PREFERENTIAL RECOGNITION OF VITRONECTIN (S-PR0TEIN) BY A MONOCLONAL ANTIBODY UPON INTERACTION WITH THROMBIN, ANTITHROMBIN AND GLYCOSAMINOGLYCANS

1987 ◽  
Author(s):  
B R Tomaslni ◽  
D F Mosher
Keyword(s):  
Monoclonal Antibody ◽  
Conformational Change ◽  
Membrane Attack Complex ◽  
Physiological Role ◽  
Keratan Sulfate ◽  
Fold Increase ◽  
Antigenic Determinants ◽  
Preferential Recognition ◽  
Relative Potencies

V1tronect1n/S-Prote1n (VN/SP) is a glycoprotein present at a concentration of 200-400 ug/ml 1n plasma and serum. It has been shown to promote cel 1-substratum adhesion and to act as an Inhibitor of the membrane attack complex of complement and of the inactivation of thrombin by antithrombin III in the presence of low levels of heparin. We have previously shown that VN/SP binds more avidly to heparln-agarose and to a monoclonal antibody (MaVN/SP)-Sepharose column when present 1n serum rather than 1n plasma. In order to examine the possibility of a serum-induced conformational change, we utilized, 1n this study, an Indirect enzyme-linked Immunosorbent system to test for the exposure of new antigenic determinants. When MaVN/SP was Incubated with plasma or serum, recognition of VN/SP 1n serum was approximately 50 fold greater than recognition of VN/SP in plasma. Since VN/SP has been shown to Interact strongly with the thromb1n-ant1thrombin complex, we examined the antigenicity of VN/SP when Incubated with thrombin and antithrombin 1n the presence and absence of heparin. Incubation of VN/SP with heparin promoted a 2.5-fold Increase 1n recognition by MaVN/SP. When MaVN/SP was Incubated with thromb1n-ant1thrombin but not thrombin or antithrombin alone, recognition was Increased by 7-fold 1n the absence of heparin and by 32-fold 1n the presence of heparin. This differential recognition of VN/SP was not observed with a second monoclonal antibody raised originally against S-Prote1n. Treatment of VN/SP with various glycosaminoglycans and polysaccharides demonstrated the following relative potencies for Induction of the partial antigenic change: dextran sulfate>fucoidan>heparin> dermatan suIfate>hyaluronic acid. No effect was detected upon Incubation of VN/SP with keratan sulfate, heparan sulfate or chondroltln sulfate. These data suggest a conformational change Induced by thrombin-antlthrombin which may allow VN/SP to Interact more avidly with other molecules such as heparin. The physiological role of this putative conformational change is under investigation.

scholarly journals Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway.

1976 ◽  
Vol 144 (4) ◽  
pp. 1076-1093 ◽  
Author(s):  
R G Medicus ◽  
O Götze ◽  
H J Müller-Eberhard
Keyword(s):  
Solid Phase ◽  
Alternative Pathway ◽  
Critical Role ◽  
Membrane Attack Complex ◽  
Physiological Role ◽  
Enzyme Formation ◽  
Degree Of Stability ◽  
Alternative Pathway Of Complement

In this study the physiological role of properdin and the differential subunit composition of the solid phase enzymes of the pathway have been explored. Cell-bound C3 and C5 convertase differ in their C3b requirement. Apparently one molecule of C3b is sufficient to allow formation of C3 convertase (C3b,B), whereas two or more are required for generation of C5 convertase (C3bn,B). This conclusion was drawn from results indicating the critical role of the spacial distribution of C3b molecules on the cell surface in enzyme formation. While the C3/C5 convertase is fully capable of acting on C5 and thereby initiating the assembly of the cytolytic membrane attack complex, it is exceedingly labile and vulnerable to destruction by the C3b inactivator. It is the apparent role of properdin to confer a degree of stability upon the labile enzyme and to protect its C3 convertase function against enzymatic destruction. To achieve these effects, precursor properdin (pre-P) is recruited in a binding-activation reaction by the labile C3/C5 convertase. Multiple C3b molecules appear to be needed for the formation of properdin-activating principle. Three modes of regulation have been described, which involve spontaneous dissociation enzymatic degradation by C3b inactivator and disassembly by beta1H. The functional differences of pre-P and activated properdin (P) were delineated, pre-P displaying a weak affinity for C3b and P the capacity of strong interaction, P generating a soluble C3 convertase in serum and pre-P being unable to do so. Because of the profound differences between native pre-P and the laboratory product P, the question was raised as to whether soluble P represents an unphysiological form of the protein. On the basis of this and other studies, the conclusion was reached that in vitro properdin recruitment constitutes the terminal event of the properdin pathway, and that properdin augments the function of C3/C5 convertase without changing its substrate specificity.

