Objective: To explore the structural domains of the CENP-E protein that interact with Mps1 protein.Methods: Two recombinant vectors named pEGFP-CENPE2(containing 674-1085 amino acids of CENP-E protein) and pEGFP-CENPE 3(containing 1200~2134 amino acids of CENP-E protein) were transfected into human embryo kidney 293(HEK293) cells respectively.The respective energy transfer efficiency(Ef) between either EGFP-CENPE2 and Mps1,or EGFP-CENPE3 and Mps1 were detected by FRET through selective photobleaching of the acceptors.Results: Both recombinant proteins expressed in HEK293 cells transfected by the recombinant plasmids were found to co-localize with the Mps1 protein as confirmed by confocal microscopy.The Ef between EGFP-CENPE3 and Mps1 protein was [(12.63?0.48)%,n=30] and that between EGFP-CENPE3 and Mps1 protein was [(3.17?0.21)%,n=30] as revealed by the results from FRET,the result of FRET was confirmed by co-Immunoprecipitate(CO-IP) method.When compared with that between the control and Mps1,the Ef between EGFP-CENPE3 and Mps1 was significantly higher(p

OBJECTIVETo study the kinetogenic effects of serum containing Semen Arecae, Dandelion, Semen raphani and Atractylodes macrocephala on the colonic smooth muscle cells of rats.

METHODSSerum containing Chinese materia medicas was made according to standard methods. Smooth muscle cells were isolated from the muscle layers of Wistar rat's colon, referred to modified Bitar's method. The contractile response of colonic smooth muscle cells to serum containing Chinese materia medicas (10%, 50%, 100% concentration) and other medicines (blank and 1 x 10(-3) mol/L acetylcholine) were separately observed. The contractility was presented by the decrease of the cell length between the drug groups and the control.

RESULTSSerum containing each Chinese materia medica can make dose-dependent contraction at different concentrations (P < 0.05), but the strongest effect of each serum had no significant difference (P > 0.05).

CONCLUSIONSerum containing Semen Arecae, Dandelion, Semen raphani and Atractylodes macrocephala can make notable contraction on colonic smooth muscle cells in rats.

Animals ; Cells, Cultured ; Colon ; cytology ; Drugs, Chinese Herbal ; pharmacology ; Medicine, Chinese Traditional ; Muscle Contraction ; drug effects ; Muscle, Smooth ; cytology ; Myocytes, Smooth Muscle ; drug effects ; Rats

By using a cell-based high throughput screening model for the CLA-1 up-regulator, Streptomyces 203909 was found to produce up-regulator of CLA-1. A novel trichostatin analogue was isolated from the rice fermentation of Streptomyces sp. CPCC 203909by a combination of various chromatographic techniques including column chromatography (CC) over silica gel, flash C18 CC, and reversed-phase HPLC. Its structure was identified as (-)-(R,2E,4Z)-7-[(4'-dimethylamino) phenyl]-4,6-dimethyl-7-oxohepta-2,4-dienoyl-L-glutamine (1) by the spectroscopic and chemical methods, and combination with the CD spectroscopy and Marfey's method. In the prelimi- nary assays, Compound 1 showed cytotoxicity against human embryonic kidney 293 cell line with IC50 value 35.3 [µmol · L(-1).
Cell Survival ; drug effects ; Fermentation ; Hep G2 Cells ; Humans ; Hydroxamic Acids ; chemistry ; isolation & purification ; metabolism ; pharmacology ; Molecular Structure ; Streptomyces ; chemistry ; metabolism

Objective To compare and validate the efficiency of four models predicting the malignancy of solitary pulmonary nodules (SPN). Methods Patients diagnosed with SPN during health check-up were selected as the research subjects. Risk assessment was conducted using four predictive models. Outcomes were obtained through prospective follow-up. Statistical description and univariate analysis were performed for all risk factors of the four models. ROC curve was applied to compare the efficiency of the four predictive models. Results A total of 479 cases were included in this study. Among these patients, 82 were diagnosed with lung tumor, and the malignant rate was 17.12%. Age, nodule diameter, smoking, family history of tumor, history of extrapulmonary tumor ≥5 years, upper lobe site, unclear boundary, and spiculation rates were higher in the malignancy group than those in the benign group (P < 0.05). The efficiency of Brock model was the best. Its AUC was 0.833, sensitivity was 80.49%, and specificity was 74.31%. Its Youden index, positive likelihood ratio, positive predictive value, and negative predictive value were the highest, and its negative likelihood ratio was the lowest. The AUC, sensitivity, and specificity of Mayo model were 0.815, 81.71%, and 67.51%, respectively; those of PKUPH model were 0.754, 69.51%, and 73.55%, respectively; and those of VA model were 0.738, 68.29%, and 67.55%, respectively. Conclusion The Brock model might be the most appropriate predictive model for the risk assessment of SPN among the health check-up population, and the VA model is the worst. The combination of Brock, Mayo, and PKUPH models requires further study.

