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1.
In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts. 相似文献
2.
J. N. Whitaker P. K. Herman S. M. Sparacio S. R. Zhou E. N. Benveniste 《Journal of neurochemistry》1991,57(2):406-414
Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were greater than 95% astrocytes and less than 3% microglia. Under serum-free conditions, leupeptin induced a 1.4- to 2.0-fold increase, maximal by 48 hours, in cathepsin D protein quantified by a radioimmunoassay. Cathepsin D enzymatic activity, inhibitable by pepstatin, also increased. Northern blot analysis demonstrated that leupeptin also increased cathepsin D mRNA expression. Kinetic analysis indicated that maximal cathepsin D mRNA levels are detected 24 h after stimulation with leupeptin. Exposure of astrocytes under the same conditions to rat recombinant interferon-gamma, human recombinant tumor necrosis factor-alpha, human recombinant interleukin-1 beta, lipopolysaccharide, calcium ionophore, or a combination of these reagents did not increase the level of cathepsin D above controls. These results indicate that astrocytic cathepsin D mRNA and protein can be induced by selected materials. Furthermore, the effects attributed to leupeptin as a proteinase inhibitor may be modified by its ability to increase cathepsin D activity. 相似文献
3.
A Aharonov I Vlodavsky R M Pruss C F Fox H R Herschman 《Journal of cellular physiology》1978,95(2):195-202
Epidermal growth factor (EGF) is a mitogen for Swiss 3T3 cells. Short incubation periods with physiological concentrations of EGF induced increased binding of Swiss 3T3 cells to Con A-coated nylon fibers. This effect was not induced in an EGF non-responsive 33 variant, in the transformed murine XC cells or in Swiss SV3T3 cells. The increase in Con A fiber-binding seems to be specific for EGF, since it was not observed in response to insulin, prostaglandin F2alpha or a higher serum concentration, which also initiate cell devision of confluent quiescent 3T3 cells. EGF also reduced Con A-mediated hemadsorption to 3T3, but had no effect on hemadsorption by the EFG non-responsive 3T3 variant. There was no change in the number of Con A-receptors on 3T3 cells after EGF treatment. Binding to WGA-coated fibers and WGA-mediated hemadsorption were not effected by preincubation with EGF. 相似文献
4.
Kakehashi H Nishioku T Tsukuba T Kadowaki T Nakamura S Yamamoto K 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(9):5728-5737
The aspartic proteinase cathepsin E is localized mainly in the endosomal structures of APCs and has been implicated in a variety of immune responses, however, the precise roles of cathepsin E in these cells remain speculative. In this study, we report the effect of disrupting the gene encoding cathepsin E on the nature and functions of dendritic cells (DCs) and macrophages derived from mouse bone marrow precursors, as well as mouse peritoneal macrophages. Whereas cathepsin E deficiency induced the accumulation of the lysosome-associated membrane protein (LAMP)-1 and LAMP-2 and elevated the lysosomal pH in macrophages, it did not have these effects on DCs. Although cathepsin E deficiency also caused a marked decrease in degradation of phagocytosed OVA and chemotactic responses to MCP-1 and fMLP by macrophages, these abilities were little affected in DCs by the absence of cathepsin E. Interestingly, cathepsin E deficiency markedly decreased the ability of macrophages to present intact OVA, as well as an OVA-derived antigenic peptide (266-281), to cognate T cells, while that of DCs was inversely enhanced by the absence of this protein. This paradox was resolved, in part, by the enhanced phagocytic activity and the increased expression of the costimulatory molecules CD86, CD80, and CD40, which amplify the response of T cells, in cathepsin E-deficient DCs compared with the wild-type cells. These results indicate that cathepsin E differentially regulates the nature and function of DCs and macrophages. 相似文献
5.
