants. EXPL proteins lack the HFD motif and have two additional Cys residues in their C- and N-terminal regions. The positions of conserved tryptophan residues at the C-terminal region are different from those of alpha- and beta-expansins. Expression of the Os-EXPL3 gene is correlated with elongation and slightly induced by applied GA. However, the expression of the Os-EXPL1 and Os-EXPL2 genes showed limited correlation with cell elongation and was not induced by GA. We found no expression of the Os-EXPR1 gene in the organs examined.

  7. Direct vs 2-stage approaches to structured motif finding

    PubMed Central

    2012-01-01

    Background The notion of DNA motif is a mathematical abstraction used to model regions of the DNA (known as Transcription Factor Binding Sites, or TFBSs) that are bound by a given Transcription Factor to regulate gene expression or repression. In turn, DNA structured motifs are a mathematical counterpart that models sets of TFBSs that work in concert in the gene regulations processes of higher eukaryotic organisms. Typically, a structured motif is composed of an ordered set of isolated (or simple) motifs, separated by a variable, but somewhat constrained number of “irrelevant” base-pairs. Discovering structured motifs in a set of DNA sequences is a computationally hard problem that has been addressed by a number of authors using either a direct approach, or via the preliminary identification and successive combination of simple motifs. Results We describe a computational tool, named SISMA, for the de-novo discovery of structured motifs in a set of DNA sequences. SISMA is an exact, enumerative algorithm, meaning that it finds all the motifs conforming to the specifications. It does so in two stages: first it discovers all the possible component simple motifs, then combines them in a way that respects the given constraints. We developed SISMA mainly with the aim of understanding the potential benefits of such a 2-stage approach w.r.t. direct methods. In fact, no 2-stage software was available for the general problem of structured motif discovery, but only a few tools that solved restricted versions of the problem. We evaluated SISMA against other published tools on a comprehensive benchmark made of both synthetic and real biological datasets. In a significant number of cases, SISMA outperformed the competitors, exhibiting a good performance also in most of the cases in which it was inferior. Conclusions A reflection on the results obtained lead us to conclude that a 2-stage approach can be implemented with many advantages over direct approaches. Some of these

  8. Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii.

    PubMed Central

    Coleman, J P; Hudson, L L; Adams, M J

    1994-01-01

    A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group. Images PMID:8050999

  9. Choline kinase-alpha by regulating cell aggressiveness and drug sensitivity is a potential druggable target for ovarian cancer

    PubMed Central

    Granata, A; Nicoletti, R; Tinaglia, V; De Cecco, L; Pisanu, M E; Ricci, A; Podo, F; Canevari, S; Iorio, E; Bagnoli, M; Mezzanzanica, D

    2014-01-01

    Background: Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). Methods: To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments. Results: In silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by 1H-magnetic spectroscopy analysis; (III) a 35–36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context. Conclusion: We showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with

  10. Ligand-dependent coactivation of the human bile acid receptor FXR by the peroxisome proliferator-activated receptor gamma coactivator-1alpha.

    PubMed

    Savkur, Rajesh S; Thomas, Jeffrey S; Bramlett, Kelli S; Gao, Yunling; Michael, Laura F; Burris, Thomas P

    2005-01-01

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been shown to play an important role in energy metabolism by coordinating transcriptional programs involved in mitochondrial biogenesis, adaptive thermogenesis, gluconeogenesis, and fatty acid oxidation. PGC-1alpha also plays a crucial role in cholesterol metabolism by serving as a coactivator of the liver X receptor-alpha and inducing the expression of cholesterol 7-alpha-hydroxylase. Here, we demonstrate that PGC-1alpha also functions as an effective coactivator of farnesoid X receptor (FXR), the bile acid receptor. Transient cotransfection assays demonstrate that PGC-1alpha enhances ligand-mediated FXR transcription when either full-length FXR or Gal4 DNA binding domain-FXR-ligand binding domain chimeras were analyzed. Mammalian two-hybrid analyses, glutathione S-transferase affinity chromatography and biochemical coactivator recruitment assays demonstrate ligand-dependent interaction between the two proteins both in vivo and in vitro. PGC-1alpha-mediated coactivation of FXR was highly ligand-dependent and absolutely required an intact activation function-2 (AF-2) domain of FXR and the LXXLL motif in PGC-1alpha. The integrity of the charge clamp was required, further illustrating the role of the ligand binding domain of FXR in PGC-1alpha recognition. Together, these results indicate that PGC-1alpha functions as a potent coactivator for FXR and further implicates its role in the regulation of genes that are involved in bile acid and lipid metabolism.

  11. Leucine-induced activation of translational initiation is partly regulated by the branched-chain {alpha}-keto acid dehydrogenase complex in C2C12 cells

    SciTech Connect

    Nakai, Naoya . E-mail: nakai@hss.osaka-u.ac.jp; Shimomura, Yoshiharu; Tamura, Tomohiro; Tamura, Noriko; Hamada, Koichiro; Kawano, Fuminori; Ohira, Yoshinobu

    2006-05-19

    Branched-chain amino acid leucine has been shown to activate the translational regulators through the mammalian target of rapamycin. However, the leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway. The irreversible and rate-limiting step in the leucine oxidation pathway is catalyzed by the branched-chain {alpha}-keto acid dehydrogenase (BCKDH) complex. The complex contains E1 ({alpha}2{beta}2), E2, and E3 subunits, and its activity is abolished by phosphorylation of the E1{alpha} subunit by BCKDH kinase. The relationship between the activity of BCKDH complex and leucine-mediated activation of the protein translation was investigated using the technique of RNA interference. The activity of BCKDH complex in C2C12 cell was modulated by transfection of small interfering RNA (siRNA) for BCKDH E2 subunit or BCKDH kinase. Transfection of siRNAs decreased the mRNA expression and protein amount of corresponding gene. Suppression of either E2 subunit or kinase produced opposite effects on the cell proliferation and the activation of translational regulators by leucine. Suppression of BCKDH kinase for 48 h resulted in decreasing cell proliferation. In contrast, E2 suppression led to increased amount of total cellular protein. The phosphorylation of p70 S6 kinase by leucine was increased in E2-siRNA transfected C2C12 cells, whereas the leucine's effect was diminished in kinase-siRNA transfected cells. These results suggest that the activation of the translational regulators by leucine was partly regulated by the activity of BCKDH complex.

