RESUMO
Twenty-four polymorphic microsatellite loci were isolated and characterized for Liza affinis using a (GT)13-enriched genomic library. The number of alleles per locus ranged from 3 to 9, with a mean number of 6.250. The observed and expected heterozygosities ranged from 0.417 to 1.000 and from 0.550 to 0.861, with an average of 0.859 and 0.779, respectively. Deviation from Hardy-Weinberg proportions was detected at three loci. Evidence of null alleles was found at two loci. These markers will be useful in further studies investigating the genetic variation and population structure of this species, and may provide insights into the maintenance and efficient management of eastern keelback mullet resources.
Assuntos
Repetições de Microssatélites , Smegmamorpha/genética , Animais , China , Polimorfismo GenéticoRESUMO
Sillago sinica is a newly identified species belonging to Sillaginidae, Perciforms, and was found along the coast of China in 2011. In the present study, 81 microsatellite loci were isolated from an enriched genomic library, and 24 positive clones containing microsatellite repeats had adequate flanking sequences for the development of PCR primers. Sixteen of these primers were monomorphic or would not amplify. Eight were polymorphic in an examined population with the number of alleles per locus ranging from 2 to 14. The number of observed and expected heterozygosities per locus varied from 0.125 to 0.958 and from 0.120 to 0.904, respectively. The polymorphism information content ranged from 0.110 to 0.721. All loci conformed to Hardy-Weinberg equilibrium (P > 0.05) after Bonferroni correction. There was no significant linkage disequilibrium between the eight polymorphic loci. These results suggest that these markers may be very useful for the characterization of natural populations of this species.
Assuntos
Repetições de Microssatélites , Perciformes/genética , Alelos , Animais , Genoma , Heterozigoto , Desequilíbrio de Ligação , Polimorfismo GenéticoRESUMO
The aim of this study was to develop a method to detect a point mutation in the ribosomal S12 protein (rpsL) gene in streptomycin-resistant strains of Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to detect a point mutation in codon 43 of the rpsL gene in X. oryzae pv. oryzicola and X. oryzae pv. oryzae. The 304-bp PCR product from the rpsL gene was digested by MboII to form two fragments (201 and 103 bp) if there was a mutation at codon 43, or three fragments (146, 103, and 55 bp) if there was no mutation. Compared with the results from nucleotide sequencing, the PCR-RFLP method was accurate in detecting the point mutation at codon 43 of the rpsL gene in streptomycin-resistant strains of X. oryzae pv. oryzicola and X. oryzae pv. oryzae. These results indicate that the PCR-RFLP is a simple, rapid and reliable method for detecting the point mutation at codon 43 of the rpsL gene.
Assuntos
Proteínas de Bactérias/genética , Códon , Mutação , Proteínas Ribossômicas/genética , Xanthomonas/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Sequência de Bases , Farmacorresistência Bacteriana , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Xanthomonas/efeitos dos fármacosRESUMO
The aim of this study was to investigate the correlation between apparent diffusion coefficients (ADCs), the relative apparent diffusion coefficient (rADC), and the pathological prognostic factor human epidermal growth factor receptor 2 (HER-2) in patients with breast cancer. A total of 64 women with breast cancer underwent breast diffusion-weighted imaging. HER-2 expression was detected in histological specimens. The ADC value, rADC value, and HER-2 level were determined. The ADC and rADC values of the breast cancer group were 1.1495 ± 0.1499 x 10(-3) and 0.6602 ± 0.0853, respectively. The differences in the ADC and rADC values between the two groups were statistically significant. There was no correlation between the ADC value and HER-2 expression in patients with breast cancer (r = -0.508, P = 0.043). However, the rADC value eliminated the individual differences to some extent. Therefore, compared to the ADC value, the rADC value had a better correlation with HER-2 expression.
