RESUMO
Glucose is essential as the main energy source for living organisms. However, excessive elevation of blood sugar levels can lead to diabetes and serious complications such as arteriosclerosis. Even though blood sugar levels as well as hypoxia associated with hyperglycemia are known to be closely related to diabetes complications, the responses of vascular endothelial cells to glucose and oxygen have not been fully investigated. In this study, using a microfluidic device that can control the oxygen concentration, we observed the behavior of vascular endothelial cell monolayers while simultaneously controlling glucose and oxygen levels. Results showed that the cell migration speed was increased by high-glucose exposure in an oxygen-rich environment, but was decreased in a hypoxic environment regardless of glucose condition. The expression of vascular endothelial-cadherin at the cell periphery, which plays a role in cell-cell adhesion, was increased by hypoxic exposure, but was largely independent of glucose condition. This suggested that cell-cell adhesion is less involved in the increase in migration caused by high glucose. Furthermore, stabilization and nuclear translocation of hypoxia-inducible factor-1α, which is involved in cellular hypoxia sensing, increased 5 h after exposure to high glucose, but decreased 3 days after the exposure. This indicated that intracellular hypoxia was generated by increased oxygen consumption in mitochondria just after the high-glucose exposure, but it was moderated within 3 days.
Assuntos
Glucose , Oxigênio , Humanos , Oxigênio/metabolismo , Glucose/farmacologia , Glucose/metabolismo , Células Endoteliais/metabolismo , Glicemia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Hipóxia , Movimento CelularRESUMO
BACKGROUND: In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair. METHODS: Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay. RESULTS: Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency. CONCLUSION: These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Pontos de Checagem do Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Fatores de Transcrição TFII/fisiologia , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Citometria de Fluxo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismoRESUMO
During organ development, local changes in gene expression govern morphogenesis and cell fate. We have generated a microanatomical atlas of epithelial gene expression of embryonic salivary glands. The mouse submandibular salivary gland first appears as a single mass of epithelial cells surrounded by mesenchyme, and it undergoes rapid branching morphogenesis to form a complex secretory organ with acini connected to an extensive ductal system. Using laser capture microdissection, we collected samples from 14 distinct epithelial locations at embryonic days 12.5, 13.5, 14, and 15, and characterized their gene expression by microarray analysis. These microarray results were evaluated by qPCR of biological replicates and by comparisons of the gene expression dataset with published expression data. Using this gene expression atlas to search for novel regulators of branching morphogenesis, we found a substantial reduction in mRNA levels of GSK3ß at the base of forming clefts. This unexpected finding was confirmed by immunostaining, and inhibition of GSK3ß activity enhanced salivary gland branching. This first microanatomical expression atlas of a developing gland characterizes changes in local gene expression during salivary gland development and differentiation, which should facilitate the identification of key genes involved in tissue morphogenesis.
Assuntos
Bases de Dados Genéticas , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/fisiologia , Organogênese/genética , Glândula Submandibular/embriologia , Animais , Mapeamento Cromossômico , Regulação para Baixo , Células Epiteliais/fisiologia , Perfilação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Endogâmicos ICR , Microdissecção/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Órgãos , Transdução de SinaisRESUMO
BACKGROUND: The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams-Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date. METHODS: A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated. RESULTS: We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.
Assuntos
Proteína BRCA1/fisiologia , Fatores de Transcrição TFII/fisiologia , Animais , Proteína BRCA1/metabolismo , Células COS , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Chlorocebus aethiops , Dano ao DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Sirtuína 1/genética , Sirtuína 1/metabolismo , Transativadores/metabolismo , Transativadores/fisiologia , Fatores de Transcrição TFII/metabolismo , Ativação Transcricional/fisiologiaRESUMO
BACKGROUND: DBC1/KIAA1967 (deleted in breast cancer 1) is a putative tumour-suppressor gene cloned from a heterozygously deleted region in breast cancer specimens. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. Hereditary breast and ovarian cancer susceptibility gene product BRCA1, by binding to the promoter region of SIRT1, is a positive regulator of SIRT1 expression. METHODS: A physical interaction between DBC1 and BRCA1 was investigated both in vivo and in vitro. To determine the pathophysiological significance of DBC1, its role as a transcriptional factor was studied. RESULTS: We found a physical interaction between the amino terminus of DBC1 and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous DBC1 and BRCA1 form a complex in the nucleus of intact cells, which is exported to the cytoplasm during ultraviolet-induced apoptosis. We also showed that the expression of DBC1 represses the transcriptional activation function of BRCT by a transient expression assay. The expression of DBC1 also inhibits the transactivation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed that DBC1 may modulate the cellular functions of BRCA1 and have important implications in the understanding of carcinogenesis in breast tissue.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteína BRCA1/metabolismo , Regulação Neoplásica da Expressão Gênica , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/genética , Proteína BRCA1/química , Proteína BRCA1/fisiologia , Células Cultivadas , Células HeLa , Humanos , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Sirtuína 1/genética , Distribuição Tecidual , Ativação Transcricional/genéticaRESUMO
BACKGROUND: The phosphatidylinositol 3'-kinase (PI3K)-AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K-AKT-activating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.