  12. 5-caffeoylquinic acid and caffeic acid down-regulate the oxidative stress- and TNF-alpha-induced secretion of interleukin-8 from Caco-2 cells.

    PubMed

    Zhao, Zhaohui; Shin, Hee Soon; Satsu, Hideo; Totsuka, Mamoru; Shimizu, Makoto

    2008-05-28

    Although chlorogenic acid (CHA) easily reaches a millimolar level in the gastrointestinal tract because of its high concentration in coffee and fruits, its effects on intestinal epithelial cells have been little reported. We investigated in this study the down-regulative effects of 5-caffeoylquinic acid (CQA), the predominant isomer of CHA, on the H(2)O(2-) or TNF-alpha-induced secretion of interleukin (IL)-8, a central pro-inflammatory chemokine involved in the pathogenesis of inflammatory bowel diseases, in human intestinal epithelial Caco-2 cells. After the cells had been pre- and simultaneously treated with CQA, the oversecretion of IL-8 and overexpression of its mRNA induced by H(2)O(2) were significantly suppressed in a dose-dependent manner in the range of 0.25-2.00 mmol/L. We further found that a metabolite of CQA, caffeic acid (CA), but not quinic acid, significantly inhibited the H(2)O(2)-induced IL-8 secretion and its mRNA expression in the same dose-dependent manner. Both CQA and CA suppressed the TNF-alpha-induced IL-8 secretion as well. Caffeic acid at 2.00 mmol/l was able to absolutely block the H(2)O(2)- or TNF-alpha-induced oversecretion of IL-8 in Caco-2 cells. However, CQA and CA did not suppress the TNF-alpha-induced increase in the IL-8 mRNA expression, indicating that the suppressive mechanisms are different between TNF-alpha-induced and H(2)O(2)-induced IL-8 production models. These results suggest that the habit of drinking coffee and/or eating fruits with a high CHA content may be beneficial to humans in preventing the genesis of inflammatory bowel diseases.

  13. Up-regulated expression of transforming growth factor-alpha in the bronchiolar-alveolar duct regions of asbestos-exposed rats.

    PubMed Central

    Liu, J. Y.; Morris, G. F.; Lei, W. H.; Corti, M.; Brody, A. R.

    1996-01-01

    It has become apparent that the numerous growth factors and cytokines are produced during the development of fibroproliferative lung disease. Investigators must sort out which combinations of these factors are playing mechanistic roles in the disease process. Here we demonstrate that transforming growth factor (TGF)-alpha, a potent epithelial and mesenchymal cell mitogen, is upregulated specifically at the sites of asbestos fiber deposition in the lungs of rats exposed for 5 hours. Unexposed animals and those exposed to high concentrations of iron spheres exhibited no increase in TGF-alpha expression at any time during the experiment. Inhaled asbestos fibers deposit initially at the bronchiolar-alveolar duct regions and alveolar macrophages accumulate at these sites within hours. Non-isotopic in situ hybridization and immunohistochemistry were used to show that the mRNA that codes for TGF-alpha along with the peptide were clearly up-regulated at the bronchiolar-alveolar duct regions by 24 hours after the single asbestos exposure. The numbers of labeled cells demonstrated that expression of the mRNA and protein remained significantly above background for at least 2 weeks after exposure along with increased cell proliferation assessed by staining for proliferating cell nuclear antigen. This, to our knowledge, is the first demonstration of TGF-alpha expression at sites of lung injury in developing fibroproliferative disease. This finding supports the hypothesis that the growth factor is involved in the dramatic epithelial and mesenchymal proliferation we documented previously, although additional experiments will be essential to establish the precise role of TGF-alpha. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8686744

  14. [IMPACT OF INDIVIDUAL PERSONALITY FEATURES ON ABILITY TO VOLUNTARY REGULATION OF EXPRESSION EEG ALPHA AND BETA FREQUENCIES].

    PubMed

    Aslanyan, E V; Kiroy, V N; Stoletniy, A S; Lazurenko, D M; Bahtin, O M; Minyaeva, N R; Kiroy, R I

    2015-05-01

    The ability to voluntary control severity of alpha- and beta-2 frequency bands in the parietal and frontal cortical areas was investigated at 17 volunteers using biofeedback. The impact of different personality traits on the effectiveness of control was evaluated. According to the data, it was easier task to decrease expression beta-2 frequency in the frontal cortex than to decline the power of alpha frequency in the parietal cortex. The effectiveness of voluntary control of brain activity is influenced by personality features as extraversion, psychoticism, neuroticism, mobility and steadiness of nerve processes, level of person anxiety. PMID:26263685

  15. Ochnaflavone inhibits TNF-alpha-induced human VSMC proliferation via regulation of cell cycle, ERK1/2, and MMP-9.

    PubMed

    Suh, Seok-Jong; Jin, Un-Ho; Kim, Sung-Hoon; Chang, Hyeun-Wook; Son, Jong-Keun; Lee, Seung Ho; Son, Kun-Ho; Kim, Cheorl-Ho

