RESUMO
1. In prior studies, it has been shown that tivantinib is extensively metabolized in humans to many oxidative metabolites and glucuronides. In order to identify the responsible enzymes, we investigated the in vitro metabolism of tivantinib and its four major circulating metabolites. 2. The primary isoforms involved in the elimination of tivantinib were CYP2C19 and CYP3A4/5. CYP2C19 showed catalytic activity for the formation of M5 (hydroxylated metabolite), but not for M4 (a stereoisomer of M5), whereas CYP3A4/5 catalyzed the formation of both metabolites. For the elimination of M4, M5 and M8 (keto-metabolite), CYP3A4/5 was the major cytochrome P450 isoform and UGT1A9 was mainly involved in the glucuronidation of M4 and M5. 3. ADH4 was identified as one of the major alcohol dehydrogenase isoforms contributing to the formation of M6 (sequential keto-metabolite of M4 and M5) and M8. The substrate preference of ADH for M4, and not M5, was observed in the formation of M6. 4. In conclusion, CYP2C19, CYP3A4/5, UGT1A9 and ADH4 were the primary drug metabolizing enzymes involved in the in vitro metabolism of tivantinib and its metabolites. The stereoselective hydroxylation by CYP2C19 and substrate stereoselectivity of ADH4-catalyzed oxidation in the in vitro metabolism of tivantinib was discovered.
Assuntos
Álcool Desidrogenase/metabolismo , Antineoplásicos/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Pirrolidinonas/metabolismo , Quinolinas/metabolismo , Humanos , Hidroxilação , OxirreduçãoRESUMO
Inhibitor of kappaB (IκB) kinase beta (IKKß) plays a critical role in nuclear factor-kappaB (NF-κB) activation and production of proinflammatory cytokines in various inflammatory diseases including rheumatoid arthritis. We previously reported a novel IKKß inhibitor Compound D, 4-[6-(cyclobutylamino)imidazo[1,2-b]pyridazin-3-yl]-2-fluoro-N-{[(2S,4R)-4-fluoropyrrolidin-2-yl]methyl}benzamide, which is efficacious in experimental arthritis models. In the present study, we characterized the pharmacological properties of Compound D and investigated the mechanisms of the anti-arthritic effect. Compound D inhibited IKKß kinase activity with 160-fold selectivity against IKKα. The cellular analyses revealed that Compound D selectively blocked NF-κB promoter activity among major cellular signaling pathways, such as the activator protein-1 pathway, consistent with inhibition of the NF-κB signaling pathway including phosphorylation of IκBα. In addition, Compound D inhibited NF-κB-driven production of tumor necrosis factor alpha (TNFα) and interleukin-6 comparably. The correlation between inhibitory effect on TNFα production and plasma concentration of the compound was observed in vivo. Consecutive administration of Compound D decreased gene expression of proinflammatory cytokines and inflammatory mediators in the paws of arthritic mice with attenuation of paw swelling. Notably, Compound D was rapidly distributed to the arthritic paws, rather than healthy paws, and where it decreased the gene expression of proinflammatory cytokines by a single oral administration. Furthermore, Compound D completely inhibited arthritis progression even when treatment occurred after disease development. These data suggest that the downregulation of proinflammatory cytokines in local inflamed joints is one of the mechanisms underlying the anti-arthritic effect of the IKKß inhibitor, Compound D.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/metabolismo , Benzamidas/farmacologia , Citocinas/metabolismo , Compostos Heterocíclicos com 2 Anéis/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Articulações/efeitos dos fármacos , Animais , Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Benzamidas/uso terapêutico , Regulação para Baixo , Feminino , Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 2 Anéis/uso terapêutico , Proteínas I-kappa B/metabolismo , Interleucina-6/metabolismo , Articulações/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Distribuição Tecidual , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We have discovered imidazo[1,2-b]pyridazine derivatives that show suppressive activity of inflammation in arthritis models. We optimized the substructures of imidazo[1,2-b]pyridazine derivatives to combine potent IKKß inhibitory activity, TNFα inhibitory activity in vivo and excellent pharmacokinetics. The compound we have acquired, which had both potent activities and good pharmacokinetic profiles based on improved physicochemical properties, demonstrated efficacy on collagen-induced arthritis models in mice and rats.
