RESUMO
It is well established that endothelial injury plays an essential role in atherosclerotic plaque formation. Accumulating evidence has shown that high glucose levels may detrimentally affect cultured endothelial cells through endoplasmic reticulum (ER) stress. In this study, we investigated the effect of rosuvastatin on high glucose-induced ER stress in human umbilical vein endothelial cells (HUVECs). HUVECs treated with 30 mM glucose were used to simulate high-glucose conditions, and rosuvastatin concentrations ranging from 0.1 to 10 nM were used. Cell viability was analyzed by thiazolyl blue tetrazolium bromide assay, and apoptosis rate was measured using flow cytometry. Expression of GRP78, IRE1α, XBP1s, and CHOP was quantified using western blot and real-time polymerase chain reaction. Compared to cells treated with high glucose alone, cell viability increased and apoptosis rate decreased significantly in the rosuvastatin + high-glucose groups. Furthermore, GRP78, IRE1α, XBP1s, and CHOP expression was downregulated as a result of rosuvastatin administration. These results suggest that rosuvastatin may protect HUVECs from injury induced by high glucose levels, through alleviation of ER stress.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana/patologia , Rosuvastatina Cálcica/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
An isolate capable of degrading paraffin wax was isolated from petroleum-contaminated sites in Daqing, China, and identified as Pseudomonas sp strain PW-1 by analyzing the 16S rDNA sequence (GenBank accession No.: KF529529) as well as the biochemical and physiological characteristics. The optimized degradation conditions of the isolate were as follows: FeSO4 metal ion concentration of 0.01 g, temperature of 30°C, (NH4)2SO4 nitrogen source concentration of 1.5 g/L, and a carbon: nitrogen ratio of 10:1. Response surface methodology-based analysis of the culture time, inoculation amount, and initial pH of the medium revealed that the optimal theoretical conditions were a culture time of 11.16 days, inoculation amount of 3.13%, and an initial pH of 9.29. The theoretical degradation rate was up to 54.68% under the optimal conditions. Taking into account the experimental conditions of a laboratory, 11.2 days of cultivating time, 3% inoculum, and a medium initial pH of 9.3 were used in practical settings. Experimental results showed that the degradation rate of paraffin wax was 52.85%, which demonstrated that this strain could degrade 1050 mg paraffin wax, using it as the sole carbon source, in a 1000-mL minimal salts medium. These results indicate that the strain PW1 can be used for application in oil wells with paraffin deposition problems in order to enhance oil recovery.
Assuntos
Biodegradação Ambiental , Parafina/química , Petróleo/metabolismo , Pseudomonas/metabolismo , Carbono/química , Carbono/metabolismo , China , Nitrogênio/química , Nitrogênio/metabolismo , Parafina/metabolismo , Petróleo/toxicidade , Filogenia , Pseudomonas/química , Pseudomonas/genética , RNA Ribossômico 16S/genéticaRESUMO
There is increasing evidence suggesting that endoplasmic reticulum stress (ERS) plays an important role in the initiation and development of atherosclerosis. This study was designed to examine the effect of probucol on cultured human umbilical vein endothelial cells (HUVECs) injured by hypoxia/reoxygenation (H/R) and the potential mechanisms involving ERS. Injured HUVECs induced by Na2S2O4 served as an H/R model in vitro. The concentration of probucol in this study ranged from 3 to 27 µM. Cell viability was analyzed using MTT and a lactate dehydrogenase (LDH) assay. The expression of GRP78, X-box-binding protein (XBP)-1, and CHOP (c/EBP-hemologous protein) were quantified using western blot. Compared to cells with H/R injury alone, the results showed that the cell viability increased significantly with probucol, while the LDH leakage rate was significantly lower as analyzed by the LDH assay. Furthermore, the expression levels of GRP78, XBP-1, and CHOP were significantly downregulated. These results indicated that probucol effectively protected HUVEC from injury induced by H/R and that the mechanism might be related to attenuation of ERS.
