<PubmedArticleSet>
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<PubmedArticle>
<MedlineID>21256213</MedlineID>
<PMID>11357136</PMID>
<ArticleTitle><Protname>LDL-receptor-related protein 6</Protname> is a receptor for Dickkopf proteins.</ArticleTitle>
<AbstractText><Protname>Wnt glycoproteins</Protname> have been implicated in diverse processes during embryonic patterning in metazoa. They signal through frizzled-type seven-transmembrane-domain receptors to stabilize <Protname>beta-catenin</Protname>. <Protname>Wnt</Protname> signalling is antagonized by the extracellular <Protname>Wnt</Protname> inhibitor <Protname>dickkopf1</Protname> (<Protname>dkk1</Protname>), which is a member of a multigene family. <Protname>dkk1</Protname> was initially identified as a head inducer in Xenopus embryos but the mechanism by which it blocks <Protname>Wnt</Protname> signalling is unknown. <Protname>LDL-receptor-related protein 6</Protname> (<Protname>LRP6</Protname>) is required during <Protname>Wnt</Protname>/<Protname>beta-catenin</Protname> signalling in Drosophila, Xenopus and mouse, possibly acting as a co-receptor for <Protname>Wnt</Protname>. Here we show that <Protname>LRP6</Protname> (ref. 7) is a specific, high-affinity receptor for <Protname>Dkk1</Protname> and <Protname>Dkk2</Protname>. <Protname>Dkk1</Protname> blocks <Protname>LRP6</Protname>-mediated <Protname>Wnt</Protname>/<Protname>beta-catenin</Protname> signalling by interacting with domains that are distinct from those required for <Protname>Wnt</Protname>/<Protname>Frizzled</Protname> interaction. <Protname>dkk1</Protname> and <Protname>LRP6</Protname> interact antagonistically during embryonic head induction in Xenopus where <Protname>LRP6</Protname> promotes the posteriorizing role of <Protname>Wnt</Protname>/<Protname>beta-catenin</Protname> signalling. Thus, DKKs inhibit <Protname>Wnt</Protname> co-receptor function, exemplifying the modulation of LRP signalling by antagonists.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21199558</MedlineID>
<PMID>11302704</PMID>
<ArticleTitle>Homo-oligomerisation and nuclear localisation of <Protname>mouse histone deacetylase 1</Protname>.</ArticleTitle>
<AbstractText>Reversible <Protname>histone</Protname> acetylation changes the chromatin structure and can modulate gene transcription. <Protname>Mammalian histone deacetylase 1</Protname> (<Protname>HDAC1</Protname>) is a nuclear protein that belongs to a growing family of evolutionarily conserved enzymes catalysing the removal of acetyl residues from core histones and other proteins. Previously, we have identified <Protname>murine HDAC1</Protname> as a growth factor-inducible protein in murine T-cells. Here, we characterise the molecular function of <Protname>mouse HDAC1</Protname> in more detail. Co-immunoprecipitation experiments with epitope-tagged <Protname>HDAC1 protein</Protname> reveal the association with endogenous <Protname>HDAC1 enzyme</Protname>. We show that <Protname>HDAC1</Protname> can homo-oligomerise and that this interaction is dependent on the N-terminal <Protname>HDAC</Protname> association domain of the protein. Furthermore, the same <Protname>HDAC1</Protname> domain is also necessary for in vitro binding of <Protname>HDAC2</Protname> and <Protname>HDAC3</Protname>, association with <Protname>RbAp48</Protname> and for catalytic activity of the enzyme. A lysine-rich sequence within the carboxy terminus of <Protname>HDAC1</Protname> is crucial for nuclear localisation of the enzyme. We identify a C-terminal nuclear localisation domain, which is sufficient for the transport of <Protname>HDAC1</Protname> and of reporter fusion proteins into the nucleus. Alternatively, <Protname>HDAC1</Protname> can be shuttled into the nucleus by association with another <Protname>HDAC1 molecule</Protname> via its N-terminal <Protname>HDAC</Protname> association domain. Our results define two domains, which are essential for the oligomerisation and nuclear localisation of <Protname>mouse HDAC1</Protname>. Copyright 2001 Academic Press.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21264574</MedlineID>
<PMID>11278544</PMID>
<ArticleTitle>Interaction of <Protname>p130</Protname> with, and consequent inhibition of, the catalytic subunit of <Protname>protein phosphatase 1alpha</Protname>.</ArticleTitle>
<AbstractText>The <Protname>protein p130</Protname> was originally isolated from rat brain as an <Protname>inositol 1,4,5-trisphosphate-binding protein</Protname> with a domain organization similar to that of <Protname>phospholipase C-delta1</Protname> but which lacks <Protname>phospholipase C</Protname> activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with <Protname>p130</Protname> has now led to the identification of the catalytic subunit of <Protname>protein phosphatase 1alpha</Protname> (<Protname>PP1calpha</Protname>) as a <Protname>p130-binding protein</Protname>. The association between <Protname>p130</Protname> and <Protname>PP1calpha</Protname> was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of <Protname>p130</Protname> with <Protname>PP1calpha</Protname> resulted in inhibition of the catalytic activity of the latter in a <Protname>p130</Protname> concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express <Protname>p130</Protname> and of mouse brain extract with antibodies to <Protname>p130</Protname> and to <Protname>PP1calpha</Protname> also detected the presence of a complex of <Protname>p130</Protname> and <Protname>PP1calpha</Protname>. The activity of <Protname>glycogen phosphorylase</Protname>, which is negatively regulated by dephosphorylation by <Protname>PP1calpha</Protname>, was higher in COS-1 cells that stably express <Protname>p130</Protname> than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca(2+) signaling, <Protname>p130</Protname> might also contribute to regulation of protein dephosphorylation through its interaction with <Protname>PP1calpha</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21133166</MedlineID>
<PMID>11242053</PMID>
<ArticleTitle>Methylation of <Protname>histone H3</Protname> lysine 9 creates a binding site for <Protname>HP1</Protname> proteins.</ArticleTitle>
<AbstractText>Distinct modifications of <Protname>histone</Protname> amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. In addition to <Protname>histone</Protname> modifications that facilitate gene activity, it is of similar importance to restrict inappropriate gene expression if cellular and developmental programmes are to proceed unperturbed. Here we show that mammalian methyltransferases that selectively methylate <Protname>histone H3</Protname> on lysine 9 (<Protname>Suv39h HMTases</Protname>) generate a binding site for <Protname>HP1</Protname> proteins--a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure. High-affinity in vitro recognition of a methylated <Protname>histone H3</Protname> peptide by <Protname>HP1</Protname> requires a functional chromo domain; thus, the <Protname>HP1</Protname> chromo domain is a specific interaction motif for the methyl epitope on lysine9 of <Protname>histone H3</Protname>. In vivo, heterochromatin association of <Protname>HP1</Protname> proteins is lost in <Protname>Suv39h</Protname> double-null primary mouse fibroblasts but is restored after the re-introduction of a catalytically active <Protname>SWUV39H1 HMTase</Protname>. Our data define a molecular mechanism through which the <Protname>SUV39H</Protname>-<Protname>HP1</Protname> methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21073547</MedlineID>
<PMID>11206060</PMID>
<ArticleTitle>Delineation of the <Protname>calcineurin</Protname>-interacting region of <Protname>cyclophilin B</Protname>.</ArticleTitle>
<AbstractText>The immunosuppressant drug cyclosporin A (<Protname>CsA</Protname>) inhibits T-cell function by blocking the phosphatase activity of <Protname>calcineurin</Protname>. This effect is mediated by formation of a complex between the drug and <Protname>cyclophilin</Protname> (<Protname>CyP</Protname>), which creates a composite surface able to make high-affinity contacts with <Protname>calcineurin</Protname>. In vitro, the <Protname>CyPB</Protname>/<Protname>CsA</Protname> complex is more effective in inhibiting <Protname>calcineurin</Protname> than the <Protname>CyPA</Protname>/<Protname>CsA</Protname> and <Protname>CyPC</Protname>/<Protname>CsA</Protname> complexes, pointing to fine structural differences in the <Protname>calcineurin</Protname>-binding region. To delineate the <Protname>calcineurin</Protname>-binding region of <Protname>CyPB</Protname>, we mutated several amino acids, located in two loops corresponding to <Protname>CyPA</Protname> regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type <Protname>CyPB</Protname>, the G77H, D155R, and D158R mutants had intact isomerase and <Protname>CsA</Protname>-binding activities, indicating that no major conformational changes had taken place. When complexed to <Protname>CsA</Protname>, they all displayed only reduced affinity for <Protname>calcineurin</Protname> and much decreased inhibition of <Protname>calcineurin</Protname> phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of <Protname>CyPB</Protname>/<Protname>CsA</Protname> with <Protname>calcineurin</Protname>, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in <Protname>CyPA</Protname> and might therefore account for the higher affinity of the <Protname>CyPB</Protname>/<Protname>CsA</Protname> complex for <Protname>calcineurin</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21092952</MedlineID>
<PMID>11171121</PMID>
<ArticleTitle>Characterization and mapping of the <Protname>12 kDa FK506-binding protein</Protname> (<Protname>FKBP12</Protname>)-binding site on different isoforms of the <Protname>ryanodine receptor</Protname> and of the <Protname>inositol 1,4,5-trisphosphate receptor</Protname>.</ArticleTitle>
<AbstractText>We investigated the interaction of the <Protname>12 kDa FK506-binding protein</Protname> (<Protname>FKBP12</Protname>) with two <Protname>ryanodine-receptor</Protname> isoforms (<Protname>RyR1</Protname> and <Protname>RyR3</Protname>) and with two <Protname>myo-inositol 1,4,5-trisphosphate (IP3) receptor</Protname> isoforms (<Protname>IP3R1</Protname> and <Protname>IP3R3</Protname>). Using <Protname>glutathione S-transferase</Protname> (<Protname>GST</Protname>)-<Protname>FKBP12</Protname> affinity chromatography, we could efficiently extract <Protname>RyR1</Protname> (42+/-7% of the solubilized <Protname>RyR1</Protname>) from terminal cisternae of skeletal muscle as well as <Protname>RyR3</Protname> (32+/-4% of the solubilized <Protname>RyR3</Protname>) from <Protname>RyR3</Protname>-overexpressing HEK-293 cells. These interactions were completely abolished by FK506 (20 microM) but were largely unaffected by <Protname>RyR-channel</Protname> modulators. In contrast, neither <Protname>IP3R1</Protname> nor <Protname>IP3R3</Protname> from various sources, including rabbit cerebellum, A7r5 smooth-muscle cells and <Protname>IP3R</Protname>-overexpressing Sf9 insect cells from Spodoptera frugiperda, were retained on the GST-<Protname>FKBP12</Protname> matrix. Moreover, immunoprecipitation experiments indicated a high-affinity interaction of <Protname>FKBP12</Protname> with <Protname>RyR1</Protname> but not with <Protname>IP3R1</Protname>. In order to determine the <Protname>FKBP12</Protname>-binding site, we fragmented both <Protname>RyR1</Protname> and <Protname>IP33R1</Protname> by limited proteolysis. We obtained a 45 kDa fragment of <Protname>RyR1</Protname> that bound to the GST-<Protname>FKBP12</Protname> matrix, indicating that it retained all requirements for <Protname>FKBP12</Protname> binding. This fragment was identified by its interaction with <Protname>antibody m34C</Protname> and must therefore contain its epitope (amino acids 2756-2803). However, no fragment of <Protname>IP3R1</Protname> was retained on the column. These molecular data are in agreement with the lack of correlation between <Protname>FKBP12</Protname> and <Protname>IP3R1</Protname> expression in various cell types. The observation that <Protname>FKBP12</Protname> did not affect IP3-induced Ca2+ release but reduced caffeine-induced Ca2+ release also indicated that mature <Protname>IP3R1</Protname> and <Protname>IP3R3</Protname>, in contrast to <Protname>RyR1</Protname> and <Protname>RyR3</Protname>, did not display a specific, high-affinity interaction with <Protname>FKBP12</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21111052</MedlineID>
<PMID>11163216</PMID>
<ArticleTitle>A <Protname>Rab11</Protname>/<Protname>Rip11</Protname> protein complex regulates apical membrane trafficking via recycling endosomes.</ArticleTitle>
<AbstractText><Protname>Rab11</Protname> is a <Protname>GTPase</Protname> that regulates endosomal trafficking to apical plasma membrane domains in polarized epithelial cells. We report the identification of a novel <Protname>Rab11</Protname> effector, <Protname>Rip11</Protname>. <Protname>Rip11</Protname> is enriched in polarized epithelial cells where, like <Protname>Rab11</Protname>, it is localized to subapical recycling endosomes (ARE) and the apical plasma membrane. Using various transport assays, we demonstrate that <Protname>Rip11</Protname> is important for protein trafficking from ARE to the apical plasma membrane. <Protname>Rip11</Protname> is recruited to ARE by binding to <Protname>Rab11</Protname> as well as through a Mg(2+)-dependent interaction of its C2 domain with neutral phospholipids. The association of <Protname>Rip11</Protname> with membranes is regulated by a phosphorylation and dephosphorylation cycle. We propose a model whereby the <Protname>Rab11</Protname>/<Protname>Rip 11</Protname> complex regulates vesicle targeting from the ARE.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21067040</MedlineID>
<PMID>11149895</PMID>
<ArticleTitle><Protname>NOSIP</Protname>, a novel modulator of <Protname>endothelial nitric oxide synthase</Protname> activity.</ArticleTitle>
<AbstractText>Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of <Protname>endothelial nitric oxide synthase</Protname> (<Protname>eNOS</Protname>) with effector proteins such as <Protname>Ca2+-calmodulin</Protname>, by posttranslational modifications such as phosphorylation via <Protname>protein kinase B</Protname>, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of <Protname>eNOS</Protname> is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed <Protname>NOSIP</Protname> (<Protname>eNOS interacting protein</Protname>), which avidly binds to the carboxyl-terminal region of the <Protname>eNOS</Protname> oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of <Protname>eNOS</Protname> and <Protname>NOSIP</Protname> in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the <Protname>caveolin-1</Protname> scaffolding domain. NO production was significantly reduced in <Protname>eNOS</Protname>-expressing CHO cells (CHO-<Protname>eNOS</Protname>) that transiently overexpressed <Protname>NOSIP</Protname>. Stimulation with the calcium ionophore A23187 induced the reversible translocation of <Protname>eNOS</Protname> from the detergent-insoluble to the detergent-soluble fractions of CHO-<Protname>eNOS</Protname>, and this translocation was completely prevented by transient coexpression of <Protname>NOSIP</Protname> in CHO-<Protname>eNOS</Protname>. Immunofluorescence studies revealed a prominent plasma membrane staining for <Protname>eNOS</Protname> in CHO-<Protname>eNOS</Protname> that was abolished in the presence of <Protname>NOSIP</Protname>. Subcellular fractionation studies identified <Protname>eNOS</Protname> in the <Protname>caveolin</Protname>-rich membrane fractions of CHO-<Protname>eNOS</Protname>, and coexpression of <Protname>NOSIP</Protname> caused a shift of <Protname>eNOS</Protname> to intracellular compartments. We conclude that <Protname>NOSIP</Protname> is a novel type of modulator that promotes translocation of <Protname>eNOS</Protname> from the plasma membrane to intracellular sites, thereby uncoupling <Protname>eNOS</Protname> from plasma membrane caveolae and inhibiting NO synthesis.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21153752</MedlineID>
<PMID>11115513</PMID>
<ArticleTitle>The <Protname>neuronal adaptor protein X11alpha</Protname> interacts with the copper chaperone for <Protname>SOD1</Protname> and regulates <Protname>SOD1</Protname> activity.</ArticleTitle>
<AbstractText>The <Protname>neuronal adaptor protein X11alpha</Protname> participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human <Protname>X11alpha</Protname>, and we isolated a clone encoding domains II and III of the copper chaperone for <Protname>Cu,Zn-superoxide dismutase-1</Protname> (<Protname>CCS</Protname>). The <Protname>X11alpha</Protname>/<Protname>CCS</Protname> interaction was confirmed in coimmunoprecipitation studies plus <Protname>glutathione S-transferase</Protname> fusion protein pull-down assays and was shown to be mediated via PDZ2 of <Protname>X11alpha</Protname> and a sequence within the carboxyl terminus of domain III of <Protname>CCS</Protname>. <Protname>CCS</Protname> delivers the copper cofactor to the antioxidant <Protname>superoxide dismutase-1</Protname> (<Protname>SOD1</Protname>) enzyme and is required for its activity. Overexpression of <Protname>X11alpha</Protname> inhibited <Protname>SOD1</Protname> activity in transfected Chinese hamster ovary cells which suggests that <Protname>X11alpha</Protname> binding to <Protname>CCS</Protname> is inhibitory to <Protname>SOD1</Protname> activation. <Protname>X11alpha</Protname> also interacts with another copper-binding protein found in neurons, the <Protname>Alzheimer's disease amyloid precursor protein</Protname>. Thus, <Protname>X11alpha</Protname> may participate in copper homeostasis within neurons.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20550571</MedlineID>
<PMID>11100870</PMID>
<ArticleTitle>Quantitation of protein binding to the capillary wall in acidic, isoelectric buffers and means for minimizing the phenomenon.</ArticleTitle>
<AbstractText>Notwithstanding the use of acidic, amphoteric, isoelectric buffers with isoelectric points (pI) in the pH 2-3 range, adsorption of proteins to the naked silica wall can be non-negligible. Two such buffers have been tested: iminodiacetic acid (IDA; pI 2.23, apparent pH 3.2 in 7 M urea) and aspartic acid (pI 2.77, apparent pH 3.7 in 7 M urea). Three potential quenchers of such interactions have been tested: hydroxyethylcellulose (HEC; number average molecular mass, Mr 27,000), TEPA (tetraethylenepentamine) and a novel, quatemarized piperazine [N(methyl-N-omega-iodobutyl)-N'-methylpiperazine] (Q-Pip), either alone or in binary and ternary mixtures. Human alpha- and beta-globin chains have been used as test proteins in capillary electrophoresis separations. It has been found that mixtures of these compounds are the worst possible remedy. E.g., a ternary mixture comprising 0.5% HEC, 0.5 mM TEPA and 1 mM Q-Pip still leaves behind 4.5% adsorbed protein onto the silica surface in runs in IDA buffer and 7 M urea (pH 3.2). Conversely, 0.5 mM TEPA or 1 mM Q-Pip, when used alone, minimize adsorption down to only 1.8% and 0.5%, respectively. When the same globin chain separations are performed in Asp and 7 M urea (pH 3.7), the situation is much worse: 44% protein is adsorbed in a ternary mixture of 0.5% HEC, 1 mM Q-Pip and 0.5 mM TEPA. However, when used alone, 0.5 mM TEPA and 1 mM Q-Pip reduce globin adsorption to levels of 8% and 5%, respectively. TEPA and Q-Pip are found to be in all cases the best quenchers of protein interaction to naked fused-silica; in addition they exhibit the unique property of smoothing the base-line and giving reproducible runs. The best method for desorbing bound protein was found to be an electrophoretic step consisting in driving sodium dodecylsulphate micelles from the cathodic reservoir.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20538492</MedlineID>
<PMID>11085749</PMID>
<ArticleTitle>Hematopoietic expression of <Protname>HOXB4</Protname> is regulated in normal and leukemic stem cells through transcriptional activation of the <Protname>HOXB4</Protname> promoter by <Protname>upstream stimulating factor (USF)-1</Protname> and <Protname>USF-2</Protname>.</ArticleTitle>
<AbstractText>The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since <Protname>HOXB4</Protname> plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of <Protname>HOXB4</Protname> expression in human hematopoietic stem cells. <Protname>HOXB4</Protname> expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary <Protname>HOXB4</Protname> regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human <Protname>CD34</Protname>(+) bone marrow cells in <Protname>thrombopoietin</Protname>/<Protname>Flt-3</Protname> ligand/stem cell factor induced <Protname>HOXB4</Protname> 3-10-fold, whereas culture in <Protname>granulocyte/macrophage colony-stimulating factor</Protname>, only increased <Protname>HOXB4</Protname>/<Protname>luciferase</Protname> expression 20-50%. Mutations within the <Protname>HOXB4</Protname> promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified <Protname>upstream stimulating factor (USF)-2</Protname> and <Protname>micropthalmia transcription factor</Protname> (<Protname>MITF</Protname>). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with <Protname>USF-1</Protname>, bind to the <Protname>HOXB4</Protname> promoter in vitro. Cotransfection assays in both K562 and <Protname>CD34</Protname>(+) cells showed that <Protname>USF-1</Protname> and <Protname>USF-2</Protname>, but not <Protname>MITF</Protname>, induce the <Protname>HOXB4</Protname> promoter in response to signals stimulating stem cell self-renewal, through activation of the <Protname>mitogen-activated protein kinase</Protname> pathway. Thus hematopoietic expression of the <Protname>human HOXB4</Protname> gene is regulated by the binding of <Protname>USF-1</Protname> and <Protname>USF-2</Protname>, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20519048</MedlineID>
<PMID>11063589</PMID>
<ArticleTitle>Critical role of a subdomain of the N-terminus of the <Protname>V1a vasopressin receptor</Protname> for binding agonists but not antagonists; functional rescue by the <Protname>oxytocin receptor</Protname> N-terminus.</ArticleTitle>
