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Biomedical subjects

Z Fishelson

Publications and source records attributed to Z Fishelson.

At least 19 recordsLinked to original sources
PubMed1995

Complement membrane attack complex, perforin, and bacterial exotoxins induce in K562 cells calcium-dependent cross-protection from lysis.

The complement membrane attack complex (MAC), the cytolytic granule protein of cytotoxic lymphocytes perforin, the streptococcal exotoxin streptolysin O (SLO), and the bee venom polypeptide melittin utilize a similar mechanism to incorporate into cell membranes, induce a Ca2+ influx and a rise in intracellular Ca2+ concentration, and produce cell lysis. At sublytic concentrations, these proteins trigger several cellular activities, including protein phosphorylation and synthesis. We have recently demonstrated that human leukemic cells treated with sublytic doses of human complement become more resistant to lytic complement doses. The study has now been extended to include three other pore-formers: murine perforin, SLO and melittin. As shown here, sublytic MAC induces in the K562 human erythroleukemic cells protection from lytic perforin, and vice versa, sublytic perforin induces protection from complement. Also, sublytic SLO and melittin increase resistance of K562 cells to lytic complement and perforin doses. The capacity of Ca2+ ionophores to induce resistance to the lytic proteins has been examined. Exposure of K562 cells to sublytic concentrations of ionomycin or A23187 for 1 h at 37 degrees C confers on them resistance to complement- and perforin-mediated lysis. The protective effects of the ionophores can be abrogated by chelation of extracellular Ca2+ and by inhibition of RNA or protein synthesis in the cells. These results indicate the following: 1) nucleated cells exposed to sublytic complement MAC, perforin, SLO, or melittin may become resistant to the four pore-formers. Physiologically, this may be regarded as an immunologic tachyphylaxis. 2) Ca2+ influx induced by these pore-formers is an essential and sufficient factor to produce this tachyphylaxis.

Bacterial Proteins↗
PubMed1995

Shedding of tyrosine and serine/threonine ecto-protein kinases from human leukemic cells.

Ecto-protein kinases (ecto-PK), primarily of the serine/threonine kinase type, have been previously described on the surface of various normal, transformed, and tumor cells. We have found that in the presence of ATP and Mg2+, exogenously added substrates such as phosvitin and poly(Glu4-Tyr) are phosphorylated by intact K562 erythroleukemia, HL60 promyelocytic leukemia, and U937 histiocytic leukemia human cells. Phosphoamino acid analysis indicated that phosvitin, histone H2B, casein, and protamine are phosphorylated on serine and threonine residues, whereas poly(Glu4-Tyr) is phosphorylated on tyrosine. We also present evidence showing that the C9 complement protein, a key component of the membranolytic protein complex of the complement system, is exclusively phosphorylated by the K562 cells on serine residues. Phosphorylation of poly(Glu4-Tyr) is markedly enhanced by Mn2+, whereas C9 phosphorylation is rather inhibited by Mn2+. It is concluded that human leukemic cells express on their surface two types of ecto-PK, one phosphorylating serines and threonines and one specific to tyrosines. The ecto-PKs are spontaneously shed from fully viable cells into the medium in a temperature-dependent manner. Upon sedimentation of cell supernatants at 100,000g, the ecto-PKs are found sedimented with small membrane vesicles. Treatment of intact K562 cells or of released membrane vesicles with bacterial phospholipase C, but not with trypsin or pronase, releases the two types of ecto-PK from the cell or vesicle membrane, respectively. This is accompanied by a marked increase in the released phosphorylating activity. It is, therefore, suggested that these ecto-PKs are either covalently linked to phospholipids or strongly attached to lipid-anchored molecules in the cell surface membrane. Several endogenous proteins in the released membranes are phosphorylated by the ecto-PKs on serines and to a lesser extend on threonines. Two proteins (PTP79 and PTP54) are phosphorylated in a manganese-dependent manner on tyrosines.

Adenosine Triphosphate↗
PubMed1995

Compound heterozygous complement C3 deficiency.

Complete deficiency of the third component of the complement system is a result of defects in the two alleles of the C3 gene. In this study a family with C3 deficiency is reported; the parents expressed a distinct abnormality of the C3 gene and their two children had compound heterozygous C3 deficiency. These are the first reported cases of compound heterozygous complement deficiency. Our results indicate that the maternal abnormality leads to synthesis of an abnormal proC3 protein which is not secreted from the cells. The paternal abnormality results in ablation of synthesis of the proC3 protein.

