RESUMO
The human discs, large homolog 1 gene (DLG1) is mapped to the schizophrenia-susceptibility locus 3q29, and it encodes a scaffold protein that interacts with the N-methyl-D-aspartate receptor presumably dysregulated in schizophrenia. In the current study, we have newly identified a splicing variant of DLG1, which is transcribed from an unreported 95-base-pair exon (exon 3b) and is labeled 3b(+). We investigated the mRNA expression of 3b(+) in the post-mortem dorsolateral prefrontal cortices of patients with psychiatric disorders, obtained from The Stanley Medical Research Institute, and examined the potential association of the expression with the genotype of the single-nucleotide polymorphism (SNP) rs3915512 located within exon 3b. A real-time quantitative reverse transcriptase-polymerase chain reaction revealed that the mRNA levels of 3b(+) were significantly reduced in patients with early-onset schizophrenia (onset at <18 years old, P=0.0003) but not in those with non-early-onset schizophrenia, early-onset or non-early-onset bipolar disorder or in the controls. Furthermore, the genotype at the rs3915512 SNP was closely associated with the levels of 3b(+) mRNA expression. It is inferred that the T allele fails to meet the exonic splicing enhancer consensus, thus resulting in skipping of exon 3b, leading to the expression of 3b(-) (the previously known DLG1 variant) but not 3b(+). Because all the subjects with early-onset schizophrenia in the current study possess the T/T genotype, the reduced level of the DLG1 3b(+) transcript may be involved in the susceptibility and/or pathophysiology of early-onset schizophrenia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Membrana/genética , Córtex Pré-Frontal/patologia , Esquizofrenia/genética , Idade de Início , Proteína 1 Homóloga a Discs-Large , Éxons , Feminino , Genes Supressores de Tumor , Predisposição Genética para Doença , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Esquizofrenia/epidemiologia , Esquizofrenia/patologiaAssuntos
Fístula Artério-Arterial/diagnóstico , Artérias Brônquicas/patologia , Anomalias dos Vasos Coronários/diagnóstico , Varizes/diagnóstico , Idoso , Broncoscopia , Angiografia Coronária , Diagnóstico Diferencial , Evolução Fatal , Feminino , Humanos , Japão , Pessoa de Meia-Idade , Artéria Pulmonar , Radiografia Torácica , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Schneckenbecken dysplasia (SBD) is an autosomal recessive lethal skeletal dysplasia that is classified into the severe spondylodysplastic dysplasias (SSDD) group in the international nosology for skeletal dysplasias. The radiological hallmark of SBD is the snail-like configuration of the hypoplastic iliac bone. SLC35D1 (solute carrier-35D1) is a nucleotide-sugar transporter involved in proteoglycan synthesis. Recently, based on human and mouse genetic studies, we showed that loss-of-function mutations of the SLC35D1 gene (SLC35D1) cause SBD. OBJECT: To explore further the range of SLC35D1 mutations in SBD and elucidate whether SLC35D1 mutations cause other skeletal dysplasias that belong to the SSDD group. METHODS AND RESULTS: We searched for SLC35D1 mutations in five families with SBD and 15 patients with other SSDD group diseases, including achodrogenesis type 1A, spondylometaphyseal dysplasia Sedaghatian type and fibrochondrogenesis. We identified four novel mutations, c.319C>T (p.R107X), IVS4+3A>G, a 4959-bp deletion causing the removal of exon 7 (p.R178fsX15), and c.193A>C (p. T65P), in three SBD families. Exon trapping assay showed IVS4+3A>G caused skipping of exon 4 and a frameshift (p.L109fsX18). Yeast complementation assay showed the T65P mutant protein lost the transporter activity of nucleotide sugars. Therefore, all these mutations result in loss of function. No SLC35D1 mutations were identified in all patients with other SSDD group diseases. CONCLUSION: Our findings suggest that SLC35D1 loss-of-function mutations result consistently in SBD and are exclusive to SBD.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Doenças Fetais/genética , Deleção de Genes , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Sequência de Bases , Clonagem Molecular , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Reação em Cadeia da Polimerase , Gravidez , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
