A review of conscious sedation with orally administered benzodiazepines.
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Anesthesia, Dental↗
Biomedical subjects
R L Stevens
Publications and source records attributed to R L Stevens.
Proteoglycans in cell-mediated cytotoxicity. Identification, localization, and exocytosis of a chondroitin sulfate proteoglycan from human cloned natural killer cells during target cell lysis.
A clone of natural killer (NK) cells (JTB18) was found to be ultrastructurally similar to peripheral blood large granular lymphocytes (LGL). These cells incorporated [35S]sulfate into cell-associated proteoglycan molecules, which were then isolated by CsCl density gradient centrifugation. As assessed by gel filtration chromatography, the native 35S-labeled proteoglycan and its beta-eliminated 35S-labeled glycosaminoglycans were of Mr approximately 200,000 and 50,000, respectively. The 35S-labeled proteoglycans were resistant to proteolysis, since their Mr were apparently not altered by incubation with either pronase or S. aureus V8 protease. The purified NK cell 35S-labeled proteoglycans were degraded by approximately 90% to 35S-labeled disaccharides with either chondroitinase ABC or AC. High performance liquid chromatographic analysis of the digests revealed these disaccharides to be composed entirely of chondroitin sulfate A (glucuronic acid----N-acetylgalactosamine-4SO4). Whole 35S-labeled cells incubated with chondroitinase ABC failed to release 35S-labeled disaccharides into the supernatant, and x-ray energy-dispersive analysis revealed that sulfur-containing molecules were present in the intracellular granules, thereby localizing the NK cell-associated proteoglycan primarily in the granules of the cell, rather than on the plasma membrane. The 35S-labeled cloned NK cells incubated for 30 min to 4 h with K562 tumor cell targets at a 0.5:1 ratio exocytosed a mean of 49% of the granular 35S-labeled proteoglycans during the first 60 min of the culture. Proteoglycan release was maximal with an effector/target cell ratio of 0.5:1 for JTB18:K562. Significant proteoglycan release from JTB18 NK cells was also obtained with other sensitive target cells such as REX, Molt4, and CEM, but not with cells such as KG1 and Laz156, which have been shown previously to be resistant to killing by this NK cell. Thus, protease-resistant intracellular proteoglycans with chondroitin sulfate A side chains are specifically exocytosed from the granules of human NK effector cells upon contact with sensitive targets, suggesting that these proteoglycans may be involved in the mechanism of cytotoxicity.
Cell Line↗
Purification and analysis of the core protein of the protease-resistant intracellular chondroitin sulfate E proteoglycan from the interleukin 3-dependent mouse mast cell.
Chondroitin sulfate E proteoglycan was extracted in the presence of protease inhibitors from 6 X 10(9) mouse bone marrow-derived, interleukin 3-dependent mast cells, of which 3 X 10(7) had been biosynthetically labeled with [35S]sulfate or [3H]glycine. Chondroitin sulfate E proteoglycan was purified to apparent homogeneity by density-gradient centrifugation, differential molecular weight dialysis, DEAE-52 ion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. Chondroitin sulfate E proteoglycan, radiolabeled with [3H]glycine or [35S]sulfate, filtered as a single peak of radioactivity on Sepharose CL-4B with a Kav of 0.41. When purified [3H]glycine-labeled proteoglycan was digested with chondroitinase ABC and subjected to gel filtration, all of the radioactivity was shifted to a lower molecular weight. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr of the peptide core obtained by chondroitinase ABC treatment was approximately 10,000. The purified proteoglycan was resistant to degradation by collagenase, clostripain, trypsin, chymotrypsin, elastase, chymopapain, V8 protease, proteinase K, and Pronase, as assessed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the core peptide of the intact proteoglycan revealed that glycine, serine, and glutamic acid/glutamine accounted for 70% of the total amino acids and were present in a molar ratio of 4.3/1.6/1.0. When analyzed for neutral hexose content by gas-liquid chromatography, the proteoglycan contained approximately 2% of its weight as mannose, fucose, galactose, and other sugars, indicating that oligosaccharides were linked to the peptide core. The mouse bone marrow-derived mast cell chondroitin sulfate E proteoglycan, like the rat serosal mast cell heparin proteoglycan, is markedly protease resistant, has highly sulfated glycosaminoglycans, and contains a peptide core that is rich in serine and glycine. These characteristics of the mast cell class of intracellular proteoglycans may contribute to their function in stimulus-induced granule secretion as well as in mediator storage, including retention of cationic neutral proteases.
