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1.
Toxicol Lett ; 318: 1-11, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31618665

RESUMO

Triptolide (TP), a principal bioactive component extracted from traditional Chinese medicine Tripterygium wilfordii Hook. F. (TWHF), has attracted wide attention of its therapeutic effects on inflammation and autoimmune diseases. However, the therapeutic application of TP is hindered by severe cardiomyocyte toxicity and narrow therapeutic window. We previously identified that the p53 was an indispensable contributor in TP-induced myocardial injury. p53 has an inhibitory effect on IKKß-NF-κB pathway that regulates glucose transporters (GLUT) expression. Based on these evidences, we speculate that p53 mediates TP-disturbed glucose uptake by blocking IKKß-NF-κB signaling. This study focused on the effect of TP on cardiac glucose uptake and the role of p53 in glucose metabolism in cardiomyocytes, and p53 -/- mice. TP treatment depressed glucose consumption and ATP production resulting in myocardial damage. Incubation with ATP (5 mM) remarkably decreased the cellular damage. Immunoblotting and immunofluorescence identified that TP suppressed glucose uptake by restricting IKKß-NF-κB signaling activation, GLUT1 and GLUT4 expression. p53 inhibition alleviated the cell damage and the compromise of glucose uptake. Mechanistically, p53 antagonist PFTα abolished TP-induced the inhibition of IKKß, IκBα phosphorylation, p65 nuclear translocation, and GLUT1, GLUT4 expression. Consistently, in acute heart injury models, p53 deficiency upregulated IKKß-NF-κB activation and GLUT1, GLUT4 protein levels which was also indicated as amelioration of heart histological injury after 1.2 mg kg-1 TP administration. The present findings indicate that TP-induced p53 overactivation suppresses glucose uptake by inhibiting IKKß-NF-κB pathway and downregulating NF-κB-dependent GLUT1 and GLUT4 expression.


Assuntos
Diterpenos/toxicidade , Glucose/metabolismo , Cardiopatias/induzido quimicamente , Quinase I-kappa B/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , NF-kappa B/metabolismo , Fenantrenos/toxicidade , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cardiotoxicidade , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Compostos de Epóxi/toxicidade , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Cardiopatias/genética , Cardiopatias/metabolismo , Cardiopatias/patologia , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
2.
Mol Biotechnol ; 61(12): 938-944, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31641996

RESUMO

The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM. We hypothesize that non-specific binding of primers at room temperature is prevented by binding of the primed template by EcoExoIIIM, which is then released from the DNA by heat denaturation before the first PCR cycle. Through this mechanism, PCR would be enhanced by reducing off-target extension at room temperature.

3.
Microbiologyopen ; 8(9): e00848, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31069963

RESUMO

The eukaryotic GINS, Cdc45, and minichromosome maintenance proteins form an essential complex that moves with the DNA replication fork. The GINS protein complex has also been reported to associate with DNA polymerase. In archaea, the third domain of life, DNA polymerase D (PolD) is essential for DNA replication, and the genes encoding PolDs exist only in the genomes of archaea. The archaeal GAN (GINS-associated nuclease) is believed to be a homolog of the eukaryotic Cdc45. In this study, we found that the Thermococcus sp. 4557 DP1 (small subunit of PolD) interacted with GINS15 in vitro, and the 3'-5' exonuclease activity of DP1 was inhibited by GINS15. We also demonstrated that the GAN, GINS15, and DP1 proteins interact to form a complex adapting a GAN-GINS15-DP1 order. The results of this study imply that the complex constitutes a core of the DNA replisome in archaea.

4.
Acta Pharmacol Sin ; 39(8): 1294-1304, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29323338

RESUMO

Ulinastatin (UTI) is a broad-spectrum serine protease inhibitor isolated and purified from human urine with strong anti-inflammatory and cytoprotective actions, which is widely used for the treatment of various diseases, such as pancreatitis and sepsis. Although the therapeutic effects of UTI are reported to be associated with a variety of mechanisms, the signaling pathways mediating the anti-inflammatory action of UTI remain to be elucidated. In the present study we carried out a systematic study on the anti-inflammatory and anti-oxidative mechanisms of UTI and their relationships in LPS-treated RAW264.7 cells. Pretreatment with UTI (1000 and 5000 U/mL) dose-dependently decreased the mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, iNOS) and upregulated anti-inflammatory cytokines (IL-10 and TGF-ß1) in LPS-treated RAW264.7 cells. UTI pretreatment significantly inhibited the nuclear translocation of NF-κB by preventing the degradation of IκB-α. UTI pretreatment only markedly inhibited the phosphorylation of JNK at Thr183, but it did not affect the phosphorylation of JNK at Tyr185, ERK-1/2 and p38 MAPK; JNK was found to function upstream of the IκB-α/NF-κB signaling pathway. Furthermore, UTI pretreatment significantly suppressed LPS-induced ROS production by activating PI3K/Akt pathways and the nuclear translocation of Nrf2 via promotion of p62-associated Keap1 degradation. However, JNK was not involved in mediating the anti-oxidative stress effects of UTI. In summary, this study shows that UTI exerts both anti-inflammatory and anti-oxidative effects by targeting the JNK/NF-κB and PI3K/Akt/Nrf2 pathways.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Glicoproteínas/farmacologia , Inflamação/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Serino Proteinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Citocinas/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Fator de Transcrição RelA/metabolismo
5.
Zhonghua Yu Fang Yi Xue Za Zhi ; 44(2): 128-33, 2010 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-20388333

