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At least 361 records · Page 20Linked to original sources
PubMed2003

Distinct subtypes of group I metabotropic glutamate receptors on rat spinal neurons mediate complex facilitatory and inhibitory effects.

While group I glutamate metabotropic (mGlu) receptors show discrete neuronal distribution in the neonatal rat spinal cord, the functional role of their distinct receptor subtypes remains uncertain. Intracellular recording from lumbar motoneurons together with extracellular recording of ventral root (VR) responses was used to investigate the differential contribution by mGlu receptor subtypes to cell excitability and network activity. The group I agonist DHPG evoked motoneuron depolarization (via the AIDA or CPCCOEt-sensitive mGlu receptor subtype 1) mainly at network level and generated sustained, network-dependent oscillations (via the MPEP-sensitive mGlu receptor subtype 5). DHPG also decreased the peak amplitude of synaptic responses induced by dorsal root stimuli, an effect unrelated to depolarization and dependent on glycinergic transmission. Synaptic responses were insensitive to AIDA or MPEP. The present results can be explained by assuming excitation of discrete classes of interneurons by group I mGlu receptor activity. Thus, the cellular distribution of those mGlu receptors at strategic circuit connections may determine the functional outcome of the network in terms of excitation or inhibition. Even if there was insufficient activation by endogenous glutamate of mGlu receptors during synaptic activity evoked by DR stimuli, it is apparent that such receptors are important pharmacological targets for powerful and rapid up- or down-regulation of spinal signal processing at network level, providing a rationale for the proposed use of mGlu receptor agonists in a variety of spinal pathological conditions.

Animals↗
PubMed1994

Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration.

Bacterial Proteins↗
PubMed1999

Cubilin, the endocytic receptor for intrinsic factor-vitamin B(12) complex, mediates high-density lipoprotein holoparticle endocytosis.

Receptors that endocytose high-density lipoproteins (HDL) have been elusive. Here yolk-sac endoderm-like cells were used to identify an endocytic receptor for HDL. The receptor was isolated by HDL affinity chromatography and identified as cubilin, the recently described endocytic receptor for intrinsic factor-vitamin B(12). Cubilin antibodies inhibit HDL endocytosis by the endoderm-like cells and in mouse embryo yolk-sac endoderm, a prominent site of cubilin expression. Cubilin-mediated HDL endocytosis is inhibitable by HDL(2), HDL(3), apolipoprotein (apo)A-I, apoA-II, apoE, and RAP, but not by low-density lipoprotein (LDL), oxidized LDL, VLDL, apoC-I, apoC-III, or heparin. These findings, coupled with the fact that cubilin is expressed in kidney proximal tubules, suggest a role for this receptor in embryonic acquisition of maternal HDL and renal catabolism of filterable forms of HDL.

Animals↗
PubMed2006

Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner.

Aldosterone is a major regulator of epithelial Na(+) absorption and acts in large part through induction of the epithelial Na(+) channel (ENaC) gene in the renal collecting duct. We previously identified Dot1a as an aldosterone early repressed gene and a repressor of ENaCalpha transcription through mediating histone H3 Lys-79 methylation associated with the ENaCalpha promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaCalpha. AF9 and Dot1a interact in vitro and in vivo as evidenced in multiple assays and colocalize in the nuclei of mIMCD3 renal collecting duct cells. Overexpression of AF9 results in hypermethylation of histone H3 Lys-79 at the endogenous ENaCalpha promoter at most, but not all subregions examined, repression of endogenous ENaCalpha mRNA expression and acts synergistically with Dot1a to inhibit ENaCalpha promoter-luciferase constructs. In contrast, RNA interference-mediated knockdown of AF9 causes the opposite effects. Chromatin immunoprecipitation assays reveal that overexpressed FLAG-AF9, endogenous AF9, and Dot1a are each associated with the ENaCalpha promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaCalpha promoter and represses ENaCalpha transcription in an aldosterone-sensitive manner. This mechanism appears to be more broadly applicable to other aldosterone-regulated genes because overexpression of AF9 alone or in combination with Dot1a inhibited mRNA levels of three other known aldosterone-inducible genes in mIMCD3 cells.

Aldosterone↗
PubMed1997

G protein betagamma complex-mediated apoptosis by familial Alzheimer's disease mutant of APP.

