AIMTo study the relationship of neurotoxicity of 6-hydroxydopamine (6-OHDA) and the function of glutamate transporter.

METHODSUsing in vivo microdialysis together with high performance liquid chromatography (HPLC) to detect the alteration of glutamate in the striatum and extracellular fluid of the PC12 cell. The rate of apoptosis and the activity of PC12 cells are read in a flow cytometer and a photometer for enzyme-labeled assays. The function of glutamate transporter is decided by detecting the ability of L-[3H]-glutamate uptake.

RESULTS6-OHDA was shown to induce apoptosis and decrease the activity of PC12 cells. Increased release of glutamate was also found in PC12 cells and the injured striatum of the PD rats. But glutamate uptake in PC12 cells and rat striatum synaptosomes are inhibited obviously.

CONCLUSIONThe neurotoxicity of 6-hydroxydopamine is associated with declined function of glutamate transporters, which may be one important pathogenesis mechanisms of Parkinson's disease.

Amino Acid Transport System X-AG ; drug effects ; Animals ; Apoptosis ; drug effects ; Corpus Striatum ; metabolism ; Glutamic Acid ; metabolism ; Male ; Oxidopamine ; toxicity ; PC12 Cells ; Parkinson Disease ; metabolism ; Rats ; Rats, Sprague-Dawley

ObjectiveTo investigate the value of 18F-FDG SPECT myocardial imaging in evaluating haemodynamic change,treatment outcome and prognosis for idiopathic pulmonary arterial hypertension (IPAH).MethodsAll 24 patients with IPAH underwent 18 F-FDG SPECT myocardial imaging.Right ventricle/left ventricle (RV/LV)-FDG uptake was calculated by ROI method drawing over the central areas of left and right ventricular free walls.All patients underwent right heart catheterization within 3 days after imaging studies.Mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance (PVR) were recorded.After six month pharmaceutical treatment,15 IPAH patients were re-examined with 18F-FDG SPECT myocardial imaging followed by repeated right heart catheterization within 3 days.Plasma N-terminal pro-brain naturetic peptide (NT-proBNP) and endothelin-1 ( ET-1 ) were measured in 17 patients using electrochemiluminescent immunoassay and enzyme immunoassay respectively.All patients were followed up for 12 months at least.Correlations between RV/LV-FDG uptake and mPAP and PVR were determined by simple linear regression analysis.Change of RV/LV-FDG before and after treatment was calculated using Student's t-test.Survival in groups with RV/LV FDG uptake ≥ 1.15 and RV/LV-FDG uptake ＜ 1.15 were compared using Log-rank test.ResultsSignificant correlations were found between RV/LV-FDG uptake and mPAP (r =0.562,P ＜ 0.01 ),and between RV/LV-FDG uptake and PVR ( r =0.574,P ＜ 0.01 ).There were no significant correlation between RV/LV-FDG uptake and NT-proBNP( r =0.18 1,P ＞ 0.05 ),but a significant correlation between RV/LV-FDG and ET-1 was observed (r =0.669,P ＜ 0.01 ).The RV/LV-FDG uptake in patients with positive treatment outcome ( n =6) decreased from 1.38 ± 0.52 to 0.92 ±0.26 (t =4.018,P ＜ 0.05) after 6 months treatment.In contrast,no significant change of RV/LV-FDG uptake was seen in those patients (n =9) with negative treatment outcome ( t =1.861,P ＞ 0.05 ).The mean follow-up time was (21 ±8) months.Mean survival time for the patients with RV/LV- FDG uptake ≥ 1.15was 28 months (95％ confidence interval:24-32 months),which was significantly lower than 34 months survival (95％ confidence interval:33-35 months) for the patients with RV/LV-FDG ＜ 1.15 (x2 =3.956,P ＜0.05 ).Conclusions Detection of right ventricle myocardial glucose metabolism level with 18F-FDG SPECT may be a practical method for evaluating haemodynamic change,treatment outcome and prognosis of IPAH.

OBJECTIVETo investigate the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-gamma) on the sperm acrosin activity and the rate of acrosome reaction and to probe into their mechanisms.