Characterization of thrombin binding to alpha 2-macroglobulin 总被引:1,自引:0,他引:1
The formation and structural characteristics of the human alpha 2-macroglobulin (alpha 2M)-thrombin complex were studied by intrinsic protein fluorescence, sulfhydryl group titration, electrophoresis in denaturing and nondenaturing polyacrylamide gel systems, and in macromolecular inhibitor assays. The interaction between alpha 2M and thrombin was also assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digests of denatured alpha 2M-125I-thrombin and alpha 2M-125I-trypsin complexes. In experiments measuring fluorescence changes and sulfhydryl group exposure caused by methylamine, we found that thrombin produced its maximum effects at a mole ratio of approximately 1.3:1 (thrombin:alpha 2M). Measurements of the ability of alpha 2M to bind trypsin after prior reaction with thrombin indicated that thrombin binds rapidly at one site on alpha 2M, but occupies the second site with some difficulty. Intrinsic fluorescence studies of trypsin binding to alpha 2M at pH 5.0, 6.5, and 8.0 not only revealed striking differences in trypsin's behavior over this pH range, but also some similarities between the behavior of thrombin and trypsin not heretofore recognized. Structural studies, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure alpha 2M-125I-thrombin covalent complex formation, indicated that covalency reached a maximum at a mole ratio of approximately 1.5:1. At this ratio, only 1 mol of thrombin is bound covalently per mol of alpha 2M. These gel studies and those of proteolytic digests of denatured alpha 2M-125I-trypsin and alpha 2M-125I-thrombin complexes suggest that proteinases form covalent bonds with uncleaved alpha 2M subunits. The sum of our results is consistent with a mechanism of proteinase binding to alpha 2M in which the affinity of the proteinase for alpha 2M during an initial reversible interaction determines its binding ratio to the inhibitor. 相似文献
6.
Summary 1-Methylguanine and 7-methylguanine, both metabolic products of tRNA degradation, are known to induce transformation of Chinese hamster fibroblasts in culture. The effects of these compounds on the cell membrane have been studied by the method of Concanavalin A-mediated hemadsorption. 1-Methylguanine or 7-methylguanine induced a 50% increase of Con A-mediated hemadsorption within 20 hours of exposure of the cells to the agent at a concentration of 10-5 M. This alteration was reversed within 13 days when the cells were grown in the control medium. Prolonged treatment with 1-methylguanine or 7-methylguanine resulted in changes which were only slowly reversed during growth of the cells in the control medium. The effect of the methylated purines on the cell membrane could be completely inhibited by simultaneous addition of dibutyryl-cAMP at a concentration of 10-5 M. The possible mechanism of cell membrane alteration by methylated purines and its relevance to transformation in vitro are discussed. 相似文献
7.
The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response. 总被引:5,自引:1,他引:4
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Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme. 相似文献
8.
Murine B16F10 melanoma cells, cultured within 0.7% agarose gels containing the fluorescent proteinase substrate acetamidofluorescein-BSA, catalyze the hydrolysis of the substrate in the region immediately surrounding the cell. Fluorescence ratio measurements on hydrolyzed substrate correlate with an average pH of 5.5 ± 0.2 in the adjacent substratum region. Enzymatic activity within the gel is partially inhibited by leupeptin, pepstatin, phenylmethylsulfonyl fluoride, EDTA and by anti-human cathepsin B, suggesting potential roles for thiol-, aspartic- and metalloproteinases. The time-course of fluorescence intensity, correlated with substratum pH measurements, suggest that substrate hydrolysis is catalyzed by enzymes with pH optima of < 5.5. Invasion by these cells through thin barriers of reconstituted basement membrane gel (Matrigel) is totally blocked by the thiol proteinase inhibitor, leupeptin. It is suggested that secreted or cell-surface acid proteinase enzymes, activated by the cell-mediated local hyperacidity, are involved in substrate hydrolysis and that these enzymes may be important in invasiveness by this cell-line. 相似文献
9.
Localization of cathepsin B in two human lung cancer cell lines 总被引:1,自引:0,他引:1
M Erdel G Trefz E Spiess S Habermaas H Spring T Lah W Ebert 《The journal of histochemistry and cytochemistry》1990,38(9):1313-1321
We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis. 相似文献
10.