Assuntos
Proteínas Sanguíneas/genética , Neoplasias do Endométrio/genética , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Linhagem Celular Tumoral , Metilação de DNA , Neoplasias do Endométrio/enzimologia , Feminino , Humanos , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Colouterine fistulae secondary to sigmoid diverticulitis are unusual. Methods for diagnosis remain to be established. We report a case with a colouterine fistula in which sonohysterography detected the flow of ultrasound contrast medium between the uterine cavity and the sigmoid colon through the posterior uterine wall, thus confirming the diagnosis. The diagnosis was further substantiated by a charcoal challenge test. The patient underwent en bloc resection of the uterus, Fallopian tubes, ovaries and sigmoid colon, the organs involved with diverticulitis. This is the first report to describe a colouterine fistula successfully diagnosed by sonohysterography using ultrasound contrast medium.
Assuntos
Diverticulose Cólica/diagnóstico por imagem , Fístula/diagnóstico por imagem , Fístula Intestinal/diagnóstico por imagem , Doenças do Colo Sigmoide/diagnóstico por imagem , Doenças Uterinas/diagnóstico por imagem , Idoso , Diverticulose Cólica/complicações , Endossonografia , Feminino , Fístula/etiologia , Humanos , Fístula Intestinal/etiologia , Cuidados Pré-Operatórios/métodos , Doenças do Colo Sigmoide/complicações , Doenças Uterinas/etiologiaRESUMO
Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.
Assuntos
Fase de Clivagem do Zigoto/efeitos dos fármacos , Fase de Clivagem do Zigoto/efeitos da radiação , Proteínas Fetais , Cloreto de Lítio/farmacologia , Proteínas Repressoras , Salamandra/embriologia , Fatores de Transcrição , Raios Ultravioleta , Animais , Padronização Corporal , Transplante de Células , Fase de Clivagem do Zigoto/citologia , Citoplasma/fisiologia , Proteínas de Ligação a DNA/biossíntese , Proteínas do Olho , Proteína Goosecoid , Proteínas de Homeodomínio/biossíntese , Hibridização In Situ , Modelos Biológicos , Morfogênese , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Proteínas com Domínio T/biossíntese , Xenopus/embriologiaRESUMO
We report here the efficacy of amantadine hydrochloride for two patients with alternating hemiplegia of childhood (AHC) that did not respond to flunarizine. Amantadine was administered to one patient at age one year and seven months and to the other at age 25 years. The frequencies and duration of the hemiplegic attacks significantly improved in both patients. However, the attacks gradually returned to the previous level after a significant reduction in seizures for three years in the younger patient with ongoing AHC. Our therapeutic results further support the hypothesis that glutamate and NMDA receptors are involved in inducing alternating hemiplegic attacks, because amantadine as well as its derivative, memantine, are clinically available non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, with neuroprotective effects. Amantadine is worth trying when treating patients with AHC as a first trial or a substitute for flunarizine once the latter agent looses effect.
Assuntos
Amantadina/farmacologia , Dopaminérgicos/farmacologia , Hemiplegia/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/fisiologia , Adulto , Feminino , Ácido Glutâmico/metabolismo , Hemiplegia/patologia , Humanos , Lactente , Masculino , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Recidiva , Convulsões/tratamento farmacológico , Resultado do TratamentoRESUMO
OBJECTIVES: Corticosteroids are considered to be contraindicated during the acute phase of Kawasaki disease (KD) based on unfavorable results in early studies. In our hospital, however, corticosteroids have been used in some cases of KD with satisfactory results. We analyzed outcomes of patients with KD treated with or without corticosteroids. STUDY DESIGN: Medical records of 299 children with KD treated with one of the 4 regimens were reviewed retrospectively. Regimen 1 consisted of aspirin, dipyridamole, and propranolol; regimen 2 was regimen 1 plus prednisolone, 2 mg/kg/d, for 1 week, followed by tapering over 2 weeks; regimen 3 was regimen 1 plus intravenous gamma-globulin (IVGG), 200 or 400 mg/kg/d, for 5 consecutive days; and regimen 4 was regimen 1 plus both prednisolone and IVGG. RESULTS: Although patients treated with regimens 2 and 4 were more ill at presentation than those treated with regimens 1 and 3, respectively, the duration of fever was shorter in the former patient groups (P =.0013). Coronary aneurysms developed least frequently in patients treated with regimen 4 and less frequently with regimen 2 than with regimen 1 (P =.0730). Multiple regression analysis showed significant reductions of fever and coronary aneurysm incidence with prednisolone (P <.0001 and P =.0307, respectively). CONCLUSION: Our data suggest a possible role of corticosteroids in the treatment of the acute phase of KD.
