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Biomedical subjects

R Snyderman

Publications and source records attributed to R Snyderman.

At least 91 records · Page 5Linked to original sources
PubMed1987

Identification of a novel inositol bisphosphate isomer formed in chemoattractant stimulated human polymorphonuclear leukocytes.

Analysis of inositol phosphate formation in chemoattractant-stimulated human polymorphonuclear leukocytes demonstrated the production of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,4-bisphosphate and another inositol bisphosphate isomer not detected in unstimulated cells. Studies in cell sonicates provided evidence that the previously unidentified inositol bisphosphate isomer is produced via the degradation of inositol 1,3,4-trisphosphate. This unidentified inositol bisphosphate peak was purified by high pressure liquid chromatography, and base hydrolyzed to form a mixture of inositol monophosphate isomers. Based on these studies, the unidentified peak was identified as inositol 3,4-bisphosphate. Identification of this isomer defines a new metabolic product derived from the initial inositol 1,4,5-trisphosphate formation, and also suggests another substrate for the inositol 1-phosphatase.

Calcium↗
PubMed1987

Chemotactic factor and P15E-related chemotaxis inhibitor in human melanoma cell lines with different macrophage content and tumorigenicity in nude mice.

The present study was designed to characterize the production of chemoattractants by human melanoma lines with high (M4Be, M3Da, NTerDa) or low tumorigenic (Doc8, M1Do) potential when heterotransplanted in nude mice. Supernatants from the Doc8 and M1Do cell lines were strongly chemotactic in vitro for mononuclear phagocytes. Chemotactic activity was destroyed by proteolytic enzymes, and upon gel filtration on Sephadex G75, it eluted in the cytochrome c region corresponding to an apparent m.w. of 12,000. Upon chromatofocusing, the Sephadex-separated tumor-derived chemotactic factor (TDCF) showed an isoelectric point of 5.5 to 6. Cell lines with high tumorigenic potential contained low or no detectable chemotactic activity. When culture supernatants of cell lines with modest (M3Da) or no (M4Be) chemotactic activity were exposed to immobilized monoclonal antibodies directed against the retroviral transmembrane protein P15E, appreciable chemotactic activity was detectable (M4Be) or preexisting levels increased (M3Da). The material eluted from Sepharose-bound anti-P15E antibodies inhibited the migration of monocytes in response to chemoattractants. These findings demonstrate the coexistence in some human melanoma cell line supernatants of factors (TDCF and P15E-related inhibitor) with opposite influence on monocyte chemotaxis. That tumor cell products play a pivotal role in regulating the extravasation of monocytes into neoplastic tissues is suggested by the close correlation observed between macrophage levels in melanomas grown in nude mice and levels of chemotactic activity detectable in culture supernatants.

Animals↗
PubMed1987

Inhibition of human natural killer cell activity by a synthetic peptide homologous to a conserved region in the retroviral protein, p15E.

It has been shown previously that the retroviral envelope protein p15E suppresses certain monocyte and lymphocyte functions. In this paper, we describe the effects on natural killer (NK) activity of a synthetic peptide (CKS-17) with homology to a region of p15E conserved among numerous retroviruses. Enriched human NK cells were assayed against K562 tumor target cells in a 51Cr-release cytotoxicity assay. Pretreatment of NK cells with CKS-17 at concentrations as low as 1.5 microM, but not with equivalent concentrations of control materials, markedly and reproducibly suppressed NK lytic activity. Prior exposure of NK cells to interferon-alpha (IFN-alpha) at 1000 U/ml did not alter their sensitivity to CKS-17-induced inhibition. Pretreating NK cells with CKS-17 almost entirely diminished their responsiveness to IFN-alpha and IFN-gamma, but not to interleukin 2 (IL 2). Kinetics experiments demonstrated that CKS-17-mediated suppression of both endogenous and activated NK cells was reversible after 18 hr at 37 degrees C. Experiments designed to examine the CKS-17 mechanism of action revealed that the peptide bound to all Leu-11+ lymphocytes, as shown by two-color flow cytometry. CKS-17 did not, however, inhibit effector cell/target cell conjugate formation. These data suggest a new mechanism for immune suppression mediated by retroviruses; inhibition of NK function. They moreover imply that the CKS-17 peptide interferes with the lytic phase of NK cytolysis.

