Objective To investigate a method for the purification of the N?terminal peptide fragment(NT)of the myocardial calcium channel Cav1.2,and characterize its interaction with calmodulin(CaM). Methods EscherichiacoliBL?21 cells were transformed with plasmid pGEX?6p?3/NT harboring the NT?GST fusion gene. The cells harboring pGEX?6p?3/NT were cultured and protein expression was induced with isopropyl?β?D?thiogalactoside(IPTG). Then,the GST?NT fusion protein was purified by using glutathione Sepharose 4B(GS?4B)beads. GST was cleaved off with the PreScission protease,and SDS?PAGE was performed to detect the purity and relative molecular weight of the purified peptide. Further, GST pull?down assay was performed to characterize the interaction of the NT peptide with CaM. Results SDS?PAGE analysis showed that the NT peptide was successfully purified,with high purity. Results of the GST pull?down assay showed that the NT peptide could interact with CaM. Conclusion This study establishes a method for the purification of the NT peptide and lays the foundation for further research on the interaction partners and biological functions of NT.

OBJECTIVETo study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment.

METHODSAn eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed.

RESULTSA stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed.

CONCLUSIONExogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estradiol ; pharmacology ; Estrogen Antagonists ; pharmacology ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Humans ; Tamoxifen ; analogs & derivatives ; pharmacology ; Transfection

OBJECTIVE: To explore the correlation between the interleukin-17 (IL-17) level of peripheral blood and aggression of bipolar mania. METHODS: Thirty-six patients of bipolar mania were selected as experimental group by DSM-IV-TR and received treatment with quetiapine and lithium. Thirty-six healthy volunteers with similar age and gender were selected as control group. The level of IL-17 at baseline in each group and the level of IL-17 in the experimental group after treatment for 2, 4 and 8 weeks were detected by ELISA. RESULTS: The level of IL-17 in experimental group at baseline, after treatment for 2 and 4 weeks were all significantly higher than that in control group. After 8 weeks treatment, there was no significant difference between the two groups (P > 0.05). After 2, 4 and 8 weeks treatment, the total score and aggression score of Young Mania Rating Score (YMRS) were significantly lower than the baseline level (P < 0.05). In experimental group, the level of IL-17 was positively correlated with the two scores of YMRS at baseline (P < 0.05). CONCLUSION Bipolar mania may be related to the up-regulation of IL-17. The level of IL-17 is related to the severity of manic symptoms at baseline, especially aggression symptom.
Aggression/drug effects* ; Antipsychotic Agents/therapeutic use* ; Biomarkers/blood* ; Bipolar Disorder/drug therapy* ; Case-Control Studies ; Diagnostic and Statistical Manual of Mental Disorders ; Double-Blind Method ; Humans ; Interleukin-17/metabolism* ; Lithium Compounds/therapeutic use* ; Quetiapine Fumarate/therapeutic use* ; Treatment Outcome

OBJECTIVETo evaluate the relation of dose intensity and efficacy, toxicity in advanced breast cancer treated with paclitaxel as a single agent.

METHODSSeventy-one patients with advanced breast cancer received paclitaxel as a single agent with different dose intensities. According to the phase I or phase II trial, the standard dose intensity of paclitaxel was defined as 58.3 mg.(m(2))(-1).week(-1). The dose of paclitaxel was 175 mg/m(2) given every three weeks, ranging 33.3 - 70.3 mg.(m(2))(-1).week(-1) [median delivered dose intensity 58.82 mg.(m(2))(-1).week(-1)]. Efficacy and toxicity was evaluated.

RESULTSThe overall response rate in this group of advanced breast cancer was 40.8%. Responses were seen in lungs, soft tissue, bone and liver, with the response rates of 52.0%, 38.0%, 12.5%, 7.7%, respectively. When the relative dose intensity (RDI) was > 1.0, 0.9 - 1.0, < 0.9, the response rates were 44.2%, 47.6%, 0, respectively. The difference between the group (RDI >/= 0.9% - 1.0%) in 7 patients and the group (RDI < 0.9) was significant (P < 0.05). Toxicity was well tolerated, with the efficacy decreased as soon as the RDI had been reduced without embarrassing the toxicity.

