AIM In order to search inhibitors of protein kinase CK2, we observed the inhibitory effects of kaempferol on recombinant human protein kinase CK2 holoenzyme and its kinetics in vitro. METHODSCloning, prokaryotic expression and purification of human protein kinase CK2 α' and β subunits by gene engineering, the two subunits were mixed at equal molar ratio to reconstitute CK2 holoenzyme and identify its biological properties. The CK2 activity was assayed by detecting incorporation of 32P of [γ-32P]ATP into the substrate. The inhibitory effect of kaempferol on CK2 was assayed in the presence of different concentrations of kaempferol. Kinetic analysis of kaempferol-induced inhibition was carried out in the condition that casein concentration was fixed at 2 g·L-1 and ATP was changed at various concentrations(10, 20, 40, 80 μmol·L-1), or ATP was fixed at 10 μmol·L-1 and casein was changed at different concentrations (1, 2, 4, 8 g·L-1). RESULTS Kaempferol was shown to strongly inhibit the holoenzyme activity of recombinant human protein kinase CK2 with IC50 of 1.9 μmol·L-1, which was more effective than chrysin, morin and genistein which are both known as CK2 special inhibitors. Kinetic studies of kaempferol on recombinant human CK2 showed that kaempferol acted as a noncompetitive inhibitor with substrate ATP(Ki=1.1 μmol·L-1) and casein (Ki=3.1 μmol·L-1). CONCLUSIONKaempferol is a novel potent inhibitor of protein kinase CK2 in vitro. Discussions indicate that flavonoid inhibitors of CK2 may adopt different orientations in theactive site of CK2 and that these are determined by the number and position of their hydroxyl groups.

Objective To observe the effect of 7 flavonoids on recombinant human protein kinase CK2 holoenzyme activity and investigate their structure-activity relationship. Methods Recombinant hu-man protein kinase CK2 α' and β subunits were mixed at equal molar ratio to reconstitute CK2 holoen-zyme. The CK2 activity was assayed by detecting incorporation of 32p of [γ-32P] ATP into the substrate for the inhibitory effect by flavonoids and calculation of IC50 was performed according to probability unit (PROBIT) method. Results Myricetin, quercetin, morin, luteolin, kaempferol, apigenin, and chrysin were shown to obviously inhibit recombinant CK2 holoenzyme activity in a concentration-dependent man-ner with IC50 values of 1.18, 0.51, 16.16, 0.86, 1.88, 1.72, and 13.63 umol/L, respectively. Myricetin, quercetin, luteolin, kaempferol, and apigenin were more effective than DRB and A3, which were known as CK2 inhibitors in vitro. Whereas morin and chrysin displayed a similar effect to DRB. Structure-activity study indicated that the major structural requirements for the potent inhibition of CK2 by these flavonoids were hydroxyl group at position 6, 3' and 4'. Different from these requirements, absence of a hydroxyl group at position 3 did not modify their inhibitory potency, while addition of hydroxyl groups at positions 2' or 5' was detrimental to the inhibitory effect on CK2. Conclusion The inhibitory effect of flavonoid on protein kinase CK2 in vitro may be determined by the position of their hydroxyl groups.

This paper describes the construction and practical experience of the key laboratory for teaching of biochemistry and molecular biology,and indicates that the laboratory promotes the development of teaching and scientific research.It is proved to be a suitable measure for sharing teaching resource,improving teaching quality and raising teacher' academic level.