Assuntos
Ditionita/efeitos adversos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Probucol/farmacologia , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Células Endoteliais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , HumanosRESUMO
Genomic imprinting is an important epigenetic mechanism that has vital effects on fetal growth and development. We observed the differences in four tissues (heart, spleen, liver, and kidney) from dead transgenic cloned goats using hematoxylin and eosin (H&E) staining. Eight imprinted genes in the tissues of dead transgenic cloned and normal goats were analyzed using reverse transcription polymerase chain reaction. H&E staining results from the abortion group indicated the lack of obvious morphological changes in heart and spleen tissues, while inflammatory cell infiltration and glomerular nephritis characteristics were observed in liver and kidney tissues, respectively. Compared to the control group, CDKN1C, H19, IGF2R, and SNRPN were significantly (P < 0.05) overexpressed in the heart tissue of the abortion group, while XIST was significantly reduced. In the liver tissues, CDKN1C and DLK1 expression decreased, while GNAS, H19, IGF2R, PEG3, and XIST expression increased significantly. In the spleen tissues, DLK1 expression increased, while GNAS, H19, IGF2R, PEG3, SNRPN, and XIST expression decreased. In the kidney tissues, CDKN1C, DLK1, GNAS, IGF2R, and PEG3 expression increased, while H19 and XIST expression decreased. The overall expression of imprinted genes was abnormal in different tissues of transgenic cloned goats, and the degree of abnormal genomic imprinting was more severe in the abortion group compared to the death and control groups. These results suggest that abnormal expression of imprinted genes may cause developmental defects in transgenic cloned goats. Moreover, abnormal epigenetic modifications may affect the reprogramming of transgenic donor cells.
Assuntos
Clonagem de Organismos/mortalidade , Epigênese Genética , Genes Letais , Impressão Genômica , Cabras/genética , Lactoferrina/genética , Animais , Animais Geneticamente Modificados , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Feminino , Perfilação da Expressão Gênica , Cabras/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lactoferrina/metabolismo , Fígado/metabolismo , Masculino , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Gravidez , Transdução de Sinais , Baço/metabolismo , TransgenesRESUMO
Dairy goat is a good model for production of transgenic proteins in milk using somatic cell nuclear transfer (SCNT). However, animals produced from SCNT are often associated with lung deficiencies. We recently produced six transgenic cloned dairy goats harboring the human lactoferrin gene, including three live transgenic clones and three deceased transgenic clones that died from respiratory failure during the perinatal period. Imprinted genes are important regulators of lung growth, and may be subjected to faulty reprogramming. In the present study, first, microsatellite analysis, PCR, and DNA sequence identification were conducted to confirm that these three dead kids were genetically identical to the transgenic donor cells. Second, the CpG island methylation profile of the imprinted insulin-like growth factor receptor (IGF2R) gene was assessed in the lungs of the three dead transgenic kids and the normally produced kids using bisulfite sequencing PCR. In addition, the relative mRNA level of IGF2R was also determined by real-time PCR. Results showed that the IGF2R gene in the lungs of the dead cloned kids showed abnormal hypermethylation and higher mRNA expression levels than the control, indicating that aberrant DNA methylation reprogramming is one of the important factors in the death of transgenic cloned animals.
