ROS is not involved in induction of cell death by Ent-11 alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid in HepG2 cells / 中国中药杂志

<p><b>OBJECTIVE</b>To identify the role of reactive oxygen species (ROS) formation on cell death induced by Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) in HepG2 cells.</p><p><b>METHOD</b>MTT assay was used to determine the effect of 5F on proliferation of HepG2 cells, and apoptotic morphological changes were assessed using Hoechst/PI assay. To evaluate intracellular ROS levels, a GENMED kit was used. HepG2 cells were treated with 5F for 24 h or with 1 mmol x L(-1) GSH for 1 h prior to treatment with 5F for 24 h, then cytoplasmic mono- and oligonucleosomes were assessed with Cell Death Detection ELISA kit.</p><p><b>RESULT</b>The cytotoxicity of 5F on HepG2 cells was elevated with increasing 5F concentrations, as evidenced by the cell viability assay, and the apoptotic changes such as chromatin condensation were confirmed by Hoechst/PI staining. The decrease in ROS generation was observed in HepG2 cells following treatment with 5F. Cytoplasmic mono- and oligonucleosomes induced by 5F were not changed by decreasing basal level of ROS-mediated signaling with GSH. Further more, induction of ROS production by cisplatinum (CDDP) was canceled by treatment with 5F and 5F revealed a additive effect to cell killing by CDDP.</p><p><b>CONCLUSION</b>5F can not only induce apoptosis through non-ROS-depandent pathway, and can abate oxidant stress.</p>

Effect of 5F from Pteris semipinnata on expression of Nr1d1 in HO-8910PM cell line / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effects of PsL5F (ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid, an extract from Pteris semipinnata L) on the expression of nuclear receptor subfamily 1, group D, member 1 (Nr1d1) in highly metastatic ovarian carcinoma HO-8910PM cells, and its mechanisms.</p><p><b>METHOD</b>Microarray Chip was used to examine the level of Nr1d1 mRNA expression on HO-8910PM cells treated with PsL5F. Fluorescent quantitative real-time PCR assay and Western blot were performed to verify the effects of PsL5F on Nr1d1 mRNA and protein expression.</p><p><b>RESULT</b>After 24 h treatment of 100 micromol x L(-1) PsL5F, the mRNA and protein levels of Nr1d1 in HO-8910PM cells were 35.34 +/- 1.07 and 7.71 +/- 0.43 times compared to those of control group (P < 0.01, P < 0.01), respectively.</p><p><b>CONCLUSION</b>PsL5F can up-regulate significantly the expression of Nr1d1 in HO-8910PM cells. Antitumor effects and its mechanisms of PsL5F in HO-8910PM cells may be involved in the up-regulation of Nr1d1 expression.</p>

Construction of the Key Laboratory for Teaching Efficiency of Biochemistry and Molecular Biology / 中华医学教育探索杂志

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.

Rapid Deadenylation: A New Mechanism of miRNA Inhibiting Gene Expression / 生物化学与生物物理进展

A paper published in Proc Natl Acad Sci USA On March 14,2006,by Ligang Wu et al, New York University School of Medicine, indicating that rapid readenylation is a new mechanism of miRNA inhibiting gene expression.

shRNA inhibits SURVIVIN expression in human lung cancer cell A549 / 基础医学与临床

Objective To construct shRNA expression vector of SURVIVIN for RNAi-mediated therapy of lung cancer.Methods DNA templates of SURVIVIN shRNA were designed,synthesized and cloned into the shuttle vector to get recombinant plasmids.After plasmids were transfected into A549 cells,the one with the most repression was screened by means of RT-PCR.Then MTT and Western blot were done to ascertain whether the proliferation of A549 cells were inhibited and SURVIVIN was down-regulated.Results The recombinants were constructed and screened successfully.The proliferation of A549 cells and SURVIVIN expression were repressed.Conclusion Mediated by RNAi,SURVIVIN expression was down-regulated,which suggested a potential gene therapy of huaman lung cancer.