Amino Acids↗
Purification and characterization of protease-resistant secretory granule proteoglycans containing chondroitin sulfate di-B and heparin-like glycosaminoglycans from rat basophilic leukemia cells.
Proteoglycans were extracted from nuclease-digested sonicates of 10(9) rat basophilic leukemia (RBL-1) cells by the addition of 0.1% Zwittergent 3-12 and 4 M guanidine hydrochloride and were purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography, and Sepharose CL-6B gel filtration chromatography under dissociative conditions. Between 0.3 and 0.8 mg of purified proteoglycan was obtained from approximately 1 g initial dry weight of cells with a purification of 200-800-fold. The purified proteoglycans had a hydrodynamic size range of Mr 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, alpha-chymotrypsin, Pronase, papain, chymopapain, collagenase, and elastase. Amino acid analysis of the peptide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%). Approximately 70% of the glycosaminoglycan side chains of RBL-1 proteoglycans were digested by chondroitinase ABC and 27% were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by beta-elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparin/heparan sulfate) glycosaminoglycans were of approximately Mr 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high performance liquid chromatography revealed that 9-29% were the unusual disulfated disaccharide chondroitin sulfate di-B (IdUA-2-SO4----GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA----GalNAc-4-SO4. Subpopulations of proteoglycans in one preparation were separated by anion exchange high performance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. However, no segregation of subpopulations without both chondroitin sulfate di-B and heparin/heparan sulfate glycosaminoglycans was achieved, suggesting that RBL-1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-6B chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hydrodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acids↗
Novel inhibition of proteoglycan synthesis and exocytosis by diethylcarbamazine in the Swarm rat chondrocyte.
Pretreatment of cultured chondrosarcoma chondrocytes at 37 degrees C for 15 min with 15 mM diethylcarbamazine (DEC) followed by a 60-min pulse with [35S] sulfate in the presence of DEC resulted in an approximate 40% inhibition of synthesis and a 75% inhibition of secretion of 35S-proteoglycan. The inhibition was dose-related and was not due to a decrease in protein synthesis. Chondrocytes exposed for 75 min to 15 mM DEC, washed, incubated for 17 h in DEC-free medium, and then pulsed with [35S]sulfate showed no inhibition in the rate of synthesis of proteoglycan or in the per cent of radiolabeled proteoglycans exocytosed into the culture medium, indicating full reversibility of the inhibitory effect. When chondrocytes were incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, secretion of beta-D-xyloside-bound 35S-glycosaminoglycan was inhibited by more than 70% despite an approximate 3-fold increase in intracellular 35S-macromolecules, as compared to cells exposed to beta-D-xyloside alone. Upon removal of DEC, the block in the secretion of beta-D-xyloside-bound 35S-glycosaminoglycans was reversed, although there was a 15-30-min lag in the initiation of exocytosis. Light and electron microscopic examination of chondrocytes after 75 min of incubation with 15 mM DEC revealed large vacuoles, a distended Golgi apparatus, and a distended endoplasmic reticulum which contained electron dense material. Upon removal of DEC, the vacuoles disappeared and distended organelles returned to their normal appearance between 15 and 30 min, coincident with the start of exocytosis of 35S-proteoglycan and beta-D-xyloside-bound 35S-glycosaminoglycan. These biochemical and morphological studies indicate that DEC treatment of chondrosarcoma chondrocytes alters the transport of molecules from the endoplasmic reticulum to the Golgi and the transport of molecules from the Golgi to the cell surface.
