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Results for “Mediator complex”

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PubMed1987

Modulation of acute immune complex-mediated tissue injury by the presence of polyionic substances.

Considerable attention has been focused on the role of electrostatic charge in the pathogenesis of immune complex-mediated tissue injury. The authors have examined the ability of cationic (histone, polyhistidine, polyarginine) and anionic (polyanetholsulfonate) polyelectrolytes to modulate acute immune complex-mediated tissue injury. Tissue injury elicited in rats by the reversed dermal Arthus reaction was increased 26-43% by addition of polyelectrolytes to antibody prior to its intradermal injection. Kinetic studies using 111In-labeled neutrophils indicated that the enhanced tissue injury was not the result of increased influx of neutrophils. Infusion of polyethylene glycol-conjugated superoxide dismutase prior to induction of the Arthus reaction resulted in 40-68% suppression of tissue injury. Concomitant in vitro functional studies (enzyme secretion, O-2 and H2O2 generation, and chemiluminescence) of rat neutrophils demonstrated that addition of polyelectrolytes to preformed immune complexes (IgG-bovine serum albumin) resulted in marked increases in O-2, H2O2, and chemiluminescence, but no increases in enzyme secretion, compared with neutrophils stimulated with immune complexes alone. The cationic polyelectrolytes did not alter the capacity of preformed immune complexes to activate complement in vitro. These studies suggest that both cationic and anionic polyelectrolytes can increase the pathogenic potential of immune complexes and that this modulation is, at least in part, mediated by enhanced generation of toxic oxygen-derived metabolites by neutrophils.

Animals↗

PubMed2006

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220.

The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.

Animals↗

PubMed1982

An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement.

In this study, an in vitro model of immune complex-mediated basement membrane zone separation caused by periphigoid antibodies, serum complement, and peripheral blood leukocytes is described. When cryostat sections of fresh-frozen normal human skin were treated with either of 4 bullous pemiphigoid sera containing complement-activating anti-basement membrane zone antibodies and subsequently incubated at 37 degrees C with normal human peripheral blood leukocytes and fresh human serum, leukocytes attached to 96% of the basement membrane zone in 100% of sections. Sixty-seven percent of the sections developed focal areas of basement membrane zone separation resembling dermal-epidermal separation described in early pemphigoid lesions. In control sections in which either leukocytes, pemphigoid antibody or fresh human serum were omitted, significantly less leukocyte attachment and basement membrane zone separation occurred. Evidence that leukocytes caused separation was supported by an absolute requirement for viable leukocytes during incubation, a high correlation between leukocyte attachment and separation and experiments showing that leukocytes attached to the basement membrane zone were activated. This study provides the first in vitro evidence directly supporting a functional role for immune-complex mediated inflammation in the pathogenesis of basement membrane zone separation and blisters in bullous pemphigoid.

Antibodies↗

PubMed1995

Three subunits of the RNA polymerase II mediator complex are involved in glucose repression.

Glucose triggers a complex response in yeast which includes induction and repression of a large number of genes. Glucose repression is in part mediated by the Mig1 repressor, a zinc finger protein that binds to the promoters of many glucose repressed genes. However, some genes that are required for gluconeogenic growth are also repressed by a Mig1-independent mechanism. We have isolated mutations in three genes that are involved in this Mig1-independent component of repression and cloned the genes by complementation. All three genes encode subunits of the recently discovered RNA polymerase II mediator complex. Two of them are yeast cyclin C and its associated kinase. Disruptions of the three genes have identical phenotypes with respect to glucose repression and show no synergism with each other. This suggests that these three subunits of the mediator complex function closely together in transmitting the transcriptional response to glucose.

Amino Acid Sequence↗

PubMed2004

The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation.