    2006-12-01

    Ochnaflavone (c-3 of apigenin-0-c-4 of apigenin; OC), a biflavonoid present in the human diet, is known to inhibit angiotensin II-induced hypertrophy and serum-induced smooth muscle cell proliferation. OC is known to have anti-fungal and anti-inflammatory activities. However, it is not known whether OC exerts similar cardioprotective effects in cells treated with tumor necrosis factor (TNF)-alpha. In this study, we isolated OC from Lonicera japonica and studied its effect on matrix metalloproteinase-9 (MMP-9) gene expression in human aortic smooth muscle cells (HASMC). Furthermore, we investigated whether OC exerts the multiple suppressive effects on cytokine TNF-alpha-induced HASMC. Treatment of OC showed its potent inhibitory effects on DNA synthesis of cultured HASMC in the presence of TNF-alpha. These inhibitory effects were associated with reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and G1 cell cycle arrest. Treatment of OC, which induced a cell cycle block in G1-phase, induced downregulation of cyclins and CDKs and upregulation of the CDK inhibitor p21(waf1) expression, whereas upregulation of p27 or p53 by OC was not observed. Because anti-atherogenic effects need not be limited to anti-proliferation, we decided to examine whether OC exerts inhibitory effects on MMP-9 activity in TNF-alpha-induced HASMC. OC inhibited TNF-alpha-induced MMP-9 secretion on HASMC in a dose-dependent manner. This inhibition was characterized by downregulation of MMP-9, which was transcriptionally regulated at nuclear factor (NF)-kappaB site and activation protein (AP)-1 site in the MMP-9 promoter. These findings indicate the efficacy of OC in inhibiting cell proliferation, G1 to S-phase cell cycle progress, and MMP-9 expression through the transcription factors NF-kappaB and AP-1 on TNF-alpha-induced HASMC. The findings of the present study may provide a potential mechanism that explains the anti-atherogenic activity of OC.

  16. Estrogen Receptor beta binds Sp1 and recruits a Corepressor Complex to the Estrogen Receptor alpha Gene Promoter

    PubMed Central

    Bartella, V; Rizza, P; Barone, I; Zito, D; Giordano, F; Giordano, C; Catalano, S; Mauro, L; Sisci, D; Panno, ML; Fuqua, SA; Andò, Sebastiano

    2015-01-01

    Human estrogen receptors (ERs) alpha and beta are crucially involved in the regulation of mammary growth and development. Normal breast tissues display a prevalently expression of ER beta than ER alpha, which drastically increases during breast tumorogenesis. So, it is reasonable to assume how a dysregulation of the two estrogen receptor subtypes may induce breast cancer development. However, the molecular mechanism underlying the opposite role played by the two estrogen receptors on tumor cell growth remains to be elucidated. In the present study, we have demonstrated that ER beta overexpression in breast cancer cells decreases cell proliferation and down-regulates ER alpha mRNA and protein content along with a concomitant repression of estrogen-regulated genes. Transient transfection experiments, using a vector containing the human ER alpha promoter region, showed that elevated levels of the ER beta down-regulated basal ER alpha promoter activity. Furthermore, side-directed mutagenesis and deletion analysis have revealed that the proximal GC-rich motifs at −223 and −214 is crucial for the ER beta-induced ER alpha down-regulation in breast cancer cells. This occurred through ER beta-Sp1 protein-protein interaction within the ER alpha promoter region and the recruitment of a corepressor complex containing NCoR/SMRT (nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor), accompanied by hypoacetylation of histone H4 and displacement of RNA polymerase II. Silencing of NCoR gene expression by RNA interference reversed the down-regulatory effect of ER beta on ER alpha gene expression and cell proliferation. Our results provide evidence for a novel mechanism by which overexpression of ER beta through NCoR is able to down regulate ER alpha gene expression, thus inhibiting ER alpha’s driving role on breast cancer cell growth. PMID:22622808

  17. Simultaneous quantification of GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in human and rat serum.

    PubMed

    Porcu, Patrizia; O'Buckley, Todd K; Alward, Sarah E; Marx, Christine E; Shampine, Lawrence J; Girdler, Susan S; Morrow, A Leslie

    2009-01-01

    The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, p<0.001), (3alpha,5alpha)-3,21-dihydroxypregnan-20-one (3alpha,5alpha-THDOC, +205%, p<0.01), (3alpha,5alpha)-3-hydroxyandrostan-17-one (3alpha,5alpha-A, +216%, p<0.001), (3alpha,5alpha,17beta)-androstane-3,17-diol (3alpha,5alpha-A-diol, +190%, p<0.01). (3alpha,5beta)-3-hydroxypregnan-20-one (3alpha,5beta-THP) and (3alpha,5beta)-3-hydroxyandrostan-17-one (3alpha,5beta-A) were not altered, while (3alpha,5beta)-3,21-dihydroxypregnan-20-one (3alpha,5beta-THDOC) and (3alpha,5beta,17beta)-androstane-3,17-diol (3alpha,5beta-A-diol) were increased from undetectable levels to 271+/-100 and 2.4+/-0.9 pg+/-SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3alpha,5alpha-THP (+1806%, p<0.0001), 3alpha,5beta-THP (+575%, p<0.001), 3alpha,5alpha

  18. CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells.

    PubMed Central

    Ren, S; Li, J; Atweh, G F

    1996-01-01

    Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter. PMID:8628660

  19. Sequential visibility-graph motifs

    NASA Astrophysics Data System (ADS)

    Iacovacci, Jacopo; Lacasa, Lucas

    2016-04-01

    Visibility algorithms transform time series into graphs and encode dynamical information in their topology, paving the way for graph-theoretical time series analysis as well as building a bridge between nonlinear dynamics and network science. In this work we introduce and study the concept of sequential visibility-graph motifs, smaller substructures of n consecutive nodes that appear with characteristic frequencies. We develop a theory to compute in an exact way the motif profiles associated with general classes of deterministic and stochastic dynamics. We find that this simple property is indeed a highly informative and computationally efficient feature capable of distinguishing among different dynamics and robust against noise contamination. We finally confirm that it can be used in practice to perform unsupervised learning, by extracting motif profiles from experimental heart-rate series and being able, accordingly, to disentangle meditative from other relaxation states. Applications of this general theory include the automatic classification and description of physical, biological, and financial time series.

  20. Unravelling daily human mobility motifs.

    PubMed

    Schneider, Christian M; Belik, Vitaly; Couronné, Thomas; Smoreda, Zbigniew; González, Marta C

    2013-07-01

    Human mobility is differentiated by time scales. While the mechanism for long time scales has been studied, the underlying mechanism on the daily scale is still unrevealed. Here, we uncover the mechanism responsible for the daily mobility patterns by analysing the temporal and spatial trajectories of thousands of persons as individual networks. Using the concept of motifs from network theory, we find only 17 unique networks are present in daily mobility and they follow simple rules. These networks, called here motifs, are sufficient to capture up to 90 per cent of the population in surveys and mobile phone datasets for different countries. Each individual exhibits a characteristic motif, which seems to be stable over several months. Consequently, daily human mobility can be reproduced by an analytically tractable framework for Markov chains by modelling periods of high-frequency trips followed by periods of lower activity as the key ingredient.