Cysteine proteinase content of rat muscle lysosomes. Evidence for an unusual proteinase activity 总被引:1,自引:0,他引:1
Lysosomal cysteine proteinases were fractionated from partially purified rat muscle lysosomes. By gel filtration on Sephadex G75, cathepsin D was separated from two thiol-requiring proteolytic fractions of Mr 25 000 and 55 000, respectively. By chromatofocusing, the first fraction (Mr = 25 000) was resolved into three isoenzymic forms of cathepsin H, eluted at pH 5.8, 6.0 and 7.2, respectively, and two isoenzymic forms of cathepsin B, eluted at pH 5.5 and 5.25. Cathepsin H isoenzymes hydrolyzed Arg-NNap and BANA, were totally inhibited by 1 mM p-CMB and only to 60% by 5.10(-5) M leupeptin. The two forms of cathepsin B which degraded Z-Phe-Arg-NMec, Z-Arg-Arg-NNap and BANA were very sensitive to p-CMB and leupeptin. In addition to cathepsins B and H, a typical cathepsin-L- like activity was found in this fraction but only as a very minor component. The high Mr fraction (Mr = 55 000) contained a cysteine proteinase hydrolyzing, at pH 6.0, Z-Phe-Arg-NMec, and to a lesser extent Z-Arg-Arg-NNap and BANA. Unlike cathepsins B and H, it was very sensitive to p-CMB and HgCl2 and was fully activated only in the presence of 10 mM DTT, and inhibited to 93% by 2.10(-8) M leupeptin. By chromatofocusing, it was resolved into several isoenzymatic forms, eluted between pH 5.8 and 4.0. 相似文献
11.
A Tsuji T Arai P S Furcinitti J P Langmore K Kurachi 《Biochimica et biophysica acta》1991,1078(1):85-93
A large, intracellular proteinase accumulated by inhibitors (PABI) was found in cultured mammalian cells as a large, multicatalytic proteinase with a greatly elevated concentration in the presence of small peptide proteinase inhibitors (Tsuji and Kurachi (1989) J. Biol. Chem. 264, 16093). Electron microscopic analysis showed that the tertiary structure of PABI highly resembled that of alpha 2-macroglobulin complexed with a proteinase(s). Isolation of the anti-PABI cross-reacting material from calf serum added to the culture media of baby hamster kidney cells further supported that the primary component of PABI was alpha 2-macroglobulin. Immunoblot analyses and the substrate specificity of PABI indicated that the major proteinase component contained in PABI was thrombin. When alpha 2-macroglobulin was added to the PABI-depleted serum, a significant accumulation or a degradation of the intracellular alpha 2-macroglobulin was observed in the presence or absence of leupeptin, respectively. Similarly, when thrombin was added to the PABI-depleted fetal calf serum supplemented with fresh alpha 2-macroglobulin, a significant amount of intracellular thrombin was found only in the presence of leupeptin. These results indicate that the major component of the intracellular PABI molecules is a complex of alpha 2-macroglobulin with thrombin which is internalized from the culture media. Intracellular accumulation of PABI, therefore, is a phenomenon primarily relevant to the culture cells. Whether or not PABI is also generated in certain physiological or pathological conditions requires further study. 相似文献
12.
M Komukai K Kobayashi S Horiuchi 《Comparative biochemistry and physiology. B, Comparative biochemistry》1986,85(1):55-59
Degradation of muscle homogenate from the metamorphosing tadpole tail of bullfrog, Rana catesbeiana, was examined at acid and neutral pHs. More rapid and complete degradation was observed at acid pH. Proteinases working at acid pH were not inhibited by pepstatin but were inhibited by leupeptin. However, the inhibition by leupeptin was enhanced by pepstatin. These results show that lysosomal proteinases, a thiol proteinase(s) rather than cathepsin D, are involved in the degradation of tail muscle proteins. 相似文献
13.
A proteinase extracted with 1M NaCl from particulate fraction of the postnuclear fraction of mouse myeloid leukemia M1 cells was partially purified by Bio-Gel HTP treatment and Sephadex G-75 gel filtration. The apparent molecular mass of the proteinase was 26,000 Da and the isoelectric point was about pH 10. The enzyme activity was inhibited by phenylmethanesulfonylfluoride, chymostatin, and soy-bean trypsin inhibitor. It hydrolysed specifically Suc-Ala2-Pro-Phe-4-methylcoumaryl-4-amide (MCA). NaCl and KCl enhanced several times the activity for Suc-Ala2-Pro-Phe-MCA, but not that for fluorescein-labeled albumin and fibrinogen. These enzymic properties of the major proteinase are similar to those of chymotrypsin and cathepsin G. The role of a cathepsin G-like proteinase in relation to M1 cell differentiation is discussed. 相似文献
14.
