RESUMO
The envelope protein (Env) of the Jaagsiekte sheep retrovirus (JSRV) is known to be a unique oncoprotein responsible for inducing ovine pulmonary adenocarcinoma (OPA). The objective of this study was to prepare a specific monoclonal antibody (mAb) against the JSRV Env protein using bioinformatic analysis. According to the structure and epitope prediction results of JSRV Env, the JSRV-Env572-615 antigen was prepared via peptide synthesis (amino acid sequence 572-615, denoted as JSRV-Env572-615). BALB/c mice were immunized to prepare the anti-JSRV-Env572-615 mAb. Spleen cells were fused with SP2/0 myeloma cells after being screened by indirect ELISA and cloned by limiting dilution. The specificity of mAb was evaluated by western blot analysis and immunohistochemistry assays. Western blot results showed that the JSRV Env protein was able to bind to mAb with high specificity. Immunohistochemistry assays demonstrated that the mAb was able to recognize JSRV Env in adenomatous hyperplasia of the lung. Furthermore, JSRV was detected in peripheral blood leukocytes during the pre-clinical period of OPA in 2 of the 25 sheep using this newly synthesized mAb. Therefore, this mAb may be a useful tool for the detection of JSRV in sheep.
Assuntos
Adenocarcinoma/diagnóstico , Adenocarcinoma/veterinária , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Retrovirus Jaagsiekte de Ovinos/imunologia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/veterinária , Adenomatose Pulmonar Ovina/diagnóstico , Adenocarcinoma/imunologia , Adenocarcinoma/virologia , Adenocarcinoma de Pulmão , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Especificidade de Anticorpos , Biologia Computacional , Diagnóstico Precoce , Epitopos/química , Epitopos/imunologia , Produtos do Gene env/química , Produtos do Gene env/imunologia , Retrovirus Jaagsiekte de Ovinos/isolamento & purificação , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Pulmão/imunologia , Pulmão/virologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/virologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/administração & dosagem , Peptídeos/síntese química , Peptídeos/imunologia , Adenomatose Pulmonar Ovina/imunologia , Adenomatose Pulmonar Ovina/virologia , Ovinos , Carneiro Doméstico , Baço/citologia , Baço/imunologiaRESUMO
We examined the aberrant microRNA (miRNA) expression profile responsible for the changes in angiogenesis observed in endometriotic lesions. This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient, and their correlation with the most important angiogenic and fibrinolytic factors. miRNA expression was quantified using a microRNA array and reverse-transcription microRNA polymerase chain reaction. Levels of vascular endothelial growth factor A (VEGFA), epidermal growth factor receptor 2 (EGFR2), phosphatase and tensin homolog (PTEN), and C-X-C chemokine receptor type 4 (CXCR4) were quantified using enzyme-linked immunosorbent assay. The endometrial tissue showed significantly lower levels of miR-200b, miR-15a-5p, miR-19b-1-5p, miR-146a-5p, and miR-200c, and higher levels of miR-16-5p, miR-106b-5p, and miR-145-5p. VEGFA was significantly upregulated, whereas EGFR2, PTEN, and CXCR4 were markedly downregulated, in the endometriotic tissues compared to that in the normal endometrial tissues. In conclusion, differences in the miRNA levels could modulate the expression of VEGFA, EGFR2, PTEN, and CXCR4, and may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in the eutopic endometrium might facilitate the implantation of endometrial cells at ectopic sites.
Assuntos
Endometriose/genética , MicroRNAs/biossíntese , PTEN Fosfo-Hidrolase/biossíntese , Receptor ErbB-2/biossíntese , Receptores CXCR4/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adulto , Endometriose/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , PTEN Fosfo-Hidrolase/genética , Receptor ErbB-2/genética , Receptores CXCR4/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Our study clarifies the role of the autocrine motility factor receptor (AMFR) gene in porcine preadipocyte differentiation. AMFR-siRNA was transfected into porcine preadipocytes and the preadipocytes were induced to differentiation. Subsequently, qRT-PCR was conducted to examine changes in mRNA expression of a series of genes in porcine preadipocytes, including AMFR, sterol-regulatory element-binding protein-1a (SREBP1a), SREBP2, insulin-induced gene 1 (Insig1), and Insig2. Expression changes in the mRNA of genes regulating adipocyte differentiation were also analyzed using qRT-PCR, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and Kruppel-like factor 2 (KLF2). Western blot analysis was conducted to examine the changes in AMFR protein expression in porcine preadipocytes. Additionally, morphological changes in differentiated porcine preadipocytes were examined by oil red O staining, and changes in optical density (OD) values were measured using an ultraviolet spectrophotometer. At 24 h after transfection with AMFR-siRNA, AMFR mRNA expression significantly reduced (P < 0.01), and AMFR protein expression markedly decreased (P < 0.05). The mRNA expression of SREBP1a, SREBP2, Insig1, and C/EBPα was significantly reduced (P < 0.01), whereas the expression of KLF2 mRNA was significantly elevated (P < 0.01). After induction of preadipocyte differentiation, the number of lipid droplets decreased in the AMFR-silenced group, and the OD value markedly reduced (P < 0.05). In addition, the expression of C/EBPα mRNA significantly decreased (P < 0.05), whereas the expression of KLF2 mRNA considerably increased (P < 0.05). Taken together, silencing of the AMFR gene inhibits the differentiation of porcine preadipocytes.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular , Receptores do Fator Autócrino de Motilidade/metabolismo , Adipócitos/citologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Células Cultivadas , Inativação Gênica , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Receptores do Fator Autócrino de Motilidade/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , SuínosRESUMO
The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin ß-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the ß-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein ß-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean ß-subunit gene. The level of 7S protein ß-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.
