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Biomedical subjects

E Pearlstein

Publications and source records attributed to E Pearlstein.

At least 37 records · Page 2Linked to original sources
PubMed1985

C1q, a subunit of the first component of complement, enhances binding of plasma fibronectin to bacteria.

The interaction of plasma fibronectin with C1q of the complement system has been demonstrated in the past several years. In addition, the antibody-independent binding of C1q to bacteria, as well as the binding of plasma fibronectin to bacteria, is well documented. This study examines whether the binding of C1q to bacteria enhances the interaction of C1q and bacteria with plasma fibronectin. Highly purified 125I-C1q bound to several species of bacteria in the absence of antibody. The binding of 125I-C1q to bacteria was saturable and specific since the addition of unlabeled C1q inhibited binding while the presence of bovine serum albumin did not. Bacteria which had been pretreated with either buffer or unlabeled C1q were tested for their ability to bind 125I-fibronectin. When bacteria were preincubated with buffer, Staphylococcus aureus bound fivefold more 125I-fibronectin than did Escherichia coli. However, preincubation of E. coli with C1q increased the binding of 125I-fibronectin by up to 20-fold, whereas pretreatment of S. aureus with C1q increased fibronectin binding by only twofold. These results were confirmed by immunoblotting studies which demonstrated the presence of C1q, as well as an increase in fibronectin antigens on the C1q-treated bacteria as compared with the level of fibronectin on buffer-treated bacteria. In addition, preincubation of 3H-labeled bacteria with C1q enhanced their attachment to fibronectin-coated surfaces but not to albumin-coated surfaces. The biological consequences of these observations are discussed.

Adhesiveness↗
PubMed1984

Primary structure of human plasma fibronectin--characterization of the 6,000 dalton C-terminal fragment containing the interchain disulfide bridges.

The carboxy terminal fragment of human plasma fibronectin has been isolated after tryptic digestion and separation by DEAE-cellulose chromatography and gel filtration on Sephadex G-50. It has a molecular weight of 6,000 which changes to 3,000 after reduction indicating that the fragment is a dimer. We have determined the amino acid sequence of the 6kDa fragment and showed that it contains 26 residues including two half-cystines which form two interchain disulfide bridges. The 6kDa fragment is not phosphorylated as in bovine fibronectin although its amino acid sequence is identical to that reported for bovine plasma fibronectin. When compared to the sequence deduced from a rat cDNA, one amino acid substitution can be found. It appears that the carboxyl end of fibronectin is highly conserved among species.

Amino Acid Sequence↗
PubMed1984

Lack of effect of in vivo prostacyclin on the development of pulmonary metastases in mice following intravenous injection of CT26 colon carcinoma, Lewis lung carcinoma, or B16 amelanotic melanoma cells.

Honn et al. [Science (Wash. DC), 212: 1270, 1981] have recently reported a 93% reduction in the development of metastases of B16 amelanotic tumor cells given i.v. following a single dose of prostacyclin (PGI2) (100 micrograms) and theophylline (100 micrograms) 30 min prior to the injection of tumor cells. We have been unable to reduce pulmonary metastases induced by the i.v. injection of CT26 colon adenocarcinoma, Lewis lung carcinoma, or B16 amelanotic melanoma cells with a similar regimen. Thus, PGI2 and theophylline given prior to injection of tumor cells and 2 hr postinjection had no effect on the number or volume of pulmonary tumor nodules for CT26 cells, using 15 experimental and 14 control animals; Lewis lung cells, using 14 experimental and 13 control animals; or B16 amelanotic cells, using 26 experimental and 12 control animals. The PGI2 used was shown to be active in vitro, inhibiting tumor-induced platelet aggregation by all three tumors at 10(-9)M; and in vivo by inhibition of Lewis lung-induced thrombocytopenia at 1 hr, using 100 micrograms PGI2 prior to the injection of tumor cells.

Animals↗
PubMed1984

Effect of antiplatelet antibody on the development of pulmonary metastases following injection of CT26 colon adenocarcinoma, Lewis lung carcinoma, and B16 amelanotic melanoma tumor cells into mice.

