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1.
Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various cells, particularly in granulocytes and monocytes . We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances : an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (-) cells and Annexin V positive (+)/7AAD positive (+) cells, respectively, in U937 cells after 6 hr of treatment. The modulation of CD93 expression on U937 cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (-) cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the cells that stained positive for CD93 mAb (mNI-11) among the treated U937 cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 beta; LFA-1beta) and CD54 (intercellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 cells identified as Annexin V positive (+)/7AAD negative (-) cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 cells. Importantly, however , the U937 cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 cells with apoptotic properties, and that the decrease in CD93 expression on U937 cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid cells. 相似文献
2.
The data on a study of the monokine-producing ability of human monocyte-like cell line U937 are presented. Antigens of Yersinia pestis EV (lipopolysaccharide and fraction 1A) induce monokine production by cell line U937. The obtained monokines essentially enhance neutrophil killer and chemotactic activities, stimulate FcR expression, increase the number of lysosomes, and the lability of lysosomal membranes in neutrophils. F1A significantly suppresses LPS in respect to the ability to induce monokine production, which stimulate neutrophil functional activity. 相似文献
3.
The possible degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100(280-288) is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100(280-288) severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general. 相似文献
4.
Characterization of the C1q receptor on a human macrophage cell line, U937. 总被引:7,自引:1,他引:6
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The binding of C1q to the human macrophage cell line U937 has been studied. Fluorescence microscopy with fluorescein-conjugated F(ab')2 anti-C1q antibody showed that 100% of the cell population is able to bind exogenous C1q. Monomeric C1q binding to U937 cells is very weak at normal ionic strength (I0.15) and was therefore investigated at I0.07, conditions which stabilize the binding. However, aggregation of C1q on dextran sulphate or a lipid A-rich lipopolysaccharide allowed a firm, binding at I0.15. Quantitative binding studies with monomeric 125I-C1q showed a concentration-dependent, saturable, specific and reversible binding involving specific membrane receptors. Scatchard plots of C1q binding indicated [1.6 +/- 0.7 (1 S.D.)] X 10(6) sites per cell with an equilibrium constant of (2.9 +/- 1.8) X 10(7) M-1 at I0.07. The location of the molecule region mediating C1q binding was established with collagen-like fragments prepared by partial pepsin digestion, confirming earlier results obtained by inhibition studies. 相似文献
5.
6.
Arthur J. Chu 《Cell biochemistry and function》1994,12(2):79-88
The effect of hemicholinium-3 (HC-3) on choline uptake and phosphatidylcholine (PC) biosynthesis was examined in human leukemic monocyte-like U937 cells. HC-3 inhibited [3H]choline uptake in a dose- and time-dependent manner. After a 3 h treatment, HC-3 (100 μM) decreased choline uptake by as much as 80 per cent (p < 0·0001; n = 4). Reduction of incorporation of label into PC was also detected in a dose-dependent manner; the extent of inhibition, however, was always 10–20 per cent less than that observed in the total uptake. At 3 h HC-3 decreased the incorporation into PC by 65 per cent (p < 0·0001; n = 5). Kinetic studies in vivo showed that HC-3 inhibited total uptake and incorporation into PC differently, suggesting that the labelling of PC is not simply dictated by [3H]choline uptake. In separate experiments, cells were pretreated with 100 μM HC-3 for 3 h. After washing, the inhibitory effect on total uptake was no longer observed, while a 20 per cent stimulation of the incorporation into PC was obtained in these pretreated cells. In pulse-chase studies, the cells were prelabelled with [3H]choline for 30 min and chased with HC-3 for up to 3 h; the results showed a significant stimulation of incorporation into PC in a longer chase with 100 μM HC-3. After a 3 h treatment, the cytosolic CTP:cholinephosphate cytidylytransferase (CT) was activated by 56 per cent, while choline kinase (CK) was inhibited slightly. The stimulation of CT was not simply due to the intact HC-3 molecule, and there was no redistribution of CT between cytosol and microsomes. Taken together, the results suggest that HC-3 activates PC biosynthesis apart from the inhibitory effect on choline uptake. 相似文献
7.