  1. alpha2 adrenoceptors are involved in the regulation of the gripping-induced immobility episodes in taiep rats.

    PubMed

    Eguibar, José R; Cortés, Ma Del Carmen; Valencia, Jaime; Arias-Montaño, José A

    2006-10-01

    In 1989 Holmgren et al. (Holmgren et al. 1989 Lab Anim Sci 39:226-228) described a new mutant rat that developed a progressive motor disturbance during its lifespan. The syndrome is characterized by a tremor in the hind limbs followed by ataxia, episodes of tonic immobility, epilepsy, and paralysis. The acronym of these symptoms (taiep) became the name of this autosomic, recessive mutant rat. The taiep rats are neurological mutant animals with a hypomyelination, followed by a progressive demyelination process. At 7-8 months of age, taiep rats develop immobility episodes (IEs) characterized by a cortical desynchronization, associated with the theta rhythm in the hippocampus and changes of the nucal electromyogram (EMG), whose pattern is like rapid-eye-movement (REM) sleep. These rats also show an altered sleep pattern with an equal REM sleep distribution. This study analyzed therole of alpha(2) adrenoceptors in the expression of gripping-induced IEs in 8-month-old male taiep rats. The alpha(2) adrenoceptor agonists clonidine and xylacine increased the frequency of gripping-induced IEs whereas the alpha(2) antagonists yohimbine and idazoxandecreased or prevented such episodes. These findings correlate with the pharmacological observations in narcoleptic dogs and humans in which alpha(2) adrenergic mechanisms are involved in the modulation of cataplexy. Unexpectedly, the repetitive administration of clonidine resulted in jumping behavior, indicative of phasic activation of extensor musculature. Taken together, our results show that alpha(2) adrenoceptors are involved in the modulation in gripping-induced IEs and after the administration of several doses of clonidine produced phasic motor activation.

  2. Hyperinsulinemia may boost both hematocrit and iron absorption by up-regulating activity of hypoxia-inducible factor-1alpha.

    PubMed

    McCarty, M F

    2003-01-01

    There is growing evidence that increases in both hematocrit and body iron stores are components of the insulin resistance syndrome. The ability of insulin and of IGF-I - whose effective activity is increased in the context of insulin resistance - to boost activity of the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha), may be at least partially responsible for this association. HIF-1alpha, which functions physiologically as a detector of both hypoxia and iron-deficiency, promotes synthesis of erythropoietin, and may also mediate the up-regulatory impact of hypoxia on intestinal iron absorption. Insulin/IGF-I may also influence erythropoiesis more directly, as they are growth factors for developing reticulocytes. Conversely, the activation of HIF-1alpha associated with iron deficiency may be responsible for the increased glucose tolerance noted in iron-deficient animals; HIF-1alpha promotes efficient glucose uptake and glycolysis - a sensible adaptation to hypoxia - by inducing increased synthesis of glucose transporters and glycolytic enzymes. Recent reports that phlebotomy can increase the efficiency of muscle glucose uptake in lean healthy omnivores are intriguing and require further confirmation. Whether increased iron stores contribute to the elevated vascular risk associated with insulin resistance is doubtful, inasmuch as most prospective studies fail to correlate serum ferritin or transferrin saturation with subsequent vascular events. However, current data are reasonably consistent with the possibility that moderately elevated iron stores are associated with increased overall risk for cancer - and for colorectal cancer in particular; free iron may play a catalytic role in 'spontaneous' mutagenesis. Thus, iron excess may mediate at least some of the increased cancer risk associated with insulin resistance and heme-rich diets. People who are insulin resistant can minimize any health risk associated with iron overload by avoiding heme

  3. Allicin inhibits SDF-1alpha-induced T cell interactions with fibronectin and endothelial cells by down-regulating cytoskeleton rearrangement, Pyk-2 phosphorylation and VLA-4 expression.

    PubMed

    Sela, Uri; Ganor, Sharon; Hecht, Iris; Brill, Alexander; Miron, Talia; Rabinkov, Aharon; Wilchek, Meir; Mirelman, David; Lider, Ofer; Hershkoviz, Rami

    2004-04-01

    Allicin, a major ingredient of fresh garlic extract that is produced during the crushing of garlic cloves, exerts various beneficial biological effects, including a broad spectrum of antimicrobial activity, antihyperlipidaemic and antihypertensive effects. However, how allicin affects the immune system is less well known, and its effect on human T cells has never been studied. Here, we examined the in-vitro effects of allicin on the functioning of T cells related to their entry to inflamed extravascular sites. We found that allicin (20-100 microm) inhibits the SDF-1alpha (CXCL12)-induced T cell migration through fibronectin (FN), and that this inhibition is mediated by the down-regulation of (i) the reorganization of cortical actin and the subsequent T cell polarization, and (ii) T cell adhesion to FN. Moreover, allicin also inhibited T cell adhesion to endothelial cells and transendothelial migration. The mechanisms underlying these inhibitory effects of allicin are associated with its ability to down-regulate the phosphorylation of Pyk2, an intracellular member of the focal adhesion kinases, and to reduce the expression of the VCAM-1- and FN-specific alpha4beta1-integrin (VLA-4). The ability of allicin to down-regulate these chemokine-induced and VLA-4-mediated T cell functions explains its beneficial biological effects in processes where T cells play an important role and suggests that allicin may be used therapeutically with chronic inflammatory diseases. PMID:15056375

  4. Estrogen receptor alpha transcriptionally activates casein kinase 2 alpha: A pivotal regulator of promyelocytic leukaemia protein (PML) and AKT in oncogenesis.

