Search PubMed⌕ Search

Biomedical subjects

Z Fishelson

Publications and source records attributed to Z Fishelson.

68 records · Page 4Linked to original sources
PubMed1984

Characterization of the initial C3 convertase of the alternative pathway of human complement.

C3(H2O),Bb, the initial C3 convertase of the alternative complement pathway, was demonstrated to be a metal-containing protein complex by sucrose density gradient ultracentrifugation. Demonstration of this labile enzyme became possible by increasing its half-life with nickel instead of magnesium ions used for enzyme formation. The enzyme was generated from C3, the internal thioester bond of which was hydrolyzed (C3(H2O)), and from 125I-Factor B and Factor D. The sedimentation coefficient of the enzyme complex was 10.7S. By using 63Ni for enzyme formation, the metal ion was detected in the enzyme complex after ultracentrifugation in the presence of 10 mM EDTA. The stoichiometry of the constituents in the C3(H2O),Bb(Ni) complex was 1:1:1. To verify that C3 is incorporated into the enzyme complex in the form of C3(H2O), the enzyme complex was adsorbed to anti-Factor B-Sepharose and subjected to decay-dissociation. Examination of the subsequently eluted protein by SDS gel electrophoresis under reducing conditions demonstrated the presence of an intact C3 alpha-chain. This work provides further evidence that a C3 convertase can be generated from noncleaved C3 that is modified at the thioester site. With the use of a fluorometric assay, the activity (kcat/Km) and the half-life of the initial C3 convertase were determined and compared to those of C3b,Bb.

Chemical Phenomena↗
PubMed1983

C3 convertase of the alternative complement pathway. Demonstration of an active, stable C3b, Bb (Ni) complex.

The purposes of this study were to demonstrate the C3 convertase complex, C3b, Bb (EC 3.4.21.47), of the alternative pathway of complement by ultracentrifugation and to determine whether the metal ion required for enzyme formation is present in the active enzyme complex. It has been shown previously that C3b,Bb formed with Ni2+ rather than Mg2+ exhibits enhanced stability. Using sucrose density gradient ultracentrifugation, an enzymatically active C3b,Bb(Ni) complex could be demonstrated which has a sedimentation coefficient of 10.7 S and which is stable in 10 mM EDTA. Upon formation of the enzyme with the radioisotope 63Ni2+, the ultracentrifugal distribution of the metal correlated with that of the enzyme complex. The molar ratio of Ni to C3b,Bb was 1:1. Displacement of Ni by Mg during formation of the enzyme indicated that both metals may bind to the same site in the enzyme. Binding of 63Ni to the catalytic site bearing fragment Bb was significantly stronger than its binding to C3b or to the zymogen, Factor B. It is proposed that there is one metal-binding site in the C3b,Bb enzyme which is not susceptible to chelation by EDTA and which is located in the Bb subunit.

Complement Activating Enzymes↗
PubMed1983

The C3/C5 convertase of the alternative pathway of complement: stabilization and restriction of control by lanthanide ions.

The alternative pathway C3 convertase (C3b,Bb) is a Mg-dependent, labile enzyme with a t/2 of 3 min at 37 degrees C and of 14 min at 24 degrees C (at half physiological ionic strength). To stabilize the enzyme, metal ions of the lanthanide series were tested. Formation and decay of the enzyme as well as binding of radiolabeled Factor B, Factor H or properdin to C3b were measured using C3b-bearing sheep erythrocytes (EC3b). Binding of Factor B to EC3b in presence of 40 microM gadolinium (Gd) was two to three times greater than in presence of 1 mM Mg. Binding of Factor H and of properdin to EC3b was partially inhibited by Gd. Although it enhanced Factor B uptake by EC3b, Gd was unable to substitute for Mg in enzyme formation by Factor D and completely inhibited (at 10 microM) Mg-dependent enzyme activation. However, the preformed enzyme was not inhibited by Gd. Instead, exposure of EC3b,Bb to 40-100 microM Gd increased the t/2 at 37 degrees C from 3 min to 12-28 min, and at 24 degrees C from 14 min to 32 min. The slow decay of the enzyme correlated with slow release of Bb. Similar enzyme stabilization was observed using terbium, ytterbium, dysprosium and lanthanum. The Gd stabilized enzyme was also less susceptible to control by Factor H and properdin than the unstabilized enzyme. Furthermore, Gd protected surface bound-C3b from being cleaved by Factor I. The Gd effects were instantaneously reversed upon addition of 10 mM EDTA. Thus, Gd is able to stabilize preformed C3b,Bb and to render the enzyme refractory to control by Factors H and I.

Cations, Divalent↗
PubMed1982

C3 convertase of human complement: enhanced formation and stability of the enzyme generated with nickel instead of magnesium.