Adolescent↗
PubMed1987

Human Gi protein alpha-subunit: deduction of amino acid structure from a cloned cDNA.

The amino acid sequence of the alpha-subunit of Gi, the human adenylate cyclase inhibiting GTP-binding protein, has been deduced from the nucleotide sequence of a DNA clone complementary to Gi alpha mRNA from differentiated U937 cells. The cDNA encodes a polypeptide of 355 amino acids (Mr 40456). The amino acid sequence homology between human Gi alpha and rat, murine, and bovine Gi alpha is 98.6, 97.7 and 87.9% respectively. Differentiation of the U937 cells from monoblasts to monocyte-like cells resulted in a 3-fold increase in Gi alpha mRNA as well as a 3.6-fold increase in the 41 kDa pertussis toxin substrate presumed to be Gi alpha. Thus, increased levels of this G-protein are associated with monocyte differentiation and appear to be regulated transcriptionally.

Adenylyl Cyclase Inhibitors↗
PubMed1987

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes.

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.

Calcium↗
PubMed1987

Altered cell-averaged microviscosity of murine peritoneal macrophages undergoing activation in vivo or in vitro.

The cell-averaged microviscosity of intact murine peritoneal mononuclear phagocytes in various stages of activation was assessed by quantifying fluorescent depolarization of 1,6-diphenyl-1,3,5-hexatriene. Macrophages activated in vivo with Mycobacterium bovis, strain BCG, were significantly more fluid than resident peritoneal macrophages, responsive macrophages elicited with thioglycollate broth, proteose peptone broth, or fetal bovine serum, or primed macrophages elicited with pyran copolymer, MVE-2. Specifically, the cell-averaged microviscosity decreased from a mean of 3.47 +/- .07 eta 25 degrees C (poise) (range of 3.32 to 3.67 p) to 2.62 eta 25 degrees C. Exposure of responsive macrophages in vitro to bacterial endotoxin plus hybridoma supernatants containing macrophage-activating factor or purified recombinant interferon gamma resulted in decreased microviscosity; the largest effect was seen after 24 hr. Macrophages primed in vivo with MVE-2 and treated in vitro with endotoxin also developed decreased microviscosity. Similar changes in microviscosity were observed in a plasma membrane-enriched fraction isolated from macrophages activated in vitro with interferon gamma and endotoxin, thus suggesting that the cell-averaged measurements reflected changes in membrane viscosity. The optimum concentration of MAF-inducing decreased overall microviscosity was identical to that for inducing tumoricidal capacity. Taken together, the data indicate activation of lytic capacity in murine macrophages is closely associated with decreased cell-averaged microviscosity and that this change reflects, at least in part, decreased microviscosity of the plasma membrane of these cells.

Animals↗
PubMed1987

Suppressive effect on polyclonal B-cell activation of a synthetic peptide homologous to a transmembrane component of oncogenic retroviruses.

Purified feline leukemia virus, UV light-inactivated feline leukemia virus, and a synthetic peptide (CKS-17) homologous to a well-conserved region of the transmembrane components of several human and animal retroviruses were each studied for their effects on IgG production by feline peripheral blood lymphocytes. Using a reverse hemolytic plaque assay, both the viable virus and the UV-inactivated feline leukemia virus, but not the CKS-17, activated B lymphocytes to secrete IgG. When staphylococcal protein A, a polyclonal B-cell activator, was used to stimulate IgG synthesis by feline lymphocytes, the viable virus, the UV-inactivated virus, and the CKS-17 peptide each strongly suppressed IgG secretion without compromising viability of the lymphocytes. These findings suggest that the immunosuppressive influences of feline leukemia virus on immunoglobulin synthesis may reside in a conserved portion of the envelope glycoprotein that includes the region homologous to CKS-17.

Amino Acid Sequence↗
PubMed1987

Recombinant hydrophilic region of murine retroviral protein p15E inhibits stimulated T-lymphocyte proliferation.