RESUMO

OBJECTIVE: To find out the perceived stress in general public during prevalence of severe acute respiratory syndrome (SARS) and its impact on health behavior. METHODS: A retrospective survey was conducted in Guangzhou, Hangzhou, and Taiyuan according to the epidemic situations of SARS, and 2532 subjects were randomly selected from constructive industry, school, and commercial business and residents in urban and rural areas. The perceive stress was measured by Chinese perceived stress scale (CPSS), and health related behavior during SARS was tested by uniform and self-made questionnaire. EpiData 2.0 was used for data management and CPSS value was calculated according to answer to 14 questions contained in the scale. Health risk stress among different population group and health related behavior among low, medium and high stress state were analyzed by SPSS 11.5. RESULTS: 2424 subjects were involved in the survey. The CPSS value was measured from 0 - 49 (22.7 +/- 6.8), M = 24.0. 39.3% (953/2379) subjects were under the health risk stress. The health related behaviors such as washing hands, opening the window for air, keeping away from others when cough and sneeze, doing exercises etc were reduced with the stress increased. Logistic regression indicated that compared with the persons with the thoughts of nothing serious of SARS, without any dread of SARS, and knowing nothing about prevention of SARS, the perceived stress was significantly related with perceiving of the thread to certain extent (beta = 0.41, Wald chi(2) = 4.84, P = 0.03), worrying little about the epidemic (beta = 0.50, Wald chi(2) = 6.69, P = 0.01), worrying about it to certain extent (beta = 1.39, Wald chi(2) = 48.59, P = 0.00) and scared so much (beta = 1.77, Wald chi(2) = 53.59, P = 0.00), and knowing little about the prevention (beta = 0.74, Wald chi(2) = 4.48, P = 0.03), knowing something about prevention (beta = -0.98, Wald chi(2) = 8.29, P = 0.00) and knowing the prevention very well (beta = -1.18, Wald chi(2) = 10.66, P = 0.00). CONCLUSION: The adoption of health related behaviors declined with increase of perceived stress. Opening connection to authority and government, enhancing the awareness of outburst affairs among general public and providing positive social support may be effective ways to reduce the population perceived stress.


Assuntos
Comportamentos Relacionados com a Saúde , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/psicologia , Percepção Social , Inquéritos e Questionários , Adolescente , Adulto , Idoso , Cultura , Feminino , Humanos , Pessoa de Meia-Idade , Ocupações , Estudos Retrospectivos , População Rural , Síndrome Respiratória Aguda Grave/prevenção & controle , População Urbana , Adulto Jovem
6.
Biochem Biophys Res Commun ; 376(2): 369-74, 2008 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-18782564

RESUMO

The relationships among three PCNA (proliferating cell nuclear antigen) subunits in the hyperthermophilic archaeon Sulfolobus tokodaii (StoPCNAs) were analyzed and the effects of two PCNA complexes on the activities of the DNA helicase Hjm, DNA Ligase I, and Holliday junction specific endonuclease Hjc were tested. There was no strong self-interaction of each StoPCNA. StoPCNA1 and StoPCNA3 interacted with each other, so did StoPCNA2 and StoPCNA3, but no interaction between StoPCNA1 and StoPCNA2 was observed. Two trimeric complexes (designed StoPCNA123 and StoPCNA323) were formed in vitro and it was determined that StoPCNA323 was composed of one StoPCNA2 and two StoPCNA3 subunits, with StoPCNA2 bridging the two StoPCNA3 subunits. Both complexes inhibited the unwinding activity of Hjm and the ligation activity of DNA Ligase I. In contrast, both stimulated the Holliday junction cleavage activity of Hjc. Our results provide further evidence that in crenarchaea, the PCNAs exhibit diversity in subunit interaction and complex formation.


