RESUMO
We previously developed a lymphocyte microwell-array system, which effectively detects antigen-specific B-cells by monitoring intracellular Ca(2+) mobilization at the single-cell level with a fluorescent Ca(2+) indicator, fluo-4. However, it is difficult for the system to perform time-lapse monitoring. Here, we developed a novel method, a lymphocyte microwell-array chip system equipped with a charge-coupled device (CCD) time-lapse scanner (MAC-CCD system), for monitoring intracellular Ca(2+) mobilization. The MAC-CCD system is able to monitor intracellular Ca(2+) mobilization of more than 15,000-20,000 individual live B-cells every 10 s. In addition, we adopted a correlation method in a MAC-CCD system, which enabled us to detect B-cells with a frequency of as few as 0.046%. Furthermore, we succeeded in obtaining six influenza nucleoprotein-specific human monoclonal antibodies from the peripheral blood of influenza-vaccinated volunteers. These results demonstrate that the MAC-CCD system with a correlation method could detect very rare antigen-specific B-cells.
Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos/imunologia , Microfluídica , Orthomyxoviridae/imunologia , Corantes Fluorescentes , Humanos , Microscopia de FluorescênciaRESUMO
BACKGROUND: The S100A8/A9 complex is expressed in a subset of activated neutrophils and macrophages in acute inflammatory lesions associated with various diseases. OBJECTIVE: To investigate (a) whether serum S100A8/A9 levels are increased in patients with unstable angina (UA); and (b) whether S100A8/A9 expression is upregulated in coronary atherosclerotic plaques of patients with UA. DESIGN: Serum S100A8/A9 levels in 39 patients with stable angina (SA) and 53 patients with UA were measured. In addition, the presence of the S100A8/A9 complex in directional coronary atherectomy specimens was studied immunohistochemically. Cell types which stain positive for S100A8/A9 were identified by immunodouble staining with neutrophils and macrophages. RESULTS: Mean (SD) serum S100A8/A9 levels were significantly higher in patients with UA than in those with SA (3.25 (3.08) microg/ml vs 0.77 (0.31) microg/ml, p<0.05). In patients with UA, immunodouble staining clearly showed that the S100A8/A9 complex was expressed in infiltrated neutrophils and occasional macrophages. The S100A8/A9-positive area was significantly higher in UA than in SA (mean (SD) 18.3 (14.2)% vs 1.3 (2.4)%, respectively, p<0.001). CONCLUSIONS: The S100A8/A9 complex may be involved in the inflammatory process of coronary atherosclerotic plaques in patients with UA.
Assuntos
Angina Instável/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Doença da Artéria Coronariana/metabolismo , Neutrófilos/metabolismo , Idoso , Angina Pectoris/diagnóstico , Angina Instável/diagnóstico , Angina Instável/etiologia , Biomarcadores/sangue , Proteína C-Reativa/análise , Calgranulina A/sangue , Calgranulina B/sangue , Estudos de Coortes , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/diagnóstico , Diagnóstico Diferencial , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Infiltração de NeutrófilosRESUMO
BACKGROUND/AIMS: Gram-positive bacterial DNA is frequently detectable in gallbladder bile of primary biliary cirrhosis (PBC) patients. To advance these findings, lipoteichoic acid (LTA) of gram-positive bacteria with high antigenicity was examined in liver specimens and bile from PBC patients and controls. METHODS: LTA was examined by Western blotting in the gallbladder bile from 15 PBC, 11 cholecystolithiasis and six normal subjects, and by immunohistochemistry in liver specimens from 16 PBC, six primary sclerosing cholangitis (PSC), eight chronic viral hepatitis C (CVH-C) and five normal subjects. RESULTS: In the gallbladder bile, there was no significant difference in the positive rate of LTA between PBC and controls. LTA-containing mononuclear cells were frequently detected in the portal tracts, particularly around the bile ducts and in hepatic sinusoids in PBC, while they were infrequent or occasional in control livers. These LTA-containing cells were sinusoidal endothelial cells and Kupffer cells, and portal monocytes, which frequently expressed scavenger receptor class B type 1. CONCLUSIONS: LTA derived from bacterial fragments may reach the bile, not only in the diseased state but also under normal conditions. Such LTA may be involved in the development and progression of portal tract lesions, particularly bile duct lesions, in PBC.