Retroviral envelope protein p15E and antigenically related proteins have been implicated as potential mediators of immune dysfunction associated with retroviral infections and with neoplasia. Due to its extreme hydrophobicity, purified p15E has not been available in a nondenatured form or in sufficient quantities for detailed studies on the mechanisms of its immunosuppressive effects. Therefore, a plasmid was constructed to direct the synthesis in Escherichia coli of the major hydrophilic region of murine p15E. The purified recombinant p15E derivative, soluble under physiological conditions, inhibited by up to 60% (EC50 = 7.5 nM) the anti-CD3-driven proliferation of human T lymphocytes but had no effect on the proliferation of the transformed T-cell line Jurkat. The recombinant protein also inhibited, by up to an average of 92% (EC50 = 2.1 microM), the proliferation of the murine T-cell line CTLL-2. These data (i) provide direct evidence that a retroviral envelope protein can itself inhibit lymphoproliferative function and (ii) map the inhibitory activity to a specific region of p15E. The availability of soluble, recombinant p15E should facilitate studies of the pathogenesis of the immunosuppression accompanying retroviral infections and neoplastic diseases.

Animals↗
PubMed1987

A conserved region at the COOH terminus of human immunodeficiency virus gp120 envelope protein contains an immunodominant epitope.

A highly immunogenic epitope from a conserved COOH-terminal region of the human immunodeficiency virus (HIV) gp120 envelope protein has been identified with antisera from HIV-seropositive subjects and a synthetic peptide (SP-22) containing 15 amino acids from this region (Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg). Peptide SP-22 absorbed up to 100% of anti-gp120 antibody reactivity from select HIV+ patient sera in immunoblot assays and up to 79% of serum anti-gp120 antibody reactivity in competition RIA. In RIA, 45% of HIV-seropositive subjects had antibodies that bound to peptide SP-22. Human anti-SP-22 antibodies that bound to and were eluted from an SP-22 affinity column reacted with gp120 in RIA and immunoblot assays but did not neutralize HIV or inhibit HIV-induced syncytium formation in vitro, even though these antibodies comprised 70% of all anti-gp120 antibodies in the test serum. In contrast, the remaining 30% of SP-22 nonreactive anti-gp120 antibodies did not react with gp120 in immunoblot assays but did not react in RIA and neutralized HIV in vitro. Thus, approximately 50% of HIV-seropositive patients make high titers of nonneutralizing antibodies to an immunodominant antigen on gp120 defined by SP-22. Moreover, the COOH terminus of gp120 contains the major antigen or antigens identified by human anti-gp120 antibodies in immunoblot assays.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Lymphoma models for B cell activation and tolerance. V. Anti-Ig mediated growth inhibition is reversed by phorbol myristate acetate but does not involve changes in cytosolic free calcium.

B cell lymphomas which can be growth inhibited by crosslinking their surface IgM receptors by anti-Ig reagents provide models for normal B cell regulation and tolerance. WEHI-231 and CH31 are two independently derived lines that are exquisitely sensitive to negative signalling by antibodies specific for mu or kappa chains, but are unaffected by antibodies against MHC class 1 or 2 antigens. In order to determine the mechanism of this growth inhibition as a model for tolerance, we have examined the roles played by protein kinase C activation and calcium mobilization/influx during negative signalling in these cells. We found that growth inhibition caused by anti-mu crosslinking was reversed in the presence of either phorbol myristate acetate (PMA) or by lipopolysaccharide (LPS) from E. coli. The effect of PMA on negative signalling was a true reversal since phorbol esters could be added after anti-mu treatment, thus allowing nearly normal cellular progression into the S phase of the cell cycle. In contrast, pretreatment with PMA did not provide protection against the growth inhibition from anti-mu. Indeed, a "desensitization" protocol demonstrated that PMA pretreatment actually decreased reversal by both PMA and LPS of the effects of anti-mu on B lymphoma growth. These studies suggest that both LPS and PMA act via at least one common intermediate, which is assumed to involve activation and translocation of protein kinase C. Analysis of changes in calcium ion concentration after treatment with anti-Ig reagents showed both mobilization from internal stores and influx via calcium channels in WEHI-231, as has been reported for normal B cells. However, these changes did not correlate with negative signalling for the several reasons. Firstly, anti-mu inhibition of the growth of WEHI-231 could be induced in the relative absence of extracellular Ca++ or in quin-2 loaded (buffered) cells. Secondly, pretreatment with high concentrations of PMA ablated calcium mobilization, yet failed to modulate growth inhibition in WEHI-231 cells. Moreover, LPS provided protection from the effects of anti-mu yet did not alter cellular [Cai++]. In addition, PMA posttreatment (under conditions causing a reversal of the effects of anti-mu) can be applied as long as four hours after the initial exposure to anti-mu and the rapid measurable changes in calcium flux. Indeed, such changes in intracellular free calcium occurred in elutriated WEHI-231 lymphoma cells at all phases of the cell cycle, although we have previously identified early G1 as the only critical period in which negative signalling can be delivered.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Coexistence of a chemotactic factor and a retroviral P15E-related chemotaxis inhibitor in human tumor cell culture supernatants.