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP- and glutamine-dependent formation of formylglycinamidine ribonucleotide, ADP, P(i), and glutamate in the fourth step of de novo purine biosynthesis. Like all amidotransferases (ATs), FGAR-AT is proposed to channel ammonia between a glutaminase and AT domain. In Gram-negative bacteria and eukaryotes, FGAR-AT is a single approximately 140 kDa protein. In archae and Gram-positive bacteria, the FGAR-AT is formed from three proteins: PurS (10 kDa), PurQ (25 kDa, a glutaminase), and smPurL (80 kDa, an AT). This is the only known AT to require a third structural component (PurS) for activity. Here we report the first purification and biochemical characterization of a three-component AT from Bacillus subtilis. Efforts to isolate an intact FGAR-AT focused initially on coexpression of PurS, smPurL, and PurQ. However, all attempts to purify the complex resulted in separation of the constituent proteins. PurS, smPurL, and PurQ were therefore separately expressed and purified to homogeneity. PurQ had a glutaminase activity of 0.002 s(-1), and smPurL had an ammonia-dependent AT activity of 0.044 s(-1). Reconstitution of PurS, smPurL, and PurQ at a ratio of 2:1:1 gave an activity of 2.49 s(-1), similar to that previously reported for the Escherichia coli 140 kDa FGAR-AT (5.00 s(-1)). PurS was essential for the glutamine-dependent FGAR-AT activity. Surprisingly, activity was found to be absolutely dependent on the presence of Mg2+ and ADP, and a stable FGAR-AT complex of 2PurS/1smPurL/1PurQ was detected only in the presence of Mg2+, ADP, and glutamine. The implications of these observations are discussed with respect to ammonia channeling.

Adenosine Diphosphate↗

PubMed1983

Immune complex-mediated disease not a factor in patients on maintenance venom immunotherapy.

Although a serum sickness-like presentation has been reported as a sequela of Hymenoptera stings, the possible role of an immune complex-mediated pathologic condition in patients receiving regular venom immunotherapy has never been addressed. To evaluate this problem, 30 adult and 15 pediatric patients receiving regular monthly doses of venom (100 micrograms of antigen) were studied. All had been receiving immunotherapy for 12 to 29 mo. At the time of venom administration, a questionnaire related to symptoms of immune complex disease was completed. A urinalysis was performed 12 hr later. In addition, blood was drawn to evaluate the presence of immune complexes by Clq and Raji cell assays. Symptom surveys revealed no clinical manifestations suggestive of immune complex pathology. All urinalyses were negative for gross and microscopic hematuria. None of the specimens was elevated on the Clq assay. Only four of the 45 patients had significantly positive Raji cell assays. Prospective reevaluation showed the presence of immune complex before venom administration, with no change in acute-phase reactants or Raji cell titers 12 hr later. Monthly administration of Hymenoptera venom appears to be unassociated with immune complex-mediated disease, by either clinical or immunologic parameters.

Adolescent↗

PubMed1984

Bowel-associated dermatosis-arthritis syndrome. Immune complex-mediated vessel damage and increased neutrophil migration.

In a recent report we described a syndrome, identical to bowel-bypass syndrome, that occurred in four patients who had not had bypass surgery. Herein, circulating immune complexes (CICs) and neutrophil migration are evaluated in three of those four patients to test the hypothesis that the cutaneous lesions might have resulted from interaction between immune complex-mediated vessel damage and increased neutrophil migration. In vitro assays indicated that CICs were present in one of two patients and "histamine trap" test evidence for CICs was present in both patients tested. Although serum from the three patients appeared to increase neutrophil movement, statistically significant increases were not observed when data were pooled in this small study group. Preliminary results suggest that immune complex-mediated vessel damage, followed by extensive accumulation of neutrophils, may cause the pustular vasculitis in the bowel-associated dermatosis-arthritis syndrome.

Adult↗

PubMed2000

Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism. The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or internalization.

