To investigate the biological role of snake venom cystatin(sv-cystatin) in tumor progression, the cDNA of sv-cystatin amplified by PCR from pUC18-cystatin plasmid was cloned into methanol-inducible expression vector pPICZ?A. The linearized recombinant plasmid pPICZ?A-cystatin was transfered into Pichia pastoris, strain GS115 by electrophoration. Transfermants with phenotype Mut+ selected were identified by PCR analysis and induced in 1.0% methanol. The reombinant sv-cystatin protein was examined by SDS-PAGE, Western blot analysis. The molecular mass of expression product was about 14 kDa and approximately 16 mg/L of recombinant sv-cystatin was produced from one of GS115-cystatin transformants. The chromatography purified protein could reduce the activity of papain. The ability of B16F1 cells treated with recombinant sv-cystatin to invade the reconstituted basement membrane decreased significantly (P

To explore the prevention effect of the joint combination of Yindanxinnaotong soft capsule (YDXNT) and exercise (swimming) on atherosclerotic rats. The method of 3 x 3 factorial design, including two factors (YDXNT and swimming) and three levels (0, 1, 2 g x kg(-1) YDXNT; 0, 0.5, 1 h swimming), was mainly adopted. The atherosclerotic rat model was established by ligating their left common carotid arteries and feeding high-fat diet. After 8 weeks, blood samples were collected from their thoracic aorta to determine blood viscosity, plasma viscosity, fibrinogen (FIB), nitric oxide (NO), 6-keto-PGF(1alpha) endothelin (ET) and thromboxane B2 (TXB2). The tissues of left common carotid arteries of the rats were collected to detect the positive expression of SM22alpha and determine the semi-quantitation through the immunohistochemical staining. The result showed that the combination of YDXNT and swimming can significantly decrease the plasma viscosity (F = 3.241, P = 0.017), the high and low shear blood viscosity (F = 6.444, P = 0.001; F = 3.002, P = 0.024) and FIB (F = 4.046, P = 0.005). The increased NO and 6-keto-PGF(1alpha) and the decreased ET and TXB2 indicated a significant interaction (P < 0.05). The swimming showed an obvious main effect in the expression of up-regulated protein SM22alpha (F = 8.088, P = 0.001). The study suggested that the combined administration of YDXNT and swimming could improve the hemorheological parameters of atherosclerotic rats, protect the vascular endothelium, inhibit the vascular remodeling in atherosclerosis and positively prevent the atherosclerosis.
Animals ; Atherosclerosis ; drug therapy ; genetics ; metabolism ; prevention & control ; Blood Viscosity ; drug effects ; Combined Modality Therapy ; Drugs, Chinese Herbal ; administration & dosage ; Exercise Therapy ; Humans ; Male ; Microfilament Proteins ; genetics ; metabolism ; Muscle Proteins ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Swimming

Objective To investigate the efficacy of using a calibrator with values assigned with the reference method for improving the comparability of serum gamma-glutamyltransferase (GGT) measurements.Methods The IFCC reference method for GGT was established and the performance was verified by testing a certified reference material (CRM).A calibrator was prepared and its value for GGT was assigned with the reference method.Forty serum samples were measured on different (including HITACHI 7600,7060,7170,7180 and BECKMAN LX20,OLYMPUS AU 400) chemistry analyzers with Zhongsheng GGT reagent kits calibrated with the calibrator.The samples were also measured on the same analyzers using a theoretical factor.Biases of results obtained with the calibration and with the theoretical factor based calculation were compared.Results The reference method resuhs on the CRM agreed the certified value within the stated uncertainty.Serum results calculated from the theoretical factor showed various biases and inter-analyzer variations.When the analyzers were calibrated with the calibrator,the number of results with biases less than 10% became significantly higher and those with biases more than 20% significantly lower.The variation of the results on 5 serum samples was reduced from 11.0%～14.0% to less than 5% by using the calibrator.Conclusion Accuracy and comparability of GGT measurements with of ZhongSheng GGT kits can be improved by using a calibrator that has a value assigned with the reference method.

OBJECTIVETo construct a subtracted cDNA library of differentially expressed genes in eosinophils from asthma patients.

METHODSSuppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes in the eosinophils of asthma patients before and after treatment. The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library, followed by amplification of the library through E. coli transformation with calcium chloride and screening of blue and white clones of the transformants. One hundred positive bacterial clones were randomly picked and identified by colony PCR.