In familial Alzheimer's disease (FAD), three missense mutations, V642I, V642F and V642G, that co-segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin-sensitive apoptosis in an FAD trait-linked manner by activating the G protein Go, which consists of G alpha(o) and G betagamma subunits. We investigated which subunit was responsible for the induction of apoptosis by V642I APP in NK1 cells. In the same system, expression of mutationally activated G alpha(o) or G alpha(i) induced little apoptosis. Apoptosis by V642I APP was antagonized by the overexpression of the carboxy-terminal amino acids 495-689 of the beta-adrenergic receptor kinase-1, which blocks the specific functions of G betagamma. Co-transfection of G beta2gamma2 cDNAs, but not that of other G beta(x)gamma(z) (x = 1-3; z = 2, 3), induced DNA fragmentation in a manner sensitive to bcl-2. These data implicate G betagamma as a cell death mediator for the FAD-associated mutant of APP.

Alzheimer Disease↗
PubMed2003

G protein and adenylate cyclase complex-mediated signal transduction in the rat heart during sepsis.

Changes in the protein level of various subunits of GTP-binding protein and the activity of adenylate cyclase in the rat heart during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The protein levels of various subunits of GTP-binding protein were determined by Western blot analysis. The activity of adenylate cyclase was measured based on the rate of formation of cAMP from [alpha-32P]ATP. The results show that protein levels of G alphas and G beta remained stable during the early and the late phases of sepsis. The protein levels of G alpha i-2 and G alpha i-3 remained relatively unaltered during the early phase of sepsis, but they were increased by 46.5% (P < 0.05) and 61.3% (P < 0.01), respectively, during the late phase of sepsis. The basal adenylate cyclase activity remained unchanged during the early phase while it was decreased by 25.7% (P < 0.05) during the late phase of sepsis. The isoproterenol-stimulated adenylate cyclase activity was unchanged during early sepsis while it was decreased by 44.6% (P < 0.01) during late sepsis. These data demonstrate that during the late hypodynamic phase of sepsis, myocardial G alpha i-2 and G alpha i-3 protein levels were increased and the increases were coupled with a reduction in adenylate cyclase activity. Because GTP-binding proteins mediate sympathetic control of cardiac function, the present findings may have a pathophysiological significance in contributing to the understanding of the pathogenesis of cardiac dysfunction during the late stage of sepsis.

Adenylyl Cyclases↗
PubMed1987

Integrin, a transmembrane glycoprotein complex mediating cell-substratum adhesion.

A monoclonal antibody, CSAT, which inhibits the adhesion of chick cells to substrata coated with fibronectin, laminin and vitronectin, has been used to identify a cell surface receptor required for cell-substratum adhesion. This receptor, termed integrin, is found on the ventral surface of cells in close contact adhesion sites, at the periphery of adhesion plaques and beneath stress fibres. It is a heterodimer consisting of non-covalently linked alpha and beta subunits. Integrin binds directly to laminin, fibronectin and vitronectin with dissociation constants in the micromolar range. The binding of integrin to matrix molecules is sensitive to peptides carrying the cell-binding sequence Arg-Gly-Asp and requires heteromeric integrity. Integrin also binds directly to the cytoskeleton-associated protein talin. Thus, integrin has the properties of a transmembrane molecule capable of bringing extracellular matrix and cytoskeleton-associated molecules in proper juxtaposition to form adhesion structures. The integrin beta subunit is phosphorylated following Rous sarcoma virus transformation. Phosphorylation alters the ability of the receptor to bind extracellular matrix molecules as well as talin, suggesting a mechanism for the alteration of cellular adhesive and morphological properties following malignant transformation. A major phosphorylation site is on the cytoplasmic domain of the beta subunit. Synthetic peptides homologous with this region of integrin inhibit integrin-talin binding. The gene for the beta subunit of integrin has been sequenced. Its structure is consistent with the membrane-spanning properties of the receptor. Integrin is structurally and serologically related to adhesion receptors from mammalian tumour cells, fibroblasts, platelets and lymphocytes. It appears to be a member of a supergene family of receptors involved in cellular adhesive interactions. Antibody and peptide inhibition experiments have suggested a role for integrin and integrin-like molecules in cell migration, neurite extension, neural differentiation, histogenesis and embryonic development in Drosophila. Thus, integrin appears representative of a set of evolutionarily conserved, biologically important adhesive molecules.

Animals↗
PubMed2004

Cell size and Cln-Cdc28 complexes mediate entry into meiosis by modulating cell growth.