<AbstractText>A fundamental issue in molecular pharmacology is to define how agonist:receptor interaction differs from that of antagonist:receptor. The <Protname>V(1a) receptor</Protname> (<Protname>V(1a)R</Protname>) is a member of a family of related G-protein-coupled receptors that are activated by the neurohypophysial peptide hormone <Protname>arginine-vasopressin</Protname> (<Protname>AVP</Protname>). Here we define a short subdomain of the N-terminus of the <Protname>V(1a)R</Protname> from Glu(37) to Asn(47) that is an absolute requirement for binding <Protname>AVP</Protname> and other agonists. In marked contrast to the situation for agonists, deleting this segment has little or no effect on the binding of either peptide or non-peptide antagonists. In addition, we established that this subdomain was crucial for receptor activation and second messenger generation. The <Protname>oxytocin receptor</Protname> (<Protname>OTR</Protname>) also binds <Protname>AVP</Protname> with high affinity but exhibits a different pharmacological profile to the <Protname>V(1a)R</Protname>. Substitution of the N-terminus of the <Protname>V(1a)R</Protname> with the corresponding sequence from the <Protname>OTR</Protname> generated a chimeric receptor (<Protname>OTR</Protname>(N)-<Protname>V(1a)R</Protname>). The presence of the <Protname>OTR</Protname> N-terminus recovered high affinity agonist binding such that the <Protname>OTR</Protname>(N)-<Protname>V(1a)R</Protname> possessed almost wild-type <Protname>V(1a)R</Protname> pharmacology and signaling. Consequently, a domain within the N-terminus is required for agonist binding but it does not provide the molecular discriminator for subtype-selective agonist recognition. Cotransfection and peptide mimetic studies demonstrated that this N-terminal subdomain had to be contiguous with the receptor polypeptide to be functional. This study establishes that a segment of the <Protname>V(1a)R</Protname> N-terminus has a pivotal role in the mechanism of agonist binding and provides molecular insight into key differences between the interaction of agonists and antagonists with a peptide receptor family.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20491939</MedlineID>
<PMID>11035087</PMID>
<ArticleTitle>Identification of a complex that binds to the <Protname>CD154</Protname> 3' untranslated region: implications for a role in message stability during T cell activation.</ArticleTitle>
<AbstractText><Protname>CD154</Protname> expression is regulated throughout a time course of <Protname>CD3</Protname>-dependent T cell activation by differential mRNA decay. To understand the molecular basis of the "stability" phase of this pathway, experiments were conducted to identify sequences and specific complexes important in this regulation. Gel retardation assays using extracts from both Jurkat T cells and <Protname>CD3</Protname>-activated <Protname>CD4</Protname>(+) T cells revealed a major complex (complex I) that bound a 65-bp highly CU-rich region of the <Protname>CD154</Protname> 3' untranslated region. The specificity of the CU-rich element for complex-I formation was confirmed by disruption of this complex by oligo(dCT) competition. Formation of complex I strongly correlated with <Protname>CD154</Protname> mRNA stability across a time course of T cell activation. UV cross-linking identified a major oligo(dCT)-sensitive species at approximately 90 kDa that showed induced and increased expression in extracts from 24- and 48-hr anti-<Protname>CD3</Protname>-activated T cells, respectively. This protein was absent in equivalent extracts from resting or 2-h-activated T cells. Using an in vitro decay assay, we found that a <Protname>CD154</Protname>-specific transcript was more rapidly degraded in 2-h-activated extract and stabilized in the 24- and 48-h extracts compared to extracts from resting T cells. Disruption of complex I resulted in the rapid decay of a <Protname>CD154</Protname>-specific transcript demonstrating a functional role for complex I in mRNA stabilization in vitro. These studies support a model of posttranscriptional regulation of <Protname>CD154</Protname> expression being controlled in part by the interaction of a poly(CU)-binding complex with a specific sequence in the 3' untranslated region.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20576405</MedlineID>
<PMID>11016930</PMID>
<ArticleTitle>The N-terminal end of <Protname>nebulin</Protname> interacts with <Protname>tropomodulin</Protname> at the pointed ends of the thin filaments.</ArticleTitle>
<AbstractText>Strict regulation of <Protname>actin</Protname> thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with <Protname>actin</Protname> filament capping proteins to regulate thin filament lengths. <Protname>Nebulin</Protname> is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. <Protname>Tropomodulin</Protname> (<Protname>Tmod</Protname>), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of <Protname>nebulin</Protname> colocalizes with <Protname>Tmod</Protname> at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of <Protname>nebulin</Protname> bind specifically to <Protname>Tmod</Protname> as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the <Protname>nebulin</Protname> molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two <Protname>Tmod</Protname> isoforms, erythrocyte <Protname>Tmod</Protname> (<Protname>E-Tmod</Protname>), expressed in embryonic and slow skeletal muscle, and <Protname>skeletal Tmod</Protname> (<Protname>Sk-Tmod</Protname>), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal <Protname>nebulin</Protname> fragments. <Protname>Sk-Tmod</Protname> binds <Protname>nebulin</Protname> with higher affinity than <Protname>E-Tmod</Protname> does, suggesting that the <Protname>Tmod</Protname>/<Protname>nebulin</Protname> interaction exhibits isoform specificity. These data provide evidence that <Protname>Tmod</Protname> and <Protname>nebulin</Protname> may work together as a linked mechanism to control thin filament lengths in skeletal muscle.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20453769</MedlineID>
<PMID>10996078</PMID>
<ArticleTitle><Protname>INCENP</Protname> binds the <Protname>Aurora-related kinase AIRK2</Protname> and is required to target it to chromosomes, the central spindle and cleavage furrow.</ArticleTitle>
<AbstractText>Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the <Protname>inner centromere protein</Protname> (<Protname>INCENP</Protname>[2]), the <Protname>Aurora-related serine-threonine protein kinase AIRK2</Protname> [3,4] and the unidentified human autoantigen <Protname>TD-60</Protname> [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of <Protname>INCENP</Protname> function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with <Protname>AIRK2</Protname> function primarily affects cytokinesis [3,8]. Here, we report that <Protname>INCENP</Protname> is stockpiled in Xenopus eggs in a complex with <Protname>Xenopus AIRK2</Protname> (<Protname>XAIRK2</Protname>), and that <Protname>INCENP</Protname> and <Protname>AIRK2 kinase</Protname> bind one another in vitro. This association was found to be evolutionarily conserved. <Protname>Sli15p</Protname>, the binding partner of <Protname>yeast Aurora kinase Ipl1p</Protname>, can be recognized as an <Protname>INCENP</Protname> family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between <Protname>INCENP</Protname> and <Protname>Aurora kinase</Protname> was found to be biologically relevant. <Protname>INCENP</Protname> and <Protname>AIRK2</Protname> colocalized exactly in human cells, and <Protname>INCENP</Protname> was required to target <Protname>AIRK2</Protname> correctly to centromeres and the central spindle.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20433074</MedlineID>
<PMID>10975987</PMID>
<ArticleTitle><Protname>IL-5</Protname>-Induced <Protname>JAB</Protname>-<Protname>JAK2</Protname> interaction.</ArticleTitle>
<AbstractText>Receptor activation by the haematopoietic growth factor proteins <Protname>interleukin 5</Protname> (<Protname>IL-5</Protname>) and <Protname>granulocyte-macrophage colony-stimulating factor</Protname> (<Protname>GM-CSF</Protname>) leads to phosphorylation of <Protname>JAK2</Protname> as a key trigger of signal transduction. <Protname>JAB</Protname> has recently been identified as a regulator of <Protname>JAK2</Protname> phosphorylation and activity by binding phosphorylated <Protname>JAK2</Protname> and inducing its degradation. As part of our effort to define molecular recognition networks that lead to signalling, we investigated the effect of <Protname>JAB</Protname> on both <Protname>JAK2</Protname> phosphorylation and <Protname>JAK2</Protname> interaction state that ensue upon <Protname>IL-5</Protname> stimulation in recombinant 293T cells cotransfected 293T cells with <Protname>IL-5R alpha</Protname>, <Protname>beta c</Protname> and <Protname>hJAK2</Protname> either with or without <Protname>JAB</Protname>. Without <Protname>JAB</Protname>, stimulation with wild-type and re-engineered single chain (sc) <Protname>IL-5</Protname> induced a time-dependent phosphorylation of <Protname>JAK2</Protname>. In the presence of <Protname>JAB</Protname> cotransfection, no <Protname>phospho-JAK2</Protname> was observed, and <Protname>JAB</Protname> was observed co-immunoprecipitated with non-phosphorylated <Protname>JAK2</Protname>. The time dependence of <Protname>JAB</Protname> co-immunoprecipitation correlated with the time dependence of <Protname>JAK2</Protname> phosphorylation when <Protname>JAB</Protname> was absent. Since <Protname>JAB</Protname> has already been shown to bind <Protname>JAK2</Protname> via a phosphorylated tyrosine, the current data suggest that <Protname>JAB</Protname> binds to phosphorylated <Protname>JAK2</Protname>, enhances <Protname>JAK2</Protname> dephosphorylation and remains associated in a complex, with dephosphorylated <Protname>JAK2</Protname>, that may be a precursor leading to irreversible <Protname>JAK2</Protname> degradation.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20549653</MedlineID>
<PMID>10961991</PMID>
<ArticleTitle>Covalent modification of <Protname>p73alpha</Protname> by <Protname>SUMO-1</Protname>. Two-hybrid screening with <Protname>p73</Protname> identifies novel <Protname>SUMO-1</Protname>-interacting proteins and a <Protname>SUMO-1</Protname> interaction motif.</ArticleTitle>
<AbstractText>Two-hybrid screening in yeast with <Protname>p73alpha</Protname> isolated <Protname>SUMO-1</Protname> (<Protname>small ubiquitin-like modifier 1</Protname>), the enzyme responsible for its conjugation, <Protname>Ubc-9</Protname>, and a number of novel <Protname>SUMO-1</Protname>-interacting proteins, including thymine <Protname>DNA glycosylase</Protname>, <Protname>PM-Scl75</Protname>, <Protname>PIASx</Protname>, <Protname>PKY</Protname>, and <Protname>CHD3/ZFH</Protname>. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with <Protname>SUMO-1</Protname> in the two-hybrid system. We show here that <Protname>p73alpha</Protname>, but not <Protname>p73beta</Protname>, can be covalently modified by <Protname>SUMO-1</Protname>. The major <Protname>SUMO-1</Protname>-modified residue in <Protname>p73alpha</Protname> is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus <Protname>SUMO-1</Protname> modification site b(X)XXhKXE, where b is a basic amino acid. <Protname>SUMO-1-modified p73</Protname> is more rapidly degraded by the proteasome than unmodified <Protname>p73</Protname>, although <Protname>SUMO-1</Protname> modification is not required for <Protname>p73</Protname> degradation. <Protname>SUMO-1</Protname> modification does not affect the transcriptional activity of <Protname>p73alpha</Protname> on an <Protname>RGC-luciferase</Protname> reporter gene in SK-N-AS cells. Instead, <Protname>SUMO-1</Protname> modification may alter the subcellular localization of <Protname>p73</Protname>, because <Protname>SUMO-1-modified p73</Protname> is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of <Protname>p73</Protname> with other proteins that are substrates for <Protname>SUMO-1</Protname> modification or which interact with <Protname>SUMO-1</Protname>, such as those identified here.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20519623</MedlineID>
<PMID>10950948</PMID>
<ArticleTitle>The <Protname>Yersinia protein kinase A</Protname> is a host factor inducible <Protname>RhoA</Protname>/<Protname>Rac</Protname>-binding virulence factor.</ArticleTitle>
<AbstractText>The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the <Protname>Yersinia protein kinase A</Protname> (<Protname>YpkA</Protname>), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of <Protname>YpkA</Protname>. The amino terminus, which contains the kinase domain, is sufficient to localize <Protname>YpkA</Protname> to the plasma membrane while the carboxyl terminus of <Protname>YpkA</Protname> is required for <Protname>YpkAs</Protname> morphological effects. <Protname>YpkAs</Protname> carboxyl-terminal region was found to affect the levels of <Protname>actin</Protname>-containing stress fibers as well as block the activation of the <Protname>GTPase RhoA</Protname> in Yersinia-infected cells. We show that the carboxyl-terminal region of <Protname>YpkA</Protname>, which contains sequences that bear similarity to the <Protname>RhoA</Protname>-binding domains of several <Protname>eukaryotic RhoA-binding kinases</Protname>, directly interacts with <Protname>RhoA</Protname> as well as <Protname>Rac</Protname> (but not <Protname>Cdc42</Protname>) and displays a slight but measurable binding preference for the GDP-bound form of <Protname>RhoA</Protname>. Surprisingly, <Protname>YpkA</Protname> binding to <Protname>RhoA(GDP)</Protname> affected neither the intrinsic nor <Protname>guanine nucleotide exchange factor</Protname>-mediated GDP/GTP exchange reaction suggesting that <Protname>YpkA</Protname> controls activated <Protname>RhoA</Protname> levels by a mechanism other than by simply blocking <Protname>guanine nucleotide exchange factor</Protname> activity. We go on to show that <Protname>YpkAs</Protname> kinase activity is neither dependent on nor promoted by its interaction with <Protname>RhoA</Protname> and <Protname>Rac</Protname> but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that <Protname>YpkA</Protname> possesses both similarities and differences with the <Protname>eukaryotic RhoA/Rac-binding kinases</Protname> and suggest that the yersiniae utilize the <Protname>Rho GTPases</Protname> for unique activities during their interaction with eukaryotic cells.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20389384</MedlineID>
<PMID>10930463</PMID>
<ArticleTitle>Specific sequence motif of 8-Cys repeats of <Protname>TGF-beta binding proteins</Protname>, <Protname>LTBPs,</Protname> creates a hydrophobic interaction surface for binding of <Protname>small latent TGF-beta</Protname>.</ArticleTitle>
<AbstractText><Protname>Transforming growth factor (TGF)-betas</Protname> are secreted in large latent complexes consisting of <Protname>TGF-beta</Protname>, its <Protname>N-terminal latency-associated peptide</Protname> (<Protname>LAP</Protname>) propeptide, and <Protname>latent TGF-beta binding protein</Protname> (<Protname>LTBP</Protname>). <Protname>LTBPs</Protname> are required for secretion and subsequent deposition of <Protname>TGF-beta</Protname> into the extracellular matrix. <Protname>TGF-beta1</Protname> associates with the 3(rd) 8-Cys repeat of <Protname>LTBP-1</Protname> by <Protname>LAP</Protname>. All <Protname>LTBPs</Protname>, as well as <Protname>fibrillins</Protname>, contain multiple 8-Cys repeats. We analyzed the abilities of <Protname>fibrillins</Protname> and <Protname>LTBPs</Protname> to bind latent <Protname>TGF-beta</Protname> by their 8-Cys repeats. 8-Cys repeat was found to interact with <Protname>TGF-beta1</Protname>*<Protname>LAP</Protname> by direct cysteine bridging. <Protname>LTBP-1</Protname> and <Protname>LTBP-3</Protname> bound efficiently all <Protname>TGF-beta</Protname> isoforms, <Protname>LTBP-4</Protname> had a much weaker binding capacity, whereas <Protname>LTBP-2</Protname> as well as <Protname>fibrillins -1 and -2</Protname> were negative. A short, specific <Protname>TGF-beta</Protname> binding motif was identified in the <Protname>TGF-beta</Protname> binding 8-Cys repeats. Deletion of this motif in the 3(rd) 8-Cys repeat of <Protname>LTBP-1</Protname> resulted in loss of <Protname>TGF-beta</Protname>*<Protname>LAP</Protname> binding ability, while its inclusion in non-<Protname>TGF-beta</Protname> binding 3(rd) 8-Cys repeat of <Protname>LTBP-2</Protname> resulted in <Protname>TGF-beta</Protname> binding. Molecular modeling of the 8-Cys repeats revealed a hydrophobic interaction surface and lack of three stabilizing hydrogen bonds introduced by the <Protname>TGF-beta</Protname> binding motif necessary for the formation of the <Protname>TGF-beta</Protname>*<Protname>LAP</Protname> - 8-Cys repeat complex inside the cells.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20493579</MedlineID>
<PMID>10913114</PMID>
<ArticleTitle><Protname>GAS41</Protname>, a highly conserved protein in eukaryotic nuclei, binds to <Protname>NuMA</Protname>.</ArticleTitle>
<AbstractText>The yeast two-hybrid system was used to identify binding partners of <Protname>NuMA</Protname>, a component of the nuclear matrix in interphase cells. By using the C-terminal half of <Protname>NuMA</Protname> as bait, a human cDNA sequence coding for a 223-amino acid protein with a non-helical N-terminal domain and a C-terminal alpha-helical portion was identified and fully sequenced. It was identical to <Protname>GAS41</Protname>, a sequence amplified in human gliomas. The sequence of the homologous Drosophila protein was established, and the alignment for <Protname>GAS41</Protname> from nine different species showed that <Protname>GAS41</Protname> is a general eukaryotic protein found in species as diverse as Arabidopsis, Drosophila, Caenorhabditis elegans, yeast, and man. Northern blot analysis showed a single transcript in eight human tissues. A polyclonal antibody to <Protname>GAS41</Protname> showed a dotted staining pattern in interphase nuclei and a uniform distribution in mitotic cells. A <Protname>GFP-GAS41 fusion protein</Protname> displayed equivalent patterns. In vitro <Protname>GAS41</Protname> bound to the C-terminal part of the rod region of <Protname>NuMA</Protname>, as shown by dot overlay and by surface plasmon resonance measurements. The K(d) of the complex was 2 x 10(-)(7) m. <Protname>GAS41</Protname> is related to the <Protname>AF-9</Protname> and <Protname>ENL</Protname> proteins, which are putative transcription factors found as fusion proteins in some acute leukemias. The <Protname>NuMA</Protname>/<Protname>GAS41</Protname> interaction may provide a link between nuclear structure and gene expression.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20353817</MedlineID>
<PMID>10895262</PMID>
<ArticleTitle>Adhesive receptors on malaria-parasitized red cells.</ArticleTitle>
<AbstractText>Antigenic variation, rosetting and cytoadhesion are key determinants in the survival and virulence of the malaria parasite Plasmodium falciparum. These properties reside in a multigene protein family called <Protname>P. falciparum erythrocyte membrane protein I</Protname> (<Protname>PfEMPI</Protname>), encoded by the large and diverse var gene family. <Protname>PfEMPI</Protname> plays a central role in the biology of P. falciparum and its interaction with the human host. The molecular mechanism and the domains involved in cytoadherence, rosetting and antigenic variation are beginning to unfold. Domains mediating rosetting and adhesion to several key host receptors have already been identified. Understanding the role of <Protname>PfEMPI</Protname> in the pathogenesis and survival of malaria parasites is the key for the development of anti-adhesion vaccines and therapeutics to reduce the mortality and morbidity of P. falciparum infections.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20329227</MedlineID>
<PMID>10872826</PMID>
<ArticleTitle><Protname>Gadd45</Protname> family proteins are coactivators of nuclear hormone receptors.</ArticleTitle>
<AbstractText><Protname>Gadd45</Protname> family genes encode nuclear acidic proteins composed of <Protname>Gadd45</Protname>, <Protname>MyD118</Protname>, and <Protname>CR6</Protname>. Sequence analysis showed that <Protname>Gadd45</Protname> family proteins (<Protname>Gadd45</Protname>, <Protname>MyD118</Protname>, and <Protname>CR6</Protname>) contain LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. Interaction between <Protname>Gadd45</Protname> or <Protname>CR6</Protname> and <Protname>RXR alpha</Protname> was confirmed by a two-hybrid test in yeast. Results from a series of <Protname>GST</Protname> pulldown assays showed that these <Protname>Gadd45</Protname> family proteins interact with several nuclear hormone receptors including <Protname>RXR alpha</Protname>, <Protname>RAR alpha</Protname>, <Protname>ER alpha</Protname>, <Protname>PPAR alpha</Protname>, <Protname>PPAR beta</Protname>, and <Protname>PPAR gamma2</Protname> in vitro. Interaction between <Protname>Gadd45</Protname> family proteins and nuclear hormone receptors resulted in modest activation of transactivating function of nuclear hormone receptors in reporter systems. When fused to DNA binding domain of <Protname>GAL4</Protname>, <Protname>Gadd45</Protname> and <Protname>CR6</Protname> activated the UAS-mediated transcription in mammalian cells. These results suggest that <Protname>Gadd45</Protname> family proteins bind to nuclear hormone receptors and act as nuclear coactivators.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20328610</MedlineID>
<PMID>10860733</PMID>
<ArticleTitle>1.9 A resolution crystal structure of the <Protname>Saccharomyces cerevisiae Ran-binding protein Mog1p</Protname>.</ArticleTitle>
<AbstractText>The 1.9 A resolution X-ray crystal structure of <Protname>Ran-binding protein Mog1p</Protname> shows that it has a unique fold based on a six-stranded antiparallel beta-sheet backed on both sides by an extensive alpha-helix. The topology of some elements of <Protname>Mog1p</Protname> secondary structure resemble a portion of <Protname>nuclear transport factor 2</Protname> (<Protname>NTF2</Protname>), but the hydrophobic cavity and surrounding negatively charged residues that are important in the <Protname>NTF2</Protname>-<Protname>RanGDP</Protname> interaction are not conserved in <Protname>Mog1p</Protname>. In addition to binding <Protname>RanGTP</Protname>, <Protname>Mog1p</Protname> forms a 1:1 complex with <Protname>RanGDP</Protname> and so binds <Protname>Ran</Protname> independent of its nucleotide state. <Protname>Mog1p</Protname> and <Protname>NTF2</Protname> compete for binding to <Protname>RanGDP</Protname> indicating that their binding sites on <Protname>RanGDP</Protname> are sufficiently close to prevent both proteins binding simultaneously. Although there may be some overlap between the <Protname>Mog1p</Protname> and <Protname>NTF2</Protname> binding sites on <Protname>RanGDP</Protname>, these sites are not identical. Sequence analysis of <Protname>Mog1p</Protname> homologues from Schizosaccharomyces pombe, human, and Caenorhabditis elegans in the context of the <Protname>Mog1p</Protname> crystal structure indicates the presence of a cluster of highly conserved surface residues consistent with an interaction site for <Protname>Ran</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20298662</MedlineID>