Autoradiography↗
PubMed1994

Inherited human complement C3 deficiency. An amino acid substitution in the beta-chain (ASP549 to ASN) impairs C3 secretion.

We recently described a case of hereditary complement C3 deficiency (C3D) in a New Zealand male who has a small amount of serum C3 (7 micrograms/ml), a normal size 5.2-kilobase C3 mRNA that is present in normal quantities, and a normal size M(r) 180,000 proC3 molecule that is synthesized in normal amounts. Secretion of C3 from this patient's cells was greatly diminished, however, and an aberrant C3 trypsin cleavage profile indicated an abnormality in the proC3 structure. To determine the primary structure of the C3D proC3 molecule, the corresponding cDNA was cloned and sequenced in the present study, revealing a normal signal peptide, tetraarginine linker, and thiolester domain. One nucleotide substitution in exon 13 (G1705 AC to AAC) was found, however, that resulted in an amino acid change in a highly conserved region of the C3 beta-chain (Asp549 to Asn). This substitution has not been described in any individual with either C3 Fast or C3 Slow phenotypes. Immunoprecipitation of C3 from L-cells transfected with full-length normal and C3D cDNAs demonstrated that C3 was secreted by the cells transfected with the normal C3 cDNA; however, only a C3 precursor was detected in the intracellular compartment of the cells transfected with the C3D cDNA and none detected extracellularly. Immunofluorescence studies revealed a perinuclear localization of C3 in the C3D transfectants, arrested early in the secretory pathway. Allele-specific polymerase chain reaction analysis demonstrated that this New Zealand family is a compound heterozygous C3D kindred, with the Asn549 point mutation being inherited from the mother and a yet undescribed C3 defect being inherited from the father. Taken together, these data indicate that 1) C3 deficiency is caused in a New Zealand kindred by two distinct molecular genetic mutations, one being an amino acid substitution in a highly conserved region of the beta-chain that results in impaired C3 secretion, and 2) the molecular basis of this deficiency has not been described in any other C3-deficient individual, providing additional evidence that multiple defects cause inherited C3 deficiency in humans.

Alleles↗
PubMed1994

Functional and antigenic similarities between a 94-kD protein of Schistosoma mansoni (SCIP-1) and human CD59.

Schistosomiasis is a parasitic disease affecting approximately 200 million people, primarily in the third world. Schistosoma mansoni, one of the causative agents of this disease, parasitize the human mesenteric and portal blood systems while successfully evading host immune responses. During parasite penetration into the mammalian host and shortly afterwards, the larvae rapidly convert from being sensitive to being resistant to C-mediated killing. Treatment of the C-resistant parasitic forms with trypsin renders the parasite susceptible to C attack, thus indicating the presence of C inhibitory protein(s) on the parasite surface. We describe here an intrinsic schistosome C inhibitory protein (SCIP-1) that exhibits antigenic and functional similarities with the human C-inhibitor CD59. Like CD59, SCIP-1 is capable of inhibiting formation of the C membrane attack complex (MAC), probably by binding to C8 and C9 of the C terminal pathway. In addition, SCIP-1 is apparently also membrane-anchored via glycosyl phosphatidylinositol as it can be specifically released with phosphatidylinositol-specific phospholipase C. Soluble SCIP-1, partially purified from Nonidet P-40 extracts of schistosome tegument is capable of inhibiting hemolysis of sensitized sheep erythrocytes and of rabbit erythrocytes by human C. Anti-human CD59 antibodies block this activity of SCIP-1 and in addition, upon binding to intact parasites, render them vulnerable to killing by human and guinea pig C. SCIP-1 is located on the surface of C-resistant forms of the parasite, i.e., 24-h cultured mechanical schistosomula and in vivo-derived adult worms as revealed by immunofluorescence and immunogold electron microscopy studies. These results identify one of the mechanisms schistosomes use to escape immune attack.

Animals↗
PubMed1994

Inherited complement C3 deficiency: a defect in C3 secretion.

