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Intestinal-type alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be a gut mucosal defense factor. In humans, a single gene (ALPI) for IAP has been isolated, and its transcription produces two kinds of alternatively spliced mRNAs (aAug10 and bAug10). Recently, we discovered that vitamin D up-regulated the expression of both types of human IAP alternative splicing variants in Caco-2 cells. However, the functional difference of protein encoded by the mRNA variants has remained elusive. In the present study, we aimed to provide further insight into the characterization and structure of IAP isoforms. To analyze the protein translated from the ALPI gene, we constructed two kinds of cDNA expression plasmids (aAug10 and bAug10), and the transfected cells were homogenized and assayed for alkaline phosphatase (ALP) activity. We also designed the homology-modeled 3D structures of the protein encoded by the mRNA variants (ALPI-aAug10 and ALPI-bAug10). The levels of ALP activity of COS-1 cells transfected with the aAug10 plasmid were increased significantly, while cells transfected with the bAug10 plasmid had undetectable ALP activity. The homology-modeled 3D structures revealed that the variant bAug10 lacks the central N-terminal α-helix and residue corresponding to Asp-42 of ALPI-aAug10 near the active site. This is the first report on the characterization and structure of alternatively spliced transcript variants of the human ALPI gene. Further studies on the regulation of aAug10 and/or bAug10 mRNA expression may identify novel physiological functions of IAP.
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Fosfatase Alcalina , Proteínas Ligadas por GPI , Intestinos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Processamento Alternativo , Células CACO-2 , Células Epiteliais/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Hypophosphatasia (HPP) is an inherited skeletal disease characterized by defective bone and tooth mineralization due to a deficiency in tissue-nonspecific alkaline phosphatase (TNALP). Patients with the severe infantile form of HPP may appear normal at birth, but their prognosis is very poor. To develop a practical gene therapy for HPP, we endeavored to phenotypically correct TNALP knockout (Akp2 -/- ) mice through adeno-associated virus type 8 (AAV8) vector-mediated, muscle-directed, TNALP expression. Following treatment of neonatal Akp2 -/- mice with a single intramuscular injection of ARU-2801 (AAV8-TNALP-D10-vector) at 1.0 × 1012 vector genomes/body, high plasma ALP levels (19.38 ± 5.02 U/mL) were detected for up to 18 months, and computed tomography analysis showed mature bone mineralization. Histochemical staining for ALP activity in the knee joint revealed ALP activity on the surface of the endosteal bone of mice. Throughout their lives, the surviving treated Akp2 -/- mice exhibited normal physical activity and a healthy appearance, whereas untreated controls died within 3 weeks. No ectopic calcification or abnormal calcium metabolism was detected in the treated mice. These findings suggest that ARU-2801-mediated neonatal intramuscular gene therapy is both safe and effective, and that this strategy could be a practical option for treatment of the severe infantile form of HPP.
