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.

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.

Human promyelocytic leukemia HL - 60 cells labelled with 3H - inositol were treated with vincristine (VCR), it was found that the radioactivity of 3H-phosphatidyl inositol - 4-phosphate (PIP) and 3H - Phosphatidyli-nositol - 4,5 - bisphosphate(PIP2) had no obvious change, but that of 3H-phosphatidyl inositol (PI) decreased rapidly, only accounted for 38. 55%~61.01% of the control during 1~ 120 min of the treatment. It showed that VCRdid not affect the activity of PI kinase and PIP kinase, but enhanced the hydrolysis of PI of HL - 60 cells. The inhibitorty effect of VCR upon cell proliferation and DNA synthesis was observed after above biochemical changes,these results suggest that VCR may inhibit HL-60 cells proliferation by interfering the metabolism of phosphoininositides.

Aim To study the effect of sodium quercetin-7-sulfate (SQMS) on activity of recombinant human phosphoinositide 3-kinase (PI3-K) p110? catalytic subunit.Methods Recombinant human PI3-K p110? catalytic subunit was expressed by gene engineering.PI3-K activity was assayed by incubating recombinant PI3-K p110? with phosphatidylinositol-4,5-bisphosphate and [?-~ ~32 P]ATP; the ~ ~32 P-radiolabeled lipids were extracted with chloroform and methanol, and assessed by scintillation counter.Results SQMS showed inhibition on the recombinant p110? catalytic subunit with IC_~50 13.14 ?mol?L~-1 .Conclusions SQMS is an inhibitor of PI3-K. The recombinant PI3-K p110? catalytic subunit might be used as a molecular target for simpler target screening more effective inhibitors of PI3-K.

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.

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.

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.

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

OBJECTIVE:To establish a method for determination of the content of quercetin in quercetin-PVP solid dis?persions.METHODS:The detection wavelength for dertermination of quercetin content was set at374nm.RESULTS:The caliberation curve was linear in the range of5.0～25.0?g/ml(r=0.9998).The average recovery was99.12%,RSD=0.70%(n=6).CONCLUSION:This method is stable,quick,accurate and simple,and is suitable for quality control of quercetin in quercetin-PVP solid dispersions.

Aim To study the effect of cantharidin on the expression of NF-?B(P65), FAK protein and the level of phosphorylated FAK, and to investigate posssible mechanism of cantharidin on the metastasis-associated ability of human highly metastatic ovarian carcinoma HO-8910PM cells. Methods MTT assay was used to examine the effect of cantharidin on proliferation of HO-8910PM cells after 24 h treatment; The expression levels of NF-?B(P65), FAK protein and the level of phosphorylated FAK were assessed by Western blot analysis.Results The expression level of of NF-?B(P65) protein decreased obviously in HO-8910PM cells treated with 5～20 ?mol?L -1 cantharidin for 24 h, and the effects appeared in a dose-dependent manner. Cantharidin significantly up-regulated the expression of FAK, down-regulated the level of phosphorylated FAK.Conclusion The antitumor activity of cantharidin is involved in the expression of NF-?B(P65) and the level of phosphorylated FAK.