Three different murine tumors, CT26 colon adenocarcinoma, Lewis lung carcinoma, and B16 amelanotic melanoma, were injected into syngeneic mice (BALB/c and C57BL/6J) to test the effect of rabbit anti-mouse platelet antibody on the development of pulmonary metastases. Antiplatelet antibody, when injected i.p., decreased the platelet count from 1.5 x 10(6)/microliters to 0.12 x 10(6)/microliters at 6 hr, which remained at this level for 24 hr. Antiplatelet antibody given 6 hr pre- and 18 hr post-i.v. injection of tumor cells decreased the mean number of CT26 tumor nodules per lung by 57% (range, 47 to 65%) and decreased the mean nodule volume of tumor per lung by 37% (range, 0 to 71%) (124 experimental animals), when compared to the effect of nonimmune serum or irrelevant anti-immunoglobulin antibody in 136 control animals. With Lewis lung carcinoma, antiplatelet antibody decreased the mean number of tumor nodules by 62% (range, 57 to 78%) and decreased the mean nodule volume of tumors by 64% (range, 60 to 77%) using 48 experimental animals and 65 control animals. When tumor cells were given s.c., antiplatelet antibody given 6 hr pre-injection, 18 hr post-injection, and every 48 hr thereafter also decreased the mean number of metastases by 42% in 14 experimental and 15 control animals. With B16 amelanotic melanoma, antiplatelet antibody given 6 hr pre- and 18 hr post-injection decreased the mean number of tumor nodules by 85% and decreased the mean nodule volume of tumors by 66% using 9 experimental and 9 control animals. Similar results were obtained when all three tumors were injected 6 hr after the injection of antiplatelet antibody. However, negative results were obtained if antiplatelet antibody was injected 6 hr after the injection of tumor cells. Since antiplatelet antibody has its maximum effect at 6 hr, it is likely that platelets play their role in the development of pulmonary metastases during the first 12 hr of tumor inoculation.

Adenocarcinoma↗
PubMed1983

Primary structure of human plasma fibronectin. The 29,000-dalton NH2-terminal domain.

The complete amino acid sequence of the NH2-terminal domain obtained after trypsin digestion of human plasma fibronectin has been determined. It contains residues 1-259 and has a Mr of 29,000. The 29-kDa fragment was isolated from the other major tryptic cleavage products of 200, 180, and 31 kDa by affinity chromatography on gelatin- and heparin-Sepharose columns. The two high Mr fragments bound to gelatin and were easily removed; the 31-kDa fragment failed to bind to heparin while the 29-kDa fragment did and was eluted with 0.15 M NaCl. The 29-kDa domain has a blocked NH2-terminal (pyrrolidone carboxylic acid) which was removed by digestion with pyroglutamate aminopeptidase, and the amino acid sequence of the first 36 residues was obtained. The sequence showed a glutamine residue at position 3 which is probably the acceptor site for transglutaminase as reported for bovine fibronectin. Extensive trypsin digestion of the completely reduced and alkylated 29-kDa fragment yielded twenty-four peptides which were separated and purified by high performance liquid chromatography; their amino acid composition and amino acid sequence has been determined and the arrangement of peptides was achieved by comparison with the sequence recently reported for bovine fibronectin. The sequences in human and bovine fibronectin were nearly identical with only nine amino acid differences which can all be explained by single base substitutions. Apparently this domain is highly conserved in the two species studied thus far.

Alkylation↗
PubMed1983

A new mechanism for tumor induced platelet aggregation. Comparison with mechanisms shared by other tumor with possible pharmacologic strategy toward prevention of metastases.