Two sets of seemingly contradictory evidence have been reported concerning the effects of tumor cell products on the regulation of monocyte migration in vitro and presumably the extravasation of macrophages into tumors in vivo. The present study was designed to explore the relationship between chemotactic and anti-chemotactic products related to tumor cells: a tumor-derived chemotactic factor (TDCF) and retroviral P15E-related inhibitor(s) of chemotaxis. Culture supernatants of the human 8387 sarcoma and SW626 ovarian carcinoma were depleted of P15E-related antigens with immobilized anti-P15E monoclonal antibodies. This treatment produced a significant and consistent increase of the polarizing and chemotactic activity in the tumor cell supernatants. The material eluted from Sepharose-bound anti-P15E antibodies was devoid of chemotactic and polarizing activity and suppressed the polarization and migration of monocytes in response to chemoattractants. These results demonstrate the coexistence in culture supernatants of two human tumor cell lines of factors with opposite influences on monocyte chemotaxis. The data suggest that the entry of monocytes into neoplastic tissue may be regulated by the interplay of chemotactic and anti-chemotactic principals produced by tumor cells.

Antigen-Antibody Complex↗
PubMed1986

A pertussis/choleratoxin-sensitive N protein may mediate chemoattractant receptor signal transduction.

Chemoattractant receptors on phagocytic leukocytes utilize a guanine nucleotide regulatory (N) protein to activate phospholipase C and subsequent biological responses. Since pertussis toxin inhibits activation of leukocytes by chemoattractants and ribosylates a ca. 40 kD protein in these cells it had generally been assumed that chemoattractant receptors are coupled to Ni. We now report that human polymorphonuclear leukocytes (PMNs), monocytes, and the myeloid HL-60 and U937 cell lines, but not erythrocytes or bovine brain contain a ca. 40 kD protein which is a substrate for ADP ribosylation by choleratoxin (CT). This N protein, termed Nc for chemotaxis-related N protein, comigrates with the ca. 40 kD PT substrate during one-dimensional gel electrophoresis. In vivo treatment of PMNs with PT or CT reduced high affinity binding of chemoattractants to membrane preparations from the cells, implying that chemoattractant receptors are coupled to an N protein which is a substrate for both PT and CT. We suggest that Nc rather than Ni couples chemoattractant receptors to phospholipase C.

Adenosine Diphosphate Ribose↗
PubMed1986

Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols.