The molecular basis of inherited complement C3 deficiency in a 20-year-old newly diagnosed male patient was studied. Using an enzyme-linked immunosorbent assay, the patient's C3 serum level was found to be approximately 7 micrograms/ml, which is less than 1% of normal. In contrast, Northern analysis indicated that the patient's C3 mRNA was of normal size and quantity. Peripheral blood monocytes (PBM) and skin fibroblast cultures (F) from the patient and from healthy donors were labeled for 2 h with [35S] methionine. Analysis of cell lysates and supernatants by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated normal levels of C3 in lysates of patient's PBM and F. However, C3 secretion in the patient's cells was extremely reduced, with pulse-chase experiments demonstrating a long delay in the disappearance of intracellular C3. Secretion of C1r and factor B by the patient's cells was normal. Lipopolysaccharide and interleukin-1 increased C3 synthesis in the patient's PBM and F, but had no effect on the secretion. SDS-PAGE analysis of trypsin-cleaved intracellular C3 revealed an aberrant cleavage profile for the patient's C3. Collectively, these data indicate that C3 deficiency in this patient is due to a defect in the C3 secretion, probably as the result of abnormality in the proC3 structure.

Adolescent↗
PubMed1994

Schistosoma mansoni: isolation and characterization of Smpi56, a novel serine protease inhibitor.

Tegumental extracts from adult worms of Schistosoma mansoni contain an inhibitory activity to the S. mansoni 28-kDa serine protease and to pancreatic elastase. By using biotinylated elastase and streptavidin-agarose, the postulated protease inhibitor has been isolated from the crude worm extract in a single step. Monospecific rabbit antibodies raised against the protease inhibitor have immunoprecipitated a 56-kDa [35S]Met-labeled serine protease inhibitor which was designated Smpi56 (S. mansoni protease inhibitor, 56 kDa). Smpi56 binds tightly to and inhibits the 28-kDa protease of S. mansoni and pancreatic and neutrophil elastase but not papain, pepsin, thrombin, trypsin, chymotrypsin, proteinase K, urokinase and acetylcholinesterase. The biological function of Smpi56 is still not known, but in view of its elastase inhibitory activity it may be speculated that the parasite is employing Smpi56 to protect itself from activated neutrophils. Smpi56 may also potentially protect the parasite from its endogenous 28-kDa protease.

Animals↗
PubMed1993

Reduced CR1 expression on aged human erythrocytes: immuno-electron microscopic and functional analysis.

Recognition and clearance of aged human erythrocytes (AE) is a complex process involving immune and non-immune reactions. Complement activation on the surface of AE and deposition of the C3b complement component appear to facilitate this process. Complement receptor type 1 (CR1, CD35) expressed on the surface of human erythrocytes binds to C3b molecules and promotes their inactivation by complement factor I. This may protect the erythrocytes from lysis by complement and by phagocytes. It has been previously reported that aging of human erythrocytes is accompanied by a decrease in the number of CR1 molecules expressed on their surface. Results presented here further support this finding with for the first time a presentation of immune-electron microscopic observation. Haemagglutination and binding assays showed that AE express significantly fewer CR1 (CD35) molecules than young erythrocytes (YE). This is associated in AE with reduced CR1-like factor I co-factor activity and increased deposition in vivo of C3, C4 and properdin as well as increased sensitivity to lysis in vitro by homologous and heterologous complement. Three different immuno-electron microscopical techniques have been used to clearly show the quantitative difference in CR1 (CD35) expression between AE and YE. Finally our results demonstrate that the previously reported clustered arrangement of CR1 (CD35) on human erythrocytes is similar on aged and young erythrocytes.

Antibodies, Monoclonal↗
PubMed1993

Hereditary properdin deficiency in three families of Tunisian Jews.

Hereditary properdin deficiency is a rare genetic disorder of the complement system. Three propositi and six additional family members with properdin deficiency have been found following analysis of the hemolytic activity of the classical (CH50) and the alternative (AP50) complement pathways in the sera of 101 survivors of meningococcal infections and 59 survivors of severe pneumococcal and Haemophilus influenza infections. All the properdin-deficient individuals had undetectable levels of properdin by radial immunodiffusion and by Western blotting. They belonged to three non-related families of Tunisian Jews who came from different parts of Tunisia. Two patients had a meningococcal infection at 15 and 16 years of age, respectively, and one had Haemophilus influenza meningitis at 1.5 years of age. In contrast to the fulminant and fatal course of meningococcal infection which was previously described in some properdin-deficient patients, our patients had a relatively mild disease. Properdin deficiency may not be as rare as previously thought. Analysis of AP50, in addition to CH50, in sera of patients who had meningococcal infection, will probably disclose many more cases of hereditary properdin deficiency. In addition, our findings indicate that, as in other complement abnormalities, hereditary properdin deficiency may also be associated with the ethnic origin of the patient.