C5b-9 membrane attack complex mediates endothelial cell apoptosis in experimental glomerulonephritis

2000 ◽  
Vol 278 (5) ◽  
pp. F747-F757 ◽  
Author(s):  
Jeremy Hughes ◽  
Masaomi Nangaku ◽  
Charles E. Alpers ◽  
Stuart J. Shankland ◽  
William G. Couser ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Regulatory Protein ◽  
Membrane Attack Complex ◽  
Fold Increase ◽  
Renal Perfusion ◽  
Con A ◽  
Immune Mediated ◽  
The Right ◽  
Endothelial Integrity

We studied the role of the C5b-9 membrane attack complex in two models of inflammatory glomerulonephritis (GN) initiated by acute glomerular endothelial injury in Piebold-viral-Glaxo (PVG) complement-sufficient rats (C+), C6-deficient rats (C6−), and rats systematically depleted of complement with cobra venom factor (CVF). GN was induced by performing a left nephrectomy and selectively perfusing the right kidney with either 1) the lectin concanavalin A (Con A) followed by complement-fixing anti-Con A (Con A GN) or 2) purified complement-fixing goat anti-rat glomerular endothelial cell (GEN) antibody [immune-mediated thrombotic microangiopathy (ITM)]. Comparable levels of GEN apoptosis were detected in C+ animals in both models. CVF administration reduced GEN apoptosis by 10- to 12-fold. GEN apoptosis was C5b-9 dependent because PVG C6− rats were protected from GEN loss. Furthermore, functional inhibition of the cell surface complement regulatory protein CD59 by renal perfusion with anti-CD59 antibody in ITM resulted in a 3.5-fold increase in GEN apoptosis. Last, in Con A GN, abrogation of GEN apoptosis preserved endothelial integrity and renal function. This study demonstrates the specific role of C5b-9 in the induction of GEN apoptosis in experimental inflammatory GN, a finding with implications for diseases associated with the presence of antiendothelial cell antibodies.

scholarly journals Evidence that mammalian phosphatidylinositol transfer protein regulates phosphatidylcholine metabolism

1998 ◽  
Vol 335 (1) ◽  
pp. 175-179 ◽  
Author(s):  
Marie E. MONACO ◽  
Richard J. ALEXANDER ◽  
Gerry T. SNOEK ◽  
Nancy H. MOLDOVER ◽  
Karel W. A. WIRTZ ◽  
...  
Keyword(s):  
Physiological Role ◽  
Fold Increase ◽  
Transfer Protein ◽  
Antisense Mrna ◽  
Cell Clones ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer proteins (PITPs) and their yeast counterpart (SEC14p) possess the ability to bind phosphatidylinositol (PtdIns) and transfer it between membranes in vitro. However, the biochemical function of these proteins in vivo is unclear. In the present study, the physiological role of PITP was investigated by determining the biochemical consequences of lowering the cellular content of this protein. WRK-1 rat mammary tumour cells were transfected with a plasmid containing a full-length rat PITPα cDNA inserted in the antisense orientation and the resultant cell clones were analysed. Three clones expressing antisense mRNA for PITPα were compared with three clones transfected with the expression vector lacking the insert. The three antisense clones had an average of 25% less PITPα protein than control clones. Two of the three antisense clones also exhibited a decreased rate of growth. All three antisense clones exhibited a significant decrease in the incorporation of labelled precursors into PtdCho during a 90-min incubation period. Under the same conditions, however, there was no change in precursor incorporation into PtdIns. Further experimentation indicated that the decrease in precursor incorporation seen in antisense clones was not due to an increased rate of turnover. When choline metabolism was analysed more extensively in one control (2-5) and one antisense (4-B) clone using equilibrium-labelling conditions (48 h of incubation), the following were observed: (1) the decrease in radioactive labelling of PtdCho seen in short-term experiments was also observed in long-term experiments, suggesting that the total amount of PtdCho was lower in antisense-transfected clones (this was confirmed by mass measurements); (2) a similar decrease was seen in cellular sphingomyelin, lysoPtdCho and glycerophosphorylcholine; (3) an average two-fold increase in cellular phosphorylcholine was observed in the antisense-transfected clone; (4) cellular choline was, on average, decreased; and (5) cellular CDPcholine was not significantly altered.

ISOLATION OF cDNA FOR TYPE-2 PLASMINOGEN ACTIVATOR INHIBITOR

1987 ◽  
Author(s):  
T Ny ◽  
L Hansson ◽  
B Åstedt
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Human Placenta ◽  
Physiological Role ◽  
Plasminogen Activator Inhibitor ◽  
Expression Library ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Activator Inhibitor

The placental type plasminogen activator inhibitor (PAI-2) has been purified from extracts of human placenta and from a histiocytic lymphoma cell line. It is mainly an uPA inhibitor but it also inhibits the two-chain form of tPA.In order to determine the factors regulating PAI-2 gene expression and thereby clarify the physiological role of PAI-2 we have undertaken the molecular cloning of PAI-2 cDNA. A λgt11 expression library prepared from placental mRNA, was screened, immunologically using a monoclonal antibody probe developed against PAI-2 purified from human placenta. When 1.7×105 recombinant phages were screened six positive clones were obtained. Hybridization experiments and comparison of restriction enzyme cleavege pattern revealed that the DNA inserts of the six clones were, related. To identify the clones as coding for PAI-2, a lysogen made from one of them was induced, and the proteins were separated by SDS-PAGE. In immuno-blotting wxperiments the recombinant fusion protein and purified PAI-2 were recognized by the monoclonal antibody and a monospecific polyclonal antibody against PAI-2, revealing an immunological similarity. The nucleotide sequence of the largest cDNA was determined. It was found to code for a protein with extensive sequence homology with members of the serine protease inhibitor family (serpins) Alignment of the active center region with other serpins indicates that PAI-2 is an arg-serpin, as expected for an inhibitor of plasminogen activators.