<PMID>10838076</PMID>
<ArticleTitle>Role of the N-terminal region of <Protname>staphylokinase</Protname> (<Protname>SAK</Protname>): evidence for the participation of the N-terminal region of <Protname>SAK</Protname> in the enzyme-substrate complex formation.</ArticleTitle>
<AbstractText><Protname>Staphylokinase</Protname> (<Protname>SAK</Protname>) forms an inactive 1:1 complex with <Protname>plasminogen</Protname> (<Protname>PG</Protname>), which requires both the conversion of <Protname>PG</Protname> to <Protname>plasmin</Protname> (<Protname>Pm</Protname>) to expose an active site in <Protname>PG</Protname>-<Protname>SAK</Protname> activator complex and the amino-terminal processing of <Protname>SAK</Protname> to expose the positively charged (Lys-11) amino-terminus after removal of the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of <Protname>SAK</Protname> affects its <Protname>PG</Protname> activation capability was investigated by generating <Protname>SAK</Protname> mutants, blocked in the native amino-terminal processing site of <Protname>SAK</Protname>, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with <Protname>PG</Protname>, <Protname>Pm</Protname>, miniplasmin and kringle structures. A ternary complex formation between <Protname>PG</Protname>-<Protname>SAK</Protname> <Protname>PG</Protname> was observed when an immobilized <Protname>PG</Protname>-<Protname>SAK</Protname> binary complex interacted with free radiolabelled <Protname>PG</Protname> in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native <Protname>SAK</Protname> or its mutant derivatives. Furthermore, the binary (activator) complex formed between <Protname>PG</Protname> and <Protname>SAK</Protname> mutant, <Protname>PRM3</Protname>, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with <Protname>PG</Protname>, <Protname>Pm</Protname> or miniplasmin substrate during ternary complex formation as compared to native <Protname>SAK</Protname>. Additionally, activator complex formed with <Protname>PRM3</Protname> failed to activate <Protname>miniplasminogen</Protname> and exhibited highly diminished activation of substrate <Protname>PG</Protname>. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation of the N-terminal region of <Protname>SAK</Protname> in accession and processing of substrate by the <Protname>SAK</Protname>-<Protname>Pm</Protname> activator complex to potentiate the <Protname>PG</Protname> activation by enhancing and/or stabilizing the interaction of free <Protname>PG</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20283899</MedlineID>
<PMID>10823934</PMID>
<ArticleTitle>Specific interaction of <Protname>CCR5</Protname> amino-terminal domain peptides containing sulfotyrosines with <Protname>HIV-1 envelope glycoprotein gp120</Protname>.</ArticleTitle>
<AbstractText>The <Protname>HIV-1 envelope glycoprotein gp120</Protname> interacts consecutively with <Protname>CD4</Protname> and the <Protname>CCR5 coreceptor</Protname> to mediate the entry of certain HIV-1 strains into target cells. Acidic residues and sulfotyrosines in the amino-terminal domain (Nt) of <Protname>CCR5</Protname> are crucial for viral fusion and entry. We tested the binding of a panel of <Protname>CCR5</Protname> Nt peptides to different soluble <Protname>gp120</Protname>/<Protname>CD4</Protname> complexes and anti-<Protname>CCR5</Protname> mAbs. The tyrosine residues in the peptides were sulfated, phosphorylated, or unmodified. None of the <Protname>gp120</Protname>/<Protname>CD4</Protname> complexes associated with peptides containing unmodified or phosphorylated tyrosines. The <Protname>gp120</Protname>/<Protname>CD4</Protname> complexes containing envelope glycoproteins from isolates that use <Protname>CCR5</Protname> as a coreceptor associated with Nt peptides containing sulfotyrosines but not with peptides containing sulfotyrosines in scrambled Nt sequences. Finally, only peptides containing sulfotyrosines inhibited the entry of an R5 isolate. Our data show that proper posttranslational modification of the <Protname>CCR5</Protname> Nt is required for <Protname>gp120</Protname> binding and viral entry. More importantly, the Nt domain determines the specificity of the interaction between <Protname>CCR5</Protname> and <Protname>gp120</Protname>s from isolates that use this coreceptor.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20278276</MedlineID>
<PMID>10816375</PMID>
<ArticleTitle>Analysis of <Protname>plasminogen-binding M proteins</Protname> of Streptococcus pyogenes.</ArticleTitle>
<AbstractText>Group A streptococci are common human pathogens that cause a variety of infections. They express <Protname>M proteins</Protname> which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express <Protname>M proteins</Protname> that bind <Protname>plasminogen</Protname> and <Protname>plasmin</Protname> with high affinity. The binding is mediated by a 13-amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these <Protname>M proteins</Protname>. This sequence binds to kringle 2 in <Protname>plasminogen</Protname>, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of <Protname>plasminogen</Protname>. It could be demonstrated that <Protname>plasminogen</Protname>, absorbed from plasma by growing group A streptococci expressing the <Protname>plasminogen-binding M proteins</Protname>, could be activated by exogenous and endogenous <Protname>streptokinase</Protname>, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20261553</MedlineID>
<PMID>10799509</PMID>
<ArticleTitle>Identification and characterization of <Protname>CKIP-1</Protname>, a novel pleckstrin homology domain-containing protein that interacts with <Protname>protein kinase CK2</Protname>.</ArticleTitle>
<AbstractText>The catalytic subunits of <Protname>protein kinase CK2</Protname>, <Protname>CK2alpha</Protname> and <Protname>CK2alpha'</Protname>, are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of <Protname>CK2alpha</Protname> and <Protname>CK2alpha'</Protname> are mediated by specific interaction partners, we used the yeast two-hybrid system to identify <Protname>CK2alpha</Protname>- or <Protname>CK2alpha'</Protname>-binding proteins. We report the identification and characterization of a novel <Protname>CK2</Protname>-interacting protein, designated <Protname>CKIP-1</Protname>, that interacts with <Protname>CK2alpha</Protname>, but not <Protname>CK2alpha'</Protname>, in the yeast two-hybrid system. <Protname>CKIP-1</Protname> also interacts with <Protname>CK2alpha</Protname> in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged <Protname>CK2alpha</Protname> and an <Protname>enhanced green fluorescent protein</Protname> fusion protein encoding <Protname>CKIP-1</Protname> (i.e. <Protname>EGFP</Protname>-<Protname>CKIP-1</Protname>) when they are co-expressed. <Protname>CK2</Protname> activity is detected in anti-<Protname>CKIP-1</Protname> immunoprecipitates performed with extracts from non-transfected cells indicating that <Protname>CKIP-1</Protname> and <Protname>CK2</Protname> interact under physiological conditions. The <Protname>CKIP-1</Protname> cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. <Protname>EGFP</Protname>-<Protname>CKIP-1</Protname> is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal <Protname>pleckstrin</Protname> homology domain. We postulate that <Protname>CKIP-1</Protname> is a non-enzymatic regulator of one isoform of <Protname>CK2</Protname> (i.e. <Protname>CK2alpha</Protname>) with a potential role in targeting <Protname>CK2alpha</Protname> to a particular cellular location.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20242066</MedlineID>
<PMID>10779362</PMID>
<ArticleTitle>Structure-function analysis of <Protname>SUV39H1</Protname> reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression.</ArticleTitle>
<AbstractText><Protname>SUV39H1</Protname>, a human homologue of the Drosophila position effect variegation modifier <Protname>Su(var)3-9</Protname> and of the <Protname>Schizosaccharomyces pombe silencing factor clr4</Protname>, encodes a novel heterochromatic protein that transiently accumulates at centromeric positions during mitosis. Using a detailed structure-function analysis of <Protname>SUV39H1</Protname> mutant proteins in transfected cells, we now show that deregulated <Protname>SUV39H1</Protname> interferes at multiple levels with mammalian higher-order chromatin organization. First, forced expression of full-length <Protname>SUV39H1</Protname> (412 amino acids) redistributes endogenous <Protname>M31</Protname> (<Protname>HP1beta</Protname>) and induces abundant associations with inter- and metaphase chromatin. These properties depend on the C-terminal <Protname>SET</Protname> domain, although the major portion of the <Protname>SUV39H1</Protname> protein (amino acids 89 to 412) does not display affinity for nuclear chromatin. By contrast, the <Protname>M31</Protname> interaction surface, which was mapped to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin. Second, cells overexpressing full-length <Protname>SUV39H1</Protname> display severe defects in mitotic progression and chromosome segregation. Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G(2)-specific distribution of phosphorylated <Protname>histone H3</Protname> at serine 10 (phosH3) is dispersed in these cells. This <Protname>phosH3</Protname> shift is not observed with C-terminally truncated mutant <Protname>SUV39H1</Protname> proteins or with deregulated <Protname>M31</Protname>. Together, our data reveal a dominant role(s) for the <Protname>SET</Protname> domain of <Protname>SUV39H1</Protname> in the distribution of prominent heterochromatic proteins and suggest a possible link between a chromosomal <Protname>SU(VAR) protein</Protname> and <Protname>histone H3</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20317050</MedlineID>
<PMID>10764735</PMID>
<ArticleTitle>Mechanisms for high affinity mannose 6-phosphate ligand binding to the <Protname>insulin-like growth factor II/mannose 6-phosphate receptor</Protname>.</ArticleTitle>
<AbstractText>The two mannose 6-phosphate (Man-6-P) binding domains of the <Protname>insulin-like growth factor II/mannose 6-phosphate receptor</Protname> (<Protname>Man-6-P/IGF2R</Protname>), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one <Protname>Man-6-P/IGF2R</Protname> molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated <Protname>FLAG</Protname> epitope-tagged <Protname>Man-6-P/IGF2R</Protname> constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-<Protname>bovine serum albumin</Protname> (PMP-<Protname>BSA</Protname>), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the <Protname>Man-6-P/IGF2R</Protname> extracytoplasmic domain bound PMP-<Protname>BSA</Protname> with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-<Protname>BSA</Protname> increased the dissociation rate of pre-formed (125)I-PMP-<Protname>BSA</Protname>/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple <Protname>Man-6-P/IGF2R</Protname> binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --&gt; Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-<Protname>BSA</Protname> binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate <Protname>Man-6-P/IGF2R</Protname> molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for <Protname>Man-6-P/IGF2R</Protname> oligomerization.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>21060443</MedlineID>
<PMID>10749676</PMID>
<ArticleTitle><Protname>Thrombospondin-1</Protname> binds to polyhistidine with high affinity and specificity.</ArticleTitle>
<AbstractText><Protname>Thrombospondin-1</Protname> (<Protname>TSP1</Protname>) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a (125)I solid-phase binding assay to demonstrate that <Protname>TSP1</Protname> binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of (125)I-labelled <Protname>TSP1</Protname> to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by <Protname>TSP1</Protname>. Heparin was a potent inhibitor of binding with a K(i) of 50 nM, suggesting that the heparin-binding domain of <Protname>TSP1</Protname> may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of <Protname>TSP1</Protname> to directly compete for <Protname>TSP1</Protname> binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that <Protname>TSP1</Protname> in platelet releasates is the major polypeptide retained on the six-histidine-peptide column. We conclude that <Protname>TSP1</Protname> contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of <Protname>TSP1</Protname> to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with <Protname>TSP1</Protname> warrants further study.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20195117</MedlineID>
<PMID>10732979</PMID>
<ArticleTitle>Generation and evaluation of putative neuroregenerative drugs. Part 2: screening virtual libraries of novel polyketides which possess the binding domain of rapamycin.</ArticleTitle>
<AbstractText>The use of computational methods to direct engineered biosynthesis toward candidates based on the desired properties of the target compounds has been explored. The objective for this study has been the modification of rapamycin in order to eliminate its immunosuppressive activity and retain its neuroregenerative abilities. We have designed analogues of rapamycin which have truncated effector domains but retain the ability to bind to <Protname>FKBP</Protname> proteins, which is a prerequisite for the neuroregenerative abilities of the drugs. The procedures described here consist of the screening of large virtual libraries of molecules which retain the binding domain of rapamycin but in which different substitute ketide units replace the effector domain. These methods have provided analogues of rapamycin that cannot retain the immunosuppressive abilities of rapamycin, have a binding affinity to <Protname>FKBP12</Protname> identical to that of rapamycin (by linear interaction energy calculations), and are suitable for synthesis by modified polyketide synthases.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20179889</MedlineID>
<PMID>10713102</PMID>
<ArticleTitle><Protname>TrkA</Protname> amino acids controlling specificity for <Protname>nerve growth factor</Protname>.</ArticleTitle>
<AbstractText><Protname>Neurotrophins</Protname> are important for the development and maintenance of the vertebrate nervous system, mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the <Protname>Trk</Protname> family. The extracellular portion of the <Protname>Trk receptors</Protname> has been previously proposed to consist of a cysteine-rich motif, a leucine-rich motif, a second cysteine-rich motif followed by two immunoglobulin-like domains. Earlier studies have shown that a major <Protname>neurotrophin</Protname>-binding site in the <Protname>Trk</Protname> receptors resides in the second <Protname>immunoglobulin</Protname>-like domain. Although the individual amino acids in <Protname>TrkA</Protname> involved in binding to <Protname>nerve growth factor</Protname> (<Protname>NGF</Protname>) and those in <Protname>TrkC</Protname> involved in binding to <Protname>neurotrophin-3</Protname> have been mapped in this domain, the <Protname>Trk</Protname> amino acids that provide specificity remained unclear. In this study, a minimum set of residues in the human <Protname>TrkC</Protname> second <Protname>immunoglobulin</Protname>-like domain, which does not bind <Protname>nerve growth factor</Protname> (<Protname>NGF</Protname>), were substituted with those from <Protname>human TrkA</Protname>. The resulting Trk variant recruited binding of <Protname>NGF</Protname> equivalent to <Protname>TrkA</Protname>, maintained <Protname>neurotrophin-3</Protname> binding equivalent to <Protname>TrkC</Protname>, and also bound brain-derived <Protname>neurotrophin</Protname>, although with lower affinity compared with <Protname>TrkB</Protname>. This implies that the amino acids in the second <Protname>immunoglobulin</Protname>-like domain that determine <Protname>Trk</Protname> specificity are distinct for each <Protname>Trk</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20158947</MedlineID>
<PMID>10692436</PMID>
<ArticleTitle>Ca(2+)-dependent and Ca(2+)-independent <Protname>calmodulin</Protname> binding sites in <Protname>erythrocyte protein 4.1</Protname>. Implications for regulation of <Protname>protein 4.1</Protname> interactions with transmembrane proteins.</ArticleTitle>
<AbstractText>In vitro protein binding assays identified two distinct <Protname>calmodulin</Protname> (<Protname>CaM</Protname>) binding sites within the NH(2)-terminal 30-kDa domain of <Protname>erythrocyte protein 4.1</Protname> (<Protname>4.1R</Protname>): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound <Protname>CaM</Protname> in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to <Protname>CaM</Protname>. Thus, <Protname>4.1R</Protname> is a unique <Protname>CaM</Protname>-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity <Protname>CaM</Protname> binding sites. <Protname>CaM</Protname> bound to <Protname>4.1R</Protname> at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one <Protname>CaM</Protname> molecule binds to two distinct sites in the same molecule of <Protname>4.1R</Protname>. Interactions of <Protname>4.1R</Protname> with membrane proteins such as band 3 is regulated by Ca(2+) and <Protname>CaM</Protname>. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both <Protname>CaM</Protname> binding sites, the ability of Ca(2+)/<Protname>CaM</Protname> to down-regulate <Protname>4. 1R</Protname>-band 3 interaction was abrogated by such deletions. Thus, regulation of <Protname>protein 4.1</Protname> binding to membrane proteins by Ca(2+) and <Protname>CaM</Protname> requires binding of <Protname>CaM</Protname> to both Ca(2+)-independent and Ca(2+)-dependent sites in <Protname>protein 4.1</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20132233</MedlineID>
<PMID>10668799</PMID>
<ArticleTitle>Interaction cloning and characterization of <Protname>RoBPI</Protname>, a novel protein binding to <Protname>human Ro ribonucleoproteins</Protname>.</ArticleTitle>
<AbstractText><Protname>Human Ro ribonucleoproteins</Protname> (<Protname>RNPs</Protname>) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (<Protname>Ro60</Protname>) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named <Protname>RNP</Protname> interaction trap assay (RITA), we cloned a novel <Protname>Ro RNP-binding protein</Protname> (<Protname>RoBPI</Protname>), based on its property to interact in vivo in yeast with an <Protname>RNP</Protname> complex made of recombinant <Protname>Ro60</Protname> (<Protname>rRo60</Protname>) protein and hY5 (rhY5) RNA. <Protname>RoBPI</Protname> cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb <Protname>RoBPI</Protname> mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that <Protname>60 kDa RoBPI</Protname> did not interact in yeast with <Protname>rRo60</Protname> alone, with rhY5 RNA alone, or with bait <Protname>RNPs</Protname> consisting of <Protname>rRo60</Protname> and recombinant hY1, hY3, or hY4 RNAs. <Protname>RoBPI</Protname> coimmunoprecipitated with <Protname>Ro RNPs</Protname> from HeLa cell extracts and partially colocalized with <Protname>Ro60</Protname> in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but <Protname>RoBPI</Protname> seems more conserved, their interaction may represent a gain of function for <Protname>Ro RNPs</Protname>. Alternatively, interaction of <Protname>RohY5 RNPs</Protname> with <Protname>RoBPI</Protname> may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these <Protname>RNPs</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20102675</MedlineID>
<PMID>10636863</PMID>
<ArticleTitle>Direct interaction of the <Protname>CD38</Protname> cytoplasmic tail and the <Protname>Lck</Protname> SH2 domain. <Protname>Cd38</Protname> transduces T cell activation signals through associated <Protname>Lck</Protname>.</ArticleTitle>
<AbstractText><Protname>CD38</Protname> ligation has been shown to induce activation of intracellular signaling cascade in T lymphocytes through a <Protname>Lck</Protname>-dependent pathway. However, it is not clear how <Protname>Lck</Protname> initiates the <Protname>CD38</Protname>-mediated signaling process. In the present study, we showed that <Protname>CD38</Protname> and <Protname>Lck</Protname> were physically associated through the cytoplasmic tail and the <Protname>Src</Protname> homology 2 domain, respectively. This was evidenced by coimmunoprecipitation of <Protname>Lck</Protname> with <Protname>CD38</Protname> and <Protname>Lck</Protname> with isolated <Protname>CD38</Protname> cytoplasmic domain from T cell lysate, cell lysate of COS-7 cells cotransfected with cDNAs of <Protname>Lck</Protname> and <Protname>CD38</Protname>, or a mixture of in vitro translated <Protname>CD38</Protname> and <Protname>Lck</Protname>. Because the <Protname>CD38</Protname> cytoplasmic domain does not contain any tyrosine residue, the interaction should be independent of phosphotyrosine. The interaction was further confirmed by in vitro interaction between a purified <Protname>Lck</Protname> <Protname>Src</Protname> homology 2 domain and a nonphosphosynthetic peptide corresponding to the membrane proximal region of the <Protname>CD38</Protname> cytoplasmic domain. In addition, <Protname>CD38</Protname> ligation resulted in an elevated tyrosine kinase activity of the <Protname>CD38</Protname>-associated <Protname>Lck</Protname> and ultimate activation of <Protname>interleukin-2</Protname> gene transcription. Furthermore, expression of a kinase-deficient <Protname>Lck</Protname> mutant suppressed <Protname>interleukin-2</Protname> gene activation in a dose-dependent manner. These results strongly suggested that <Protname>CD38</Protname> ligation indeed tranduced signals for T cell activation using its associated <Protname>Lck</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20079562</MedlineID>
<PMID>10611293</PMID>
