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At least 307 records · Page 17Linked to original sources
PubMed2006

Smad-Sp1 complexes mediate TGFbeta-induced early transcription of oncogenic Smad7 in pancreatic cancer cells.

The transcription factor Sp1 has been implicated in cell-type-specific activation of transforming growth factor-beta (TGFbeta) target genes in normal epithelial cells as well as in aberrant gene activation by TGFbeta in epithelial tumor cells. Here, we have examined the interaction of Sp1 with components of the Smad signaling cascade and its role in TGFbeta-induced early gene expression in pancreatic cancer cells. Gene expression profiling was carried out in mithramycin-A-treated cells to identify Sp1-regulated TGFbeta early response genes. We found that in pancreatic cancer cells Smad proteins and Sp1 cooperatively regulate expression of a distinct set of TGFbeta target genes potentially involved in tumor progression, including MMP-11, cyclin D1 and Smad7. Mechanistically, TGFbeta rapidly induces nuclear translocation of Smad proteins and subsequently stimulates Smad-Sp1 complex formation. Using the Smad7 promoter as a model for Smad-/Sp1-induced early gene activation, we demonstrated that this interaction increases Sp1 binding to GC-rich promoter boxes and results in superinduction of Sp1-mediated transcription. Moreover, inhibition of Sp1-DNA binding or transfection of Sp1-specific siRNA prevents TGFbeta-induced Smad7 expression and consequently enhances Smad signaling in pancreatic cancer cells, as indicated by increased receptor-mediated phosphorylation of Smad3. We thus conclude that Sp1 strongly contributes to the aberrant transcriptional response of transformed epithelial cells to TGFbeta stimulation.

Base Sequence↗
PubMed1991

Azathioprine reduces extravasation and neutrophil trafficking in immune complex-mediated inflammation in the rat colon.

Azathioprine (AZ) has been used in the treatment of refractory inflammatory bowel disease. The mechanism by which AZ decrease colonic inflammation is not known. It is alluded that AZ may be effective in the maintenance of remission. We examined whether AZ in non-immunosuppressive doses reduces extravasation and neutrophil trafficking in a rat model of colonic inflammation. Rats were treated with I.P. injection of AZ (1 mg/kg) for 6 weeks. At the end of 2 and 6 weeks rats were injected I.V. immune complex and on the following day the proximal colon was perfused with 2.5% formaldehyde (local irritant 3 ml/hour for 5 mins). Extravasation was measured by Evans' blue technique and neutrophil concentration in the tissue was determined by measuring myeloperoxidase (MPO). AZ did not inhibit extravasation and MPO after 2 weeks of therapy. However, after 6 weeks, AZ reduced extravasation to 20 +/- 2 micrograms/gm compared to untreated animals (51 +/- 6 micrograms/gm tissue) and MPO levels to 0.3 +/- 13 compared to untreated rats (0.8 +/- 0.32 mU/gm). There was a good correlation between extravasation and MPO levels. These results suggest that long-term treatment with AZ may prevent extravasation and cause reduction in neutrophil trafficking. Such an effect may be beneficial for maintaining remission in IBD.

Aminosalicylic Acids↗
PubMed1989

A nucleoprotein complex mediates the integration of retroviral DNA.

The integration of viral DNA into the host genome is an essential step in the retrovirus life cycle. To understand this process better, we have examined the native state of viral DNA in cells acutely infected by murine leukemia virus (MLV), using both a physical assay for viral DNA and a functional assay for integration activity (Brown et al. 1987). The viral DNA and integration activity copurify during velocity sedimentation, gel filtration, and density equilibrium centrifugation, indicating that viral DNA is in a large (approximately 160S) nucleoprotein complex that includes all functions required for integration activity in vitro. Analysis by immunoprecipitation shows that the viral capsid protein is part of the active nucleoprotein complex, but recognition of the complex by only a subset of anti-capsid sera implies that the protein is constrained conformationally. The viral DNA within this structure is accessible to nucleases; the effects of nucleases on the integrity of the complex suggest that the integration-competent particle is derived from and similar to the core of extracellular virions.

Animals↗
PubMed1990

Koebnerization as a cutaneous manifestation of immune complex-mediated vasculitis.