Assuntos
Ductos Biliares Intra-Hepáticos/metabolismo , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/patogenicidade , Lipopolissacarídeos/metabolismo , Cirrose Hepática Biliar/etiologia , Cirrose Hepática Biliar/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Ácidos Teicoicos/metabolismo , Adulto , Idoso , Bile/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Antígenos CD36/metabolismo , Estudos de Casos e Controles , Feminino , Granuloma/metabolismo , Granuloma/patologia , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/patologia , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Receptores Depuradores , Receptores Depuradores Classe BRESUMO
It is urgently necessary to clarify functions of uncharacterized proteins. To understand life processes, we must investigate the functions and networks of proteins expressed from genomic DNA. In the near future, microinfusion experiments will become a more important method for analyzing uncharacterized function of proteins in vivo. Here we provide a practical manual for performing microinfusion experiments in mice. We also describe our experiment in which we performed a single injection of morphine following Secreted Protein Acidic and Rich in Cysteine (SPARC) infusion into the basolateral amygdala of previously uninjected mice and found markedly enhanced locomotor activity. We discuss the utility of microinfusion experiments in mice.
Assuntos
Microinjeções/métodos , Farmacologia/métodos , Animais , Camundongos , Morfina/administração & dosagem , Morfina/farmacologia , Atividade Motora/efeitos dos fármacos , Osteonectina/administração & dosagem , Osteonectina/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
OBJECTIVES: To develop a new ELISA system for liver-type arginase using monoclonal antibodies against the enzyme, and to verify the utility of the arginase in diagnosis of hepatic disorders. DESIGN AND METHODS: We have developed an enzyme-linked immunosorbent assay (ELISA), using two kinds of monoclonal antibodies (Mo6G3 and Mo9C5) for human liver-type arginase as the first and second antibodies respectively. We have also developed a new method to eliminate the influence of erythrocyte-derived arginase contamination in hemolytic samples. This ELISA was applied to specimens received from patients with acute and chronic hepatic disease and also patients who had undergone partial hepatectomy. RESULTS: This assay is sensitive and reproducible for the measurement of liver-type arginase in the sera of patients with liver dysfunction, and enabled us to detect enzyme concentrations as low as 27 pmol/L without any processing of the samples. The assay showed within-run coefficients of variation (CV) ranging from 1.9 to 4.1% and between-day CV from 3.6 to 5.1% for arginase concentrations varying from 57.1 to 1200 pmol/L. The recovery was 113% (mean) with a range of 96 to 129%. These antibodies reacted strongly with both recombinant and native liver-type arginases, while, to some extent, with erythrocyte-derived arginase. Correction for erythrocyte-derived arginase contamination in hemolytic samples was, however, easily made by assaying peroxidase-like activity of hemoglobin. From the view of a limited localization of arginase in the liver, the marked increase of the enzyme in serum reflects initiation of liver injury, while the rapid decrease reflects termination of the damage. Such quick normalization in circulating liver-type arginase indicated another merit of the enzyme in diagnosis of liver diseases. CONCLUSIONS: The changes in circulating liver-type arginase level could be helpful not only in the diagnosis of liver diseases but also subsequent treatment of the patients with liver damage.