Animals↗
Heterogeneity of mammalian mast cells differentiated in vivo and in vitro.
The evidence for mast cell heterogeneity in vivo has grown in the last several years from the initial morphologic and histochemical observations to biochemical and functional criteria. In several species, there appear to be at least two distinct classes of tissue mast cells that differ in their content of intragranular amines, proteoglycans, and proteases and in their metabolism of arachidonic acid after cell activation. With the recent discovery of differentiation/growth factors for mast cells in vitro, it has been shown that a discrete subpopulation of cells resembling one of the two in vivo classes can be derived in culture. The diversity of mast cell mediators synthesized by these various populations and their potential functions in vivo imply that mast cells may be involved not only in allergic responses but also in other inflammatory processes, including tissue repair and growth.
Amines↗
Homology of the rat basophilic leukemia cell and the rat mucosal mast cell.
Secretory granules of the rat basophilic leukemia (RBL-1) cell, a chemically generated tumor cell line maintained in tissue culture, were shown to stain with alcian blue but not with safranin counterstain and to have sparse, small, electron-dense granules. A Mr 25,000 protein was the major [3H]diisopropyl fluorophosphate-binding protein in extracts of RBL-1 cells. Double-immunodiffusion analysis of extracts revealed immunoreactivity for rat mast cell protease (RMCP)-II, a Mr 25,000 neutral protease present in the secretory granules of rat mucosal mast cells and cultured rat bone marrow-derived mast cells, but no immunoreactivity for RMCP-I, the predominant neutral protease of rat connective tissue mast cells. By radial immunodiffusion, there was 66.8 ng of RMCP-II per 10(6) cells. Whereas rat connective tissue mast cells stain with alcian blue and safranin and contain heparin proteoglycan, rat mucosal and rat bone marrow-derived mast cells stain with alcian blue only and contain a non-heparin proteoglycan and lesser amounts of histamine. Proliferation of rat mucosal mast cells in vivo and rat bone marrow-derived mast cells in vitro requires T-cell factors, whereas no comparable requirement has been observed for connective tissue mast cells. The transformed RBL-1 tumor cells, whose growth is independent of factors other than those present in standard tissue culture medium, has previously been shown to contain predominantly chondroitin sulfate di-B proteoglycans and low amounts of histamine. The similar histology and secretory granule biochemistry of the rat mucosal mast cells, rat culture-derived mast cell, and RBL-1 cell suggest that they comprise a single mast cell subclass distinct from the rat connective tissue mast cell.
Animals↗
Unique features of human basophilic granulocytes developed in in vitro culture.
Human basophilic granulocytes were obtained in suspension culture of mononuclear cells from umbilical cord blood. Precursors of basophils in the cord blood are nonadherent cells and bear neither surface Ig nor T cell marker. Cultured basophils contain chondroitin-4-sulfate proteoglycan, 1.74 micrograms histamine per 10(6) cells in average, and bear 120,000-380,000 IgE receptors per cell. The IgE receptor molecule has a molecular weight of approximately 64,000 daltons. Human, rat and mouse IgE bind to the receptors with comparable high affinity and passively sensitize the cells for mediator release. Challenge of sensitized basophils with anti-IgE resulted in stimulation of phospholipid methylation, 45Ca uptake, release of both histamine and free arachidonic acid, and enhancement of phosphatidylinositol (PI) turnover. Evidence was obtained that the activation of membrane-associated proteolytic enzyme and methyltransferases is involved in subsequent IgE-mediated biochemical cascades such as PI turnover, Ca2+ uptake and mediator release.
Antibodies, Anti-Idiotypic↗
Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro.
