The structural protein VP6 of rotavirus form the middle layer of the triple-layered viral capsid, playing a key role in the organization of the virion. The gene of structural protein 6 of rotavirus strain TB-Chen isolated from a clinic sample was amplified using PCR from the reverse transcription product of RV genome RNA, using pET as expression vector, a recombinant plasmid pET-VP6 containing coding sequence of VP6 protein was constructed. The results showed that the VP6 was highly efficiently expressed in E. coli BL21(DE3) cells which were transformed with the recombinant plasmid pET-VP6.The expressed VP6 protein possessed 27.4% of total cells protein, with an approximately 45kDa of molecular weight, and could be recognized by guinea pig anti-SA11 antibody on Western blot. The results obtained provide important basis for further study on structure and function of the VP6 protein.

AIM: To study the effects of Radix Saposhnikoviae(RL) and Feutus Trbuli(FI) used singly and combinedly on antagonizing type Ⅰ allergy,such as passive cutaneous anaphylaxis(PCA),released model from mast cell granules,Schulz-Dale respond. METHODS: Mouse and rat models mainly induced by IgE were used in experiments. RESULTS: RL and FI used singly and combinedly could significantly inhibit PCA of same species of mice,the degranulation of mast cells of skull in rats and Schulz-Dale response. CONCLUSION: Both RL and FI respectively have antagonistic effect on type Ⅰ allergy and RL in combination with FI will possess more satisactory effect

Objective To identify the different proteins of pancreatic cancer with and without diabetes mellitus (DM) ,so as to provide some clues for understanding the molecular mechanisms of DM in the development .of pancreatic cancer. Methods Samples were divided into pancreatic cancer with or without DM and their normal adjacent tissue with 5 each. The protein expression profiles were analyzed using 2-DE in combination with MALDI-TOF/TOF MS/MS. Western blot was used to verify the expression of a candidate protein CapG. Results Seven proteins were obviously up-regulated in pancreatic cancer with DM(P＜0.05). These proteins were involved in cell motility, metabolism, oxidative stress, etc. Additionally, CapG implicated a functional contribution of cancer invasion and metastasis. Conclusion The results also provide evidence to support the hypothesis that DM plays an important role in the development of pancreatic cancer.

Objective: To establish the optimum preparation process for Xuesaitong Dropping Pills(total saponin of Radix Notoginseng). Methods: On the basis of orthogonal design, preparation conditions were selected among sort of polyglycol (as substance), consumption of polyglycol and processing temperature of usoline (as cooling agent). Results: Under the condition of using polyglycol 6000, and processing temperature at 10～15 ?C , the quality of Xuesaitong Dropping Pills is good.

The aim of this study was to investigate the roles of chemokine CCL20 in development of CD4(+)CD25(+) thymocytes by means of fetal thymus organ culture. Fetal mouse thymus lobes were removed at the fetus age of 14.5 days and cultured in complete RPMI 1640 with 20% FBS in vitro. Phenotypes of the thymocytes were analyzed by FACS and the number of cells per lobe was counted. The results revealed that from day 14.5 to day 19, the absolute and relative numbers of the CD4(+)CD25(+) thymocytes varied similarly as their development as in vitro culture at 6 days. Data showed that during the 6 days in vitro culture the CD4(+)CD25(+) cell percentage out of CD4(+) cells was 58.29%, 12.14%, 6.08%, 17.78%, 9.06%, 4.04% and the CD4(+)CD25(+) cell percentage out of CD25(+) cells was 3.75%, 10.81%, 17.20%, 51.93%, 61.64%, 80.06%. All these data indicated similar characters to their development in vivo. Moreover, at interference with CCL20, the percentage of CD4(+)CD25(+) T cells in thymocytes significantly decreased at the 3 and 6 days from 3.24+/-0.18 and 3.96+/-0.24 to 1.27+/-0.11 (p<0.001) and 1.76+/-0.22 (p<0.001) respectively. It is concluded that the development of CD4(+)CD25(+) thymocytes is similar both in vitro and in vivo, interfering with CCL20 significantly downregulate the expression of CD4(+)CD25(+) T cells. The above data may help to understand the development of naturally arising CD4(+)CD25(+) regulatory T cells.
Animals ; Chemokine CCL20 ; pharmacology ; Embryo, Mammalian ; Embryonic Development ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Organ Culture Techniques ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Thymus Gland ; cytology ; embryology