Assuntos
Arginase/análise , Ensaio de Imunoadsorção Enzimática/métodos , Hepatopatias/diagnóstico , Hepatopatias/enzimologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/enzimologia , Fígado/enzimologia , Doença Aguda , Adulto , Animais , Anticorpos Monoclonais/metabolismo , Arginase/metabolismo , Western Blotting , Calibragem , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/enzimologia , Colangiocarcinoma/sangue , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/enzimologia , Neoplasias do Colo/sangue , Neoplasias do Colo/enzimologia , Convalescença , Eritrócitos/enzimologia , Feminino , Hepatite/sangue , Hepatite/diagnóstico , Hepatite/enzimologia , Humanos , Hepatopatias/sangue , Neoplasias Hepáticas/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Autoantibody against human liver-type arginase was detected in blood of patients treated with partial liver transplantation and consisted of all subclasses of IgG, i.e., IgG1, IgG2, IgG3 and IgG4, and IgM. We newly constructed an ELISA system for the antibodies by the aid of arginase protein immunopurified from extracts of human liver tissues. Addition of 2.0 mol/l urea in 0.1 mol/l citrate buffer(pH 4.5) was effective for elimination of immunoglobulins, such as IgG and IgM, and rheumatoid factors adsorped non-specifically to liver-type arginase-autoantibody complexes on the plate. We found that, during a short period of about two months after operation, in successful cases, liver-type arginase increased, remarkably and repeatedly, in blood of recipients followed by elevation of IgM level within a week and also IgG2 level two or three weeks later. Thus the change in IgG2 level seemed to depend on those of the arginase and/or IgM. However, in unsuccessful cases, such fluctuation was not so clear as the successful cases. To be noteworthy was production of autoantibodies directed to liver-type arginase in blood of patients with liver injury although the arginase, as well as AST and ALT, is an enzyme which leaks out of liver tissue. Appearance of the autoantibodies in blood supports occurrence of liver injury, in part, in graft liver because the enzyme exists exclusively in the liver. Among immunoglobulins to liver-type arginase, IgG2 seemed to be the most helpful index to know rightly postoperative conditions of recipients of liver transplantation, and its measurement could be useful for long-term follow-up of the patients.
Assuntos
Arginase/imunologia , Autoanticorpos/sangue , Rejeição de Enxerto/diagnóstico , Sobrevivência de Enxerto , Transplante de Fígado , Monitorização Fisiológica , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática , Seguimentos , Humanos , Imunoglobulina G/sangue , Fígado/enzimologiaRESUMO
To clarify actions of vitamin A on mucosal immunity associated with interleukin-5 (IL-5), we examined effects of vitamin A on mucosal IgA level in IL-5 receptor alpha-chain-knockout (IL-5Ralpha(-/-)) mice. Daily supplementation of retinyl acetate (1 mg/mouse) increased Th2 cytokine levels and a number of their positive cells in the small intestinal mucosa of IL-5Ralpha(-/-) mice, as observed in wild-type or IL-5Ralpha(+/-) mice. Wild-type and heterozygous mice increased the IgA level and a number of IgA-containing cells in the mucosa in response to the vitamin A treatment, but not in IL-5Ralpha(-/-) mice. Retinyl acetate increased anti-cholera toxin (CT) IgA level in the mucosa of wild-type mice, improving their survival rate after an exposure to 0.4 mg of CT. However, retinyl acetate failed to induce resistance to CT toxicity in IL-5Ralpha(-/-) mice. Our results suggest that IL-5 may play an important role in an action of vitamin A on mucosal IgA system.
Assuntos
Imunidade nas Mucosas , Imunoglobulina A Secretora/biossíntese , Mucosa Intestinal/imunologia , Receptores de Interleucina/fisiologia , Vitamina A/farmacologia , Animais , Cruzamentos Genéticos , Citocinas/imunologia , Diterpenos , Heterozigoto , Imunidade nas Mucosas/efeitos dos fármacos , Intestino Delgado/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Subunidades Proteicas , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Receptores de Interleucina-5 , Ésteres de Retinil , Células Th2/imunologia , Vitamina A/análogos & derivadosRESUMO
[reaction: see text] 3-Fluoro-5-methylene-2-cyclopentenone is treated with appropriate nucleophiles and Lewis acids to undergo regioselective 1,2-addition, exocyclic 1,4-addition, and endocyclic 1,4-addition, leading to 3-substituted 4-methylene-2-cyclopentenones, 5-substituted 3-fluoro-2-cyclopentenones, and 3-substituted 5-methylene-2-cyclopentenones in good yields, respectively.