    PubMed

    Das, Nilanjana; Datta, Neerajana; Chatterjee, Uttara; Ghosh, Mrinal Kanti

    2016-06-01

    Protein kinase CK2α is frequently upregulated in different cancers. Alteration of CK2α expression and its activity is sufficient to induce dramatic changes in cell fate. It has been established that CK2α induces oncogenesis through modulation of both AKT and PML. CK2α has been found to be overexpressed in breast cancer. In contrary, statistical reports have shown low level of PML. However, the regulation of CK2α gene expression is not fully understood. In the current study, we found that CK2α and activated AKT positively correlate with ERα, whereas PML follows an inverse correlation in human breast cancer tissues. Modulation of ERα signalling leads to recruitment of activated ERα on the ERE sites of CK2α promoter, resulting in CK2α transactivation. Furthermore, the DMBA induced tumours in rat showed elevated level of active CK2α. Consequently it mediates enhancement of AKT activity and PML degradation, resulting in increased cellular proliferation, migration and metastasis. Syngeneic ERα overexpressing stable mouse 4T1 cells produce larger primary tumours and metastatic lung nodules in mice, corroborating our in vitro findings. Hence, our study provides a novel route of ERα dependent CK2α mediated oncogenesis that causes upregulation and consequent AKT activation along with degradation of tumour suppressor PML. PMID:27012497

  5. Selective activation of p38alpha and p38gamma by hypoxia. Role in regulation of cyclin D1 by hypoxia in PC12 cells.

    PubMed

    Conrad, P W; Rust, R T; Han, J; Millhorn, D E; Beitner-Johnson, D

    1999-08-13

    Hypoxic/ischemic trauma is a primary factor in the pathology of a multitude of disease states. The effects of hypoxia on the stress- and mitogen-activated protein kinase signaling pathways were studied in PC12 cells. Exposure to moderate hypoxia (5% O(2)) progressively stimulated phosphorylation and activation of p38gamma in particular, and also p38alpha, two stress-activated protein kinases. In contrast, hypoxia had no effect on enzyme activity of p38beta, p38beta(2), p38delta, or on c-Jun N-terminal kinase, another stress-activated protein kinase. Prolonged hypoxia also induced phosphorylation and activation of p42/p44 mitogen-activated protein kinase, although this activation was modest compared with nerve growth factor- and ultraviolet light-induced activation. Hypoxia also dramatically down-regulated immunoreactivity of cyclin D1, a gene that is known to be regulated negatively by p38 at the level of gene expression (Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996) J. Biol. Chem. 271, 20608-20616). This effect was partially blocked by SB203580, an inhibitor of p38alpha but not p38gamma. Overexpression of a kinase-inactive form of p38gamma was also able to reverse in part the effect of hypoxia on cyclin D1 levels, suggesting that p38alpha and p38gamma converge to regulate cyclin D1 during hypoxia. These studies demonstrate that an extremely typical physiological stress (hypoxia) causes selective activation of specific p38 signaling elements; and they also identify a downstream target of these pathways. PMID:10438538

  6. Comparative genomic analysis of upstream miRNA regulatory motifs in Caenorhabditis.

    PubMed

    Jovelin, Richard; Krizus, Aldis; Taghizada, Bakhtiyar; Gray, Jeremy C; Phillips, Patrick C; Claycomb, Julie M; Cutter, Asher D

    2016-07-01

    MicroRNAs (miRNAs) comprise a class of short noncoding RNA molecules that play diverse developmental and physiological roles by controlling mRNA abundance and protein output of the vast majority of transcripts. Despite the importance of miRNAs in regulating gene function, we still lack a complete understanding of how miRNAs themselves are transcriptionally regulated. To fill this gap, we predicted regulatory sequences by searching for abundant short motifs located upstream of miRNAs in eight species of Caenorhabditis nematodes. We identified three conserved motifs across the Caenorhabditis phylogeny that show clear signatures of purifying selection from comparative genomics, patterns of nucleotide changes in motifs of orthologous miRNAs, and correlation between motif incidence and miRNA expression. We then validated our predictions with transgenic green fluorescent protein reporters and site-directed mutagenesis for a subset of motifs located in an enhancer region upstream of let-7 We demonstrate that a CT-dinucleotide motif is sufficient for proper expression of GFP in the seam cells of adult C. elegans, and that two other motifs play incremental roles in combination with the CT-rich motif. Thus, functional tests of sequence motifs identified through analysis of molecular evolutionary signatures provide a powerful path for efficiently characterizing the transcriptional regulation of miRNA genes. PMID:27140965

  7. Improvement of cloned [alpha]-amylase gene expression in fed-batch culture of recombinant Saccharomyces cerevisiae by regulating both glucose and ethanol concentrations using a fuzzy controller

    SciTech Connect

    Shiba, Sumihisa; Nishida, Yoshio; Park, Y.S.; Iijima, Shinji; Kobayashi, Takeshi . Dept. of Biotechnology)

    1994-11-05

    The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B-12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the [alpha]-amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. To increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy controller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed-batch culture. The synthesis of [alpha]-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory [alpha]-amylase activities of cells harboring plasmids pNA3 and pNA7 in fed-batch culture were 175 and 392 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled.

  8. A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha.

    PubMed

    Lee, Kyung Sun; Kim, So Ri; Park, Hee Sun; Park, Seoung Ju; Min, Kyung Hoon; Lee, Ka Young; Choe, Yeong Hun; Hong, Sang Hyun; Han, Hyo Jin; Lee, Young Rae; Kim, Jong Suk; Atlas, Daphne; Lee, Yong Chul

    2007-12-31

    Reactive oxygen species (ROS) play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. Recent studies have demonstrated that antioxidants are able to reduce airway inflammation and hyperreactivity in animal models of allergic airway disease. A newly developed antioxidant, small molecular weight thiol compound, N-acetylcysteine amide (AD4) has been shown to increase cellular levels of glutathione and to attenuate oxidative stress related disorders such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. However, the effects of AD4 on allergic airway disease such as asthma are unknown. We used ovalbumin (OVA)-inhaled mice to evaluate the role of AD4 in allergic airway disease. In this study with OVA-inhaled mice, the increased ROS generation, the increased levels of Th2 cytokines and VEGF, the increased vascular permeability, the increased mucus production, and the increased airway resistance in the lungs were significantly reduced by the administration of AD4. We also found that the administration of AD4 decreased the increases of the NF-kappaB and hypoxia-inducible factor-1alpha (HIF-1alpha) levels in nuclear protein extracts of lung tissues after OVA inhalation. These results suggest that AD4 attenuates airway inflammation and hyperresponsiveness by regulating activation of NF-kappaB and HIF-1alpha as well as reducing ROS generation in allergic airway disease. PMID:18160846

  9. Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation.