Objective:To assess the prognostic value of methylation of interferon regulatory factor 6 ( IRF6) gene promoter in patients diagnosed with Kidney renal clear cell carcinoma (KIRC). Methods:The primary lesions of fifty KIRC patients who were diagnosed at the First Affiliated Hospital of Nanjing Medical University from January 2016 to January 2020 were collected. The expression of IRF6 protein was determined with an immunohistochemical method. The correlation between the level of IRF6 expression and survival and/or metastasis status was analyzed. The mRNA and protein levels of the IRF6 in KIRC and normal renal tissues were compared by using bioinformatic tools. The difference in the methylation rate of the IRF6 gene promoter between tumor and adjacent tissues was analyzed by searching the online databases. Statistical analysis was carried out for the methylation status of the IRF6 gene promoter region to select those negatively correlated with the overall survival (OS) among the patients. In vitro experiments were conducted with cell lines to verify the correlation between the status of promoter methylation and transcription level of the IRF6 gene. Results:The mRNA and protein levels of the IRF6 gene in KIRC tissues were significantly lower than those of the normal controls, and this was more prominent in patients who had died or developed metastasis. The extent of IRF6 gene promoter methylation in the KIRC tissues was much higher compared with that of the adjacent normal renal tissues. There was a significant negative correlation between the methylation of the IRF6 gene promoter and mRNA level of the IRF6 ( R= -0.52). The higher methylation degree in the IRF6 gene promoter regions cg12034118 and cg16030177, the shorter the OS and worse prognosis in the patients. Only twenty CpG sites in cg12034118 were confirmed to be highly methylated in KIRC cell lines. The transcription level of the IRF6 gene was upregulated in a time- and dose-dependent manner after the treatment with demethylation reagent 5-azadeoxycytidine. Conclusion:The methylation of IRF6 gene promoter in the renal tissues of KIRC patients is closely correlated with the OS. Cg12034118 may provide a promising biomarker for laboratory detection, and its high methylation rate has certain reference value for the prognosis.

This study was to investigate the effect of phenylhexyl isothiocyanate (PHI) on drug resistance and sensitivity on K562/A02 cell line to adriamycin (ADM) and to elucidate the possible mechanisms. The inhibition rates of ADM and PHI + ADM on growth of K562/A02 cell line were measured by MTT assay, and K562/A02 cell resistant multiple was calculated. The apoptosis rate of K562/A02 cell line, the changes of intracellular ADM concentrations and MRP1 protein level were detected by flow cytometry (FCM). Intracellular reoxidized GSH level was determined by spectrometric enzyme assay and MRP1 mRNA was assayed by semiquantitative RT-PCR before and after using PHI. The results indicated that the survival rate of K562/A02 cell line decreased with the increasing concentration of PHI. Apoptosis rate increased after treatment in combination with two above drugs, the changes of drug resistance multiple and intracellular ADM level had statistical significance between K562/A02 and K562 cells (p < 0.05), when the concentration of PHI was more than 20 micromol/L. Intracellular GSH level in K562/A02 cell line reduced 5% when 1 microg/ml ADM was used alone, and it increased slightly at first, then decreased when more than 10 micromol/L PHI was used. When more than 20 micromol/L PHI was used in combination with 1 microg/ml ADM, intracellular GSH level in K562/A02 cell line decreased progressively with increasing the concentration of PHI. The expressions of MRP1 mRNA and protein had no statistical significance between K562/A02 and K562 cells (p > 0.05) after or before PHI was used. It is concluded that the cyto-toxicity of PHI to K562/A02 cell line does not associate with the depletion of the intracellular GSH. PHI not only enhances the sensitivity of K562/A02 cell line to ADM, but also partially reverses effect of K562/A02 cell resistance to ADM. ADM combined with PHI can diminish side effect and dosage of ADM.
Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; Isothiocyanates ; pharmacology ; K562 Cells

OBJECTIVETo investigate if CYP3A5 is involved in drug resistances mechanisms of acute leukemia.