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Medial arterial calcification (MAC) is a major complication of chronic kidney disease (CKD) and an indicator of poor prognosis. Aortic overexpression of tissue-nonspecific alkaline phosphatase (TNAP) accelerates MAC formation. The present study aimed to assess whether a TNAP inhibitor, SBI-425, protects against MAC and improves survival probability in a CKD-mineral and bone disorder (MBD) mouse model. CKD-MBD mice were divided in three groups: vehicle, SBI-10, and SBI-30. They were fed a 0.2% adenine and 0.8% phosphorus diet from 14 to 20 weeks of age to induce CKD, followed by a high-phosphorus (0.2% adenine and 1.8% phosphorus) diet for another 6 weeks. At 14-20 weeks of age, mice in the SBI-10 and SBI-30 groups were given 10 and 30 mg/kg SBI-425 by gavage once a day, respectively, while vehicle-group mice were given distilled water as vehicle. Control mice were fed a standard chow (0.8% phosphorus) between the ages of 8 and 20 weeks. Computed tomography imaging, histology, and aortic tissue calcium content revealed that, compared to vehicle animals, SBI-425 nearly halted the formation of MAC. Mice in the control, SBI-10 and SBI-30 groups exhibited 100% survival, which was significantly better than vehicle-treated mice (57.1%). Aortic mRNA expression of Alpl, encoding TNAP, as well as plasma and aortic tissue TNAP activity, were suppressed by SBI-425 administration, whereas plasma pyrophosphate increased. We conclude that a TNAP inhibitor successfully protected the vasculature from MAC and improved survival rate in a mouse CKD-MBD model, without causing any adverse effects on normal skeletal formation and residual renal function. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Fosfatase Alcalina/antagonistas & inibidores , Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Distúrbio Mineral e Ósseo na Doença Renal Crônica/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Niacinamida/análogos & derivados , Sulfonamidas/farmacologia , Calcificação Vascular/prevenção & controle , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Aorta/enzimologia , Aorta/patologia , Doenças da Aorta/enzimologia , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/complicações , Distúrbio Mineral e Ósseo na Doença Renal Crônica/enzimologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Niacinamida/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteoblastos/patologia , Fatores de Tempo , Calcificação Vascular/enzimologia , Calcificação Vascular/etiologia , Calcificação Vascular/patologiaRESUMO
BACKGROUND: Hypophosphatasia (HPP) is an inherited metabolic bone disease characterized by reduced mineralization due to mutations in the tissue non-specific alkaline phosphatase (ALPL) gene. HPP is clinically variable with extensive allelic heterogeneity in the ALPL gene. We report the findings of in vitro functional studies following site-directed mutagenesis in bi-allelic mutations causing extreme clinical phenotypes; severe perinatal and asymptomatic HPP. AIMS: Elucidate genotype-phenotype correlation using in vitro functional studies and 3 dimensional (3D) ALP modelling. METHODS: Clinical, biochemical and radiological features were recorded in two children with extreme HPP phenotypes: Subject 1 (S1): Perinatal HPP with compound heterozygous mutations (c.110T>C; c.532T>C); Subject 2 (S2): asymptomatic with homozygous missense mutation (c.715G>T). Plasmids created for mutants 1 c.110T>C (L37P), 2 c.532T>C (Y178H) and 3 c.715G>T (D239Y) using in vitro mutagenesis were transfected into human osteosarcoma (U2OS) cells and compared to wildtype (WT) and mock cDNA. ALP activity was measured using enzyme kinetics with p-nitrophenylphosphate. Mineral deposition was evaluated photometrically with Alizarin Red S staining after culture with mineralization medium. Western blot analysis was performed to identify the mature type protein expression (80â¯kDa). Mutations were located on a 3D ALP model. Co-transfection was performed to identify dominant negative effect of the mutants. RESULTS: Phenotype: S1, had typical perinatal HPP phenotype at birth; extremely under-mineralized bones and pulmonary hypoplasia. S2, diagnosed incidentally by laboratory tests at 4â¯years, had normal growth, development, dentition and radiology. All S2's siblings (3 homozygous, 1 heterozygous) were asymptomatic. All subjects had typical biochemical features of HPP (low ALP, high serum pyridoxal-5'-phosphate), except the heterozygous sibling (normal ALP). Functional assay: Mutants 1 and 2 demonstrated negligible ALP activity and mineralization was 7.9% and 9.3% of WT, respectively. Mutant 3 demonstrated about 50% ALP activity and 15.5% mineralization of WT. On Western blot analysis, mutants 1 and 2 were detected as faint bands indicating reduced expression and mutant 3 was expressed as mature form protein with 50% of WT expression. Mutant 1 was located near the Glycosylphosphatidylinositol anchor, 2 at the core structure of the ALP protein and 3 at the periphery of the protein structure. Co-transfection did not reveal a dominant negative effect in any of the mutants. CONCLUSION: Our findings expand the current knowledge of functional effect of individual mutations and the importance of their location in the ALP structure.