Kaempferol is a potent inhibitor of recombinant human protein kinase CK2 holoenzyme in vitro / 中国药理学与毒理学杂志

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.

Expression,purification and activity assay of recombinant mouse protein kinase CK2? subunit from escherichia coli / 中国药理学通报

AIM To study the expression, purification and activity assay of recombinant mouse protein kinase CK2? subunit from Escherichia coli. METHEDS The recombinant plasmid containing mouse protein kinase CK2? subunit cDNA constructed successfully was transformed into Escherichia coli BL21 (DE3) and specifically induced by IPTG. The recombinant mouse CK2? subunit was sequentially purified by DE 52, P11 phosphocellulose and Heparin Sepharose chromatography. The purified recombinant protein was analysed by SDS PAGE. RESULTS One protein with molecular mass of 42 ku was overexpressed by inducing ITPG. The recombinant protein was composed of approximately 30 6% of the total bacterial proteins. From 278 mg soluble proteins, the yield of the CK2? protein was 4 7 mg. SDS PAGE analysis of the purified recombinant protein showed only one band in agreement with native mouse CK2? subunit. The recombinant mouse CK2? and ? subunits were mixed at the same molar ratio. The produced CK2 holoenzyme displayed full activity. The characteristics and functions of reconstituted CK2 holoenzyme were consistent with those of the given native CK2. CONCLUSION The recombinant protein is mouse protein kinase CK2? subunit.

Purification of 5F from Pteris semipinnata and its enhanced cytotoxicity in vitro / 中国药理学通报

AIM To find a new method for purifying the active compound 5F isolated from Pteris semipinnata L.(PsL ) and observe its enhanced cytotoxicity when combined with other antitumor agents. METHODS Silica gel combined with AgNO 3 was made to purify 5F. Cytotoxicity was detected with trypan blue dye exclusion assay. RESULTS After purification, the concentration of compound 5F in purified products was higher than 99%. The inhibitory rates of 5F combined with 5 Fu or CDDP or VCR were higher than that of these drugs used alone. CONCLUSIONS Compound 5F could be purified effectively using silica gel combined with AgNO 3. 5F could enhance the cytotoxicity on HL 60 and K562 cells of the drugs mentioned above. The different effect of these agents on the various phases of cell cycle kinetics may explain the enhanced effect of 5F.

The anti-inflammatory effect of total flavonoids isolated from Platychadus orientalis and its mechanism / 中国药理学通报

AIM To investigate the anti-inflammatory eff ec t of total flavonoids isolated from Platychadus orientalis and its mechanism . METHODS The inflammatory models in mice and swelling of rats hind paws were induced by dimethylbenzene and carrageenan respectivel y. The biosynthesis of leukotriene and ?-glucuronidase release in the cells we re measured by HPLC and fluorescence spectrophotometer respectively. RES ULTS Total flavonoids isolated from Platychadus orientalis remarkably inhibited the ear edema induced by dimethylbenzene in mice at the dose of 12.5 ～50.0 mg?kg -1 and the swelling of rats hind paws induced by carrageenan at the dose of 25.0～50.0 mg?kg -1 . Total flavonoids at final concentrati on of 5.0～125.0 mg?L -1 inhibited 5-HETE and LTB 4 synthesis by 30.5%～ 87.3% and 27.8%～84.3% respectively. In comparision with controls, Total flavono ids at final concentration of 5.0～45.0 mg?L -1 inhibited ?-glucuronidas e release by 13.4%～32.2%. CONCLUSION Total flavonoids isolated f rom Platychadus orientalis has an anti-inflammatory effect which may be rel ated to inhibiting 5-HETE and LTB 4 production and ?-glucuronidase release.