METHODSBy using RT-PCR, immunohistochemistry and MTT assay, CYP3A5 mRNA and protein were detected in leukemia cell lines and acute leukemia patients, meanwhile transcriptional regulation of CYP3A5 induced by daunorubicin was observed. A pcDNA3-CYP3A5 reconstituted plasmid and its stably transfected cell line HL-60/CYP3A5 were both established.

RESULTSCYP3A5 mRNA was detected in K562 and U937 cells, whose IC(50) values of daunorubicin were 2.1-fold higher than those of NB4 and HL-60 cells. Bone marrow CYP3A5 positive blast cell percentage at the time of diagnosis in primary drug resistance group (17.2%) was significantly higher than that of continuous complete remission (CCR) group (0.4%) and secondary drug resistance group (5.4%). In their first complete remission of the early relapsed group, the positive rate had been 23.9% as compared with that of CCR group (1.3%). Daunorubicin increased CYP3A5 mRNA level in K562/A02 and activated its transcription in HL-60/ADR. HL-60/CYP3A5 cell was significantly resistant to daunorubicin and vincristine than HL-60 cells did (3.0 and 4.0 times, respectively).

CONCLUSIONCYP3A5 expressed in leukemia cells may cause in situ metabolization of many kinds of anticancer drugs, thus led to drug resistance.

Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System ; genetics ; physiology ; Daunorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Leukemia ; drug therapy ; enzymology ; RNA, Messenger ; analysis ; Tumor Cells, Cultured

OBJECTIVETo investigate the protective effects of selenium on rat hippocampal neurons against ischemia-reperfusion (IR) injury.

METHODSThirty-two rats were randomly divided into sham-operated group, IR group and selenium-treated group, and in the latter two groups, cerebral IR injury was induced by middle cerebral artery occlusion; Na2SeO3 treatment was administer in selenium-treated group. At 14 days after reperfusion, the brain tissues were harvested from the rats and hippocampal neuron injuries were observed by TUNEL and Methylene Blue staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nerve growth factor (NGF) in the hippocampal tissues were measured by ELISA.

RESULTSCompared with IR group, the rats in selenium-treated group showed no significant increase in the expression of m-NGF (P>0.05), but pro-NGF expression was significantly increased (P<0.05) in the hippocampal tissue. Na2SeO3 treatment significantly inhibited the expressions of TNF-α and IL-1β and decreased the apoptosis of hippocampal neurons following cerebral IR injury (P<0.05).

CONCLUSIONSelenium produces antiapoptotic effect to protect the hippocampal neurons following cerebral IR injury possibly not by increasing the level of m-NGF but by decreasing the expressions of the inflammatory factors.

Animals ; Apoptosis ; Brain Ischemia ; metabolism ; pathology ; Hippocampus ; cytology ; metabolism ; pathology ; Interleukin-1beta ; metabolism ; Male ; Nerve Growth Factor ; metabolism ; Neurons ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology ; Sodium Selenite ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

Objective To investigate the acute effect of local vibration stimulus with low frequency on hand control ability, flexibility, grip strength, and muscle EMG activity. Methods 10 subjects in this study held a vibrator for 20 seconds with isometric contraction at the vibration frequency of 12.5 Hz (vibration group, VIB) or with only isometric contraction (control group, CON). The same procedure measurement, including control ability, flexibility, grip strength, and muscle activity, were recorded both at pre-stimulus (pre-test) and 60-second after stimulus (post-test), respectively. Paired-t test was used to compare the values between pre-test and post-test and the changing rate between groups. Results The control ability and flexibility in pre-post test showed significant improvement after VIB intervention in VIB (P＜0.05). The changing rate of grip strength and EMG in VIB was significantly higher than those in CON (P＜0.05). Conclusions Acute local vibration stimulus with low frequency can be used to improve the hand control ability and flexibility for those who need better hand functional ability in the future.