Physical interaction between the MADS box of serum response factor and the TEA/ATTS DNA-binding domain of transcription enhancer factor-1.
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Serum response factor is a MADS box transcription factor that binds to consensus sequences CC(A/T)(6)GG found in the promoter region of several serum-inducible and muscle-specific genes. In skeletal myocytes serum response factor (SRF) has been shown to heterodimerize with the myogenic basic helix-loop-helix family of factors, related to MyoD, for control of muscle gene regulation. Here we report that SRF binds to another myogenic factor, TEF-1, that has been implicated in the regulation of a variety of cardiac muscle genes. By using different biochemical assays such as affinity precipitation of protein, GST-pulldown assay, and coimmunoprecipitation of proteins, we show that SRF binds to TEF-1 both in in vitro and in vivo assay conditions. A strong interaction of SRF with TEF-1 was seen even when one protein was denatured and immobilized on nitrocellulose membrane, indicating a direct and stable interaction between SRF and TEF-1, which occurs without a cofactor. This interaction is mediated through the C-terminal subdomain of MADS box of SRF encompassing amino acids 204-244 and the putative 2nd and 3rd alpha-helix/beta-sheet configuration of the TEA/ATTS DNA-binding domain of TEF-1. In the transient transfection assay, a positive cooperative effect of SRF and TEF-1 was observed when DNA-binding sites for both factors, serum response element and M-CAT respectively, were intact; mutation of either site abolished their synergistic effect. Similarly, an SRF mutant, SRFpm-1, defective in DNA binding failed to collaborate with TEF-1 for gene regulation, indicating that the synergistic trans-activation function of SRF and TEF-1 occurs via their binding to cognate DNA-binding sites. Our results demonstrate a novel association between SRF and TEF-1 for cardiac muscle gene regulation and disclose a general mechanism by which these two super families of factors are likely to control diversified biological functions. (+info)
Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera.
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Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79-63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection. (+info)
Anti-neutrophil cytoplasmic antibodies (ANCA) from patients with systemic vasculitis recognize restricted epitopes of proteinase 3 involving the catalytic site.
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ANCA with specificity for proteinase 3 (PR3), a neutrophil primary granule enzyme, are of diagnostic value in Wegener's granulomatosis (WG) and certain other forms of systemic vasculitis. There is evidence to suggest that they play a pathogenic role in disease, and that the interaction of ANCA with PR3 is likely to be important. We showed, using a resonant mirror biosensor, that C-ANCA from different patients recognized the same or closely related epitopes on PR3. Studies using linear peptides in the SPOT system confirmed the highly restricted nature of this interaction and identified five linear epitopes. Fluid-phase inhibition studies, using a different set of peptides, validated the sequences involved. Using a computer-generated model of the structure of PR3, four of five epitopes were shown to be intimately linked with the catalytic site. The restricted number of epitopes, and their location at the catalytic site, has important implications for the role of C-ANCA in the pathogenesis of vasculitis. (+info)
Application of the transition state theory to water transport across cell membranes.
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We have applied the transition state theory of Eyring et al. (The Theory of Rate Processes, McGraw-Hill, 1941) to water transport across cell membranes. We have then evaluated free energy (Delta F(not equal)), enthalpy (Delta H(not equal)) and entropy (Delta S(not equal)) of activation for water permeation across membranes, such as Arbacia eggs, Xenopus oocytes with or without aquaporin water channels, mammalian erythrocytes, aquaporin proteoliposomes, liposomes and collodion membrane. Delta H(not equal) was found to be correlated with Delta S(not equal). This is so-called Delta H(not equal) and Delta S(not equal) compensation over the ranges of Delta H(not equal) and Delta S(not equal) from 2 to 22 kcal/mol and from -26 to 45 e.u., respectively, indicating that low Delta H(not equal) values correspond to negative Delta S(not equal). Large positive Delta S(not equal) and high Delta H(not equal) values might be accompanied by reversible breakage of secondary bonds in the membrane, presumably in membrane lipid bilayer. Largely negative Delta S(not equal) and low Delta H(not equal) values for aquaporin water channels, aquaporin proteoliposomes and porous collodion membrane could be explained by the immobilization of permeating water molecules in the membrane, i.e., the partial loss of rotational and/or translational freedoms of water molecules in water channels. (+info)
A therapeutic target for hormone-independent estrogen receptor-positive breast cancers.