In the yeast Saccharomyces cerevisiae, mitotic cell cycle progression depends upon the G(1)-phase cyclin-dependent kinase Cln-Cdc28 and cell growth to a minimum cell size. In contrast, Cln-Cdc28 inhibits entry into meiosis, and a cell growth requirement for sporulation has not been established. Here, we report that entry into meiosis also depends upon cell growth. Moreover, sporulation and cell growth rates were proportional to cell size; large cells grew rapidly and sporulated sooner while smaller cells grew slowly and sporulated later. In addition, Cln2 protein levels were higher in smaller cells suggesting that Cln-Cdc28 activity represses meiosis in smaller cells by preventing cell growth. In support of this hypothesis, loss of Clns, or the presence of a cdc28 mutation increased cell growth specifically in smaller cells and accelerated meiosis in these cells. Finally, overexpression of CLNs repressed meiosis in smaller cells, but not in large cells. Taken together, these results demonstrate that Cln-Cdc28 represses entry into meiosis in part by inhibiting cell growth.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1979

In vivo responses to inhaled proteins. II. Induction of interstitial pneumonitis and enhancement of immune complex-mediated alveolitis by inhaled concanavalin A.

An animal model of environmental lung disease is described in which phytomitogen, antigen, or both, are administered in aerosol form to previously immunized or immunologically naive rabbits. Inhalation of concanavalin A alone induced an interstitial pneumonitis in nonimmunized rabbits. Inhalation of concanavalin A alone induced an interstitial pneumonitis in nonimmunized rabbits. Inhalation of bovine serum albumin (BSA) alone typically produced only focal eosinophilic granulomas in BSA-immunized animals, and no injury whatever in nonimmune animals. However, simultaneous administration of BSA-concanavalin A aerosol mixtures to BSA-immunized rabbits induced a severe interstitial pneumonitis and granulomatous vasculitis, together with areas of frank parenchymal necrosis. When repeated on a chronic basis over a 4- or 8-week interval, challenge with BSA-concanavalin A aerosols resulted in both acute necrotic lesions as well as areas of frank interstitial fibrosis. Necrotic foci in acutely injured lungs were associated with interstitial deposits of BSA, rabbit anti-BSA antibody, and complement. Electron microscopy revealed numerous neutrophils within the pulmonary interstitial spaces of these animals, often in association with collagen and elastin fibers. The pattern of injury in immune rabbits induced by antigen-concanavalin A aerosols, in its nonnecrotizing form, is consistent with that of an extrinsic allergic alveolitis. However, the severe, necrotizing form of acute injury closely resembles changes seen in Wegener's granulomatosis. Possible mechanisms of injury produced by antigen and phytomitogen inhalation are discussed.

Aerosols↗
PubMed1994

Estrogen receptor-Sp1 complexes mediate estrogen-induced cathepsin D gene expression in MCF-7 human breast cancer cells.

Cathepsin D is an estrogen (17 beta-estradiol, E2)-inducible lysosomal protease. A putative estrogen receptor (ER)-Sp1-like sequence (GGGCGG(n)23ACGGG) has been identified in the non-coding strand of the cathepsin D promoter (-199 to -165), and electromobility shift assays of nuclear extracts from MCF-7 and HeLa cells confirm that both the ER and Sp1 protein bind to 32P-labeled ER/Sp1 oligo. For example, nuclear extracts from MCF-7 cells bind to the 32P-labeled ER/Sp1 oligo; however, ER/Sp1 binding can be decreased by selective competition with excess unlabeled estrogen responsive element and Sp1 oligos, immunodepletion with ER or Sp1 antibodies, and by treating cells with ICI 164,384, an antiestrogen which inhibits formation of ER homodimer. Moreover, E2-induced chloramphenicol acetyltransferase (CAT) activity in MCF-7 cells cotransfected with a human estrogen receptor expression plasmid and a plasmid containing an ER/Sp1 sequence cloned upstream to a thymidine kinase promoter and a CAT reporter. In cotreatment studies, ICI 164,384 inhibited E2-induced CAT activity. In contrast, E2 did not induce CAT activity in MCF-7 cells transfected with plasmids containing mutations in the ER or Sp1 segments of the ER/Sp1 oligo, thus confirming that both cognate binding sites are required for estrogen responsiveness.

Adenosine Triphosphate↗
PubMed2006

Zebrafish Trap230/Med12 is required as a coactivator for Sox9-dependent neural crest, cartilage and ear development.

The vertebrate Sox9 transcription factor directs the development of neural crest, otic placodes, cartilage and bone. In zebrafish, there are two Sox9 orthologs, Sox9a and Sox9b, which together perform the functions of the single-copy tetrapod Sox9. In a large-scale genetic screen, we have identified a novel zebrafish mutant that strongly resembles the Sox9a/Sox9b double mutant phenotype. We show that this mutation disrupts the zebrafish Trap230/Med12 ortholog, a member of the Mediator complex. Mediator is a coactivator complex transducing the interaction of DNA-binding transcription factors with RNA polymerase II, and our results reveal a critical function of the Trap230 subunit as a coactivator for Sox9.

Animals↗
PubMed1999

Intestinal ischemia-reperfusion injury is mediated by the membrane attack complex.