Two unusual examples of the cutaneous manifestations of vasculitis are presented. In both cases lesions occurred on previously traumatized skin and on normal skin of the dependent areas. Lesional skin biopsy specimens obtained from the koebnerized sites and from the other dependent sites revealed evidence of vascular injury in both patients. A diagnosis of leukocytoclastic vasculitis was made in one patient and pityriasis lichenoides et varioliformis acuta in the other. Direct immunofluorescence microscopy of lesional skin specimens from both patients demonstrated dermal vascular immune deposits. Raji cell assay detected a significant elevation of circulating immune complexes in the serum of both patients. Neither koebnerizing leukocytoclastic vasculitis nor koebnerizing pityriasis lichenoides et varioliformis acuta has been reported previously.

Adult↗
PubMed1997

Molecular Recognition Effects in Metal Complex Mediated Double-Strand Cleavage of DNA: Reactivity and Binding Studies with Model Substrates.

Double-strand breaks in duplex DNA are thought to be significant sources of cell lethality because they appear to be less readily repaired by DNA repair mechanisms. We recently described the design and cleavage chemistry of ((2S,8R)-5-amino-2,8-dibenzyl-5-methyl-3,7-diazanonanedioato)copper(II) (1), which effects nonrandom double-strand cleavage of duplex DNA. After DNA nicking by generation of hydroxyl radicals, the key step in this process appears to occur through recognition by the metal complex of the nicked-abasic site on duplex DNA, followed by delivery of OH(*) to cleave at the opposing strand, forming a double-strand lesion. Through the use of model nucleic acid substrates and comparison to DNA scission chemistry, we have investigated the electrostatic and hydrophobic contributions to DNA binding by complex 1. We have complemented these reactivity studies with studies on the binding of 1 to a model nucleic acid substrate, using (2)H NMR spectroscopy with deuterated 1 and HDO T(1)()()relaxation enhancement methods to study the binding of 1 to nucleotide substrates. With these methods, we have estimated that the association constant for the 1(+).5'-AMP(2)(-) complex is approximately 16 M(-)(1) and that the binding interaction involves both electrostatic and aromatic stacking interactions between the nucleic acid base and the pendant aromatic side chains of 1.

Journal Article↗
PubMed2006

A Ferrous-Triapine complex mediates formation of reactive oxygen species that inactivate human ribonucleotide reductase.

Ribonucleotide reductase plays a central role in cell proliferation by supplying deoxyribonucleotide precursors for DNA synthesis and repair. The holoenzyme is a protein tetramer that features two large (hRRM1) and two small (hRRM2 or p53R2) subunits. The small subunit contains a di-iron cluster/tyrosyl radical cofactor that is essential for enzyme activity. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a new, potent ribonucleotide reductase inhibitor currently in phase II clinical trials for cancer chemotherapy. Ferric chloride readily reacts with Triapine to form an Fe(III)-(3-AP) complex, which is reduced to Fe(II)-(3-AP) by DTT. Spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide prove that Fe(II)-(3-AP) reduces O2 to give oxygen reactive species (ROS). In vitro activity assays show that Fe(II)-(3-AP) is a much more potent inhibitor of hRRM2/hRRM1 and p53R2/hRRM1 than Triapine. Electron paramagnetic resonance measurements on frozen solutions of hRRM2 and p53R2 show that their tyrosyl radicals are completely quenched by incubation with Fe(II)-(3-AP). However, the enzyme activity is maintained in protein samples supplemented with catalase alone or in combination with superoxide dismutase. Furthermore, catalase alone or in combination with superoxide dismutase markedly decreases the antiproliferative effect of Triapine in cytotoxicity assays. These results indicate that Triapine-induced inhibition of ribonucleotide reductase is caused by ROS. We suggest that ROS may ultimately be responsible for the pharmacologic effects of Triapine in vivo.

Cell Line, Tumor↗
PubMed2004

A multicomponent protein complex mediates environmental stress signaling in Bacillus subtilis.