Assuntos
Agrobacterium tumefaciens/genética , Antígenos de Plantas/genética , Globulinas/genética , Glycine max/genética , Interferência de RNA , Proteínas de Armazenamento de Sementes/genética , Proteínas de Soja/genética , Antígenos de Plantas/metabolismo , Cotilédone/citologia , Cotilédone/genética , Cotilédone/metabolismo , Técnicas de Transferência de Genes , Genoma de Planta , Globulinas/metabolismo , Recombinação Genética , Proteínas de Armazenamento de Sementes/metabolismo , Proteínas de Soja/metabolismo , TransgenesRESUMO
Hypertension affects one-fifth of the world population. Genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) that correlated with hyper-tension in large samples. However, the genetic mutations leading to hypertension might differ among various populations, as they have different origins and are subjected to different environmental pressures. Therefore, additional studies are urgently needed to verify the GWAS findings across different populations. This study focused on the natriuretic peptide receptor C gene (NPR3), one of the hypertension-positive genes identified in a GWAS of an East Asian population. The correlation analysis between NPR3 and hypertension was replicated in 450 Chinese Dai (235 patients vs 215 controls) and 484 Chinese Mongolian (211 patients vs 273 controls) individuals. The positive SNP identified by GWAS analysis and three other tag SNPs representing the NPR3 linkage disequilibrium (LD) block regions were selected for genotyping. The results revealed that the rs1173766 polymorphism was associated with the occurrence of hypertension (χ(2) = 6.87, P = 0.0088), and that the T allele should be protective in the Dai ethnic group. Consider-ing a close LD block at the 3' end of the NPR3 gene in the East Asian population, we speculate that there might be a mutation in the last five exons or the 3' untranslated region of NPR3 that could change the structure or expression of the NPR3 gene. However, in the Mongolian ethnic group, these SNPs were not associated with the incidence of hypertension, suggesting population heterogeneity for the genetic factors that contribute to hypertension.
Assuntos
Hipertensão/genética , Receptores do Fator Natriurético Atrial/genética , Adulto , Idoso , Povo Asiático/genética , China , Etnicidade/genética , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genética Populacional , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo GenéticoRESUMO
Plant traits are important indices for regulating and controlling yield ability in soybean varieties. It is important to comprehensively study the quantitative trait locus (QTL) mapping for soybean plant traits, cloning related genes, and marker assistant breeding. In this study, 236 F2 generation plants and a derivative group were constructed by using Jiyu50 and Jinong18, obtained from Jilin Province. A total of 102 simple sequence repeat markers were used to construct a genetic linkage map. With 2 years of molecular and phenotypic data, QTL analyses and mapping were conducted for soybean maturity, plant height, main stem node, main stem branch, seed weight per plant, and more. Five main plant traits were analyzed via inclusive composite interval mapping using QTL IciMapping v2.2. Using one-dimensional scanning, a total of 30 QTLs were detected and distributed across 1 (A1), 4 (C2), and 12 (G). There were 9 linkage groups, including 16 major QTLs. Using two-dimensional scanning, 7 pairs of epistatic QTL interactions for maturity and plant height were detected in the soybean.
Assuntos
Mapeamento Cromossômico/métodos , Glycine max/genética , Locos de Características Quantitativas , Cromossomos de Plantas/genética , DNA de Plantas/análise , Ligação Genética , Hibridização Genética , Repetições de MicrossatélitesRESUMO
Calpain-3 (CAPN3) is a member of the calpain family of Ca(2+)-regulated cysteine proteases, which play an important role in sarcomere remodeling and mitochondrial protein turnover, and thus, regulating beef tenderness in cattle. Currently, multiple CAPN3 transcripts have been detected in human, monkey, rat, and rabbit. However, whether this transcript is present in cattle remains unknown. In this study, we identified 2 CAPN3 transcripts in the skeletal muscle individuals of local black cattle from Jilin, China. One transcript corresponded to the known full-length protein and was referred to as CAPN3a, while the second transcript did not contain exons 2-19 and contained a single-nucleotide insert in the penultimate base of exon 1 compared to CAPN3a; this protein was referred to as CAPN3b. The expression level of CAPN3b was approximately 50-fold lower than that of CAPN3a. Moreover, CAPN3b mRNA was not translated into a functional protein because it had lost essential domains according to bioinformatic analysis. Our results not provide a foundation for understanding the function of CAPN3, but also are useful for further elucidating the effect of CAPN3 on meat quality in cattle.