Objective To observe the mortality of fragile hip fractures and evaluate the death-associated risk factors.Methods 100 men and 186 women aged 50 to 97 (mean,77.09± 10.65) years old who had fragile hip fracture over 50 years old from 2010 to 2012 were followed up,and the clinical data were retrospectively analyzed.Three months,one year and the total mortality of following time were calculated.Mortality-related risk factors were evaluated including age,gender,and surgery,duration from injury to operation,pulmonary infection,number and kind of complications.Results The 286 patients were followed up between 6 months and 42 months,with 21.42±9.88 months in average.The three month mortality was 7.69％,the patients who were followed up over one year were 231 cases,the one year mortality was 16.02％,and the total mortality of following time was 17.48％.The mortality was associated with age,gender,surgery,duration from injury to operation,number of complications,pre-injury cardiovascular disease and respiratory system diseases,and pulmonary infection.A Binary Logistic Regression analysis revealed that the independent risk factors affecting the mortality included age (OR=5.385,P=0.003),surgery (OR=21.217,P=0.000),number of complications (OR=9.038,P=0.000),pre-injury cardiovascular disease (OR=3.201,P=0.041).Conclusion The early mortality of fragile hip fractures was high and was associated with many risk factors.Age,surgery,number of complications and pre-injury cardiovascular disease were the independent risk factors affecting the mortality of fragile hip fractures.The positive treatment with complications,early surgery in condition allowed,can lower the early mortality.

Objective To construct single chain antibody specific to membrane protein of Schistosoma japonicum by gonetic engineering technique. Methods The V\-H (heavy-chain variable region) and V\-L (light-chain variable region) genes were amplified by PCR from the genomic DNA of NP11-4 cell line, and sequenced by Sanger's method. The ScFv was constructed in pTHA90 vector using V\-H and V\-L genes, then expressed by IPTG. Results The V\-H and V\-L genes were obtained through PCR. The DNA sequences showed that V\-H and V\-L were new variable region genes of antibody. They were registered by GenBank. A ScFv gene with (Gly4Ser) 3 intralinker in the pTHA90 vector was successfully constructed. The ScFv was expressed as thioredoxin-fused proteins about 36\^2 kDa. Conclusion A specific ScFv against the membrane protein of Schistosoma japonicum was constructed and expressed.

Objective To fabricate a novel tissue-engineered cartilage with adipose-derived stem cells (ADSCs) seeded on the chitosan hydrogel scaffold to repair articular cartilage defect.Methods Adipose tissue and costal cartilage were harvested from New Zealand rabbits,and ADSCs in passage one and chondrocytes were obtained after the samples were digested and cultured in vitro.ADSCs were digested,suspended,seeded onto the sterile chitosan gel,and cultured in vitro for 1 week to fabricate the tissue-engineered cartilage.The defects were respectively filled with the tissue-engineered cartilage (composite group),chondrocyte suspension (cell group),chitosan gel (material group) and nothing at all (control group).At postoperative 12 weeks,cartilage repair was evaluated using the gross examination,histological staining,immunohistochemical staining and international cartilage repair society (ICRS) histological score.Results Effect of cartilage repair in composite group was significantly better compared to other groups.The regenerated tissue in composite group seemed tightly bound in normal tissue,with similar structure and extracellular matrix secretion.ICRS histological score in composite group was (13.89 ± 0.14) points,which differed significantly from (7.06 ± 0.19) points in control group,(7.14 ± 0.22) points in cell group and (7.46 ± 0.26) points in material group (P ＜0.01).Conclusion The tissue-engineered cartilage with ADSCs seeded onto the chitosan hydrogel is effective for repair of articular cartilage defect.