METHODSThirty-six nearly normal semen samples were treated with IFN-gamma and/or TNF-alpha after isolated by 75% Percoll. The sperm acrosin activity was tested by the method of BAEE/ADH Unity, the rate of acrosome reaction observed by Triple-stain technique, the NO concentration measured by HPLC and the activities of Na+ -K+ -ATPase, Ca2+ -ATPase and SOD assayed by kit method.

RESULTSBoth IFN-gamma and TNF-gamma could decrease sperm acrosin activity and acrosome reaction (P < 0.05 or P < 0.01). TNF-alpha showed stronger inhibiting effect, IFN-gamma markedly reduced the activities of Na+ -K+ -ATPase, Ca2+ -ATPase and SOD in sperm (P < 0.01), and their synergistic action was weaker. However TNF-alpha produced hardly any effect on Na+ -K+ -ATPase and Ca2+ -ATPase. The NO concentration in sperm was significantly increased by IFN-gamma and/or TNF-alpha (P < 0.01).

CONCLUSIONIFN-gamma and TNF-alpha have some inhibiting effect on sperm acrosin activity and the rate of acrosome reaction, which could be attributed to their influence on the activities of Na+ -K+ -ATPase, Ca2+ -ATPase and SOD, the NO concentration and so on.

Acrosome Reaction ; drug effects ; Adult ; Calcium-Transporting ATPases ; metabolism ; Chromatography, High Pressure Liquid ; Humans ; Interferon-gamma ; pharmacology ; Male ; Middle Aged ; Nitric Oxide ; metabolism ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Spermatozoa ; drug effects ; enzymology ; metabolism ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.

METHODSTwo specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.

RESULTSAfter reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.

CONCLUSIONSCleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.

5' Untranslated Regions ; metabolism ; Base Sequence ; DNA, Catalytic ; genetics ; metabolism ; Hepacivirus ; enzymology ; genetics ; Humans ; Molecular Sequence Data ; RNA Processing, Post-Transcriptional ; RNA, Catalytic ; metabolism ; RNA, Viral ; metabolism ; mRNA Cleavage and Polyadenylation Factors ; genetics ; metabolism

Objective　To study the probability of using hepatitis D virus (HDV) ribozyme as a kind of anti-hepatitis-C-virus (HCV) gene thera-py drugs. Methods　The natural HDV genomic ribozyme′s stem Ⅳ was optimized and its substrate-binding region reconstructed, thus three recombinant HCV-specific HDV genomic ribozymes RzC1, RzC2 and RzC3 were obtained. HCV RNA 5'-noncoding region and 5'-fragment of C region (HCV RNA5'-NCR-C) were transcribed from plasmid pHCV-neo by T7 phage RNA polymerase in vitro, and radiolabelled at its 5'-end. The trans-cleaving reaction was performed by mixing the ribozymes and substrate at mol ratio 100∶1 under conditions as follows: 37℃, pH7.5, Mg2+ 20 mmol/L and deionized formamide 2.5 mol/L. Percentage of trans-cleaved products were calculated at different time points and used as the activity indicator of the three ribozymes. Results　RzC1, RzC2 trans-cleaved more substrate when the time extended, and got to 24.9%,20.3% after reac-ting for 90 minutes respectively; RzC3 was not able to trans-cleave its substrate. Conclusion　Recombinant HDV genomic ribozymes have the ability to trans-cleave HCV RNA, but the appropriate target sequence should be selected.

