BACKGROUND: One of the key neuropathological changes in Alzheimer disease is that neurofibrils over phosphorylated cytoskeletal protein (such as r and neurofilaments) composed of entwist together, and the phosphorylation of τ protein can be catalyzed by cyclin dependent kinase 5 (CDK5),however whether the phosphorylation of neurofilaments can be catalyzed by CDK5, as well as its role in the pathogenesis of Alzheimer diseases is less acknowledged.OBJECTIVE: To explore the role of over-expression of intracellular CDK5 in the phosphorylation of neurofilamentsDESIGN: Randomized controlled study.SETTING: Biochemical and Molecular Biological Department of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: This study was conduced at Biochemical and Molecular Biological Department of Tongji Medical College, Huazhong University of Science and Technology between February and May 2001. In vitro cultured rat neuroblastoma cell strain (N2a) was adopted as subjects.METHODS: In vitro cultured N2a cells were divided into 2 groups, namely transfection group and non-transfection group. In transfection group,CDK5 gene was transfected into N2a cell line by using liposome transfection technique so as to obtain N2a/CDK5 cell line stably expressing CDK5, immune-precipitation and enzyme activity assay was used to detect the CDK5 activity, meanwhile immunofluorescence technique and immuneblot assay was used to detect CDK5 expression and phosphorylation of neurofilaments.MAIN OUTCOME MEASURES: Phosphorylation of neurofilaments in both groups.RESULTS: In transfection group of N2a cell line, CDK5 expression increased presented by deep coloration of SMI31 antibody and weak coloration of SMI32 antibody, implying hyper-phosphorylation of neurofilaments. Meanwhile, the activity of CDK5 was 3.5 times higher than that in non-transfection group.CONCLUSION: Intracellular over-expressison of CDK5 would lead to hyperactivity of CDK5 and hyper-phosphorylation of neurofilaments, however the hyper-phosphorylation of neurofilamentsmight invlove in the pathological development of AD.

AIM: To explore the effect of receptor tyrosine kinase system mediated by phosphotyrosine phosphatase (PTP) on tau phosphorylation in rat hippocampus. METHODS: Pervanadate (PVN), inhibitor of PTP or inhibitor of glycogen synthase kinase-3 (GSK-3), LiCl were injected into rat hippocampus by stereotaxy technique. The level of tau phosphorylation was detected by Western blot and immunohistochemistry after 24 h of injection. RESULTS: PVN significantly inhibited tau phosphorylation at PHF-1 epitope and the inhibition of tau phosphorylation by PVN was stronger than that of LiCl (P

To study the prevention of dauricine (Dau) on bradykinin (BK) induced alteration of intracellular calcium homeostasis and tau phosphorylation, fluorescence spectrophotometer with dual excitation was utilized to measure the intracellular calcium concentration ([Ca2+]i), MTT to detect cell viability and immuncytochemistry to examine tau phosphorylation. The results showed (1) cells treated with BK 1 μmol/L induced a transit increase in [Ca2+]i in all the cell lines detected, among them, the sustained increase of [Ca2+]i level was only seen in PS1Δ9/APPswe cell at 2 h and 24 h after the treatment. Dau (3μmol/L or 6 μmol/L) prevented BK-induced transit and sustained elevation and fluctuation of [Ca2+]i;(2) BK treatment decreased the cell metabolism detected at 2 h in PS1Δ9/APPswe and Dau antagonized the effect; (3) BK induces Alzheimer-like tau hyperphosphorylation at tau-1 epitope and Dau partially antagonized this effect. In conclusion,Dau inhibits BK-induced disturbance in intracellular calcium homeostasis and tau hyperphosphorylation at tau-1 sites.

Aim To explore the effect of prenatal expo-sure to lipopolysaccharide ( LPS ) on lipid metabolism in mice offspring from the starting point of FAT/CD36 expression.Methods 8-week old C57 mice mated 2∶1, then they were caged separately , marked as preg-nancy 0 d.The pregnant mice were given single intrap-eritoneal injection of 75 μg? kg -1 LPS, and the con-trol received injections of 0.2 mL saline .The perirenal adipose of female mice and epididymis adipose of male mice were collected in 4 w,8 w,12 w,respectively. The weight of visceral adipose tissue and the free fatty acid( FFA) and triglyceride ( TG) of adipose tissue and FAT/CD36 of offspring mice were quantitated .Results The body weight of offspring of LPS group was also significantly higher than that of NS group , and LPS group offspring displayed increased adipose tissue wet weights , the expression of TG and FFA was increased in LPS group compared with NS .Especially , prenatal exposure to inflammatory stimulation resulted in marked increase of FAT/CD36 and abnormal adipocyte development .Conclusions Inflammation induced by prenatal exposure to LPS results in increased body weight , adipose coefficient and FAT/CD36 that might develop into obesity in adult mice .These results are relevant in that anomalous local adipose tissue and FAT/CD36 regulation may be an important mechanism underlying obesity .