Because tumor-induced platelet aggregation appears to play a role in the development of certain experimental tumor metastases, we examined the mechanism(s) of tumor-induced platelet aggregation as well as the effect of various anti-platelets agents. Two mechanisms for tumor-induced platelets aggregation have been previously described: (1) a mechanism which requires complement, a stable plasma factor, divalent cation and a sialo-lipo-protein vesicular component of the tumor membrane for platelet aggregation; and (2) a mechanism which operates via the generation of thrombin and requires a phospholipid component of the tumor membrane. We now report a new mechanism of tumor-induced platelet aggregation which is shared by three different tumors: a spontaneously metastatic human melanoma, HM29, a murine melanoma, B16F10, and a carcinogen-induced metastatic murine colon carcinoma, CT26. These tumors do not require cell-surface sialic acid or serum complement as does the first mechanism. They do not require cell-surface phospholipid, as do the tumors representing the other two mechanism. They do not aggregate platelets via the generation of thrombin as do tumors representing the second mechanism. These tumors are unique in that they require a trypsin-sensitive surface protein for activity. The ability of the thrombin-generating tumors to aggregate platelets is uniquely sensitive to two highly specific, synthetic thrombin-competitive inhibitors: DAPA and No. 805. The other two groups of tumors are at least 10 times more sensitive to inhibition of platelet aggregation by elevation of cyclic AMP levels (prostacyclin, 6-keto-PGE1, dibutyryl cyclic AMP) and at least 10 times more sensitive to inhibition of prostaglandin synthesis (indomethacin, ibuprofen). Thus, tumor-induced platelet aggregation is heterogeneous with respect to mechanism of action as well as inhibition by anti-platelet pharmacologic agents. Sensitivity to anti-platelet agents correlates with the mechanism by which tumor cells aggregate platelets.

Adenocarcinoma↗
PubMed1983

Requirements for the binding of human plasma fibronectin to the C1q subunit of the first component of complement.

We have shown previously that [125I]fibronectin (Fn) binds to solid-phase C1q in a dose-dependent manner. When C1r and C1s were added, the binding of Fn to C1q was abolished; removal of C1r and C1s restored Fn binding to C1q. In this report, we have systematically examined the optimal conditions that favor the Fn-C1q interaction. Our studies show that purified native 125I-Fn binds to C1q in a specific, saturable manner. Maximal 125I-Fn binding to C1q and gelatin occurs at low ionic strength (mu = 0.05) and drops sharply as the ionic strength is increased. At mu = 0.20, the binding to C1q is inhibited by 95%, whereas the binding to gelatin is inhibited by 50%. Optimal binding of Fn to C1q and gelatin occurs between pH 5.5 and 7.5, is decreased by 45% at 4 degrees C, and increases with incubation time; saturation of binding occurs in 60 min at 37 degrees C. Scatchard analysis of binding at mu = 0.05 indicates a single class of high affinity binding sites (Kd = 3.7 X 10(-8) M +/- 0.36 SD). The Kd of the reaction when C1q is bound to either plastic or immune complexes is essentially the same, and, in both cases, increases as the ionic strength of the medium is increased. Finally, significant binding of Fn to C1q can be demonstrated at physiologic ionic strength employing either insoluble immune complexes containing C1q or chemically cross-linked C1q.

Animals↗
PubMed1982

The interaction of human plasma fibronectin with a subunit of the first component of complement, C1q.

Fibronectin is a normal plasma protein that enhances reticuloendothelial system functioning, and may participate in immune complex clearance. The interaction of 125I-fibronectin with human C1 and C1q in vitro was investigated by employing a highly reproducible solid-phase binding assay in microtiter wells. We demonstrated that although fibronectin does not bind to antigen-antibody complex (BSA-anti-BSA) or immune complexes containing C1, a 20-fold increase in binding was obtained when the complexes contained C1q alone. In the absence of antigen-antibody complexes, fibronectin binds to the C1q fixed to the wells in a dose-response fashion but not to intact C1. C1q in the fluid phase inhibits 85% of the fibronectin binding to immobilized C1q. The amount of fibronectin bound by immobilized C1q or gelatin is approximately equal. The binding of fibronectin to C1q could be inhibited by the restoration of C1r + C1s to the C1 macromolecular complex before the addition of fibronectin. The inhibition was dependent on the concentration of C1r + C1s and achieved a maximum of 70% at 100 micrograms/ml. This inhibition could be reversed by the removal of C1r and C1s subunits with EDTA or C1 inhibitor. Digestion of C1q with pepsin resulted in an 85% loss of fibronectin binding. It therefore appears that at least one site of fibronectin binding to C1q is in the globular portion of this complement component.