<ArticleTitle>The alpha-helical FXXPhiPhi motif in <Protname>p53</Protname>: TAF interaction and discrimination by <Protname>MDM2</Protname>.</ArticleTitle>
<AbstractText>Transcriptional activation domains share little sequence homology and generally lack folded structures in the absence of their targets, aspects that have rendered activation domains difficult to characterize. Here, a combination of biochemical and nuclear magnetic resonance experiments demonstrates that the activation domain of the tumor suppressor <Protname>p53</Protname> has an FXXPhiPhi motif (F, Phe; X, any amino acids; Phi, hydrophobic residues) that folds into an alpha-helix upon binding to one of its targets, <Protname>hTAF(II)31</Protname> (a <Protname>human TFIID TATA box-binding protein-associated factor</Protname>). <Protname>MDM2</Protname>, the cellular attenuator of <Protname>p53</Protname>, discriminates the FXXPhiPhi motif of <Protname>p53</Protname> from those of <Protname>NF-kappaB</Protname> <Protname>p65</Protname> and <Protname>VP16</Protname> and specifically inhibits <Protname>p53</Protname> activity. Our studies support the notion that the FXXPhiPhi sequence is a general alpha-helical recognition motif for <Protname>hTAF(II)31</Protname> and provide insights into the mechanistic basis for regulation of <Protname>p53</Protname> function.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20057142</MedlineID>
<PMID>10591185</PMID>
<ArticleTitle>The inhibitory receptor <Protname>LIR-1</Protname> uses a common binding interaction to recognize class I MHC molecules and the viral homolog <Protname>UL18</Protname>.</ArticleTitle>
<AbstractText><Protname>LIR-1</Protname> is a class I MHC receptor related to <Protname>natural killer inhibitory receptors</Protname> (<Protname>KIRs</Protname>). Binding of <Protname>LIR-1</Protname> or <Protname>KIRs</Protname> to class I molecules results in inhibitory signals. Unlike individual <Protname>KIRs</Protname>, <Protname>LIR-1</Protname> recognizes many class I alleles and also binds <Protname>UL18</Protname>, a human cytomegalovirus class I MHC homolog. Here, we show that <Protname>LIR-1</Protname> interacts with the relatively nonpolymorphic alpha3 domain of class I proteins and the analogous region of <Protname>UL18</Protname> using its N-terminal <Protname>immunoglobulin</Protname>-like domain. The &gt;1000-fold higher affinity of <Protname>LIR-1</Protname> for <Protname>UL18</Protname> than for class I illustrates how a viral protein competes with host proteins to subvert the host immune response. <Protname>LIR-1</Protname> recognition of class I molecules resembles the <Protname>CD4</Protname>-class II MHC interaction more than the <Protname>KIR</Protname>-class I interaction, implying a functional distinction between <Protname>LIR-1</Protname> and <Protname>KIRs</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20036539</MedlineID>
<PMID>10567369</PMID>
<ArticleTitle>The N-terminal <Protname>ERK</Protname>-binding site of <Protname>MEK1</Protname> is required for efficient feedback phosphorylation by <Protname>ERK2</Protname> in vitro and <Protname>ERK</Protname> activation in vivo.</ArticleTitle>
<AbstractText>An <Protname>ERK2</Protname>-binding site at the N terminus of <Protname>MEK1</Protname> was reported to mediate their stable association. We examined the importance of this binding site in the feedback phosphorylation of <Protname>MEK1</Protname> on Thr(292) and Thr(386) by <Protname>ERK2</Protname>, the phosphorylation and activation of <Protname>ERK2</Protname> by <Protname>MEK1</Protname>, and the interaction of <Protname>MEK1</Protname> with <Protname>ERK2</Protname> and <Protname>Raf-1</Protname>. Deletion of the binding site from <Protname>MEK1</Protname> reduced its phosphorylation by <Protname>ERK2</Protname>, but had no effect on its phosphorylation by <Protname>p21-activated protein kinase-1</Protname> (<Protname>PAK1</Protname>). A <Protname>MEK1</Protname> N-terminal peptide containing the binding site inhibited <Protname>MEK1</Protname> phosphorylation by <Protname>ERK2</Protname>. However, it did not affect <Protname>MEK1</Protname> phosphorylation by <Protname>p21-activated protein kinase</Protname> or <Protname>myelin basic protein</Protname> phosphorylation by <Protname>ERK2</Protname>. Deletion of the N-terminal <Protname>ERK</Protname>-binding domain of <Protname>MEK1</Protname> also reduced its ability to phosphorylate <Protname>ERK2</Protname> in vitro, to co-immunoprecipitate with <Protname>ERK2</Protname>, and to stimulate <Protname>ERK2</Protname> activation in transfected cells, but it did not alter the association with endogenous <Protname>Raf-1</Protname>. Using <Protname>ERK2</Protname>-<Protname>p38</Protname> chimeras and an <Protname>ERK2</Protname> deletion mutant, a <Protname>MEK1</Protname>-binding site of <Protname>ERK2</Protname> was localized to its N terminus.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>20005856</MedlineID>
<PMID>10536032</PMID>
<ArticleTitle>Further characterization of the combining sites of <Protname>Bandeiraea (Griffonia) simplicifolia lectin-I</Protname>, <Protname>isolectin A(4)</Protname>.</ArticleTitle>
<AbstractText><Protname>Bandeiraea (Griffonia) simplicifolia lectin-I</Protname>, <Protname>isolectin A(4)</Protname>(<Protname>GS I-A(4)</Protname>), which is cytotoxic to the human colon cancer cell lines, is one of two lectin families derived from its seed extract. It contains only a homo-oligomer of subunit A, and is most specific for GalNAcalpha1. In order to elucidate the <Protname>GS I-A(4)</Protname>-glycoconjugate interactions in greater detail, the combining site of this <Protname>lectin</Protname> was further characterized by enzyme linked lectino-sorbent assay (ELLSA) and by inhibition of <Protname>lectin</Protname>-glycoprotein interactions. This study has demonstrated that the Tn-containing glycoproteins tested, consisting of mammalian salivary glycoproteins (armadillo, asialo-hamster sublingual, asialo-ovine, -bovine, and -porcine submandibular), are bound strongly by <Protname>GS I-A(4)</Protname>. Among monovalent inhibitors so far tested, p-NO2-phenylalphaGalNAc is the most potent, suggesting that hydrophobic forces are important in the interaction of this <Protname>lectin</Protname>. <Protname>GS I-A(4)</Protname>is able to accommodate the monosaccharide GalNAc at the nonreducing end of oligosaccharides. This suggests that the combining site of the lectin is a shallow cavity. Among oligosaccharides and monosaccharides tested as inhibitors of the binding of <Protname>GS I-A(4)</Protname>, the hierarchy of potencies are: GalNAcalpha1--&gt;3GalNAcbeta1--&gt;3Galalpha1--&gt;4Galbeta 1--&gt;4Glc (Forssman pentasaccharide) &gt; GalNAcalpha1--&gt;3(LFucalpha1--&gt;2)Gal (blood group A)()> GalNAc > Galalpha1--&gt;4Gal > Galalpha1--&gt;3Gal (blood group B-like)> Gal.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99442054</MedlineID>
<PMID>10513896</PMID>
<ArticleTitle>Interaction of newly synthesized <Protname>apolipoprotein B</Protname> with <Protname>calnexin</Protname> and <Protname>calreticulin</Protname> requires glucose trimming in the endoplasmic reticulum.</ArticleTitle>
<AbstractText><Protname>Apolipoprotein B</Protname> (<Protname>ApoB</Protname>) is the only protein component of the low density lipoproteins (LDL) in plasma. It is a glycoprotein with a molecular mass of about 550 kDa (4536 amino acids) containing 16 N-glycans. We have studied the interaction of <Protname>ApoB</Protname> with two <Protname>lectin</Protname>-like chaperones of the Endoplasmic Reticulum (ER)--<Protname>Calnexin</Protname> (<Protname>CN</Protname>) and <Protname>Calreticulin</Protname> (<Protname>CR</Protname>). Using a co-immunoprecipitation approach we observed that newly synthesized <Protname>ApoB</Protname> associates with <Protname>CN</Protname> and <Protname>CR</Protname>. The interaction was transient; within 30-60 min after synthesis bulk of newly formed <Protname>ApoB</Protname> dissociated. Using McA Rh7777 cells expressing an N-terminal fragment of <Protname>ApoB</Protname> we found that inhibition of glucosidases in the ER prevented the association of <Protname>CN</Protname> and <Protname>CR</Protname> to newly synthesized <Protname>ApoB</Protname>. The results showed that like for association with other glycoprotein substrates, trimming of glucose residues was essential for <Protname>ApoB</Protname> binding to <Protname>CN</Protname> and <Protname>CR</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99420955</MedlineID>
<PMID>10493212</PMID>
<ArticleTitle>Monoclonal antibody specific for <Protname>tissue factor pathway inhibitor</Protname>-<Protname>factor Xa</Protname> complex: its characterization and application to plasmas from patients with disseminated intravascular coagulation and pre-disseminated intravascular coagulation.</ArticleTitle>
<AbstractText><Protname>Tissue factor pathway inhibitor</Protname> (<Protname>TFPI</Protname>), a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2 and K3), inhibits the initial reactions of the extrinsic blood coagulation pathway through its K1 and K2 domains. We prepared and characterized a monoclonal antibody (<Protname>Mab8-1</Protname>) against <Protname>TFPI</Protname>-<Protname>factor Xa</Protname> (<Protname>TFPI</Protname>-<Protname>Xa</Protname>) complex. The reactivities of <Protname>Mab8-1</Protname> toward <Protname>TFPI</Protname>-<Protname>Xa</Protname> complex, <Protname>TFPI</Protname> without C-terminal (<Protname>TFPI</Protname>-C)-<Protname>Xa</Protname> complex, K1K2-<Protname>Xa</Protname> complex and K2K3-<Protname>Xa</Protname> complex were examined using a surface plasmon resonance analysis (Biacore). The Biacore system allowed a quantitative analysis of antibody-antigen interaction, in real time, from which the association and dissociation rate constants could readily be obtained. The bindings of <Protname>Mab8-1</Protname> to <Protname>TFPI</Protname>-<Protname>Xa</Protname> complex, <Protname>TFPI</Protname>-C-<Protname>Xa</Protname> complex and K2K3-Xa complex were each concentration-dependent. However, no binding of <Protname>Mab8-1</Protname> to the K1K2-Xa complex was observed. The binding of <Protname>Mab8-1</Protname> to <Protname>TFPI</Protname> or <Protname>Xa</Protname> was also not observed. These results suggested that the epitope for <Protname>Mab8-1</Protname> was exposed in the K3 domain of <Protname>TFPI</Protname>, which was generated by the conformational change after the formation of <Protname>TFPI</Protname>-<Protname>Xa</Protname> complex. We then developed an enzyme-linked immunosorbent assay method specific for <Protname>TFPI</Protname>-<Protname>Xa</Protname> complex using <Protname>Mab8-1</Protname>, and we used this assay to measure plasma levels of <Protname>TFPI</Protname>-<Protname>Xa</Protname>. The normal range assessed from analyses of plasma from 30 normal healthy volunteers was 17.7-66.7 with a mean of 35.5 +/- 11.7 pmol/l. In order to asses the clinical implication of <Protname>TFPI</Protname>-<Protname>Xa</Protname> complex in the plasma of patients with thrombotic disorders, plasma concentrations were measured in 37 patients with disseminated intravascular coagulation (DIC) caused by a variety of underlying diseases. The <Protname>TFPI</Protname>-<Protname>Xa</Protname> antigen levels were significantly higher in the patients with DIC (51.9 +/- 21.6 pmol/l) and the 36 patients with pre-DIC (55.1 +/- 20.2 pmol/l) than in the 137 non-DIC patients (37.9 +/- 13.1 pmol/l). In the patients with DIC or pre-DIC, there was no significant correlation between <Protname>TFPI</Protname>-<Protname>Xa</Protname> complex and the elevated levels of <Protname>thrombin</Protname>-<Protname>antithrombin</Protname> complex, <Protname>plasmin</Protname>-<Protname>alpha2 plasmin inhibitor</Protname> complex, D-dimer, soluble <Protname>fibrin</Protname> monomer, soluble <Protname>thrombomodulin</Protname> or <Protname>tissue factor</Protname>. These data indicate that the plasma level of <Protname>TFPI</Protname>-<Protname>Xa</Protname> seems to be a novel independent molecular marker of DIC and pre-DIC.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99400624</MedlineID>
<PMID>10469658</PMID>
<ArticleTitle>Control of glycosylation of MHC class II-associated invariant chain by translocon-associated <Protname>RAMP4</Protname>.</ArticleTitle>
<AbstractText>Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the <Protname>ribosome-associated membrane protein 4</Protname> (<Protname>RAMP4</Protname>). <Protname>RAMP4</Protname> is a small, tail-anchored protein of 66 amino acid residues that is homologous to the <Protname>yeast YSY6 protein</Protname>. <Protname>YSY6</Protname> suppresses a secretion defect of a <Protname>secY</Protname> mutant in Escherichia coli. The interaction of <Protname>RAMP4</Protname> with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with <Protname>RAMP4</Protname> contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with <Protname>RAMP4</Protname> and inefficient N-glycosylation. We propose that <Protname>RAMP4</Protname> controls modification of Ii and possibly also of other secretory and membrane proteins containing specific <Protname>RAMP4</Protname>-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99367435</MedlineID>
<PMID>10438481</PMID>
<ArticleTitle><Protname>beta(1) integrin</Protname> binds the 16-kDa subunit of <Protname>vacuolar H(+)-ATPase</Protname> at a site important for <Protname>human papillomavirus E5</Protname> and <Protname>platelet-derived growth factor</Protname> signaling.</ArticleTitle>
<AbstractText><Protname>Integrins</Protname> mediate adhesive interactions between cells and the extracellular matrix, and play a role in cell migration, proliferation, differentiation, cytoskeletal organization, and signal transduction. We have identified an interaction between the <Protname>beta(1) integrin</Protname> and the 16-kDa subunit of <Protname>vacuolar H(+)-ATPase</Protname> (16K). This interaction was first isolated in a yeast two-hybrid screen and confirmed by coimmunoprecipitation and in in vitro binding assays using bacterially expressed proteins. Immunofluorescent studies performed in L6 myoblasts expressing both native and epitope-tagged 16K demonstrate co-localization with <Protname>beta(1) integrin</Protname> in focal adhesions. Deletion of the fourth of four transmembrane helices in 16K results in loss of interaction with <Protname>beta(1) integrin</Protname> in vitro and in the two-hybrid system, and less prominent staining in focal adhesions. This helix is also required for ligand-independent activation of <Protname>platelet-derived growth factor-beta receptor</Protname> signaling by the <Protname>human papillomavirus E5 oncoprotein</Protname>. Overexpression of 16K or expression of 16K lacking this helix alters the morphology of myoblasts and fibroblasts, suggesting that the interaction of 16K with integrins could be important for cell growth control. We also discuss the possible role 16K might play in integrin movement.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99340247</MedlineID>
<PMID>10409738</PMID>
<ArticleTitle><Protname>Human TAF(II)55</Protname> interacts with the vitamin D(3) and <Protname>thyroid hormone receptors</Protname> and with derivatives of the <Protname>retinoid X receptor</Protname> that have altered transactivation properties.</ArticleTitle>
<AbstractText>We have identified novel interactions between the human <Protname>(h)TATA-binding protein-associated factor TAF(II)55</Protname> and the ligand-binding domains (LBDs) of the nuclear receptors for vitamin D(3) (<Protname>VDR</Protname>) and thyroid hormone (<Protname>TRalpha</Protname>). Following expression in Cos cells, <Protname>hTAF(II)55</Protname> interacts with the <Protname>VDR</Protname> and <Protname>TRalpha</Protname> LBDs in a ligand-independent manner whereas no interactions with the <Protname>retinoid X receptors</Protname> (<Protname>RXRs</Protname>) or with other receptors were observed. Deletion mapping indicates that <Protname>hTAF(II)55</Protname> interacts with a 40-amino-acid region spanning alpha-helices H3 to H5 of the <Protname>VDR</Protname> and <Protname>TRalpha</Protname> LBDs but not with the equivalent highly related region of <Protname>RXRgamma</Protname>. <Protname>TAF(II)55</Protname> also interacts with chimeric receptors in which the H3-to-H5 region of <Protname>RXRgamma</Protname> has been replaced with that of the <Protname>VDR</Protname> or <Protname>TRalpha</Protname>. Furthermore, replacement of two single amino acids of the <Protname>RXRgamma</Protname> LBD with their <Protname>VDR</Protname> counterparts allows the <Protname>RXRgamma</Protname> LBD to interact with <Protname>hTAF(II)55</Protname> while the corresponding double substitution allows a much stronger interaction. In transfection experiments, the single mutated <Protname>RXRgamma</Protname> LBDs activate transcription to fivefold higher levels than wild-type <Protname>RXRgamma</Protname> while the double mutation activates transcription to a level comparable to that observed with the <Protname>VDR</Protname>. There is therefore a correlation between the ability of the modified <Protname>RXRs</Protname> to interact with <Protname>hTAF(II)55</Protname> and transactivation. These results strongly suggest that the <Protname>TAF(II)55</Protname> interactions with the modified <Protname>RXR</Protname> LBDs modulate transcriptional activation.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99318134</MedlineID>
<PMID>10391250</PMID>
<ArticleTitle>Interaction of <Protname>c-Abl</Protname> and <Protname>p73alpha</Protname> and their collaboration to induce apoptosis.</ArticleTitle>
<AbstractText><Protname>c-Abl</Protname>, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of <Protname>c-Abl</Protname>. <Protname>p73</Protname>, a member of the <Protname>p53</Protname> family of tumour-suppressor proteins, can also induce apoptosis. Here we show that the apoptotic activity of <Protname>p73alpha</Protname> requires the presence of functional, kinase-competent <Protname>c-Abl</Protname>. Furthermore, <Protname>p73</Protname> and <Protname>c-Abl</Protname> can associate with each other, andthis binding is mediated by a PxxP motif in <Protname>p73</Protname> and the SH3 domain of <Protname>c-Abl</Protname>. We find that <Protname>p73</Protname> is a substrate of the <Protname>c-Abl kinase</Protname> and that the ability of <Protname>c-Abl</Protname> to phosphorylate <Protname>p73</Protname> is markedly increased by gamma-irradiation. Moreover, <Protname>p73</Protname> is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving <Protname>p73</Protname> and <Protname>c-Abl</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99296607</MedlineID>
<PMID>10366588</PMID>
<ArticleTitle><Protname>Transportin-SR</Protname>, a nuclear import receptor for SR proteins.</ArticleTitle>
<AbstractText>The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain-bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed <Protname>transportin-SR</Protname> (<Protname>TRN-SR</Protname>), binds specifically and directly to the RS domains of <Protname>ASF/SF2</Protname> and <Protname>SC35</Protname> as well as several other SR proteins. The nuclear transport regulator <Protname>RanGTP</Protname> abolishes this interaction. Recombinant <Protname>TRN-SR</Protname> mediates nuclear import of RS domain- bearing proteins in vitro. <Protname>TRN-SR</Protname> has amino acid sequence similarity to several members of the <Protname>importin beta</Protname>/<Protname>transportin</Protname> family. These findings strongly suggest that <Protname>TRN-SR</Protname> is a nuclear import receptor for the SR protein family.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99263023</MedlineID>
<PMID>10330192</PMID>
<ArticleTitle>Identification of <Protname>CHIP</Protname>, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.</ArticleTitle>
<AbstractText>The chaperone function of the <Protname>mammalian 70-kDa heat shock proteins Hsc70</Protname> and <Protname>Hsp70</Protname> is modulated by physical interactions with four previously identified chaperone cofactors: <Protname>Hsp40</Protname>, <Protname>BAG-1</Protname>, the <Protname>Hsc70-interacting protein Hip</Protname>, and the <Protname>Hsc70-Hsp90-organizing protein Hop</Protname>. <Protname>Hip</Protname> and <Protname>Hop</Protname> interact with <Protname>Hsc70</Protname> via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, <Protname>carboxyl terminus of Hsc70-interacting protein</Protname> (<Protname>CHIP</Protname>). <Protname>CHIP</Protname> is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. <Protname>Hsc70</Protname> and <Protname>Hsp70</Protname> were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between <Protname>CHIP</Protname> and both <Protname>Hsc70</Protname> and <Protname>Hsp70</Protname>, and complexes containing <Protname>CHIP</Protname> and <Protname>Hsc70</Protname> were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using <Protname>glutathione S-transferase</Protname> fusions, we found that <Protname>CHIP</Protname> interacted with the carboxy-terminal residues 540 to 650 of <Protname>Hsc70</Protname>, whereas <Protname>Hsc70</Protname> interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of <Protname>CHIP</Protname>. Recombinant <Protname>CHIP</Protname> inhibited <Protname>Hsp40-stimulated ATPase</Protname> activity of <Protname>Hsc70</Protname> and <Protname>Hsp70</Protname>, suggesting that <Protname>CHIP</Protname> blocks the forward reaction of the <Protname>Hsc70</Protname>-<Protname>Hsp70</Protname> substrate-binding cycle. Consistent with this observation, both <Protname>luciferase</Protname> refolding and substrate binding in the presence of <Protname>Hsp40</Protname> and <Protname>Hsp70</Protname> were inhibited by <Protname>CHIP</Protname>. Taken together, these results indicate that <Protname>CHIP</Protname> decreases net ATPase activity and reduces chaperone efficiency, and they implicate <Protname>CHIP</Protname> in the negative regulation of the forward reaction of the <Protname>Hsc70</Protname>-<Protname>Hsp70</Protname> substrate-binding cycle.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99242826</MedlineID>
<PMID>10225901</PMID>
<ArticleTitle>Molecular interactions of Porphyromonas gingivalis fimbriae with host proteins: kinetic analyses based on surface plasmon resonance.</ArticleTitle>