CONCLUSIONPaclitaxel as a single agent therapy with standard dose intensity is effective and well tolerated by patients with advanced breast cancer.

Adult ; Aged ; Antineoplastic Agents, Phytogenic ; administration & dosage ; adverse effects ; Bone Neoplasms ; drug therapy ; secondary ; Breast Neoplasms ; drug therapy ; pathology ; Dose-Response Relationship, Drug ; Female ; Humans ; Leukopenia ; chemically induced ; Liver Neoplasms ; drug therapy ; secondary ; Middle Aged ; Neoplasm Staging ; Paclitaxel ; administration & dosage ; adverse effects ; Remission Induction

OBJECTIVE: To investigate the protective effects of procyanidin on periprosthetic osteolysis caused by tricalcium phosphate (TCP) wear particles in the mouse calvaria and its mechanism. METHODS: Forty-eight male ICR mice were randomly divided into sham group, TCP group, and procyanidin (0.2 mg/kg, 1 mg/kg, 5 mg/kg)-treated group (n=12). A periprosthetic osteolysis model in the mouse calvaria was established by implanting 30 mg of TCP wear particles onto the surface of bilateral parietal bones following removal of the periosteum. On the 2 day post-operation, procyanidin (1 mg/kg, 5 mg/kg) was locally injected to the calvaria under the periosteum every other day. After 2 weeks, all the mice were sacrificed to collect the blood samples and the calvaria. Periprosthetic osteolysis and osteoclastogenesis in the mouse calvaria were observed by tartrate resistant acid phosphatase (TRAP) staining and HE staining. mRNA levels of TRAP, capthesin K, c-Fos and NFATc1 in the periprosthestic bone tissue were examined by real-time fluorescence quantitative PCR. Serum contents of total anti-oxidation capacity (T-AOC) and MDA, and superoxide dismutase (SOD) activity were determined by chemical colorimetry. Protein expressions of autophagic biomarkers such as Beclin-1 and LC-3 in periprosthetic bone tissue of the calvaria were examined by Western blot. RESULTS: Compared with sham group, periprosthetic osteolysis, osteoclastogenesis, mRNA levels of TRAP, capthesin K, c-Fos and NFATc1, and serum MDA content were increased significantly in the TCP group (P＜0.05), whereas serum T-AOC level and SOD activity were decreased. The protein expressions of Beclin-1 and LC-3, and the conversion of LC3-II from LC3-I were both up-regulated markedly in the mouse calvaria of TCP group (P＜0.05). Compared with TCP group, osteolysis, osteoclastogenesis, mRNA levels of TRAP, capthesin K, c-Fos and NFATc1 and serum MDA content were decreased obviously in the procyanidine group (P＜0.05), serum T-AOC level and SOD activity were increased, the expressions of Beclin-1 and LC-3, and the conversion of LC3-II from LC3-I were down-regulated obviously in the mouse calvaria of procyanidin group (P＜0.05). CONCLUSION Procyanidin has a protective effect of periprosthetic osteolysis caused by TCP wear particles in the mouse calvaia, its mechanism may be mediated by inhibition of oxidative stress and autophagy.
Animals ; Autophagy ; Biflavonoids ; pharmacology ; Calcium Phosphates ; adverse effects ; Catechin ; pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Osteolysis ; Oxidative Stress ; Proanthocyanidins ; pharmacology ; Prostheses and Implants ; adverse effects ; Random Allocation ; Skull

Obesity-associated protein (FTO) is an important m6A demethylase that regulates RNA methylation modification and can promote the proliferation of acute myeloid leukemia(AML) cells. FTO regulates the methylation level of AML through multiple cellular signaling pathways such as FTO/RARA/ASB2, FTO/m6A/CEBPA, and PDGFRB/ERK, and participates in the occurrence, development, treatment and prognosis of AML. At present, studies have found that a variety of inhibitors targeting FTO have shown good anti-leukemia effects, and the study of FTO will provide new ideas for the treatment of AML. This review focus on the mechanism of action of FTO in AML and the research progress of FTO inhibitors in AML.
Humans ; Methylation ; Leukemia, Myeloid, Acute/genetics* ; Prognosis ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism*