Activation of RAS signalling induced by K-ras/BRAF mutations is a hallmark of colorectal tumours. In addition, Ras association domain families 1 and 2 (RASSF1 and RASSF2), the negative regulators of K-ras, are often inactivated by methylation of the promoter region in those tumours. However, reports showing differences in the occurrence of these alterations on the basis of tumour characteristics have been scarce. We analysed K-ras/BRAF mutations and the methylation status of RASSF1 and RASSF2 promoter regions in 120 colorectal adenomas with respect to their clinicopathological features. K-ras/BRAF mutations and RASSF2 methylation were observed in 49 (41%) and 30 (25%) of the samples, respectively, while RASSF1 methylation was observed in only 3 (2.5%). Adenomas with RASSF2 methylation often carried K-ras/BRAF mutations simultaneously (22 out of 30, P<0.01). Multivariate analysis revealed that the concomitance of these alterations was frequently observed in serrated adenomas (odds ratio (OR) 11.11; 95% confidence interval (CI) 1.96-63.00), but rarely in adenomas located in the sigmoid or descending colon (OR 0.13; 95% CI 0.03-0.58). A comparison between adenomas and cancers showed a significantly higher prevalence of these alterations in cancers than in adenomas in the proximal colon (58 vs 27%, P=0.02). Frequency and the time point of the occurrence of Ras signalling disorders differ according to colorectal neoplasia's characteristics, particularly the location.
Assuntos
Adenoma/genética , Adenoma/patologia , Aberrações Cromossômicas , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/genética , Adenoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/metabolismo , Análise Mutacional de DNA , Feminino , Genes ras/genética , Humanos , Masculino , Metilação , Pessoa de Meia-Idade , Análise Multivariada , Mutação , Proteínas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: We previously reported that gastrin induces expression of CXC chemokines through activation of nuclear factor kappaB (NFkappaB) in gastric epithelial cells that express gastrin receptor. AIMS: To clarify gastrin receptor mediated signals leading to activation of NFkappaB. METHODS: MKGR26 cells were created by transfecting gastrin receptor cDNA into MKN-28 cells. Degradation of inhibitor kappaB (IkappaB) and phosphorylation of protein kinase C (PKC)-delta were both detected by western blot analysis. NFkappaB activation was determined by luciferase assay and electrophoretic mobility shift analysis. RESULTS: Gastrin induced degradation of IkappaB-alpha and activation of NFkappaB, which was abolished by the selective gastrin receptor antagonist L-740,093 and the general PKC inhibitor GF109203X. Gastrin induced phosphorylation of PKC-delta, and its inhibitor rottlerin partially suppressed NFkappaB activation. However, the mitogen activated protein kinase (MAPK) kinase inhibitor PD98059, p38 MAPK inhibitor SB203580, and tyrphostin AG1478 had no effect on NFkappaB activation. Introduction of the dominant negative mutant of IkappaB kinase, of NFkappaB inducing kinase, and of tumour necrosis factor receptor associated factor 6 (TRAF6), but not that of TRAF2, inhibited gastrin induced activation of NFkappaB. CONCLUSIONS: Gastrin activates NFkappaB via a PKC dependent pathway which involves IkappaB kinase, NFkappaB inducing kinase, and TRAF6.