Arthur J. Chu 《Cell biochemistry and function》1994,12(1):45-55
A previous study showing that ethanol (ETOH) blocked [3H]choline incorporation into phosphatidylcholine (PC) suggested an inhibition of PC biosynthesis in human leukemic monocyte-like U937 cells. The mechanism of the inhibitory action of ETOH was investigated. Cells were pulsed with [3H]choline for 30 min and chased in the presence or absence of ETOH for up to 6 h. PC biosynthesis was inhibited drastically within 1 h after exposure to ETOH which increased intracellular cAMP appreciably. After a 3-h treatment, ETOH significantly inhibited both choline kinase (CK) and the cytosolic CTP: cholinephosphate cytidylyltransferase (CT). The inactivated CT was no longer stimulated by exogenous phosphatidylglycerol (PG). There was no evidence for redistribution of CT activity between cytosol and microsomes. When cells were exposed to 8-Bromo-cAMP ranging from 100 to 300 μM, PC biosynthesis remained unaffected despite the drastically elevated cAMP. These results seem to suggest that the raised cAMP is not a prerequisite for the inhibition of PC biosynthesis in U937 cells. Following pretreatment with protein kinase inhibitors (H-89 and K-252a), PC biosynthesis was decreased significantly and the inhibitory effect of ETOH was potentiated. Taken together, our results suggest that the inhibition of PC biosynthesis and the inhibitory effect of ETOH are independent of the activation of cAMP-dependent protein kinase. Unlike protein kinase inhibitors, pretreatment with tyrosine kinase inhibitors (erbstatin, genistein and tyrphostin 25) resulted in differential effects on PC biosynthesis and on the inhibitory action of ETOH. Genistein stimulated PC biosynthesis by 30 per cent as well as partially preventing /reversing the ETOH action, while tyrphostin 25 produced a synergistic inhibition. The relevance of tyrosine phosphorylation/dephosphorylation to the regulation of PC biosynthesis and ETOH action remains to be established. 相似文献
8.
Secretion, cleavage and binding of complement component C3 by the human monocytic cell line U937. 总被引:7,自引:0,他引:7
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Secretion of complement component C3 by U937 cells was studied. Preliminary evidence for a cell-associated proteolytic activity specific for C3 is given, as well as for a covalent-like binding of C3 fragments to the cell membranes. Secretion of C3, in the presence of 10 ng of phorbol 12-myristate 13-acetate/ml, is 120-140 ng/10(6) cells per 24 h on the third day after addition of the activator. As shown by SDS/polyacrylamide-gel electrophoresis, the intracellular pro-C3 (200 kDa) and the extracellular secreted C3 (alpha-chain 110 kDa and beta-chain 75 kDa) are identical with the forms of C3 previously characterized from human serum. Incubation of U937 cells in the presence of exogenous radiolabelled C3 shows that membrane-bound proteinase(s), not related to the classical-pathway or the alternative-pathway C3 convertases, is (are) able to cleave C3; this cleavage leads to the binding of the resulting C3 fragments to the cell membrane through reaction of membrane acceptors with the carbonyl group of C3 revealed after disruption of the intramolecular thioester bond. The proteolysis appears to be fairly specific to C3, as C4, which also possesses an intramolecular thioester bond, is not cleaved and does not bind to the cells. p-Nitrophenyl p'-guanidinobenzoate (1 mM) and di-isopropyl phosphorofluoridate (2 mM) are potent inhibitors of the proteolysis, whereas soya-bean trypsin inhibitor (1 mM), leupeptin (0.1 mg/ml) and 1,10-phenanthroline (1 mM) were ineffective. Immunological characterization of the cell-bound C3 fragments with monoclonal antibodies shows an evolution of the proteolysis of the fragments from iC3b to C3dg epitopes. Extraction of membrane-bound fragments by detergent, followed by SDS/polyacrylamide-gel electrophoresis, shows two fragments, of 43 kDa and 46 kDa, with C3dg-like characteristics. 相似文献
9.
