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2.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 35(4): 289-292, 2019 Jul 28.
Artigo em Chinês | MEDLINE | ID: mdl-31701707

RESUMO

OBJECTIVE: To investigate the protective effects of Sestrin2 protein on lung epithelial Beas-2B cells in the heat-exposure environment and its mechanism. METHODS: Lung epithelial Beas-2B cells were cultured at 37℃, 39℃, 40℃ and 41℃ respectively. Cells were harvested at different times (0, 3, 6 and 12 h) after pancreatin digestion. The expressions of Sestrin2, superoxide dismutase(SOD), reactive oxygen species(ROS), cell mitochondrial membrane potential and apoptosis rate of cells were detected by Western blot, fluorescence spectrophotometer and flow cytometry, respectively. Gene expression sequence was cloned into high expression plasmid pcDNA3.1+. Beas-2B cells were transfected by Lipfectamine 2000 to construct Sestrin2 and SOD high expression cells. The changes of mitochondrial membrane potential and cell apoptosis were observed in the Sestrin2 and SOD high expression cells. RESULTS: With the increase of temperature, the expression level of Sestrin2 protein in heat treatment group was decreased compared with the control group. When Beas-2B cells were exposed to 41℃, the ROS level was increased, mitochondrial membrane potential was decreased significantly and apoptosis rate was increased at different time points. After high expression of Sestrin2 and SOD in the Beas-2B cells, the expression level of ROS was decreased and the change tendency of mitochondrial membrane potential was decreased, and the apoptosis rate was reduced at 41℃ exposure. CONCLUSION: Sestrin2 can alleviate the apoptosis of lung epithelial cells induced by heat exposure through mitochondrial membrane potential and SOD, which has protective effect on lung epithelial Beas-2B cells.


Assuntos
Apoptose , Células Epiteliais/patologia , Temperatura Alta , Proteínas Nucleares/metabolismo , Linhagem Celular , Humanos , Potencial da Membrana Mitocondrial , Proteínas Nucleares/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Transfecção
3.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 35(5): 422-427, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31894674

RESUMO

OBJECTIVE: To investigate the effects of mitochondrial ATPase inhibitory factor 1 (Atpif1) on hemoglobin synthesis. METHODS: Firstly, the K562 cells were divided into 2 groups, hypoxia-treated group and normoxic control group. The K562 cells in hypoxia-treated group were treated with 2% oxygen. The K562 cells in the two groups were collected after cultured for 24, 48 and 72 hours. The proliferation-inhibitory rates of cells were detected by CCK-8 assay. The apoptosis rates of K562 cells were analyzed by flow cytometry. The hemoglobin synthesis of K562 cells was induced by hemin. The gene expressions of Atpif1, Aladelta-aminolevulinate synthase 2 (Alas2) and nuclear factor kappa B (NF-κB) were detected by qRT-PCR. Then, the K562 cells were cultured in hypoxic incubator and divided into blank control group, negative control group and si-Atpif1 group. After sliencing Atpif1 gene, the hemoglobin synthesis and the levels of NF-κB and Alas2 were determined. RESULTS: Compared with the normoxic control group, the proliferation activity of K562 cells was inhibited, the apoptosis rate was increased, and the hemoglobin synthesis was also increased in hypoxia-treated groups. The expressions of Atpif1, Alas2 and NF-κB mRNA of K562 cells were upregulated. Compared with blank control group and negative control group, the content of hemoglobin was decreased, and the levels of NF-κB and Alas2 mRNA were also decreased in si-Atpif1 group. CONCLUSION: Atpif1 gene is involved in the regulation of hemoglobin synthesis. Exploring its roles in the development of high altitude polycythemia (HAPC) can provide new ideas and therapeutic targets for the prevention and treatment of HAPC.