·AIM: To evaluate the effects of two series of enantiomers [(R, R)-XY-1 through (R, R)-XY-12 and (S,S)-XY-1 through (S, S)-XY-12] on ocular blood flow in rabbits.·METHODS; Colored microsphere technique was used for in vivo experiments to determine the ocular blood flow in various tissues of ocular hypertensive (40mmHg) rabbit eyes.·RESULTS; Of the twelve compounds of ( R, R)-XY series examined, four increased choroidal blood flow at 10g/L, 50uL instilled into eyes. All compounds of (S, S)-XY series were not effective on ocular blood flow.·CONCLUSION; Some compounds of (R, R)-XY series increased the ocular blood flow, which might be useful for the prevention and treatment of ocular blood flow related eye diseases. Among all twenty-four compounds, (R, R)-XY-1and (R, R)-XY-9 seem to be the most potent ones.KEYWORDS; ocular blood flow; ischemia; enantiomer

AIM: The effects of ZX-5, as nitric oxide (NO) donor, on ocular blood flow has been investigated using colored microsphere technique in previous study. The relationship between the production of NO by ZX-5 and ocular blood flow has been evaluated. ZX-5 has been shown to have strong positive effect on increasing choroidal blood flow. However,the effect of ZX-5 on retinal function recovery, the effects of its optical isomers, (R, R)-ZX-5 and (S, S)-ZX-5, on choroidal blood flow and retinal function recovery have not been studied and merit investigation.METHODS: Colored microsphere technique was used for in vivo experiments to determine choroidal blood flow of ocular hypertension (40mmHg) in rabbit eyes. Electroretinography was used to measure the b-wave recovery as an indication of retinal function recovery.RESULTS: (R, R)-ZX-5 increased choroidal blood flow at 10g/L, 50μL instillation into eyes at all time points (P＜0.05).(S, S)-ZX-5 was not effective in increasing choroidal blood flow. ZX-5 and (R, R)-ZX-5 showed significant effects in retinal function recovery after ischemia of the retina at all time points (P＜0.05); whereas (S, S)-ZX-5 did not show significant effect on recovery of b-wave after ischemia at most time points except at 120 and 240 minutes.CONCLUSION: ZX-5 and (R, R)-ZX-5 have high potency in increasing the choroidal blood flow and improving the retinal function recovery. It is hoped that they could be used for the prevention/treatment of ocular blood flow related eye diseases.

Objective To investigate the effect of 131I on apoptosis of thyrocytes in patients with Graves disease. Methods Forty-seven patients with Graves disease were divided into two groups, two week group (G2w) and four week group (G4w). All patients underwent thyoid needle biopsy before 131I treatment and the repeated biopsy at two weeks (G2w) or four weeks (G4w) after 131I treatment. The positive units of pro-apoptotic proteins (Fas, FasL) and anti-apoptotic protein (Bcl-2) were studied with immunohistochemistry staining. The differences of the two groups were compared with t-test. Liner correlation analysis was applied to study the correlation between 131I dose and apoptosis-related proteins and that between serum sTSH after 131I treatment and apoptosis-related proteins. Results Fas, FasL and Bcl-2 expression (positive units) were significantly increased in both groups after 131I treatment, G2w :22.84 ± 9.31 vs 16.20 ± 6.75,21.13±6.29vs 14.56±4.06, 21.69±7.83 vs 15.22 ±5.94, t= -3.08, -3.73, -4.05 (allP<0.05); G4w:21.69 ±4.52 vs 15.83 ±5.03, 19. 11 ±3.75 vs 14.02 ±4.98, 19.06 ±3.44 vs 16.63 ±4. 73, t = - 5.26, - 5.00, - 2.41 (all P<0.05). However, no statistical differences were found between G2w and G4w (t = 0. 53, 0. 82, 1.46, all P > 0.05). Significant correlation was found between 131I 0. 727, rFasL = 0. 763 (both P<0.05)), but not between the dose and Bcl-2, rBcl-2 = - 0. 094, 0. 102(both P > 0.05). There were significant correlation between serum sTSH three months after 131I treatment and apoptosis-related proteins, rFas = 0.433, rFasL = 0. 601, rBcln2 = - 0. 397, (all P<0. 05). Conclusions 131I can induce thyrocytes to express the pro-apoptotic proteins in patients with Graves disease.