Animals↗
PubMed1982

Platelet aggregating material (PAM) of two virally-transformed tumors: SV3T3 mouse fibroblast and PW20 rat renal sarcoma. Role of cell surface sialylation.

Platelets are required for certain experimental tumor metastases and several lines of tumor cells have been shown to aggregate platelets. We have extracted a sedimentable sialolipoprotein, platelet aggregating material (PAM) from the cell surface of SV40 transformed Balb C3T3 fibroblasts which aggregates heparinized PRP at 2.5 micrograms/ml via the release reaction, following a one minute lag period. A similar extract from non-transformed 3T3 cells has barely measurable activity at 40 micrograms/ml. Gel-filtered platelets (GFP) do not aggregate with PAM. However, PAM aggregation can be restored by addition of 5% plasma but not by fibrinogen. Two plasma components are required: a heat-labile complement component which is activated during the lag period; and a heat-stable factor which is required for platelet aggregation. The pathophysiologic significance of PAM has been examined in ten variant cell lines derived from a spontaneously metastatic renal cell sarcoma of rats, initially induced with polyoma virus (PW20 Wistar-Furth parental lines). These lines were selected in vitro and in vivo from a single line and differed in their capacity to form distant tumors in various organs after subcutaneous injection. These cells were examined for cell surface sialylation, PAM and PAM sialic acid content, since cell surface sialic acid is increased in a variety of tumor tissues and PAM is inhibited by neuraminidase. A good correlation was obtained between in vivo metastatic potential and cell surface sialic acid, r = 0.83, p less than 0.003; cell surface sialic acid and PAM, r = 0.85, p less than 0.002; in vivo metastatic potential and sialic acid content of PAM, r = 0.69, p less than 0.03; and in vivo metastatic potential and PAM, r = 0.68, p less than 0.03. We conclude that platelets may play a role in hematogenous metastasis via the ability of tumor cells to aggregate platelets by cell surface constituents containing sialic acid. The platelet-tumor cell interaction requires activation of the alternate complement pathway and a heat stable plasma factor.

Animals↗
PubMed1981

Fibonectin is a component of the surface coat of human neutrophils.

Although adherence to surfaces is central to neutrophil function many of the determinants of neutrophil adherence are still unknown. The possible involvement of cell surface material, fibronectin in particular, was therefore studied. Surface coat material was visualized ultrastructurally by the ferrocyanide--reduced osmium technique of Karnovsky (1971). Loosely attached surface coat material was seen distributed uniformly on cells in suspension. Indirect immunofluorescence indicated the presence of fibronectin on the neutrophil surface. Distribution of fibronectin as determined by indirect immunoferritin localization corresponded with the distribution of cell coat material. Some, if not all, of this fibronectin was synthesized by neutrophils themselves since metabolically labelled fibronectin could be obtained by immunoprecipitation after short-term culture with [36S]methionine. Neutrophils also adhere to Sepharose beads to which gelatin is covalently linked (GS) but not to plain Sepharose beads (PS). In the process they transfer surface coat material to GS but not PS. Similar transfer was seen when cells were permitted to adhere to glass or plastic coverslips. Indirect immunofluorescence showed that fibronectin-containing material was transferred from neutrophils to GS but not PS. Parallel studies with antisera to 2 other plasma proteins, factor VIIIR and alpha 1-antitrypsin showed that neutrophils did not transfer these to either GS or PS beads. The data suggest that material antigenically and functionally related to fibronectin is associated with the extracellular coat of neutrophils and is transferred with cell surface material to surfaces to which neutrophils adhere.

Cell Adhesion↗
PubMed1981

Role of platelets in tumor cell metastases.

