scholarly journals Interaction of heparin with fibronectin and isolated fibronectin domains

1990 ◽  
Vol 272 (3) ◽  
pp. 605-611 ◽  
Author(s):  
K C Ingham ◽  
S A Brew ◽  
D H Atha
Keyword(s):  
Spatial Distribution ◽  
Heavy Chain ◽  
Broad Spectrum ◽  
Binding Sites ◽  
Fluid Phase ◽  
Phase Interaction ◽  
Plasma Fibronectin ◽  
Lower Affinity ◽  
Exclusion Chromatography ◽  
Fibronectin Domains

Fluorescence polarization, gel exclusion chromatography and affinity chromatography were used to characterize the interaction of heparins of different size with human plasma fibronectin (Fn) and several of its isolated domains. The fluid-phase interaction of Fn with heparin was dominated by the 30 kDa and 40 kDa Hep-2 domains located near the C-terminal ends of the A and B chains respectively. The 30 kDa Hep-2A domain from the heavy chain was indistinguishable from the 40 kDa Hep-2B domain in this respect; the presence of an additional type III homology unit in the latter had no effect on the binding. Evidence was provided that each Hep-2 domain has two binding sites for heparin. The N-terminal Hep-1 domain reacted weakly in fluid phase even though it binds strongly to immobilized heparin. Fn and Hep-2 fragments were rather undiscriminating in their reaction with fluoresceinamine-labelled heparins of different sizes. However, oligosaccharides smaller than the tetradecasaccharide (14-mer) bound Fn with a 5-10-fold lower affinity. These results suggest that the Hep-2 domains of Fn are able to recognize a broad spectrum of oligosaccharides that presumably vary significantly with respect to the amount and spatial distribution of charge.

scholarly journals Fibronectin binding to complement subcomponent C1q. Localization of their respective binding sites

1985 ◽  
Vol 226 (1) ◽  
pp. 207-215 ◽  
Author(s):  
J Sorvillo ◽  
I Gigli ◽  
E Pearlstein
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Binding Sites ◽  
Solid Phase ◽  
Fluid Phase ◽  
Globular Domain ◽  
Plasma Fibronectin ◽  
Fibronectin Binding ◽  
Inhibition Studies ◽  
Radiobinding Assay

The interaction of purified human plasma fibronectin with the C1q subcomponent of complement was investigated by using a solid-phase radiobinding assay. 125I-fibronectin binding to native C1q, purified collagen domain (C1q-c) or globular domain (C1q-g) was compared. When the purified domains were insolubilized by binding to plastic, the C1q-c exhibited 59% of the binding demonstrated with intact C1q, whereas the C1q-g exhibited 35% of the binding. N-Terminal sequencing of the globular domain showed that a sequence of seven collagen-like amino acids was retained on each chain of the C1q-g fragment. 125I-fibronectin binding to C1q could be inhibited equally well by fluid-phase C1q and C1q-c, but not by fluid-phase C1q-g, implying that the collagen-like region retained on the C1q-g is masked in the fluid phase. In addition, studies were performed to determine which subunit(s) of C1q bind(s) fibronectin. The percentages of fibronectin bound by the A, B, and C chain of C1q were found to be 38, 21 and 41% respectively. Inhibition studies with purified 200-180 kDa, 50 kDa or 29 kDa fragments of fibronectin show that the binding site on fibronectin for C1q is the 50 kDa gelatin-binding domain.

scholarly journals Tissue-Restricted Expression of the Cardiac α-Myosin Heavy Chain Gene Is Controlled by a Downstream Repressor Element Containing a Palindrome of Two Ets-Binding Sites

1998 ◽  
Vol 18 (12) ◽  
pp. 7243-7258 ◽  
Author(s):  
Madhu Gupta ◽  
Radovan Zak ◽  
Towia A. Libermann ◽  
Mahesh P. Gupta
Keyword(s):  
Gene Regulation ◽  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Binding Sites ◽  
Cardiac Myocyte ◽  
Specific Expression ◽  
Tissue Specific Expression ◽  
Nonmuscle Cells ◽  
Muscle Gene

ABSTRACT The expression of the α-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in α-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac α-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the α-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the α-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the α-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the α-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the α-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the α-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the α-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.