Assuntos
Artrite Experimental/tratamento farmacológico , Quinase I-kappa B/antagonistas & inibidores , Imidazóis/química , Inibidores de Proteínas Quinases/química , Piridazinas/química , Administração Oral , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Quinase I-kappa B/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Piridazinas/farmacocinética , Piridazinas/uso terapêutico , Ratos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The localization of vasa homolog protein in the spermatogenic cells of mice, rats, and guinea pigs was studied by immunofluorescence and electron microscopies with the antibody against mouse vasa homolog (MVH) protein. By immunofluorescence microscopy, four types of granular staining patterns were identified: (1) fine particles observed in diplotene and meiotic cells, (2) small granules associated with a mitochondrial marker and appearing in pachytene spermatocytes after stage V, (3) strands lacking the mitochondrial marker in late spermatocytes, and (4) large irregularly shaped granules in round spermatids. Immunoelectron microscopy defined the ultrastructural profiles of these MVH protein-positive granules: the first type consisted of small dense particles, the second had intermitochondrial cement (IMC), the third type, consisting of strands, had loose aggregates of either material dissociated from IMC or 70-90-nm particles, and the fourth had typical chromatoid bodies (CBs). The results suggest that MVH proteins function in these components of nuage. MVH protein-positive structures other than CBs disappeared during meiosis and CB appeared first in early spermatids. The results suggest that the formation of nuage is discontinued between spermatocytes and spermatids. The formation of nuage in spermatocytes and of CB in spermatids is discussed.
Assuntos
Cromátides/química , RNA Helicases DEAD-box/química , Espermatócitos/química , Espermatogênese , Animais , Western Blotting , RNA Helicases DEAD-box/ultraestrutura , Imunofluorescência , Cobaias , Masculino , Camundongos , Coelhos , Ratos , Ratos WistarRESUMO
Imidazo[1,2-b]pyridazine derivatives from high-throughput screening were developed as IKKbeta inhibitors. By the optimization of the 3- and 6-position of imidazo[1,2-b]pyridazine scaffold, cell-free IKKbeta inhibitory activity and TNFalpha inhibitory activity in THP-1 cell increased. Also, these compounds showed high kinase selectivity. The structure-activity relationship was revealed and the interaction model of imidazo[1,2-b]pyridazine compounds with IKKbeta was constructed.
Assuntos
Quinase I-kappa B/antagonistas & inibidores , Imidazóis/química , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/química , Piridazinas/farmacologia , Descoberta de Drogas , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Rheumatoid arthritis (RA) is the most common inflammatory arthritis, characterized by marked infiltration of mononuclear cells including B cells into the inflamed synovium. Anti-glucose-6-phosphate isomerase (GPI) antibody (Ab) is an arthritogenic Ab in K/BxN T cell receptor transgenic mice, and is also present in some patients with RA. To characterize synovial B cells from anti-GPI Ab-positive RA, synovial immunoglobulin (Ig) heavy chain variable regions (VH) were compared with those of negative individuals. Synovial tissues were obtained from six RA patients (three anti-GPI Ab-positive and three anti-GPI Ab-negative). Ig-VH genes were amplified by PCR using family-specific primers and were subsequently sequenced. In synovial B cells from anti-GPI Ab-positive RA patients, VH4 and JH4 were predominantly expressed (p<0.0001). The immunoglobulin heavy chain complementarity-determining region 3 (IgH-CDR3) length in the synovium of anti-GPI Ab-positive individuals was shorter than that in anti-GPI Ab-negative individuals (p=0.0005). In addition, the IgH-CDR3 of anti-GPI Ab-positive patients was rich in basic-ionized amino acids (arginine, histidine, and lysine) near their central position, suggesting a high affinity. Our results support the notion that Ig-VH4 B cells in RA synovium with anti-GPI Ab are affinity-matured and that anti-GPI Ab might be associated with the skewed IgH-CDR3.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/genética , Genes de Cadeia Pesada de Imunoglobulina , Glucose-6-Fosfato Isomerase/imunologia , Região Variável de Imunoglobulina/genética , Líquido Sinovial/imunologia , Idoso , Sequência de Aminoácidos , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Autoanticorpos/metabolismo , Feminino , Amplificação de Genes , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/administração & dosagem , Glucose-6-Fosfato Isomerase/imunologia , Imunoglobulina G/administração & dosagem , Sinovite/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Complexo Antígeno-Anticorpo/fisiologia , Artrite Reumatoide/enzimologia , Autoanticorpos/metabolismo , Cartilagem Articular/imunologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Humanos , Imunoglobulina G/metabolismo , Macaca fascicularis , Receptor da Anafilatoxina C5a/biossíntese , Receptor da Anafilatoxina C5a/genética , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Sinovite/enzimologia , Sinovite/metabolismoRESUMO