Platelets may have a role in the development of animal tumor metastases. Ultrastructural studies in vivo have shown arrested tumor emboli surrounded by platelets. Several tumor cell lines induce thrombocytopenia in vivo. Certain tumor cells aggregate platelets in vitro. Correlations exist between the ability of some tumor cells to aggregate platelets in vitro and their metastatic potential in vivo. Antiplatelet agents have impaired or altered the spread of certain tumor metastases. It is suggested that platelets have a role in the sequestration, adherence, and penetration of tumor cells through the blood vessel endothelial cell barrier, thus preventing their rapid clearance from the circulation and allowing extravascular formation of nests of cells. Antiplatelet agents, particularly prostaglandins, may prove useful in preventing experimental animal metastases when administered before the inoculation of tumor cells. Their potential in human malignancy, where the patient presents with an established tumor, remains to be established.

Animals↗
PubMed1981

Inhibition of the platelet-aggregating activity of two human adenocarcinomas of the colon and an anaplastic murine tumor with a specific thrombin inhibitor, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide.

Platelets are required for certain experimental metastases. Several lines of animal tumor cells aggregate platelets in vitro and in vivo. Previous studies with one of these lines, an SV40-transformed 3T3 mouse fibroblast (SV3T3) have revealed that the platelet-aggregating material is an extractable membrane-associated sialolipoprotein which requires divalent cation, complement, and a heat-stable plasma component for activity. Little information is available on the interaction of human tumors with platelets. We now report on the ability of two human adenocarcinomas of the colon (LoVo and HCT-8) and an anaplastic mouse tumor (Hut-20) to aggregate platelets by a different mechanism, the generation of thrombin. These spontaneous cell lines aggregate human or rabbit platelet-rich plasma after a 1- to 2-min lag period. This is often followed by a visible clot. Unlike SV3T3 cells, aggregation by LoVo, HCT-8, and Hut-20 cells is not inhibited by neuraminidase, trypsin, or cobra venom factor. These three cell lines markedly shorten the recalcification time of citrated plasma, whereas SV3T3 cells do not. Phospholipase A2 treatment inhibits the shortening of the recalcification time for the three tumors; this parallels its inhibitory effect on platelet aggregation. LoVo, HCT-8, and Hut-20 cells generate thrombin via the "tissue factor" coagulation pathway (using coagulation factor-deficient substrates). Dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, a highly specific, potent antithrombin antagonist, inhibits LoVo-, HCT-8-, and Hut-20-induced platelet aggregation at 4 to 15 microM, whereas its effect on SV3T3 cells is negligible. If platelets are required for certain human tumor metastases, dansylarginine-N-(3-ethyl-1, 5-pentanediyl)amide, or other antithrombin agents, may prove to be valuable therapeutic agents.

Adenocarcinoma↗
PubMed1980

Fibronectin-collagen binding and requirement during cellular adhesion.

Fibronectin isolated from human plasma and from the extracellular matrices of cell monolayers mediates the attachment in vitro and spreading of trypsin-treated cells on a collagen substratum. Fibronectin-dependent kinetics of cellular attachment to collagen were studied for several adherent cell types. It was shown that trypsin-treated human umbilical-cord cells, mouse sarcoma CMT81 cells, endothelial cells, and human fibroblasts from a patient with Glanzmann's disease were completely dependent on fibronectin for their attachment to collagen, whereas guinea-pig and monkey smooth-muscle cells and chick-embryo secondary fibroblasts displayed varying degrees of dependence on fibronectin for their attachment. Radiolabelled human plasma fibronectin possessed similar affinity for collagen types I, II and III from a variety of sources. The fibronectin bound equally well to the collagens with or without prior urea treatment. However, in the fibronectin-mediated adhesion assay using PyBHK fibroblasts, a greater number of cells adhered and more spreading was observed on urea-treated collagen. Fibronectin extracted from the extracellular matrix of chick-embryo fibroblasts and that purified from human plasma demonstrated very similar kinetics of complexing to collagencoated tissue-culture dishes. Fibronectin from both sources bound to collagen in the presence of 0.05-4.0m-NaCl and over the pH range 2.6-10.6. The binding was inhibited when fibronectin was incubated with 40-80% ethylene glycol, the ionic detergents sodium dodecyl sulphate and deoxycholate, and the non-ionic detergents Nonidet P-40, Tween 80 and Triton X-100, all at a concentration of 0.1%. From these results we proposed that fibronectin-collagen complexing is mainly attributable to hydrophobic interactions.

Animals↗