    PubMed Central

    Weyrich, A S; McIntyre, T M; McEver, R P; Prescott, S M; Zimmerman, G A

    1995-01-01

    Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues. Images PMID:7537762

  10. A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha.

    PubMed

    Lee, Kyung Sun; Kim, So Ri; Park, Hee Sun; Park, Seoung Ju; Min, Kyung Hoon; Lee, Ka Young; Choe, Yeong Hun; Hong, Sang Hyun; Han, Hyo Jin; Lee, Young Rae; Kim, Jong Suk; Atlas, Daphne; Lee, Yong Chul

    2007-12-31

    Reactive oxygen species (ROS) play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. Recent studies have demonstrated that antioxidants are able to reduce airway inflammation and hyperreactivity in animal models of allergic airway disease. A newly developed antioxidant, small molecular weight thiol compound, N-acetylcysteine amide (AD4) has been shown to increase cellular levels of glutathione and to attenuate oxidative stress related disorders such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. However, the effects of AD4 on allergic airway disease such as asthma are unknown. We used ovalbumin (OVA)-inhaled mice to evaluate the role of AD4 in allergic airway disease. In this study with OVA-inhaled mice, the increased ROS generation, the increased levels of Th2 cytokines and VEGF, the increased vascular permeability, the increased mucus production, and the increased airway resistance in the lungs were significantly reduced by the administration of AD4. We also found that the administration of AD4 decreased the increases of the NF-kappaB and hypoxia-inducible factor-1alpha (HIF-1alpha) levels in nuclear protein extracts of lung tissues after OVA inhalation. These results suggest that AD4 attenuates airway inflammation and hyperresponsiveness by regulating activation of NF-kappaB and HIF-1alpha as well as reducing ROS generation in allergic airway disease.

  11. Interconnected network motifs control podocyte morphology and kidney function.

    PubMed

    Azeloglu, Evren U; Hardy, Simon V; Eungdamrong, Narat John; Chen, Yibang; Jayaraman, Gomathi; Chuang, Peter Y; Fang, Wei; Xiong, Huabao; Neves, Susana R; Jain, Mohit R; Li, Hong; Ma'ayan, Avi; Gordon, Ronald E; He, John Cijiang; Iyengar, Ravi

    2014-02-01

    Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease. PMID:24497609

  12. Relationship between expression of serendipity alpha and cellularisation of the Drosophila embryo as revealed by interspecific transformation.

    PubMed

    Ibnsouda, S; Schweisguth, F; de Billy, G; Vincent, A

    1993-10-01

    A dramatic reorganization of the cytoskeleton underlies the cellularisation of the syncytial Drosophila embryo. Formation of a regular network of acto-myosin filaments, providing a structural framework, and possibly a contractile force as well, appears essential for the synchronous invagination of the plasma membrane between adjacent nuclei. The serendipity alpha (sry alpha) gene is required for this complete reorganization of the microfilaments at the onset of membrane invagination. We compare here the structure and expression of sry alpha between D. pseudoobscura, D. subobscura and D. melanogaster. Interspersion of evolutionarily highly conserved and divergent regions is observed in the protein. One such highly conserved region shows sequence similarities to a motif found in proteins of the ezrin-radixin-moesin (ERM) family. Four 7-13 bp motifs are conserved in the 5' promoter region; two of these are also found, and at the same position relative to the TATA box, in nullo, another zygotic gene recently shown to be involved in cellularisation. The compared patterns of expression of D. melanogaster sry alpha and nullo, and D. pseudoobscura sry alpha reveal a complex regulation of the spatiotemporal accumulation of their transcripts. The D. pseudoobscura sry alpha gene is able to rescue the cellularisation defects associated with a complete loss of sry alpha function in D. melanogaster embryos, even though species-specific aspects of its expression are maintained. Despite their functional homologies, the D. melanogaster and D. pseudoobscura sry alpha RNAs have different subcellular localisations, suggesting that this specific localization has no conserved role in targeting the sry alpha protein to the apical membranes. PMID:8287797

  13. Neural Circuits: Male Mating Motifs.

    PubMed

    Benton, Richard

    2015-09-01

    Characterizing microcircuit motifs in intact nervous systems is essential to relate neural computations to behavior. In this issue of Neuron, Clowney et al. (2015) identify recurring, parallel feedforward excitatory and inhibitory pathways in male Drosophila's courtship circuitry, which might explain decisive mate choice.

  14. Redox active motifs in selenoproteins

    PubMed Central

    Li, Fei; Lutz, Patricia B.; Pepelyayeva, Yuliya; Arnér, Elias S. J.; Bayse, Craig A.; Rozovsky, Sharon

    2014-01-01

    Selenoproteins use the rare amino acid selenocysteine (Sec) to act as the first line of defense against oxidants, which are linked to aging, cancer, and neurodegenerative diseases. Many selenoproteins are oxidoreductases in which the reactive Sec is connected to a neighboring Cys and able to form a ring. These Sec-containing redox motifs govern much of the reactivity of selenoproteins. To study their fundamental properties, we have used 77Se NMR spectroscopy in concert with theoretical calculations to determine the conformational preferences and mobility of representative motifs. This use of 77Se as a probe enables the direct recording of the properties of Sec as its environment is systematically changed. We find that all motifs have several ring conformations in their oxidized state. These ring structures are most likely stabilized by weak, nonbonding interactions between the selenium and the amide carbon. To examine how the presence of selenium and ring geometric strain governs the motifs’ reactivity, we measured the redox potentials of Sec-containing motifs and their corresponding Cys-only variants. The comparisons reveal that for C-terminal motifs the redox potentials increased between 20–25 mV when the selenenylsulfide bond was changed to a disulfide bond. Changes of similar magnitude arose when we varied ring size or the motifs’ flanking residues. This suggests that the presence of Sec is not tied to unusually low redox potentials. The unique roles of selenoproteins in human health and their chemical reactivities may therefore not necessarily be explained by lower redox potentials, as has often been claimed. PMID:24769567

  15. E-cadherin and alpha-, beta-, and gamma-catenin protein expression is up-regulated in ovarian carcinoma cells in serous effusions.