<AbstractText>Fimbriae of Porphyromonas gingivalis are thought to play an important role in the colonization and invasion of periodontal tissues. In this study, we analyzed the interactions of P. gingivalis fimbriae with <Protname>human hemoglobin</Protname>, <Protname>fibrinogen</Protname>, and salivary components (i.e., <Protname>proline-rich protein</Protname> [<Protname>PRP</Protname>], <Protname>proline-rich glycoprotein</Protname> [<Protname>PRG</Protname>], and <Protname>statherin</Protname>) based on surface plasmon resonance (SPR) spectroscopy with a biomolecular interaction analyzing system (BIAcore). The real-time observation showed that the fimbriae interacted more quickly with <Protname>hemoglobin</Protname> and <Protname>PRG</Protname> than with other proteins and more intensely with <Protname>fibrinogen</Protname>. The significant association constant (ka) values obtained by BIAcore demonstrated that the interactions between fimbriae and these host proteins are specific. These estimated Ka values were not too different; however, the Ka values for <Protname>hemoglobin</Protname> (2.43 x 10(6)) and <Protname>fibrinogen</Protname> (2.16 x 10(6)) were statistically greater than those for the salivary proteins (1.48 x 10(6) to 1.63 x 10(6)). The Ka value of anti-fimbriae <Protname>immunoglobulin G</Protname> for fimbriae was estimated to be 1. 22 x 10(7), which was 6.55-fold higher than the mean Ka value of the host proteins. Peptide PRP-C, a potent inhibitor of <Protname>PRP</Protname>-fimbriae interaction, dramatically inhibited fimbrial association to <Protname>PRP</Protname> and <Protname>PRG</Protname> and was also inhibitory against other host proteins by BIAcore. The binding of fimbriae to these proteins was also evaluated by other methods with hydroxyapatite beads or polystyrene microtiter plates. The estimated binding abilities differed considerably, depending on the assay method that was used. It was noted that the binding capacity of <Protname>PRP</Protname> was strongly diminished by immobilization on a polystyrene surface. Taken together, these findings suggest that P. gingivalis fimbriae possess a strong ability to interact with the host proteins which promote bacterial adherence to the oral cavity and that SPR spectroscopy is a useful method for analyzing specific protein-fimbriae interactions.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99230304</MedlineID>
<PMID>10212261</PMID>
<ArticleTitle>Inhibition of <Protname>Ets-1</Protname> DNA binding and ternary complex formation between <Protname>Ets-1</Protname>, <Protname>NF-kappaB</Protname>, and DNA by a designed DNA-binding ligand.</ArticleTitle>
<AbstractText>Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for <Protname>TBP</Protname>, <Protname>Ets-1</Protname>, and <Protname>LEF-1</Protname> in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor <Protname>Ets-1</Protname> is specifically inhibited by a polyamide bound in the minor groove. <Protname>Ets-1</Protname> is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by <Protname>Ets-1</Protname>, by occupying the adjacent minor groove. Full-length <Protname>Ets-1</Protname> binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of <Protname>NF-kappaB</Protname>, and the Ets-inhibitory polyamide also blocks formation of ternary <Protname>Ets-1</Protname>. <Protname>NF-kappaB</Protname>.DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for <Protname>NF-kappaB</Protname> also inhibits <Protname>NF-kappaB</Protname> binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99214605</MedlineID>
<PMID>10187830</PMID>
<ArticleTitle>Mechanisms for <Protname>GroEL</Protname>/<Protname>GroES</Protname>-mediated folding of a large 86-kDa fusion polypeptide in vitro.</ArticleTitle>
<AbstractText>Our understanding of mechanisms for <Protname>GroEL</Protname>/<Protname>GroES</Protname>-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated <Protname>chaperonin</Protname>-dependent folding of a large 86-kDa fusion polypeptide, in which the mature <Protname>maltose-binding protein</Protname> (<Protname>MBP</Protname>) sequence was linked to the N terminus of the alpha subunit of the <Protname>decarboxylase</Protname> (<Protname>E1</Protname>) component of the <Protname>human mitochondrial branched-chain alpha-ketoacid dehydrogenase</Protname> complex. The fusion polypeptide, <Protname>MBP-alpha</Protname>, when co-expressed with the beta subunit of <Protname>E1</Protname>, produced a chimeric protein <Protname>MBP</Protname>-<Protname>E1</Protname> with an (<Protname>MBP-alpha</Protname>)2beta2 structure, similar to the alpha2 beta2 structure in native <Protname>E1</Protname>. Reactivation of <Protname>MBP</Protname>-<Protname>E1</Protname> denatured in 8 M urea was absolutely dependent on <Protname>GroEL</Protname>/<Protname>GroES</Protname> and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged <Protname>E1</Protname>. <Protname>Chaperonin</Protname>-mediated refolding of the <Protname>MBP-alpha</Protname> fusion polypeptide showed that the folding of the <Protname>MBP</Protname> moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively. This explained the occurrence of an <Protname>MBP-alpha</Protname>. <Protname>GroEL</Protname> binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same <Protname>GroEL</Protname> scaffold. Moreover, we show that when the <Protname>alpha.GroEL</Protname> complex (molar ratio 1:1) was incubated with <Protname>GroES</Protname>, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the <Protname>MBP-alpha.GroEL</Protname> (1:1) complex was capped by <Protname>GroES</Protname> exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the <Protname>GroEL</Protname> cavity that is not sequestered by <Protname>GroES</Protname>.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99173985</MedlineID>
<PMID>10074138</PMID>
<ArticleTitle>Interaction of the human immunodeficiency virus type 1 nucleocapsid with <Protname>actin</Protname>.</ArticleTitle>
<AbstractText>The nucleocapsid (NC) domain of the <Protname>retrovirus Gag protein</Protname> plays several important roles in the viral life cycle, including virus assembly, viral genomic RNA encapsidation, primer tRNA placement, and enhancement of viral reverse transcription. In this study, deletion of NC domain of <Protname>human immunodeficiency virus type 1 (HIV-1) Gag</Protname> was found to drastically reduce virus particle production in <Protname>CD4</Protname>(+) T cells. Cellular fractionation experiments showed that although most of the uncleaved wild-type <Protname>HIV-1 Gag</Protname>, unmyristylated <Protname>Gag</Protname>, and p6(<Protname>Gag</Protname>) domain-truncated <Protname>Gag</Protname> molecules copurified with the host cell cytoskeleton, most of the mutant <Protname>Gag</Protname> molecules lacking both the NC and p6(<Protname>Gag</Protname>) domains failed to cofractionate with cytoskeleton. In wild-type virus-infected cells, in which the viral protease was active, the cleaved NCp7 copurified with the cytoskeleton, whereas most of the MAp17 and CAp24 did not. Monoclonal antibody against <Protname>actin</Protname> coimmunoprecipitated full-length <Protname>Gag</Protname> and p6(<Protname>Gag</Protname>) domain-truncated <Protname>Gag</Protname> molecules from cell lysates but failed to precipitate the truncated mutant <Protname>Gag</Protname> molecules lacking NC plus p6(<Protname>Gag</Protname>). Purified recombinant NCp7, but not CAp24, was able to bind <Protname>F-actin</Protname> in cosedimentation experiments. Furthermore, wild-type NCp7 and a zinc finger mutant NCp7(F16A), like a cellular <Protname>actin</Protname>-binding protein (<Protname>the villin headpiece</Protname>), bound <Protname>F-actin</Protname> in a dose-dependent fashion in vitro. Taken together, these results suggest that HIV-1 NCp7 can bind <Protname>F-actin</Protname> directly and that interaction between <Protname>HIV-1 Gag</Protname> and the <Protname>actin</Protname> cytoskeleton through the NC domain may play an important role in HIV-1 assembly and/or other steps of the viral life cycle.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99151984</MedlineID>
<PMID>10026279</PMID>
<ArticleTitle>Importance of <Protname>factor VIIa</Protname> Gla-domain residue Arg-36 for recognition of the macromolecular substrate <Protname>factor X</Protname> Gla-domain.</ArticleTitle>
<AbstractText>Macromolecular substrate docking with coagulation enzyme-cofactor complexes involves multiple contacts distant from the enzyme's catalytic cleft. Here we characterize the binding of the Gla-domain of macromolecular substrate <Protname>coagulation factor X</Protname> to the complex of <Protname>tissue factor</Protname> (<Protname>TF</Protname>) and <Protname>VIIa</Protname>. Site-directed mutagenesis of charged residue side chains in the VIIa Gla-domain identified Arg-36 as being important for macromolecular substrate docking. Ala substitution for Arg-36 resulted in an increased KM and a decreased rate of X activation. X with a truncated Gla-domain was activated by mutant and wild-type VIIa at indistinguishable rates, demonstrating that Arg-36 interactions require a properly folded Gla-domain of the macromolecular substrate. VIIa Arg-36 was also required for effective docking of the X Gla-domain in the absence of phospholipid, demonstrating that the Gla-domain of VIIa participates in protein-protein interactions with X. In the absence of <Protname>TF</Protname>, the mutant VIIa had essentially normal function, indicating that the cofactor positions VIIa's Gla-domain for optimal macromolecular substrate docking. Computational docking suggests multiple charge complementary contacts of the X Gla-domain with <Protname>TF</Protname>.VIIa. A prominent interaction is made by the functionally important X residue Gla-14 with the center of the extended docking site created by residues in the carboxyl module of <Protname>TF</Protname> and the contiguous VIIa Gla-domain. These data demonstrate the functional importance of interactions of the Gla-domains of enzyme and substrate, and begin to elucidate the molecular details of the ternary <Protname>TF</Protname>.VIIa.X complex.</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99121109</MedlineID>
<PMID>9920917</PMID>
<ArticleTitle><Protname>Smad1</Protname> recognition and activation by the <Protname>ALK1</Protname> group of <Protname>transforming growth factor-beta</Protname> family receptors.</ArticleTitle>
<AbstractText>Two structural elements, the L45 loop on the kinase domain of the <Protname>transforming growth factor-beta</Protname> (<Protname>TGF-beta</Protname>) family type I receptors and the L3 loop on the MH2 domain of <Protname>Smad</Protname> proteins, determine the specificity of the interactions between these receptors and <Protname>Smad</Protname> proteins. The L45 sequence of the <Protname>TGF-beta type I receptor</Protname> (<Protname>TbetaR-I</Protname>) specifies <Protname>Smad2</Protname> interaction, whereas the related L45 sequence of the <Protname>bone morphogenetic protein (BMP) type I receptor</Protname> (<Protname>BMPR-I</Protname>) specifies <Protname>Smad1</Protname> interactions. Here we report that members of a third receptor group, which includes <Protname>ALK1</Protname> and <Protname>ALK2</Protname> from vertebrates and <Protname>Saxophone</Protname> from Drosophila, specifically phosphorylate and activate <Protname>Smad1</Protname> even though the L45 sequence of this group is very divergent from that of <Protname>BMPR-I</Protname>. We investigated the structural elements that determine the specific recognition of <Protname>Smad1</Protname> by <Protname>ALK1</Protname> and <Protname>ALK2</Protname>. In addition to the receptor L45 loop and the <Protname>Smad1</Protname> L3 loop, the specificity of this recognition requires the alpha-helix 1 of <Protname>Smad1</Protname>. The alpha-helix 1 is a conserved structural element located in the vicinity of the L3 loop on the surface of the <Protname>Smad</Protname> MH2 domain. Thus, <Protname>Smad1</Protname> recognizes two distinct groups of receptors, the <Protname>BMPR-I</Protname> group and the <Protname>ALK1</Protname> group, through different L45 sequences on the receptor kinase domain and a differential use of two surface structures on the <Protname>Smad1</Protname> MH2 domain.</AbstractText>
</PubmedArticle>
<!-- GENIA_48 -->
<PubmedArticle>
<MedlineID>94129004</MedlineID>
<PMID>94129004</PMID>
<ArticleTitle>Retinoic acid downmodulates erythroid differentiation and <Protname>GATA1</Protname> expression in purified adult-progenitor culture.</ArticleTitle>
<AbstractText>
All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia.
We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of <Protname>interleukin-3</Protname> (<Protname>IL-3</Protname>) <Protname>granulocyte-macrophage colony-stimulating factor</Protname> (<Protname>GM-CSF</Protname>) and <Protname>erythropoietin</Protname> (<Protname>Ep</Protname>) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones.
This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS-liquid-suspension culture supplemented with saturating <Protname>Ep</Protname> level and low-dose <Protname>IL-3</Protname>/<Protname>GM-CSF</Protname>, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master <Protname>GATA1</Protname> gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of <Protname>GATA1</Protname> mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of <Protname>GATA1</Protname> induction in the early stages of erythropoietic differentiation.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>97175006</MedlineID>
<PMID>97175006</PMID>
<ArticleTitle>Pancreatic development and maturation of the islet B cell.</ArticleTitle>
<AbstractText>
Studies of pluripotent islet cultures. Pancreas organogenesis is a highly regulated process, in which two anlage evaginate from the primitive gut.
They later fuse, and, under the influence of the surrounding mesenchyme, the mature organ develops, being mainly composed of ductal, exocrine and endocrine compartments. Early buds are characterized by a branching morphogenesis of the ductal epithelium from which endocrine and exocrine precursor cells bud to eventually form the two other compartments. The three compartments are thought to be of common endodermal origin; in contrast to earlier hypotheses, which suggested that the endocrine compartment was of neuroectodermal origin.
It is thus generally believed that the pancreatic endocrine-lineage possesses the ability to mature along a differentiation pathway that shares many characteristics with those of neuronal differentiation. During recent years, studies of <Protname>insulin</Protname>-gene regulation and, in particular, the tissue-specific transcriptional control of <Protname>insulin</Protname>-gene activity have provided information on pancreas development in general.
The present review summarizes these findings, with a special focus on our own studies on pluripotent endocrine cultures of rat pancreas.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>97087515</MedlineID>
<PMID>97087515</PMID>
<ArticleTitle>Tissue and cell-type specific expression of the <Protname>tuberous sclerosis gene</Protname>, <Protname>TSC2</Protname>, in human tissues.</ArticleTitle>
<AbstractText>
<Protname>TSC2</Protname> is a gene on chromosome 16p13.3 associated with the autosomal dominant neurocutaneous disorder, tuberous sclerosis complex (TSC).
By using a partial nucleotide sequence from the cloned <Protname>TSC2</Protname> and polymerase chain reaction methodology, we constructed a digoxigenin-labeled complementary DNA probe to examine <Protname>TSC2</Protname> gene expression in autopsy- or biopsy-derived human tissues by in situ hybridization. <Protname>TSC2</Protname> messenger RNA was widely expressed in various cell types throughout the body, including epithelia, lymphocytes, and cells with endocrine functions, e.g., adrenal cortex and anterior pituitary.
It was prominently and selectively (within the central nervous system) expressed in pyramidal cells of the cerebral cortex and other motor neurons, e.g., in spinal cord and brainstem nuclei. Visceral <Protname>TSC2</Protname> expression was comparable in autopsy tissues from patients with and without TSC; <Protname>TSC2</Protname> messenger RNA expression was most prominent in cells with a rapid mitotic rate and turnover, e.g., epithelia and lymphocytes, with central nervous system pyramidal cells and other neurons being an obvious exception, and/or in cells with important secretory/transport functions. This widespread expression of the <Protname>TSC2</Protname> gene supports the view that it encodes a protein vital to cell growth and metabolism or one that functions as a tumor/growth suppressor.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>94110342</MedlineID>
<PMID>94110342</PMID>
<ArticleTitle>Steroid-resistant asthma.</ArticleTitle>
<AbstractText>
Cellular mechanisms contributing to inadequate response to glucocorticoid therapy. The current study examined whether alterations in <Protname>glucocorticoid receptor</Protname> (<Protname>GR</Protname>) binding contribute to poor response to glucocorticoid therapy in asthma. 29 asthma patients with forced expiratory volume in 1 s (FEV1) &lt; 70% predicted were studied. Patients were classified as steroid sensitive (SS) if their morning FEV1 increased &gt; 30% after a 1-wk course of oral prednisone 20 mg twice daily and steroid resistant (SR) if they failed to increase &gt; 15%. PBMC obtained from these two groups, 17 SR and 12 SS, as well as 12 normal controls were analyzed. SR patients had two distinguishable GR binding abnormalities: 15 of the 17 SR patients demonstrated a significantly reduced GR binding affinity, as compared with SS patients (P = 0.0001) and normal controls (P = 0.0001). This defect was localized to T cells and reverted to normal after 48 h in culture media. However, incubation with a combination of <Protname>IL-2</Protname> and <Protname>IL-4</Protname> sustained this abnormality. The other two SR patients had an abnormally low GR number with normal binding affinity that was not limited to T cells. Furthermore, GR number failed to normalize after incubation in media alone or <Protname>IL-2</Protname> and <Protname>IL-4</Protname>. Therefore, SR asthma may be due to more than one abnormality, the majority related to a reversible cytokine-induced reduction in GR binding affinity and the second related to an irreversible reduction in GR number. These findings may have important implications for the design of alternative treatment approaches for recalcitrant asthma.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>97098661</MedlineID>
<PMID>97098661</PMID>
<ArticleTitle><Protname>Elf-1</Protname> and <Protname>Stat5</Protname> bind to a critical element in a new enhancer of the human <Protname>interleukin-2 receptor alpha</Protname> gene [published erratum appears in Mol Cell Biol 1997 Apr;17(4):2351]</ArticleTitle>
<AbstractText>
The <Protname>interleukin 2 receptor alpha</Protname>-chain (<Protname>IL-2R alpha</Protname>) gene is a key regulator of lymphocyte proliferation. <Protname>IL-2R alpha</Protname> is rapidly and potently induced in T cells in response to mitogenic stimuli. <Protname>Interleukin 2</Protname> (<Protname>IL-2</Protname>) stimulates <Protname>IL-2R alpha</Protname>. transcription, thereby amplifying expression of its own high-affinity receptor.
<Protname>IL-2R alpha</Protname> transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains <Protname>NF-kappaB</Protname> and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific <Protname>Ets protein Elf-1</Protname> and <Protname>HMG-I(Y)</Protname> proteins. However, none of these proximal regions account for the induction of <Protname>IL-2R alpha</Protname> transcription by <Protname>IL-2</Protname>. To find new regulatory regions of the <Protname>IL-2R alpha</Protname> gene, 8.5 kb of the 5' end noncoding sequence of the <Protname>IL-2R alpha</Protname> gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine <Protname>IL-2</Protname>-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers <Protname>IL-2</Protname> responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for <Protname>IL-2</Protname> inducibility of PRRIII/CAT reporter constructs. <Protname>IL-2</Protname> induced the binding of <Protname>Stat5a</Protname> and <Protname>b</Protname> proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of <Protname>IL-2R alpha</Protname> expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in <Protname>IL-2R alpha</Protname> regulation and suggest that the T-cell-specific <Protname>Elf-1</Protname> factor can serve as a transcriptional repressor.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>94149008</MedlineID>
<PMID>94149008</PMID>
<ArticleTitle><Protname>rel</Protname> Is rapidly tyrosine-phosphorylated following <Protname>granulocyte-colony stimulating factor</Protname> treatment of human neutrophils.</ArticleTitle>
<AbstractText>
Stimulation of neutrophils with <Protname>granulocyte-colony stimulating factor</Protname> (<Protname>G-CSF</Protname>) results in an enhanced respiratory burst, prolonged survival, and increased tumor cell killing. The effects of <Protname>G-CSF</Protname> are mediated by binding to specific, high affinity receptors. <Protname>G-CSF receptors</Protname> lack intrinsic tyrosine kinase activity, but activation of the receptor results in the rapid induction of tyrosine kinase activity. Antiphosphotyrosine immunoblots of whole cell lysates prepared from neutrophils show that the <Protname>G-CSF</Protname> rapidly induces prominent tyrosine phosphorylation of a protein of a relative molecular mass of 80 kDa. Using monospecific antibodies, the 80-kDa tyrosine-phosphorylated protein has been shown to be <Protname>p80c-rel</Protname>, a proto-oncogene belonging to a family of transcriptional regulators which include <Protname>NF-kB</Protname>. The induction of tyrosine phosphorylation of <Protname>p80c-rel</Protname> was unique to <Protname>G-CSF</Protname> in that <Protname>granulocyte-macrophage colony stimulating factor</Protname> which also stimulates neutrophils and induces tyrosine phosphorylation does not result in tyrosine phosphorylation of <Protname>p80c-rel</Protname>. The consequences of <Protname>p80c-rel</Protname> tyrosine phosphorylation are not yet known; however, tyrosine-phosphorylated <Protname>p80c-rel</Protname> is capable of binding to DNA, and <Protname>G-CSF</Protname> stimulation results in an increase in the amount of <Protname>p80c-rel</Protname> which binds to DNA. These results demonstrate that one of the first biochemical events which occurs in neutrophils following <Protname>G-CSF</Protname> stimulation, activation of a tyrosine kinase, leads directly to the tyrosine phosphorylation of <Protname>p80c-rel</Protname>.