Assuntos
Fosfatase Alcalina/genética , Alelos , Calcificação Fisiológica , Hipofosfatasia/enzimologia , Hipofosfatasia/genética , Mutagênese/genética , Mutação/genética , Fosfatase Alcalina/sangue , Fosfatase Alcalina/química , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Humanos , Hipofosfatasia/sangue , Hipofosfatasia/diagnóstico por imagem , Recém-Nascido , MasculinoRESUMO
Immunoproteomic analysis was performed to identify unknown, pathology-related molecules in patients with seronegative (SN) obstetric antiphospholipid syndrome (APS) who clinically satisfied the diagnostic criteria for APS, but not the serological criteria. We collected peripheral blood from 13 SN-APS outpatients with known thrombotic predisposition, 13 with no known thrombotic predisposition, and four multiparous women with no history of miscarriage (control). Plasma proteins from volunteers were purified and used as plasma protein antigens. Two-dimensional immunoblotting was performed using pooled control or SN-APS serum samples as the primary antibodies. Mass spectrometry of reactive spots specific to SN-APS serum led to the identification of complement molecule C9. Western blotting using commercial purified alkylated C9 was performed to detect autoantibodies. Examination of individual patient serum identified reactivity in one patient with, and in two patients without known thrombotic predisposition. This study suggests that SN-APS pathologies were associated with autoantibodies that react to specific C9 epitopes.
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Síndrome Antifosfolipídica/imunologia , Autoanticorpos/sangue , Complemento C9/imunologia , Proteômica/métodos , Adulto , Síndrome Antifosfolipídica/sangue , Biomarcadores/sangue , Eletroforese em Gel Bidimensional , Epitopos/imunologia , Feminino , Humanos , Masculino , Espectrometria de MassasRESUMO
HOTAIR is a lncRNA that plays critical role in gene regulation and chromatin dynamics through epigenetic mechanisms. In this work we studied the physiological role of HOTAIR during the process of mineralization using osteoblastic osteosarcoma cells focusing in ALPL (Tissue Non-Specific Alkaline Phosphatase), a pivotal gene that controls bone formation. HOTAIR knockdown resulted in upregulation of ALPL, increase of alkaline phosphatase (ALP) activity, and enhanced mineralization in osteoblastic SaOS-2 cells cultured in mineralizing medium. Luciferase assays using reporter vectors containing ALPL promoter showed that HOTAIR repression increases ALPL promoter activity. Furthermore, HOTAIR knockdown increased histone H3K4 methylation levels at ALPL promoter region, suggesting that ALPL repression by HOTAIR is regulated by epigenetic mechanisms. This work supports that physiological bone formation is epigenetically regulated by a lncRNA.
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Fosfatase Alcalina/biossíntese , Calcificação Fisiológica/fisiologia , Regulação da Expressão Gênica/fisiologia , Osteoblastos/metabolismo , RNA Longo não Codificante/metabolismo , Fosfatase Alcalina/genética , Linhagem Celular Tumoral , Metilação de DNA , Epigênese Genética , Técnicas de Silenciamento de Genes , Humanos , Osteossarcoma , RNA Longo não Codificante/genéticaRESUMO
PROBLEM: The effectiveness of progesterone (P4) treatment for preventing preterm births is unclear. Its effects on the uterine cervix were tested using cultured human uterine cervical fibroblasts (UCFs). METHOD OF STUDY: UCFs were incubated with lipopolysaccharide (LPS) in the presence or absence of P4 under various conditions. mRNA was subjected to PCR arrays and real-time RT-PCR to assess IL-6, IL-8, IL-1beta, PTGS2, MMP-1, and CXCL10 expression. RESULTS: When exposed to a high-LPS concentration (2.0 µg/mL), expression of these genes was not suppressed by simultaneous P4 (1.0 µmol/L) treatment, but it was significantly inhibited when P4 was administered 1 hour prior to LPS, with the exception of the chemokines IL-8 and CXCL10. Expression of all genes was restricted by P4 under low-level LPS (0.2 µg/mL) stimulation, especially when administered prior to LPS treatment. CONCLUSION: These data suggest that early or prophylactic P4 administration is an effective and important measure for reducing preterm birth risk.
