RESUMO
BACKGROUND AND AIM: Serologic markers such as myeloperoxidase (MPO) antineutrophil cytoplasmic antibodies (ANCA) (MPO-ANCA) have been used to screen patients for ulcerative colitis (UC). However, MPO-ANCA shows limited accuracy in Asians. Proteinase 3 ANCA (PR3-ANCA) has performed better at UC diagnosis in Japanese adults than MPO-ANCA. The present study aimed to evaluate usefulness of PR3-ANCA for diagnosis of UC in Japanese pediatric practice. METHODS: Patients under 17 years old undergoing assessment at 12 Japanese pediatric centers between November 2016 and February 2018 were prospectively enrolled and divided into groups with UC, Crohn's disease (CD), intestinal disease control (IC), and healthy control (HC). Serum PR3-ANCA and MPO-ANCA were analyzed using chemiluminescence enzyme immunoassay kits. RESULTS: Sera from 367 patients (148 with UC at a median age of 12 years; 120 with CD, 13 years; 56 with IC, 10.5 years; and 43 with HC, 10 years) were examined. Median PR3-ANCA values in UC (1.6 U/mL) were greater than in CD (0.2; P < 0.001), IC (0.15; P < 0.001), and HC (0.1; P < 0.001). In receiver operating characteristic curve analyses, the area under the curve for PR3-ANCA was 0.79, significantly greater than for MPO-ANCA (0.58; P < 0.001). Using a cut-off value of 0.8 U/mL determined from the receiver operating characteristic analyses, PR3-ANCA showed significantly greater sensitivity (64.9%) than MPO-ANCA (cut-off, 0.2 U/mL; sensitivity, 19.6%; P < 0.001) and good specificity (83.6%). CONCLUSIONS: In Japanese children and adolescents, PR3-ANCA performed better as a serologic marker for diagnosis of UC than MPO-ANCA. To our knowledge, this is the first report of such a comparison.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/sangue , Colite Ulcerativa/diagnóstico , Mieloblastina/imunologia , Adolescente , Biomarcadores/sangue , Criança , Feminino , Humanos , Masculino , Peroxidase/imunologia , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Diagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available. RESULTS: Fifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (esophageal carcinoma SEREX antigen)-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a). Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA). The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues. CONCLUSIONS: We have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.
RESUMO
BACKGROUND: Various serologic markers such as anti-glycoprotein 2 antibodies and anti-Saccharomyces cerevisiae antibodies have been reported to be diagnostically useful in Crohn's disease. Mitsuyama et al. reported that antibodies to Crohn's disease peptide 353, a newly proposed serologic marker, were more useful in Japanese adults than anti-Saccharomyces. We addressed the same issue in Japanese children and adolescents. METHODS: Prospectively enrolled subjects under 17 years old assessed and treated at 12 pediatric centers in Japan included groups with Crohn's disease, ulcerative colitis, other intestinal diseases, or good health. The 3 serum markers were analyzed by enzyme-linked immunosorbent assays. RESULTS: Enrolled subjects, numbering 367, included 120 with Crohn's disease, 148 with ulcerative colitis, 56 with other intestinal diseases, and 43 healthy subjects. In Crohn's disease, anti-Crohn's disease peptide 353, anti-glycoprotein 2, and anti-Saccharomyces concentrations (median, 2.25, 3.0, and 8.9 U/mL) were significantly greater than in ulcerative colitis (1.1, 1.9, and 3.4; all P < 0.001), other intestinal diseases (1.1, 1.85, and 2.95; all P < 0.001), and healthy controls (1.1, 1.7, and 2.8; all P < 0.001), respectively. At 95% specificity, sensitivity of anti-Crohn's disease peptide (45.0%) was significantly higher than for anti-glycoprotein 2 (30.8%; P < 0.05) or anti-Saccharomyces (26.7%; P < 0.01). CONCLUSIONS: Anti-Crohn's disease peptide 353 proved more useful for diagnosis of Crohn's disease in Japanese children than the other 2 markers. To our knowledge, this is the first pediatric report to that effect.