Assuntos
Cabras/genética , Lactoferrina/genética , Pulmão/metabolismo , Receptor IGF Tipo 2/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Clonagem de Organismos , Metilação de DNA , Transferência Embrionária , Feminino , Expressão Gênica , Impressão Genômica , Humanos , Repetições de Microssatélites , Dados de Sequência Molecular , Receptor IGF Tipo 2/metabolismo , Análise de Sequência de DNARESUMO
The genetic resources and the mechanism of miniaturization in the Tibet Mini-pig have not been comprehensively studied. Polymorphisms in genes related to the insulin-like growth factor (IGF) axis have been investigated for years, but few on the polymorphism of IGF-binding protein-5 (IGFBP-5) in the Tibetan pig. In this study, allele-specific polymerase chain reaction (AS-PCR) was used to analyze polymorphisms in exon 1 of the IGFBP-5 gene in two pig breeds, Tibet Mini-pigs and Junmu No. 1 White pigs. A BLAST analysis of the expressed sequence tags in the porcine IGFBP-5 gene revealed that exon 1 of this gene has two single nucleotide polymorphisms (SNPs), G188T and G503A. The AS-PCR results demonstrated that in both pig breeds examined, the TT, GT, and GG genotypes existed at the G188T locus, with GT as the most common genotype. At the G503A locus, GG, GA, and AA genotypes existed in Junmu No. 1 White pigs, with the GA genotype as the most frequently occurring. By contrast, at this locus, only the GA and AA genotypes were observed in the Tibetan pigs, and AA was more common than GA. There was a significant difference (P < 0.01) in allele distribution between the two breeds at the G503A locus but not the G188T locus, and there was a lower polymorphism information content for the two polymorphic loci in Tibet Mini-pigs than in Junmu No. 1 White pigs. The present study revealed SNPs in exon 1 of IGFBP-5 gene in the Tibet Mini-pig, possibly providing more understanding of the mechanism of miniaturization.
Assuntos
Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Polimorfismo Genético , Porco Miniatura/genética , Alelos , Animais , Cruzamento , Éxons/genética , Genótipo , Polimorfismo de Nucleotídeo Único , Suínos , Porco Miniatura/crescimento & desenvolvimento , TibetRESUMO
Mesenchymal stem cells derived from bone marrow (BMSCs) are a population of self-renewing multipotent cells that are capable of differentiating into various cellular lineages, and are widely employed in tissue engineering and cell therapy. Recently, clinical research involving BMSCs has become increasingly popular. In order to conduct appropriate research, it is first necessary to amplify large amounts of functional BMSCs in vitro. However, after several passages of expanding in vitro, the proliferation and differentiation potential of BMSCs gradually decline. To determine whether overexpression of Oct4 or Sox2 might prevent this decline, we transfected Oct4 or Sox2, which are essential for the pluripotency and self-renewal of embryonic stem cells, into BMSCs of Xiaomeishan porcine by a lentivirus. The results showed that overexpression of Sox2 or Oct4 BMSCs in culture media containing a basic fibroblast growth factor resulted in higher proliferation and differentiation compared to controls, suggesting that genetic modification of stemness-related genes is an efficient way to maintain the proliferation and differentiation potential of BMSCs.
Assuntos
Adipogenia , Proliferação de Células , Células-Tronco Mesenquimais/fisiologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Células Cultivadas , Fatores de Crescimento de Fibroblastos/fisiologia , Expressão Gênica , Células HEK293 , Humanos , Fator 3 de Transcrição de Octâmero/genética , Osteogênese , Fatores de Transcrição SOXB1/genética , Sus scrofaRESUMO
Alkaline phosphatase (ALP) of Helicoverpa armigera Hub. (Lepidoptera; Noctuidae) (GenBank accession No. EU729322) was cloned and expressed. The target gene H.a-ALP, having an open reading frame of 1608 bp, was reverse-transcribed from cDNA by the polymerase chain reaction. The open reading frame of the target gene was cloned into the pET-32a expression vector to obtain recombinant protein in Escherichia coli DE-3 cells for the subsequent production of polyclonal antibody. New Zealand white rabbits were used for production of anti-pET-32a-H.a-ALP. The production of antibody was also optimized by employing ELISA for titer determination. The produced antiserum was processed and used as an antibody. Western blot results showed that the polyclonal antibody produced was capable of effectively binding target protein not only from H. armigera but also from other lepidopterans such as Mythimna separata and Plutella xylostella. This antibody was also used to detect levels of ALP within different instars of H. armigera. Thus, it is concluded that this antibody-based assay is very useful for the effective detection of gene-specific expression. Furthermore, it may also be used to detect the expression levels and tissue localization of ALP, as well as in other physiological studies involving this enzyme.