Assuntos
Hemoglobinas , Proteínas , Apoptose/genética , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Hemoglobinas/genética , Humanos , Células K562 , NF-kappa B/genética , Proteínas/genética , RNA Mensageiro/genética
4.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 34(1): 39-42, 2018 Jan 08.
Artigo em Chinês | MEDLINE | ID: mdl-29926657

RESUMO

OBJECTIVE: To establish an animal model for loaded swimming, so as to investigate the energy metabolism effects of soybean isoflavones (SI) on swimming mice. METHODS: Thirty male Kunming mice were randomly divided into three groups:normal control, swimming group, and swimming+SI group. The normal control group mice were fed a basic AIN-93M diet, the SI groups were supplied with soybean isoflavones(4 g/kg).Two weeks later, the mice were forced to swim for an hour,and then all the mice were killed, the samples of blood, liver and muscles of hind were collected.The serum contents of lactic acid(Lac), the activities of lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), creatine kinase (CK) and ATPase were measured. RESULTS: Compared with normal control,the serum content of Lac was significantly improved in the group of the swimming control and SI(P<0.05),the activity of LDH in the serum was obviously improved in the group of the swimming control and SI, and the activity of CK and SDH were both significantly improved in the group of the swimming control and SI except the activity of SDH in the liver of the group SI; compared with the swimming control,the serum contents of Lac,the activities of LDH, ATPase, SDH, CK were obviously improved(P<0.05). CONCLUSIONS: Soybean isoflavones can improve the energy metabolism,antioxidant capacity of the swimming mice.


Assuntos
Metabolismo Energético , Isoflavonas/farmacologia , Soja/química , Natação , Adenosina Trifosfatases/sangue , Animais , Creatina Quinase/sangue , L-Lactato Desidrogenase/sangue , Ácido Láctico/sangue , Masculino , Camundongos , Distribuição Aleatória , Succinato Desidrogenase/sangue
5.
Hepatology ; 66(4): 1045-1057, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28407288

RESUMO

The development of pathogenic mechanisms, specific antiviral treatments and preventive vaccines for hepatitis C virus (HCV) infection has been limited due to lack of cell culture models that can naturally imitate the entire HCV life cycle. Here, we established an HCV cell culture model based on human fetal liver stem cells (hFLSCs) that supports the entire blood-borne hepatitis C virus (bbHCV) life cycle. More than 90% of cells remained infected by various genotypes. bbHCV was efficiently propagated, and progeny virus were infectious to hFLSCs. The virus could be passed efficiently between cells. The viral infectivity was partially blocked by specific antibodies or small interfering RNA against HCV entry factors, whereas HCV replication was inhibited by antiviral drugs. We observed viral particles of approximately 55 nm in diameter in both cell culture media and infected cells after bbHCV infection. CONCLUSION: Our data show that the entire bbHCV life cycle could be naturally imitated in hFLSCs. This model is expected to provide a powerful tool for exploring the process and the mechanism of bbHCV infection at the cellular level and for evaluating the treatment and preventive strategies of bbHCV infection. (Hepatology 2017;66:1045-1057).


Assuntos
Células-Tronco Fetais , Hepacivirus/fisiologia , Fígado/citologia , Modelos Biológicos , Replicação Viral , Humanos , Fígado/virologia , Cultura Primária de Células , Proteínas Virais/biossíntese , Liberação de Vírus
6.
Aging (Albany NY) ; 8(10): 2337-2354, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27713146

RESUMO

The corneal epithelium plays important roles in the maintenance of corneal transparency for good vision, and acts as a protective barrier against foreign insults. Structural and functional changes with aging in the corneal epithelium have been documented. Here we found that transforming growth factor-ß (TGF-ß) is highly expressed in the elderly donor corneal epithelium, as are senescence-associated genes, such as p16 and p21. In human corneal epithelial cell (HCEC) models, TGF-ß induces cellular senescence, characterized by increased SA-ß-gal positive cells and elevated expression of p16 and p21. Pharmacological inhibition of TGF-ß signaling alleviates TGF-ß-induced cellular senescence. In addition, we determined that senescence-associated inflammation was significantly aggravated in TGF-ß-induced cellular senescence by detecting the expression of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNFα). Both genetic and pharmacological approaches revealed that blocking nuclear factor-κB (NF-κB) signaling not only inhibited the production of inflammatory factors, but also rescued the senescent phenotype induced by TGF-ß in HCECs. Mechanistically, TGF-ß induced an atypical RNA stress responses, leading to accelerated mRNA degradation of IκBα, an inhibitor of NF-κB. Together, our data indicate that TGF-ß-driven NF-κB activation contributes to corneal epithelial senescence via RNA metabolism and the inflammation blockade can attenuate TGF-ß-induced senescence.