Assuntos
Anticorpos/imunologia , Colite Ulcerativa/diagnóstico , Doença de Crohn/diagnóstico , Peptídeos/imunologia , Adolescente , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lactente , Japão , Masculino , Estudos Prospectivos , Saccharomyces cerevisiae/imunologia , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Autoantibodies against transcriptional intermediary factor 1 (TIF1) and Mi-2 are selectively detected in patients with dermatomyositis (DM). To measure these antibodies readily, the development of reliable ELISA systems has been needed. OBJECTIVE: This study aimed to establish enzyme-linked immunosorbent assays (ELISAs) for anti-TIF1γ and anti-Mi-2ß antibodies (Abs) and to assess their utility. METHODS: Serum samples were obtained from 104 patients with classic DM, 68 with clinically amyopathic DM (CADM) and 70 with polymyositis, who were followed up at 8 medical centers across Japan. Serum samples from 190 patients with other connective tissue diseases (CTDs) and 123 healthy individuals were also assessed. Serum antibody levels were examined by ELISAs coated with full-length TIF1γ or Mi-2ß proteins produced by a baculovirus expression system. To assess the cross-reactivity, partial-length Mi-2ß proteins with or without mutations were produced and examined for reactivity. RESULTS: When compared with immunoprecipitation assay, anti-TIF1γ Ab ELISA showed 100% sensitivity and 100% specificity, while anti-Mi-2ß Ab ELISA showed 100% sensitivity and 99.6% specificity. Anti-TIF1γ Ab was positive in 30 (28.8%) with classic DM and 4 (5.9%) with CADM, whereas 14 (13.5%) with classic DM, but none with CADM, were positive for anti-Mi-2ß Ab. Of 30 anti-TIF1γ Ab-positive DM patients, 23 (67.6%) had malignancy. Anti-Mi-2ß Ab-positive serum samples exhibited modest cross-reactivity with the TIF1γ protein due to the homologous amino acid sequence containing cysteines in their plant homeodomains. CONCLUSION: The current study demonstrates the utility of newly established ELISAs for anti-TIF1γ and anti-Mi-2ß Abs, which can serve as easier detection systems for routine testing.
Assuntos
Dermatomiosite/sangue , Dermatomiosite/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Fatores de Transcrição/imunologia , Adulto , Idoso , Autoanticorpos , Cisteína/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Conformação Proteica , Domínios Proteicos , Curva ROC , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol lambda directly binds to proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA replication and repair enzymes, both in vitro and in vivo. A pull-down assay using deletion mutants of Pol lambda showed that the confined C-terminal region of Pol lambda directly binds to PCNA. Furthermore, a synthetic peptide of 20-mers derived from the C-terminal region of Pol lambda competes with full-length Pol lambda for binding to PCNA. The residues between amino acids 518 and 537 of Pol lambda are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol lambda associates with PCNA in vivo by immunoprecipitation analysis and EGFP-tagged Pol lambda co-localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol lambda. Pol micro, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue.
Assuntos
Núcleo Celular/metabolismo , DNA Polimerase beta/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sítios de Ligação , DNA Nucleotidilexotransferase/metabolismo , DNA Polimerase beta/genética , Replicação do DNA , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Rad51 paralogs belong to the Rad52 epistasis group of proteins and are involved in homologous recombination (HR), especially the assembly and stabilization of Rad51, which is a homolog of RecA in eukaryotes. We previously cloned and characterized two RAD51 paralogous genes in Arabidopsis, named AtRAD51C and AtXRCC3, which are considered the counterparts of human RAD51C and XRCC3, respectively. Here we describe the identification of RAD51B homologue in Arabidopsis, AtRAD51B. We found a higher expression of AtRAD51B in flower buds and roots. Expression of AtRAD51B was induced by genotoxic stresses such as ionizing irradiation and treatment with a cross-linking reagent, cisplatin. Yeast two-hybrid analysis showed that AtRad51B interacted with AtRad51C. We also found and characterized T-DNA insertion mutant lines. The mutant lines were devoid of AtRAD51B expression, viable and fertile. The mutants were moderately sensitive to gamma-ray and hypersensitive to cisplatin. Our results suggest that AtRAD51B gene product is involved in the repair of double-strand DNA breaks (DSBs) via HR.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Reparo do DNA , DNA/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Northern Blotting , Cisplatino/farmacologia , Clonagem Molecular , DNA/genética , Dano ao DNA , DNA Bacteriano/genética , DNA Complementar/química , DNA Complementar/genética , Raios gama , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Filogenia , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
We identified a novel mutation of a nuclear-encoded gene, designated as CRUMPLED LEAF (CRL), of Arabidopsis thaliana that affects the morphogenesis of all plant organs and division of plastids. Histological analysis revealed that planes of cell division were distorted in shoot apical meristems (SAMs), root tips, and embryos in plants that possess the crl mutation. Furthermore, we observed that differentiation patterns of cortex and endodermis cells in inflorescence stems and root endodermis cells were disturbed in the crl mutant. These results suggest that morphological abnormalities observed in the crl mutant were because of aberrant cell division and differentiation. In addition, cells of the crl mutant contained a reduced number of enlarged plastids, indicating that the division of plastids was inhibited in the crl. The CRL gene encodes a novel protein with a molecular mass of 30 kDa that is localized in the plastid envelope. The CRL protein is conserved in various plant species, including a fern, and in cyanobacteria, but not in other organisms. These data suggest that the CRL protein is required for plastid division, and it also plays an important role in cell differentiation and the regulation of the cell division plane in plants. A possible function of the CRL protein is discussed.