U937 cells were found to be activated by an antibacterial peptide, KLKLLLLLKLK-NH2 (L5), to generate superoxide anion (O2-)-like peripheral neutrophils. However, the state of cell surface calreticulin, a possible receptor for L5, was suggested to differ between neutrophils and U937 cells. Unlike the former, the latter ones were activated by anti-C-domain peptide antibody of calreticulin even in the absence of L5 and generated O2- in a GTP-binding protein (G-protein)-dependent manner. 相似文献
10.
Purification and characterization of a unique human interleukin 1 from the tumor cell line U937 总被引:2,自引:0,他引:2
P J Knudsen C A Dinarello T B Strom 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(9):3311-3316
Interleukin 1 (IL 1) produced by a human tumor cell line was purified to homogeneity by a three-step chromatographic method and was tested in various assays for multiple biologic properties. The purified IL 1 stimulated the proliferative response of the D10.G4.1 cell line, a mouse IL 1 indicator T cell; caused the release of prostaglandin E2 and prostacyclin from cultured human foreskin fibroblasts and from primary human umbilical vein endothelial cells; and elicited characteristic endogenous pyrogen fever in rabbits. To stimulate IL 1 production, the histiocytic lymphoma cell line U937 was incubated with the exotoxin from toxic shock strains of Staphylococcus aureus. Supernatants from stimulated U937 cells were concentrated, and were applied to a reverse-phase HPLC column. IL 1 activity was eluted from the column at high acetonitrile concentration. Subsequent chromatography over hydroxyapatite yielded a single IL 1 species with a pI of 5.5. IL 1 was then purified to homogeneity by gel exclusion HPLC migrating as a 14 kDa species. The molecular size was confirmed by SDS-PAGE and was visualized as a single molecule by silver staining; biologic activity was recovered from the same region of the gel. Limited N-terminal sequence analysis suggested some homology to the pI 7 form of the human blood monocyte IL 1. The pI 5.5 IL 1 produced by U937 cells was only partially neutralized with anti-human monocyte IL 1 antibody, suggesting that U937-derived IL 1 is structurally related to one of the molecularly cloned IL 1 species. IL 1 from stimulated U937 cells possesses the functional characteristics of monocyte IL 1 but may represent a structurally unique IL 1 species, as determined by sequence analysis, size, and antibody reactivity. 相似文献
11.
12.
The effect of ethanol (ETOH) on the incorporation of [14C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [14C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [3H]choline or [14C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [14C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [14C] ethanolamine into [14C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway. 相似文献
13.
Experimental gene transfer has permitted a wide variety of studies on gene regulation and function. However, possible effects of the introduced DNA on cellular metabolism are not well understood. Here we demonstrated that introduction of DNA into a promonocytic cell line, U937, by electroporation caused extensive cell death. The toxicity of DNA was concentration-dependent. Various DNAs including plasmid and genomic DNAs all caused cell death, indicating that the toxicity is nucleotide sequence-independent. DNA-induced cell death was associated with internucleosomal DNA fragmentation, a decrease in cell size, and a considerable proportion of cells outside cell cycle. From these results, we concluded that cells died by apoptosis. Our findings have experimental implication for an important issue concerning the interpretation of experiments using gene transfer. In addition, we propose that our observed phenomenon may be relevant to an important immune defense mechanism in monocytes/macrophages that facilitates a response to certain viral infections. 相似文献
14.