Assuntos
Proteínas Arqueais/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sulfolobus/metabolismo , Proteínas Arqueais/genética , Sequência de Bases , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/antagonistas & inibidores , DNA Ligases/metabolismo , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Técnicas do Sistema de Duplo-Híbrido
7.
J Bacteriol ; 190(8): 3006-17, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18296528

RESUMO

Hjm and Hel308a are novel, RecQ-like DNA helicases recently identified in the euryarchaeotes Pyrococcus furiosus and Methanothermobacter thermautotrophicus, respectively. In this study, an Hjm/Hel308 homologue (designated StoHjm) from Sulfolobus tokodaii, a hyperthermophilic archaeon belonging to the Crenarchaeota subdomain of archaea, was cloned, purified, and characterized. Unlike Hjm and Hel308a, which unwind DNA in a 3'-to-5' direction, StoHjm unwound DNA in both 3'-to-5' and 5'-to-3' directions. Remarkably, StoHjm exhibited structure-specific single-stranded-DNA-annealing and fork regression activities in vitro. In addition, gel filtration, affinity pulldown, and yeast two-hybrid analyses revealed that StoHjm physically interacted with StoHjc, the Holliday junction-specific endonuclease from S. tokodaii. This interaction may have functional significance, because the unwinding activity of StoHjm was inhibited by StoHjc in vitro. These results may suggest that the Hjm/Hel308 family helicases, in association with Hjc endonucleases, are involved in processing of stalled replication forks.


Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , DNA Arqueal/metabolismo , Endonucleases/metabolismo , Sulfolobus/enzimologia , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Cromatografia em Gel , Clonagem Molecular , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA de Cadeia Simples/metabolismo , Eletroforese em Gel de Poliacrilamida , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
8.
Zhonghua Yu Fang Yi Xue Za Zhi ; 41(4): 254-7, 2007 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-17959042

RESUMO

OBJECTIVE: To describe the changes of health related behavior and influencing factor during and after the prevalence of severe acute respiratory syndrome (SARS), as to providing evidence for inducing public health related behavior to cope with the emergent infectious public health hazards. METHODS: According to the epidemic strength of SARS, Guangzhou, Taiyuan, Hangzhou were selected to be the research areas, and convenience sampling was used to identify 200 city residents, 200 country residents and 400 occupational populations from each area. The uniform and self-made questionnaire was carried out by about 2400 residents. RESULTS: All 2424 subjects completed the questionnaire. The correlation coefficient of scores of health related behavior during and after SARS was 0.782 (P < 0.01). And 39.3% subjects were considered as under the health risked stress by CPSS. Multiple linear regression indicated that the scores of health related behavior were significantly related to the perceived stress, demographic characteristics and recognition of SARS. CONCLUSIONS: The public health related behavior should be developing in the SARS, and part of health related behavior had been kept after SARS and part been decreased. Behavior change should depends on the prevalence of the disease, cultural heritage and behavior variability. More attention should be paid to induce the public psychology and behavior and enhance the degree of recognition on related knowledge through health education as to relieving the stress in populations when we conduct to cope with the emergent public health hazards.


Assuntos
Comportamentos Relacionados com a Saúde , Conhecimentos, Atitudes e Prática em Saúde , Síndrome Respiratória Aguda Grave/epidemiologia , Adulto , Feminino , Educação em Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Risco , População Rural , Amostragem , Inquéritos e Questionários , População Urbana
9.
Hepatobiliary Pancreat Dis Int ; 3(4): 612-5, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15567757

RESUMO

BACKGROUND: The incidence of carcinoma of the pancreas is increasing in the world. Pancreatic carcinoma is characterized by early local extension to contiguous structures and metastases to regional lymph nodes and the liver. This study was conducted to increase the rate of pancreatoduodenectomy combined with vascular reconstruction. METHODS: Pancreatoduodenectomy with vascular reconstruction was performed for 79 patients at a number of hospitals in Fujian Province, Zhejiang Province, Shanghai and Xinjiang Uyghur Autonomous Region from April 1994 to December 2003. One of these patients also underwent right hemicolectomy; but all received through superior mesenteric vein (SMV)-portal vein (PV) reconstruction. The reconstructions of the superior mesenteric artery (SMA) and hepatic artery (HA) were performed in 4 patients, and reconstructions of the SMA or HA were carried out in 7 and 4 patients respectively. Partial reconstruction of the inferior vena cava (ICV) was done in 2 patients when the tumor was adhering to the wall of the inferior caval vein. RESULTS: Four patients died during the peri-operative period, with a mortality rate of 5%. No complications such as biliary or pancreatic fistulae or artificial blood vessel infection were noted. Histological examination showed one patient with neuroendocrine cancer and the other 78 patients with adenocarcinoma of the pancreatic head. Resected endothelia and vascular margins proved to be microscopically tumor-free. Follow-up for 3 months to 10 years for all except two patients showed 7 of the 9 patients who had undergone resection and reconstruction of the SMA and HA died 7 months or 4 years after operation and 37 survived for over 3 years and 12 for more than 5 years. The rest are still under follow-up. CONCLUSION: Pancreatoduodenectomy with vascular reconstruction for carefully selected patients with carcinoma of the pancreatic head has proved to be a safe and reliable treatment, capable of raising the rates of tumor resection and survival.


Assuntos
Adenocarcinoma/cirurgia , Carcinoma Neuroendócrino/cirurgia , Neoplasias Pancreáticas/cirurgia , Pancreaticoduodenectomia , Procedimentos Cirúrgicos Vasculares , Adenocarcinoma/mortalidade , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/mortalidade , Análise de Sobrevida
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