Objective To investigate the long-term follow-up results of patients treated with radical nephrectomy for small renal carcinoma.Methods Between January 1992 and June 2004,a total of 56 pa- tients(41 men and 15 women;mean age,54 years;age range,19-71 years)underwent radical nephrectomy for small renal carcinoma.The clinical data and long-term follow-up results of the 56 cases were analyzed ret- rospectively.All the patients were followed by questionnaire;and the 5-and 10-year survival rates were calcu- lated by Kaplan-Meier method.Results None of the patients was found to have metastasis preoperatively. Postoperative pathology showed renal clear cell carcinoma in 44 cases,granular cell carcinoma in 7,and mixed cell carcinoma in 5.Among these cases,ipsilateral adrenal metastasis was found in 1 case,local lymph- aden metastasis in 2,and perirenal fat metastasis in 4.Postoperatively,49 of the 56 patients(87.5%)were followed for 11-155 months with a mean of 71 months.The 5-and 10-year survival rates were 81.7% and 66.9%,respectively.Five patients died of metastasis from renal carcinoma.Conclusions Radical ne- phrectomy for small renal carcinoma has favorable long-term effects,therefore it is an optimal surgical proce- dure for small renal carcinoma.

OBJECTIVETo establish fluorescence resonance energy transfer (FRET) assay method of detecting proteolytic activity of non-structural protein 3-4A (NS3-4A) serine protease of hepatitis C virus (HCV) for high throughput screening inhibitors against HCV in vitro.

METHODSHCV recombinant plasmid pMAL~c2/NS3-4A was transformed into the E.coli strain K12TB1. Maltose-binding-protein (MBP) NS3-4A fusion protein expression was induced by adding isopropyl-β-D-thiogalacto-pyranoside (IPTG) and purified by affinity chromatography. The proteolytic activity of MBP-NS3-4A protease was analyzed by FRET with the special protease substrate. The reaction system in this model was optimized, and the reliability of the model was evaluated.

RESULTSHigh throughput screening model for HCV NS3-4A protease inhibitors was established, and the best concentrations of enzyme and substrate were optimized. In the model, the Km value of protease was 4.74 μmol/L, Z factor was up to 0.80, and coefficient of variation (CV) was 1.91%. BILN 2061, one of the known HCV protease inhibitors, was measured with the Ki of 0.30 nmol/L.

CONCLUSIONThe assay model using FRET method for HCV NS3 4A serine protease is stable and reliable, and the model is suitable for high throughput screening for HCV NS3 4A protease inhibitors.

Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; Fluorescence Resonance Energy Transfer ; Hepacivirus ; enzymology ; High-Throughput Screening Assays ; methods ; Protease Inhibitors ; pharmacology ; Viral Nonstructural Proteins ; antagonists & inhibitors ; genetics

Heart failure (HF) is the end stage of various kinds of cardiovascular diseases and leads to a high mortality worldwide.Numerous studies have demonstrated that frequencies of CD4+CD25+Foxp3+ regulatory T cells (Tregs) are reduced in HF patients and properly expanding Tregs attenuates HF progression.Histone deacetylase (HDAC) 9 has been revealed to contribute to several cardiovascular and cerebrovascular diseases.Plenty of studies showed that HDAC9 negatively regulated the number and function of Tregs.Thus,we aim to investigate the expression of HDAC 9 in patients with chronic heart failure (CHF) and the relationship among HDAC9,Tregs and CHF.Our research showed a reduced number of Tregs and an increased expression of HDAC9 mRNA in CHF patients.Patients with CHF were divided into two groups by heart function grade of New York Heart Association (NYHA),we found that the HDAC9 mRNA expression level in NYHA grade Ⅱ-Ⅲ group were lower than that in NYHA grade Ⅳ group.More importantly,the correlation study suggested that the expression of HDAC9 mRNA was negatively correlated to Tregs frequency and left ventricular ejection fraction (LVEF),whereas positively correlated to larger left ventricular end-diastolic dimension (LVEDD) and B-type natriuretic peptide (BNP) in patients with CHF.The correlation studies also showed a positive correlation between HDAC9 and the severity of CHF.Our research suggests that HDAC9 may be a new indicator for assessing CHF and it may offer a new direction for research of CHF.