RESUMO
Magnesium, a promising biodegradable metal, has been reported in several studies to increase bone formation. Although there is some information regarding the concentrations of magnesium ions that affect bone remodeling at a cellular level, little is known about the effect of magnesium ions on cell gap junctions. Therefore, this study aimed to systematically investigate the effects of different concentrations of magnesium on bone cells, and further evaluate its effect on gap junctions of osteoblasts. Cultures of normal human osteoblasts were treated with magnesium ions at concentrations of 1, 2 and 3 mM, for 24, 48 and 72 h. The effects of magnesium ions on viability and function of normal human osteoblasts and on gap junction intercellular communication (GJIC) in osteoblasts were investigated. Magnesium ions induced significant (P<0.05) increases in cell viability, alkaline phosphate activity and osteocalcin levels of human osteoblasts. These stimulatory actions were positively associated with the concentration of magnesium and the time of exposure. Furthermore, the GJIC of osteoblasts was significantly promoted by magnesium ions. In conclusion, this study demonstrated that magnesium ions induced the activity of osteoblasts by enhancing GJIC between cells, and influenced bone formation. These findings may contribute to a better understanding of the influence of magnesium on bone remodeling and to the advance of its application in clinical practice.
Assuntos
Magnésio/farmacologia , Osteoblastos/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Junções Comunicantes/efeitos dos fármacos , Humanos , Íons/farmacologia , Magnésio/química , Osteoblastos/fisiologia , Osteocalcina/análise , Osteogênese/efeitos dos fármacos , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Magnesium, a promising biodegradable metal, has been reported in several studies to increase bone formation. Although there is some information regarding the concentrations of magnesium ions that affect bone remodeling at a cellular level, little is known about the effect of magnesium ions on cell gap junctions. Therefore, this study aimed to systematically investigate the effects of different concentrations of magnesium on bone cells, and further evaluate its effect on gap junctions of osteoblasts. Cultures of normal human osteoblasts were treated with magnesium ions at concentrations of 1, 2 and 3 mM, for 24, 48 and 72 h. The effects of magnesium ions on viability and function of normal human osteoblasts and on gap junction intercellular communication (GJIC) in osteoblasts were investigated. Magnesium ions induced significant (P<0.05) increases in cell viability, alkaline phosphate activity and osteocalcin levels of human osteoblasts. These stimulatory actions were positively associated with the concentration of magnesium and the time of exposure. Furthermore, the GJIC of osteoblasts was significantly promoted by magnesium ions. In conclusion, this study demonstrated that magnesium ions induced the activity of osteoblasts by enhancing GJIC between cells, and influenced bone formation. These findings may contribute to a better understanding of the influence of magnesium on bone remodeling and to the advance of its application in clinical practice.
Assuntos
Humanos , Osteoblastos/efeitos dos fármacos , Magnésio/farmacologia , Fatores de Tempo , Ensaio de Imunoadsorção Enzimática , Comunicação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Reprodutibilidade dos Testes , Junções Comunicantes/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Íons/farmacologia , Magnésio/químicaRESUMO
Considerable attention is currently being directed toward methods for producing recombinant human proteins in the mammary glands of genetically modified transgenic livestock. However, the expression of inserted genes in transgenic animals is variable and often very low because of the randomness of the site of transgene integration. One possible strategy to avoid the expression problem associated with random integration is to use site-specific integration by targeting integration to a high expression locus and, thereby, to improve expression of the transferred gene. In the present study, we focused on glial cell line-derived neurotrophic factor (GDNF), a novel type of neurotrophic factor first cloned in 1993. Research has shown that GDNF may have potential applications in the treatment of Parkinson's disease and other diseases of the central nervous system since it acts as a protective factor for central dopaminergic neurons. Here, we constructed a gene targeting vector to knock-in the human GDNF gene at the bovine beta-casein gene locus as a first step to producing transgenic animals with a high level of expression of human GDNF protein in their mammary glands. Bovine fetal fibroblast cells were transfected with linearized pNRTCNbG by electroporation. Three cell clones were identified with successful targeting to the beta-casein locus; and were confirmed using both polymerase chain reaction analysis and sequencing. Gene-targeted cells were used as nuclear donors; a total of 161 embryos were reconstructed, 23 of which developed to the blastocyst stage. These blastocysts were transferred to 8 recipient cows, but no offspring were obtained.