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Colo do Útero/patologia , Fibroblastos/fisiologia , Inflamação/tratamento farmacológico , Nascimento Prematuro/tratamento farmacológico , Progesterona/farmacologia , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Gravidez , Nascimento Prematuro/imunologia , RNA Mensageiro/análiseRESUMO
Medial arterial calcification (MAC) and renal osteodystrophy are complications of mineral bone disease (MBD) associated with chronic kidney disease (CKD). Our aim was to develop a novel mouse model to investigate the clinical course of CKD-MBD. Eight-week-old C57BL/6 J male mice were assigned to the following groups: the control group, fed a standard chow for 6 or 12 weeks; the CKD-normal phosphorus (NP) group, fed a chow containing 0.2% adenine, with normal (0.8%) phosphorus, for 6 or 12 weeks; and the CKD-high phosphorus (HP) group, fed 6 weeks with the 0.2% adenine/0.8% phosphorus diet, followed by a chow with 1.8% phosphorus for 2 weeks, 4 weeks or 6 weeks. Serum phosphorus was significantly increased in the CKD-HP group, and associated with MAC formation; the volume of calcification increased with longer exposure to the high phosphorus feed. MAC was associated with upregulated expression of runt-related transcription factor 2, alkaline phosphatase, and osteopontin, indicative of osteoblastic trans-differentiation of vascular smooth muscle cells. A significant mineral density depletion of cortical bone was observed. We describe the feasibility of developing a model of CKD-MBD and provide findings of a direct association between elevated serum phosphorus and the formation of MAC and renal osteodystrophy.
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Distúrbio Mineral e Ósseo na Doença Renal Crônica/etiologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/metabolismo , Hiperfosfatemia/etiologia , Hiperfosfatemia/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/patologia , Animais , Biomarcadores , Distúrbio Mineral e Ósseo na Doença Renal Crônica/patologia , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Camundongos , Miócitos de Músculo Liso/metabolismo , Osteoblastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tomografia Computadorizada por Raios XRESUMO
Hypophosphatasia (HPP) is an inherited systemic bone disease that is characterized by bone hypomineralization. HPP is classified into six forms according to the age of onset and severity as perinatal (lethal), perinatal benign, infantile, childhood, adult, and odontohypophosphatasia. The causative gene of the disease is the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP). TNAP is expressed ubiquitously, and its physiological role is apparent in bone mineralization. A defect in bone mineralization can manifest in several ways, including rickets or osteomalacia in HPP patients. Patients with severe forms suffer from respiratory failure because of hypoplastic chest, which is the main cause of death. They sometimes present with seizures due to a defect in vitamin B6 metabolism resulting from the lack of alkaline phosphatase activity in neuronal cells, which is also lethal. Patients with a mild form of the disease exhibit rickets or osteomalacia and a functional defect of exercise. Odontohypophosphatasia shows only dental manifestations. To date, 302 mutations in the ALPL gene have been reported, mainly single-nucleotide substitutions, and the relationships between phenotype and genotype have been partially elucidated. An established treatment for HPP was not available until the recent development of enzyme replacement therapy. The first successful enzyme replacement therapy in model mice using a modified human TNAP protein (asfotase alfa) was reported in 2008, and subsequently success in patients with severe form of the disease was reported in 2012. In 2015, asfotase alfa was approved in Japan in July, followed by in the EU and Canada in August, and then by the US Food and Drug Administration in the USA in October. It is expected that therapy with asfotase alfa will drastically change treatments and prognosis of HPP.