Assuntos
Mucosa Gástrica/efeitos dos fármacos , Gastrinas/farmacologia , NF-kappa B/metabolismo , Proteína Quinase C/fisiologia , Proteínas/fisiologia , Animais , Western Blotting , Linhagem Celular , DNA Complementar/genética , Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Cobaias , Humanos , NF-kappa B/antagonistas & inibidores , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Receptores da Colecistocinina/antagonistas & inibidores , Receptores da Colecistocinina/genética , Receptores da Colecistocinina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF , Transfecção , Células Tumorais Cultivadas , Quinase Induzida por NF-kappaBRESUMO
Peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits the growth of several types of cancer cells. However, the mechanisms by which this occurs are poorly understood. The goal of the present study was to investigate the effects of PPARgamma on mutated ras-induced cell growth, activation of transcription factors and expression of genes associated with cellular transformation in rat intestinal epithelial cells. A human PPARgamma cDNA was introduced to the activated H-ras-transfected IEC-6 cells (IECras) and 1 clone (IECrasPR82) that stably expresses both activated ras and PPARgamma was obtained. Thiazolidinedione derivatives such as troglitazone and rosiglitazone, selective ligands for PPARgamma, inhibited the cellular growth of IECrasPR82 cells in a time-dependent manner and induced G1 cell cycle arrest. Treatment with troglitazone (20 microM) decreased the expression of cyclin D1, heparin-binding epidermal growth factor-like growth factor (HB-EGF) and amphiregulin and suppressed the promoter activities of cyclin D1 and HB-EGF. Furthermore, a luciferase assay and an electrophoretic mobility shift assay showed that thiazolidinedione derivatives suppressed the transcriptional activities of AP-1 and Ets, both of which play crucial roles in the expression of cyclin D1 and HB-EGF. These findings suggest that reduction of EGF-like growth factors and cyclin D1 through the suppression of AP-1 and Ets may be 1 mechanism whereby PPARgamma inhibits their growth.
Assuntos
Ciclina D1/biossíntese , Fator de Crescimento Epidérmico/biossíntese , Receptores Citoplasmáticos e Nucleares/agonistas , Tiazolidinedionas , Fatores de Transcrição/agonistas , Proteínas ras/metabolismo , Animais , Northern Blotting , Western Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Cromanos/farmacologia , Ciclina D1/antagonistas & inibidores , Ciclina D1/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/antagonistas & inibidores , Células Epiteliais/metabolismo , Citometria de Fluxo , Humanos , Hipoglicemiantes/farmacologia , Intestinos/citologia , Ligantes , Luciferases/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ratos , Rosiglitazona , Tiazóis/farmacologia , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , TroglitazonaRESUMO
Although hypergastrinemia is frequently observed in individuals with a chronic Helicobacter pylori infection, its pathophysiological significance in gastric mucosal inflammation is unclear. The present study was designed to determine if gastrin induces the expression of CXC chemokines in gastric epithelial cells. Human and rat gastric epithelial cells, transfected with gastrin receptor, were stimulated with gastrin. The expression of mRNAs for human interleukin-8 (IL-8) and rat cytokine-induced neutrophil chemoattractant-1 and release of human IL-8 protein were then determined by Northern blot analysis and ELISA, respectively. Gastrin not only induced the expression of mRNAs for these chemokines but also stimulated IL-8 protein release. A luciferase assay using IL-8 promoter genes showed that nuclear factor (NF)-kappaB is absolutely required and activator protein-1 (AP-1) is partly required for the maximum induction of IL-8 by gastrin. An electrophoretic mobility shift assay revealed that gastrin is capable of activating both NF-kappaB and AP-1. In addition, the inhibition of NF-kappaB abrogated gastrin-induced chemokine expression. These results suggest that gastrin is capable of upregulating CXC chemokines in gastric epithelial cells and therefore may contribute to the progression of the inflammatory process in the stomach.