Objective To explore the responses and mechanisms of peripheral primary afferent neurons to adenosine 5'-triphosphate (ATP) and bradykinin (BK) applied separately or in combination by electrophysiological recording and behavioral observation. Methods The experiments were done on samples of acutely isolated rat dorsal root ganglion (DRG) neurons by the whole-cell patch clamp recording technique, to record ATP-activated current (IATP) and the regulating effect of BK on IATP and to observe the global behavior with pain behavioral experiment. Results ATP added after the pretreatment of BK in the majority of detected cells, IATP would be reinforced significantly, the degree of increment depending on the concentration of BK (BK 10-6 -10-4 mol/L), while the EC50 values of the concentration-response curve with and without pretreatment of BK were very close to each other (1.65×10-5 mol/L vs 2.0×10-5 mol/L). In the behavioral experiment, subcutaneously intraplantar injection of BK and ATP separately in hind limbs of rats both induced concentration-dependent pain behavioral (paw lifting) responses, while the duration of hindpaw lifting was prolonged dramatically with the increase in the ATP concentration, when BK (10-6 mol/L) was injected in combination with ATP (10-5, 10-4 and 10-3 mol/L). Conclusion Inflammatory mediators like BK and ATP etc play an important role in the production, transmission and modulation of pain information in peripheral sensory nerve endings. Both electrophysiological and behavioral experiments demonstrate that there is a synergic effect between ATP and BK, which is thought to be non-competitive. BK may reinforce IATP remarkably, and the pain responses induced by the increment in ATP concentration increase with the existence of BK.

Adolescent ; Aneurysm ; complications ; pathology ; Heart Neoplasms ; complications ; Heart Ventricles ; Hemangioma, Capillary ; complications ; Humans ; Male ; Pulmonary Artery ; Thrombosis ; complications ; pathology

OBJECTIVETo establish an HPLC method for determining nine triterpenes contained in Ganoderma lucidum.

METHODChromatography conditions: Alltima C18 (4.6 mm x 150 mm, 5 microm) was adopted as the chromatographic column, with acetonitrile-0.04% formic acid solution as the mobile phase. The detective wavelength was set at 254 nm, and the column temperature was 15 degrees C.

RESULTThe linearities of ganoderic acid C2, ganoderic acid G, ganoderenic acid B, ganoderic acid B, ganoderenic acid A, ganoderic acid A, lucideric acid A, ganoderenic acid D, and ganoderic acid C1 ranged between 6.81-40.88, 6.38-38.25, 6.75-40.50, 6.38-38.25, 5.95-35.65, 5.90-35.25, 7.00-42.00, 6.20-37.15 and 6.05-36.4 mg x L(-1) (r = 0.999 4, 0.999 2, 0.999 4, 0.999 2, 0.999 2, 0.994 5, 0.999 0, 0.999 2 and 0.998 4). Their recoveries were 102.1%, 102.3%, 100.6%, 103.3%, 104.1%, 103.2%, 96.42%, 102.5% and 101.5%, with RSD being 1.5%, 0.96%, 1.9%, 1.3%, 1.7%, 2.5%, 0.62%, 2.9% and 1.3%. The content of triterpenes contained in G. lucidum samples from 31 different areas and under different cultivation conditions.

CONCLUSIONThe method is so feasible and highly reproducible that it can be used for quantitatie determination of the content of triterpenoid acid contained in G. lucidum.

China ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; Reishi ; chemistry ; Triterpenes ; analysis

OBJECTIVETo explore the demographic characteristics and clinical features of patients with idiopathic pulmonary arterial hypertension (IPAH) in China.

METHODSBetween March 2007 and September 2010, IPAH diagnosis was confirmed by right heart catheterization in 150 adult patients from 31 clinical centers in China. Clinical and hemodynamic data were analyzed and patients were divided into WHO functional class I/II and WHO functional class III/IV group.

RESULTSThe mean age of 150 patients were 36 ± 13 years with female patient/male patient ratio of 2:1, and mean BMI was (21.3 ± 3.5) kg/m(2). Fatigue (n = 123, 82.0%) and dyspnea (n = 112, 74.7%) are the most common symptoms. Accentuated pulmonic second sound (P(2)) was detected in 92.0% (n = 138) of patients during physical examination, which was also the most common sign. About 49.0% (n = 73) patients were WHO functional class I/II patients and 46.0% (n = 68) patients were WHO functional class III/IV patients. Six minutes walking distance (6MWD) and Borg dyspnea score was (337 ± 101) m and 2.0 (2.0, 4.0), respectively. Right ventricular hypertrophy was suggested by ECG in 93.1% (n = 140) patients. Right atrial pressure was (10 ± 6) mm Hg, mean pulmonary artery pressure was (61 ± 16) mm Hg, cardiac index was (2.3 ± 0.8) L×min(-1)×m(-2) and pulmonary vascular resistance (1484 ± 699) dyn×s(-1)×cm(-5) in this cohort. 6 MWD (305 m ± 89 m vs. 377 m ± 88 m) was significantly shorter while Borg dyspnea score [3.0 (3.0, 5.0) vs. 2.0 (2.0, 3.0)] was significantly higher in WHO functional class III/IV patients than in WHO functional class I/II patients. Similarly hemodynamic parameters were also worse in WHO functional class III/IV patients than in WHO functional class I/II patients (all P < 0.05).