The role of protein kinase CK2 in cell cycle control / 中国药理学通报

Research of cell cycle is the bas is of investigating the organism growth,development,heredity and other medical associated events. The molecular basis of cell cycle control is the regulator control system with CDK-Cyclin-CKI as the core.Protein kinase CK2 is one of the most conservative protein kinase during evolution and more and more researches have proved that protein kinase CK2 plays an important role in cell cycle control.

Inhibitory effect of tyrphostin AG114 on recombinant human protein kinase CK2 holoenzyme / 中国药理学与毒理学杂志

AIM　To study the direct effect of tyrphostin AG114 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS　Recombinant human protein kinase CK2 α and β subunits were cloned and expressed by genetic engineering, and purified to homogeneity. The two subunits were mixed at equal molar ratio and reconstituted CK2 holoenzyme, which exerted the maximum biological activity. The CK2 activity was assayed by detecting incorporation of 32P of ［γ-32P］ATP or ［γ-32P］GTP into the substrate in various conditions. RESULTS　The recombinant human CK2 was a second messenger (Ca2+, cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG114 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 20.8 μmol·L-1, which lay between IC50 of 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole(DRB) and N-(2-aminoethyl)-5-chloronaphthalene-1-sulfonamide(A3), known as CK2 special inhibitors. Kinetic studies of AG114 inhibition on recombinant human CK2 showed that the inhibition was mixed competitive with GTP and noncompetitive with casein. CONCLUSION　AG114 not only is an effective inhibitor of protein tyrosine kinases, but also is a novel potent inhibitor of protein kinase CK2. The recombinant human protein kinase CK2 might be used as a molecular target for simpler screening method and development of more effective inhibitors of CK2.

Study on the Anti-inflammatory Effect of Biota orientalis / 中国药科大学学报

AIM　The purpose is to investigate the Anti-inflammatory mechanism of Biota orientalis. METHODS　Using rat leukocytes and rabbit platelets as experimental material the biosynthesis of leukotriene, 12-hydroxyheptadecatrienoic acid(12-HHT) and 12-hydroxyeicosatetraenoic acid(12-HETE) in the cells was determined with HPLC. RESULTS　Biota orientalis was shown to inhibit the biosynthesis of leukotriene B4(LTB4) and 5-hydroxyeicosatetraenoic acid(5-HETE) with IC50 of 0.40 and 0.41　mg/ml crude herb, respectively. It was also displayed to inhibit biosynthesis of 12-HHT in the platelets. CONCLUSION　Biota orientalis leaves contain anti-inflammatory components and the anti-inflammatory mechanism was related with inhibiting metabolism of arachidonic acid.

Study on Solid Dispersion of Quercetin / 中国药房

OBJECTIVE:To prepare solid dispersion for increasing solubility of quercetin METHODS:Using polyvinyl pyrrolidone(PVP) as the carrier,solid dispersion of quercetin was prepared by solvent -volatilization method The solubility of the solid dispersion was detected IR spectrometer and UV spectrometer were used to investigate the physical and chemical properties of the solid dispersion RESULTS:The solubility of solid dispersion was significantly increased compared with quercetin and the physical mixture Quercetin existed as ultrafine crystalline or molecular form in solid dispersion,so there was no chemical reaction between quercetin and PVP CONCLUSION:PVP could be used as the carrier of solid dispersion to increase the solubility of quercetin in water

Role of calcium ion in the cyotoxicity and DNA fragment induction in HL-60 cells by 6F isolated from Pteris semipinnata L / 中国病理生理杂志