scholarly journals Determination of Phase-Eigenvalues by Rational Factorization and Enhanced Simulation of Two-Phase Mass Flow

2019 ◽  
Vol 2 (2) ◽  
pp. 61-77
Author(s):  
Puskar R. Pokhrel ◽  
Bhadra Man Tuladhar
Keyword(s):  
Fluid Dynamics ◽  
Debris Flows ◽  
Lateral Pressure ◽  
Solid Phase ◽  
Fluid Phase ◽  
Factorization Method ◽  
Phase Interaction ◽  
Two Phase ◽  
Simulation Results

In this paper, we present simple and exact eigenvalues for both the solid- and fluid-phases of the real two-phase general model developed by Pudasaini (2012); we call these phase-eigenvalues, the solid- phase-eigenvalues and the fluid-phase-eigenvalues. Results are compared by applying the derived phase- eigenvalues that incorporate the phase-interactions in the two-phase debris movements against the simple and classical solid and fluid eigenvalues without any phase interaction. We have constructed several different set of eigenvalues including the coupled phase eigenvalues by using rational factorization method. At first, we consider for general debris height; factorizing the solid and fluid lateral pressure contributions by considering the negligible pressure gradient; negligible solid lateral pressure; negligible fluid lateral pressure; negligible solid and fluid lateral pressure. Secondly, for a thin debris ow height, we also construct the fourth set of eigenvalues in three different cases. These phase-eigenvalues incorporate strong interaction between the solid and fluid dynamics. The simulation results are produced by taking all these different sets of coupled phase-eigenvalues and are compared with the classical uncoupled set of solid and fluid eigenvalues. The results indicate the importance of phase-eigenvalues and supports for a complete description of the phase- eigenvalues for the enhanced description of real two-phase debris flows and landslide motions.

scholarly journals Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene.

1994 ◽  
Vol 14 (7) ◽  
pp. 4947-4957 ◽  
Author(s):  
J D Molkentin ◽  
D V Kalvakolanu ◽  
B E Markham
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Retinoic Acid ◽  
Cardiac Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Binding Sites ◽  
Ectopic Expression ◽  
Specific Expression ◽  
Cardiac Phenotype

The alpha-myosin heavy-chain (alpha-MHC) gene is the major structural protein in the adult rodent myocardium. Its expression is restricted to the heart by a complex interplay of trans-acting factors and their cis-acting sites. However, to date, the factors that have been shown to regulate expression of this gene have also been found in skeletal muscle cells. Recently, transcription factor GATA-4, which has a tissue distribution limited to the heart and endodermally derived tissues, was identified. We recently found two putative GATA-binding sites within the proximal enhancer of the alpha-MHC gene, suggesting that GATA-4 might regulate its expression. In this study, we establish that GATA-4 interacts with the alpha-MHC GATA sites to stimulate cardiac muscle-specific expression. Mutation of the GATA-4-binding sites either individually or together decreased activity by 50 and 88% in the adult myocardium, respectively. GATA-4-dependent enhancement of activity from a heterologous promoter was mediated through the alpha-MHC GATA sites. Coinjection of an alpha-MHC promoter construct with a GATA-4 expression vector permitted ectopic expression in skeletal muscle but not in fibroblasts. Thus, the lack of alpha-MHC expression in skeletal muscle correlates with a lack of GATA-4. GATA-4 DNA binding activity was significantly up-regulated in triiodothyronine- or retinoic acid-treated cardiomyocytes. Putative GATA-4-binding sites are also found in the regulatory regions of other cardiac muscle-expressed structural genes. This indicates a mechanism whereby triiodothyronine and retinoic acid can exert coordinate control of the cardiac phenotype through a trans-acting regulatory factor.

scholarly journals Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain

1974 ◽  
Vol 139 (1) ◽  
pp. 135-149 ◽  
Author(s):  
Christopher E. Fisher ◽  
Elizabeth M. Press
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Binding Sites ◽  
Light Chains ◽  
Rabbit Antibody ◽  
Heavy Chains ◽  
Group 4 ◽  
Hypervariable Regions ◽  
Affinity Labelling ◽  
Antibody Complex

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten–antibody complex. The extent of covalent labelling was 0.5–0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3–5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29–34 and 95–114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.

scholarly journals Effect of hepatocyte-stimulating factor and glucocorticoids on plasma fibronectin levels