Activity of the general stress transcription factor sigma(B) of Bacillus subtilis is regulated directly by a partner-switching mechanism in which key protein interactions are governed by serine phosphorylation. Signals of energy or environmental stress are conveyed to sigma(B) by independent pathways, each terminating with a differentially regulated serine phosphatase, whose activity is required to control the partner-switching regulators. We present genetic and biochemical evidence that activation of the RsbU environmental signaling phosphatase is modulated by a second, atypical partner switch that comprises redundant negative regulatory proteins in a large, multicomponent signaling complex. In the current model, negative regulation of the RsbU phosphatase depends solely on the RsbS antagonist protein. Here, we perform a critical genetic test that invalidates this model and demonstrates that the RsbS antagonist alone is insufficient to prevent environmental signaling. Also required is one of a family of four co-antagonist proteins, here renamed RsbRA, RsbRB, RsbRC, and RsbRD, each with a carboxyl-terminal domain closely resembling the entire RsbS protein. Because any single member of the RsbR family, together with RsbS, was sufficient for environmental signaling, we conclude that the RsbR proteins serve as redundant co-antagonists necessary for RsbS antagonist function. Moreover, purification of RsbRA from cell extracts by nickel affinity and gel-filtration chromatography found a multicomponent complex containing the RsbRA and RsbRB co-antagonists together with the RsbS antagonist. We propose that this complex serves as a machine to transmit stress signals to sigma(B), and that the properties of the complex may contribute to environmental stress sensing.

Amino Acid Substitution↗
PubMed1982

Concurrent antiglomerular basement membrane antibody and immune complex mediated glomerulonephritis.

A teenage male, with Goodpasture's syndrome and serum antiglomerular basement membrane (anti-GBM) antibodies, had a focal proliferative glomerulonephritis with crescents. Immunofluorescence microscopy of his glomeruli using anti-IgG antibodies demonstrated both intense linear GBM staining, and granular subepithelial staining. Electron microscopy revealed numerous subepithelial electron-dense deposits. Identical IgG subclass restriction (dominance of IgG1 and IgG4) of both types of glomerular deposits in this patient supports, but does not prove, a postulate that the linear staining was due to anti-GBM antibodies bound to intact GBM, and that the granular staining was due to anti-GBM antibodies complexed with freed GBM antigens.

Adolescent↗
PubMed1987

On the formation of a dicloxacillin-p-hydroxymercuribenzoate suicide complex mediated by beta-lactamase I from Bacillus cereus.

p-Hydroxymercuribenzoate is a non-competitive inhibitor of beta-lactamase I from Bacillus cereus and also, after preliminary preincubation, an inactivator of the enzyme. Submitted to the simultaneous action of PCMB plus dicloxacillin, the enzyme completely loses its activity. Extensive dialysis can restore the enzymatic activity only if preincubation had been carried out with either PCMB or dicloxacillin but not if both inhibitors had been simultaneously present. Mercaptoethanol protects the enzyme from the action of PCMB, but not from the severe inactivation caused by dicloxacillin-PCMB mixtures. All these data suggest the formation of a complex between PCMB and the acyl-enzyme intermediate generated upon hydrolysis of the beta-lactam bond of dicloxacillin.

Bacillus cereus↗
PubMed1999

Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation.

The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system. This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well. As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery. Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon. In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded. This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system. When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in Delta(hrd3) cells. Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules.

Amino Acid Sequence↗
PubMed2000

Plexin/neuropilin complexes mediate repulsion by the axonal guidance signal semaphorin 3A.

In the developing nervous system axons navigate with great precision over large distances to reach their target areas. Chemorepulsive signals such as the semaphorins play an essential role in this process. The effects of one of these repulsive cues, semaphorin 3A (Sema3A), are mediated by the membrane protein neuropilin-1 (Npn-1). Recent work has shown that neuropilin-1 is essential but not sufficient to form functional Sema3A receptors and indicates that additional components are required to transduce signals from the cell surface to the cytoskeleton. Here we show that members of the plexin family interact with the neuropilins and act as co-receptors for Sema3A. Neuropilin/plexin interaction restricts the binding specificity of neuropilin-1 and allows the receptor complex to discriminate between two different semaphorins. Deletion of the highly conserved cytoplasmic domain of Plexin-A1 or -A2 creates a dominant negative Sema3A receptor that renders sensory axons resistant to the repulsive effects of Sema3A when expressed in sensory ganglia. These data suggest that functional semaphorin receptors contain plexins as signal-transducing and neuropilins as ligand-binding subunits.

