Objective:To prepare and test tetrameric sH-2K~d-HBc complex for the further measurement of the specific CTL response.Methods:PE labled streptavidin with 4 biotinylated binding sites can bind to 4 biotinylated monomer to form the corresponding tetramer.Mice were immunized via different methods of genetic immunization by use of the construted pcDNA3-C plasmid to get the specific CTLs.Then our prepared tetramer was applied to stain the specific CTLs by the analysis of flow cytometry.Results:We applied our prepared tetramer to stain the cells from the experimental groups and control group.The results showed the tetramer was able to discriminate the frequencies of specific CTL induced by the three immunol methods(0.24%,0.26%,0.36% vs 0.07%,P≤0.05).This demonstrated that the prepared tetramer could bind its targets specifically and efficiently.The three immunol methods induced different levels of immune responses.Compared with the traditional muscle injection,gene gun induced weaker humoral immune response and stronger cellular immune response,and hydrodynamic injection induced the strongest humoral and cellular immune responses.Conclusion:Have successfully constructed the sH-2K~d-HBc tetramer.The techniques and methods can be used for preparation of tetramers of other types of MHCⅠ molecules.

ObjectiveTo investigate the effect of MK-801 on levodopa-induced dyskinesia (LID)in Parkinson' s disease (PD). MethodsRat models ( n = 25) of Parkinsonism related motor complications were established and were randomly divided into levodopa-induced dyskinesia (LID) group (n = 10), MK801 treatment group (n = 10) and PD group (n =5). Another 5 rats were served as control group. The behaviors of LID rats treated with MK-801 were observed. Immunohistochemistry and Western blot analysis were used to determine the expression of β-arrestin1 in the striate of rats.Results After MK-801 treatment, abnormalinvoluntarymovementscores and peakturning weredecreasedin LIDrats.Immunohistochemistry showed that β-arrestin1-positive cells of the lesioned side ((2. 95 ± 0. 44) × 104) in LID rats were decreased compared to the contralateral side ( ( 3.78 ± 0. 37 ) × 104, t = 5. 415, P ＜ 0. 05 ).Western blot showed that the levels of β-arrestinl in PD group ( presented as lesioned side/contralateral side) were ( 81.02％ ± 2. 23％ ). The levels of β-arrestin1 (64. 88％ ± 3. 10％ ) were deceased in LID rats compared to PD rats ( t = 9.47, P ＜ 0. 01 ). However, the levels of β-arrestin1 ( 89. 26％ ± 1.90％ )were increased in MK-801-treated rats (t = 14. 82, P ＜0. 01). ConclusionsMK-801 reduces LID in PD rats. The beneficial effect of MK-801 may be mediated through the increased expression of β-arrestinl which in turninhibits the overactivation of glutamate receptors.

Objective To investigate the relationship between G protein-coupled receptor kinase 6 (GRK6) and N-methyl-D-aspartate (NMDA) receptor in the mechanism study underlying motor complications in Parkinson's disease (PD).Methods The rat models (n= 25) of Parkinsonism related motor complications were established and were randomly divided into levodopa-induced dyskinesia (LID) group (n= 10,intraperitoneal injection of levodopa for 23 d),MK-801 treatment group (n= 10,intraperitoneal injection of MK-801 at day23 after intraperitoneal injection with levodopa for 22 days) and PD group (n= 5,intraperitoneal injection of vitamin C).Another 5 rats were served as controls (sham-operation group).The behavior changes of rats in MK-801 treatment group were observed,and the expression of GRK6 in the striate of rats was detected by immunohistochemistry and Western blot.Results After the chronic treatment with levodopa methyl ester,PD rats displayed abnormal involuntary movements,which was similar to levodopainduced dyskinesia in PD patients.Immunohistochemistry showed that GRK6-positive cells of lesion side were decreased in LID rats as compared with PD rats [(3.23±0.41 ) × 103 vs.(4.81 ± 1.31 ) ×103,P＜0.01].Rats in MK-801 treatment group displayed the decreased AIM scores and increased peak rotation,and the increased GRK6-positive cells of lesion side as compared with LID rats (P＜0.05).Western blot showed that the levels of GRK6 was 83.7% ±2.1% in PD group (presented as lesion side/unlesion side),76.8% ± 2.2% in LID group and 91.1% ± 2.7% in MK-801 treatment group (intergroups comparison:all P＜0.05).These results were in accordance with the results of immunohistochemistry.Conclusions Antagonist of NMDA receptor can be used to reduce the motor complications in rats.It may be due to increased GRK6 which inhibits the overactivation of glutamic acid receptors.