RESULTSThe amplified library contained more than 3,000 positive bacterial clones. Analysis of the randomly selected 100 white clones by PCR showed that 90% of the clones contained 100-500 bp inserts, which might be the cDNA fragments of differentially expressed genes in eosinophils of asthma patients before treatment.

CONCLUSIONA subtracted cDNA library of differentially expressed genes in the eosinophils of asthma patients before and after treatment is constructed successfully by SSH and T/A cloning techniques, which lays a solid foundation for screening and cloning new specific differentially.expressed genes in the eosinophils of asthma patients.

Asthma ; blood ; genetics ; DNA, Complementary ; genetics ; Eosinophils ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation ; genetics ; Gene Library ; Humans ; Male ; Middle Aged ; Nucleic Acid Hybridization

OBJECTIVETo study the effects of interferon-gamma (IFN-gamma) on the adhesiveness and invasiveness of prostate cancer cells.

METHODSWe determined the effect of IFN-gamma on the adhesiveness of prostate cancer cell lines LNCaP and PC-3 by treating the plate with fibronectin and laminin, on the invasiveness of prostate cancer cells into the artificial basal membrane consisting of Matrigel and fibronectin using the Transwell chamber, and on the expression of annexin-2 in these cells by Western blot.

RESULTSThe adhesion rates of prostate cancer cell lines LNCaP and PC-3 were 21% and 23% in the IFN-gamma treatment group, significantly lower than 46% and 40% in the untreated group (P < 0.05). The invasiveness of the cells was markedly decreased in the former as compared with the latter (P < 0.05). IFN-gamma significantly inhibited the expression of annexin-2 in prostate cancer cell lines LNCaP and PC-3 (P < 0.05).

CONCLUSIONIFN-gamma can decrease the adhesiveness and invasiveness of the prostate cancer cell line by down-regulating the expression of annexin-2.

Annexin A2 ; biosynthesis ; Blotting, Western ; Cell Adhesion ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Humans ; Interferon-gamma ; pharmacology ; Male ; Neoplasm Invasiveness ; Prostatic Neoplasms ; metabolism ; pathology

OBJECTIVETo construct and identification of a lentiviral vector for RNA interference (RNAi) targeting STUB1 gene.

METHODSA pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting STUB1 gene was designed, synthesized and inserted into linearized pMagic 4.0 vector. The recombinant plasmid was identified by double restriction digestion with Age I/EcoR I and DNA sequencing.

RESULTPCR and DNA sequencing showed that the shRNA sequence was successfully inserted into pMagic 4.0 vector. The pMagic 4.0 vector was successfully packaged into lentivirus particles.

CONCLUSIONA lentiviral shRNA expression vector and particles targeting STUB1 gene has been successfully constructed for the further study of the STUB1 gene.

Gene Targeting ; Genetic Vectors ; Lentivirus ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Ubiquitin-Protein Ligases ; genetics

OBJECTIVETo study the protected effect of Yindan Xinnaotong capsule (YDXNTC) and main components compatibility on myocardium ischemia/reperfusion injury.

METHODGlobal ischemia/reperfusion was adopted to induce myocardial ischemia/reperfusion injury (MIRI) in isolated rat heart. Sprague-Dawley (SD) rats were divided into control, model, YDXNTC, Ginkgo biloba extract (GBE) group, ethanol extract of Salvia miltiorrhiza (SM-E) group, aqueous extract of Salvia miltiorrhiza (SM-H) group, mixed compatibility of other components in YDXNTC (MC), GBE and SM-E compatibility (GSEC), GBE and SM-H compatibility (GSHC), and SM-E and SM-H compatibility (SEHC). During the experiment, electrocardiogram was recorded to observe cardiac arrest time, heart resuscitation time, regaining normal rhythm time, the incidence and duration of arrhythmias (VT/VF). At the end of reperfusion, hearts were arrested and homogenated to assay the activity of superoxide dismutase (SOD), and the content of malondialdehyde (MDA), lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), cardiac troponin I.