CONCLUSIONIdiopathic pulmonary arterial hypertension patients in this cohort affect mostly young adults, dominated by female gender and lower body mass index. Fatigue and dyspnea are the most common symptoms and accentuated pulmonic second sound (P(2)) is the most common sign. IPAH patients are often displaying severe functional and hemodynamic disturbance at first visit to hospitals. Dyspnea and hemodynamic impairment are related to 6MWD and WHO functional class.

Adolescent ; Adult ; Aged ; Familial Primary Pulmonary Hypertension ; Female ; Hemodynamics ; Humans ; Hypertension, Pulmonary ; diagnosis ; physiopathology ; Male ; Middle Aged ; Ventricular Function ; Young Adult

OBJECTIVETo investigate the influence of anthropometric measures of obesity, including body mass index (BMI), abdominal subcutaneous adipose tissue and visceral adipose tissue, on pathological characteristics in patients with clinically localized prostate cancer.

METHODSFrom January 2006 to March 2013, the 413 patients of prostate cancer who received radical prostatectomy (RP) and their clinical and pathological data had been collected. The median age for the entire cohort was 68 years, which ranged from 48 to 78 years. All patients were diagnosed with prostate cancer before surgery and the Gleason score ranged from 4 to 10 (median 7). Anthropometric measures of abdominal adiposity including anterior abdominal fat, posterior abdominal fat and anteroposterior diameter were measured from the T2 weighted sagittal localization images of MRI scans and subcutaneous adipose tissue and the percentage of visceral adipose tissue were calculated. The patients' clinical and pathologic characteristics across BMI groups were compared used Student's t test for continuous variables or chi-squared test for categorical variables. Moreover, univariable and multivariable logistic regression models were used to address the influence of anthropometric measures of obesity on pathological outcomes.

RESULTSThe BMI ranged from 14.2 to 34.0 kg/m(2) and the median value was 23.8 kg/m(2). The abdominal subcutaneous adipose tissue ranged from 12.6 to 60.3 mm and the median value was 31.4 mm. The percentage of visceral adipose tissue ranged from 71.1% to 92.1% and the median value was 83.8%. In RP specimens, Gleason score ≥ 8 was observed in 141 patients (34.1%), pathological tumor stage was T3a in 69 patients (16.7%) and pathological tumor stage was T3b in 78 patients (18.9%). Positive surgical margin and lymph node involvement were observed in 71(17.2%) and 38(9.2%) patients, respectively. Although univariate analysis showed that BMI ≥ 25 kg/m(2) was associated with pathological Gleason score ≥ 8 (OR = 1.413, P = 0.035), this positive correlation disappeared in multivariate analysis(P = 0.095). In multivariate analysis, the percentage of visceral adipose tissue was significantly associated with pathological Gleason score (OR = 9.618, P = 0.000), extracapsular extension (OR = 6.750, P = 0.002) and seminal vesicle invasion (OR = 4.419, P = 0.007) after adjusting for patient age, PSA level, clinical stage and biopsy Gleason score.

CONCLUSIONSAnthropometric measures of abdominal adiposity was more sophisticated than simple BMI to evaluate the risk of obesity with regard to the aggressiveness of prostate cancer. The percentage of visceral adipose tissue was an independent factor for pathological Gleason score, extracapsular extension and seminal vesicle invasion in RP specimens.

Adiposity ; Aged ; Anthropometry ; Body Mass Index ; Humans ; Intra-Abdominal Fat ; Logistic Models ; Male ; Middle Aged ; Obesity ; complications ; Prostate ; pathology ; Prostatectomy ; Prostatic Neoplasms ; pathology ; Risk Factors