Assuntos
Glucocorticoides/administração & dosagem , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Prednisolona/administração & dosagem , Doença Aguda , Aspirina/administração & dosagem , Pré-Escolar , Aneurisma Coronário/complicações , Dipiridamol/administração & dosagem , Quimioterapia Combinada , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Propranolol/administração & dosagem , Estudos RetrospectivosRESUMO
Several T-box genes are considered to play important roles in developing limbs, tails and neural retinae. Five novel T-box genes in the Japanese newt were isolated and their expression was analyzed, together with another T-box gene of brachyury, during embryogenesis and in the developing and regenerating limbs and tail. Four are designated CpTbx2, CpTbx3, CpTbx6R and CpEomesodermin based on molecular phylogenetic analyses, and the other is named CpUbiqT from its ubiquitous expression. While all were expressed during embryogenesis, only four of them (CpTbx2, CpTbx3, CpUbiqT and brachyury) were detected in developing limbs and/or tails. Except for brachyury, they were continuously expressed in normal adult appendages and showed elevated expression levels in regenerating limbs, whereas only CpTbx2 showed significant up-regulation in regenerating tails. Compared with orthologous genes in other species, CpTbx2, CpTbx3 and CpEomesodermin showed several notable differences such as an abundance of maternal transcripts of CpEomesodermin, a unique insertion sequence within the T-box domain of CpTbx2, and a lack of visible expression of CpTbx2and CpTbx3 in the apical ectodermal region of developing limbs. In view of the uniqueness of the newt, these results are discussed with respect to the possibility of their involvement in regeneration.
Assuntos
Proteínas de Ligação a DNA/genética , Extremidades/embriologia , Proteínas Fetais , Regulação da Expressão Gênica no Desenvolvimento , Regeneração/genética , Salamandridae/embriologia , Proteínas com Domínio T , Cauda/embriologia , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Extremidades/crescimento & desenvolvimento , Dados de Sequência Molecular , Salamandridae/genética , Homologia de Sequência de Aminoácidos , Cauda/crescimento & desenvolvimentoAssuntos
Fator Estimulador de Colônias de Granulócitos/sangue , Síndrome de Linfonodos Mucocutâneos/sangue , Síndrome de Linfonodos Mucocutâneos/imunologia , Doença Aguda , Estudos de Casos e Controles , Pré-Escolar , Convalescença , Doença das Coronárias/diagnóstico por imagem , Doença das Coronárias/etiologia , Feminino , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Contagem de Leucócitos , Peróxidos Lipídicos/sangue , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Síndrome de Linfonodos Mucocutâneos/metabolismo , Síndrome de Linfonodos Mucocutâneos/terapia , Neutrófilos , UltrassonografiaRESUMO
The long-term outcome of coronary aneurysm after Kawasaki disease was investigated using dipyridamole-loading angiography in 33 children with coronary aneurysms after Kawasaki disease. Morphologic regression may not accompany functional improvement in the dilated coronary arteries.
Assuntos
Aneurisma Coronário/diagnóstico por imagem , Angiografia Coronária , Vasos Coronários/patologia , Dipiridamol , Síndrome de Linfonodos Mucocutâneos/complicações , Vasodilatadores , Adolescente , Cateterismo Cardíaco , Criança , Pré-Escolar , Aneurisma Coronário/etiologia , Aneurisma Coronário/fisiopatologia , Vasos Coronários/fisiopatologia , Dipiridamol/administração & dosagem , Feminino , Seguimentos , Humanos , Processamento de Imagem Assistida por Computador , Lactente , Infusões Intravenosas , Masculino , Índice de Gravidade de Doença , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagemRESUMO
In a previous study, we investigated otorhinolaryngological care by the general practitioners in medical rural areas and demonstrated that a close relationship between general practitioners and otorhinolaryngologists was necessary. In the present study, a survey of prefectural health administration's views on otorhinolaryngological care in medical rural areas was performed. A questionnaire for otorhinolaryngological care in medical rural areas was sent to the divisions of health administration in the 47 prefectures of Japan. Of these prefectures, 46 (97.9%) responded. About 80% of respondents have medical rural areas, and the otorhinolaryngological care is inadequate in most areas. General practitioners of internal medicine or surgery are primarily demanded in about 75% of the prefectures. Otorhinolaryngological care in medical rural areas has never been considered in most prefectures. Because the need for otorhinolaryngological medical care will increase, prefectural health administrations need to pay more attention to otorhinolaryngological care in medical rural areas, and a close relationship between general practitioners, otorhinolaryngologists, and the administration should be established.
Assuntos
Serviços de Saúde Comunitária , Administração de Serviços de Saúde , Área Carente de Assistência Médica , Otolaringologia , Medicina de Família e Comunidade , Humanos , Japão , Saúde da População Rural , Inquéritos e Questionários , Recursos HumanosRESUMO
This is a report of two-year-old female monozygotic twins, both having a ventricular septal defect (VSD, Kirklin type II) and accompanying moderate pulmonary hypertension (PH). The two patients underwent a patch closure procedure to repair the VSD at the same time, and their postoperative courses were uneventful. Rubenstein and Weaver have reported the only case of surgical treatment for monozygotic twins having both VSD and PH. Our patients are the second such case of surgical treatment of VSD and PH in monozygotic twins.