We demonstrate that nickel++ (Ni) can replace Mg in the formation of the C3b,Bb enzyme, and that Ni is more efficient in enzyme formation than Mg. C3b-bearing sheep erythrocytes (EC3b) were used to measure radiolabeled Factor B uptake and C3 convertase activity. Up to nine times more Factor B was specifically bound to EC3b in the presence of Ni than in the presence of Mg under identical conditions. To form one effective hemolytic site (1 Z) per EC3b cell with Ni, three times less Factor B, 12 times less Factor D, and 66 times less metal ion were required than when using Mg. The C3b,Bb formed with Ni (C3b,Bb(Ni)) had a 5 to 10 times longer half-life at different temperatures than the enzyme formed with Mg (C3b,Bb(Mg)). Native properdin stabilized C3b,Bb(Ni) to the same extent as C3b,Bb(Mg) (four to five times) and Factor H accelerated similarly the decay of both enzymes. Ni could also replace Mg in the formation of the C4b,2a enzyme. C4b,2a formation was measured by using C1 and C4b-bearing sheep erythrocytes (EAC1,4b) and native C2. To form 1 Z per EAC1,4b cell with Ni, two times less C2 and 60 times less metal ion were required than when using Mg. The half-life of the C4b,2a formed with Ni was two times longer than that formed with Mg. Decay of both C3b,Bb and C4b,2a enzymes formed either with Mg or with Ni was unaffected by EDTA. These results show that Ni is more efficient than Mg in the generation of both enzymes and suggest that Ni binds to the Mg-binding sites of the enzymes with a higher affinity than Mg.

Complement Activating Enzymes↗
PubMed1981

Tumor cell destruction by cytotoxic T lymphocytes: the basis of reduced antitumor cell activity in syngeneic hosts.

Tumor overgrowth in spite of an ongoing antitumor immune response may be due to a basic immunologic defect in T cell-mediated responses against the potentially immunogenic tumor cells. To further understand T cell-mediated responses in syngeneic tumor-host systems, we have analyzed the interaction of cytotoxic T lymphocytes (CTL) with syngeneic tumor cells and have compared it with CTL-allogeneic tumor cell interaction. The major conclusions of this study are: 1) Syngeneic and allogeneic CTL lyse target cells through a similar mechanism. 2) The reduced reactivity in the syngeneic system is due to the low content of effector cells capable of binding to and killing tumor cells. 3) The avidity of CTL-syngeneic tumor cell binding is lower than CTL-allogeneic tumor cell binding. We suggest that the latter 2 observations result from a low immunogenicity of tumor cells in the syngeneic host.

Animals↗
PubMed1978

T lymphocyte-mediated cytolysis: dissociation of the binding and lytic mechanisms of the effector cell.

To determine functional relationships between the cytotoxic T lymphocyte (CTL) receptor for target binding and the lytic mechanism, we have studied the reaction between two immunized CTL populations (AalphaB and BalphaA), both at the population and the single-cell level. When studied at the population level, the reaction of AalphaB with BalphaA (bidirectional system) resulted in formation of AalphaB/BalphaA conjugates and bidirectional cytolysis. However, when the viability of cells in individual AalphaB/BalphaA conjugates was analyzed, unidirectional instead of bidirectional lysis occurred. These results indicate that under conditions that are conducive to lysis, binding of a potentially lytic cell to its target does not necessarily result in target lysis. Short heat treatment of CTL (44 degrees C, 10 min) totally abolished their lytic activity, without affecting their capacity to bind specifically, thus dissociating the binding from the lytic activity of the CTL. The cytolytic activity is probably associated with, or triggered by the CTL-binding unit. The binding unit, on the other hand, appears to be a functional receptor of the CTL, which is involved in but not sufficient to bring about lysis.

Animals↗
PubMed1976

Possible role of nucleus-membrane interaction in capping of surface membrane receptors.

Interaction of multivalent ligands and cell surface receptors can induce redistribution of these receptors to form patches and caps. In this study, we have investigated the role of nucleus-membrane interaction in the capping of membrane components. Mouse L cells and leukemia EL4 cells were enucleated with the aid of cytochalasin B, yielding cytoplasts and karyoplasts. Capping of surface receptors was induced by allo- and hetero-immune sera followed by fluorescein-conjugated antiglobulin serum, or by the plant lectin concanavalin A. Capping could easily be induced in intact cells, but virtually no capping was detected in the nucleus-free cytoplasts. Interestingly, karyoplasts, which posses cell-membrane components but very little cytoplasm, could be easily induced to cap their surface antigens. Hence, cap formation of membrane components seems not to be an autonomous membrane process. The data suggest that interaction of surface membranes and inner cell components associated with the nucleus is involved in the movement of surface membrane receptors.