Assuntos
Caseínas/genética , Bovinos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Animais , Animais Geneticamente Modificados , Blastocisto , Bovinos/metabolismo , Feminino , Marcação de Genes , Genes Reporter , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , TransfecçãoRESUMO
A variety of molecular epidemiological studies have been conducted to examine the association between the DNMT3B -149C/T polymorphism and cancer susceptibility; however, there has been no study investigating the association between the DNMT3B -149C/T polymorphism and the risk of laryngeal squamous cell carcinoma (LSCC) until now. To determine the role of the DNMT3B -149C/T polymorphism in LSCC, we genotyped 113 patients with LSCC and 110 controls from a Chinese population using polymerase chain reaction-restriction fragment length polymorphism analysis. The chi-square test was used to examine differences in the distributions of genotypes studied between patients and controls. The association between the DNMT3B -149C/T polymorphism and the risk of LSCC was estimated using ORs and their 95%CIs. Genotypic frequencies in the patients with LSCC were not similar to those of the controls, with the differences being statistically significant (P = 0.001). When the DNMT3B -149 CC genotype was used as the reference group, the CT genotype was not associated with LSCC risk (adjusted OR, 2.12; 95%CI = 0.89-5.19; P = 0.07), but the TT genotype was associated with significantly increased risk for LSCC (adjusted OR = 3.27; 95%CI = 1.79-10.66; P = 0.009). Under the recessive model of inheritance, the TT genotype was associated with significantly increased risk for LSCC (adjusted OR = 1.98; 95%CI = 1.12-5.95; P = 0.012), compared with other genotypes. These results suggested that the DNMT3B -149C/T polymorphism is associated with a genetic susceptibility for developing LSCC in a Chinese population.
Assuntos
Carcinoma de Células Escamosas/genética , DNA (Citosina-5-)-Metiltransferases/genética , Neoplasias de Cabeça e Pescoço/genética , Povo Asiático , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição/genética , Polimorfismo de Nucleotídeo Único , Carcinoma de Células Escamosas de Cabeça e Pescoço , DNA Metiltransferase 3BRESUMO
Rspo1 belongs to the Rspo family, which is composed of 4 members (Rspo1-4) that share 40 to 60% sequence homology and similar domain organizations, and regulate the WNT signaling pathway via a common mechanism. Rspo1 plays a key role in vertebrate development and is an effective mitogenic factor of gastrointestinal epithelial cells. We report the cloning of chicken Rspo1 and its gene expression distribution among tissues. It contained an open reading frame of 783 bp encoding a protein of 260 amino acids, and its molecular weight was predicted to be 28.80 kDa. Reverse transcription-polymerase chain reaction-based gene expression analysis indicated that chicken Rspo1 was highly expressed in the stomach muscle tissue, but was expressed at low levels in the lung, brain, jejunum, cecum, ileum, spleen, pancreas, kidney, and glandular stomach. These results suggest that Rspo1 plays a major role in muscular immune protection.
Assuntos
Galinhas/genética , Trombospondinas/genética , Animais , Clonagem Molecular/métodos , Feminino , Masculino , Análise de Sequência de DNA , Distribuição Tecidual , Via de Sinalização WntRESUMO
In the present study, we examined whether the ARNTL (BMAL1) rs2278749 T/C polymorphism was associated with the susceptibility to Alzheimer disease (AD). This case-control study examined the genotypes of apolipoprotein E (APOE e4) and BMAL1 rs2278749 T/C using restriction fragment length polymorphism and the TaqMan assay, respectively. A total of 296 unrelated AD patients and 423 control subjects were included. Both in the entire sample and in APOE e4 non-carriers, the prevalence of T carriers in BMAL1 rs2278749 T/C in AD patients was significantly higher than that in control subjects (entire sample: χ(2) = 12.950, P < 0.0001; APOE e4 non-carriers: χ(2) = 13.094, P < 0.0001). Both in the entire sample and in APOE e4 non-carriers, the prevalence of TT genotypes 2278749 in AD patients was also significantly higher than that in control subjects (entire sample: χ(2) = 7.765, P = 0.024; APOE e4 non-carriers: χ(2) = 13.062, P < 0.0001). However, among APOE e4 carriers, the difference in the prevalence of T carriers or TT genotypes in the BMAL1 rs2278749 T/C between patients and control subjects presents was not significant (T carriers: χ(2) = 0.078, P = 0.851 or TT genotypes: χ(2) = 2.576, P = 0.325). Among APOE e4 non-carriers, T carriers in the BMAL1 rs2278749 T/C were associated with a high susceptibility to AD, but among APOE e4 carriers, the association between AD and BMAL1 rs2278749 T/C was not significant.