Assuntos
Envelhecimento/metabolismo , Senescência Celular/fisiologia , Epitélio Corneano/metabolismo , NF-kappa B/metabolismo , RNA , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Criança , Epitélio Corneano/citologia , Feminino , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem
7.
PLoS One ; 11(10): e0165580, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27783701

RESUMO

Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10-24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10-3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10-3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10-3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10-3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10-4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10-5). KC corneas also had increased mtDNA damage (P = 3.63×10-10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC.


Assuntos
DNA Mitocondrial/metabolismo , Ceratocone/fisiopatologia , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Células Cultivadas , Criança , Córnea/citologia , Córnea/metabolismo , DNA Mitocondrial/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Ceratocone/diagnóstico , Potencial da Membrana Mitocondrial , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adulto Jovem
10.
Ophthalmic Genet ; 36(2): 132-6, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25675348

RESUMO

BACKGROUND: Keratoconus (KC) is a complex degenerative disorder of the cornea. Genetic, environmental, and lifestyle factors may all contribute to the pathogenesis of KC. Most of the reported KC-associated SNPs have been detected in Caucasians and Australians. To investigate whether the reported associated SNPs can be found in a Chinese population, we performed a replication study of the significantly associated SNPs. MATERIALS AND METHODS: A total of 210 unrelated Chinese KC patients and 191 unrelated controls were included in the present study. SNPs rs4954218 (Near RAB3GAP1 (5')), rs4894535 (FNDC3B), rs2956540 (LOX), rs3735520 (Near HGF (5')), rs1324183 (MPDZ-NF1B), rs1536482 (RXRA-COL5A1), rs7044529 (COL5A1), rs2721051 (Near FOXO1 (3')), rs9938149 (BANP-ZNF469) and rs6050307 (VSX1) were assessed for their association with KC. The genotype of each SNP was detected using the Sequenom MassARRAY-Assay. RESULTS: SNP rs1324183 located in MPDZ-NF1B was associated with an increased risk of KC (OR=3.108, 95% CI=1.366-7.072, p=0.005), and SNP rs2956540 in the LOX gene may confer a reduced risk of KC with a borderline p value in our population (OR=0.664, 95% CI=0.447-0.986, p=0.042). No significant difference was observed between patients and controls in the other eight SNP genotypes and allele frequencies. CONCLUSIONS: The replication association of rs1324183 (MPDZ-NF1B) with KC in our population and the results, which are identical to those in different populations, suggest that rs1324183 (MPDZ-NF1B) is a common genetic risk for KC and should be further investigated.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Proteínas de Transporte/genética , Loci Gênicos , Ceratocone/genética , Neurofibromina 1/genética , Polimorfismo de Nucleotídeo Único , Adulto , China/epidemiologia , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Técnicas de Genotipagem , Humanos , Masculino , Proteínas de Membrana , Adulto Jovem
11.
Rejuvenation Res ; 18(3): 211-24, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25556695

RESUMO

Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Células-Tronco Fetais/citologia , Feto/citologia , Hepatócitos/citologia , Fígado/citologia , Adulto , Moléculas de Adesão Celular/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Conexinas , Feminino , Células-Tronco Fetais/metabolismo , Feto/metabolismo , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunofenotipagem , Fígado/metabolismo , Fenótipo , Gravidez , Segundo Trimestre da Gravidez , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 31(6): 498-503, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27215016

RESUMO

Resveratrol, as a natural polyphenolic compound, has a wide range of beneficial effects, which includes anti-tumor, cardiovascular protection, anti-oxidant and estrogen-like effects, and so on. Its various physiological properties are closely related to the therapeutic principle for prevention and treatment of high altitude hypoxia injury. Resveratrol may play an important role in relieving or curing high altitude diseases, especially high altitude polycythemia(HAPC). However, the literature about study and application of resveratrol in plateau medicine field is rarely reported up to now. In this review, we summarized the physiological effects of resveratrol, discussed the possible main principle of resveratrol for HAPC therapy, and looked forward to resveratrol's perspective or potential application in high altitude medicine.