Animals↗
PubMedDate not supplied

'Immune complex' mediated intravascular hemolysis due to IgM cephalosporin-dependent antibody.

Immune hemolytic anemia (IHA) related to cephalosporins is rare and generally considered to be the result of a drug-adsorption mechanism. In previously reported cases, the hemolysis was usually extravascular and the causative antibodies were IgG, incapable of activating complement, and demonstrable by the direct or indirect antiglobulin test using red cells (RBCs) pretreated in vitro with cephalosporin. The authors report a patient in whom acute intravascular hemolysis developed while she was receiving cefotaxime (a cephalosporin as yet not reported to cause IHA). The patient's RBCs were coated only with complement fragments (C3d), even at the peak of the hemolytic episode. Her serum and eluates repeatedly yielded negative results when tested against normal or cephalosporin-coated RBCs. However, strong hemagglutination and C5b-9-mediated hemolysis were observed if the patient's serum was tested against RBCs in the presence of the drug, its ex vivo antigen and, to a lesser degree, cephalothin and ceftriaxon, but not in the presence of penicillin and other related cephalosporins. The positive reactions were not changed by preincubating the serum with different amounts of the drugs. All of these findings reflect the typical picture of drug-induced IHA by the so-called "immune complex" mechanism and not by the drug-adsorption mechanism. The authors conclude that cephalosporin can cause immune hemolysis in two ways: the drug-adsorption mechanism and, as described here, the "immune complex" mechanism.

Aged↗
PubMed2004

Metal-protein complex-mediated transport and delivery of Ni2+ to TCR/MHC contact sites in nickel-specific human T cell activation.

Nickel allergy clearly involves the activation of HLA-restricted, skin-homing, Ni-specific T cells by professional APCs. Nevertheless, knowledge concerning the molecular details of metal-protein interactions underlying the transport and delivery of metal ions to APC during the early sensitization phase and their interactions with HLA and TCRs is still fragmentary. This study investigates the role of human serum albumin (HSA), a known shuttling molecule for Ni(2+) and an often-disregarded, major component of skin, in these processes. We show that Ni-saturated HSA complexes (HSA-Ni) induce and activate Ni-specific human T cells as potently as Ni salt solutions when present at equimolar concentrations classically used for in vitro T cell stimulation. However, neither HSA itself nor its Ni-binding N-terminal peptide are involved in determining the specificity of antigenic determinants. In fact, HSA could be replaced by xenogeneic albumins exhibiting sufficient affinity for Ni(2+) as determined by surface plasmon resonance (Biacore technology) or atomic absorption spectroscopy. Moreover, despite rapid internalization of HSA-Ni by APC, it was not processed into HLA-associated epitopes recognizable by Ni-specific T cells. In contrast, the presence of HSA-Ni in the vicinity of transient contacts between TCR and APC-exposed HLA molecules appeared to facilitate a specific transfer of Ni(2+) from HSA to high-affinity coordination sites created at the TCR/HLA-interface.

Antigen-Presenting Cells↗
PubMed2004

PAF1-complex-mediated histone methylation of FLOWERING LOCUS C chromatin is required for the vernalization-responsive, winter-annual habit in Arabidopsis.

The winter-annual habit (which typically involves a requirement for exposure to the cold of winter to flower in the spring) in Arabidopsis thaliana is mainly due to the repression of flowering by relatively high levels of FLC expression. Exposure to prolonged cold attenuates FLC expression through a process known as vernalization and thus permits flowering to occur in the spring. Here we show that the elevated FLC expression characteristic of nonvernalized winter annuals requires two genes, EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING 8 (ELF8), that are homologs of components of the PAF1 complex of Saccharomyces cerevisiae. Furthermore, ELF7 and ELF8 are also required for the expression of other genes in the FLC clade of flowering repressors such as MAF2 and FLM. FLC, FLM, and MAF2 are involved in multiple flowering pathways that account for the broad effects of elf7 and elf8 mutations on flowering behavior. ELF7 and ELF8 are required for the enhancement of histone 3 trimethylation at Lys 4 in FLC chromatin. This modification of FLC chromatin appears to be required to elevate FLC expression to levels that can delay flowering in plants that have not been vernalized. A model of the role of ELF7, ELF8, and other previously described genes in the modification of the chromatin of flowering repressors is presented.