Using highly purified T-kininogen and cathepsin E-like proteinase and 72 kDa proteinase in rat spleen, the release of T-kinin from T-kininogen was found to occur by consecutive cleavage by cathepsin E-like proteinase and 72 kDa proteinase. 72 kDa proteinase seems to be serine proteinase, because it was completely inhibited by diisopropyl fluorophosphate but not by pepstatin, leupeptin and bestatin. 相似文献
15.
Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.
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D Gullberg K R Gehlsen D C Turner K Ahln L S Zijenah M J Barnes K Rubin 《The EMBO journal》1992,11(11):3865-3873
Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Estrogen action at endometrial membranes: alterations in luminal surface detectable within seconds
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The morphological effects of estrogen on the luminal surfaces of rat endometrial cells were investigated by scanning electron microscopy. Ovariectomized rats were injected intravenously with estradiol-17 beta (E2 beta), 0.5 micrograms/0.25 ml per 100 g body wt. At various intervals thereafter, the lumen of a uterine horn was flushed with buffered 2% glutaraldehyde and then prepared for scanning electron microscopy by conventional methods. In control rats that had received an equivalent volume of placebo vehicle, the luminal cell surface was characterized by short, sparse microvilli (MV) and, in most cells, a single, central cilium. At 30 s after E2 beta injection, the number of MV was significantly increased. By 1 min, MV density was further increased and MV were frequently clustered; also, the central cilium of many cells was no longer evident. Similar results were obtained after exposure to diethylstilbestrol for 30 s to 1 min, whereas neither a subthreshold dose of E2 beta nor a dose of the relatively inactive congener E2 alpha equivalent to a saturating concentration of E2 beta gave statistically significant responses in surface changes by the present criteria. After 3-7 min of E2 beta exposure, MV had increased greatly in length and density. These effects underwent dramatic regression by 15-30 min after E2 beta treatment, with distinct diminution of microvillar lengths and numbers, reduction of clustering, and reappearance of the central cilium in many cells. This was succeeded at 1 h by a renewed surge of surface activity. These results are consistent with cumulative evidence for rapid alterations of the surface membrane of estrogen-sensitive cells in response to physiological levels of active hormone. Whether these responses in the luminal surfaces are primary, or are secondary reflections of receptor- mediated membrane alterations at the basolateral blood-front, remains to be determined. 相似文献
17.
Chain BM Free P Medd P Swetman C Tabor AB Terrazzini N 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(4):1791-1800
Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells. 相似文献
18.
Abstract: The cysteine proteinase cathepsin B (CB) was isolated from immortalized murine BV-2 microglial cells and examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to establish physicochemical properties of CB from what is generally considered the resident CNS macrophage. Microglial proteases have been implicated in several pathological processes occurring in the CNS, including neurodegeneration. Murine microglial CB was observed to consist of two major single-chain species of 32 and 34 kDa, with pls of 5.5-5.2 and 5.1-4.5, respectively. In addition, a minor 24-kDa CB species was also observed in some microglial preparations. The major CB isozymes in microglia differed from those observed in murine liver and brain, which consisted of both single- and double-chain CB variants of 31 and 24–25 kDa/5 kDa, respectively, with pl values of 5.5-4.5. A microglial pro-CB of 37 kDa was also isolated, which could be processed to the 34-kDa single-chain CB species. Cystatin was observed to prevent pro-CB processing, whereas E-64 and leupeptin were only partially inhibitory. The 37-kDa pro-CB species was observed to undergo processing into the 34-kDa CB species when incubated at pH 5.5 but remained stable with respect to molecular mass when incubated at pH 7.0. In contrast, the 34-kDa single-chain CB species was observed to autodegrade when incubated at pH 7.0, whereas incubation at pH 5.5 did not affect the integrity of the species as monitored by immunoblotting. Both pro-CB and 32-kDa single-chain CB species were observed extracellularly following lipopolysaccharide activation of BV-2 microglial cells. 相似文献
19.