Rat serosal heparin-containing mast cells (HP-MC) were maintained in vitro for as long as 30 days when co-cultured with mouse skin-derived 3T3 fibroblasts. In contrast, when the mast cells were cultured alone, on fibronectin-, gelatin-, or dermal-collagen-coated dishes, on acid and heat-killed fibroblasts in the presence or absence of 24 hr fibroblast-conditioned medium, or on a monolayer of mouse serosal macrophages, they failed to adhere to the dishes, released significant amounts of their histamine and lactate dehydrogenase, and stained with trypan blue, indicating a loss of viability. The rat serosal HP-MC cultured with the 3T3 fibroblasts became so adherent to the fibroblasts that the two cell types could be separated from one another only by trypsinization. The cultured HP-MC stained with both alcian blue and safranin and continued to synthesize proteoglycan at a rate comparable to that of freshly isolated cells. The 35S-labeled proteoglycan synthesized by these cultured cells, like that produced by freshly isolated rat serosal HP-MC, was a 750,000 to 1,000,000 m.w. proteoglycan containing only heparin glycosaminoglycans of 50,000 to 100,000 m.w. When HP-MC were cultured for 1 wk with the fibroblasts and were then incubated for 5 min with a 1/20 dilution of rabbit anti-rat IgE, they generated and released an average of 22 +/- 10 ng (mean +/- SD, n = 5) of prostaglandin D2 per 10(6) cells and exocytosed a higher net percentage of their total histamine content (44 +/- 11% [mean +/- SD, n = 8]) than did cells just isolated from the animal (6 +/- 4% [mean +/- SD, n = 4]). As assessed by electron microscopy, many of the cultured HP-MC resembled freshly isolated cells except that some secretory granules had fused with one another in some cells. Morphologically, after activation the cultured HP-MC underwent compound exocytosis like freshly isolated cells. These results demonstrate that the in vivo differentiated rat HP-MC maintain their histology, morphology, immunologic responsiveness, histamine content, and ability to synthesize heparin proteoglycan when co-cultured with living fibroblasts.
Animals↗
Identification of the neutral glycosphingolipids of murine mast cells: expression of Forssman glycolipid by the serosal but not the bone marrow-derived subclass.
The expression of neutral glycosphingolipids by mouse T cell-dependent, bone marrow-derived mast cells (BMMC) obtained in vitro was determined by chromatographic and immunochemical criteria. Neutral glycosphingolipids were isolated from BMMC by extraction of 3 to 5 X 10(8) cells in chloroform/methanol (1/1, v/v) and chromatography on DEAE-Sephadex, and were analyzed by thin layer chromatography with orcinol staining. The predominant neutral glycosphingolipids of BMMC were glucosylceramide (CMH), lactosylceramide (CDH), globotriosylceramide (CTH), globotetraosylceramide (globoside), and a molecule migrating slightly faster than gangliotetraosylceramide (asialo GM1) and slower than globopentaosylceramide (Forssman glycolipid). The profiles on thin layer chromatograms of the neutral glycosphingolipids were the same for BMMC derived from BALB/c, C57BL/6, WBBF1-W/Wv, and WBBF1-+/+ mice, and for cells differentiated in either WEHI-3 conditioned medium or concanavalin A-splenocyte conditioned medium. High performance liquid chromatography of benzoylated neutral glycosphingolipids of BMMC on a Zipax column confirmed the identity of the four neutral glycosphingolipids identified by thin layer chromatography. The fifth major glycosphingolipid had an elution time greater than that of globotetraosylceramide and did not co-elute with any of the standards tested. Direct biochemical analyses of the neutral glycosphingolipids of mouse serosal mast cells (SMC) were not feasible because only 2 X 10(6) SMC could be isolated per 100 mice. However, mouse SMC bound a rat monoclonal anti-globopentaosylceramide antibody (M1/87.27.7) and rat monoclonal B1.1 antibody, as assessed by indirect immunofluorescence and flow cytometry, whereas mouse BMMC did not. The binding of B1.1 antibody to SMC could be blocked by the anti-globopentaosylceramide antibody, and the specificity of B1.1 antibody for globopentaosylceramide was confirmed immunochemically with the use of a solid phase radioimmunoassay. As estimated immunochemically, the amount of globopentaosylceramide in mouse SMC was 62 ng/10(6) cells, whereas BMMC contained less than 8 ng/10(6) cells. Thus, the expression of globopentaosylceramide is a characteristic of the mouse SMC that is lacking in the T cell-dependent BMMC.