Rheumatoid arthritis (RA) is a chronic disease of unknown pathogenesis. To identify abnormally expressed genes in synovium from RA patients, we performed column gel electrophoresis-coupled subtractive hybridization (CGESH). CGESH is a newly developed subtractive hybridization technique to achieve sufficient enrichment of DNA sequences. CGESH was performed using restricted enzyme digested cDNA synthesized from mRNA of synovial tissues from one RA patient and one osteoarthritis (OA) patient. The obtained subtraction libraries (RA-OA) were screened by dot blot hybridization. The clones showing higher hybridization with the RA-OA probe were identified by sequence analysis and homology search. Their DNA sequencing revealed that the genes of HLA-DRB1, sequestosome 1, elongation factor 1alpha were included. Furthermore, a functionally unknown gene (FLJ00133) was also identified. It is reported that sequestosome 1 is a scaffold in the signal transduction of TNFalpha and interleukin 1, which are the important cytokines involved in the pathogenesis of RA. It is possible that other genes identified by the CGESH technique would be associated with the pathogenesis of RA, although there is no direct evidence yet. Our results imply that the CGESH technique is a useful tool to detect genes involved in the pathogenesis of RA. Further investigation of the functional roles of candidate genes should shed light on the pathogenesis of RA.
Assuntos
Artrite Reumatoide/genética , Eletroforese/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Hibridização de Ácido Nucleico/métodos , Membrana Sinovial/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Humanos , Regulação para Cima/genéticaRESUMO
The pathogenic role of autoantibodies in rheumatoid arthritis (RA) remains elusive. Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) are candidates for arthritogenic Abs because they directly induce arthritis in mice. High titers of anti-GPI Abs are found in some RA patients with severe forms. The aim of this study was to analyze the role of IgG, including anti-GPI Abs, in the joints of RA patients. Synovial tissue was obtained from 6 patients with RA (3 anti-GPI Abs- positive and 3 anti-GPI Abs- negative) and compared histologically and immunohistochemically for IgG and C3 deposition. IgG fractions were separated from the sera of anti-GPI Abs-positive RA patients and healthy subjects, and injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. On day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of the C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from the monkeys' joints. The synovia of anti-GPI Abs-positive RA patients showed diffuse infiltration of cells, including mast cells, and strong deposition of IgG and C3. In monkeys, IgG from RA patients, including anti-GPI Abs, resulted in recruitment of granulocytes and mononuclear cells, strong deposition of IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients, including that of anti-GPI Abs, may play a role in the synovitis of RA, although the pathogenesis of human anti-GPI Abs is still uncertain.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/toxicidade , Glucose-6-Fosfato Isomerase/imunologia , Imunoglobulina G/toxicidade , Articulações/imunologia , Proteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Animais , Artrite Reumatoide/enzimologia , Artrite Reumatoide/genética , Autoanticorpos/sangue , Autoanticorpos/imunologia , Sequência de Bases , Complemento C3/análise , Complemento C3/imunologia , Granulócitos/imunologia , Haplorrinos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Articulações/efeitos dos fármacos , Articulações/patologia , Leucócitos Mononucleares/imunologia , Mastócitos/imunologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Líquido Sinovial/citologia , Líquido Sinovial/imunologiaRESUMO
INTRODUCTION: Immunization with glucose-6-phosphate isomerase (GPI) induces severe arthritis in DBA/1 mice. The present study was designed to identify the cytokines and co-stimulatory molecules involved in the development of GPI-induced arthritis. METHODS: Arthritis was induced in DBA/1 mice with 300 microg human recombinant GPI. CD4+ T cells and antigen-presenting cells from splenocytes of arthritic mice were cultured in the presence of GPI. Tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12 levels were assessed using cytometric bead array. Monoclonal antibodies to TNF-alpha, IFN-gamma, IL-12, CD40L, inducible co-stimulator (ICOS), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA-4Ig) were used to block TNF-alpha and IFN-gamma production, examine clinical index in mice with GPI-induced arthritis, and determine anti-GPI antibody production. RESULTS: Large amounts of TNF-alpha and IFN-gamma and small amounts of IL-2 and IL-6 were produced by splenocytes from mice with GPI-induced arthritis. Anti-TNF-alpha mAbs and CTLA-4Ig suppressed TNF-alpha production, whereas anti-IFN-gamma mAbs, anti-IL-12 mAbs, and CTLA-4 Ig inhibited IFN-gamma production. A single injection of anti-TNF-alpha and anti-IL-6 mAbs and two injections of CTLA-4Ig reduced the severity of arthritis in mice, whereas injections of anti-IFN-gamma and anti-IL-12 mAbs tended to exacerbate arthritis. Therapeutic efficacy tended to correlate with reduction in anti-GPI antibodies. CONCLUSION: TNF-alpha and IL-6 play an important role in GPI-induced arthritis, whereas IFN-gamma appears to function as a regulator of arthritis. Because the therapeutic effects of the tested molecules used in this study are similar to those in patients with rheumatoid arthritis, GPI-induced arthritis appears to be a suitable tool with which to examine the effect of various therapies on rheumatoid arthritis.