AIM: To investigate the changes of cytosolic free calcium concentration ([Ca 2+ ] i) and expression of Bcl-2 in HL-60 cells treated by 6F isolated from Pteris semipinnata L. (PSL), and to discuss the relations between calcium ion and cytotoxicity and DNA fragment induction effects of 6F. METHODS: HL-60 cells were used as in vitro model. [Ca 2+ ] i was measured on fluorescent spectrophotometry using Fura-2/AM as Ca 2+ indicator. Bcl-2 expressing level was measured by flow cytometry. Tetrazolium salt (MTT) and diphenylamine staining methods were applied for cytotoxicity assay and DNA fragmentation detection, respectively. RESULTS: [Ca 2+ ] i increased obviously in a dose and time dependent manner after treated HL-60 cells with 6F. 6F decreased the expressing level of Bcl-2. Adding 2 mmol/L Ca 2+ to the medium, or 1 mmol/L EDTA to chelate Ca 2+ , or 4 ?mol/L calcium ionophore A 23187 to increase the concentration of cytosolic Ca 2+ , the DNA fragment induction by 6F was not affected, whereas the cytotoxicity of 6F was enhanced. 250 ?mol/L Zn 2+ attenuated the DNA fragment induction, and the cytotoxicity of 6F against HL-60 cells was enhanced significantly. CONCLUSION: It was speculated that the decreased expressing of Bcl-2 by compound 6F was related to increased [Ca 2+ ] i in HL-60 cells, and DNA fragment induction was possibly catalyzed by Ca 2+ - independent DNase.

Separation and identification of quercetin,genistein and their sulphate derivatives with micellar paper chromatography / 中国药学杂志

OBJECTIVE：To investigate the use of micellar paper chromatography on the isolation and identification of flavonoids and their soluble derivatives.METHOD:Quercetin,Genistein and their sulphate derivatives were separated and identified by SDS solution above critical micelle concentration (CMC) on paper chromatography.The concentration of SDS,ratio of organic modifier and their concentration,fluorescence were investigated.RESULTS:The best concentration of SDS micelle was 0.01～0.02mol*L-1,and the spot effects of micellar chromatography could be improved by adding 2%～4% isopropanol (or n-amyl alcohol)into the mobile phase.CONCLUSION:The simple,rapid,accurate and qualitative method could be used to analyze and isolate flavonoids and their soluble derivatives.

Extraction of diterpenoids from Pteris semipinnata by supercritical CO_2 fluid and their analysis with HPLC-MS / 中草药

Object To establish a high efficient and reliable method for extraction and analysis of the diterpenoids in Pteris semipinnata L. Methods Supercritical CO 2 fluid modified with alcohol was used to extract the diterpenoids in P. semipinnata, the extracting conditions were optimized by orthogonal design method, and the modifying solvent was investigated through total ions chromatograghy normalization. A quadrupole mass spectrometer coupled with atmospheric pressure chemical ionization interface was employed as a detector for HPLC, the -1 ion was used as selective ion for the detecting of ent 11 ? hydroxy 15 oxo kaur 16 en 19 olic acid (5F) in selective ion monitoring (SIM) mode. The peak area of SIM and tolal ion chromatogram (TIC) were used for quantitative determination. As an example of its application, this method was used to determinate the content of 5F as antitumor diterpenoids in P. semipinnata. Results The optimized conditions for supercritical CO 2 fluid extraction are 25 MPa, 60 ℃, 15% methanol, flow rate 3 0 mL/min; analytical column was Diamonsil ODS (150 mm?4 6 mm, 5 ?m); the mobile phase of HPLC was CH 3CN 2 mmol/L NH 4AC (35∶65), flow rate 1 0 mL/min, injection volume 5 ?L; the standard curve showed good linearity over the range of 0 05-2 5 ?g; the limit of detection is 0 4 ng; the recovery is 97 8% (n=3). Conclusion This method is highly efficient, sensitive, and selective, which can be applied to study the antitumor drug of diterpenoids in P. semipinnata and to establish the drug standard.

