Objective To investigate the toxic effect of the carboxyl-terminal peptide of β-amyloid precursor protein (APPC31) on Neuro2a cells as well as its role in the toxic process in Neuro2a cells induced by Aβ42 in vitro.Methods The plasmid vector and the APPC31 construct were transiently transfected into Neuro2a cells respectively by lipofectamine 2000.The viability of the cells was measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 48 h after transfection,and their morphocytology was observed by 4', 6-diamidino-2-phenylindole (DAPI) nucleus staining.Afterword different constructs including vector, WTAPP695, APP( D664A), the amino-terminal peptide of β-amyloid precursor protein (APP△C31) and APPC31 were transiently transfected into Neuro2a cells respectively via the same method.At 24 h after transfection Aβ42 was added into the culture medium of Neuro2a cells with the desired concentration for another 24 h for cell studies.The viabihty and morphocytology of the cells were measured by using the MTT assay and DAPI nucleus staining, respectively.Results When incubated in the absence of Aβ42, the viability of cells transfected with vector and APPC31construct were 0.81 ±0.10 and 0.88 ±0.12 respectively, and accordingly there was no significant difference between the these two groups (t = - 0.78, P = 0.48 ); meanwhile no obvious cell nuclear morphological changes of apoptosis or death occurred.However when incubated in the presence of Aβ42, the viability of cells transfected with vector, WTAPP695, APP( D664A), APP△C31 and APPC31 constructs were 0.82 ±0.01, 0.78 ±0.03, 0.55 ±0.04, 0.81 ±0.04, 0.78 ±0.02 and 0.54 ±0.02 respectively.The viability of cells transfected with WTAPP construct and APPC31 construct decreased significantly ( F = 47.53, P ＜0.05) compared with the control group, meanwhile cells displayed condensed nuclear and even nuclear fragmentation.Conclusions In vitro, over-expression of a certain level of APPC31 in Neuro2a cells fails in causing cell death, but this short peptide enhances cytotoxicity induced by Aβ42 in Neuro2a cells.Thus,these results provide the experimental basis for the further explication of the pathogenesis of Alzheimer's disease.

Objective To investigate the expression of aquaporins-3 (AQP3) in amniotic epithelial cells regulated by cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal pathway and to explore the mechanisms of its expression.Methods The amniotic epithelial cells were collected from 30 patients who underwent elective caesarean sections at term with normal amniotic fluid volume and primarily cultured.The cultured cells were treated with (1) forskolin groups: different concentration (0,2.5,5,50 or 100 μmol/L) of forskolin treated cells for 2 hours,and the optimal concentration of forskolin treated cells with different time (0,1,2,10 or 20 hours) ; (2)SP-cAMP groups: different concentration (0,2.5,5,50 or 100 μmol/L) of SP-cAMP treated cells for 2 hours,and the optimal concentration of SP-cAMP treated cells with different time (0,1,2,10 or 20 hours); (3)H-89 groups: different concentration (0,5,10,50 or 100 μmol/L) of H-89 treated cells for 2 hours,and the optimal concentration of H-89 treated cells with different time (0,1,2,10 or 20 hours).The level of intracellular cAMP and activity of PKA were detected by using ELISA,and immunohistochemistry was used to detect the localization of AQP3,the protein expression of total cAMP-response element binding protein (CREB) and phospho-CREB (p-CREB) and AQP3 were assessed by western blot analysis.Cell proliferation was assessed by cell counting kit-8 (CCK-8)assay.Results (1) The brown staining of AQP3 was detected in both cell membrane and cytoplasm in each group.(2) There was no significant change of the cell proliferation rate among groups with different concentration of forskolin,SP-cAMP and H-89 treatment (P ＞ 0.05).(3) After different concentration of forskolin treated 2 hours,the expression of total CREB had no significant difference among them(P ＞ 0.05).While the expression of cAMP level,PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 2.5 μmol/L,5 μmol/L,50 μmol/L forskolin group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 5 μmol/L forskolin group were higher than that in 2.5 μmol/L and 50 μmol/L (P ＜ 0.05).The optimal forskolin concentration was 5 μmol/L.(4) After different concentration of SP-cAMP treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 5 μμmol/L,50 μmol/L SP-cAMP group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 50 μmol/L SP-cAMP group were higher than that in 5 μmol/L (P ＜0.05).The optimal SP-cAMP concentration was 50 μmol/L (5) After different concentration of H-89 treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were lower in 10 μmol/L,50 μmol/L and 100 μmol/L H-89 group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 10 μmol/L H-89 group were lower than that in 50 μmol/L,100 μmol/L (P ＜ 0.05).The optimal H-89 concentration was 10 μmol/L.(6) p-CREB and AQP3 protein expression were significantly lower in 5 μmol/L forskolin combined 10 μmol/L H-89 incubating 2 hours group when compared with 5 μmol/L forskolin,but higher than that in 10 μmol/L H-89 treated group (P ＜ 0.05).Total CREB was no significant difference among the three groups (P ＞ 0.05).Conclusion cAMP-PKA signal transduction pathway may regulate AQP3 protein expression in human amniotic epithelial cells.