Assuntos
Quimiocinas CXC/biossíntese , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Gastrinas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , NF-kappa B/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL1 , Quimiocinas CXC/genética , Fatores Quimiotáticos/biossíntese , Fatores Quimiotáticos/genética , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Mucosa Gástrica/citologia , Mucosa Gástrica/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Humanos , Interleucina-1/farmacologia , Interleucina-8/biossíntese , Interleucina-8/genética , NF-kappa B/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , Pirrolidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores da Colecistocinina/genética , Receptores da Colecistocinina/metabolismo , Tiocarbamatos/farmacologia , Fator de Transcrição AP-1/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND & AIMS: Epidermal growth factor (EGF)-like growth factors are induced after acute gastric injury and may play an important role in mucosal repair. However, the mechanisms that trigger these growth factors are poorly understood. We determined the role of EGF receptor (EGFR) in stress-induced expression of heparin-binding EGF-like growth factor (HB-EGF) in a rat gastric epithelial cell line (RGM1 cells). METHODS: RGM1 cells were transfected with a plasmid containing complementary DNA encoding a dominant-negative human EGFR (HERCD533). Cells were treated with hydrogen peroxide (0-400 micromol/L) or sorbitol (600 mmol/L). Tyrosine phosphorylation of EGFR was determined by immunoprecipitation and Western blotting with an antiphosphotyrosine antibody. HB-EGF messenger RNA and protein were determined with Northern and Western blotting, respectively. Cell growth was evaluated by cell number and [(3)H]thymidine incorporation. RESULTS: Oxidative stress and osmotic stress induced tyrosine phosphorylation of EGFR within 2 minutes, followed by a marked increase in HB-EGF and amphiregulin transcripts in RGM1 cells. Introduction of HERCD533 into the cells inhibited not only tyrosine phosphorylation of EGFR but also growth response to EGF. Furthermore, oxidative stress-induced HB-EGF messenger RNA expression was impaired in HERCD533-expressing cells. CONCLUSIONS: EGFR plays a crucial role in the stress-induced expression of EGF-like growth factors in gastrointestinal epithelial cells.
Assuntos
Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Mucosa Gástrica/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Estresse Oxidativo/fisiologia , Anfirregulina , Animais , Western Blotting , Meios de Cultura Livres de Soro/farmacologia , DNA Complementar , Família de Proteínas EGF , Fator de Crescimento Epidérmico/análise , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Mucosa Gástrica/citologia , Mucosa Gástrica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Glicoproteínas/genética , Substâncias de Crescimento/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Ligantes , Fosforilação , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transfecção , Fator de Crescimento Transformador alfa/genética , Tirosina/metabolismoRESUMO
The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor. In the present study, we investigated the role of met expression on the modulation of apoptosis in colorectal tumours. The gene expressions of c-met and the anti-apoptotic bcl-2 family, including bcl-2, bcl-x(L)and bcl-w, were analysed in human colorectal adenomas and adenocarcinomas by using a quantitative polymerase chain-reaction combined with reverse transcription. In seven of 12 adenomas and seven of 11 carcinomas, the c-met gene was overexpressed. The bcl-w, bcl-2 and bcl-x(L)genes were over-expressed in nine, five and six of 12 adenomas and in five, two and seven of 11 carcinomas, respectively. The c-met mRNA level in human colorectal adenomas and carcinomas was correlated with bcl-w but not with bcl-2 or with bcl-x(L)mRNA level. The administration of c-met-antisense oligonucleotides decreased Met protein levels in the LoVo human colon cancer cell line. In the case of c- met -antisense-treated cells, apoptotic cell death induced by serum deprivation was more prominent, compared to control or c-met -nonsense-treated cells. Treatment with c-met-antisense oligonucleotides inhibits the gene expression of bcl-w in LoVo cells. On the other hand, the gene expression of bcl-2 or bcl-x(L)was not affected by treatment with c-met-antisense oligonucleotides. These findings suggest that Met expression modulates apoptosis through bcl -w expression in colorectal tumours.