Assuntos
Doenças Mamárias/diagnóstico , Neoplasias da Mama/diagnóstico , Imagem de Difusão por Ressonância Magnética/métodos , Adulto , Doenças Mamárias/metabolismo , Neoplasias da Mama/metabolismo , Diagnóstico Diferencial , Feminino , Humanos , Pessoa de Meia-Idade , Receptor ErbB-2/metabolismoRESUMO
BACKGROUND: H19 gene has been proved to be essential for human tumor growth which contains CpG rich regions. Imprinted gene expression in many cancers is usually associated with the function of methylation. We performed this study to better understand wether H19 DMR methylation correlates to the progression of esophageal squamous cell carcinoma through IGF2 imprinting pathway. METHODS: LOI of IGF2 was detected in 276 samples, which were determined as heterozygote with ApaI polymorphism in exon 9 of IGF2 by PCR-RFLP and RT-PCR-RFLP. Methylation status of H19 DMR in informative samples was analyzed by bisulfite sequencing PCR. IGF2 expression was examined by real-time PCR and IHC. RESULTS: 208 ESCC patients were informative for ApaI polymorphism. 92 tumor and 30 normal tissues showed IGF2 LOI. Methylation status of H19 CBS6 was higher in patients with IGF2 LOI compared to patients with IGF2 MOI (p < 0.05). IGF2 expression in patients with IGF2 LOI was higher than patients with IGF2 MOI (p < 0.05) which was correlated with lymph node involvement, neoplastic grade and metastasis (p < 0.05). CONCLUSIONS: Our results suggested that H19 CBS6 hypermethylation is related to the LOI of IGF2 which usually leads to an overexpression of IGF2, playing important roles in the occurrence, development as well as metastasis of ESCC. Therefore, H19 CBS6 methylation potentially represents a novel clinically relevant epigenetic marker to identify individuals at increased risk for the occurrence, progression and prognosis of ESCC.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Metilação de DNA , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Impressão Genômica/genética , Fator de Crescimento Insulin-Like II/genética , RNA Longo não Codificante/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Metilação de DNA/genética , Progressão da Doença , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologiaRESUMO
Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P = 0.003, Student t-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control = 6.3 ± 0.9; HOC = 3.5 ± 1.5; P = 0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Transplante de Células/métodos , Hepatócitos/citologia , Transplante de Fígado/métodos , Animais , Feminino , Citometria de Fluxo , Rejeição de Enxerto/diagnóstico , Hepatectomia , Imuno-Histoquímica , Fígado/anatomia & histologia , Fígado/cirurgia , Masculino , Cultura Primária de Células , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase em Tempo Real/métodos , Taxa de SobrevidaRESUMO
Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Student t-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.
Assuntos
Animais , Feminino , Masculino , Ratos , Proliferação de Células , Diferenciação Celular/fisiologia , Transplante de Células/métodos , Hepatócitos/citologia , Transplante de Fígado/métodos , Citometria de Fluxo , Rejeição de Enxerto/diagnóstico , Hepatectomia , Imuno-Histoquímica , Fígado/anatomia & histologia , Fígado/cirurgia , Cultura Primária de Células , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase em Tempo Real/métodos , Taxa de SobrevidaRESUMO
Mastitis affects the concentrations of potassium and sodium in milk. Since sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase) is critical for maintaining the homeostasis of these two ions, and is involved in cell apoptosis and pathogenesis, we presumed that polymorphism of the ATP1A1 gene, which encodes the bovine Na(+), K(+)-ATPase α1 subunit could be associated with mastitis. The ATP1A1 gene was analyzed in 320 Holstein cows using PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods. A C/A SNP was identified at nucleotide position -15,739 in exon 17 of the ATP1A1 gene, but it did not induce any change in amino acids. We examined a possible association of polymorphism of the ATP1A1 gene with somatic cell score and 305-day milk yields. Individuals with genotype CC in ATP1A1 had significantly lower somatic cell scores and 305-day milk yields than those with genotype CA. We also examined changes in Na(+), K(+)-ATPase activity of red cell membranes. The Na(+), K(+)-ATPase activity was significantly higher in dairy cows with genotype CC compared to the other two genotypes, and the Na(+), K(+)-ATPase activity of the resistant group was significantly higher than that of the susceptible group in dairy cows. We conclude that this polymorphism has potential as a marker for mastitis resistance in dairy cattle.
Assuntos
Eritrócitos/enzimologia , Predisposição Genética para Doença , Mastite Bovina/genética , Polimorfismo de Nucleotídeo Único , ATPase Trocadora de Sódio-Potássio/genética , Alelos , Animais , Sequência de Bases , Bovinos , Membrana Celular/enzimologia , Ativação Enzimática , Feminino , Frequência do Gene , Genótipo , Mastite Bovina/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100 percent; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6 percent, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83 percent, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7 percent ethanol (62 vs 62 percent, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62 percent, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.
Assuntos
Animais , Masculino , Camundongos , Cálcio/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citocalasina B/farmacologia , Células-Tronco Mesenquimais , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Microscopia Confocal , Oócitos/fisiologia , Partenogênese/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca(2)+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca(2)+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 microg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca(2)+]i oscillations, completion of meiosis and parthenogenetic development.
Assuntos
Cálcio/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citocalasina B/farmacologia , Células-Tronco Mesenquimais , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Animais , Masculino , Camundongos , Microscopia Confocal , Oócitos/fisiologia , Partenogênese/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Animais , Células do Cúmulo/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização in vitro , Meiose/fisiologia , Camundongos , Folículo Ovariano/crescimento & desenvolvimento , GravidezRESUMO
Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.