Difference between transcranial magnetic stimulation and transcranial direct current stimulation in theory, safety, detection of brain function and clinic treatment were reviewed in order to help reasonably select and effectively apply them in clinic.

A highly sensitive electrochemical deoxyribonucleic acid(DNA) biosensor based on multi-walled carbon nanotubes(MWNT)/Ag-TiO_2 composite film was developed. The solution containing Ag-TiO_2-MWNT composite was casted on the carbon paste electrode surface to form a robust film, which combine the advantages of the good biocompatibility of Ag-TiO_2 naocomposite and the fine conductivity, as well as the large active surface area of carbon nanotubes. The composite could greatly improve the immobilization capacity of the probe DNA. The morphologies and electrochemistry of the nanocomposite film were investigated by scanning electron microscopy and electrochemical techniques including electrochemical impedance spectroscopy and cyclic voltammetry, respectively. DNA hybridization events were monitored by a label-free method of electrochemical impedance spectroscopy. This label-free electrochemical impedance DNA biosensor showed high sensitivity and selectivity for phosphinothricin acetyltransferase gene sequence assay. The multicomponents films also displayed a high stability during repeated regeneration and hybridization process.

Gas chromatography with mass spectrum detector ( GC-MSD) coupled with purge-and-trap system was set up to analyze the concentration of isoprene in natural waters. The best experimental conditions were established, including purge gas flow rate ( 50 mL/min ) , purge time ( 15 min ) , the optimum capillary column ( Rt-Alumina BOND/KCl) and the appropriate condition of temperature programming. When analyzing isoprene in natural waters, the precision was <4% (n=6), the detection limit was 0. 5 pmol/L and the recovery was 91%-102%. The preservative experiment showed that there was no obvious variation in sample concentrations of isoprene within 60 days. The concentrations of isoprene measured with the method ranged from 60 . 8 to 278 . 7 pmol/L in the Jiaozhou Bay and its adjacent river estuaries and from 44 . 7 to 77 . 2 pmol/L in Yellow River estuary, which was in good accord with those results reported in literatures in other coastal waters. In conclusion, the analytical method could meet the requirements of the analysis of concentration of isoprene in natural waters.

Objective To study the relationship between oxidative stress and endothelial progenitor cells(EPCs)count in the first-degree relatives of diabetes mellitus(FDRs).Methods Three groups were evaluated with 40 type 2 diabetes mellitus(T2DM)patients,38 FDRs and 30 healthy individuals as the control(NC).Fasting plasma glucose(FPG),glycosylated hemoglobin(HbA1c),TG,TC and fasting plasma insulin concentrations were measured and homeostasis model assessment-insulin resistance(HOMAIR)was calculated.Quantity of EPCs and flow-mediated dilation(FMD)were evaluated.Malonaldehyde(MDA),glutathion peroxidase(GSH-Px),erythrocuprein(SOD)and total anti-oxidative capacity(TAO-C)were measured.Results In T2DM group FPG[(7.86 ±0.77)mmol/L]and HbA1c[(7.24 ±0.20)％]were significantly higher than those in NC[FPG(4.90 ± 0.35)mmol/L,HbA1 c(5.34 ± 0.37)％]and FDRsgroup[FPG(5.13±0.95)mmol/L,HbA1c(5.36 ±0.36)％](all P values ＜0.05).TC in T2DM group[(5.88 ±0.76)mmol/L]was higher than in NC[(4.66±0.90)mmol/L]and FDRs [(4.95 ± 0.76)mmol/L].HOMA-IR was 0.48 ± 0.25 in NC,0.81 ± 0.46 in FDRs and 1.47 ± 0.24 in T2DM group,P ＜ 0.01.In T2DM group,the plasma levels of SOD[(69.30 ± 2.21)U/ml],TAO-C [(7.30 ± 0.29)U/ml]and GSH-Px[(856.5 ± 9.01)U/ml]were significantly lower than those in NC [SOD(75.33 ±3.63)U/ml,TAO-C(8.17 ±0.58)U/ml and GSH-Px(938.1 ± 19.35)U/ml]and FDRs group[SOD(74.91 ±4.53)U/ml,TAO-C(8.24 ±0.46)U/ml and GSH-Px(936.9 ± 15.78)U/ml](all P values ＜ 0.01).Serum level of MDA was(2.87 ± 0.63)μmol/L in NC,(3.28 ± 0.71)μmol/L in FDRs and(3.69 ± 0.39)μmol/L in T2DM group(P ＜ 0.01).The quantity of EPCs and FMD％ were 96.75 ±8.11 and 8.36 ± 2.21 in NC,83.34 ± 12.43 and 6.78 ± 0.98 in FDRs and 58.45 ± 7.58 and 2.86 ± 0.35 in T2DM group with statistical differences between different groups(all P values ＜ 0.05).Pearson correlation analysis showed that lnHOMA-IR was positively correlated with MDA(r =0.486,P ＜0.05)and negatively correlated with SOD,TAO-C,GSH-Px(r =-0.426,-0.601,-0.524,all P values ＜ 0.05)in FDRs group.Conclusions Insulin resistance,oxidative stress,decreased quantity of EPCs and impairment of endovascular function have already occurred in the FDRs of T2DM with normal glucose tolerance and they are correlated with each other.