In this study, we studied the effect of glycogen synthase kinase-3 (GSK-3) overactivation on neurofilament phosphorylation in cultured cells. After N2a cells were treated with the specific inhibitor (wortmannin) of phosphoinositol-3 kinase (PI-3K) or treated with wortmannin and the specific inhibitor (LiCl) of glycogen synthase kinase-3 (GSK-3), GSK-3 activity and neurofilament phosphorylation were detected by using GSK-3 activity assay, Western blots and immunofluoresence. Our results showed that after treatment of N2a cells with wortmannin for 1 h, overactivation of GSK-3 caused a reduced staining with antibody SMI32 and an enhanced staining with antibody SMI31. When N2a cells were treated with wortmannin and LiCl, the activity of GSK-3 was reduced substantially. At the same time, the phosphorylation of neurofilament was also reduced. The study demonstrated that overactivation of GSK-3 induced hyperphosphorylation of neurofilament and suggested that in vitro overactivation of GSK-3 resulted in neurofilament hyperphosphorylation and this may be the underlying mechanism for Alzheimer's disease.

Objective To analyze the dTP value in the patients with coronary heart disease (CHD) complicating diabetes mellitus (DM) and its relationship with major adverse cardiovascular events (MACE) and rehospitalization.Methods Two hundreds and seventy CHD patients were selected as the research subjects,including 136 cases of non-MD and 134 cases of DM.Their clinical condition was recorded.The indicators such as height,body mass,blood pressure and heart rate were measured.ECG,echocardiography,coronary angiography and other examiantions were carried out.The various indicators were detected.11-dh-TXB2 and 6-k-PGF1a levels were detected in the two groups and then dTP value was calculated.The 1-year follow-up was performed,MACE and rehospitalization were recorded.Epdate software was used for building a database and SPSS 17.0 software was applied for conducting the statistical analysis.Results The dTP level in the f non-DM and DM patients were 1.8 ± 0.6 and 2.0 ± 0.7 respectively,the difference was statistically significant (P＜ 0.05).For the non-DM CHD group,hs-CRP,systolic blood pressure,diastolic pressure,lesions number and severe lesions number were correlated with dTP level(P＜0.05).For the complicating DM CHD group,hs CRP,blood glucose,CHO level,lesions number and severe lesions number were correlated with dTP level(P＜0.05).After 1-year follow-up,MACE had 33 cases (24.3％) in the non-DM group and 44 cases (32.8％) in the DM group respectively,the difference was not statistically significant (P＞0.05).The rehospitalized cases had 12 cases (8.8％) in the non-DM group and 24 cases (17.9 ％).in the DM group respectively,the difference was statistically significant (P＜ 0.05).The dTP levels of MACE occurrence and non-MACE occurrence were 2.3 ± 0.8 and 1.8 ± 0.6 respectively,the difference was statistically significant (P＜0.05).The dTP levels of rehospitalized patients and non-rehospitalized patients were 2.4 ± 1.0 and 1.9 ±-0.6 respectively,the difference was statistically significant(P＜0.05).Conclusion The dTP level in the patients with CHD complicating DM is significantly increased,suggesting that platelet is obviously activated,moreover higher dTP level increases the risk of MACE and rehospitalization.So the anti-platelet therapy should be strengthened.

Objective To study the expression of interleukin-6 (IL-6) and hepcidin in patients with diffuse large B-cell lymphoma (DLBCL) and their significance in anemia. Methods 45 DLBCL patients with or without anemia were analyzed. Peripheral blood samples were collected during diagnosis, and the concentrations of IL-6, hepcidin, serum ferritin and hemoglobin (Hb) were measured. 24 healthy volunteers were collected as controls. Results The levels of plasma hepcidin and IL-6 in patients with DLBCL were (347±171)μg/L and 0.27 ng/L (0-9.61 ng/L), respectively, and compared with those [(175 ± 92)μg/L] and 0 ng/L in healthy controls, the differences were statistically significant (both P<0.001). Plasma hepcidin levels in patients with high lactate dehydrogenase (LDH) (P=0.003), B symptoms (P=0.040) or age-adjusted international prognostic index (IPI)>1 (P=0.010) were increased. The levels of IL-6 in patients of male (P=0.003), stage Ⅲ-Ⅳ (P=0.008) or IPI>1 (P=0.004) were significantly higher. The level of hepcidin was highly correlated with serum ferritin (r=0.77, P<0.001), weakly correlated with IL-6 (r=0.31, P=0.030), and not correlated with Hb (r=-0.12, P=0.3). There was a negative correlation between IL-6 expression and Hb (r=-0.35, P=0.009). Multivariate analysis showed that IL-6 could predict anemia (P=0.03), whereas hepcidin could not (P=0.89). Conclusion The elevated hepcidin level is frequent in DLBCL, and the elevated IL-6 plays the major role in the development of anemia.