We have previously described thrombin (Th)-protease nexin 1 (PN1) inhibitory complex binding to cell surface heparins and subsequent low density lipid receptor-related protein (LRP)-mediated internalization. Our present studies examine the catabolism of urinary plasminogen activator (uPA)-PN1 inhibitory complexes, which, unlike Th.PN1 complexes, bind almost exclusively through the uPA receptor. In addition, the binding site in PN1 required for the LRP-mediated internalization of Th.PN1 complexes is not required for the LRP-mediated internalization of uPA.PN1 complexes. Thus, the protease moiety of the complex partially determines the mechanistic route of entry. Because cell surface heparins are only minimally involved in the binding and internalization of uPA.PN1 complexes, we then predicted that complexes between uPA and the heparin binding-deficient PN1 variant, PN1(K7E), should be catabolized at the same rate as complexes formed with native PN1. Surprisingly, the uPA.PN1(K7E) complexes were degraded at only a fraction of the rate of native complexes. Internalization studies revealed that both uPA. PN1(K7E) and native uPA.PN1 complexes were initially internalized at the same rate, but uPA.PN1(K7E) complexes were rapidly retro-endocytosed in an intact form. By examining the pH dependence of complex binding in the range of 4.0-7.0, it was determined that the uPA.PN1 inhibitory complexes must specifically bind to endosomal heparins at pH 5.5 to be retained and sorted to lysosomes. These studies are the first to document a role for heparins in the catabolism of SERPIN-protease complexes at a point further in the pathway than cell surface binding, and this role may extend to other heparin-binding LRP-internalized ligands.

Amyloid beta-Protein Precursor↗

PubMed1999

Caenorhabditis elegans mediator complexes are required for developmental-specific transcriptional activation.

Mediator proteins are required for transcriptional regulation of most genes in yeast. Mammalian Mediator homologs also function as transcriptional coactivators in vitro; however, their physiological role in gene-specific transcription is not yet known. To determine the role of Mediator proteins in the development of complex organisms, we purified putative Mediator complexes from Caenorhabditis elegans and analyzed their phenotypes in vivo. C. elegans Mediator homologs were assembled into two multiprotein complexes. RNA interference assays showed that the CeMed6, CeMed7, and CeMed10/CeNut2 gene products are required for the expression of developmentally regulated genes, but are dispensable for expression of the ubiquitously expressed genes tested in this study. Therefore, the gene-specific function of Mediator as an integrator of transcriptional regulatory signals is evolutionarily conserved and is essential for C. elegans development.

Amino Acid Sequence↗

PubMed2003

[Potential causes of antigenemia in the patients with an immune complex-mediated membranoproliferative glomerulonephritis in Lithuania].

UNLABELLED: The pathogenesis of an immune complex-mediated membranoproliferative glomerulonephritis (IMPGN) involves persistent deposition of circulating immune complexes in the glomeruli caused by persistent antigenemia. We have previously reported relatively high incidence of IMPGN in Lithuania. The objective of our study was to evaluate potential causes of persistent antigenemia in the patients with IMPGN. MATERIAL AND METHODS: Forty-five patients with IMPGN diagnosed on renal biopsy during 2000-2002 were retrospectively evaluated for the presence of persistent bacterial or viral infections, autoimmune diseases and other associated medical conditions. Patients with established diagnosis of systemic lupus erythematosus (SLE) before the biopsy were not included in the study. RESULTS: A great majority (20; 44%) of the patients were found to have persistent bacterial infections of various localization. Four patients (9%) were infected with hepatitis B virus (HBV). Three (7%) patients were eventually diagnosed with SLE and another 3 (7%) had other associated pathology. In the remaining 15 (33%) patients, IMPGN remained idiopathic. Testing for hepatitis C virus (HCV) antibody was performed in 36 patients (12 of them with idiopathic IMPGN) and was negative in all patients. Testing for HCV RNA was not performed. Patients with bacterial infections were significantly younger compared to the group of idiopathic IMPGN (36.5+/-19.1 and 53.8+/-16.4, respectively, p=0.01). We conclude that persistent bacterial infection was a major potential source of antigenemia in our patients with IMPGN, particularly in the younger patients, while HBV and HCV infection was rarely detected.