Adolescent↗
PubMed1992

Sublytic complement attack protects tumor cells from lytic doses of antibody and complement.

Sublytic doses of the membrane attack complex (MAC) of complement are known to exert multiple stimulatory effects on metabolically active cells. Results presented herewith demonstrate that pretreatment of the human leukemic cells K562 and HL-60 with sublytic doses of antibody and normal human serum protects them from lytic complement concentrations, a phenomenon proposed to be called "complement-induced protection". C7- and C8-deficient human sera are ineffective in inducing resistance unless they are reconstituted with purified human C7 and C8, respectively. The complement-induced protection is inhibitable by actinomycin D and cycloheximide indicating that the increased complement resistance depends on RNA and protein synthesis triggered by the sublytic complement doses. Free extracellular Ca2+ is also required to achieve maximal protection, indicating a role for Ca2+ ions in the cell stimulatory events which culminate in increased complement resistance. Quantitative analysis of bound complement components indicated that similar amounts of C3 and C9 molecules are deposited on "protected" and control cells during complement activation. The "protected" K562 and HL-60 cells regain sensitivity to lytic MAC doses after about 8 or 3 h, respectively, of culture in growth medium, in the absence or presence of actinomycin D and cycloheximide. The "induced protection" is not species restricted and protection from human complement can be induced in K562 cells by treatment with sublytic doses of antibody and rabbit or guinea pig sera.

Animals↗
PubMed1992

Schistosoma mansoni: cell-specific expression and secretion of a serine protease during development of cercariae.

Eukaryotic serine proteases are an important family of enzymes whose functions include fertilization, tissue degradation by neutrophils, and host invasion by parasites. To avoid damaging the cells or organisms that produced them, serine proteases must be tightly regulated and sequestered. This study elucidates how the parasitic blood fluke Schistosoma mansoni synthesizes, stores, and releases a serine protease during differentiation of its invasive larvae. In situ hybridization with a cDNA probe localized the protease mRNA to acetabular cells, the first morphologically distinguishable parasite cells that differentiate from the embryonic cell masses present in the intermediate host snail. The acetabular cells contained vimentin but not cytokeratins, consistent with a mesenchymal, not epithelial, origin. Antiprotease antibodies, localized by immunoperoxidase, showed that the protease progressively accumulated in these cells and was packaged in vesicles of three morphologic types. Extension of cytoplasmic processes containing protease vesicles formed "ducts" which reached the anterior end of fully differentiated larvae. During invasion of human skin, groups of intact vesicles were released through the acetabular cytoplasmic processes and ruptured within the host tissue. Ruptured protease vesicles were noted adjacent to degraded epidermal cells and dermal-epidermal basement membrane, as well as along the surface of the penetrating larvae themselves. These observations are consistent with the proposed dual role for the enzyme in facilitating invasion of host skin by larvae and helping to release the larval surface glycocalyx during metamorphosis to the next stage of the parasite.

Animals↗
PubMed1992

Complement membrane attack complexes induce in human leukemic cells rapid expression of large proteins (L-CIP).

The effect of sublytic doses of the complement membrane attack complexes (MAC) on protein synthesis in human leukemic cells was examined. As shown herein, rapid protein synthesis is evident in K562 erythroleukemic cells upon exposure to sublytic complement doses. Analysis of cell extracts by SDS-PAGE revealed high molecular weight proteins which appeared in the cells already after 15 min treatment with complement at 37 degrees C, reaching a maximal level after 40-50 min. These large complement-induced proteins (L-CIP) were clearly observed in gels stained by Coomassie blue and in autoradiograms following [35S]-Met or [3H]-Leu incorporation. Rabbit antibodies prepared against L-CIP were reactive in immunoassays with extracts of MAC-treated cells but not of non treated cells. They also bound to the surface of intact K562 cells (as determined by immunofluorescence), but only after treatment of the cells with complement. Both heterologous (rabbit and guinea pig) and homologous (human) sera induced L-CIP synthesis. The induction of L-CIP was indeed mediated by the complement MAC since L-CIP could not be detected in K562 cells exposed to heat-inactivated human serum or C6-deficient rabbit serum. Similarly, C7- or C8-deficient human sera could not induce L-CIP production unless they were reconstituted with purified human C7 or C8, respectively. The synthesis of L-CIP was largely inhibited by the protein synthesis inhibitors cycloheximide and puromycin and partially inhibited by the RNA synthesis inhibitor actinomycin D. L-CIP was similarly induced in two other human leukemic cell lines, U937 and HL-60, but not in K562/S, a subline of K562 which is highly sensitive to complement damage. These results are discussed with respect to the resistance of leukemic cells, and nucleated cells in general, to complement-mediated immune damage.