Assuntos
Apoptose , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Biossíntese de Proteínas , Proteínas , Proteínas Proto-Oncogênicas c-met/metabolismo , Adenocarcinoma/metabolismo , Adenoma/metabolismo , Adulto , Idoso , Proteínas Reguladoras de Apoptose , Western Blotting , Carcinoma/metabolismo , Estudos de Casos e Controles , Colo/metabolismo , Meios de Cultura Livres de Soro , DNA/metabolismo , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Células Tumorais Cultivadas , Regulação para Cima , Proteína bcl-XRESUMO
Many articles pertaining to quality of life (QOL) following stem cell transplantation have been published in the US and western Europe. However, since the actions of health insurance systems and overall cultural aspects are strongly associated with QOL, investigations into QOL should be carried out within all countries. Therefore, we have investigated the QOL of adult patients following stem cell transplantation at 31 hospitals in Japan. The survivors, who were surveyed by mail questionnaire, were 20 years or older at the time of this study. The underlying diseases were acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, myelodysplastic syndrome, and multiple myeloma. Median age at the time of the study was 36 years, and median interval after transplantation was 35.3 months. Of 383 patients surveyed, 282 (73.6%) responded to the questionnaire. One hundred and ninety-two patients were treated with an allogeneic-related transplantation, 52 with allogeneic-unrelated, and 38 with an autologous transplantation. Our data revealed that the length of time since transplantation and the diagnosis of chronic GVHD were associated with QOL. When unrelated and related transplantation recipients were compared, ratings on relief from pain, stability in weight, and confidence in dealing with daily life were lower among unrelated transplantation patients.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/psicologia , Qualidade de Vida , Adolescente , Adulto , Idoso , Estudos de Avaliação como Assunto , Feminino , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/psicologia , Neoplasias Hematológicas/terapia , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Inquéritos e Questionários , Transplante Autólogo/efeitos adversos , Transplante Autólogo/psicologia , Transplante Homólogo/efeitos adversos , Transplante Homólogo/psicologiaRESUMO
Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) is shown to inhibit the growth of MKN-45 cells, a human gastric cancer cell line, which overexpresses c-Met tyrosine kinase. The aim of the present study was to investigate whether PPARgamma regulates the expression of c-Met. Two days after the activation of PPARgamma by troglitazone, a potent and selective PPARgamma ligand, a dramatic reduction of c-MET transcripts and the c-Met protein in MKN45 cells was observed. The luciferase assay showed that the activation of PPARgamma suppressed -249 to +330 c-MET promoter activity, driven by cotransfection of ETS-1 expression vector. These data demonstrate that PPARgamma activation is capable of suppressing Ets-induced c-MET gene transcription. Thus, it is possible that the growth inhibitory effect of PPARgamma on MKN-45 cells is related to the suppression of c-MET transcription.
Assuntos
Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Tiazolidinedionas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Divisão Celular/efeitos dos fármacos , Cromanos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas c-ets , Neoplasias Gástricas/tratamento farmacológico , Supressão Genética , Tiazóis/farmacologia , Ativação Transcricional , Troglitazona , Células Tumorais CultivadasRESUMO
BACKGROUND AND STUDY AIMS: The aim of the present study was to evaluate a new endoscope disinfector (WM-1) that uses acidic electrolytic water (AEW). MATERIALS AND METHODS: AEW was produced by electrolysis of a 0.05% NaCl-water mixture, with a redox potential greater than 1000 mV and a pH lower than 2.7. In the first study, an endoscope artificially contaminated with 15 species of bacteria and four strains of viruses was treated using the WM-1. In the second study, endoscopic contamination after clinical use was examined by culture for Helicobacter pylori and other bacteria, and by polymerase chain reaction for the H. pylori urease gene and hepatitis C virus. The extent of contamination was then examined after exposing the WM-1 to AEW. The safety of AEW was examined using both in vivo and in vitro studies. RESULTS: All of the bacteria and viruses were destroyed or inactivated after the instrument had been exposed to AEW. Clinical contamination was detected from the instrument in 19 of 30 endoscopic procedures, whereas no bacteria or viruses were detected after five minutes' exposure to AEW. AEW was found to be nonirritant, nontoxic to cells, and nonmutagenic. CONCLUSION: The WM-1 successfully and safely disinfected the endoscopes. With running costs of yen 24 per day ($0.21 per day), the WM-1 provides an effective and inexpensive alternative to conventional disinfection equipment.