Adolescent↗

PubMed1982

Immune complex-mediated glomerulonephritis and interstitial pneumonia simulating Goodpasture's syndrome.

An autopsy case of what was clinically considered to be Goodpasture's syndrome was investigated. The lung had hemorrhagic interstitial pneumonia, showing granular patterns of IgG and C3 along the alveoli by the immunofluorescent method and electron-dense subepithelial deposits by electron microscopy. The kidney had crescentic and segmental necrotizing glomerulonephritis associated with membranous nephropathy. Uneven, continuous patterns of immunofluorescent IgG and C3 along the GBM were noted. Electron microscopy showed numerous subepithelial deposits, and immunoelectron microscopy revealed that IgG was not present in the GBM itself but present in the subepithelial deposits. Anti-GBM antibody activity was not detected in the serum or the kidney eluate. It was suggested that renal and pulmonary lesions occurred through the same mechanism and in association with immune deposits. We propose that there is a disease having immune complex-mediated deposits. We propose that there is a disease having immune complex-mediated renal and pulmonary lesions which clinically resembles the conventional Goodpasture's syndrome.

Adult↗

PubMed1994

Intraglomerular expressions of IL-1 alpha and platelet-derived growth factor (PDGF-B) mRNA in experimental immune complex-mediated glomerulonephritis.

Both PDGF and IL-1 play important roles as autocrine growth factors for cultured mesangial cells, and may be closely associated with the progression of glomerulonephritis. In this study we investigated intraglomerular expressions of PDGF-B and IL-1 alpha mRNA in mice with bovine serum albumin (BSA) nephritis, a model of immune complex-mediated glomerulonephritis, using the reverse transcription-polymerase chain reaction (RT-PCR) method. We also quantified intraglomerular PDGF-B mRNA by the competitive PCR and studied the correlation between the level of intraglomerular PDGF-B mRNA expression and the degree of observed glomerular injury. While expression of neither PDGF-B nor IL-1 alpha mRNA was detected in glomeruli from control mice, both were strongly expressed in glomeruli from mice with BSA nephritis. IL-1 alpha mRNA in glomeruli showed low accumulation in mice with mild glomerular injury, and was increased in mice with moderate glomerular injury. In contrast, high intraglomerular expression of PDGF-B mRNA occurred in all mice with mild glomerular injury and continued throughout the course of the disease. We observed no correlation between the level of PDGF-B mRNA expression and the histologic grade of renal damage. These results suggest that PDGF and IL-1 have different growth properties, and PDGF might play a role as a competence factor rather than a progression factor in the pathogenesis of immune complex-mediated glomerulonephritis.

Animals↗

PubMed2002

In vivo association of adenovirus large E1A protein with the human mediator complex in adenovirus-infected and -transformed cells.

The adenovirus large E1A protein activates transcription from early viral promoters by a mechanism that requires a forty amino acid zinc finger activation domain in E1A conserved region 3 (CR3). Recent results indicate that activation by a Gal4 DNA-binding domain-E1A-CR3 fusion requires an interaction between the E1A-CR3 zinc finger and the Sur2 subunit of the mammalian Mediator (of transcription) complex. Although several host proteins have been shown to bind stably to E1A proteins in adenovirus-infected and -transformed cells, an in vivo interaction with Mediator complex subunits has not been described previously. Using immunoprecipitation and gel filtration analyses of nuclear extracts prepared from HeLa cells infected with adenovirus 5 or mutants that express either large or small E1A specifically and from adenovirus 5-transformed cells, we report here that large E1A, but not small E1A, binds to Mediator complex in vivo. Only approximately 1 to 10% of large E1A is bound to Mediator complex at 18 h postinfection and in transformed cells, probably explaining why Mediator complex subunits were not identified among cellular E1A-binding proteins described earlier. Surprisingly, even though extracted Mediator can quantitatively bind to an E1A-CR3 affinity column, only on the order of 1% of cellular Mediator complex is bound by E1A in vivo. Much of the large E1A bound to Mediator in 293 cells is in a stable complex that includes RNA polymerase II, leading us to suggest that the interaction of E1A-CR3 with Mediator stabilizes the interaction of Mediator with the polymerase. This stabilization of the interaction between Mediator and RNA polymerase II may contribute to the mechanism of activation by E1A-CR3.