To investigate the effects of Tianqi Pingchan (TQPC) Granule, a compound traditional Chinese herbal medicine with antitremor activity, on levodopa-induced dyskinesia and the expression of G protein-coupled receptor kinase 6 (GRK6) in rats with Parkinson disease (PD).

Objective: To study the inhibitory effect of total alkaloids from lotus seed on human hepatoma HepG2 cells.Methods: The effect of total alkaloids from lotus seed on the growth of HepG2 cells was studied by CCK-8 kit.The apoptosis rate of HepG2 cells was detected by flow cytometry.Results: When the action time was the same, with the increase of drug concentration, the inhibitory rate of total alkaloids from lotus seed on HepG2 cells increased, in a dose-dependent manner.At 72 h, the half inhibitory concentration (IC50) of total alkaloids from lotus seed on HepG2 cells was 1.501 μg·ml-1.At the same concentration, the inhibitory rate of the total alkaloids from lotus seed on HepG2 cells increased with the extension of the action time.At 72 h, the inhibition rate of 10 μg·ml-1 total alkaloids from lotus seed reached 72%.After treated with the total alkaloids from lotus seed at different concentrations, the apoptosis rate of HepG2 cells significantly increased in a dose-dependent manner.Compared with the blank control group, the difference was statistically significant (P <0.05), and the apoptosis rate of HepG2 cells was 85.6% treated with 20 μg·ml-1 total alkaloids from lotus seed.Conclusion: The total alkaloids from lotus seed can induce cell apoptosis and inhibit the proliferation of human hepatoma HepG2 cells.

Tripterygium glycosides preparation which extracted from the traditional Chinese herb Tripterygium wilfordii (TWHY), was widely used to treat the autoimmune diseases. Previous works demonstrated that TWHF had potent anti-inflammatory and immunosuppressive properties. But the different quality and high incident rate of side effects of different manufactures inhibited its clinical application. Since TWHF had been generally known to play a therapeutical effect by synergism of multiple constituents, it was necessary to build the relationship between the HPLC fingerprint and bioactivity so as to ensure the quality safety and efficacy. The HPLC fingerprint showed that description and content of peaks from different manufactures were diverse. Only 11 common peaks were found. In this study, mice spleen cells stimulated by Con A were used to test the proliferation inhibition bioactivity of TWHF preparations, which were incubated with 30, 15, 7.5, 3.75, 1.88 and 0.94 mg x L(-1) TWHF preparations for 48 h. The results showed that mice spleen cells proliferation was inhibited by all TWHF preparations significantly compared with the control group, which suggested the TWHF preparations showed immune suppress activity. The TWHF preparations from 7 manufacture showed different IC50 value, which might belong to different contents which showed in the HPLC fingerprint. Moreover, a relationship between the HPLC fingerprint and the bioactivity were established to identify important constituents by grey relational analysis (GRA). The result showed that all the contents were relative with the IC50, especially No. 5 and 10 peaks, but No. 1 peak, which was proved to be triptolide, had few contribute to the inhibition of mice spleen cells proliferation. The study of relationship between the HPLC fingerprint and the IC50 by GRA could help to investigate mechanism of bioactive and provide an evidence for the quantification of multi-constituents.
Animals ; Anti-Inflammatory Agents ; analysis ; pharmacology ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Glycosides ; analysis ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; drug effects ; Tripterygium ; chemistry