Animals↗
Analysis of polysulfated chondroitin disaccharides by high-performance liquid chromatography.
A high-performance liquid chromatography method for analyzing disaccharides derived from chondroitin sulfate glycosaminoglycans has been developed which employs a Whatman Partisil-10 PAC amino-cyano column and an acetonitrile/methanol/ammonium acetate solvent to resolve disulfated, monosulfated, and unsulfated disaccharides in a chromatographic run of less than 20 min. The single known trisulfated chrondroitin disaccharide can be eluted in an alternate solvent system containing the same mobile phase components in different proportions. Disaccharides were prepared for chromatography from glycosaminoglycans and proteoglycans of known compositions by digestion with chondroitinase ABC, with the exception of king crab cartilage glycosaminoglycan which was incubated sequentially with hyaluronidase and chondroitinase ABC. Disaccharides were extracted from the digestion mixtures in 80% ethanol, dried over nitrogen, resuspended in the HPLC solvent, and chromatographed at a flow rate of 1 ml/min. Unsaturated disaccharides in the column eluate were detected by continuous ultraviolet absorbance monitoring at 232 nm; alternatively, fractions were collected and assayed for uronic acid content or radioactivity. By utilizing the HPLC technique in conjunction with chondroitinase ABC and AC digestion and sulfatase hydrolysis, the epimeric structures of chondroitin sulfates E and H were confirmed. With this technique, rapid and reproducible analyses of chondroitin sulfate disaccharides generated from mouse mast cell proteoglycan and from glycosaminoglycans of squid cranial cartilage, shark skin, hagfish skin, and hagfish notocord were in close agreement with compositions obtained by other techniques.
Animals↗
Interleukin 3: A differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan.
Mouse mast cells were differentiated and grown by culturing bone marrow cells in medium containing 2 X 10(-10) M purified interleukin 3 (IL 3). The cells obtained were similar in ultrastructure, membrane antigen phenotype, proteoglycan type, and lipid products generated upon immunologic activation to mast cells differentiated in culture by WEHI-3-conditioned medium (WEHI-3-CM) and by concanavalin A (Con A) splenocyte-conditioned medium. Phenotypically, these cells expressed IgE receptors and H-2 antigens and were recognized by a monoclonal antibody (B23.1) that did not react with mouse serosal heparin-containing mast cells. The classic phenotypic markers of mouse T cells or macrophages were not detected. The mouse mast cells differentiated with IL 3 as well as those differentiated in WEHI-3-CM incorporated [35S]sulfate into a nonheparin proteoglycan of 150,000 to 200,000 m.w. Most of the 35S-labeled macromolecules were degraded by chondroitinase ABC to yield only two disaccharides, which co-chromatographed on ascending thin layer chromatography with delta Di-4S and delta Di-diSE; thus, the proteoglycan in these cells is composed of chondroitin sulfate E glycosaminoglycans. After sensitization with monoclonal IgE, washing, and antigen activation, the IL 3 differentiated cells released the preformed mediator beta-hexosaminidase and generated and released two major classes of lipid mediators. The quantities of leukotriene C4 (LTC4), leukotriene B4 (LTB4), and platelet-activating factor (PAF-acether) generated/10(6) cells were 17, 3.0, and 3.1 ng, respectively. The ratio of these three lipid mediators was similar to that obtained from mast cells differentiated in WEHI-3-CM and in Con A-conditioned medium. Thus, T cell-derived IL 3 is the component present in the conditioned media that is required for differentiation and growth of the subclass of mast cells containing chondroitin sulfate E proteoglycan, designated E-MC. The IL 3-dependent E-MC may represent the in vitro counterpart of the T-cell-dependent mucosal mast cell, suggesting in turn that the production of LTC4 and LTB4 and of PAF-acether may play a role in adaptive intestinal immunity to helminthic parasites.