Immunodissection of mitochondria-rich cells from rabbit outer medullary collecting tubule

1988 ◽  
Vol 254 (6) ◽  
pp. F907-F911
Author(s):  
M. A. Burnatowska-Hledin ◽  
W. S. Spielman
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Lectin Binding ◽  
Physiological Role ◽  
Histochemical Staining ◽  
Positive Staining ◽  
Camp Production ◽  
Outer Medulla ◽  
Collecting Tubule

Two types of mitochondria-rich (MR) cells have been identified in the rabbit collecting tubule based on differences in immuno- and lectin cytochemistry. We have produced a monoclonal antibody, immunoglobulin (Ig) G1 (mr-mct), that reacts specifically with the MR cells (identified by positive histochemical staining for succinate dehydrogenase) found predominantly in the outer medulla (OM) and cells of the proximal tubule. IgG1 (mr-mct) reacted with 18 +/- 2% of the cells of the outer medullary collecting tubule (OMCT) and did not colocalize with peanut lectin-binding MR cells in the cortex. To isolate MR-OMCT cells, collecting tubule cells from collagenase dispersions of the OM were first adsorbed onto plates treated with a monoclonal antibody reactive against all of the OMCT cells. Of the isolated OMCT cells, 17% reacted with IgG1 (mr-mct). Cells were then detached from the plate and transferred to plates coated with IgG1. Greater than 70% of the adsorbed cells were MR as determined by positive staining with IgG1 (mr-mct). This enrichment of MR-OMCT cells was associated with a severalfold increase in adenosine 3',5' cyclic monophosphate (cAMP) production in response to isoproterenol and an attenuated increase in cAMP production to vasopressin. In summary, we report the isolation of highly enriched populations of MR cells from the OM using two-stage solid-phase immunoadsorption. This approach should provide a useful and convenient method for further investigations of the physiological role of these poorly understood tubular cells.

Hormonal modulation of mouse plasma concentration of epidermal growth factor

1984 ◽  
Vol 107 (4) ◽  
pp. 571-576 ◽  
Author(s):  
J. Perheentupa ◽  
J. Lakshmanan ◽  
S. B. Hoath ◽  
D. A. Fisher
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Vena Cava ◽  
Physiological Role ◽  
Fold Increase ◽  
Submandibular Salivary Gland ◽  
Sham Operation ◽  
Hormonal Modulation ◽  
Epidermal Growth

Abstract. To understand the physiological role of plasma epidermal growth factor (EGF) we studied in adult female mice the effect of endocrine manipulations on serum EGF concentration (S-EGF). Starting 1 month after sham operation (sh) or excision (SX) of the submandibular salivary gland (SMG), groups of 6 mice were given sc injections of T4 (0.4 μg/g daily), testosterone propionate (TP, 25 μg/g every 48 h), T4 + TP, or SMG extract (20 μg EGF daily) for 10 days. They were exsanguinated via the abdominal vena cava on day 11. During treatment the SX mice gained 55% less weight and their S-EGF was 25% lower compared with the sh mice. Hormone effects were clear in the SX mice: T4 caused a 35% decrease in S-EGF and a 5-fold increase in SMG-EGF concentration. TP caused a 1.6-fold increase in S-EGF and a 6-fold increase in SMG-EGF. Twenty-four hours after the last injection of SMG extract S-EGF remained 1.6-fold elevated. We conclude that plasma EGF is largely independent of SMG, and is modified by hormones in ways different from known tissue effects. This is consistent with an endocrine role of EGF.

scholarly journals Physiological role of the ouabain receptor protein (31.5 KD) from cat cardiac muscle, using a monoclonal antibody against the protein

1990 ◽  
Vol 52 ◽  
pp. 176
Author(s):  
Sumiko Fujino ◽  
Kazuo Togashi ◽  
Tohru Nakai ◽  
Kumi Satoh ◽  
Tetsuo Kado ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cardiac Muscle ◽  
Physiological Role ◽  
Receptor Protein

Plasminogen Activation In Vivo upon Intravenous Infusion of DDAVP

1992 ◽  
Vol 67 (01) ◽  
pp. 111-116 ◽  
Author(s):  
Marcel Levi ◽  
Jan Paul de Boer ◽  
Dorina Roem ◽  
Jan Wouter ten Cate ◽  
C Erik Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Plasma Levels ◽  
Fold Increase ◽  
Fibrinolytic System ◽  
Plasminogen Activation ◽  
Plasminogen Activator Activity ◽  
Insight Into

SummaryInfusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established.A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor α2 antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated α2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system.Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex.We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

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