Assuntos
Processamento Alternativo/genética , Calpaína/genética , Bovinos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Calpaína/química , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Seed number per pod is an important component of yield traits in soybean (Glycine max L.). In 2010, we identified a natural mutant with an increased number of four-seed pods from a soybean variety named 'Jinong 18' (JN18). Subsequent observations indicated that the trait was stably inherited. To identify and understand the function of genes associated with this mutant trait, we analyzed the genetic differences between the mutant (JN18MT01) and source variety (JN18) by transcriptome sequencing. Three types of tissues, axillary buds, unfertilized ovaries, and young pods at three different growth stages, V6, R1, and R3, were analyzed, respectively. The sequencing results yielded 55,582 expressed genes and 4183 differentially expressed genes (DEGs). Among these, the log2 ratio value of 162 DEGs was >10, and 13 DEGs had overlapping expression at three different growth stages. Comparisons of DEGs among three different growth stages yielded similar results in terms of the percentage of genes classified into each gene ontology (GO) category. DEGs were classified into 25 different functional groups in clusters of orthologous groups analysis. Proportions of the main functional genes differed significantly over developmental stages. A comparison of enriched pathways among the three developmental stages revealed that 646 unigenes were involved in 103 metabolic pathways. These results show that the development of four-seed pods is associated with a complex network involving multiple physiological and metabolic pathways. This study lays the foundation for further research on cloning and on the molecular regulation of genes related to the four-seed pod mutation.
Assuntos
Frutas/genética , Regulação da Expressão Gênica de Plantas , Glycine max/genética , Proteínas de Plantas/genética , Característica Quantitativa Herdável , Sementes/genética , Transcriptoma , Frutas/anatomia & histologia , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Mutação , Fenótipo , Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Glycine max/anatomia & histologia , Glycine max/crescimento & desenvolvimentoRESUMO
The luteinizing hormone receptor (LHR) plays a key role in testosterone production through its interaction with the gonadotropins, LH and chorionic gonadotropin. We examined the LHR splicing pattern in bovine Leydig cells; LH-induced expression of eight cloned splicing variants was detected by real-time PCR. Luteinizing hormone applied to cultured Leydig cells resulted in expression of full-length LHR and the A and B isoforms, as well as secretion of testosterone, which first increased, then declined, and then increased further, with increased LH levels. The secretion of testosterone progressively increased with increasing LH, but the expression levels of LHR (FL, A, and B) did not increase correspondingly. We conclude that the LHR splicing pattern is complex in bovine Leydig cells, and that expression of full-length LHR and isoforms A and B changes when induced with LH.
Assuntos
Células Intersticiais do Testículo/metabolismo , Receptores do LH/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Análise de Variância , Animais , Bovinos , Células Cultivadas , Éxons , Expressão Gênica , Hormônio Luteinizante/fisiologia , Masculino , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores do LH/genética , Testosterona/metabolismoRESUMO
The molecular mechanisms and potential clinical applications of neural precursor cells have recently been the subject of intensive study. Dlx5, a homeobox transcription factor related to the distal-less gene in Drosophila, was shown to play an important role during forebrain development. The subventricular zone (SVZ) in the adult brain harbors the largest abundance of neural precursors. The anterior SVZ (SVZa) contains the most representative neural precursors in the SVZ. Further research is necessary to elucidate how Dlx5-related genes regulate the differentiation of SVZa neural precursors. Here, we employed immunohistochemistry and molecular biology techniques to study the expression of Dlx5 and related homeobox genes Er81 and Islet1 in neonatal rat brain and in in vitro cultured SVZa neural precursors. Our results show that Dlx5 and Er81 are also highly expressed in the SVZa, rostral migratory stream, and olfactory bulb. Islet1 is only expressed in the striatum. In cultured SVZa neural precursors, Dlx5 mRNA expression gradually decreased with subsequent cell passages and was completely lost by passage four. We also transfected a Dlx5 recombinant plasmid and found that Dlx5 overexpression promoted neuronal differentiation of in vitro cultured SVZa neural precursors. Taken together, our data suggest that Dlx5 plays an important role during neuronal differentiation.