Thus, the tyrosine kinase activated by <Protname>G-CSF</Protname> appears to directly transduce a signal to a protein which functions as a transcriptional regulator.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>97115792</MedlineID>
<PMID>97115792</PMID>
<ArticleTitle>Mutation of tyrosines 492/493 in the kinase domain of <Protname>ZAP-70</Protname> affects multiple T-cell receptor signaling pathways.</ArticleTitle>
<AbstractText>
The protein-tyrosine kinase <Protname>ZAP-70</Protname> is implicated, together with the <Protname>Src kinase p56(lck)</Protname>, in controlling the early steps of the <Protname>T-cell antigen receptor</Protname> (<Protname>TCR</Protname>) signaling cascade. To help elucidate further the mechanism by which <Protname>ZAP-70</Protname> regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line <Protname>ZAP-70</Protname> mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited <Protname>TCR</Protname>-induced activation of <Protname>nuclear factor of activated T cells</Protname> by interfering with both intracellular calcium increase and <Protname>Ras</Protname>-regulated activation of extracellular signal-regulated kinases. Moreover, <Protname>TCR</Protname>-induced phosphorylation of <Protname>pp36-38</Protname>, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type <Protname>ZAP-70</Protname> induced constitutive activation of <Protname>nuclear factor of activated T cells</Protname>. The <Protname>ZAP-70</Protname> mutant studied here could be phosphorylated on tyrosine when associated to the <Protname>TCR</Protname> zeta chain and was able to bind <Protname>p56(lck)</Protname>. This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the <Protname>Src</Protname> homology domain 2-mediated association of <Protname>p56(lck)</Protname> with <Protname>ZAP-70</Protname>. Our data are most consistent with a model in which recruitment to the <Protname>TCR</Protname> allows <Protname>ZAP-70</Protname> autophosphorylation and binding to <Protname>p56(lck)</Protname>, which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the <Protname>ZAP-70</Protname> kinase activity. <Protname>ZAP-70</Protname> will then be able to effectively control phosphorylation of its substrates and lead to gene activation.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>97066996</MedlineID>
<PMID>97066996</PMID>
<ArticleTitle><Protname>Stat3</Protname> recruitment by two distinct ligand-induced, tyrosine-phosphorylated docking sites in the <Protname>interleukin-10 receptor</Protname> intracellular domain.</ArticleTitle>
<AbstractText>
Recent work has shown that <Protname>IL-10</Protname> induces activation of the <Protname>JAK</Protname>-<Protname>STAT</Protname> signaling pathway. To define the mechanism underlying <Protname>signal transducer and activator of transcription</Protname> (<Protname>STAT</Protname>) protein recruitment to the <Protname>interleukin 10 (IL-10) receptor</Protname>, the <Protname>STAT</Protname> proteins activated by <Protname>IL-10</Protname> in different cell populations were first defined using electrophoretic mobility shift assays. In all cells tested, <Protname>IL-10</Protname> activated <Protname>Stat1</Protname> and <Protname>Stat3</Protname> and induced the formation of three distinct DNA binding complexes that contained different combinations of these two transcription factors. <Protname>IL-10</Protname> also activated <Protname>Stat5</Protname> in Ba/F3 cells that stably expressed the <Protname>murine IL-10 receptor</Protname>. Using a structure-function mutagenesis approach, two tyrosine residues (Tyr427 and Tyr477) in the intracellular domain of the <Protname>murine IL-10 receptor</Protname> were found to be redundantly required for receptor function and for activation of <Protname>Stat3</Protname> but not for <Protname>Stat1</Protname> or <Protname>Stat5</Protname>. Twelve amino acid peptides encompassing either of these two tyrosine residues in phosphorylated form coprecipitated  <Protname>Stat3</Protname> but not <Protname>Stat1</Protname> and blocked <Protname>IL-10</Protname>-induced <Protname>Stat3</Protname> phosphorylation in a cell-free system. In contrast, tyrosine-phosphorylated peptides containing Tyr374 or Tyr396 did not interact with <Protname>Stat3</Protname> or block <Protname>Stat3</Protname> activation. These data demonstrate that <Protname>Stat3</Protname> but not <Protname>Stat1</Protname> or <Protname>Stat5</Protname> is directly recruited to the ligand-activated <Protname>IL-10 receptor</Protname> by binding to specific but redundant receptor intracellular domain sequences containing phosphotyrosine. This study thus supports the concept that utilization of distinct <Protname>STAT</Protname> proteins by different cytokine receptors is dependent on the expression of particular ligand-activatable, tyrosine-containing <Protname>STAT</Protname> docking sites in receptor intracellular domains.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96420243</MedlineID>
<PMID>96420243</PMID>
<ArticleTitle><Protname>Interleukin-6</Protname> promotes multiple myeloma cell growth via phosphorylation of <Protname>retinoblastoma protein</Protname>.</ArticleTitle>
<AbstractText>
<Protname>Interleukin-6</Protname> (<Protname>IL-6</Protname>) mediates autocrine and paracrine growth of multiple myeloma (MM) cells and inhibits tumor cell apoptosis. Abnormalities of <Protname>retinoblastoma protein</Protname> (<Protname>pRB</Protname>) and mutations of <Protname>RB</Protname> gene have been reported in up to 70% of MM patients and 80% of MM-derived cell lines. Because dephosphorylated (activated) <Protname>pRB</Protname> blocks transition from G1 to S phase of the cell cycle whereas phosphorylated (inactivated) <Protname>pRB</Protname> releases this growth arrest, we characterized the role of <Protname>pRB</Protname> in <Protname>IL-6</Protname>-mediated MM cell growth. Both phosphorylated and dephosphorylated <Protname>pRB</Protname> were expressed in all serum-starved MM patient cells and MM-derived cell lines, but <Protname>pRB</Protname> was predominantly in its phosphorylated form. In MM cells that proliferated in response to <Protname>IL-6</Protname>, exogenous <Protname>IL-6</Protname> downregulated dephosphorylated <Protname>pRB</Protname> and decreased dephosphorylated <Protname>pRB</Protname>-<Protname>E2F</Protname> complexes. Importantly, culture of MM cells with <Protname>RB</Protname> antisense, but not <Protname>RB</Protname> sense, oligonucleotide (ODN) triggered <Protname>IL-6</Protname> secretion and proliferation in MM cells; however, proliferation was only partially inhibited by neutralizing <Protname>anti-IL-6 monoclonal antibody</Protname> (<Protname>MoAb</Protname>). In contrast to MM cells, normal splenic B cells express dephosphorylated <Protname>pRB</Protname>. Although <Protname>CD40</Protname> ligand (CD40L) triggers a shift from dephosphorylated to phosphorylated <Protname>pRB</Protname> and proliferation of B cells, the addition of exogenous <Protname>IL-6</Protname> to CD40L-treated B cells does not alter either <Protname>pRB</Protname> or proliferation, as observed in MM cells.
These results suggest that phosphorylated <Protname>pRB</Protname> is constitutively expressed in MM cells and that <Protname>IL-6</Protname> further shifts <Protname>pRB</Protname> from its dephosphorylated to its phosphorylated form, thereby promoting MM cell growth via two mechanisms; by decreasing the amount of E2F bound by dephosphorylated <Protname>pRB</Protname> due to reduced dephosphorylated <Protname>pRB</Protname>, thereby releasing growth arrest; and by upregulating <Protname>IL-6</Protname> secretion by MM cells and related <Protname>IL-6</Protname>-mediated autocrine tumor cell growth.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>97031819</MedlineID>
<PMID>97031819</PMID>
<ArticleTitle>Regulation of <Protname>interferon-gamma</Protname> gene expression.</ArticleTitle>
<AbstractText>
<Protname>Interferon-gamma</Protname> (<Protname>IFN-gamma</Protname>), also known as <Protname>type II interferon</Protname>, is an important immunoregulatory gene that has multiple effects on the development, maturation, and function of the immune system. <Protname>IFN-gamma</Protname> mRNA and protein are expressed predominantly by T cells and large granular lymphocytes.
The <Protname>IFN-gamma</Protname> mRNA is induced/inhibited in these cell types by a wide variety of extracellular signals, thus implicating a number of diverse, yet convergent signal transduction pathways in its transcriptional control. In this review, I describe how DNA methylation and specific DNA binding proteins may regulate transcription of the <Protname>IFN-gamma</Protname> gene in response to extracellular signals.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>97028169</MedlineID>
<PMID>97028169</PMID>
<ArticleTitle>Dual action of retinoic acid on human embryonic/fetal hematopoiesis: blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program.</ArticleTitle>
<AbstractText>
In preliminary studies, we have analyzed the <Protname>hematopoietic growth factor</Protname> (<Protname>HGF</Protname>) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum-supplemented (FCS+) clonogenic culture. The key role of <Protname>erythropoietin</Protname> (<Protname>Epo</Protname>) for colony formation by early erythroid progenitors (burst-forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous <Protname>HGFs</Protname>, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS- clonogenic culture.
On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose-dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high-proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation.
It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative <Protname>ATRA receptor</Protname> (<Protname>RAR</Protname>) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the <Protname>RAR</Protname>/<Protname>RXR</Protname> heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU-GEMM/ BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96355009</MedlineID>
<PMID>96355009</PMID>
<ArticleTitle>Defective transcription of the <Protname>IL-2</Protname> gene is associated with impaired expression of <Protname>c-Fos</Protname>, <Protname>FosB</Protname>, and <Protname>JunB</Protname> in anergic T helper 1 cells.</ArticleTitle>
<AbstractText>
Anergic <Protname>CD4</Protname>+ Th cells do not produce <Protname>IL-2</Protname> when challenged with Ag-pulsed accessory cells because of a transcriptional defect. In this work, we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical <Protname>IL-2</Protname> DNA enhancer elements: <Protname>NF-AT</Protname> (<Protname>nuclear factor of activated T cells</Protname>; a sequence that binds a heterotrimeric <Protname>NFATp</Protname>, <Protname>Fos</Protname>, and <Protname>Jun</Protname> protein complex) and <Protname>Activator Protein-1</Protname> (<Protname>AP-1</Protname>) (that binds <Protname>Fos</Protname> and <Protname>Jun</Protname> heterodimers). Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible <Protname>AP-1</Protname> family members <Protname>c- Fos</Protname>, <Protname>FosB</Protname>, and <Protname>JunB</Protname>. However, the reduced expression of these proteins was not the result of a global <Protname>TCR</Protname>/<Protname>CD3</Protname>-signaling defect because <Protname>CD3</Protname> cross-linking induced an equivalent increase in intracellular-free calcium ions, as well as <Protname>NFATp</Protname> dephosphorylation, translocation to the nucleus, and DNA binding in both normal and anergic T cells.Thus, defective <Protname>IL-2</Protname> gene transcription appears to be due, at least in part, to a selective block in the expression of the <Protname>AP-1 Fos</Protname> and <Protname>Jun</Protname> family members in anergic T cells.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96405269</MedlineID>
<PMID>96405269</PMID>
<ArticleTitle>Regulation of <Protname>GM-CSF</Protname> gene transcription by <Protname>core-binding factor</Protname>.</ArticleTitle>
<AbstractText>
<Protname>GM-CSF</Protname> gene activation in T cells is known to involve the transcription factors <Protname>nuclear factor-kappa B</Protname>, <Protname>AP-1</Protname>, <Protname>NFAT</Protname>, and <Protname>Sp1</Protname>. Here we demonstrate that the <Protname>human GM-CSF</Protname> promoter and enhancer also encompass binding sites for <Protname>core-binding factor</Protname> (<Protname>CBF</Protname>). Significantly, the <Protname>CBF</Protname> sites are in each case contained within the minimum essential core regions required for inducible activation of transcription. Furthermore, these core regions of the enhancer and promoter each encompass closely linked binding sites for <Protname>CBF</Protname>, <Protname>AP-1</Protname>, and <Protname>NFATp</Protname>. The <Protname>GM-CSF</Protname> promoter <Protname>CBF</Protname> site TGTGGTCA is located 51 bp upstream of the transcription start site and also overlaps a YY-1 binding site.
A 2-bp mutation within the CBF site resulted in a 2-3-fold decrease in the activities of both a 69-bp proximal promoter fragment and a 627-bp full-length promoter fragment. Stepwise deletions into the proximal promoter also revealed that the <Protname>CBF</Protname> site, but not the YY-1 site, was required for efficient induction of transcriptional activation. The <Protname>AML1</Protname> and <Protname>CBF beta</Protname> genes that encode <Protname>CBF</Protname> each have the ability to influence cell growth and differentiation and have been implicated as proto-oncogenes in acute myeloid leukemia. This study adds <Protname>GM-CSF</Protname> to a growing list of cytokines and receptors that are regulated by <Protname>CBF</Protname> and which control the growth, differentiation, and activation of hemopoietic cells. The <Protname>GM-CSF</Protname> locus may represent one of several target genes that are dysregulated in acute myeloid leukemia.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96427516</MedlineID>
<PMID>96427516</PMID>
<ArticleTitle>Cytomegalovirus modulates <Protname>interleukin-6</Protname> gene expression.</ArticleTitle>
<AbstractText>
Complications after lung transplantation include the development of rejection and an increased incidence of infection, particularly with cytomegalovirus (CMV). Several recent studies have suggested that <Protname>interleukin (IL)-6</Protname> may be used to detect both infection and rejection after lung transplantation. In addition, <Protname>IL-6</Protname> may play a role in the development of bronchiolitis obliterans after transplantation. Because CMV is also associated with the development of bronchiolitis obliterans after transplantation, we determined whether CMV induces <Protname>IL-6</Protname> gene expression. We demonstrated that CMV infection increased both <Protname>IL-6</Protname> protein and mRNA in peripheral blood mononuclear cells. We also demonstrated that the <Protname>CMV immediate early 1 gene product</Protname> increased expression of the <Protname>IL-6</Protname> promoter. This effect of the <Protname>CMV immediate early 1 gene product</Protname> was dependent upon the presence of specific transcription factor binding sites in the <Protname>IL-6</Protname> promoter. These studies demonstrate that CMV may be an important cofactor in the development of rejection and infection after transplantation through its effects on <Protname>IL-6</Protname>.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96186497</MedlineID>
<PMID>96186497</PMID>
<ArticleTitle>Effects of <Protname>interleukin-10</Protname> on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide.</ArticleTitle>
<AbstractText>
Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection.
We compared the effects of the deactivating cytokine <Protname>interleukin 10</Protname> (<Protname>IL-10</Protname>) on human peripheral blood mononuclear cell (PBMC) responses to lipopolysaccharide (LPS), Cryptococcus neoformans, and Candida albicans. <Protname>IL-10</Protname> effected dose-dependent inhibition of <Protname>tumor necrosis factor alpha</Protname> (<Protname>TNF-alpha</Protname>) release in PBMC stimulated by LPS and C. neoformans, with significant inhibition seen with 0.1 U/ml and greater than 90% inhibition noted with 10 U/ml. In contrast, even at doses as high as 100 U/ml, <Protname>IL-10</Protname> inhibited <Protname>TNF-alpha</Protname> release in response to C. albicans by only 50%. <Protname>IL-10</Protname> profoundly inhibited release of <Protname>IL-1beta</Protname> from PBMC stimulated by all three stimuli. <Protname>TNF-alpha</Protname> mRNA and release was inhibited even if <Protname>IL-10</Protname> was added up to 8 h after cryptococcal stimulation.
In contrast, inhibition of <Protname>IL-1 beta</Protname> mRNA was of lesser magnitude and occurred only when <Protname>IL-10</Protname> was added within 2 h of cryptococcal stimulation. <Protname>IL-10</Protname> inhibited translocation of <Protname>NF-kappaB</Protname> in response to LPS but not the fungal stimuli.
All three stimuli induced <Protname>IL-10</Protname> production in PBMC, although over 10-fold less <Protname>IL-10</Protname> was released in response to C. neoformans compared with LPS and C. albicans. Thus, while <Protname>IL-10</Protname> has deactivating effects on PBMC responses to all three stimuli, disparate stimulus- and response-specific patterns of deactivation are seen.
Inhibition by <Protname>IL-10</Protname> of proinflammatory cytokine release appears to occur at the level of gene transcription for <Protname>TNF-alpha</Protname> and both transcriptionally and posttranscriptionally for <Protname>IL-1beta</Protname>.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96279111</MedlineID>
<PMID>96279111</PMID>
<ArticleTitle>Multiple transcription factors are required for activation of <Protname>human interleukin 9</Protname> gene in T cells.</ArticleTitle>
<AbstractText>
The genetic elements and regulatory mechanisms responsible for <Protname>human interleukin 9</Protname> (<Protname>IL-9</Protname>) gene expression in a human T cell leukemia virus type I-transformed human T cell line, C5MJ2, were investigated. We demonstrated that <Protname>IL-9</Protname> gene expression is controlled, at least in part, by transcriptional activation. Transient expression of the <Protname>luciferase</Protname> reporter gene linked to serially deleted sequences of the 5'-flanking region of the <Protname>IL-9</Protname> gene has revealed several positive and negative regulatory elements involved in the basal and inducible expression of the <Protname>IL-9</Protname> gene in C5MJ2 cells. An <Protname>AP-1</Protname> site at -146 to -140 was shown to be involved in the expression of the <Protname>IL-9</Protname> gene.
A proximal region between -46 and -80 was identified as the minimum sequence for the basal and inducible expression of the <Protname>IL-9</Protname> gene in C5MJ2 cells. Within this region, an <Protname>NF-kappaB</Protname> site at -59 to -50 and its adjacent 20-base pair upstream sequence were demonstrated to play a critical role for the <Protname>IL-9</Protname> promoter activity.
DNA-protein binding studies indicated that <Protname>NF-kappaB</Protname>, <Protname>c-Jun</Protname>, and potentially novel proteins (around 35 kDa) can bind to this important sequence. Mutations at different sites within this proximal promoter region abolished the promoter activity as well as the DNA binding. Taken together, these results suggest that the cooperation of different transcription factors is essential for <Protname>IL-9</Protname> gene expression in T cells.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96198880</MedlineID>
<PMID>96198880</PMID>
<ArticleTitle>Soluble <Protname>tumor necrosis factor</Protname> receptors inhibit phorbol myristate acetate and cytokine-induced HIV-1 expression chronically infected U1 cells.</ArticleTitle>
<AbstractText>
Recombinant <Protname>human tumor necrosis factor (TNF) binding protein-1</Protname> (<Protname>r-h TBP-1</Protname>) and recombinant <Protname>human soluble dimeric TNF receptor</Protname> (<Protname>rhu TNFR:Fc</Protname>) were used to determine the relative contributions of <Protname>TNF</Protname> to phorbol myristate acetate (PMA) and cytokine-induced human immunodeficiency virus type 1 (HIV-1) replication in chronically infected cell lines. Treatment of HIV-1-infected promonocytic U1 cells with <Protname>r-h-TBP-1</Protname> or <Protname>rhu TNFR:Fc</Protname> reduced PMA-induced <Protname>HIV-1 p24 antigen</Protname> production in a concentration-dependent manner, with a maximal inhibition of approximately 90%. Maximal inhibition of <Protname>p24 antigen</Protname> production in T-lymphocytic ACH-2 cells was 47% with <Protname>r-hTBP-1</Protname> and 42% with <Protname>rhu TNFR:Fc</Protname>. <Protname>r-hTBP-1</Protname> and <Protname>rhu TNFR:Fc</Protname> also decreased <Protname>p24 antigen</Protname> synthesized by U1 cells in response to other stimuli, including <Protname>phytohemagglutinin</Protname> (<Protname>PHA</Protname>)-induced supernatant, <Protname>granulocyte-macrophage colony-stimulating factor</Protname>, <Protname>interleukin-6</Protname>, and <Protname>TNF</Protname>. Addition of <Protname>r-hTBP-1</Protname> to U1 cells during the last 4 h of a 24 h incubation with PMA still inhibited <Protname>p24 antigen</Protname> production by 15%. U1 cells stimulated with 10(-7) M PMA released approximately 1 ng/ml endogenous <Protname>TBP-1</Protname> with an initial peak observed at 1 h and a second peak at 24 h after PMA stimulation.
<Protname>r-hTBP-1</Protname> also partially reversed inhibition of U1 cellular proliferation caused by PMA. Both <Protname>r-hTBP-1</Protname> and <Protname>rhu TNFR:Fc</Protname> blocked PMA induction of <Protname>nuclear factor (NK)-kappa B</Protname> DNA-binding activity in U1 cells in association with decreases in HIV-1 replication. We conclude that soluble <Protname>TNF</Protname> receptors can inhibit stimuli-induced HIV-1 expression and <Protname>NK-kappa B</Protname> DNA-binding activity in chronically infected U1 cells.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96216453</MedlineID>
<PMID>96216453</PMID>
<ArticleTitle><Protname>Granulocyte-macrophage colony-stimulating factor</Protname> stimulates <Protname>JAK2</Protname> signaling pathway and rapidly activates <Protname>p93fes</Protname>, <Protname>STAT1 p91</Protname>, and <Protname>STAT3 p92</Protname> in polymorphonuclear leukocytes.</ArticleTitle>
<AbstractText>
<Protname>Granulocyte-macrophage colony-stimulating factor</Protname> (<Protname>GM-CSF</Protname>), supports proliferation, differentiation, and functional activation of hemopoietic cells by its interaction with a heterodimeric receptor. Although <Protname>GM-CSF receptor</Protname> is devoid of tyrosine kinase enzymatic activity, <Protname>GM-CSF</Protname>-induced peripheral blood polymorphonuclear leukocytes (PMN) functional activation is mediated by the phosphorylation of a large number of intracellular signaling molecules. We have previously shown that <Protname>JAK2</Protname> becomes tyrosine-phosphorylated in response to <Protname>GM-CSF</Protname> in PMN. In the present study we demonstrate that also the <Protname>signal transducers and activators of transcription</Protname> (<Protname>STAT</Protname>) family members <Protname>STAT1 p91</Protname> and <Protname>STAT3 p92</Protname> and the product of the <Protname>c-fps</Protname>/<Protname>fes</Protname> protooncogene become tyrosine-phosphorylated upon <Protname>GM-CSF</Protname> stimulation and physically associated with both <Protname>GM-CSF receptor</Protname> beta common subunit and <Protname>JAK2</Protname>. Moreover <Protname>GM-CSF</Protname> was able to induce <Protname>JAK2</Protname> and <Protname>p93fes</Protname> catalytic activity. We also demonstrate that the association of the <Protname>GM-CSF receptor beta</Protname> common subunit with <Protname>JAK2</Protname> is ligand-dependent. Finally we demonstrate that <Protname>GM-CSF</Protname> induces a DNA-binding complex that contains both <Protname>p91</Protname> and <Protname>p92</Protname>. These results identify a new signal transduction pathway activated by <Protname>GM-CSF</Protname> and provide a mechanism for rapid activation of gene expression in <Protname>GM-CSF</Protname>-stimulated PMN.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96189089</MedlineID>
<PMID>96189089</PMID>
<ArticleTitle>Inactivation of <Protname>IkappaBbeta</Protname> by the <Protname>tax protein</Protname> of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of <Protname>NF-kappaB</Protname>.</ArticleTitle>
<AbstractText>
In resting T lymphocytes, the transcription factor <Protname>NF-kappaB</Protname> is sequestered in the cytoplasm via interactions with members of the <Protname>I kappa B</Protname> family of inhibitors, including <Protname>IkappaBalpha</Protname> and <Protname>IkappaBbeta</Protname>. During normal T-cell activation, <Protname>IkappaBalpha</Protname> is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional <Protname>NF-kappaB</Protname>. In contrast to its transient pattern of nuclear induction during an immune response, <Protname>NF-kappaB</Protname> is constitutively activated in cells expressing the <Protname>Tax transforming protein</Protname> of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that <Protname>HTLV-1 Tax</Protname> targets <Protname>IkappaBalpha</Protname> to the ubiquitin-proteasome pathway. However, it remains unclear how this viral protein induces a persistent rather than transient <Protname>NF-kappaB</Protname> response. In this report, we provide evidence that in addition to acting on <Protname>IkappaBalpha</Protname>, <Protname>Tax</Protname> stimulates the turnover Of <Protname>IkappaBbeta</Protname> via a related targeting mechanism.