Adenovirus E1A Proteins↗

PubMed2003

Two subunits of the Drosophila mediator complex act together to control cell affinity.

The organizing centers for Drosophila imaginal disc development are created at straight boundaries between compartments; these are maintained by differences in cell affinity controlled by selector genes and intercellular signals. skuld and kohtalo encode homologs of TRAP240 and TRAP230, the two largest subunits of the Drosophila mediator complex; mutations in either gene cause identical phenotypes. We show here that both genes are required to establish normal cell affinity differences at the anterior-posterior and dorsal-ventral compartment boundaries of the wing disc. Mutant cells cross from the anterior to the posterior compartment, and can distort the dorsal-ventral boundary in either the dorsal or ventral direction. The Skuld and Kohtalo proteins physically interact in vivo and have synergistic effects when overexpressed, consistent with a skuld kohtalo double-mutant phenotype that is indistinguishable from either single mutant. We suggest that these two subunits do not participate in all of the activities of the mediator complex, but form a submodule that is required to regulate specific target genes, including those that control cell affinity.

Animals↗

PubMed1986

Tocainide suppression of immune-complex-mediated dermal inflammation: comparison with prostaglandin E1.

Local anesthetic agents have been shown to alter a variety of polymorphonuclear leukocyte (PMN) functions and may be useful as anti-inflammatory agents. We compared the anti-inflammatory effects of therapeutic doses of the recently released local anesthetic-antiarrythmic drug tocainide to pharmacologic doses of prostaglandin E1 (PGE1) on immune-complex-mediated dermal inflammation in female Sprague-Dawley rats. Intense dermal inflammation was produced using a classic reverse passive Arthus reaction, and the inhibition of PMN accumulation in the subdermis was quantitated in biopsy samples taken 2.5 hr after the reaction was initiated and the drug was given. Using a light microscope with a counting grid, biopsy sections were randomly sampled in a blinded fashion and an inflammation index equal to the ratio of PMNs to fibroblasts was determined for each animal. The mean inflammation index in 10 animals given 25 mg of tocainide (mean serum level = 14.6 micrograms/ml) was 9.3 +/- 1.2 (+/- SEM), which was significantly less than the index of 17.7 +/- 2.5 in 10 control animals (P less than 0.025). Similarly, the five animals that received either 500 or 250 micrograms of PGE1 had a significantly reduced index, with the effect of 250 micrograms PGE1 comparable to the effect of the tocainide. These findings suggest that therapeutic levels of tocainide reduce the accumulation of PMNs in immune-complex-mediated dermal inflammation; thus, local anesthetic agents may be useful in the treatment of certain inflammatory disorders.

Alprostadil↗

PubMed1983

Regulation of immune complex-mediated complement activation by autoantibodies (F-42) isolated from sera of patients with systemic lupus erythematosus.

Sera of some patients with systematic lupus erythematosus (SLE) contain IgG autoantibodies (F-42) which have been shown to stabilize the cell bound classical pathway C3 convertase of complement, C42. C42 is susceptible to inactivation by the plasma protein C4bp while stabilized C42 is relatively resistant to C4bp. The present study demonstrates that F-42 by itself does not induce activation of the classical pathway in vitro but that it is able to modulate the immune complex-induced consumption of C2 and C3 in whole serum. Incubation of incremental concentrations of F-42 with normal human serum (NHS) for 30 min at 30 degrees C did not result in detectable consumption of C1q, C4, C2 and C3. However, when soluble immune aggregates or immune complexes were incubated in NHS together with 100 u/ml of F-42, a significant increase in consumption of C3 was seen as compared to the reaction mixture containing immune complexes or F-42 alone. In addition the presence of F-42 during the immune complex mediated consumption of complement was associated with relative protection of C2 consumption. (Fab)'2 and Fab' fragments of F-42 behaved as intact F-42, except that their activities on a molar basis were less than that of intact F-42. The results presented in this paper suggest that F-42 may play a regulatory role in the immune complex-mediated consumption at C2 and C3 in vivo.