Assuntos
Altitude , Hipóxia/tratamento farmacológico , Estilbenos/farmacologia , Humanos , Policitemia/tratamento farmacológico , Resveratrol
13.
Nat Genet ; 46(10): 1097-102, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25151357

RESUMO

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers. We performed exome sequencing on 113 tumor-normal pairs, yielding a mean of 82 non-silent mutations per tumor, and 8 cell lines. The mutational profile of ESCC closely resembles those of squamous cell carcinomas of other tissues but differs from that of esophageal adenocarcinoma. Genes involved in cell cycle and apoptosis regulation were mutated in 99% of cases by somatic alterations of TP53 (93%), CCND1 (33%), CDKN2A (20%), NFE2L2 (10%) and RB1 (9%). Histone modifier genes were frequently mutated, including KMT2D (also called MLL2; 19%), KMT2C (MLL3; 6%), KDM6A (7%), EP300 (10%) and CREBBP (6%). EP300 mutations were associated with poor survival. The Hippo and Notch pathways were dysregulated by mutations in FAT1, FAT2, FAT3 or FAT4 (27%) or AJUBA (JUB; 7%) and NOTCH1, NOTCH2 or NOTCH3 (22%) or FBXW7 (5%), respectively. These results define the mutational landscape of ESCC and highlight mutations in epigenetic modulators with prognostic and potentially therapeutic implications.


Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Predisposição Genética para Doença/genética , Mutação , Apoptose/genética , Carcinoma de Células Escamosas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Epigênese Genética/genética , Neoplasias Esofágicas/patologia , Exoma/genética , Humanos , Prognóstico , Análise de Sequência de DNA , Transdução de Sinais/genética , Análise de Sobrevida
14.
Environ Sci Technol ; 48(12): 6947-56, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24865258

RESUMO

Exposure to various infectious viruses in environmental drinking water can constitute a public health risk. However, it is difficult to detect viruses in water due to their low concentration. In this study, we have developed a novel filter cartridge system containing electropositive granule media (EGM). Viruses present in large volumes of environmental samples were adsorbed onto the EGM, and then recovered by elution and poly(ethylene glycol) (PEG) concentration. To evaluate the system's efficiency in viral recovery, poliovirus (PV-1), a surrogate for enteric viruses, was used to artificially contaminate river water samples which were then assayed by quantitative real-time PCR. To optimize the concentration procedure, the eluent type, water flow rate and properties (e.g., pH, bacterial, and viral loads), were evaluated. The highest virus recovery was obtained by pumping river water at a flow rate of 300 mL/min and then pushing 3 L of an eluent containing 3× broth [1.5% (w/v) NaCl, 3% (w/v) tryptone, 1.5% (w/v) beef powder] with 0.05 mol/L glycine through the filter. Using this procedure, the recovery efficiencies of PV-1 from 10 to 100 L of spiked river water were up to 99%. In addition, this method is virus load and pH dependent. Virus recovery was maximal at a load of between 10(3.5) and 10(5.5) TCID50 and a pH ranging from 5 to 7. The bacterial load in the water has no effect on virus recovery. Different types of viruses and surface water were tested to validate the system's applicability. Results revealed that the EGM filter cartridge was able to concentrate PV-1, human adenoviruses (HAdVs) and noroviruses (HuNoVs) with high efficiency from river, lake, and reservoir water. Furthermore, it showed more efficient recovery than glass wool and 1MDS filters. These data suggest that this system provides rapid and efficient virus recovery from a large volume of natural surface water and, as such, could be a useful tool in revealing the presence of viruses in surface water.


Assuntos
Filtração/instrumentação , Filtração/métodos , Vírus/isolamento & purificação , Microbiologia da Água , Adsorção , Óxido de Alumínio/química , Animais , Linhagem Celular , Precipitação Química , Eletrodos , Escherichia coli/crescimento & desenvolvimento , Humanos , Concentração de Íons de Hidrogênio , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Reologia , Rios/virologia , Vírus/genética , Qualidade da Água
15.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 30(6): 526-31, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26016362