Biochemical responses in activated human neutrophils mediated by protein kinase C and a Ca2+-requiring proteinase 总被引:8,自引:0,他引:8
S Pontremoli E Melloni M Michetti O Sacco F Salamino B Sparatore B L Horecker 《The Journal of biological chemistry》1986,261(18):8309-8313
Low concentrations of phorbol 12-myristate 13-acetate (PMA) elicit a specific response in human neutrophils, characterized by the production of oxygen radicals and the release into the medium of a membrane-bound serine proteinase (Pontremoli, S., Melloni, E., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., Damiani, G. and Horecker, B. L. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 1685-1689). The following evidence indicates that this response is mediated by membrane-bound protein kinase C: 1) it is blocked by inhibitors of protein kinase C; and 2) it is enhanced in cells preloaded with leupeptin which prevents proteolysis of protein kinase C and its subsequent dissociation from the cell membrane. This response is not accompanied by significant exocytosis of granule enzymes. With higher concentrations of PMA, and more particularly on stimulation with formylmethionyl-leucyl-phenylalanine (fMLP) plus cytochalasin B, a substantial exocytosis of constituents of both specific and azurophil granules is observed. With fMLP, exocytosis of granule enzymes is the predominant event, with little production of H2O2 and negligible release of membrane-bound serine proteinase. Exocytosis promoted either by a high concentration of PMA or by fMLP is inhibited by leupeptin, indicating that it is due to the action of an intracellular Ca2+-dependent thiol proteinase (calpain), either directly or by conversion by calpain of membrane-bound protein kinase C to the soluble Ca2+/phospholipid-independent form. Intracellular mobilization of Ca2+ is also observed following stimulation with either PMA or fMLP, but only the latter results in a net increase in the intracellular concentration of free Ca2+; under these conditions maximum exocytosis of granule contents is observed. 相似文献
20.
F Shirakawa Y Tanaka T Ota H Suzuki S Eto U Yamashita 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(12):4243-4248
The expression of interleukin 1 receptors (IL 1R) on human peripheral T cells was studied by the binding assay with 125I-labeled recombinant human interleukin 1 (IL 1) alpha and IL 1 beta and by the flow cytofluorometry with the fluorescein isothiocyanate (FITC)-conjugated IL 1 alpha. Peripheral blood lymphocytes expressed only few IL 1R without any stimulations. When they were stimulated with concanavalin A (Con A), IL 1R-positive cells began to increase by 4 hr, reached the maximum level at 48 hr, and then gradually decreased. The kinetics of the expression of IL 1 alpha R and IL 1 beta R showed the same pattern. Furthermore the binding of 125I-labeled IL 1 alpha to IL 1R on T cells was inhibited by the addition of either cold IL 1 alpha or IL beta, but not by interleukin 2 or interferons. The similar results were observed in the binding of 125I-labeled IL 1 beta. These results suggest that IL 1R on human peripheral T cells reactive for IL 1 alpha and IL 1 beta were identical. By Scatchard plot analysis, the numbers of IL 1R were estimated as 40 and 350 molecules per cell before and after Con A stimulation, respectively, and their Kd values were 3.1 X 10(-10) M and 2.8 X 10(-10) M. When purified T cells alone were stimulated with Con A, IL 1R were only marginally expressed. However, by the addition of monocytes, IL 1R were expressed on T cells in a dose-dependent manner. The maximum response was induced in the presence of 10% monocytes. The maximum IL 1R-positive T cells were approximately 30% by the detection of the flow cytofluorometry with FITC-conjugated IL 1 alpha. This enhancing activity of IL 1R expression on T cells by monocytes was inhibited by the addition of an anti-HLA-DR antibody or by the treatment of monocytes with the anti-HLA-DR antibody and complement. Furthermore T cell proliferative responses induced with IL 1 and Con A were also enhanced by the addition of HLA-DR-positive monocytes. These results suggest that IL 1R are expressed as the result of monocyte-T cell interaction in the early stage of T cell activation, and the expression of IL 1R on T cells and the responsiveness of T cells for IL 1 require the accessory function of HLA-DR-positive monocytes. 相似文献