Assuntos
Fosfatase Alcalina/metabolismo , Anticorpos/química , Proteínas de Insetos/metabolismo , Mariposas/enzimologia , Fosfatase Alcalina/imunologia , Animais , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Proteínas de Insetos/imunologia , Coelhos , TitulometriaRESUMO
The molecular and biochemical effects of an insecticidal toxin extracted from Meloidae beetles were investigated on Helicoverpa armigera. The toxin was identified as cantharidin, a well-known natural compound produced by beetles of family Meloidae and Oedemeridae. Furthermore, the effect of the toxin on the metabolic enzymes alkaline phosphatase (ALP) and glutathione S-transferase (GST), responsible for the metabolism of insecticides, was also investigated. Results of a diet incorporation bioassay performed under laboratory conditions showed that the LC50 value of cantharidin was 0.068 mg/g. The body weight of the insect was also significantly reduced by cantharidin treatment. The LC10 concentration of cantharidin, 0.01 mg/g, was also tested to determine its effect on ALP and GST. Our results showed that cantharidin significantly inhibited ALP activity after 48 h, whereas GST activity was significantly inhibited after 24 h. The decline of ALP and GST transcript levels was also validated by semiquantitative RT-PCR analysis. It may be concluded from the results that ALPs and GSTs may be targets of the cantharidin intoxication mechanism. Moreover, the inability of ALP and GST to metabolize cantharidin shows that the mechanism of detoxification for cantharidin is different from that for conventional insecticides. On the basis of our investigations, the chemical structure of insecticides may be modified using a model structure of cantharidin, to avoid metabolism by metabolic enzymes.
Assuntos
Cantaridina/farmacologia , Besouros/química , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Cantaridina/química , Clonagem Molecular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Repressão Enzimática , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/química , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/crescimento & desenvolvimento , Dose Letal Mediana , Mariposas/enzimologia , Mariposas/crescimento & desenvolvimento , Análise de Sequência de DNARESUMO
OBJECTIVE: To discuss the positive rate of ventricular late potential (VLP) between patients with acute ST-segment elevation myocardial infarction (STEMI) and patients with acute non NSTEMI. METHODS: One hundred and sixty-three cases of acute myocardial infarction (90 patients with STEMI and 73 with NSTEMI), admitted to the first hospital of China Medical University between June 2011 and August 2011, underwent VLP examination. RESULTS: The VLP positive rate of the STEMI group was 54.4%, while that of the NSTEMI group was 38.4%, and the differences have statistical meaning (χ2 = 4.186, p < 0.05). The occurrence rate of ventricular arrhythmia in VLP positive patients was 11.7%, while in VLP negative patients it was 3.5% (χ2 = 4.005, p < 0.05). CONCLUSION: The VLP positive rate of the STEMI group is higher than that of the NSTEMI group.
OBJETIVO: Analizar la tasa positiva del potencial tardío ventricular (PTV) entre pacientes con infarto agudo del miocardio sin elevación del segmento ST (NSTEMI por sus siglas en inglés) y el infarto agudo del miocardio con elevación del segmento ST (STEMI por sus siglas en inglés). MÉTODOS: Ciento sesenta y tres casos de infarto agudo de miocardio (90pacientes con STEMI) y 73 con NSTEMI, ingresados en la Universidad primer hospital de Medicina China entre junio y agosto de 2011, fueron sometidos a examen de PTV. RESULTADOS: La tasa positiva PVT del grupo STEMI fue 54.4%, mientras que la del grupo NSTEMI fue 38.4%, y las diferencias tienen significado estadístico (χ² = 4.186, p < 0.05). La tasa de ocurrencia de arritmia ventricular en pacientes PVTpositivos fue 11.7%, mientras que en los pacientes PVT negativos fue 3.5% (χ² = 4.005, p < 0.05). CONCLUSIÓN: La tasa PTV positiva del grupo STEMI es mayor que la del grupo NSTEMI.