RESUMO

OBJECTIVE: To investigate the effects of simple hypobaric hypoxia on parameters of hematology and blood rheology in order to establish a rat model of simulated high altitude polycythemia (HAPC) for the study of pathophysiologic mechanisms and medical prevention and treatment of HAPC. METHODS: Forty-eight male Wistar rats were randomly divided into three normal control groups and three hypoxia model groups. Normal control group rats were bred in normoxia conditions, and hypoxia group rats were subjected to hypoxic exposure for 8 hours per day at simulated 5 500 m high altitude in a hypobaric chamber. After hypoxic exposure for 2, 4, 12 weeks, one group of normal control and hypoxia model rats were killed and blood was collected, respectively. Then parameters of erythrocyte and blood rheology were examined. RESULTS: Mucous membrane of hypoxia model rats showed obviously cyanosis after 2 weeks hypoxic exposure. Hemoglobin concentration of hypoxia model rats were beyond 210 g/L after 2 weeks, 4 weeks and 12 weeks hypoxia exposure and significantly increased than that of normal control rats respectively. Besides, RBC counts, hematocrit, whole blood viscosity, erythrocyte aggregation index of hypoxia model rats were all notably higher than those of normal control rats respectively. CONCLUSION: A rat model of high altitude polycythemia can be rapidly established by hypobaric hypoxia exposure at simulated 5 500 m high altitude for 8 hours daily.


Assuntos
Altitude , Hipóxia , Policitemia/patologia , Doença da Altitude , Animais , Modelos Animais de Doenças , Contagem de Eritrócitos , Hematócrito , Masculino , Ratos , Ratos Wistar
16.
Chin J Cancer ; 32(7): 403-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23470146

RESUMO

Biomarker identification is crucial for the selection of patients who might benefit from radiotherapy. To explore potential markers for response and prognosis in patients with locally advanced esophageal carcinoma treated with radiotherapy followed by surgery, we evaluated the expression of cell cycle checkpoint-related proteins Chk2, Cdc25C, and Cyclin D1. A total of 56 patients with locally advanced esophageal squamous cell carcinoma were treated with radiotherapy followed by surgery. Pretreatment tumor biopsy specimens were analyzed for Chk2, Cdc25C, and Cyclin D1 expression by immunohistochemistry. High expression of Chk2, Cyclin D1, and Cdc25C was observed in 44 (78.6%), 15 (26.8%), and 27 (48.2%) patients, respectively. The median survival was 16 months (range, 3-154 months), with a 5-year overall survival rate of 19.6%. Overexpression of Chk2 was associated with smoking (P = 0.021), overexpression of Cdc25C was associated with patient age (P = 0.033) and tumor length (P = 0.001), and overexpression of Cdc25C was associated with pathologic complete response (P = 0.038). Univariate analysis demonstrated that overexpression of Cdc25C and pathologic complete response was associated with better survival. In multivariate analysis, Cdc25C was the most significant independent predictor of better survival (P = 0.014) for patients treated with radiotherapy followed by surgery. Overexpression of Cdc25C was significantly associated with pathologic complete response and better survival of patients with locally advanced esophageal cancer treated with radiotherapy followed by surgery. These results suggest that Cdc25C may be a biomarker of treatment response and good prognosis for esophageal carcinoma patients. Thus, immunohistochemical staining of Cdc25C in a pretreatment specimen may be a useful method of identifying optimal treatment for patients with esophageal carcinoma.


Assuntos
Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/cirurgia , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/cirurgia , Fosfatases cdc25/metabolismo , Adulto , Idoso , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Quinase do Ponto de Checagem 2/metabolismo , Terapia Combinada , Ciclina D1/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Aceleradores de Partículas , Modelos de Riscos Proporcionais , Fumar , Taxa de Sobrevida
17.
Environ Sci Technol ; 46(24): 13448-54, 2012 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-23215020