Animals↗
PubMedDate not supplied

Complement C3: a molecular mosaic of binding sites.

Many of the biological activities of the complement system are mediated by C3, the third complement component, and its proteolytic fragments. At the same time, several of the molecules which regulate complement activation target their action at the C3 molecule. Accordingly, the C3 molecule is equipped with multiple binding sites for at least 14 other complement or complement-related proteins. As described in this review, major progress has been made recently in the identification of the C3 binding sites and the residues involved. Yet this has exposed only the "tip of the iceberg". A novel technique which may facilitate the elucidation of the active sites in C3 is presented. Finally, based on the current knowledge on the C3 molecule, a hypothetical model of the molecular organization of this molecule and its binding sites is presented.

Animals↗
PubMedDate not supplied

Complement resistance of tumor cells: basal and induced mechanisms.

Clinical and experimental studies have suggested that complement may play a role in tumor cytotoxicity. However, the efficiency of complement-mediated tumor cell lysis is hampered by various protective mechanisms, which may be divided into two categories: basal and induced mechanisms. The basal mechanisms are spontaneously expressed in cells without a need for prior activation, whereas the induced mechanisms develop in cells subjected to stimulation with cytokines, hormones, drugs or with sublytic doses of complement and other pore-formers. Membrane-associated complement regulatory proteins, such as CD55 (DAF, Decay-Accelerating Factor), CD46 (MCP, Membrane Cofactor Protein), CD35 (CR1, Complement Receptor type 1) and CD59, which serve as an important mechanism of self protection and render autologous cells insensitive to the action of complement. appear to be over-expressed on certain tumors. Furthermore, tumor cells secrete several soluble complement inhibitors. Tumor cells may also express proteases that degrade complement proteins, such as C3, or ecto-protein kinases which can phosphorylate complement components, such as C9. Besides this basal resistance, nucleated cells resist, to some extent, complement damage by removing the membrane attack complexes (MAC) from their surface. Several biochemical pathways, including protein phosphorylation, activation of G-proteins and turnover of phosphoinositides have been implicated in resistance to complement. Calcium ion influx and activation of protein kinase C (PKC) and of mitogen-activated protein kinase (MAPK) have also been demonstrated to be associated with the complement-induced enhanced resistance to lysis. The complete elucidation of the molecular mechanisms involved in basal and induced tumor cell resistance will enable the development of strategies for interfering with these evasion mechanisms and the use of the cytotoxic complement system against tumor cells.

Animals↗
PubMedDate not supplied

Novel mechanisms of immune evasion by Schistosoma mansoni.

The interaction of Schistosoma mansoni with its host's immune system is largely affected by multiple specific and non-specific evasion mechanisms employed by the parasite to reduce the host's immune reactivity. Only little is known about these mechanisms on the molecular level. The four molecules described below are intrinsic parasitic proteins recently identified and studied in our laboratory. 1. m28--A 28kDa membrane serine protease. m28 cleaves iC3b and can thus restrict attack by effector cells utilizing complement receptors (especially CR3). Treatment with protease inhibitors potentiates killing of schistosomula by complement plus neutrophils. 2. Smpi56--A 56kDa serine protease inhibitor. Smpi56 binds covalently to m28 and to neutrophil's elastase and blocks their proteolytic activity. 3. P70--A 70kDa C3b binding protein. The postulated activity of P70 includes binding to C3b and blocking of complement activation of the C3 step. 4. SCIP-1--A 94kDa schistosome complement inhibitor. SCIP-1 shows antigenic and functional similarities to the human 18kDa complement inhibitor CD59. Like CD59, SCIP-1 binds to C8 and C9 and blocks formation of the complement membrane attack complex. Antibodies directed to human CD59 bind to schistosomula and potentiate their killing by complement. The structure and function of these four proteins as well as their capacity to induce protection from infection with S. mansoni are under investigation.

Animals↗
PubMedDate not supplied

Induction of complement factor B activity in human fibroblasts by IL-6/IFN-beta 2 and IFN-gamma.

Human skin fibroblasts synthesize and secrete complement Factor B, a component of the complement alternative pathway, when stimulated by mediators of inflammation such as lipopolysaccharide and various cytokines. Recombinant IL-6/IFN-beta 2 (E. coli) stimulates Factor B synthesis in fibroblasts but the effect is strongly potentiated by the addition of IFN-gamma. When both cytokines are added, the skin fibroblasts secrete significant amounts of biologically active Factor B detectable in a hemolysis test. This cooperative effect of IL-6, which is made by most tissue cells and monocytes and of IFN-gamma which is made by T-lymphocytes may play a role in local inflammatory processes. IL-6 and IFN-gamma also cooperate in the induction of (2'-5') A synthetase, a mediator of IFN action.

2',5'-Oligoadenylate Synthetase↗