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Sonodynamic therapy (SDT) has shown great potential as an approach for cancer treatment, and hyperthermotherapy (HT) is also a promising cancer therapy. Here, we investigate whether HT could improve the efficacy of SDT and to make a preliminary exploration on potential mechanism. Xenograft tumor was established in nude mice model, and SNB19 and U87MG glioma cell lines were utilized for in vitro experiment. Alamar blue assay was performed to assess cell viability. Optical microscope was used to characterize the morphology changes of the glioma cells induced by SDT and HT treatments. Apoptotic rate, mitochondrial membrane potential (MMP), and intracellular production of reactive oxygen species (ROS) were examined by flow cytometer. The cell apoptosis of tumor tissues were detected by TUNEL assay. Furthermore, the expression of apoptosis-related proteins was detected with Western blot in vitro and immunohistochemistry in vivo. SDT plus HT group could significantly reduce the cell viability with circular-cell morphological change, compared with SDT group, and cell viability was decreased depending on raise of 5-ALA concentration, ultrasound exposure time, and temperature. The results also indicate that HT increased a conspicuous apoptosis, ROS production, and a remarkable loss in MMP induced by 5-ALA-SDT in vitro. Meanwhile, our data also demonstrated that the combined treatment could significantly induce apoptosis and delay tumor growth in vivo. Furthermore, in both in vitro and in vivo experiments, SDT plus HT group expressed significantly higher protein levels of Bax and cleaved caspase-3, 8, and 9 compared to SDT, HT, and control groups and significantly lower protein level of bcl-2 than the other three groups, while the expression of these proteins was unchanged between HT and control groups. HT may provide an important promotion on 5-ALA-SDT and further propose that SDT in combination with HT is a new potential application for the treatment of human glioma.
Assuntos
Ácido Aminolevulínico/uso terapêutico , Glioma/terapia , Hipertermia Induzida , Terapia por Ultrassom , Ensaios Antitumorais Modelo de Xenoenxerto , Ácido Aminolevulínico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/terapia , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Protoporfirinas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Osteopontin (OPN), a secreted glycoprotein, has multiple physiological functions. This study investigated the regulation and roles of OPN in the mouse ovary during the periovulatory stages. Immature female mice were treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to simulate follicle maturation and ovulation. In situ hybridization and real-time RT-PCR were performed to assess expression of Opn in the periovulatory ovary. Granulosa cells (GCs) from PMSG-primed immature mice were cultured with or without hCG in the presence or absence of OPN, and effects on expression of Opn, progesterone synthesis, and vascular endothelial growth factor (VEGF) signaling were assessed by real-time RT-PCR, ELISA, and western blotting analysis. Opn transcripts were significantly upregulated 3âh after hCG treatment, followed by a peak at 16âh, and the transcripts localized to GCs. Incubation with hCG significantly increased quantities of Opn transcripts in GCs and OPN levels in the culture medium at 12 and 24âh. Furthermore, OPN treatment caused a significant increase in the levels of Star protein, P 450 cholesterol side-chain cleavage enzyme (p450scc), 3-beta-hydroxysteroid dehydrogenase (Hsd3b), and progesterone in the culture medium. OPN treatment promoted Vegf expression in GCs, which was significantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor. In addition, OPN treatment stimulated phosphorylation of AKT, a downstream PI3K signaling molecule. In conclusion, expression of Opn was upregulated in mouse ovarian GCs in response to a gonadotropin surge through epidermal growth factor receptor signaling, which enhances progesterone synthesis and Vegf expression during the early-luteal phase.
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Gonadotropinas/metabolismo , Osteopontina/metabolismo , Ovulação/genética , Animais , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas/farmacologia , Gonadotropinas Equinas/farmacologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteopontina/sangue , Osteopontina/genética , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovulação/efeitos dos fármacos , Ovulação/metabolismoRESUMO
Hypophosphatasia (HPP) is an inherited disorder characterized by defective bone mineralization caused by mutations in the alkaline phosphatase gene (ALPL). Clinically, the disease spans a great continuum of disease severity and six forms can be distinguished according to the age of onset. The most severe is the autosomal recessive perinatal form, a major prenatal skeletal dysplasia in Japan. The ALPL mutation c.1559delT causes perinatal HPP and occurs frequently in the Japanese. Most patients with perinatal HPP in Japan are homozygous for c.1559delT, and their parents are usually heterozygous with no evidence of consanguinity. Here we identified a fetus with perinatal HPP resulting from an unusual mechanism known as paternal uniparental isodisomy (UPD) of chromosome 1. Sequence analysis of ALPL in the patient revealed the presence of the homozygous mutation c.1559delT. We suspected UPD because the father and mother were heterozygous and wild type, respectively. Analysis of polymorphic microsatellite markers spanning chromosome 1 and whole-genome arrays revealed a uniparental inheritance from the father and excluded deletions or de novo mutations. This is the first description of perinatal HPP caused by UPD. This report also emphasizes the low recurrence risk of a non-Mendelian inheritance pattern in UPD and the value of determining parental genotypes with homozygous mutations in a patient to confirm whether the condition is caused by UPD or not, even when the mutation is detected as a hot spot, as described in the literature.