Objective To explore the correlation between hypokalemia and sudden death in young and middle-aged people. Methods One hundred and twenty-nine young and middle-aged patients with sudden death during treatment were selected as observation group. Then 100 cases of healthy volunteers were randomly selected as control group. The incidence of cardiovascular disease, incidence of hypokalemia and intake of potassium were compared between 2 groups. Results The incidences of hypertension, arrhythmia, myocardial infarction, heart failure and hypokalemia in observation group were significantly higher than those in control group: 17.05% (22/129) vs. 5.00%(5/100), 13.18% (17/129) vs. 2.00% (2/100), 26.36% (34/129) vs. 9.00% (9/100), 9.30% (12/129) vs. 1.00% (1/100) and 55.04% (71/129) vs. 12.00% (12/100), and there were statistical differences (P<0.01). The ratios of higher, common, lower intake of potassium in observation group were 1.55%(2/129), 27.91% (36/129) and 70.54%(91/129), in control group were 15.00% (15/100), 58.00% (58/100) and 27.00% (27/100), and there was statistical difference (P<0.01). Logistic regression analysis result showed that hypertension, arrhythmia, myocardial infarction, heart failure, hypokalemia and lower intake of potassium were the risk factor for sudden death (P<0.01). The incidences of hypertension, arrhythmia, myocardial infarction and heart failure in hypokalemia patients were significantly higher than those in normokalemia patients: 28.92% (24/83) vs. 2.05% (3/146), 19.28% (16/83) vs. 2.05% (3/146), 44.58%(37/83) vs. 4.11% (6/146) and 13.25% (11/83) vs. 1.37% (2/146), and there were statistical differences (P<0.01). The incidence of hypokalemia in people with lower intake of potassium was significantly higher than that in people with higher and common intake of potassium: 56.78% (67/118) vs. 2/17 and 14.89%(14/94), and there was statistical difference (P<0.01). Conclusions There is a significant correlation between hypokalemia and sudden death in young and middle-aged people. Preventive measures of sudden death should be made according to serum potassium level in clinic. People should pay attention to the uptake of potassium in daily life.

BACKGROUND:At present,the most frequently agents used for neural induction of bone marrow stromal cells(BMSCs)in vitro are anti-oxidants,such as beta-mercaptoethanol and all trans-retinoids.The majorities of induction from BMSCs are neuron-like cells in these protocols;however,whether it has neuronal function or not should be further studied.OBJECTIVE:TO investigate the differentiated characteristics of inducing human BMSCs into neural cells in serum-free medium.DESIGN:Observational study.SETTING:Chaozhou Central Hospital.MATERIALS:The experiment was carried out in the Chaozhou Central Hospital from April 2004 to December 2005.Adult bone marrows were derived from femoral and tibial bone marrow of three patients with fracture.All patients provided the confirmed consent and were approved by the Ethics Committee of Chaozhou Central Hospital.DMEMIF12 medium(1:1),fetal bovine serum (FBS), glutamine, N2 supplements and B27 Supplements were from GIBCO/BRL Company;recombinant basic fibroblast growth factor(bFGF)and recombinant epidermal growth factor(EGF)from Sigma Company;monoclonal antibody for vimentin(1:100),monoclonal antibody for myelin basic protein(MBP) (1:100),monoclonal antibody for S1 00(1:1 00),monoclonal antibody for neuron specific enolase(NSE)(1:1 00),and monoclonal antibody for neurofilament 200(NF200)(1:1 00)from Beijing Zhongshan Company;monoclonal antibody for glial fibrillary acidic protein (GFAP)(1:200)and polyclonal antibody for nestin(1:100)from Boster Company(Wuhan);mouse monoclonal antibody for beta-tubulin 3(1:1 000)from Sigma Company;SP-9000 kits and quick AEC from Beijing Zhongshan Company; culture dishes and flasks from Coming Company.METHODS:BMSCs from human bone marrow were cultured in serum-containing medium.When the primary culture was established, BMSCs were transferred into serum-free medium containing N2 or B27 supplement with 20 μg/L basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF),and cells were cultured in an incubator containing C02of 0.05 volume fraction at 37℃. Morphological changes of BMSCs in serum-free medium were observed under phase contrast microscope. And two days after culture. Expression of relative markers of BMSCs was detected withimmunocytochemistry.MAIN OUTCOME MEASURES:Morphological changes of BMSCs and expression of relative markers of nerve cells.RESULTS:A population of BMSCs could be isolated from adult human bone marrow,and they were processed to obtain a fibroblast-like population and were expanded as undifferentiated cells in culture for more than 10 passages.indicating their proliferative capacity.They could form spheroid state when they were sub-cultured in serum-free media supplemented with bFGF and EGF.these cells could express the markers for neural stem cells such as vimentin and nestin;they could expressed neuron specific enolase(NSE),beta-tubulin 3,TrkC and neurofilament 200(NF200)when they were plated on dishes with serum-containing medium; some cells exhibited the phenotypes for astrocytes.expressing gilal fibrillary acidic protein(GFAP)and S100 protein.CONCLUSION:The morphology,protein expression and differentiation ability of BMSCs in serum-free medium was similar to those of neural stem cells.The data support the hypothesis that adult bone marrow contains stem cells capable of differentiating into neural cells,the serum-free media make BMSCs overcome their mesenchymal commitment,showing the phenotypes for neural stem cells.