Binding of chemoattractants to receptors on human polymorphonuclear leukocytes (PMN) stimulates the phosphodiesteric cleavage of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and 1,2-diacylglycerols. To investigate the possible second messenger function of diacylglycerols in PMN activation, we tested the ability of a series of synthetic sn 1,2-diacylglycerols, known to stimulate protein kinase C in other systems, to promote superoxide anion release, oxygen consumption, lysosomal enzyme secretion, and chemotaxis. None of the diacylglycerols initiated the chemotactic migration of PMN. Several of the diacylglycerols however, were, active in stimulating superoxide anion release and lysozyme secretion, with dioctanoylglycerol (diC8) being the most potent. Unexpectedly, didecanoylglycerol (diC10) induced lysosomal enzyme secretion, but failed to stimulate superoxide production or oxygen consumption. All other biologically active diacylglycerols tested displayed similar EC50 for stimulating lysozyme secretion and superoxide production. The ability of the diacylglycerols to compete for phorbol dibutyrate (PDBu) binding in intact PMN suggested a mechanism for the divergent biological activity of diC10. Although the compounds that stimulated both superoxide production and lysosomal enzyme secretion competed for essentially all [3H]PDBu binding from its receptor, diC10, which only stimulated secretion, competed for 45% of the bound [3H]PDBu. Thus diacylglycerols can selectively activate certain functions of leukocyte chemoattractant receptor. The data suggest that a discrete pool of protein kinase C may mediate activation of the respiratory burst in PMN.

Binding, Competitive↗
PubMed1986

Extensive hydrolysis of N-formyl-L-methionyl-L-leucyl-L-[3H] phenylalanine by human polymorphonuclear leukocytes. A potential mechanism for modulation of the chemoattractant signal.

Chemoattractant receptors on human polymorphonuclear leukocytes (PMNs) stimulate a series of important biological responses. In an attempt to better understand the mechanism of stimulus response coupling of chemoattractant receptors, the kinetics of N-formyl-L-methionyl-L-leucyl-L-[3H]phenylalanine (fMet-Leu-[3H]Phe) binding to PMNs was evaluated. Unexpectedly, extensive degradation of the ligand was found to occur rapidly at 37 degrees C. Exposure of 10(7) cells/ml to 10 nM fMet-Leu-[3H]Phe led to the specific uptake of approximately 1% of the ligand within 10 min, while approximately 50% of the extracellular chemoattractant was hydrolyzed to free amino acids. Under the same conditions, isolated plasma membranes equivalent to 2.5 X 10(7) PMNs/ml bound specifically approximately 1% of the ligand and degraded about one-half of it primarily to Leu-[3H]Phe. The fMet-Leu-[3H]Phe hydrolysis commenced with no apparent latency, yet disobeyed first order kinetics as a 100-fold excess unlabeled ligand enhanced the initial consumption rate of 10 nM fMet-Leu-[3H]Phe by 500-fold, yielding an enhancement of the relative hydrolysis to approximately 70%. 1-butanol at 0.25%, which accelerates chemotaxis but inhibits superoxide anion and lysosomal enzyme secretion, reduced the hydrolysis to about 15% independent of the fMet-Leu-[3H]Phe specific activity. Lysosomal secretion could not mediate the hydrolysis process, since the supernatants of PMNs exposed either to 10 nM of 1 microM fMet-Leu-Phe reveal no degradation capacity toward fMet-Leu-[3H]Phe. These data indicate that the hydrolysis of the chemoattractant occurs at the cell surface and is dependent on the plasma membrane physical state. This phenomenon may well modulate the chemotactic signal due to its ability to profoundly alter the level of the chemoattractant proximate to the cell surface.

1-Butanol↗
PubMed1986

Receptor-coupled activation of phosphoinositide-specific phospholipase C by an N protein.

Cleavage of phosphatidylinositol 4,5-bisphosphate by phospholipase C results in the production of two important second messengers: inositol-1,4,5-trisphosphate and 1,2-diacylglycerol. Although several receptors promote this cleavage, the molecular details of phospholipase C activation have remained unresolved. In this study, occupancy of a Ca2+-mobilizing receptor, the oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte plasma membranes, was found to lead to the activation of a guanine nucleotide regulatory (N) protein by guanosine 5'-triphosphate. The activated N protein then stimulated a polyphosphoinositide-specific phospholipase C by reducing the Ca2+ requirement for expression of this activity from superphysiological to normal intracellular concentrations. Therefore, the N protein-mediated activation of phospholipase C may be a key step in the pathway of cellular activation by chemoattractants and certain other hormones.

Adenosine Triphosphate↗