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Hipofosfatasia/complicações , Dissomia Uniparental/genética , Adulto , Fosfatase Alcalina/genética , Sequência de Bases , Cromossomos Humanos Par 1/genética , Análise Citogenética , Análise Mutacional de DNA , Feminino , Humanos , Hipofosfatasia/diagnóstico por imagem , Hipofosfatasia/enzimologia , Recém-Nascido , Perda de Heterozigosidade , Masculino , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Radiografia , UltrassonografiaRESUMO
A questionnaire survey was used to determine the status of nutrition education in Japanese medical schools in 2009. A similar survey was conducted in 2004, at which time nutritional education was determined to be inadequate in Japanese medical schools. The current questionnaire was sent to the directors of Centers for Medical Education of 80 medical schools, who represented all medical schools in Japan. Sixty-seven medical schools (83.8%) responded, of which 25 schools (37.3%) offered dedicated nutrition courses and 36 schools (53.7%) did not offer dedicated nutrition courses but offered something related to nutrition in other courses; six schools (9.0%) did not offer any nutrition education. Overall, 61 schools (91.0%) offered at least some nutritional topics in their undergraduate education. Nevertheless, only 11 schools (16.4%) seem to dedicate more than 5 hours to substantial nutrition education as judged by their syllabi. Although the mean length of the course was 11 hours, substantial nutrition education accounted for only 4.2 hours. Of the 25 medical schools that offered dedicated nutrition courses, seven schools offered the nutrition course as a stand-alone course and 18 schools offered it as an integrated course. In conclusion, the status of nutrition education in Japan has improved slightly but is still inadequate.
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Ciências da Nutrição/educação , Faculdades de Medicina/estatística & dados numéricos , Povo Asiático , Currículo , Atenção à Saúde/estatística & dados numéricos , Educação de Graduação em Medicina/estatística & dados numéricos , Seguimentos , Guias como Assunto , Educação em Saúde/estatística & dados numéricos , Humanos , Japão , Ciências da Nutrição/estatística & dados numéricos , Estado Nutricional , Inquéritos e QuestionáriosRESUMO
INTRODUCTION: We had demonstrated that single nucleotide polymorphism (787T>C) in the tissue-nonspecific ALP (TNSALP) gene was associated with the bone mineral density (BMD). BMD was the lowest among TNSALP 787T homozygotes (TT-type) and highest among TNSALP 787T>C homozygotes (CC-type) in postmenopausal women. In the present study, we investigated the effects of the TNSALP genotype on associations among serum bonespecific alkaline phosphatase (BAP), serum calcium, and phosphorus in healthy young Japanese subjects. METHODS: Young healthy adult subjects (n=193) were genotyped for the polymorphism, and we measured the levels of serum BAP, serum calcium, and phosphorus. Dietary nutrient intakes were calculated based on 3-day food records before the day of blood examinations. RESULTS: Grouped by the TNSALP genotype, a significant negative correlation between serum BAP and phosphorus was observed in 787T>C homozygotes (CC-type), but not in heterozygotes (TCtype), nor in 787T homozygotes (TT-type). CONCLUSIONS: In the present study, we revealed that the single nucleotide polymorphism 787T>C in the TNSALP gene had effects on the correlation between serum BAP and phosphorus in young adult subjects. These results suggest that variation in TNSALP may be an important determinant of phosphate metabolism. Our data may be useful for planning strategies to prevent osteoporosis.