RESUMO

Antibiotic resistance poses a significant challenge to human health and its rate continues to rise globally. While antibiotic-selectable synthetic plasmid vectors have proved invaluable tools of genetic engineering, this class of artificial recombinant DNA sequences with high expression of antibiotic resistance genes presents an unknown risk beyond the laboratory setting. Contamination of environmental microbes with synthetic plasmid vector-sourced antibiotic resistance genes may represent a yet unrecognized source of antibiotic resistance. In this study, PCR and real-time quantitative PCR were used to investigate the synthetic plasmid vector-originated ampicillin resistance gene, ß-lactam antibiotic (blá), in microbes from six Chinese rivers with significant human interactions. Various levels of blá were detected in all six rivers, with the highest levels in the Pearl and Haihe rivers. To validate the blá pollution, environmental plasmids in the river samples were captured by the E. coli transformants from the community plasmid metagenome. The resultant plasmid library of 205 ampicillin-resistant E. coli (transformants) showed a blá-positive rate of 27.3% by PCR. Sequencing results confirmed the synthetic plasmid vector sources. In addition, results of the Kirby-Bauer disc-diffusion test reinforced the ampicillin-resistant functions of the environmental plasmids. The resistance spectrum of transformants from the Pearl and Haihe rivers, in particular, had expanded to the third- and fourth-generation of cephalosporin drugs, while that of other transformants mainly involved first- and second-generation cephalosporins. This study not only reveals environmental contamination of synthetic plasmid vector-sourced blá drug resistance genes in Chinese rivers, but also suggests that synthetic plasmid vectors may represent a source of antibiotic resistance in humans.


Assuntos
Coleta de Dados , Genes Bacterianos/genética , Vetores Genéticos/genética , Plasmídeos/genética , Rios , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Sequência de Bases , China , DNA Recombinante/genética , Poluição Ambiental/análise , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Geografia , Humanos , Metagenoma/genética , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Análise de Sequência de DNA
18.
PLoS One ; 7(2): e31352, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22347466

RESUMO

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1:10(6) with E. coli O157:H7 and affinity constants ranged from 1.57 × 10(10) to 2.79 × 10(10) L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food.


Assuntos
Anticorpos Monoclonais/biossíntese , Escherichia coli O157/isolamento & purificação , Contaminação de Alimentos/análise , Imunização/métodos , Animais , Escherichia coli O157/imunologia , Hibridomas , Imunoensaio/métodos , Métodos , Camundongos , Especificidade da Espécie
19.
J Microbiol Biotechnol ; 22(2): 256-63, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22370359

RESUMO

A strain of bacterium producing antifungal antibiotic was isolated and identification of the strain was attempted. We could identify the bacterium as being a Bacillus sp., based on morphological observation, physiological characteristics, and 16S rDNA sequence analysis, thus leading us to designate the strain as Bacillus sp. AH-E-1. The strain showed potent antibiotic activity against phytopathogenic and human pathogenic fungi by inducing mycelial distortion and swelling and inhibiting spore germination. The antibiotic metabolite produced by the strain demonstrated excellent thermal and pH (2-11) stability, but was labile to autoclaving. From these results, we could find a broader antifungal activity of Bacillus genus. Isolation and characterization of the active agent produced by the strain are under progress.


Assuntos
Antifúngicos/metabolismo , Bacillus/isolamento & purificação , Bacillus/metabolismo , Fungos/efeitos dos fármacos , Bacillus/classificação , Bacillus/genética , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Fungos/isolamento & purificação , Humanos , Dados de Sequência Molecular , Micoses/microbiologia , Filogenia , Doenças das Plantas/microbiologia , Plantas , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
20.
J Med Virol ; 84(3): 526-35, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22246842

RESUMO

Chemical disinfection is the most common method used to inactivate viruses from drinking water throughout the world. In this study, cell culture, ELISA, RT-PCR, and spot hybridization were employed to investigate the mechanism underlying chlorine dioxide (ClO(2) )-induced inactivation of Poliovirus type 1 (PV1), which was also confirmed by recombinant viral genome RNA infection models. The results suggested that ClO(2) inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ClO(2) degraded specifically the 40-80 nucleotides (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 RNA lacking this 40-80 nt region was not infectious. This study not only elucidated the mechanism of PV1 inactivation by ClO(2), but also defined the critical genetic target for the disinfectant to inactivate Poliovirus. This study also provides a strategy by which rapid, accurate, and molecular methods based on sensitive genetic targets may be established for evaluating the effects of disinfectants on viruses.


Assuntos
Regiões 5' não Traduzidas , Compostos Clorados/farmacologia , Desinfetantes/farmacologia , Genoma Viral , Óxidos/farmacologia , Poliovirus/efeitos dos fármacos , Poliovirus/genética , Inativação de Vírus/efeitos dos fármacos , Desinfecção , Células HeLa , Humanos , Poliovirus/imunologia
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