Antigen-Antibody Complex↗

PubMed2002

Walleye dermal sarcoma virus cyclin interacts with components of the mediator complex and the RNA polymerase II holoenzyme.

Walleye dermal sarcoma virus (WDSV) encodes an accessory protein, OrfA, with sequence homology to cyclins (retrovirus cyclin). In cells transfected with an expression construct, OrfA was localized to the nucleus and was concentrated in interchromatin granule clusters (IGCs), sites where splicing factors are concentrated. Other proteins identified in IGCs include transcription factors, the large subunit of RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8). cdk8 is the kinase partner of cyclin C and a component of the mediator complex, associated with the Pol II holoenzyme. cdk8 and cyclin C can regulate transcription via phosphorylation of cyclin H and the carboxy-terminal domain of Pol II. OrfA in transfected HeLa cells was found to colocalize and copurify with hyperphosphorylated forms of Pol II (Pol IIO) in IGCs, and OrfA was coimmunoprecipitated from lysates of transfected cells with an antibody against Pol IIO. Likewise, Pol IIO could be coprecipitated with an antibody against OrfA. A survey with antibodies against several different cdks resulted in coimmunoprecipitation of OrfA with anti-cdk8, and antiserum against OrfA was able to coprecipitate cdk8 from lysates of cells that express OrfA. Coprecipitation of OrfA with anti-cyclin C demonstrated that it was included in complexes with OrfA and cdk8. OrfA has sequence and structural similarities to cyclin C, and, functionally, OrfA appears to have the capacity to both enhance and inhibit the activity of promoters in a cell-specific manner, similar to functions of the mediator complex. These data suggest that WDSV OrfA functions through its interactions with these large, transcription complexes. Further investigations will clarify the role of the retrovirus cyclin in control of virus expression and transformation.

3T3 Cells↗

PubMed2006

Pathogenesis of B-cell superantigen-induced immune complex-mediated inflammation.

Staphylococcal protein A (SpA) is representative of a new class of antigens, the B-cell superantigens (SAgs). These antigens bind to the Fab regions of immunoglobulin molecules outside their complementarity-determining regions. SpA, the best-studied B-cell SAg, reacts with the Fabs of most VH3+ immunoglobulins, which are expressed on 30 to 60% of human peripheral B cells. Therefore, B-cell SAgs like SpA have great potential to elicit inflammatory responses in vivo. We previously reported that the interaction of SpA with VH3+ immunoglobulin molecules leads to activation of the complement cascade and produces a histologic pattern of inflammation in the skin of a rabbit indicative of immune complex injury. To elucidate the cellular and molecular events contributing to this type of unconventional immune complex-mediated inflammation, we established a mouse peritoneal Arthus reaction model. Mice treated intravenously with human polyclonal immunoglobulin G (IgG), followed by intraperitoneal injection of SpA, showed neutrophil influx into the peritoneal cavity with peak numbers appearing at 8 h. This inflammatory reaction was dependent on the interaction of SpA with VH3+ IgG. Mast cells, FcgammaRIII, complement components, and tumor necrosis factor alpha play obligatory roles, and the reaction is associated with the local release of the CXC chemokines macrophage inflammatory protein 2 and KC. The data provide further compelling evidence for the induction of immune complex-mediated injury by a B-cell SAg and highlight important factors contributing to the pathogenesis of this novel type of inflammatory reaction.

Animals↗