Assuntos
Anticorpos Monoclonais/farmacologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/terapia , Glucose-6-Fosfato Isomerase/toxicidade , Imunoconjugados/metabolismo , Interleucina-6/imunologia , Fator de Necrose Tumoral alfa/imunologia , Abatacepte , Animais , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/enzimologia , Humanos , Interleucina-6/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos DBA , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
To clarify the role of T cells in the kidneys of MRL/MpJ-lpr (MRL/lpr) mice, cytokine mRNA expression was analyzed, and tissue localization of T cells was examined by immunohistochemistry. Cells infiltrating the glomeruli, glomerular circumference, and perivascular areas in ten female MRL/lpr mice were captured by laser microdissection (LMD). Nested reverse transcription polymerase chain reaction (RT-PCR) of samples was performed with primers specific for beta-actin, T-cell receptor beta chain (TCR-Cbeta), Thy-1, B220, CD4, CD8, interleukin (IL)-2, IL-4, IL-10, IL-13, IL-17, and interferon (IFN)-gamma. Frozen sections of lesions were also stained immunohistochemically. B220, MAC-1, Thy-1, CD4, and CD8 staining was observed in glomeruli and perivascular areas, especially in glomerular circumference areas. T cells infiltrating the glomeruli, glomerular circumference areas, and perivascular areas produce INF-gamma, IL-13, and IL-17 predominately. IL-10 positivity was identified in 60% of perivascular T cells but not in a substantial number of glomerular or periglomerular T cells. The results of our study suggest that the pathogenesis of renal lesions in MRL/lpr mice is complex and not due simply to the Th1 and Th2 balance. These findings also support the concept of different molecular mechanisms for glomerulonephritis and vasculitis in these mice.
Assuntos
Nefrite Lúpica/patologia , Linfócitos T/patologia , Vasculite/patologia , Animais , Citocinas/genética , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Terapia a Laser , Nefrite Lúpica/imunologia , Camundongos , Microdissecção/métodos , Linfócitos T/imunologiaRESUMO
OBJECTIVE: To clarify the molecular mechanisms of Sjögren's syndrome (SS), we analyzed the functional role of the STAT-1 gene, one of the interferon-gamma (IFNgamma)-inducible genes, in labial salivary glands (LSGs) from SS patients. METHODS: The expression of STAT-1 messenger RNA (mRNA) was examined by real-time polymerase chain reaction (PCR) analysis, and the phosphorylation of STAT-1 protein (Tyr(701) and Ser(727) pSTAT-1) was investigated by Western blot and immunohistochemical analyses. The expression of IFNgamma-inducible 10-kd protein (IP-10), IFN regulatory factor 1 (IRF-1), and Fas was also examined by real-time PCR and immunohistochemical analyses. RESULTS: STAT-1alpha and STAT-1beta mRNA were highly expressed in LSGs from SS patients. The level of STAT-1alpha protein in SS LSGs was higher than that in 3 control LSGs, whereas STAT-1beta protein was not clearly detected by Western blot analysis. Moreover, Tyr(701) and Ser(727) pSTAT-1alpha proteins were specifically detected in SS LSGs. Immunohistochemical analysis showed localization of Tyr(701) pSTAT-1 in infiltrating lymphocytes and the adjacent ductal epithelium from SS patients. Ser(727) pSTAT-1 was localized only in the ductal epithelium of SS LSGs. The STAT-1-inducible genes IP-10 and IRF-1 and the Fas genes were highly expressed in SS LSGs and were colocalized with Ser(727) pSTAT-1-positive, but not Tyr(701) pSTAT-1-positive, cells. CONCLUSION: We found evidence of the up-regulation of STAT-1alpha mRNA and protein in LSGs from SS patients, as well as the presence of pSTAT-1alpha in ductal epithelium from SS patients. Our findings suggest that STAT-1alpha, especially Ser(727) pSTAT-1, may function as a key molecule in the pathogenesis of SS.