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BACKGROUND: The action of the steroid hormone estradiol (E2) is mediated via interaction with a specific receptor (ER) that initiates a series of events downstream, leading to the modulation of hormone-responsive genes and cell proliferation. Antihormones also bind, but do not confer the active configuration to ER, thereby, blocking the transmission of E2-ER-initiated signals for cell proliferation. Although these compounds qualify for successful therapy of ER-positive [ER (+)] breast cancer patients, only a fraction of patients responds to antihormone treatment. In this study, the functional status of ER is determined to identify alternative targets for therapy of antihormone-resistant ER (+) breast cancers. METHOD: The interaction of ER with a specific DNA sequence, designated as E2 response element (ERE), was targeted to assess the functional state of ER. ER-ERE complex formation was measured by electrophoretic mobility shift assay (EMSA) and by a newly developed technique, based on the preferential binding of DNA-protein complex to a nitrocellulose membrane (NMBA) that measures both total and functional fraction of ER. RESULTS: The NMBA assay identified functional variants of ER among ER (+) breast cancer cell lines and breast tumor biopsy specimens. ER of (21PT) cells did not bind E2 and these cells were tamoxifen (TAM) resistant. However 21PT cells were sensitive to a calmodulin (CaM) antagonist, W7, that blocked ERE-ER complex formation. CONCLUSIONS: ER variants of the 21PT type were detected among breast cancer biopsy specimens, emphasizing the significance of an alternative therapeutic target for TAM-resistant ER (+) human breast cancers with compounds such as W7. (+info)
Localization of microsomal triglyceride transfer protein in the Golgi: possible role in the assembly of chylomicrons.
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Although a critical role of microsomal transfer protein (MTP) has been recognized in the assembly of nascent apolipoprotein B (apoB)-containing lipoproteins, it remains unclear where and how MTP transfers lipids in the secretory pathway during the maturational process of apoB lipidation. The aims of this study were to determine whether MTP functions in the secretory pathway as well as in the endoplasmic reticulum and whether its large 97-kDa subunit interacts with the small 58-kDa protein disulfide isomerase (PDI) subunit and apoB, particularly in the Golgi apparatus. Using a high resolution immunogold approach combined with specific polyclonal antibodies, the large and small subunits of MTP were observed over the rough endoplasmic reticulum and the Golgi. Double immunocytochemical detection unraveled the colocalization of MTP and PDI as well as MTP and apoB in these same subcellular compartments. To confirm the spatial contact of these proteins, Golgi fractions were isolated, homogenized, and incubated with an anti-MTP large subunit antibody. Immunoprecipitates were applied on SDS-PAGE and then transferred on to nitrocellulose. Immunoblotting the membrane with PDI and apoB antibodies confirmed the colocalization of these proteins with MTP. Furthermore, MTP activity assay disclosed a substantial triglyceride transfer in the Golgi fractions. The occurrence of membrane-associated apoB in the Golgi, coupled with its interaction with active MTP, suggests an important role for the Golgi in the biogenesis of apoB-containing lipoproteins. (+info)
Immunostaining of von Willebrand factor multimers on agarose gels and nitrocellulose filters.
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Human von Willebrand factor (VWF) multimeric analysis is commonly performed by agarose gel electrophoresis, electroblotting, and immunoenzymatic staining; however, high molecular weight (HMW) multimers are poorly transferred on nitrocellulose and should be visualized by direct gel staining with radiolabeled anti-VWF antibody and autoradiography or luminography. (+info)
Real-time kinetic analysis of hCG-monoclonal antibody interaction using radiolabeled hCG probe: presence of two forms of Ag-mAb complex as revealed by solid phase dissociation studies.
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Real-time kinetics of ligand-ligate interaction has predominantly been studied by either fluorescence or surface plasmon resonance based methods. Almost all such studies are based on association between the ligand and the ligate. This paper reports our analysis of dissociation data of monoclonal antibody-antigen (hCG) system using radio-iodinated hCG as a probe and nitrocellulose as a solid support to immobilize mAb. The data was analyzed quantitatively for a one-step and a two-step model. The data fits well into the two-step model. We also found that a fraction of what is bound is non-dissociable (tight-binding portion (TBP)). The TBP was neither an artifact of immobilization nor does it interfere with analysis. It was present when the reaction was carried out in homogeneous solution in liquid phase. The rate constants obtained from the two methods were comparable. The work reported here shows that real-time kinetics of other ligand-ligate interaction can be studied using nitrocellulose as a solid support. (+info)