Assuntos
Desinfetantes , Desinfecção/métodos , Endoscópios , Contaminação de Equipamentos , Ácidos , Animais , Desinfecção/instrumentação , Endoscopia Gastrointestinal , Estudos de Avaliação como Assunto , Masculino , Coelhos , ÁguaRESUMO
The high-affinity IgE receptor (Fc epsilonRI) and the low-affinity IgG receptor (Fc gammaRIII) on mast cells are the key molecules involved in triggering the allergic reaction. These receptors share the common beta subunit (FcRbeta) which contains an immunoreceptor tyrosine-based activation motif and transduces the signals of these receptors' aggregation. In rodents, FcRbeta is essential for the cell surface expression of the Fc epsilonRI. In humans, the FcRbeta gene was reported to be one of the candidate genes causing atopic diseases. However, the role of FcRbeta in vivo still remains ambiguous. To elucidate the functions of FcRbeta, we developed the mice lacking FcRbeta [FcRbeta(-/-)]. The FcRbeta(-/-) mice lacked the expression of the Fc epsilonRI on mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) was not induced in FcRbeta(-/-) mice as was expected. In these mice, the expression of IgG receptors on mast cells was augmented but the IgG-mediated PCA reaction was attenuated. Although with bone marrow-derived cultured mast cells from FcRbeta(-/-), adhesion to fibronectin and Ca2+ flux upon aggregation of IgG receptors were enhanced, mast cells co-cultured with 3T3 fibroblasts exhibited impaired degranulation on receptor aggregation. These observations indicate that FcRbeta accelerates the degranulation of mature mast cells via the IgG receptor in connective tissues.
Assuntos
Mastócitos/imunologia , Receptores Fc/metabolismo , Células 3T3 , Animais , Células da Medula Óssea , Células Cultivadas , Técnicas de Cocultura , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Anafilaxia Cutânea Passiva/imunologia , Receptores Fc/genética , Receptores de IgE/metabolismo , Receptores de IgG/metabolismo , Transdução de Sinais , Tirosina/metabolismoRESUMO
Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, is generally used as a specific inhibitor of protein kinase C. It is known that 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, induces expression of mRNA for amphiregulin (AR), a member of EGF-related polypeptides, in mammalian epithelial cells. In this work, we determined the effect of calphostin C on AR mRNA expression in IEC-6 cells, a rat intestinal epithelial cell line, and unexpectedly found that this compound enhanced the TPA-induced expression of AR mRNA. Moreover, calphostin C alone induced expression of AR mRNA in a light-dependent manner, and this effect was abrogated by pretreatment with N-acetylcysteine. These results suggest that calphostin C can upregulate expression of AR mRNA via reactive oxygen species.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicoproteínas/biossíntese , Substâncias de Crescimento/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Naftalenos/farmacologia , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Anfirregulina , Animais , Linhagem Celular , Interações Medicamentosas , Família de Proteínas EGF , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Sequestradores de Radicais Livres/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Naftalenos/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The present study was designed to investigate whether heparin-binding epidermal growth factor-like growth factor and its related peptides are expressed in response to gastrin in rat stomach. Rat gastrin-17I (2.5 nmol/kg/hour) or gastrin-17I plus gastrin receptor antagonist, L-740,093 (2.0 mg/kg/hour), was injected intravenously into male Sprague-Dawley rats. RNA was extracted from oxyntic mucosa, and heparin-binding epidermal growth factor-like growth factor and related peptide gene expression was estimated using a ribonuclease protection assay. The level of transforming growth factor-alpha mRNA did not change at any time point during the experiment. In contrast, the levels of heparin-binding epidermal growth factor-like growth factor and amphiregulin mRNA were significantly increased within 3 hours following gastrin infusion and reached maximum levels 6 and 12 hours later, respectively. Continuous infusion of gastrin significantly increased oxyntic mucosal proliferation. Gastrin receptor antagonist significantly inhibited gastrin-induced heparin-binding epidermal growth factor-like growth factor and amphiregulin gene expression and gastrin-induced oxyntic mucosal proliferation. These findings indicate that heparin-binding epidermal growth factor-like growth factor and amphiregulin genes are induced by gastrin and that they play a role in the trophic action of gastrin on oxyntic mucosa.