Objective·To explore plasma immune and inflammatory proteins that could serve as potential screening markers for Alzheimer's disease (AD).Methods·Healthy controls (n=19) and AD patients (n=19) were enrolled.Plasma samples were collected and 70 kinds of immune and inflammatory proteins were detected.The immune and inflammatory proteins associated with AD were screened by Mann-Whitney U test and partial correlation analysis.Discriminant analysis was used to develop multi-protein combined algorithm to distinguish plasma samples of AD patients from those of healthy controls.Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficacy for the multi-protein combined algorithm.Results·Among the 70 proteins analyzed,23 were significantly higher in AD patients (P＜0.05),among which 19 were strongly correlated with AD (P＜0.05).These 19 proteins were analyzed with Wilks' lambda stepwise analysis to develop discriminant algorithm for detecting plasma samples of AD.Finally,the discriminant algorithm established by 11 plasma immune and inflammatory proteins (EGF,GRO,MDC,MCP-1,MCP-2,MCP-4,TARC,SCF,TRAIL,CTACK,GCP-2) was found to have an optimal diagnostic efficacy (AUC=0.994).The optimal cutoff value of the algorithm was-0.609.When the optimal cutoff value was obtained,the sensitivity of the equation could reach 100％ and the specificity could reach 94.7％.Conclusion·The discriminant equation composed of the above 11 plasma immune and inflammatory proteins has the potential to assist AD screening.

Objective To evaluate the efficacy and tolerability of zonisamide (ZNS) as add-on therapy in patients with refractory partial seizures.Methods In this Chinese muiticenter, double-blind, randomized, placebo-contrclled trial, ZNS was compared with placebo add-on therapy in 217 patients (intent-to treat (ITT) population) with uncontrolled partial-onset seizures.All patients entered a 3-month baseline period followed by a 4-week titration interval and a 12-week maintenance period.The starting dose of ZNS group was 100 mg/d, increased by 100 mg/d every week and reached the goal of 400 mg/d.The main outcome was measured by the median of the percentage of decreased seizure frequency.The secondary ouwomes points included the percentage of patients who had seizure attacks decreased by more than 50%,and adverse events.Results The median of the percentage of decreased seizure frequency in ZNS group was 33.33%, and the placebo group was 0.Thirty-eight patients (34.23%) experienced more than 50% reduction in the seizure frequency in ZNS group, compared with 19.81% of patients (21 cases) in the placebo group (χ2 =5.7159,P =0.0168) ; Moreover, 13 (11.71%) patients in ZNS group and 5 patients in placebo group were seizure free, 25 patients in ZNS group and 16 patients in placebo group who had seizure attacks decreased by more than 50%.The availability rate in ZNS group was higher than placebo group (34.23% vs 19.81%, U=2.4701, P=0.0135).The most common adverse events in ZNS group were drowsiness, fatigue, decreased appetite, gastrointestinal complaints, insomnia and constipation.Conclusion Zonisamide treatment was generally well tolerated and was associated with significant reductions in seizure frequency as adjunctive treatment for partial-onset seizures.