Like <Protname>IkappaBalpha</Protname>, Tax-mediated breakdown of <Protname>IkappaBbeta</Protname> in transfected T lymphocytes is blocked either by cell-permeable proteasome inhibitors or by mutation Of <Protname>IkappaBbeta</Protname> at two serine residues present within its N-terminal region. Despite the dual specificity of <Protname>HTLV-1 Tax</Protname> for <Protname>IkappaBalpha</Protname> and <Protname>IkappaBbeta</Protname> at the protein level, <Protname>Tax</Protname> selectively stimulates <Protname>NF-kappaB</Protname>-directed transcription of the <Protname>IkappaBalpha</Protname> gene. Consequently, <Protname>IkappaBbeta</Protname> protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with <Protname>IkappaBbeta</Protname> provide a potential mechanism for the constitutive activation of <Protname>NF-kappaB</Protname> in <Protname>Tax</Protname>-expressing cells.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96182568</MedlineID>
<PMID>96182568</PMID>
<ArticleTitle>A model of latent adenovirus 5 infection in the guinea pig (Cavia porcellus).</ArticleTitle>
<AbstractText>
A model of adenovirus 5 (Ad5) infection was developed in guinea pigs to begin to study its role in the pathogenesis of peripheral lung inflammation. Forty animals were inoculated intranasally with 10(7.0) pfu of Ad5/animal, and 15 animals inoculated with sterile culture media served as controls. Viral titres were 10(4.4), 10(6.1), 10(5.2), and 10(2.9) pfu/animal, on days 1, 3, 4, and 7 after infection, respectively. In situ hybridization to viral DNA and immunocytochemistry for <Protname>Ad5 E1A protein</Protname> localized the virus to airway and alveolar epithelial cells. Histologic examination showed an extensive inflammatory cell infiltration around the airways, with epithelial necrosis and an alveolar exudate that caused localized alveolar collapse in the infected areas. Immunocytochemistry identified the cells in the infiltrate as cytotoxic T cells. Although all animals 20 and 47 days after infection had seroconverted to Ad5, virus was not detected in these groups either by viral plaque assay or in situ hybridization. <Protname>Ad5 E1A</Protname> DNA was detected by polymerase chain reaction in five of six animals 20 days after infection and in five of five animals 47 days after infection. In these same animals, <Protname>E1A protein</Protname> was detected 20 days after infection in two and 47 days after infection in one while persistent bronchiolitis was observed in four and three animals 20 and 47 days after infection, respectively.
These results demonstrate that the guinea pig provides a useful model to study the role of Ad5 infection in chronic airway inflammation.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96278860</MedlineID>
<PMID>96278860</PMID>
<ArticleTitle>In vivo protein-DNA interactions of the human <Protname>beta-globin</Protname> locus in erythroid cells expressing the fetal or the adult <Protname>globin</Protname> gene program.</ArticleTitle>
<AbstractText>
To characterize the protein-DNA interactions important for the developmental control of the human <Protname>beta-globin</Protname> locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult <Protname>globin</Protname> developmental program. In the locus control region (LCR) of the <Protname>beta-globin</Protname> locus, in vivo footprints on <Protname>NF-E2</Protname> (or <Protname>AP-1</Protname>) and <Protname>GATA-1</Protname> motifs remained the same regardless of whether the fetal or the adult <Protname>globin</Protname> genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult <Protname>globin</Protname> program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult <Protname>globin</Protname> expressing cells displayed more extensive footprints than the fetal <Protname>globin</Protname> expressing cells in the 3' regulatory sequences of both the <Protname>Agamma</Protname>- and the <Protname>beta-globin</Protname> genes, suggesting a role of these 3' elements in <Protname>beta-globin</Protname> gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of <Protname>globin</Protname> genes takes place in the <Protname>gamma-</Protname> and <Protname>beta-globin</Protname> gene promoters, and that GT motifs of the <Protname>beta-globin</Protname> locus LCR may play a role in the developmental regulation of <Protname>human beta-globin</Protname> gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult <Protname>globin</Protname> gene.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99401563</MedlineID>
<PMID>99401563</PMID>
<ArticleTitle>Polyamines in human breast cancer and its relations to classical prognostic features: clinical implications.</ArticleTitle>
<AbstractText>
Experimental evidence suggest an important role of polyamines in breast cancer development. Polyamines have been determined in tissue and erythrocyte samples from 100 patients with primary invasive breast cancer and 30 patients with fibroadenomas. Statistical analysis was performed in order to determine the prognostic value of the polyamine patterns of tumor tissues and erythrocytes in comparison with clinical and histological prognostic factors. In malignant tissues, polyamine levels were significantly higher than in benign tissues.
They correlated with markers of tumor aggressivity (axillary node involvement and especially with markers of high mitotic rate as Ki-67 staining, histological grade). No correlation was found between estrogen and progesterone status, tumor size and polyamine concentrations. Erythrocyte polyamines levels were identical between cancer patients and controls. The knowledge of the polyamine pattern in breast cancer could become useful in clinical practice particularly if polyamine metabolism is targeted as a therapeutic approach. </AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99353322</MedlineID>
<PMID>99353322</PMID>
<ArticleTitle>Peptide binding affinity and pH variation establish functional thresholds for activation of HLA-DQ-restricted T cell recognition.</ArticleTitle>
<AbstractText>
Peptides derived from the <Protname>HSV-2 VP16 protein</Protname> were utilized for studies of peptide binding to DQ0302 molecules and T cell activation at both neutral and acidic pH. The native peptide <Protname>VP16</Protname> 430-444 contains an Asp at position 442, binds to DQ0302 strongly, with a Kd value of 50nM at acidic pH and very weakly, with a Kd value of greater than 10 microM at neutral pH. A truncated version of 430-444, i.e., <Protname>VP16</Protname> 433-442, binds with an affinity 10-fold lower compared to 430-444 at acidic pH, and binding at neutral pH was barely detectable. The homologous peptide 430-444,442A has an Asp to Ala substitution at position 442 and binds to DQ0302 with a Kd similar to 433-442. The short truncated analog 433-442A binds very poorly at both acidic and neutral pH. Both the wild type 430-444 and 433-442 peptides stimulated a HSV-specific T cell clone after a brief incubation with antigen presenting cells (APC) expressing DQ0302 at acidic pH. Much higher concentrations of wild type peptides were needed to activate T cells at neutral pH. In contrast, APC pulsed with Ala-substituted peptides 430-444,442A or 433-442A at neutral pH failed to stimulate the T cell clone, while APC pulsed at acidic pH and subsequently washed led to successful T cell activation. The Ala-substituted peptide was recognized by the T cell clone at neutral pH only when it was present in the APC culture throughout the stimulation process. While the MHC-peptide complexes formed with the native peptide are stable, complexes formed with the Ala-substituted peptide had a functional t1/2 of less than 4 hr at neutral pH.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96320762</MedlineID>
<PMID>96320762</PMID>
<ArticleTitle>Involvement of tyrosine phosphorylation in <Protname>endothelial adhesion molecule</Protname> induction.</ArticleTitle>
<AbstractText>
Induction of <Protname>endothelial adhesion molecules</Protname> by the cytokine <Protname>tumor necrosis factor-alpha</Protname> (<Protname>TNF</Protname>) can occur independently of <Protname>protein kinase C</Protname> and activation of a protein tyrosine kinase (PTK) has recently been implicated in the upregulation of <Protname>vascular cell adhesion molecule 1</Protname> (<Protname>VCAM-1</Protname>) by <Protname>interleukin-4</Protname> (<Protname>IL-4</Protname>) on endothelial cells. We demonstrate that the PTK inhibitors herbimycin A or genistein suppress induction of endothelial <Protname>VCAM-1</Protname> and <Protname>E-selectin</Protname>, as well as subsequent monocytic cell adhesion to endothelial cells stimulated by <Protname>TNF</Protname>. Inhibition studies indicate that specific tyrosine phosphorylation following PTK activation is involved in the mobilization of the transcription factor, <Protname>nuclear factor kappa B</Protname>, and <Protname>VCAM-1</Protname> mRNA expression. This may have implications for pathophysiological conditions that involve the upregulation of these molecules (e.g. inflammation and atherosclerosis).
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99376409</MedlineID>
<PMID>99376409</PMID>
<ArticleTitle>Dendritic cells and the pathogenesis of rheumatoid arthritis.</ArticleTitle>
<AbstractText>
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease in which unknown arthrogenic autoantigen is presented to <Protname>CD4</Protname>+ T cells. The strong association of the disease with an epitope within the <Protname>HLA-DR</Protname> chain shared between various alleles of <Protname>HLA-DR4</Protname> and <Protname>DR1</Protname> emphasizes the importance of antigen presentation. This immune response predominantly occurs in the synovial tissue and fluid of the joints and autoreactive T cells are readily demonstrable in both the synovial compartment and blood. Circulating dendritic cells (DC) are phenotypically and functionally identical with normal peripheral blood (PB) DC. In the synovial tissue, fully differentiated perivascular DC are found in close association with T cells and with B cell follicles, sometimes containing follicular DC. These perivascular DC migrate across the activated endothelium from blood and receive differentiative signals within the joint from monocyte-derived cytokines and <Protname>CD40</Protname>-ligand+ T cells. In the SF, DC manifest an intermediate phenotype, similar to that of monocyte-derived DC in vitro.
Like a delayed-type hypersensitivity response, the rheumatoid synovium represents an effector site. DC at many effector sites have a characteristic pattern of infiltration and differentiation. It is important to note that the effector response is not self-limiting in RA autoimmune inflammation. In this article, we argue that the presentation of self-antigen by DC and by autoantibody-producing B cells is critical for the perpetuation of the autoimmune response. Permanently arresting this ongoing immune response with either pharmaceutical agents or immunotherapy is a major challenge for immunology.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>96144699</MedlineID>
<PMID>96144699</PMID>
<ArticleTitle>Inhibition of <Protname>NF-AT</Protname> signal transduction events by a dominant-negative form of <Protname>calcineurin</Protname>.</ArticleTitle>
<AbstractText>
An inhibitory, "dominant-negative," form of the <Protname>calcineurin</Protname> catalytic (A) subunit was prepared, which lacks the <Protname>calmodulin</Protname>-binding domain, autoinhibitory domain and most of its catalytic core but possesses the regulatory (B) subunit binding domain. When tested for its ability to block <Protname>calcineurin</Protname>-dependent signaling in Jurkat cells, expression of this "B-subunit knock-out" (BKO) construct suppressed reporter gene activity driven by <Protname>NF-AT</Protname>, the pivotal promoter element for <Protname>interleukin (IL)-2</Protname> gene induction. Immunoprecipitation of epitope-labeled BKO demonstrated for the formation of a tight complex with endogenous B subunit in Jurkat cells, consistent with an inhibitory mechanism that involves the sequestration of the B subunit. Furthermore, the sharply reduced <Protname>NF-AT</Protname> activity produced by co-transfecting BKO could be "rescued" by overexpression of transfected B subunit, suggesting that depletion of this subunit was responsible for the inhibition. These data suggest the potential utility of agents that disrupt <Protname>calcineurin</Protname>-mediated signal transduction pathways by blocking formation of the catalytically active dimer of <Protname>calcineurin</Protname> A and B subunits.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99409030</MedlineID>
<PMID>99409030</PMID>
<ArticleTitle><Protname>c-Maf</Protname> induces monocytic differentiation and apoptosis in bipotent myeloid progenitors.</ArticleTitle>
<AbstractText>
The transcriptional mechanisms that drive colony-forming unit granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not fully understood. We have shown that the <Protname>c-Maf</Protname> and <Protname>c-Myb</Protname> transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of <Protname>c-Myb</Protname> target genes through direct binding to <Protname>Myb</Protname> consensus sites. These complexes arise in a developmentally regulated pattern, peaking at the promyelocyte stage, or in cell model systems, appearing soon after the induction of monocytic differentiation.
We wished to determine if this developmentally related interaction is a consequence of myeloid differentiation or an intrinsic differentiating stimulus. Because the elevated <Protname>Myb</Protname>:<Protname>Maf</Protname> status seen in differentiating cells can be recapitulated by overexpression of <Protname>c-Maf</Protname> in myeloid cell lines, we inducibly expressed the <Protname>c-Maf</Protname> cDNA in 2 bipotent human myeloid progenitor cells. Elevated levels of <Protname>c-Maf protein</Protname> led to marked increases in <Protname>Myb</Protname>:<Protname>Maf</Protname> complexes and the accumulation of monocyte/macrophage cells, followed by eventual programmed cell death. Analysis of targets that could mediate these phenotypic changes indicated that <Protname>c-Maf</Protname> likely plays a key role in myeloid cell development through dual mechanisms; inhibition of a select set of <Protname>c-Myb</Protname> regulated targets, such as <Protname>Bcl-2</Protname> and <Protname>CD13/APN</Protname>, coupled with the activation of as yet undefined differentiation-promoting genes.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>99357866</MedlineID>
<PMID>99357866</PMID>
<ArticleTitle>Host defense mechanisms triggered by microbial lipoproteins through <Protname>toll-like receptors</Protname>.</ArticleTitle>
<AbstractText>
The generation of cell-mediated immunity against many infectious pathogens involves the production of <Protname>interleukin-12</Protname> (<Protname>IL-12</Protname>), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce <Protname>IL-12</Protname> production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of <Protname>IL-12</Protname> production by human macrophages, and that induction is mediated by <Protname>Toll-like receptors</Protname> (<Protname>TLRs</Protname>). Several lipoproteins stimulated <Protname>TLR</Protname>-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of <Protname>TLRs</Protname> by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93268332</MedlineID>
<PMID>93268332</PMID>
<ArticleTitle>Regulation of the <Protname>interleukin-1 beta</Protname> (<Protname>IL-1 beta</Protname>) gene by mycobacterial components and lipopolysaccharide is mediated by two <Protname>nuclear factor-IL6</Protname> motifs.</ArticleTitle>
<AbstractText>
The cytokines <Protname>interleukin-1 beta</Protname> (<Protname>IL-1 beta</Protname>) and <Protname>tumor necrosis factor alpha</Protname> (<Protname>TNF-alpha</Protname>) are released by mononuclear phagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events, including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM) is a very potent inducer of <Protname>IL-1 beta</Protname> gene expression in human monocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-, and <Protname>TNF-alpha</Protname>-inducible promoter activity to a -131/+15 (positions -131 to +15) DNA fragment of the <Protname>IL-1 beta</Protname> gene by deletion analysis and <Protname>chloramphenicol acetyltransferase</Protname> assay. Within this DNA fragment, there were two novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the <Protname>nuclear factor-IL6</Protname> (<Protname>NF-IL6</Protname>) binding site.
Site-directed mutagenesis demonstrated that the two <Protname>NF-IL-6</Protname> motifs could be independently activated by LAM, LPS, or <Protname>TNF-alpha</Protname> and that they acted in an orientation-independent manner.
DNA mobility shift assay revealed specific binding of nuclear protein(s) from LAM-, LPS-, or <Protname>TNF-alpha</Protname>-stimulated THP-1 cells to the <Protname>NF-IL6</Protname> motifs. We conclude that the two <Protname>NF-IL6</Protname> sites mediate induction of <Protname>IL-1 beta</Protname> in response to the stimuli LAM, LPS, and <Protname>TNF-alpha</Protname>.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93264305</MedlineID>
<PMID>93264305</PMID>
<ArticleTitle>Activation of primary human T-lymphocytes through <Protname>CD2</Protname> plus <Protname>CD28</Protname> adhesion molecules induces long-term nuclear expression of <Protname>NF-kappa B</Protname>.</ArticleTitle>
<AbstractText>
Stimulation of highly purified human T-cells via <Protname>CD2</Protname> and <Protname>CD28</Protname> adhesion molecules induces and maintains proliferation for more than 3 weeks. This potent <Protname>interleukin 2</Protname> (<Protname>IL-2</Protname>)-dependent activation does not require monocytes or accessory cells. Long-lasting <Protname>IL-2</Protname> receptivity is associated with high-level expression of the inducible <Protname>IL-2 receptor alpha</Protname> chain (<Protname>IL-2R alpha</Protname>) gene that is regulated at both transcriptional and posttranscriptional levels. Increase of <Protname>IL-2R alpha</Protname> gene transcription involves the enhanced binding of the transcription factor <Protname>NF-kappa B</Protname> to its consensus sequence in the 5'-regulatory region of the <Protname>IL-2R alpha</Protname> gene. To dissect the molecular basis for the unusually persistent transcription of the <Protname>IL-2R alpha</Protname> gene, we analyzed nuclear <Protname>NF-kappa B</Protname> binding to a radiolabeled <Protname>IL-2R alpha</Protname> kappa B-specific oligonucleotide probe during the time course of <Protname>CD2</Protname> + <Protname>CD28</Protname> activation. Resting T-cell nuclear extracts contained <Protname>KBF1/p50</Protname> homodimer. After stimulation, two new kappa B-specific complexes were identified as <Protname>NF-kappa B p50-p65</Protname> heterodimer and putative <Protname>c-Rel</Protname> homodimer or <Protname>c-Rel</Protname>-<Protname>p65</Protname> heterodimer. Both inducible complexes persisted for at least 3 weeks. Their relative levels were very similar for the duration of proliferation. In parallel, <Protname>CD2</Protname> + <Protname>CD28</Protname> activation triggered a significant intracellular thiol decrease, suggesting that oxygen radicals are involved in the signaling pathway of adhesion molecules. Finally, micromolar amounts of pyrrolidine dithiocarbamate, an oxygen radical scavenger that efficiently blocked the nuclear appearance of <Protname>NF-kappa B</Protname> in T-lymphocytes, also inhibited <Protname>IL-2</Protname> secretion, <Protname>IL-2</Protname>R alpha cell surface expression, and T-cell proliferation. Together, these results suggest that <Protname>NF-kappa B</Protname> plays an important role in long-term activation of human primary T-lymphocytes via <Protname>CD2</Protname> + <Protname>CD28</Protname>.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93320446</MedlineID>
<PMID>93320446</PMID>
<ArticleTitle>Antisense oligonucleotides to the <Protname>p65</Protname> subunit of <Protname>NF-kappa B</Protname> block <Protname>CD11b</Protname> expression and alter adhesion properties of differentiated HL-60 granulocytes.</ArticleTitle>
<AbstractText>
<Protname>NF-kappa B</Protname> is a pleiotropic regulator of a variety of genes implicated in the cellular response to injury. This function has been attributed to the coordinated binding of subunits of <Protname>NF-kappa B</Protname> to distinct regions of the promoter elements of numerous genes, including cytokines, growth factor receptors, and adhesion molecules. Antisense phosphorothioate oligonucleotides to the <Protname>p50</Protname> and <Protname>p65</Protname> subunits of the <Protname>NF-kappa B</Protname> complex were used to define the physiologic role of this transcription factor in resting and stimulated granulocytes. A reduction in the expression of <Protname>p65</Protname> was produced by treatment with the phosphorothioate antisense oligodeoxynucleotide. This reduction was accompanied by rapid changes in the cellular adhesion of dimethyl sulfoxide-differentiated HL-60 leukemia cells stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These effects were characterized by a marked reduction in <Protname>CD11b integrin</Protname> expression on the surface of treated cells. Furthermore, the <Protname>p65</Protname> antisense oligomer effectively abolished an upregulation of <Protname>CD11b</Protname> that was produced by formyl-met-leu-phe and TPA. However, the <Protname>p65</Protname> antisense phosphorothioate oligodeoxynucleotide had no significant effect on the production of reactive oxygen intermediates or on phagocytosis by these cells. These findings indicate that antisense oligomers to <Protname>p65</Protname> can be used to define the role of <Protname>NF-kappa B</Protname> in the activation pathways of neutrophils.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93224733</MedlineID>
<PMID>93224733</PMID>
<ArticleTitle>A protein of the <Protname>AP-1</Protname> family is a component of <Protname>nuclear factor of activated T cells</Protname>.</ArticleTitle>
<AbstractText>
<Protname>Nuclear factor of activated T cells</Protname> (<Protname>NF-AT</Protname>) is a transcriptional activator involved in the induction of <Protname>IL-2</Protname> gene expression. The response element for <Protname>NF-AT</Protname> is a sequence localized between -285/-254 in the <Protname>IL-2</Protname> regulatory region. The composition of <Protname>NF-AT protein</Protname> is still not fully elucidated. We demonstrate that, in normal human T cells, an <Protname>AP-1 protein</Protname> is a component of the <Protname>NF-AT protein</Protname> complex. This was evidenced by the ability of the <Protname>AP-1</Protname> site to compete with the <Protname>NF-AT</Protname> site for binding to <Protname>NF-AT</Protname> and by the capacity of immobilized anti-<Protname>Jun</Protname> and anti-<Protname>Fos</Protname> antibodies to deplete <Protname>NF-AT</Protname>-binding activity from nuclear extracts of activated T cells. There was no detectable binding of in vitro translated <Protname>Jun</Protname>/<Protname>Fos</Protname> heterodimer (<Protname>AP-1</Protname>) to the <Protname>NF-AT</Protname> sequence, and the <Protname>NF-AT</Protname> sequence was unable to inhibit the binding of <Protname>Jun</Protname>/<Protname>Fos</Protname> to the <Protname>AP-1</Protname> sequence. The presence of an <Protname>AP-1 protein</Protname> in the <Protname>NF-AT protein</Protname> complex may regulate <Protname>NF-AT</Protname>-binding activity through protein-protein interaction.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93152834</MedlineID>
<PMID>93152834</PMID>
<ArticleTitle>Expression of <Protname>tal-1</Protname> and <Protname>GATA-binding proteins</Protname> during human hematopoiesis.</ArticleTitle>
<AbstractText>
<Protname>Tal-1</Protname> rearrangements are associated with nearly 30% of human T acute lymphoblastic leukemia. <Protname>Tal-1</Protname> gene encodes a putative transcription factor with a basic helix-loop-helix domain and is known to be predominantly expressed in hematopoietic cells. We investigated the pattern of <Protname>tal-1</Protname> expression in purified human hematopoietic cells by in situ hybridization and reverse transcriptase polymerase chain reaction analysis. Both methods demonstrated that the <Protname>tal-1</Protname> gene is expressed in megakaryocytes and erythroblasts as well as in basophilic granulocytes. In addition, our results indicate that the <Protname>tal-1</Protname> 1A promoter, which contains two consensus GATA-binding sites, is active mainly in these lineages. Because the <Protname>GATA-1</Protname> gene is known to transactivate several genes specific for the erythroid, megakaryocytic, and mastocytic/basophilic lineages, we studied <Protname>GATA-1</Protname> expression in these purified hematopoietic cells. We found that <Protname>GATA-1</Protname> and <Protname>tal-1</Protname> genes are coexpressed in these three lineages. Remarkably, the expression of both genes is downmodulated during erythroid and megakaryocytic terminal maturation. In immature hematopoietic cells, <Protname>tal-1</Protname> and <Protname>GATA-1</Protname> genes are coexpressed in committed progenitors cells (<Protname>CD34</Protname>+/<Protname>CD38</Protname>(2+)), whereas they are not detectable in the most primitive cells (<Protname>CD34</Protname>(2+)/<Protname>CD38</Protname>-). In contrast, <Protname>GATA-2</Protname> is strongly expressed in both most primitive and committed progenitors cells, whereas <Protname>GATA-3</Protname> is mostly detected in most primitive ones. Altogether our results strongly suggest that <Protname>GATA-1</Protname> modulates the transcription of <Protname>tal-1</Protname> during the differentiation of the erythroid, megakaryocytic, and basosophilic lineages.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93350203</MedlineID>
<PMID>93350203</PMID>
<ArticleTitle>Cytokine modulation of HIV expression.</ArticleTitle>
<AbstractText>
Cytokines, the peptide hormones which control the homeostasis of the immune system and also play a fundamental role in inflammatory and immune mediated reactions, have been involved at multiple levels in the pathogenesis of the acquired immune deficiency syndrome (AIDS). Infection with the human immunodeficiency virus (HIV) has been shown to induce production of several cytokines both in vitro and in vivo. Conversely, several cytokines modulate the levels of HIV expression in infected cells of both T lymphocytic and mononuclear phagocytic lineage. Activated mononuclear cells, particularly B cells which are in a state of chronic activation in HIV infected individuals, release HIV-inductive cytokines and thus play a potentially important role in the pathogenesis of HIV infection.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>92193465</MedlineID>
<PMID>92193465</PMID>
<ArticleTitle>Mineralocorticoids and <Protname>mineralocorticoid receptors</Protname> in mononuclear leukocytes in patients with pregnancy-induced hypertension.</ArticleTitle>
<AbstractText>
To examine the role of mineralocorticoids in the pathophysiology of pregnancy-induced hypertension (PIH), we studied plasma aldosterone and 18-hydroxycorticosterone levels in 25 women with PIH and 25 normal pregnant women, as controls. Furthermore, we evaluated the <Protname>mineralocorticoid receptor</Protname> (<Protname>MR</Protname>) status in mononuclear leukocytes in the 2 groups. <Protname>MR</Protname> count was significantly (P less than 0.0005) decreased in the PIH group (148 +/- 9 binding sites/cell) compared with the control group (300 +/- 17 binding sites/cell; mean +/- SEM). Plasma aldosterone in women with PIH was 281 +/- 61 pmol/L; in normal pregnant women it was 697 +/- 172 pmol/L (P less than 0.025). Plasma 18-hydroxycorticosterone was also significantly (P less than 0.025) lower (PIH, 1071 +/- 149 pmol/L; controls, 1907 +/- 318 pmol/L). These values were determined at the onset of clinical symptoms of PIH. These results cannot be explained by receptor down-regulation due to higher levels of mineralocorticoids in PIH; a hitherto unknown mineralocorticoid may, thus, be responsible for the hypertension and altered <Protname>MR</Protname> status.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93129248</MedlineID>
<PMID>93129248</PMID>
<ArticleTitle>Alpha-lipoic acid is a potent inhibitor of <Protname>NF-kappa B</Protname> activation in human T cells.</ArticleTitle>
<AbstractText>
Acquired immunodeficiency syndrome (AIDS) results from infection with a human immunodeficiency virus (HIV). The long terminal repeat (LTR) region of HIV proviral DNA contains binding sites for <Protname>nuclear factor kappa B</Protname> (<Protname>NF-kappa B</Protname>), and this transcriptional activator appears to regulate HIV activation. Recent findings suggest an involvement of reactive oxygen species (ROS) in signal transduction pathways leading to <Protname>NF-kappa B</Protname> activation. The present study was based on reports that antioxidants which eliminate ROS should block the activation of <Protname>NF-kappa B</Protname> and subsequently HIV transcription, and thus antioxidants can be used as therapeutic agents for AIDS.