Objective To determine the mass concentration in human plasma hydromorphone by established HPLC -MS/MS method.Methods The treatment of plasma with solid-phase extraction, selec-tion Kromasil 100 -5 SIL, E97915 column with 95% acetonitrile -5 mmol· L-1 ammonium acetate as the mobile phase gradient elution with positive ion, multiple reaction monitoring mode mass concentration of the sample was measured.Results The plasma samples, hydromor-phone in good ( r >0.999 8 ) 0.05 -10.00 ng · mL-1 linear relation-ship, the minimum detectable concentration was 0.05 ng· mL-1.Blood samples day and inter-day RSD were less than 15%, the average recovery >70%, and stability were good.Conclusion The method is simple, rapid, sensitive and accurate, specific, kinetics in vivo pharma-cokinetics of hydromorphone apply.

Objective To develop a HPLC -MS/MS method for the determination of hydromorphone -3 -glucuronide in human plasma.Methods The plasma was treated with solid -phase extraction.The column was XTerra?MS C18 column (2.1 mm ×50 mm,5 μm), the mobile phase consisted of 95% acetonitrile -5 mmol · L-1 ammonium bicarbonate with a gradient elution and the flow rate was 0.3 mL· min -1 , column temperature was 20 ℃ with positive ion scan and multiple reaction monitoring mode.The method was validated by investigation of the method specificity ,the standard curve and lower limit of quantification , precision and recovery , matrix effects and stability.Results Hydromorphone -3 -glucuronide had a good linear relationships in the range of 0.5-100.0 ng · mL-1 ( r>0.999 5 ) , the limit of quantification was 0.5 ng · mL-1.Intra -day and inter -day RSD were less than 15%, the average recovery were >60%, and stability was good.Conclusion The method was simple , rapid, sensitive and accurate , suitable for measuring the concentration of hydromorphone -3-glucuronide in human plasma.

Objective To develop a HPLC-MS/MS method for s deter-mination of milnacipran in human plasma.Methods The plasma sam-ples were prepared by protein precipitation.The separation was achieved on ZORBAX SB-Aq column(2.1 mm ×50 mm,3.5μm), eluted with a linear gradient using methanol -0.01% formic acid at the flow rate of 0.4 mL· min -1.The column temperature was 30 ℃.Detection of the analyte was achieved by positive ion electrospray ionization in the multi-ple reaction monitoring ( MRM ) mode.The specificity, lower limit of quantitation and standard curve, precision and recovery rate and stability as well as the matrix effect were investigated.Results The concentration of milnacipran was ranged from 20.0 to 5 00.0 ng · mL-1 ( r>0.999 8 ) , with the lower limit of quantitation of 2.0 ng · mL-1.Intra -day and inter-day relative standard deviations ( RSD ) were both below 15%.The recoveries of low, middle and high concentrations were >96%. Conclusion The method is simple, rapid, sensitive and accurate, specific, suitable for determination of milnacipran in human plasma.

Objective To determine the arbidol concentration in human plasma by established HPLC-MS/MS method.Methods The plasma was treated with protein precipitation,arbidol-d6 was used as internal standard,chromatographic column was Agilent Eclipse XDB-C18 column (2.1 mm × 50 mm,5 μm),column temperature was 30 ℃,mobile phase was methanol-0.1％ formic acid,isocratic elution,flow rate was 0.25 mL · min-1,mass concentration of the sample was measured with positive ion scan and multiple reaction monitoring mode.The specificity,lower limit of quantitation and standard curve,precision and recovery rate and stability as well as the matrix effect were investigated.Results The standard curve of arbidol was y =1.52 × 10-1x-1.38 × 10-3 (r =0.999 7),and in 10-1000 ng· mL-1 good linear relationship,the lowest concentration was 10 ng · mL-1.Intra-day and inter-day RSD of blood samples were less than 15％,the average recovery was ＞95％,and stability was good.Conclusion The method is simple,rapid,sensitive and accurate,specific,suitable for the determination of arbidol concentration in human plasma.