1986 ◽  
Vol 238 (2) ◽  
pp. 365-371 ◽  
Author(s):  
D L Amrani ◽  
D Mauzy-Melitz ◽  
M W Mosesson
Keyword(s):  
Acute Phase Protein ◽  
Plasma Fibronectin ◽  
Phagocytic Cells ◽  
Serum Samples ◽  
Exclusion Chromatography ◽  
A Minor ◽  
Two Peaks ◽  
Stimulating Factor

We evaluated the effects of hepatocyte-stimulating factor (HSF) and a glucocorticoid (dexamethasone) on changes in the levels, in vivo and in vitro, of plasma fibronectin (Fn), a glycoprotein that is synthesized and secreted by hepatocytes. In turpentine-treated chickens, plasma levels of Fn, which peaked at 48 h (whereas fibrinogen levels were maximum at 72 h) rose 200-250% over basal levels, whereas albumin levels decreased by 20-40%. Corticosterone levels in serum samples taken between 5 and 48 h after injection revealed a 124% increase in hormone levels at 24 h in turpentine-treated chickens. We also showed that circulating HSF levels were maximal 8 to 12 h after injection and that HSF activity, as assessed by molecular-exclusion chromatography, was eluted in the 30-45 kDa range. Addition of either serum-derived HSF or dexamethasone (2 nM) to chick hepatocyte cultures resulted in a 130-150% increase in secreted Fn as well as in fibrinogen. When HSF and dexamethasone were added together, a 360-489% increase in the secreted levels of both proteins was found. Chicken mononuclear phagocytic cells treated with lipopolysaccharide secreted an HSF activity that was eluted in two peaks, a minor peak at approximately 70 kDa and a major peak in the 25-40 kDa range. Addition of mononuclear-cell-derived HSF resulted in a greater increase in Fn levels than did the addition of serum HSF. These findings indicate that Fn, like fibrinogen, is an acute-phase protein, the production of which, at least in chickens, is stimulated by HSF and glucocorticoids in an additive manner.

Corticosterone's metabolite is an agonist for Na+ transport stimulation in A6 cells

1988 ◽  
Vol 255 (4) ◽  
pp. F736-F748 ◽  
Author(s):  
R. L. Duncan ◽  
W. M. Grogan ◽  
L. B. Kramer ◽  
C. O. Watlington
Keyword(s):  
Binding Sites ◽  
Short Circuit ◽  
Type I ◽  
Type Ii ◽  
Na Transport ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Lower Affinity ◽  
A6 Cells ◽  
Nuclear Binding

This study tests the hypothesis, in A6 epithelia, that 1) corticosterone stimulates active Na+ transport (short-circuit current, Isc) by an additional receptor mechanism to the type I (mineralocorticoid) and type II (glucocorticoid) mechanisms shared with aldosterone (Aldo) and 2) that the agonist may be 6 beta-OH-corticosterone made in the effector cell. The dose-response relationship of corticosterone at 24 h resolves into two components, by curve fitting, with a 50% effective concentration (EC50) for 10% of maximum Isc stimulation of 2 X 10(-9) M and an EC50 for the other 90% of 3 X 10(-7) M. The EC50 of the smaller component correlates with the apparent dissociation constant (K'd) of corticosterone for high affinity (type II) nuclear binding sites shared with Aldo. In unlabeled analogue competition studies Aldo and corticosterone displaced nuclear binding equally below 10(-8) M [3H]corticosterone, indicating only shared sites. However, nonshared saturable sites (displaced by corticosterone but not by Aldo) were found at [3H]-corticosterone concentrations above 10(-8) M. Concentration-binding curves performed with [3H]corticosterone, in presence of 1,000 X Aldo to displace shared sites, revealed a single class of binding sites with a half-maximal saturation of 2 X 10(-7) M, which is quite similar to the EC50 of the lower affinity component of Isc stimulation by corticosterone at 24 h. Reversed phase high-pressure liquid chromatography of nuclear extracts indicates that the saturable component of bound [3H] was 6 beta-OH-[3H]corticosterone derived from [3H]corticosterone. Thus, A6 cells metabolize corticosterone to 6 beta-OH-corticosterone, which in turn occupies lower-affinity receptors not shared with Aldo or corticosterone, to mediate most of the active Na+ transport stimulation by corticosterone.

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