    PubMed

    Davidson, B; Berner, A; Nesland, J M; Risberg, B; Berner, H S; Tropè, C G; Kristensen, G B; Bryne, M; Ann Florenes, V

    2000-12-01

    The aims of this study were firstly, to investigate the expression of E-cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty-seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E-cadherin and alpha-, beta-, and gamma-catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E-cadherin and alpha-, beta-, and gamma-catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E-cadherin and alpha-catenin expression, in both effusions and solid tumours, and for beta-catenin in solid tumours (range p<0. 001 to p=0.014). Up-regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up-regulation of alpha-, beta-, and gamma-catenin when compared with primary carcinomas (p=0.002-0. 015). Carcinoma cells in effusions showed in addition elevated levels of E-cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co-precipitation of E-cadherin and beta-catenin was seen in ten specimens examined. E-cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with

  16. Expression of hepatitis B virus S gene by herpes simplex virus type 1 vectors carrying alpha- and beta-regulated gene chimeras.

    PubMed

    Shih, M F; Arsenakis, M; Tiollais, P; Roizman, B

    1984-09-01

    The domain of the hepatitis B virus (HBV) S gene specifying the HBV surface antigen (HBsAg) and comprising 25 base pairs of the 5'-transcribed noncoding region, the structural gene sequences, and the 3'-noncoding gene sequences including the polyadenylylation site was fused to the promoter-regulatory regions of the beta-thymidine kinase and of the alpha 4 gene of herpes simplex virus type 1 (HSV-1). The chimeric constructs were then inserted into the HSV-1 genome and specifically into the thymidine kinase gene by homologous recombination through flanking sequences. Cells infected with recombinants carrying the chimeric genes produced and excreted the HBsAg into the extracellular medium for at least 12 hr concurrently with the multiplication of the HSV-1 vector. The temporal patterns of expression and the observation that HBV S gene linked to the HSV-1 alpha promoter-regulatory region was regulated as an HSV-1 alpha gene indicate that the HBsAg gene chimeras inserted into the virus were regulated as viral genes. The HBsAg banded in isopycnic CsCl density gradients at a density of 1.17 g/cm3. Electron microscopic studies revealed that HBsAg harvested from the extracellular medium and banded in CsCl density gradients contained spherical particles 15-22 nm in diameter, characteristic of empty HBV envelopes. The results indicate that HSV-1 is a suitable vector for the expression of foreign genes placed under the control of HSV promoter-regulatory regions.

  17. Receptor-selective retinoids implicate retinoic acid receptor alpha and gamma in the regulation of bmp-2 and bmp-4 in F9 embryonal carcinoma cells.

    PubMed

    Rogers, M B

    1996-01-01

    The effect of retinoids on malignant cells and embryos indicates that retinoids influence the expression of growth factors or alter the response of cells to growth factors. The bone morphogenetic proteins, Bmp-2 and Bmp-4, are candidates for such growth factors because retinoic acid (RA) treatment of F9 embryonal carcinoma cells induced Bmp-2 mRNA, while simultaneously repressing Bmp-4 levels. Also, recombinant Bmp-2 affected the growth and differentiation of these cells. Regulation of each gene was concentration dependent and required continuous RA treatment. The short half-lives of the Bmp-2 (75 +/- 11 min) and Bmp-4 (70 +/- 4 min) mRNAs suggest that their abundance is primarily controlled at the transcriptional level. To determine which RA receptor (RAR) controls bmp-2 and bmp-4 expression, F9 cells were exposed to various receptor-selective retinoids. RAR alpha- and gamma-selective retinoids induced Bmp-2 and repressed Bmp-4 equally as well as all-trans RA. In contrast, a RAR beta-selective retinoid had little effect on Bmp-2 induction but repressed Bmp-4. A RAR alpha-selective antagonist inhibited all-trans RA stimulation of Bmp-2, although not as dramatically as a RAR beta gamma-selective antagonist. No differences were observed between Bmp levels in all-trans RA and 9-cis RA-treated cells, indicating that the RXRs play little part in controlling these genes. The results are consistent with RAR alpha and gamma-controlled Bmp-2 and Bmp-4 regulation.

  18. ISOLATION OF AN ACTIVE LV1 GENE FROM CATTLE INDICATES THAT TRIPARTITE MOTIF PROTEIN-MEDIATED INNATE IMMUNITY TO RETROVIRAL INFECTION IS WIDESPREAD AMONG MAMMALS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lv1/TRIM5alpha (tripartite motif 5alpha) has recently emerged as an important factor influencing species-specific permissivity to retroviral infection in a range of primates, including humans. Old World monkey TRIM5alpha blocks human immunodeficiency virus type 1 (HIV-1) infectivity, and the human a...

  19. Functional whole-genome analysis identifies Polo-like kinase 2 and poliovirus receptor as essential for neuronal differentiation upstream of the negative regulator alphaB-crystallin.