Assuntos
Fator de Crescimento Epidérmico/biossíntese , Mucosa Gástrica/metabolismo , Gastrinas/farmacologia , Glicoproteínas/biossíntese , Substâncias de Crescimento/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Transcrição Gênica/efeitos dos fármacos , Anfirregulina , Animais , Benzodiazepinonas/farmacologia , DNA Complementar , Família de Proteínas EGF , Mucosa Gástrica/citologia , Mucosa Gástrica/efeitos dos fármacos , Gastrinas/administração & dosagem , Heparina/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Infusões Intravenosas , Cinética , Masculino , Células Parietais Gástricas/efeitos dos fármacos , Células Parietais Gástricas/metabolismo , Compostos de Fenilureia/farmacologia , Sondas RNA , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptor de Colecistocinina B , Receptores da Colecistocinina/antagonistas & inibidoresRESUMO
We isolated a cDNA encoding ribosomal protein S2 in sockeye salmon, Oncorhynchus nerka, using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. The cDNA encoding ribosomal protein S2 is composed of 933 nucleotides, and has a 5'-noncoding sequence of 9 bases, a 885 base open reading frame coding for a 294 amino acid polypeptide, and a 39 base 3'noncoding sequence. The amino acid sequence of sockeye salmon S2 protein deduced from the nucleotide sequence is highly homologous to those from the rat (86.1%) and Drosophila melanogaster (73.6%). The N-terminal region of S2 protein is rich in arginine-glycine sites, including eight tandem repeats, and has two consecutive copies of the RGGF motif. The sequences are considered to be requisites for nucleolar localization and binding to RNA for nucleolar proteins. Southern blot analysis indicates that there may be only a single copy of the S2 gene, which is a multiple copy gene in the rat and the fruit fly. Northern blot analysis shows that the S2 gene is expressed in the brain, pituitary, heart, liver kidney, muscle, testis and ovary of sockeye salmon.
Assuntos
DNA Complementar/genética , Proteínas Ribossômicas/genética , Salmão/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Primers do DNA/genética , Drosophila melanogaster/genética , Feminino , Expressão Gênica , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sequências Repetitivas de Ácido Nucleico , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Salmão/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Salmonid fish have pairs of genes for various hypothalamic and pituitary hormones including neurohypophysial hormones, vasotocin, and isotocin, probably because they are tetraploid. The problem here is whether two genes for the same hormone are expressed equally or differently. We therefore examined expression patterns of vasotocin and isotocin genes in four salmonid species using Northern blot analysis with chum salmon cDNAs as hybridization probes. The presence of two vasotocin and also two isotocin genes was confirmed by Southern blot analysis in rainbow trout and sockeye salmon which were not examined previously. Prior to Northern blot analysis, isotocin-I cDNA of sockeye salmon was determined and compared to those of chum salmon and masu salmon, since molecular probes are so specific that cross-species hybridization often leads misinterpretation in a quantitative study. The nucleotide sequence of sockeye salmon isotocin-I cDNA showed sufficiently high homology (> 96.0%) to those of chum salmon and masu salmon for cross-species hybridization among salmonids. Northern blot analysis showed that both isotocin-I and isotocin-II genes were well expressed in all species examined. Expression of isotocin-I gene tended to be relatively higher than that of isotocin-II gene in all species. However, expression pattern of vasotocin-I and vasotocin-II genes did not coincide among species. Expression of vasotocin-II genes was very weak or scarce in masu salmon and rainbow trout, while that in sockeye salmon was stronger than vasotocin-I gene expression. The present result may reflect complicated molecular evolution of salmonid vasotocin genes probably both in regulatory and coding regions.