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Fosfatase Alcalina/sangue , Fosfatase Alcalina/genética , Povo Asiático/genética , Osso e Ossos/química , Polimorfismo de Nucleotídeo Único , Cálcio da Dieta/sangue , Ingestão de Energia , Feminino , Heterozigoto , Homozigoto , Humanos , Japão , Masculino , Osteoporose/genética , Osteoporose/prevenção & controle , Fósforo na Dieta/sangue , Vitamina D/sangue , Adulto JovemRESUMO
Evidence indicates that the balance between osteoblastogenesis and adipogenesis of mesenchymal stem cells (MSCs) is regulated by several hormones, growth factors, and their downstream signaling cascades. Previous studies suggest that retinoic acid (RA) plays a role in osteoblastogenesis and adipogenesis. However, it is unknown whether RA regulates commitment of MSCs into osteoblasts and adipocytes. In this study, we investigated the role of RA in differentiation of MSCs using the C3H10T1/2 cell line. RA stimulated activity and expression of alkaline phosphatase (ALP) and upregulated activity of the ALP gene promoter. The effects of RA were further enhanced by bone morphogenetic protein 2 (BMP2) and resultant Smad signaling. Furthermore, overexpression of Runx2 and Msx2, critical transcription factors for bone formation and BMP2-dependent osteoblastogenesis, enhanced RA-dependent ALP activity. In view of these findings, RA likely stimulates osteoblast differentiation through the BMP2-Smad-Runx2/Msx2 pathway. In contrast, RA markedly inhibited BMP2-induced adipocyte differentiation, suppressing expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein (C/EBP)α and C/EBPδ, and inhibiting adipogenic function of C/EBPß, C/EBPδ, and PPARγ. In conclusion, our data suggest that RA regulates commitment of MSCs into osteoblasts and adipocytes by controlling transcriptional regulators.
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Adipócitos/metabolismo , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Tretinoína/farmacologia , Adipócitos/citologia , Fosfatase Alcalina/biossíntese , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Homeodomínio/biossíntese , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Osteoblastos/citologia , PPAR gama/metabolismo , Proteínas Smad/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
PROBLEM: This study aimed to investigate the regulation of expression, localization and physiological role of the CCL11/CCR3 axis in mouse ovary during the periovulatory period. METHOD OF STUDY: CCL11/CCR3 expression in the mouse ovary after treatment with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) 48 hr later was assessed in vivo and in 3-dimensional cultures in vitro. RESULTS: Real-time RT-PCR analyses revealed transient CCL11 mRNA upregulation 6 hr after hCG treatment. Immunohistochemical staining of serial ovarian sections demonstrated overlapping expression of CCL11, CCR3 and CD31 endothelial cell marker in the theca-interstitial layer at 10 hr after hCG treatment. In vitro 3-dimensional cultures of periovulatory ovarian tissues demonstrated that treatment with anti-CCL11 neutralizing antibody significantly decreased CD31 transcript. CONCLUSIONS: Gonadotropin surge leads to transient CCL11/CCR3 axis upregulation in the ovarian theca-interstitial layer, suggesting that it is involved in periovulatory physiological processes by affecting follicular vessels.