RESULT(1) YDXNTC, SM-E, SM-H and MC elevated cardiac arrest time, also reduced rebeating time, restoring normal rhythm time as well as the duration of arrhythmia, but no remarkable impact on VT/VF occurrence. GBE was effective for incidence of VT/VF, also achieved good effect on shortening rebeating time, restoring normal rhythm time and arrhythmia duration. Likewise, obviously reduced rebeating time, restoring normal rhythm time and arrhythmia duration, and evaluated cardiac arrest time were also exhibited in compatibility groups except that no lengthened cardiac arrest time was detected in GSHC. And the incidence of VT/VF was decreased by GSEC. (2) YDXNT, ginkgo biloba extract (GBE), ethanol extract of salvia miltiorrhiza (SM-E), GBE and SM-E compatibility (GSEC), and SM-E and aqueous extract of salvia miltiorrhiza (SM-H) compatibility (SEHC) could improved SOD and decreased MDA level SM-H, mixed compatibility of other elements in YDXNTC (MC) and GBE and SM-H compatibility (GSHC) showed a role on MDA reduction. (3) LDH was declined by YDXNT and SM-H. CK-MB was reduced by GBE, SM-E, SM-H, and GSEC. (4) The release of cTnI was only inhibited by GSEC.

CONCLUSIONYDXNTC, primary materials and main components compatibility has a certain protection effect on MIRI, its mechanism may be related to antioxidant and calcium overload reduction.

Animals ; Arrhythmias, Cardiac ; physiopathology ; prevention & control ; Capsules ; Creatine Kinase, MB Form ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Electrocardiography ; Ginkgo biloba ; chemistry ; Heart ; drug effects ; physiopathology ; In Vitro Techniques ; L-Lactate Dehydrogenase ; metabolism ; Male ; Malondialdehyde ; metabolism ; Myocardial Reperfusion Injury ; metabolism ; physiopathology ; prevention & control ; Myocardium ; metabolism ; pathology ; Phytotherapy ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; chemistry ; Superoxide Dismutase ; metabolism ; Troponin I ; metabolism

OBJECTIVETo investigate the effects of ischemic postconditioning (IPostC) on pneumocyte apoptosis after lung ischemia/reperfusion injury in rats.

METHODSAdult male SD rats were randomly divided into 3 groups based upon the intervention (n = 8): control group (C), lung ischemic reperfusion group (LIR), LIR+ IPostC group (IPostC). At the end of the experiment, blood specimens drawn from the arteria carotis were tested for the content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO); the pneumocyte apoptosis index (AI) was achieved by tennrminal deoxynucleotidyl transferase mediated dUTP nick end abeling (TUNEL); the expression of Bcl-2, Bax protein in lung tissue was accessed by quantitative immunohistochemistry (MHC) and Bcl-2, Bax mRNA by RT-PCR.

RESULTSIPostC could significantly attenuate the MDA level, MPO activity and improve SOD activity in blood serum which was comparable to I/R and significantly reduced the number of TUNEL-positive cells compared with I/R group, expressed as Al (% total nuclei) from (39.0 +/- 3.46) to (8.0 +/- 0.88) (P < 0.01). The protein and mRNA expression of Bcl-2 and Bax showed that IPO significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein but improved the expression of Bcl-2 that improved the Bcl-2/Bax ratio (P < 0.01) .

CONCLUSIONIPostC may attenuate pneumocyte apoptosis in LIRI by up-regulating expression of Bcl-2/Bax ratio and by inhibiting oxidant generation and neutrophils filtration.

Alveolar Epithelial Cells ; cytology ; Animals ; Apoptosis ; Ischemic Postconditioning ; Lung ; metabolism ; pathology ; Lung Injury ; physiopathology ; Male ; Malondialdehyde ; metabolism ; Peroxidase ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; physiopathology ; Superoxide Dismutase ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the clinical effect of Yangyin Yiqi Huoxue Recipe (YYHR, the basic recipe of Yangyin Tongnao Granule) in treatment of ischemic stroke patients of deficiency of qi and yin syndrome (DQYS) and static blood obstructing collaterals syndrome (SBOCS).

METHODSTotally 312 patients were assigned to the control group (86 cases) and the treatment group (226 cases) using strati- fied randomized allocation method. Patients in the treatment group were treated with modified YYHR, while those in the control group took Xueshuan Xinmaining. The treatment course was 4 weeks for all. Constituent ratios of the acute stage and the recovery stage of DQYS and SBOCS and their complicated syndromes were observed in the two groups. Changes of the clinical curative effect, clinical symptoms integral, whole blood viscosity ratio, plasma viscosity ratio, hematocrit, erythrocyte sedimentation rate (ESR), total cho- lesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) were detected in the two groups before and after treatment.