Incubation of Jurkat T cells (1 x 10(6) cells/ml) with a natural thiol antioxidant, alpha-lipoic acid, prior to the stimulation of cells was found to inhibit <Protname>NF-kappa B</Protname> activation induced by <Protname>tumor necrosis factor</Protname>-alpha (25 ng/ml) or by phorbol 12-myristate 13-acetate (50 ng/ml). The inhibitory action of alpha-lipoic acid was found to be very potent as only 4 mM was needed for a complete inhibition, whereas 20 mM was required for N-acetylcysteine. These results indicate that alpha-lipoic acid may be effective in AIDS therapeutics.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93041375</MedlineID>
<PMID>93041375</PMID>
<ArticleTitle>Activation of <Protname>NF-kappa B</Protname> by <Protname>interleukin 2</Protname> in human blood monocytes.</ArticleTitle>
<AbstractText>
We report here that <Protname>interleukin 2</Protname> (<Protname>IL-2</Protname>) acts on human blood monocytes by enhancing binding activity of the transcription factor <Protname>NF-kappa B</Protname> to its consensus sequence in the 5' regulatory enhancer region of the <Protname>IL-2 receptor alpha chain</Protname> (<Protname>p55</Protname>). Similarly, <Protname>IL-2</Protname> activates <Protname>NF-kappa B</Protname> in the human monocytic cell line U 937, but not in resting human T-cells. This effect is detectable within 15 min and peaks 1 h after exposure to <Protname>IL-2</Protname>. Enhanced <Protname>NF-kappa B</Protname> binding activity is followed by functional activation in that inducibility of the <Protname>IL-2 receptor alpha chain</Protname> is mediated by enhanced <Protname>NF-kappa B</Protname> binding and that a heterologous promoter containing the <Protname>NF-kappa B</Protname> consensus sequence (-291 to -245) of the <Protname>IL-2 receptor alpha chain</Protname> gene is activated. In addition, <Protname>IL-2</Protname> is capable of increasing transcript levels of the <Protname>p50</Protname> gene coding for the <Protname>p50</Protname> subunit of the <Protname>NF-kappa B transcription factor</Protname>, whereas mRNA levels of the <Protname>p65 NF-kappa B</Protname> gene remained unchanged.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>92195648</MedlineID>
<PMID>92195648</PMID>
<ArticleTitle>Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for <Protname>v-erbA</Protname> oncogene action.</ArticleTitle>
<AbstractText>
The <Protname>v-erbA</Protname> oncogene, a mutated version of the <Protname>thyroid hormone receptor alpha</Protname> (<Protname>c-erbA</Protname>/<Protname>TR-alpha</Protname>), inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes, suggesting a normal function of the proto-oncogene <Protname>c-erbA</Protname> in erythropoiesis. Here we demonstrate that the endogenous <Protname>thyroid hormone receptor alpha</Protname> (<Protname>c-erbA</Protname>/<Protname>TR-alpha</Protname>) and the closely related <Protname>retinoic acid receptor alpha</Protname> (<Protname>RAR-alpha</Protname>) play a role in the regulation of normal erythroid differentiation. Retinoic acid (RA) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene. When added pulsewise to immature cells, differentiation was accelerated while more mature cells underwent premature cell death. Thyroid hormone (T3) alone caused similar but weaker effects. Interestingly, T3 strongly enhanced the action of RA, suggesting cooperative action of the two receptors in modulating erythroid differentiation. Expression of the human <Protname>RAR-alpha</Protname> in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells, thus showing that the <Protname>RAR-alpha</Protname> is essential for the RA effect.
Likewise, enhanced expression of exogenous <Protname>c-erbA</Protname>/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3, suggesting a similar function of both receptors.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93011470</MedlineID>
<PMID>93011470</PMID>
<ArticleTitle>Leukotriene B4 transcriptionally activates <Protname>interleukin-6</Protname> expression involving <Protname>NK-chi B</Protname> and <Protname>NF-IL6</Protname>.</ArticleTitle>
<AbstractText>
Leukotriene B4 (LTB4) is a notable participant in inflammation and chemotaxis.
It is, however, still unclear whether LTB4 acts in this regard directly or indirectly by stimulating the release of chemotactic and inflammatory cytokines.
Here we report that LTB4 induces synthesis of <Protname>interleukin (IL)-6</Protname> by human blood monocytes through transcriptional activation of the <Protname>IL-6</Protname> gene. We furthermore demonstrate that this process involves activation of the <Protname>transcription factor NF-chi B</Protname> and, to a lesser extent, of <Protname>NF-IL6</Protname>, while the activity of the <Protname>transcription factor AP-1</Protname>, shown to otherwise confer <Protname>IL-6</Protname> inducibility, appeared to be unaffected by LTB4. Involvement of <Protname>NF-chi B</Protname> and <Protname>NF-IL6</Protname> in induction of <Protname>IL-6</Protname> transcription by monocytes was demonstrated using deleted forms of the <Protname>IL-6</Protname> promoter. Activation of the <Protname>IL-6</Protname> promoter by LTB4 was not only associated with accumulation of the respective transcripts but resulted in synthesis of functional <Protname>IL-6</Protname> protein as well. In addition, LTB4 mediated transactivation of a heterologous promoter construct containing the <Protname>NF-chi B</Protname> or the <Protname>NF-IL6</Protname> enhancer, but not the <Protname>AP-1</Protname> enhancer. The signaling events mediating this effect appeared to involve the release of H2O2, since LTB4 failed to induce <Protname>NF-chi B</Protname> or <Protname>NF-IL6</Protname> in the presence of the scavenger of H2O2, N-acetyl-L-cysteine.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>92195320</MedlineID>
<PMID>92195320</PMID>
<ArticleTitle>A lymphoid cell-specific nuclear factor containing <Protname>c-Rel</Protname>-like proteins preferentially interacts with <Protname>interleukin-6 kappa B</Protname>-related motifs whose activities are repressed in lymphoid cells.</ArticleTitle>
<AbstractText>
The <Protname>proto-oncoprotein c-Rel</Protname> is a member of the <Protname>nuclear factor kappa B transcription factor</Protname> family, which includes the <Protname>p50</Protname> and <Protname>p65</Protname> subunits of <Protname>nuclear factor kappa B</Protname>. We show here that <Protname>c-Rel</Protname> binds to <Protname>kappa B</Protname> sites as homodimers as well as heterodimers with <Protname>p50</Protname>. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various <Protname>kappa B</Protname> motifs. In particular, the <Protname>c-Rel</Protname> homodimer has a high affinity for <Protname>interleukin-6</Protname> (<Protname>IL-6</Protname>) and <Protname>beta interferon kappa B</Protname> sites. In spite of its association with <Protname>p50</Protname> in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains <Protname>c-Rel</Protname> but not <Protname>p50</Protname> epitopes; this factor, termed <Protname>IL-6 kappa B binding factor II</Protname>, appears to contain the <Protname>c-Rel</Protname> homodimer and preferentially recognizes several <Protname>IL-6 kappa B</Protname>-related <Protname>kappa B</Protname> motifs. Although it has been previously shown that the <Protname>IL-6 kappa B</Protname> motif functions as a potent <Protname>IL-1</Protname>/<Protname>tumor necrosis factor</Protname>-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that <Protname>IL-6 kappa B binding factor II</Protname> functions as a repressor specific for <Protname>IL-6 kappa B</Protname>-related <Protname>kappa B</Protname> motifs in lymphoid cells.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>93087498</MedlineID>
<PMID>93087498</PMID>
<ArticleTitle>Transcription of the hypersensitive site HS2 enhancer in erythroid cells.</ArticleTitle>
<AbstractText>
In the human genome, the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream <Protname>beta-like globin</Protname> genes 10-50 kilobases away. The mechanism of HS2 enhancer function is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer transcripts are not detectable. Splitting the HS2 enhancer between two tandem <Protname>Ap1</Protname> sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene. In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism, the HS2 enhancer may (i) open up the chromatin structure of a gene domain and (ii) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>91292530</MedlineID>
<PMID>91292530</PMID>
<ArticleTitle>Characterization of an <Protname>immediate-early</Protname> gene induced in adherent monocytes that encodes <Protname>I kappa B</Protname>-like activity.</ArticleTitle>
<AbstractText>
We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (<Protname>MAD-3</Protname>) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the <Protname>cdc10</Protname>/<Protname>ankyrin</Protname> motif, which is 60% similar (46% identical) to the <Protname>ankyrin</Protname> repeat region of the precursor of <Protname>NF-kappa B</Protname>/<Protname>KBF1 p50</Protname>. The C-terminus has a putative <Protname>protein kinase C</Protname> phosphorylation site. In vitro translated <Protname>MAD-3</Protname> protein was found to specifically inhibit the DNA-binding activity of the <Protname>p50</Protname>/<Protname>p65 NF-kappa B</Protname> complex but not that of the <Protname>p50</Protname>/<Protname>p50 KBF1 factor</Protname> or of other DNA-binding proteins. The <Protname>MAD-3</Protname> cDNA encodes an <Protname>I kappa B</Protname>-like protein that is likely to be involved in regulation of transcriptional responses to <Protname>NF-kappa B</Protname>, including adhesion-dependent pathways of monocyte activation.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>91282927</MedlineID>
<PMID>91282927</PMID>
<ArticleTitle>Towards a molecular understanding of T-cell differentiation.</ArticleTitle>
<AbstractText>
Lymphoid differentiation is one of the best studied examples of mammalian development. Here Hans Clevers and Michael Owen describe how the cloning of the genes that encode T-cell-specific membrane proteins allows the identification of transcription factors that control the expression of these T-cell genes. Such transcription factors play a key role in the development of the mature T-cell phenotype by functioning as 'master regulators of T-cell differentiation'.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>92042309</MedlineID>
<PMID>92042309</PMID>
<ArticleTitle>Inhibition of phorbol ester-induced monocytic differentiation by dexamethasone is associated with down-regulation of <Protname>c-fos</Protname> and <Protname>c-jun</Protname> (<Protname>AP-1</Protname>).</ArticleTitle>
<AbstractText>
Previous studies have shown that treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of monocytic differentiation and expression of the <Protname>c-jun</Protname> and <Protname>c-fos</Protname> early response genes. The present work demonstrates that the glucocorticoid dexamethasone inhibits TPA-induced increases in <Protname>c-jun</Protname> and <Protname>c-fos</Protname> mRNA levels in U-937 leukemia cells. These findings were associated with a block in appearance of the monocytic phenotype, including inhibition of TPA-induced increases in <Protname>lamin A</Protname>, <Protname>lamin C</Protname>, and <Protname>vimentin</Protname> transcripts. Other studies have demonstrated that TPA-induced monocytic differentiation and expression of the <Protname>c-jun</Protname> and <Protname>c-fos</Protname> genes in myeloid leukemia cells are regulated by <Protname>protein kinase C</Protname> (<Protname>PKC</Protname>). The finding that dexamethasone has no effect on TPA-induced activation of <Protname>PKC</Protname> suggests that this glucocorticoid inhibits signals downstream or parallel to this enzyme. Nuclear run-on assays demonstrate that: (1) induction of <Protname>c-jun</Protname> and <Protname>c-fos</Protname> expression by TPA is regulated by transcriptional mechanisms, (2) TPA-induced expression of <Protname>c-jun</Protname> and <Protname>c-fos</Protname> does not require protein synthesis, and (3) TPA-induced expression of both genes is inhibited at the transcriptional level by dexamethasone. To further define the effects of dexamethasone at the molecular level, we prepared a series of deleted <Protname>c-jun</Protname> promoter fragments linked to the <Protname>chloramphenicol acetyltransferase</Protname> (<Protname>CAT</Protname>) gene. Increases in <Protname>CAT</Protname> activity during transient expression of these constructs in TPA-treated U-937 cells could be assigned to the region (-97 to -20) of the promoter that contains the <Protname>AP-1</Protname> binding site. This induction of <Protname>CAT</Protname> activity was sensitive to dexamethasone. These findings suggest that dexamethasone down-regulates TPA-induced transcription of the <Protname>c-jun</Protname> gene during monocytic differentiation by inhibiting activation of the <Protname>AP-1</Protname> site.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>91321794</MedlineID>
<PMID>91321794</PMID>
<ArticleTitle>Inhibition of HIV-1 replication and <Protname>NF-kappa B</Protname> activity by cysteine and cysteine derivatives.</ArticleTitle>
<AbstractText>
HIV-1 proviral DNA contains two binding sites for the <Protname>transcription factor NF-kappa B</Protname>. HIV-1-infected individuals have, on average, abnormally high levels of <Protname>tumour necrosis factor alpha</Protname> (<Protname>TNF alpha</Protname>) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and <Protname>NF-kappa B</Protname> expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit <Protname>NF-kappa B</Protname> activity as determined by electrophoretic mobility shift assays and <Protname>chloramphenicol acetyl-transferase</Protname> (<Protname>CAT</Protname>) gene expression under control of <Protname>NF-kappa B</Protname> binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of <Protname>NF-kappa B</Protname>-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>92068245</MedlineID>
<PMID>92068245</PMID>
<ArticleTitle>Constitutive activation of <Protname>NF-kB</Protname> in human thymocytes.</ArticleTitle>
<AbstractText>
<Protname>NF-kB</Protname> is a eukaryotic transcription regulatory factor. In T cells and T cell lines, <Protname>NF-kB</Protname> is bound to a cytoplasmic proteic inhibitor, the <Protname>IkB</Protname>. Treatment of T cells with mitogens (phorbol esters) or cytokines (<Protname>TNF alpha</Protname>) induces <Protname>NF-kB</Protname> nuclear translocation and the subsequent expression of <Protname>NF-kB</Protname> dependent T cell genes. Here we examined the activation of <Protname>NF-kB</Protname> in human T cell thymic progenitors. We report differences in (Ca2+)i requirement for <Protname>NF-kB</Protname> activation in thymocytes as compared to mature T cells. Furthermore, our results indicated that thymocytes have a constitutively active form of <Protname>NF-kB</Protname>, suggesting that they are activated in vivo.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>92049367</MedlineID>
<PMID>92049367</PMID>
<ArticleTitle>Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized <Protname>human glucocorticoid receptor</Protname>.</ArticleTitle>
<AbstractText>
We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of <Protname>human glucocorticoid receptor</Protname> (<Protname>hGR</Protname>), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated <Protname>hGR</Protname> was indistinguishable from native <Protname>hGR</Protname>, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against <Protname>hGR</Protname>. In addition, cell-free synthesized <Protname>hGR</Protname> was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these <Protname>glucocorticoid receptors</Protname>. In vitro translated <Protname>hGR</Protname> was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized <Protname>GR</Protname> was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized <Protname>hGR</Protname> was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that <Protname>hGR</Protname> synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for <Protname>glucocorticoid receptor</Protname> present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding.
</AbstractText>
</PubmedArticle>
<PubmedArticle>
<MedlineID>91132035</MedlineID>
<PMID>91132035</PMID>
<ArticleTitle><Protname>Platelet-activating factor</Protname> induces phospholipid turnover, calcium flux, arachidonic acid liberation, eicosanoid generation, and oncogene expression in a human B cell line.</ArticleTitle>
<AbstractText>
<Protname>Platelet-activating factor</Protname> is a potent mediator of the inflammatory response. Studies of the actions of <Protname>platelet-activating factor</Protname> have centered mainly around neutrophils, monocytes, and platelets. In this report we begin to uncover the influence of <Protname>platelet-activating factor</Protname> on B lymphocytes. Employing the EBV-transformed human B cell line SKW6.4, we demonstrate that <Protname>platelet-activating factor</Protname> significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine, phosphatidylinositol, and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10(-9) to 10(-6) M. The inactive precursor, <Protname>lyso-platelet-activating factor</Protname>, at a concentration as high as 10(-7) M had no effect on any of the membrane phospholipids. We also show that <Protname>platelet-activating factor</Protname> from 10(-12) to 10(-6) M induced rapid and significant elevation in intracellular calcium levels, whereas <Protname>lyso-platelet-activating factor</Protname> was again ineffective. We further demonstrate the impact of <Protname>platelet-activating factor</Protname> binding to B cells by measuring <Protname>platelet-activating factor</Protname> induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production. Moreover, <Protname>platelet-activating factor</Protname> was capable of inducing transcription of the nuclear proto-oncogenes <Protname>c-fos</Protname> and <Protname>c-jun</Protname>. Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates <Protname>platelet-activating factor</Protname> induced arachidonic acid release perhaps acting at the level of phospholipase A2. In summary, <Protname>platelet-activating factor</Protname> is shown here to have a direct and profound effect on a pure B cell line.
</AbstractText>
</PubmedArticle>
</PubmedArticleSet>