Assuntos
Fator Gênico 3 Estimulado por Interferon/genética , Fator Gênico 3 Estimulado por Interferon/metabolismo , Glândulas Salivares Menores/metabolismo , Síndrome de Sjogren/genética , Síndrome de Sjogren/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Humanos , Fosforilação , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Ductos Salivares/metabolismo , Ductos Salivares/patologia , Glândulas Salivares Menores/patologia , Serina/metabolismo , Síndrome de Sjogren/patologia , Tirosina/metabolismoRESUMO
Anti-glucose-6-phosphate isomerase (GPI) antibodies are known to be arthritogenic autoantibodies in K/BxN mice, although some groups have reported that few healthy humans retain these antibodies. The expression of Fcgamma receptors (FcgammaRs) is genetically regulated and has strong implications for the development of experimental arthritis. The interaction between immune complexes and FcgammaRs might therefore be involved in the pathogenesis of some arthritic conditions. To explore the relationship between functional polymorphisms in FcgammaRs (FCGR3A-158V/F and FCGR2A-131H/R) and arthritis in individuals positive for anti-GPI antibodies, we evaluated these individuals with respect to FCGR genotype. Genotyping for FCGR3A-158V/F and FCGR2A-131H/R was performed by PCR amplification of the polymorphic site, followed by site specific restriction digestion using the genome of 187 Japanese patients with rheumatoid arthritis (including 23 who were anti-GPI antibody positive) and 158 Japanese healthy individuals (including nine who were anti-GPI antibody positive). We report here on the association of FCGR3A-158V/F functional polymorphism with anti-GPI antibody positive status. Eight out of nine healthy individuals who were positive for anti-GPI antibodies possessed the homozygous, low affinity genotype FCGR3A-158F (odds ratio = 0.09, 95% confidence interval 0.01-0.89; P = 0.0199), and probably were 'protected' from arthritogenic antibodies. Moreover, among those who were homozygous for the high affinity genotype FCGR3A-158V/V, there were clear differences in anti-human and anti-rabbit GPI titres between patients with rheumatoid arthritis and healthy subjects (P = 0.0027 and P = 0.0015, respectively). Our findings provide a molecular model of the genetic regulation of autoantibody-induced arthritis by allele-specific affinity of the FcgammaRs.
Assuntos
Antígenos CD/genética , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Predisposição Genética para Doença , Glucose-6-Fosfato Isomerase/imunologia , Polimorfismo Genético/imunologia , Receptores de IgG/genética , Adulto , Artrite Reumatoide/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas Ligadas por GPI , Genótipo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We have developed a DNA subtractive hybridization technique especially designed for mammalian genome comparison. The core of this protocol is a newly devised denaturant-containing polyacrylamide gel formed in a glass-column. In this gel system, the following DNA manipulation steps are carried out sequentially: size separation by electrophoresis, heat-denaturation, renaturation, and recovery. In the first step, a mixture of tester and driver DNA fragments are segregated according to their size whilst keeping their double-stranded forms. This reduces the complexity of the original genomic DNA fragments and also segregates DNA fragments having closely related sequences. In the second step, fractionated DNA fragments are quickly denatured and subjected to successive subtractive hybridization in situ by controlling gel temperature in a water bath. In the third step, DNA fragments are recovered by electrophoresis towards the reverse-orientation and are adsorbed onto ion-exchange beads. Two lines of experiments show that our protocol is able to highly enrich or directly extract differences among genomic DNA samples.
Assuntos
DNA/química , Eletroforese/métodos , Genoma , Hibridização de Ácido Nucleico/métodos , HumanosRESUMO
We have improved the protocol of In-Gel Competitive Reassociation (IGCR) technique, one of genome subtraction methods, and developed the apparatus for this technique. The protocol obtained by the fluorescence monitor that we had reported on this symposium last year was added to the improved IGCR technique, which made IGCR method simple and less time consuming for handling. The comprehensive subtractions with genes from twins, one of whom with rheumatoid arthritis, were employed and IGCR libraries were constructed. The analysis of the library clones will be reported.