Assuntos
Ciclopentanos/síntese química , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , EstereoisomerismoAssuntos
Cisteína Endopeptidases/metabolismo , Endopeptidases , Leucina/análogos & derivados , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Voo Espacial , Ubiquitinas/metabolismo , Ausência de Peso , Animais , Calpaína/metabolismo , Catepsina L , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Precursores Enzimáticos/metabolismo , Elevação dos Membros Posteriores , Leucina/farmacologia , Ligases/metabolismo , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simulação de Ausência de PesoAssuntos
Antígenos CD36/fisiologia , Proteínas de Transporte , Proteínas de Membrana , Proteínas de Ligação a RNA/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos , Receptores de Lipoproteínas/classificação , Animais , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL , Ratos , Receptores Depuradores , Receptores Depuradores Classe BAssuntos
Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Xenobióticos/metabolismo , Doença de Alzheimer/etiologia , Animais , Apoptose , Arteriosclerose/etiologia , Diabetes Mellitus/etiologia , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Hiperlipidemias/etiologia , Lipoproteínas LDL/metabolismo , Receptores Imunológicos/classificação , Receptores Imunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores Classe BAssuntos
Arginase/sangue , Hepatopatias/diagnóstico , Fígado/enzimologia , Animais , Biomarcadores/sangue , Intoxicação por Tetracloreto de Carbono/diagnóstico , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Fígado/irrigação sanguínea , Masculino , Ratos , Ratos Wistar , Traumatismo por Reperfusão/diagnósticoRESUMO
Cellular interactions of advanced glycation end products (AGE) are mediated by AGE receptors. We demonstrated previously that class A scavenger receptor types I and II (SR-A) and CD36, a member of class B scavenger receptor family, serve as the AGE receptors. In this study, we investigated whether scavenger receptor class B type I (SR-BI), another receptor belonging to class B scavenger receptor family, was also an AGE receptor. We used Chinese hamster ovary (CHO) cells overexpressed hamster SR-BI (CHO-SR-BI cells). (125)I-AGE-bovine serum albumin (AGE-BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-SR-BI cells. (125)I-AGE-BSA exhibited saturable binding to CHO-SR-BI cells (K(d) = 8.3 microg/ml). Endocytic uptake of (125)I-AGE-BSA by CHO-SR-BI cells was completely inhibited by oxidized low density lipoprotein (LDL) and acetylated LDL, whereas LDL exerted only a weak inhibitory effect (<20%). Cross-competition experiments showed that AGE-BSA had no effect on HDL binding to these cells and vice versa. Interestingly, however, SR-BI-mediated selective uptake of HDL-CE was completely inhibited by AGE-BSA in a dose-dependent manner (IC(50) <10 microg/ml). Furthermore, AGE-BSA partially inhibited (by <30%) the selective uptake of HDL-CE in human hepatocarcinoma HepG2 cells (IC(50) <30 microg/ml). In addition, [(3)H]cholesterol efflux from CHO-SR-BI cells to HDL was significantly inhibited by AGE-BSA in a dose-dependent manner (IC(50) <30 microg/ml). Our results indicate that AGE proteins, as ligands for SR-BI, effectively inhibit both SR-BI-mediated selective uptake of HDL-CE and cholesterol efflux from peripheral cells to HDL, suggesting that AGE proteins might modulate SR-BI-mediated cholesterol metabolism in vivo.