Objective To investigate the expression of livin of kidney tissues in rats with acute kidney injury (AKI) induced by endotoxin ,and the role of livin in the cell apoptosis of AKI .Methods The rat models with AKI were induced by endotoxin .The de-gree of kidney injury was observed by hematoxylin and eosin stain ,measuring the levels of the serum creatinine and urea nitrogen . The expression of livin and caspase-3 in kidney tissue at different time points was analyzed by immunohistochemistry assay ,and the relationship between the expression of livin and caspase-3 and kidney injury was analyzed .Results Compared to the control group , the rats injected endotoxin had the performance of AKI ,with obviously pathomorphological damage in the kidney tissues ,signifi-cantly increasing in the serum creatinine and urea nitrogen (P<0 .01) .The livin and caspase-3 of kidney tissues in rats with AKI caused by endotoxin were positive expression (P<0 .01) .With the lapse of time ,the trend of increasing of caspase-3 showed gradu-ally slow after the highest expression of livin .Conclusion The livin involved in the pathogenesis of endotoxin-induced AKI ,it might relieve the kidney injury and protect renal function by inhibiting casepase-3 important apoptotic effector protein .

Objective To observe the efficacy and safety of rituximab in the treatment of systemic sclerosis (SSc).Methods This was a prospective,non-randomized controlled trial.All patients with SSc were hospitalized from January 2011 to August 2015.Fifty-two patients were enrolled,including 15 patients in the intervention group and 37 patients in the control group.Both groups were given cyclophos-phamide and prednisone.The intervention group was also given rituximab 375 mg/m2,once every 4 weeks,a total of 4 treatments.The erythrocyte sedimentation rate (ESR),C-reactive protein (CRP),intedeukin 6 (IL-6),modified Rodnan skin score (mRSS),HRCT,forced vital capacity (FVC％),1％ forced expiratory volume percentage (FEV 1％),percentage of carbon monoxide dispersion percentage (DLCO％) were assessed alternatively before treatment,6 months and 12 months after treatment.The repeated measures analysis of variance and t-test were used for statistical analysis.Results Both groups showed improvement in ESR,CRP,IL-6,mRSS,HRCT,FVC％,FEV1％ and DLCO％ (P＜0.05),but ESR [6 month after treatment (33±9) mm/1 h,12 month after treatment (20±4) mm/1 h],CRP [6 month after treatment (12.7±3.4) mg/L,12 months after treatment (12.7± 3.4) mg/L],IL-6 [6 month after treatment (73±10) pg/ml,12 month after treatment (57±11) pg/ml],mRSS [6 months after treatment (22.2±1.3),12 month after treatment (18.4±3.1)],HRCT score [6 month after treatment (11.9±1.4),12 month after treatment (11.3±1.0)],in patients of the intervention group were decreased more significantly than patients of the control group (P＜0.05),and FVC％ [6 month after treatment (69.0± 3.4)％,12 month after treatment (77.2±4.1)％],FEV1％[6 month after treatment (73.3±3.4)％,12 month after treatment (78.4±1.6)％] and DLCO％ [6 month after treatment (60.6±2.7)％,12 month after treatment (70.7± 3.0)％] were increased more significantly than patients of the control group (P＜0.05).It showed that the efficacy of the intervention group was better than the control group.There was no infusion reactions,infection,hepatitis B virus reactivation and other side effects in the treatment were observed.Conclusion Rituximab can reduce the level of inflammation in patients with SSc and improve skin sclerosis and lung function.Rituximab combined cyclophosphamide is expected to be a new,safe and effective treatment of SSc.