Acclimatization↗
PubMed2005

Interaction of SPIN90 with the Arp2/3 complex mediates lamellipodia and actin comet tail formation.

The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.

Actin Cytoskeleton↗
PubMed1989

The leucine repeat motif in Fos protein mediates complex formation with Jun/AP-1 and is required for transformation.

Cellular and viral Fos proteins form a tight complex with other nuclear proteins, including the transcription factor and proto-oncogene AP-1/Jun. We have mapped the c-Jun binding site in Fos to a region containing regularly spaced leucine residues recently suggested to interdigitate with a similar structure in Jun. Substitution of single or multiple leucine residues or the alteration of leucine phasing by insertion of additional amino acids reduces or abolishes the binding to Jun, while the substitution of other amino acids has no noticeable effect. These results strongly suggest that the formation of a "leucine zipper" mediates the interaction between Fos and Jun. We also show that the differential binding of the various Fos mutants correlates with their potential to trans-activate AP-1-dependent transcription and to induce morphological transformation.

Animals↗
PubMed2005

Visible-light-induced photocatalytic degradation of 4-chlorophenol and phenolic compounds in aqueous suspension of pure titania: demonstrating the existence of a surface-complex-mediated path.

The visible-light-induced degradation reaction of 4-chlorophenol (4-CP) was investigated in aqueous suspension of pure TiO2. Contrary to common expectations, 4-CP could be degraded under visible illumination (lambda > 420 nm), generating chlorides and CO2 concomitantly. The observed visible reactivity was not due to the presence of trace UV light since the visible-light-induced reactions exhibited behaviors distinguished from those of UV-induced reactions. Dichloroacetate could not be degraded under visible light, whereas it degraded with a much faster rate than 4-CP under UV irradiation. The addition of tert-butyl alcohol, a common OH radical scavenger, did not affect the visible reactivity of 4-CP, which indicates that OH radicals are not involved. Other phenolic compounds such as phenol and 2,4-dichlorophenol were similarly degraded under visible light. The surface complexation between phenolic compounds and TiO2 appears to be responsible for the visible light reactivity. Diffuse reflectance UV-vis spectra showed that 4-CP adsorbed on TiO2 powder induced visible light absorption. The visible light reactivity among several TiO2 samples was apparently correlated with the surface area of TiO2. The visible-light-induced photocurrents on a TiO2 electrode could be obtained only in the presence of 4-CP. It is proposed that a direct electron transfer from surface-complexed phenol to the conduction band of TiO2 upon absorbing visible light (through ligand-to-metal charge transfer) initiates the oxidative degradation of phenolic compounds. When the surface complex formation was hindered by surface fluorination, surface platinization, and high pH, the visible-light-induced degradation of 4-CP was inhibited. The evidence of visible-light-induced reactions and the experimental conditions affecting the visible reactivity were discussed in detail.

Journal Article↗
PubMed1984

Acute immune complex mediated glomerulonephritis in a Chinese girl with Wiskott-Aldrich syndrome variant.

This study presents a 12-year-old girl with Wiskott-Aldrich syndrome variant, who developed acute glomerulonephritis without history of transfer factor therapy and the efficacy of splenectomy for the control of the patient's thrombocytopenia. The patient presented with eczema, severe thrombocytopenia and immunodeficiency. The impaired immunity was featured by impaired delayed hypersensitivity and lymphoproliferative response to nonspecific mitogen, low serum IgM, low isohaemagglutinins, recurrent infections and high IgE. She developed hematuria about one month prior to admission. For her severe thrombocytopenia splenectomy was performed and proved to be effective. At the time of splenectomy, renal biopsy was done and showed proliferative glomerulonephritis with coarse granular deposition of immunoglobulins (IgA and IgM) and C3. Electron microscopy demonstrated granular electron-dense deposits in the glomerulus, indicating an immune complex glomerulonephritis.

Antibody Formation↗