U937 cells were differentiated into macrophages after being treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) for the first two days and dedifferentiated with daily medium renewal for 10 days. Cell proliferation slowed down and the number of cells reached the maximum level on day 2. By day 4, all of the cells had spread and attached firmly to the culture dish, and more than 90% of the cells expressed the Fc-receptor and produced superoxide anion. From there on, the number of adherent, living cells decreased gradually to about half the initial count. Most of the cells eliminated from the culture by cell death were in the S phase at the time of TPA treatment. After day 8, the number of cells expressing macrophage-specific phenotypes gradually decreased, cell adhesion was weakened, and at the same time, DNA synthesis was initiated anew. The cells became round and began to proliferate as floating cells on days 9 to 10, and thereafter they became sensitive to the second round of TPA treatment. On the basis of all the results taken together, it is suggested that fully differentiated U937 cells were dedifferentiated after being cultured with frequent medium renewal. 相似文献
15.
Yamauchi Y Kuroki M Imakiire T Abe H Uchida H Beppu R Yamashita Y Kuroki M Shirakusa T 《Biochemical and biophysical research communications》2002,290(5):1551-1557
We investigated possible regulatory effects of thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, on cytokine release from macrophages. Immobilized TSP-1 enhanced IL-6 release from the human monocytic U937 cells stimulated with phorbol myristate acetate and LPS, whereas it inhibited IL-10 release. The 70-kDa fragment of TSP-1 containing the type 1 repeats showed the same regulatory effects. The enhanced IL-6 release by TSP-1 was inhibited by anti-CD36 antibody or antibody against the sequence of the binding site to CD36 in the type 1 repeats of TSP-1. Conversely, the decrease in IL-10 release by TSP-1 was strengthened by the blocking of the interaction between CD36 and TSP-1. Furthermore, the involvement of TGF-beta1 in the inhibition of IL-10 release by TSP-1 was indicated by the facts that (i) TSP-1 induced activation of TGF-beta1 produced by the U937 cells, (ii) exogenously added TGF-beta1 inhibited IL-10 release, and (iii) antibody against TGF-beta1 blocked the inhibition of IL-10 release by TSP-1. Together, the present findings suggest that TSP-1 enhances IL-6 release from macrophages by interaction with CD36, whereas IL-10 release is regulated by the balance between the enhancing effect of TSP-1 via CD36 and the suppressive effect by TSP-1-activated TGF-beta1. 相似文献
16.
Synthesis and secretion of protein C inhibitor by the human hepatoma-derived cell line, Hep G2 总被引:1,自引:0,他引:1
The site of synthesis of protein C inhibitor, a recently identified human plasma inhibitor against activated protein C, is not known. We have studied the production and secretion of protein C inhibitor by an established human liver cell line derived from hepatocellular carcinoma (Hep G2). The concentration of protein C inhibitor, as measured by a specific radioimmunoassay, increased in the medium of Hep G2 cells with time. There was no evidence for a significant intracellular pool of this protein. Protein C inhibitor secreted from Hep G2 cells (G2 protein C inhibitor) inhibited the activity of purified activated protein C in a functional assay. De novo synthesis of protein C inhibitor was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 h of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by SDS-polyacrylamide gel electrophoresis showed a peak of radioactivity corresponding to Mr 57 000. These results indicate that the liver is a site of protein C inhibitor production. 相似文献
17.
Cytofluorometric isolation of I937, an Ia antigen-bearing variant of the Ia-negative human monocytic cell line U937 总被引:2,自引:0,他引:2
Cells of the human monocyte cell line U937 are generally considered devoid of any Ia antigens on their surface. In analyzing U937 cells with a large panel of monoclonal anti-human Ia antibodies by flow cytometry, we detected a small number of cells that appeared to react with antibodies to HLA-DR and HLA-DS/DC molecules. These Ia-positive cells were isolated and were cloned, resulting in a human monocyte cell line that expresses high levels of Ia antigens. We analyzed these antigens by one- and two-dimensional polyacrylamide gel electrophoresis, after radiolabeling and immunoprecipitation. Three distinct Ia molecules, alpha 1 beta 1, alpha 1 beta 3 (HLA-DR-like), and alpha 2 beta 2 (HLA-DC/DS-like) are synthesized by I937 cells, alpha 1 beta 3 molecule being the predominant species. The Ia antigen-bearing human monocyte cell line is expected to be useful for studying events involved in antigen presentation. 相似文献
18.