Assuntos
Quimiocina CCL11/biossíntese , Gonadotropina Coriônica/farmacologia , Gonadotropinas Equinas/farmacologia , Interleucina-1beta/farmacologia , Células Tecais/efeitos dos fármacos , Animais , Quimiocina CCL11/genética , Feminino , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Receptores CCR3/biossíntese , Células Tecais/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para CimaRESUMO
Hypophosphatasia (HPP) is an inherited disease caused by a deficiency of tissue-nonspecific alkaline phosphatase (TNALP). The major symptom of human HPP is hypomineralization, rickets, or osteomalacia, although the clinical severity is highly variable. The phenotypes of TNALP knockout (Akp2(-/-)) mice mimic those of the severe infantile form of HPP. Akp2(-/-) mice appear normal at birth, but they develop growth failure, epileptic seizures, and hypomineralization and die by 20 days of age. Previously, we have shown that the phenotype of Akp2(-/-) mice can be prevented by enzyme replacement of bone-targeted TNALP in which deca-aspartates are linked to the C-terminus of soluble TNALP (TNALP-D10). In the present study, we evaluated the therapeutic effects of adeno-associated virus serotype 8 (AAV8) vectors that express various forms of TNALP, including TNALP-D10, soluble TNALP tagged with the Flag epitopes (TNALP-F), and native glycosylphosphatidylinositol-anchored TNALP (TNALP-N). A single intravenous injection of 5×10(10) vector genomes of AAV8-TNALP-D10 into Akp2(-/-) mice at day 1 resulted in prolonged survival and phenotypic correction. When AAV8-TNALP-F was injected into neonatal Akp2(-/-) mice, they also survived without epileptic seizures. Interestingly, survival effects were observed in some animals treated with AAV8-TNALP-N. All surviving Akp2(-/-) mice showed a healthy appearance and a normal activity with mature bone mineralization on X-rays. These results suggest that sustained alkaline phosphatase activity in plasma is essential and sufficient for the rescue of Akp2(-/-) mice. AAV8-mediated systemic gene therapy appears to be an effective treatment for the infantile form of human HPP.
Assuntos
Fosfatase Alcalina/genética , Dependovirus/genética , Hipofosfatasia/genética , Hipofosfatasia/terapia , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Terapia Genética , Humanos , Hipofosfatasia/enzimologia , Camundongos , Camundongos Knockout , FenótipoRESUMO
Hypophosphatasia (HPP) is an inherited systemic skeletal disease caused by mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNALP) isozyme. The clinical severity of HPP varies widely, with symptoms including rickets and osteomalacia. TNALP knockout (Akp2(-/-)) mice phenotypically mimic the severe infantile form of HPP; that is, TNALP-deficient mice are born with a normal appearance but die by 20 days of age owing to growth failure, hypomineralization, and epileptic seizures. In this study, a lentiviral vector expressing a bone-targeted form of TNALP was injected into the jugular vein of newborn Akp2(-/-) mice. We found that alkaline phosphatase activity in the plasma of treated Akp2(-/-) mice increased and remained at high levels throughout the life of the animals. The treated Akp2(-/-) mice survived for more than 10 months and demonstrated normal physical activity and a healthy appearance. Epileptic seizures were completely inhibited in the treated Akp2(-/-) mice, and X-ray examination of the skeleton showed that mineralization was significantly improved by the gene therapy. These results show that severe infantile HPP in TNALP knockout mice can be treated with a single injection of lentiviral vector during the neonatal period.
Assuntos
Fosfatase Alcalina/deficiência , Fosfatase Alcalina/uso terapêutico , Terapia Genética , Hipofosfatasia/genética , Hipofosfatasia/terapia , Lentivirus/genética , Fosfatase Alcalina/genética , Animais , Pé/diagnóstico por imagem , Hipofosfatasia/diagnóstico por imagem , Camundongos , Fenótipo , Radiografia , Análise de Sobrevida , Tíbia/enzimologia , Tíbia/patologiaRESUMO
Hypophosphatasia (HPP) is an inherited disorder caused by mutations in ALPL that encodes an isozyme of alkaline phosphatase (ALP), TNSALP. One of the most frequent ALPL mutations is c.1559delT, which causes the most severe HPP, the perinatal (lethal) form (pl-HPP). c.1559delT has been found only in Japanese and its prevalence is suspected to be high; however, the allele frequency of c.1559delT in Japanese remains unknown. We designed a screening system for the mutation based on high-resolution melting curve analysis, and examined the frequency of c.1559delT. We found that the c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1562-1/284). This indicates that â¼1 in 900 000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. In our analysis, the majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine phosphoethanolamine. Our results indicate that the only way to reliably detect whether individuals are pl-HPP carriers is to perform the ALPL mutation analysis.