Animals↗
In vitro activation of the contact (Hageman factor) system of plasma by heparin and chondroitin sulfate E.
A large number of negatively charged macromolecules, including DNA, glycosaminoglycans, and proteoglycans, were tested as possible activators of the contact (Hageman factor) system in vitro. Activation was assessed by conversion of prekallikrein to kallikrein, as determined by amidolytic assay and by cleavage of 125I-Hageman factor into 52,000- and 28,000-dalton fragments. Of particular interest to these studies, heparin proteoglycan and glycosaminoglycan from rat peritoneal mast cells, and squid chondroitin sulfate E, which is representative of the glycosaminoglycan from cultured mouse bone marrow derived mast cells, induced the reciprocal activation between Hageman factor and prekallikrein. In addition, naturally occurring heparin glycosaminoglycans from pig mucosa, bovine lung, and rat mast cells also induced activation. In contrast, native connective tissue matrix glycosaminoglycans and proteoglycans from several sources were inactive, although when one such chondroitin sulfate was further sulfated in vitro, it gained activity. When the negative charge of the activating agents was blocked by the addition of hexadimethrine bromide, the cleavage of 125I-Hageman factor in the presence of prekallikrein was prevented. The active negatively charged macromolecules induced cleavage of 125I-high molecular weight kininogen in normal plasma but not in Hageman factor-deficient or prekallikrein-deficient plasmas. Reconstitution of prekallikrein-deficient plasma with purified prekallikrein restored the kininogen cleavage upon addition of the active proteoglycans. These results suggest that both heparin from connective tissue mast cells and highly sulfated chondroitin sulfate E from cultured mouse bone marrow derived mast cells (which are considered synonomous with mucosal mast cells) could activate the contact system of plasma subsequent to an activation secretion response.
Animals↗
Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade fibronectin: their increase after sodium butyrate treatment of the cells.
Mouse bone marrow-derived mast cells, differentiated in vitro with concanavalin A splenocyte-conditioned medium and sensitized with monoclonal IgE, release neutral serine proteases after activation with specific antigen. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the supernatants from immunologically activated mast cells revealed the presence of four prominent proteins of 27,000, 29,000, 30,000 and 31,000 m.w. When the supernatants and sonicated residual cells from antigen-challenged or nonactivated IgE-sensitized mast cells were incubated with [3H]diisopropylfluorophosphate ([3H]DFP) and the proteins were subjected to SDS-PAGE followed by autoradiography, proteins of 27,000 to 31,000 m.w. were labeled with [3H]DFP. The antigen-dependent release of labeled proteins was accompanied by a corresponding depletion of similarly sized [3H]DFP-labeled proteins from these cell pellets relative to unactivated cells. The SDS gels were also stained with Coomassie Blue and were sectioned to separate the individual proteins for measurement of their incorporated radioactivity; the net percent antigen-dependent release of all four [3H]DFP-labeled proteins ranged from 64 to 68% and was comparable to that of the secretory granule markers, beta-hexosaminidase and histamine. That the [3H]DFP-labeled proteins were derived from the secretory granules of the cells was supported by studies in which mast cells were cultured for 4 days in the presence of 1 mM sodium butyrate. This treatment produced a differential increase in their cellular content of histamine (10-fold), [3H]DFP binding proteins (two- to fourfold), and beta-hexosaminidase (minimally), while the net percent antigen-dependent release of each of these constituents was unchanged. After sensitization and antigen activation, the net percent release of histamine, beta-hexosaminidase, and the four [3H]DFP-labeled proteins was 51, 59, and 53 to 61%, respectively, for sodium butyrate-treated cells, and 53, 60, and 64 to 68%, respectively, for cells not exposed to sodium butyrate. Human plasma fibronectin was used as a substrate to demonstrate that the exocytosed proteins possessed proteolytic activity. As assessed by optical density scanning of stained SDS-PAGE gels of the substrate, the proteases present in the supernatants of antigen-activated cells, but not of sensitized unchallenged cells, rapidly degraded native fibronectin at pH 7.0. This degradation was prevented by pretreatment of the exocytosed proteins from immunologically activated cells for 90 min at 37 degrees C with 2 mM DFP.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Inhibition of proliferation of mouse T cell-dependent bone marrow-derived mast cells by rat serum does not change their unique phenotype.