Assuntos
Fatores de Transcrição ARNTL/genética , Doença de Alzheimer/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Alelos , Doença de Alzheimer/diagnóstico , Apolipoproteínas E/genética , Povo Asiático/genética , Estudos de Casos e Controles , China , Feminino , Frequência do Gene , Estudos de Associação Genética , Genótipo , Humanos , MasculinoRESUMO
MicroRNA-200 family members are expressed in the developing mouse inner ear and in zebrafish (Danio rerio) olfactory epithelia, taste buds, and neuromasts, and have also been shown to be associated with differentiation of olfactory and taste buds. However, the role of the miR-200 family in the inner ear of zebrafish had not been studied. We investigated the expression and function of the miR-200 family in the zebrafish inner ear via in situ hybridization and loss-of-function methods. Expression of the miR-200 family was weak and dispersed throughout the developing zebrafish inner ear. After knockdown of miR-200 family members in the developing inner ear, no significant differences in development were observed compared to the controls. Otic vesicles, otoliths, and semicircular canals appeared normal. Compared with less differentiated olfactory filaments in olfactory epithelia, the development of hair cells and statoacoustic ganglion neurons were normal. The kinocilia and stereocilia of hair cells, the innervation of hair cells, and the formation of ribbon synapses were also unaffected. Overall, we conclude that the miR-200 family has a negligible role in the development of zebrafish inner ear; the functions of the miR- 200 family may be organ-specific.
Assuntos
Orelha Interna/embriologia , Células Ciliadas Auditivas Internas/metabolismo , MicroRNAs/genética , Peixe-Zebra/genética , Animais , Orelha Interna/citologia , Orelha Interna/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , MicroRNAs/metabolismo , Família Multigênica , Especificidade de Órgãos , Peixe-Zebra/embriologiaRESUMO
Insulin-like growth factor binding protein-3 (IGFBP-3) exerts anti-proliferative or pro-apoptotic effects through IGF-dependent as well as IGF-independent mechanisms in vitro. The purpose of this study was to examine the association between genetic variants in IGFBP-3 (rs2270628) and the risk of esophageal squamous cell carcinoma (ESCC) in a Chinese Han population. Five hundred ESCC cases and 500 cancer-free controls of the Chinese Han population were involved in this study. The IGFBP-3 single-nucleotide polymorphism (SNP) rs2270628 was genotyped and the estimated adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for its association with the risk of ESCC were determined using unconditional logistic regression analysis. Compared with the rs2270628 CC genotype, TT genotype was associated with a significantly increased ESCC risk with OR (95%CI) of 2.07 (1.05-4.09), but CT genotype was not (OR = 1.25, 95%CI =0.94-1.66). IGFBP-3 SNP rs2270628 may contribute to the risk of ESCC in the Chinese Han population.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Idoso , Povo Asiático , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.
Assuntos
Animais , Masculino , Camundongos , Envelhecimento/sangue , Apolipoproteína A-II/sangue , Autoanticorpos/sangue , Estresse Oxidativo/genética , alfa-Fetoproteínas/metabolismo , Envelhecimento/genética , Apolipoproteína A-II/genética , Autoanticorpos/genética , Biomarcadores/sangue , Biomarcadores/metabolismo , Oxirredução , Proteômica , Baço/citologia , alfa-Fetoproteínas/genéticaRESUMO
We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2â m, -6â m, -12â m, -15â m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12â m sera. Two proteins decreased in the sera from SAMP8-2â m, -6â m, and -12â m mice, and divided into 2 spots each in SAMP8-15â m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2â m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.
Assuntos
Envelhecimento/sangue , Apolipoproteína A-II/sangue , Autoanticorpos/sangue , Estresse Oxidativo/genética , alfa-Fetoproteínas/metabolismo , Envelhecimento/genética , Animais , Apolipoproteína A-II/genética , Autoanticorpos/genética , Biomarcadores/sangue , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos , Masculino , Camundongos , Oxirredução , Proteômica , Baço/citologia , alfa-Fetoproteínas/genéticaRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most intensively investigated Cl- channels. Different mutations in the CFTR gene cause the disease cystic fibrosis (CF). CFTR is expressed in the apical membrane of various epithelial cells including the intestine. The major organ affected in CF patients is the lung, but it also causes an important dysfunction of intestinal ion transport. The modulation of CFTR mRNA expression by atrial natriuretic peptide (ANP) was investigated in rat proximal colon and in human intestinal CaCo-2 cells by RNase protection assay and semi-quantitative reverse transcriptase PCR techniques. Groups of rats subjected to volume expansion or intravenous infusion of synthetic ANP showed respective increases of 60 and 50% of CFTR mRNA expression in proximal colon. CFTR mRNA was also increased in cells treated with ANP, reaching a maximum effect at 10(-9) M ANP, probably via cGMP. ANP at 10(-9) M was also able to stimulate both the CFTR promoter region (by luciferase assay) and protein expression in CaCo-2 cells (by Western blot and immunoprecipitation/phosphorylation). These results suggested the involvement of ANP, a hormone involved with extracellular volume, in the expression of CFTR in rat proximal colon and CaCo-2 intestinal cells.