Assuntos
Antígenos CD36/fisiologia , Colesterol/metabolismo , Endocitose/fisiologia , Produtos Finais de Glicação Avançada/farmacocinética , Fígado/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Soroalbumina Bovina/farmacocinética , Animais , Ligação Competitiva , Antígenos CD36/genética , Células CHO , Cricetinae , Endocitose/efeitos dos fármacos , Radioisótopos do Iodo/farmacocinética , Cinética , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/farmacologia , Fígado/citologia , Masculino , Ratos , Ratos Wistar , Receptores de Lipoproteínas/fisiologia , Receptores Depuradores , Proteínas Recombinantes/metabolismo , Receptores Depuradores Classe A , Receptores Depuradores Classe BRESUMO
We previously showed that vitamin A upregulated the expression of bone-type alkaline phosphatase (ALP) in fetal rat small intestine and rat intestinal IEC-6 cells. In this study, we examined interactions between retinoic acid (RA) and several growth factors/cytokines on the isozyme expression in IEC-6 cells. Epidermal growth factor and interleukins (ILs)-2, -4, -5, and -6 completely blocked the RA-mediated increase in ALP activity. In contrast, IL-1beta markedly increased the activity, protein, and mRNA of the bone-type ALP only when RA was present. IL-1beta and/or RA did not change the type 1 IL-1 receptor transcript level, whereas IL-1beta enhanced the RA-induced expressions of retinoic acid receptor-beta (RAR-beta) and retinoid X receptor-beta (RXR-beta) mRNAs and RA-mediated RXR response element binding. The synergism of IL-1beta and RA on ALP activity was completely blocked by protein kinase C (PKC) inhibitors. Our results suggest that IL-1beta may modify the ALP isozyme expression in small intestinal epithelial cells by stimulating PKC-dependent, RAR-beta- and/or RXR-beta-mediated signaling pathways.
Assuntos
Fosfatase Alcalina/biossíntese , Osso e Ossos/enzimologia , Interleucina-1/farmacologia , Intestino Delgado/enzimologia , Tretinoína/farmacologia , Fosfatase Alcalina/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Interleucina-5/farmacologia , Interleucina-6/farmacologia , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Isoenzimas/biossíntese , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Receptores do Ácido Retinoico/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Interaction of advanced glycation end products (AGE) with AGE-receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE-receptors identified so far are RAGE (receptor for AGE), 80 K-H, OST-48, galectin-3, and SR-A (macrophage scavenger receptor type I and II). Since SR-A belongs to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to the class B scavenger receptor family (SR-B) can recognize AGE-proteins as a ligand. This was tested in the present study at the cellular level using CHO (Chinese hamster ovary) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-BSA (bovine serum albumin) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36 but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized LDL (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE-proteins. Because CD36 is one of the major Ox-LDL receptors and is upregulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE-proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.
Assuntos
Antígenos CD36/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetinae , Biblioteca Gênica , Humanos , Lipoproteínas LDL/metabolismo , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Abstract Mizoribine, an immunosuppressive drug, has been used for treatment in organ transplantation, lupus nephritis, and rheumatoid arthritis (RA). On the basis of in vitro experiments, mizoribine has been postulated to be an inhibitor of inosine monophosphate (IMP) dehydrogenase, a pivotal enzyme in the formation of guanine ribonucleotides from IMP. To further characterize the mechanism of the antirheumatic action of this drug, we examined the effect of mizoribine on the production of interleukin (IL)-6, a major inflammatory cytokine in rheumatoid synovia, by freshly prepared rheumatoid synovial cells (RSC). Mizoribine (1.25-5 µg/ml) was able to inhibit the spontaneous production of IL-6 by fresh RSC in a dose-response fashion. The addition of guanosine monophosphate (GMP) reversed its inhibitory effects. In addition, mizoribine inhibited the enhanced production of IL-6 by the IL-1α and/or tumor necrosis factor α-stimulated RSC. Inhibition was also observed at the mRNA level, determined by Northern blot analysis. In contrast, mizoribine did not affect IL-8 production by these cells. These data suggest that mizoribine inhibits IL-6 production by fresh RSC, possibly owing to the depletion of intracellular GMP, and that this inhibitory effect of the drug on rheumatoid synovial cells may be related to its efficacy in RA.