Both mouse and rat sera have been found to inhibit proliferation in vitro of interleukin 3-dependent chondroitin sulfate E proteoglycan-containing mouse bone marrow-derived mast cells (BMMC), as assessed by quantitation of 3H-labeled thymidine incorporation into DNA, cell cycle analysis, and cell number. Rat serum (9%) inhibited 3H-labeled thymidine incorporation within 60 min of exposure in a culture medium composed of 1% fetal calf serum (FCS) and 16% concanavalin A splenocyte-conditioned medium. The anti-proliferative effect of rat serum did not alter cell viability for 17 hr of subsequent culture, was dose related, with a maximal effect at 7% rat serum, and was reversible. Cytofluorographic analysis of relative DNA content per cell revealed that the proportion of cells in the S + G2 + M phases of the cell cycle was decreased in cells treated with 9% rat serum compared with cells cultured in either 1% or 10% FCS. These rat serum-treated BMMC exhibited no change in plasma membrane antigen phenotype as assessed by 15 monoclonal antibodies, and continued to synthesize chondroitin sulfate E proteoglycan. When sensitized with monoclonal IgE antibody, washed, and challenged with specific antigen, the rat serum-treated BMMC released the preformed secretory granule-associated mediators beta-hexosaminidase and histamine, and the newly generated lipid mediators leukotriene C4 (LTC4) and leukotriene B4 (LTB4) in amounts comparable to BMMC cultured in 10% FCS. Thus, the unique cell surface phenotype, the presence of chondroitin sulfate E proteoglycan rather than heparin proteoglycan, and the generation of LTC4 and LTB4 in a ratio of approximately 6:1 upon perturbation of the IgE receptor are distinctive characteristics of the interleukin 3-dependent mouse BMMC subclass, and not a functional consequence of the rapid proliferation of the cell.
Animals↗
Inhibition of the function of activated properdin by squid chondroitin sulfate E glycosaminoglycan and murine bone marrow-derived mast cell chondroitin sulfate E proteoglycan.
We compared the relative capacities of two over-sulfated glycosaminoglycans, heparin and chondroitin sulfate E, to alter the function of native properdin (nP) and activated properdin (aP) in the formation and stabilization of the amplification C3 convertase (C3b,Bb). Heparin was more active on a weight basis than chondroitin sulfate E in inhibiting the formation of C3b,Bb without or with nP, but had no influence on the decay of a pre-formed convertase, either unstabilized or stabilized with nP or aP. In contrast, chondroitin sulfate E was over 10-fold more active than heparin in preventing the formation of C3b,Bb in the presence of aP, and gave dose-related acceleration of decay of pre-formed C3b,Bb,aP but not of unstabilized or nP-stabilized pre-formed convertase. The inhibitory effect of both glycosaminoglycans on the formation of C3b,Bb in the presence of nP or aP was less when the number of C3b sites per target cell was increased. The preferential action of chondroitin sulfate E on the function of aP during the formation and decay of C3b,Bb,aP as compared to C3b,Bb,nP implies functional differences in the two forms of P even when they have been incorporated into C3b,Bb. The equal potency, when adjusted for uronic acid content, of chondroitin sulfate E proteoglycan isolated from the T cell-dependent, bone marrow-derived murine mast cell and of chondroitin sulfate E glycosaminoglycan from squid reveals that the linkage of the glycosaminoglycan to a peptide core does not diminish its regulatory action on the alternative complement pathway.