    PubMed

    Draghetti, Cristina; Salvat, Catherine; Zanoguera, Francisca; Curchod, Marie-Laure; Vignaud, Chloé; Peixoto, Helene; Di Cara, Alessandro; Fischer, David; Dhanabal, Mohanraj; Andreas, Goutopoulos; Abderrahim, Hadi; Rommel, Christian; Camps, Montserrat

    2009-11-13

    This study aimed at identifying transcriptional changes associated to neuronal differentiation induced by six distinct stimuli using whole-genome microarray hybridization analysis. Bioinformatics analyses revealed the clustering of these six stimuli into two categories, suggesting separate gene/pathway dependence. Treatment with specific inhibitors demonstrated the requirement of both Janus kinase and microtubule-associated protein kinase activation to trigger differentiation with nerve growth factor (NGF) and dibutyryl cAMP. Conversely, activation of protein kinase A, phosphatidylinositol-3-kinase alpha, and mammalian target of rapamycin, although required for dibutyryl cAMP-induced differentiation, exerted a negative feedback on NGF-induced differentiation. We identified Polo-like kinase 2 (Plk2) and poliovirus receptor (PVR) as indispensable for NGF-driven neuronal differentiation and alphaB-crystallin (Cryab) as an inhibitor of this process. Silencing of Plk2 or PVR blocked NGF-triggered differentiation and Cryab down-regulation, while silencing of Cryab enhanced NGF-induced differentiation. Our results position both Plk2 and PVR upstream of the negative regulator Cryab in the pathway(s) leading to neuronal differentiation triggered by NGF.

  20. Proteomic identification of non-erythrocytic alpha-spectrin-1 down-regulation in the pre-optic area of neonatally estradiol-17β treated female adult rats.

    PubMed

    Govindaraj, Vijayakumar; Rao, Addicam Jagannadha

    2016-06-01

    It is well established that sexually dimorphic brain regions, which are critical for reproductive physiology and behavior, are organized by steroid hormones during the first 2 weeks after birth in the rodents. In our recent observation, neonatal exposure to estradiol-17β (E2) in the female rat revealed increase in cyclooxygenase 2 (COX-2) level, sexually dimorphic nucleus (SDN)-pre-optic area (POA) size and down-regulation of synaptogenesis related genes in POA in the adult stage. In the present study, using the same animal model, the protein profile of control and neonatally E2-treated POA was compared by 1D-SDS-PAGE, and the protein that shows a change in abundance was identified by LC-MS/MS analysis. Results indicated that there was a single protein band, which was down-regulation in E2-treated POA and it was identified as spectrin alpha chain, non-erythrocytic 1 (SPTAN1). Consistently, the down-regulation of SPTAN1 expression was also confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The SPTAN1 was identified as a cytoskeletal protein that is involved in stabilization of the plasma membrane and organizes intracellular organelles, and it has been implicated in cellular functions including DNA repair and cell cycle regulation. The evidence shows that any mutation in spectrins causes impairment of synaptogenesis and other neurological disorders. Also, protein-protein interaction analysis of SPTAN1 revealed a strong association with proteins such as kirrel, actinin, alpha 4 (ACTN4) and vinculin (VCL) which are implicated in sexual behavior, masculinization and defeminization. Our results indicate that SPTAN1 expression in the developing rat brain is sexually dimorphic, and we suggest that this gene may mediate E2-17β-induced masculinization and defeminization, and disrupted reproductive function in the adult stage. PMID:27166725

  1. Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

    PubMed

    Rastaldi, M P; Candiano, G; Musante, L; Bruschi, M; Armelloni, S; Rimoldi, L; Tardanico, R; Sanna-Cherchi, S; Cherchi, S Sanna; Ferrario, F; Montinaro, V; Haupt, R; Parodi, S; Carnevali, M L; Allegri, L; Camussi, G; Gesualdo, L; Scolari, F; Ghiggeri, G M

    2006-08-01

    Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

  2. Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells.

    PubMed

    Seo, Hye Sook; Leclercq, Guy

    2002-01-01

    The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the

  3. Alpha-hederin, but not hederacoside C and hederagenin from Hedera helix, affects the binding behavior, dynamics, and regulation of beta 2-adrenergic receptors.

    PubMed

    Sieben, Anne; Prenner, Lars; Sorkalla, Thomas; Wolf, Anne; Jakobs, Daniel; Runkel, Frank; Häberlein, Hanns

    2009-04-21

    Hederacoside C, alpha-hederin, and hederagenin are saponins of dry extracts obtained from the leaves of ivy (Hedera helix L.). Internalization of beta(2)-adrenergic receptor-GFP fusion proteins after stimulation with 1 microM terbutaline was inhibited by preincubation of stably transfected HEK293 cells with 1 microM alpha-hederin for 24 h, whereas neither hederacoside C nor hederagenin (1 microM each) influenced this receptor regulation. After incubation of A549 cells with 5 nM Alexa532-NA, two different diffusion time constants were found for beta(2)AR-Alexa532-NA complexes by fluorescence correlation spectroscopy. Evaluation of the autocorrelation curve revealed diffusion time constants: tau(bound1) = 1.4 +/- 1.1 ms (n = 6) found for receptor-ligand complexes with unrestricted lateral mobility, and tau(bound2) = 34.7 +/- 14.1 ms (n = 6) for receptor-ligand complexes with hindered mobility. The distribution of diffusion time constants was 24.3 +/- 2.5% for tau(bound1) and 8.7 +/- 4.3% for tau(bound2) (n = 6). A549 cells pretreated with 1 microM alpha-hederin for 24 h showed dose-dependent alterations in this distribution with 37.1 +/- 5.5% for tau(bound1) and 4.1 +/- 1.1% for tau(bound2). Simultaneously, the level of Alexa532-NA binding was significantly increased from 33.0 +/- 6.8 to 41.2 +/- 4.6%. In saturation experiments, alpha-hederin did not influence the beta(2)-adrenergic receptor density (B(max)), whereas the K(D) value for Alexa532-NA binding decreased from 36.1 +/- 9.2 to 24.3 +/- 11.1 nM. Pretreatment of HASM cells with alpha-hederin (1 microM, 24 h) revealed an increased intracellular cAMP level of 13.5 +/- 7.0% under stimulating conditions. Remarkably, structure-related saponins like hederacoside C and hederagenin did not influence either the binding behavior of beta(2)AR or the intracellular cAMP level.

  4. Annotating RNA motifs in sequences and alignments

    PubMed Central

    Gardner, Paul P.; Eldai, Hisham

    2015-01-01

    RNA performs a diverse array of important functions across all cellular life. These functions include important roles in translation, building translational machinery and maturing messenger RNA. Mor