BACKGROUND: The dependence of intestinal ischemia-reperfusion injury on the classical complement pathway has been shown with the complement antagonist (sCR1) and complement-specific knockout mice. Using C5 deficient mice, we show that the membrane attack complex mediates local injury. METHODS: Mice underwent intestinal ischemia-reperfusion. Albumin leak and histologic evidence were compared in wildtype mice, wildtypes treated with sCR1, neutrophil-depleted wildtypes, C5-deficient mice, and C5-deficient mice reconstituted with wildtype serum. Neutrophil tissue levels in injured C5-deficient and wildtype intestines were compared. RESULTS: C5-deficient mice had a reduction in injury similar to mice treated with sCR1. Injury was restored by reconstitution with wildtype serum. Wildtype injury was unaffected by neutrophil depletion. Injured intestines of C5-deficient and wildtype mice had similar neutrophil levels. Immunohistochemistry of wildtype and reconstituted C5-deficient mice demonstrated injured intestinal epithelium although C5-deficient mice and sCr1-treated mice were similar to sham mice. CONCLUSIONS: C5-deficient animals are protected from local injury. Injury is unaffected by neutrophil depletion, and the presence of neutrophils in injured tissue is independent of C5. Local injury is C5 dependent, but the action of C5a on granulocytes is not required. Therefore the membrane attack complex mediates local injury.

Animals↗
PubMed1982

The specificity of extracellular glycoprotein complexes in mediating cellular adhesion.

Complexes containing glycoproteins and glycosaminoglycans were released into the culture medium by both smooth and skeletal muscle cells. The particles, termed adherons, from three smooth muscle cell lines promoted cell-substratum adhesion of a clonal sympathetic nerve cell line, PC12. In contrast, the glycoprotein complexes from three skeletal muscle preparations inhibited the adhesion of PC12 cells to Petri dish surfaces. The skeletal muscle adherons all sedimented in calcium-free sucrose gradients with an S value of 16, while the smooth muscle particles had a sedimentation value of 12. Although both classes of adherons contained fibronectin and collagen, hyaluronic acid was present only in those from skeletal muscle. An antiserum prepared against skeletal muscle adherons blocked myoblast adhesion to skeletal muscle adherons but did not alter PC12 adhesion to smooth muscle adherons. These data suggest that the glycoprotein complexes released by muscle have some intrinsic specificity with respect to their ability to mediate cellular adhesion.

Animals↗
PubMed2002

TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA.

Activator-mediated transcription complex assembly on templates lacking chromatin requires the interaction of activators with two major coactivator complexes: TFIID and mediator. Here we employed immobilized template assays to correlate transcriptional activation with mediator and TFIID recruitment. In reactions reconstituted with purified proteins, we found that activator, TFIID, and mediator engage in reciprocal cooperative interactions to form a complex on promoter DNA. Preassembly of the coactivator complex accelerates the rate of transcription in a cell-free system depleted of TFIID and mediator. Our data argue that this coactivator complex is an intermediate in the assembly of an active transcription complex. Furthermore, the reciprocity of the interactions demonstrates that the complex could in principle be nucleated with either TFIID or mediator, implying that alternative pathways could be utilized to generate diversity in the way activators function in vivo.

Carrier Proteins↗
PubMed1990

Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor.

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.

Carcinoma, Squamous Cell↗
PubMed2003

Activator-independent functions of the yeast mediator sin4 complex in preinitiation complex formation and transcription reinitiation.

RNA polymerase II (Pol II) Mediator plays an essential role in both basal and activated transcription. Previously, subunits of the Sin4 Mediator complex (Sin4, Pgd1, Gal11, and Med2) have been implicated in both positive and negative transcriptional regulation. Furthermore, it was proposed that this subcomplex constitutes an activator-binding domain. A yeast nuclear-extract system was used to investigate the biochemical role of the Sin4 complex. In contrast to previous findings, we found at least two general activator-independent roles for the Sin4 complex. First, mutations in sin4 and pgd1 destabilized the Pol II-Med complex, leading to a reduced rate and extent of preinitiation complex (PIC) formation both in the presence and absence of activators. Although reduced in amount compared with the wild type, PICs that are formed lacking the Sin4 complex are stable and can initiate transcription normally. Second, mutation of pgd1 causes partial disruption of the Sin4 complex and leads to a defect in transcription reinitiation. This defect is caused by dissociation of mutant Mediator from promoters after initiation, leading to nonfunctional Scaffold complexes. These results show that function of the Sin4 complex is not essential for transcription activation in a crude in vitro system but that it plays key roles in the general transcription mechanism.

Cell Extracts↗