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To explore the molecular mechanism of human papillomavirus subtype 16(HPV-16)E7 oncogene-induced DNA re-replication in response to DNA damage. Flow cytometry was performed to examine the cell cycle changes in RPE1 E7 cells stably expressing HPV-16 E7 and its control cell RPE1 Vector after DNA damage.Immunoblotting assay was used to evaluate the early mitotic inhibitor 1(Emi1)expression in RPE1 E7 and RPE1 Vector cells with or without DNA damage.The changes of the proportion of polyploidy was detected by flow cytometry in DNA-damaged RPE1 E7 cells interfered by Emi1 small interfering RNA. Compared with the control cells,the proportion of polyploids in RPE1 E7 cells was significantly increased in response to DNA damage(=6.397,=0.0031).Emi1 protein expression was significantly increased in DNA damaged RPE1 E7 cells(=8.241,=0.0012).The polyploid ratio of RPE1 E7 cells was significantly reduced after Emi1 was interfered by two independent small interfering RNAs(=2.916,=0.0434;=3.452,=0.0260). In response to DNA damage,Emi1 promoted DNA re-replication caused by HPV-16 E7.
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Dano ao DNA , Replicação do DNA , Papillomavirus Humano 16 , Mitose , Proteínas Oncogênicas ViraisRESUMO
Transient receptor potential vanilloid member 3 (TRPV3) is a temperature-sensitive cation channel protein, which contributes to nociception, itch, hair growth, emotional control and the pathophysiology of migraine. However, research progress on TRPV3 fundamental molecular biology is rather slow, compared to other TRP channels due to the lack of its selective antagonists. It's necessary to identify TRPV3 selective antagonists for the study on TRPV3 physiological functions. In this study, several selective TRPV3 antagonists were identified by ligand-based virtual screening of shape-based similarity and electrostatic matching. The most potent one (V-39) blocked 2-APB-activated currents in a stable human TRPV3 expressed HEK293T cell line with IC50=18.0 ±1.1 μmol·L-1 (n=4). Besides, the interaction pattern between TRPV3 and its antagonists were studied through docking the antagonists into a homology model (TRPV3_HM4) generated from the crystal structure of TPRV1. The docking results show that the binding site of TRPV3 locates between linker domain (of N-terminus and TM1) and TRP Box. There are a π-π stacking interaction and hydrogen bonding interactions between compound V-39 and residues His-310, His-314 and Arg-577 of the pocket. Identification of these antagonists provides new probes for understanding the pharmacological function of TRPV3 channel.
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<p><b>OBJECTIVE</b>To evaluate anti-melanoma effect of ethanol extract of Ilex hainanensis Merr. (IME) and elucidate its underlying mechanism.</p><p><b>METHODS</b>Thirty-six tumor-bearing mice were randomized into 6 groups (n=6) as follows: model group, IME 25, 50, 100, and 200 mg/kg groups and dacarbazine (DTIC) 70 mg/kg group. The mice in the IME treatment groups were intragastrically administered with IME 25, 50, 100 or 200 mg/kg per day, respectively. The mice in the DTIC group were intraperitoneally injected with DTIC 70 mg/kg every 2 days. The drug administration was lasting for 14 days. The cell viability was evaluated by 3-(4,5-dime-thylthylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect cell cycle and apoptosis. The gene and protein expressions of nuclear factor κB-p65 (NF-κB-p65), Bcl-2, B-cell lymphomaextra large (Bcl-xL) and Bax were detected by quantitative real-time polymerase chain reaction and Western blot analyses. Caspases-3, -8, and -9 activities were detected using the colorimetric method. In addition, a B16-F10 melanoma xenograft mouse model was used to evaluate the anti-cancer activity of IME in vivo. Furthermore, a survival experiment of tumor-bearing mice was also performed to evaluate the possible toxicity of IME.</p><p><b>RESULTS</b>IME significantly inhibited the proliferation of B16-F10 cells (P<0.01). Flow cytometric analysis showed that IME induced G/S cell cycle arrest and apoptosis (both P<0.01). IME inhibited activation of NF-κB, decreased the gene and protein expressions of Bcl-2, Bcl-xL, and increased the gene and protein expressions of Bax (all P<0.01). In addition, IME induced the activation of Caspases-3, -8, and -9 in B16-F10 cells. The study in vivo showed that IME significantly reduced tumor volume (P<0.01), and the inhibitory rate came up to 68.62%. IME also induced large areas of necrosis and intra-tumoral apoptosis that correlated with a reduction in tumor volume. Survival experiment showed that treatment with IME for 14 days significantly prolonged survival time and 20% of mice in the IME 200 mg/kg group were still alive until the 50th day. Notably, IME showed no apparent side-effects during the treatment period.</p><p><b>CONCLUSION</b>IME exhibited significant anti-melanoma activity in vitro and in vivo, suggesting that IME might be a promising effective candidate with lower toxic for malignant melanoma therapy.</p>
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<p><b>OBJECTIVE</b>To study the expression of gene associated with retinoid-interferon-induced mortality-19(GRIM-19) in preimplantation embryo of mice and explore its role in embryonic development.</p><p><b>METHODS</b>The protein and mRNA expressions of GRIM-19 in 2-cell, 4-cell, 8-cell, morula, and blastocyst phases of mice preimplantation embryo were detected by Western blot analysis and Real-time polymerase chain reaction (PCR).</p><p><b>RESULTS</b>GRIM-19 was continuously expressed in every stage of preimplantation embryo of mice. Western blot analysis and Real-time PCR demonstrated a gradual increase of GRIM-19 expression from 2-cell, which reached a peak in 8-cell phase and then decreased progressively.</p><p><b>CONCLUSIONS</b>The expression of GRIM-19 in mouse preimplantation embryos changes as at different developmental phases. GRIM-19 may play an important role during embryonic development.</p>
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Animais , Feminino , Camundongos , Gravidez , Blastocisto , Metabolismo , Interferons , Farmacologia , NADH NADPH Oxirredutases , Genética , Metabolismo , RNA Mensageiro , Genética , Tretinoína , FarmacologiaRESUMO
OBJECTIVE To evaluate the use of antibiotics in general hospitals of Ningbo. METHODS Totally 4 391 case history records in April,2004 of 12 hospitals were investigated on the use of antibiotics. RESULTS Medicines incomes accounted for 55.16% in total revenue in a hospital,and antibiotics accounted for 32.66% among medicines incomes.Antibiotics using rate was 62.11% in internal medicine departments.The percentage of antibiotics using without evidence was 23.92%,the combined antibiotics using rate was 49.89%.The average duration for antibiotics using was 10.83 days.Antibiotics using rate in surgery departments before operation was 71.00%,during operation was 20.48%,the combined antibiotics using rate during operation was 14.50%,antibiotics using rate after operation was 96.55%.Antibiotics using for treatment accounted for 62.37% and for prevention was 24.17%. CONCLUSIONS Antibiotics using rate is high in hospitals in Ningbo.Income of medicine is also an important part of total revenue in a hospital.We should pay more attention to management on clinical use of antibiotics.
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OBJECTIVE To understand the current situation of infection management in outpatient stomatology department of our city,and to further strengthen the hospital infection standardization management. METHODS The hospital infection management was investigated in outpatient stomatology department of 18 medical institutions. RESULTS The qualified rate of the city level hospital infection management was 75-100%,and that of the county area level was 40-80%.The disinfection and isolation were not standardized,the standardized prevention consciousness among medical staff was weak,and the hand cleaning and disinfection was insufficient. CONCLUSIONS The stomatology department has a hidden danger to emergence of hospital infection,its infection management should be further strengthened.