Preparation of quercetin-arginine complex / 中草药

Object To prepare the water soluble quercetin arginine complex (QAC) and widen the administration path of quercetin (QUE). Methods Definite QUE and L arginine were refluxed in alcohol to prepare QAC. The QAC structure was identified by micellar paper chromatography, UV spectrometry, IR spectrometry, and X ray diffraction. Results QAC was prepared from QUE and L arginine in molar ratio 1∶1. The inhibitory activity of QAC that existed stably in room temperature on cancer cell growth was as strong as that of QUE, and the solubility of QAC in water was remarkably enhanced. Conclusion The above preparation method is simple and available, and it is suitable to improve the bioavailability.

Inhibitory effect of quercetin on recombinant human protein kinase CK2 holoenzyme and its kinetics analysis / 中草药

Object To study the direct effect of quercetin on recombinant human protein kinase CK2 holoenzyme and its kinetics. Methods Recombinant human protein kinase CK2? and ? subunits were cloned and expressed by gene engineering, and were purified. The two subunits were mixed at the same molar ratio, thus reconstituting CK2 holoenzyme, which displayed the maximum bioactivity. The CK2 activity was assayed by detecting incorporation of 32 P of [? 32 P] ATP into the substrate in the various conditions. Results The recombinant human protein kinase CK2 was the second messenger (Ca 2+ , cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. It was found that quercetin strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 522 nmol/L, which was much more effective than DRB and A3, known as CK2 special inhibitors. Kinetic studies of quercetin on recombinant human protein kinase CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein. Conclusion Quercetin is a potent inhibitor of recombinant human protein kinase CK2. The inhibition may be another molecular mechanism of antitumor effect of quercetin. This study provides a simple and rapid screening method for the development of more effective inhibitors of recombinant human protein kinase CK2.

Effect of disodium quercetin-7,4′-disulfate on enzyme activity of recombinant human phosphoinositide 3-kinase p110 catalytic subunit / 中草药

Objective To study the effect of disodium quercetin-7,4′-disulfate (SQDS) on activity ofrecombinant human phosphoinositide 3-kinase (PI3-K) p110?catalytic subunit. Methods Recombinanthuman PI3-K p110?catalytic subunit was expressed by gene engineering. PI3-K activity was assayed byincubating recombinant PI3-K p110?with phosphatidylinositol-4,5-bisphosphate and [?-32P] ATP;the32P-radiolabeled lipids were extracted with chloroform and methanol,then assessed by scintillation counter.Results SQDS showed inhibition on the recombinant p110?catalytic subunit with IC5014.88?mol/L.Conclusion SQDS is an inhibitor of PI3-K. The recombinant PI3-K p110?catalytic subunit might be usedas a molecular target for simpler screening and development of more effective inhibitors of PI3-K.

Effect of a diterpenoid 5F from Pteris semipinnata on expression of NF-?B (P65), Smad3 protein and NF-?B (P65), ETS-1 mRNA in highly metastatic ovarian carcinoma HO-8910PM cells / 中草药

Objective To study the effect of a diterpenoid 5F (ent-11?-hydroxy-15-oxo-kaur-16-en-19-oic acid) isolated from Pteris semipinnata on the expressions of NF-?B (P65), Smad3 protein and NF-?B (P65), ETS-1 mRNA, and to investigate the antitumor mechanisms of 5F. Methods MTT assay was used to examine the effect of 5F on proliferation of HO-8910PM cells. The expressions levels of NF-?B (P65) and Smad3 proteins were assessed by Western blot analysis. RT-PCR assay was used to assess the expression levels of NF-?B (P65) and ETS-1 mRNA. Results The proliferation of HO-8910PM cells can be inhibited by 5F in a dose-dependent manner. The expression levels of NF-?B (P65) and Smad3 proteins decreased obviously in HO-8910PM cells after treatment with 25—100 ?mol/L 5F for 24 h, and the effects appeared in a dose-dependent manner. 5F down-regulated significantly the expression of NF-?B (P65) mRNA, down-regulated gently the expression of ETS-1 mRNA. Conclusion The antitumor activity of 5F is involved in the expression of NF-?B (P65), Smad3 protein and NF-?B (P65), ETS-1 mRNA.