Cell Death↗
PubMed1992

The human lymphokine leukoregulin induces cell resistance to complement-mediated lysis.

Leukoregulin (LR) is a lymphokine secreted by human natural killer (NK) cells. Its effect on the susceptibility of K562 human erythroleukemic cells to lysis by antibody and complement was examined. As reported here, treatment of K562 cells with LR for 60 min at 37 degrees C confers on them resistance to complement damage. The LR-induced state of complement resistance is transient and the cells recover within 4-6 h unless a second dose of LR is added. The protective action of LR was observed using both conventional 51Cr-release and trypan blue inclusion assays. The protein synthesis inhibitors puromycin and cycloheximide and the protein kinase inhibitors tamoxifen, polymyxin B and W-7, could each block this action of LR. Fewer membrane attack complexes were found, following complement activation, on LR-treated than control cells. These results suggest that LR increases the capacity of K562 cells to down-regulate complement activation or repair the complement damage, possibly by inducing synthesis of defense proteins and/or activation of protective protein kinases.

Antibodies, Neoplasm↗
PubMed1991

Leishmanial protein kinases phosphorylate components of the complement system.

Externally oriented protein kinases are present on the plasma membrane of the human parasite, Leishmania. Since activation of complement plays an important role in the survival of these parasites, we examined the ability of protein kinases from Leishmania major to phosphorylate components of the human complement system. The leishmanial protein kinase-1 (LPK-1) isolated from promastigotes of L. major was able to phosphorylate purified human C3, C5 and C9. Only the alpha-chain of C3 and C5 was phosphorylated. The beta-chain appeared not to be a substrate for this enzyme. C3b which is formed by proteolytic cleavage of C3 was not phosphorylated by LPK-1. Trypsin treatment of phosphorylated C3 (P-C3) resulted in the disappearance of 32P from the alpha-chain. This was correlated with the conversion of the C3 alpha-chain to the alpha'-chain of C3b, and the appearance of a 9 kDa 32P fragment comigrating with the C3a fragment of C3. P-C3 was more resistant to cleavage by trypsin than nonphosphorylated C3. LPK-1 phosphorylated purified C3a and two synthetic peptides, C3a21R and YA-C3a10R, derived from its COOH-terminal end, which contain the C3a binding site to leukocytes and platelets. LPK-1 did not phosphorylate C3a8R. Phosphoamino acid analysis of the synthetic peptides indicated that serine 71 of C3a was phosphorylated by LPK-1. Treatment of C3 with either methylamine or freeze-thaw C3 (H2O) prevented phosphorylation by the LPK-1 suggesting that substrate conformation may be involved in recognition by the leishmanial enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Resistance to NK cell-mediated cytotoxicity (in K-562 cells) does not correlate with class I MHC antigen levels.

Natural Killer (NK) cells probably function as an early line of defense against virus-infected cells and tumor cells. In all cases, the killing by NK cell-mediated cytotoxicity (NK-CMC) is not MHC-restricted and the factors which determine the sensitivity to NK-CMC have not yet been identified. A positive correlation between resistance to NK-CMC and the level of class I MHC antigen (MHC I) expression on target cells has been reported in many studies, and in some cases a functional linkage between the two has been claimed. Several other studies have shown that there is no such correlation. By employing several experimental systems, we demonstrate here a lack of correlation between the level of MHC I and the sensitivity of K-562 cells to NK-CMC. Transfer of MHC I to MHC I-negative cells via vesicles had no effect on their resistance to NK-CMC. In addition, a decrease in resistance to NK-CMC and increase of MHC I levels was observed following target-cell membrane modulation by both application of cholesterol and hydrostatic pressure. Finally, no correlation between sensitivity to NK-CMC and MHC I expression was found in three sublines of K-562 cells. Since NK-CMC is a multistage process, it is concluded that components other than class I MHC antigens have a more prominent role in modulating the sensitivity of target cells to NK-CMC.

Cell Death↗