RESUMO
Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. Five AGE receptors identified so far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger receptor class A types I and II). Since SRA is known to belong to the class A scavenger receptor family, and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36, although belonging to the class B scavenger receptor family, can recognize AGE proteins as ligands. This was tested at the cellular level in this study using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells). Cellular expression of CD36 was confirmed by immunoblotting and immunofluorescent microscopy using anti-CD36 antibody. Upon incubation at 37 degrees C, (125)I-AGE-bovine serum albumin (AGE-BSA) and (125)I-oxidized low density lipoprotein (LDL), an authentic ligand for CD36, were endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CD36-CHO cells, but not wild-type CHO cells. In binding experiments at 4 degrees C, (125)I-AGE-BSA exhibited specific and saturable binding to CD36-CHO cells (K(d) = 5.6 microg/ml). The endocytic uptake of (125)I-AGE-BSA by these cells was inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of oxidized LDL. Our results indicate that CD36 expressed by these cells mediates the endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major oxidized LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, these results suggest that, like oxidized LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.
Assuntos
Antígenos CD36/química , Antígenos CD36/metabolismo , Endocitose/fisiologia , Produtos Finais de Glicação Avançada/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Animais , Anticorpos/farmacologia , Antígenos CD36/imunologia , Células CHO , Cricetinae , Endocitose/efeitos dos fármacos , Humanos , Radioisótopos do Iodo , Lipoproteínas LDL/metabolismo , Receptores Depuradores , Receptores Depuradores Classe A , Receptores Depuradores Classe B , Soroalbumina Bovina/metabolismo , TransfecçãoRESUMO
The removal of cholesterol from macrophages is important for reversing foam cell formation. In a previous study, we demonstrated that mouse peritoneal macrophages in culture secrete significant amounts of unesterified cholesterol from the lysosomes into the medium during endocytosis and subsequent metabolism of cholesterol-containing liposomes [Furuchi, T., Aikawa, K., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 27345-27348]. In this study, we found that at least two distinct mechanisms are involved in this process. The efflux of unesterified cholesterol into the medium was greatly suppressed by pregnenolone, an inhibitor of lysosomal cholesterol transport, but an appreciable proportion of the unesterified cholesterol was still released into the medium. Analysis of the medium containing the secreted cholesterol by NaBr density gradient ultracentrifugation revealed that the unesterified cholesterol was distributed in two different density peaks (bottom and d =/ approximately 1.1). The d =/ approximately 1.1 peak material formed high-density lipoprotein (HDL)-like particles that were produced and secreted by the macrophages. The lipid components of these particles were phosphatidylcholine and sphingomyelin, while the sole protein component was apolipoprotein E (apo E). Treatment with pregnenolone completely abolished the production of these HDL-like particles but had little effect on the bottom fractions. These data indicate that macrophages release lysosomal cholesterol via both pregnenolone-sensitive and -insensitive pathways, and that only the cholesterol secreted through the pregnenolone-sensitive pathway is associated with endogenously synthesized apo E-containing HDL-like particles. Moreover, we found that the pregnenolone-sensitive pathway operated independently of the presence or absence of exogenous HDL, whereas secretion via the pregnenolone-insensitive pathway was greatly stimulated by exogenously added HDL.
Assuntos
Colesterol/metabolismo , Lisossomos/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração , Ésteres do Colesterol/metabolismo , Endocitose , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Fosfolipídeos/metabolismo , Pregnenolona/farmacologiaRESUMO
Repeated administration of morphine substantially increases its locomotor-enhancing activity, a phenomenon termed locomotor sensitization. Here we show that secreted protein acidic and rich in cysteine (SPARC), an anti-adhesive glycoprotein present in the basolateral amygdala, contributes to the establishment of locomotor sensitization. The morphine-induced increase in SPARC levels in the basolateral amygdala persisted after morphine withdrawal and coincided with the duration of locomotor sensitization. Moreover, a single injection of morphine after SPARC infusion into the basolateral amygdala of previously uninjected mice substantially enhanced locomotor activity. Thus, SPARC may be an important element for establishing locomotor sensitization to morphine.