RESULTSThere was statistical difference in constituent ratios of the acute stage and the recovery stage of DQYS SBOCS and its complicated syndromes between the two groups (P < 0.01). DQYS and SBOCS was basic syndrome types of the two groups. The cured and markedly effective rate was 71.24%(161/226) in the treatment group and 43.02% (37/86) in the control group. The total effective rate was 91.15% (206/226) in the treatment group, higher than that of the control group (76.74%, 66/86) with statistical difference (P < 0.01). There was statistical difference in the clinical symptoms integral, whole blood viscosity ratio, plasma viscosity ratio, hematocrit, ESR, TC, TG,HDL-C, and LDL-C (P < 0.05, P < 0.01).

CONCLUSIONSSymptoms of ischemic stroke patients could be improved by modified YYHR. Indices such as the whole blood viscosity, plasma viscosity ratio, hematocrit, ESR, abnormal metabolism of blood lipids were also significantly improved. Pathological changes of blood stasis induced by qi-yin deficiency exist in ischemic stroke patients, and DQYS and SBOCS were basic syndrome types.

Aged ; Biomedical Research ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Hematocrit ; Humans ; Male ; Middle Aged ; Qi ; Stroke ; drug therapy ; Triglycerides ; blood ; Yin Deficiency ; therapy

Objective:To screen the clinical parameters in predicting continuous renal replacement therapy (CRRT)-related hypotension in the patients with renal failure.Methods:A retrospective analysis was conducted. Patients with renal failure received CRRT admitted to Qingdao Municipal Hospital from July 1st, 2012 to June 30th 2019 were enrolled. Clinical data was recorded for the patients, including gender, age, weight, parameters before CRRT [systolic pressure (SBP), diastolic pressure (DBP), mean arterial pressure (MAP), central venous pressure (CVP), heart rate, blood routine examination, blood biochemistry, B-type natriuretic peptide (BNP), cardiothoracic ratio, left ventricular ejection fraction (LVEF)], CRRT duration, blood flow velocity, replacement fluid volume, net ultrafiltration volume, ultrafiltration rate and 30-day prognosis. The patients who had CRRT-related hypotension or whose net ultrafiltration was zero were enrolled as intolerance ultrafiltration group. Others were enrolled in normal ultrafiltration group. The parameters of the patients in the two groups were compared, and their predictive values in CRRT-related hypotension were evaluated by receiver operating characteristic (ROC) curve analysis.Results:There was significant difference in age, BNP, CVP, CRRT duration, net ultrafiltration volume, and ultrafiltration rate between the normal ultrafiltration group ( n 98) and intolerance ultrafiltration group [ n 13; age (years old): 66.21±12.21 vs. 74.54±5.93, BNP (ng/L): 1 617.57 (755.00, 2 296.25) vs. 398.95 (76.80, 703.00), CVP (cmH 2O, 1 cmH 2O = 0.098 kPa): 14.99±2.28 vs. 12.60±1.14, CRRT duration (hours): 23.71±11.48 vs. 14.51±8.99, net ultrafiltration volume (mL): 3 120.98 (1 863.75, 3 515.00) vs. 793.85 (0, 1 125.00), ultrafiltration rate (mL/h): 133.44 (98.72, 156.64) vs. 68.47 (0, 105.21), all P < 0.05]. ROC curve analysis showed that age, BNP, CVP, CRRT duration, net ultrafiltration volume and ultrafiltration rate had good predictive value for the CRRT-related hypotension [the area under ROC curve (AUC) and 95% confidence interval (95% CI): age was 0.734 (0.626-0.842), BNP was 0.868 (0.776-0.960), CVP was 0.846 (0.757-0.935), CRRT duration was 0.746 (0.595-0.897), net ultrafiltration volume was 0.926 (0.845-1.000), and ultrafiltration rate was 0.794 (0.620-0.969), respectively]. Furthermore, according to the cut-off point of ultrafiltration rate, the patients were divided into ≥99.50 mL/h group and < 99.50 mL/h group. There were statistical differences in BNP, CVP and net ultrafiltration volume between the two groups. ROC curve analysis showed that BNP and CVP had good prediction value for hypotension when ultrafiltration rate exceeds ≥ 99.50 mL/h. The AUC (95% CI) of BNP and CVP were 0.716 (0.604-0.828) and 0.749 (0.651-0.847), the sensitivity was 70.0%, 78.8%, and the specificity was 35.5%, 45.2%, respectively. Conclusion:The values of age, BNP and CVP are more useful than other parameters in predicting CRRT-related hypotension before the start.