Effects of supplementation of saturated fatty acids (16:0 and 18:0) on metabolism of the cytotoxic n-6 fatty acids in cultured human monocyte-like cells (U937) have been examined. U937 cells were incubated in 5% delipidated fetal bovine serum containing 16:0 and 18:0. Supplementation of either 16:0 or 18:0 has no significant effect on the uptake of 18:2n-6 and 18:3n-6. However, addition of 16:0 to the medium increased whereas 18:0 suppressed the cytotoxic effects of 18:2n-6 and 18:3n-6. In addition, 16:0 supplementation reduced the incorporation of n-6 fatty acids in cellular phospholipid fraction, and enhanced the metabolism of n-6 fatty acids, particularly the conversion of 20:3n-6 to 20: 4n-6 in U937 cells. Results with microsomes prepared from U937 cells also showed that 16:0 supplementation increased the 5 desaturase activity. This may be related in part to an increase in the availability of 20:3n-6, since results obtained in a separate study have shown that 16:0 competed with 20:3n-6 for incorporaton into the phospholipid molecule at sn-2 position. Increasing the availability and formation of long chain n-6 fatty acids, which are cytotoxic, might also be responsible for increasing cytotoxicity of 16:0 supplementation. 相似文献
19.
Garcia JE de Cabo MR Rodríguez FM Losada JP López AJ Arellano JL 《Mediators of inflammation》2000,9(3-4):169-174
Cyclosporin A (CsA) is an immunosuppresor drug that has been used in the treatment of several types of inflammatory diseases. In some of them the inhibition of T-lymphocyte activation does not suitably account for the observed beneficial effect, suggesting that CsA could act on other types of cells. The present study was undertaken to determine the effect of CsA on inflammatory cytokine secretion by U937 monocyte cells. Undifferentiated and dimethylsulfoxide (DMSO) differentiated U937 cells were incubated with different concentrations of CsA (200, 20 and 2 ng/mL) in the presence or absence of phorbol-myristate-acetate (PMA). Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-6 and IL-8 levels were measured in supernatants using specific enzyme-linked immunosorbent assays. At the highest concentration used (200 ng/mL) CsA decreased the basal and stimulated secretion of all the inflammatory cytokines studied in both undifferentiated and differentiated cells, with the only exception of PMA-stimulated IL-1 secretion by undifferentiated cells. However, only basal secretion of interleukin-8 in both undifferentiated and DMSO-differentiated U937 cells was significantly reduced by CsA at the highest concentration (200 ng/ mL). At therapeutic concentrations in vivo, CsA exerts a predominant effect on IL-8 secretion by human mononuclear phagocytes. 相似文献
20.
The human monocyte-like cell line U937, which is a cholesterol auxotroph, does not grow on mevalonate, squalene, or 4,4-dimethyl cholest-7-en-3 beta-ol. It grows on cholest-7-en-3 beta-ol and converts it to cholesterol. When deprived of an exogenous source of cholesterol, the cells accumulate 4 alpha-methyl-cholest-8-en-3-one. The cell-free extracts of U937 are also devoid of 3-ketoreductase activity. The present studies indicate that the lesion in cholesterol synthesis by these cells is located at 3-ketosteroid reductase, making this the first report of a deficiency of this enzyme. In contrast, another U937 strain (U937-N) synthesizes cholesterol, does not accumulate 4 alpha-methyl-cholest-8-en-3-one, and has 3-ketosteroid reductase activity. The two strains should be valuable in studies of the regulation of cholesterol metabolism and of the role of cholesterol in membrane structure and function. 相似文献