Animals↗
Cloned mouse mast cells derived from immunized lymph node cells and from foetal liver cells exhibit characteristics of bone marrow-derived mast cells containing chondroitin sulphate E proteoglycan.
Cloned mouse mast cells which were T cell growth-dependent were derived both from immunized lymph node and from foetal liver, and were found to be morphologically and biochemically similar to mast cells previously differentiated in vitro from mouse bone marrow (BMMC). These two T cell growth-dependent mouse mast cell clones were identical to the BMMC in their preferential synthesis of chondroitin sulphate E proteoglycan rather than heparin proteoglycan. The hydrodynamic size of the cell-associated proteoglycan from each of the three mast cell sources was 150,000-250,000 mol. wt.; and that of the covalently bound glycosaminoglycans was 13,000-25,000 mol. wt. Chondroitinase ABC digestion of the [35S]proteoglycans from both cloned mast cells, as well as the BMMC, yielded only two disaccharides which comigrated on ascending thin layer chromatography with delta Di-4S and delta Di-diSE standards, respectively. Quantification of the radioactivity in the enzyme digests revealed that one-sixth to one-half of the resulting disaccharides were disulphated, similar to that found in BMMC containing chondroitin sulphate E. When sensitized with monoclonal IgE, washed, and subsequently challenged with specific antigen, each of the two cloned mast cells generated more than 100 ng of leukotriene C4 (LTC4)/10(6) cells, but only 3-12 ng leukotriene B4 (LTB4)/10(6) cells, characteristics also observed for the BMMC. Based upon these observations, it is concluded that the cloned mast cells from lymph node and liver and the bone marrow-derived mast cell belong to a distinct subclass of mast cells. These mast cells have been designated E-mast cells (E-MC) in order to distinguish them from heparin-containing mast cells (H-MC).
Animals↗
Formation of proteoglycan aggregates in rat chondrosarcoma chondrocyte cultures treated with tunicamycin.
Proteoglycan monomer and link protein isolated from the Swarm rat chondrosarcoma both contain glycosylamine-linked oligosaccharides. In monomer, these N-linked oligosaccharides are concentrated in a region of the protein core which interacts specifically with both hyaluronate and link protein to form proteoglycan aggregates present in cartilage matrix. Chondrocyte cultures were treated with tunicamycin to inhibit synthesis of the N-linked oligosaccharides, and the ability of the deficient proteoglycan and link protein to form aggregates was studied. Cultures were pretreated with tunicamycin for 3 h and then labeled with either [3H]mannose, [3H]glucosamine, [3H]serine, or with [35S]sulfate for 6 h in the presence of tunicamycin. Formation of link protein-stabilized proteoglycan aggregates in the culture medium was inhibited by up to 40% when the cells were treated with 3 micrograms of tunicamycin/ml, a concentration which inhibited 3H incorporation with mannose as a precursor by about 90%, but by only 15% with glucosamine as a precursor. When exogenous proteoglycan aggregate was added to the culture medium, however, it was found that both endogenous monomer and link protein synthesized in the presence of tunicamycin were fully able to form link-stabilized aggregates. This suggests that glycosylamine-linked oligosaccharides on monomer and on link protein are not necessary for their specific interactions with hyaluronate and with each other. Further, although tunicamycin did not inhibit net synthesis of hyaluronate, transfer of hyaluronate from the cell layer to the culture medium was retarded. This phenomenon accounted for most if not all of the decrease in the amount of proteoglycan which formed aggregates in the medium of cultures treated with tunicamycin.
Animals↗