Objective To analyze Clopidogrel Resistance (CR) and influencing factors of coronary heart disease (CHD) with diabetes (DM) patients and evaluatc the relationship of CR and major adverse cardiovascular events (MACE) and readmission of CHD with DM patients.Methods 270 CHD patients were enrolled.Clinical conditions of CR were measured by adenosine diphosphate (ADP) induced maximum platelet aggregation rate (MPAR).After 1-year follow-up,MACE events and rehospitalization were recorded.Results CR of NDM and DM patients were 45 (33.1％) and 78 cases (58.2％) respectively,and the difference was significant (P ＜ 0.001).Factors of CR of CHD DM patients included heart rate,TG level,the number of severe coronary artery disease.MACE events of CS and CR patients were 35 (23.8％) and 47 patients (38.2％) respectively,and the difference was significant (P =0.010).The readmitted patients of CS and CR groups were 15 cases (10.2％) and 27 patients (22.0％) respectively,and the differcnce was significant (P =0.008).The MACE of CR and CS patients in DM group were 32 (41.0％) and 12 cases (21.4％) respectively,and thc difference was significant (P ＜ 0.05).The Readmitted cases of CR and CS patients in DM group were 19 (24.4％) and 5 (8.9％) respectively,and the diffcrcnce was significant (P ＜ 0.05).Conclusions CR of CHD DM patients increased significantly.The influencing factors of CR of CHDDM are including heart rate,TG level,the number of severe coronary artery disease.MACE events and rehospitalization rate were significantly increased in CHD patients with DM AR.Therefore,it should be further strengthened the anti-platelet therapy for CHD patients with DM.

Objective To explore expression changes of myocardial renin angiotensin system induced by prenatal exposure to lipopolysaccharide in offspring rats.Methods Twelve pregnant SD rats were randomly divided into three groups:LPS model group:intraperitoneal injection of LPS (0.79 mg/kg)at 8,10,12 days of pregnancy ; control group:intraperitoneal injection of sterile saline (0.5 ml) at 8,10,12 days of pregnancy; LPS + PDTC group:intraperitoneal injection of LPS (0.79 mg/kg) at 8,10,12 days of pregnancy plus daily intraperitoneal injection of NF-κB inhibitor-pyrrolidine dithiocarbamate (PDTC,100 mg/kg) on day 8 to 14 pregnancy day.Protein expression of Angiotensin Ⅱ (Ang Ⅱ) in heart was detected by immunohistochemistry; myocardial ACE,ACE2 mRNA expression was detected by real-time PCR; protein expression of ACE and ACE2 in heart was detected by Western blot in offspring rats of various groups.Results Compared with control group (0.07 ± 0.02,0.11 ± 0.01),Ang Ⅱ protein levels (0.14 ± 0.04) were significantly increased at 6 weeks (P ＜ 0.01) and 16 weeks (0.17 ± 0.04,P ＜ 0.05) in offspring rats of LPS model group,which could be significantly attenuated by PDTC intervention (0.10 ± 0.01,0.13 ± 0.03,respectively,all P ＜ 0.05).Similarly,myocardial ACE mRNA expression in 16 weeks offspring rats of LPS model group was significantly upregulated compared with control group (1.10 ± 0.26 vs.0.72 ± 0.22,P ＜ 0.05),which was significantly attenuated by PDTC intervention (0.67 ± 0.01,P ＜ 0.01 vs.LPS group).Myocardial protein expression ACE2 in 16 weeks offspring rats of LPS model group was significantly downregulated compared to control group,which was slightly upregulated by PDTC intervention (P ＞ 0.05).Conclusion Pregnancy exposure to lipopolysaccharide increases myocardial ACE and Ang Ⅱexpression while reduces myocardial ACE2 expression in offspring rats,which might be one of the pathomechanisms of offspring hypertension.

Objective:To investigate the relationship between neutrophil/lymphocyte ratio (NLR) and bone mineral density (BMD) in rheumatoid arthritis (RA), and to evaluate its diagnostic value in RA with osteoporosis.Methods:134 RA patients and 69 healthy subjects were screened and NLR levels were compared between the two groups. Bone mineral density of lumbar L1-4 and femoral neck was measured by dual energy X-ray absorption (DXA), and the patients were divided into normal bone mass group (44 cases), reduced bone mass group (47 cases) and osteoporosis group (43 cases). Height, weight, course of disease, mean platelet volume, erythrocyte sedimentation rate (ESR), C-reactionprotein (CRP), complement C3, complement C4, rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibody, bone mineral density and other related indicators were recorded. The differences of NLR, body mass index(BMI) gender, age and other indicators among the three groups were compared by One-way Analysis of Variance (ANOVA) and Kruskal-Wallis test, or χ2 test. Correlation analysis was conducted to detect the correlation between NLR, bone mass and each indicator, and ordered multi-classification Logistic regression analysis was used to evaluate the im-pact of each indicator on osteoporosis, and receiver operating characteristic curve (ROC) was used to predict the diagnostic value of NLR and combined related indicators on osteoporosis. Results:NLR of RA patients (3.1±1.7) was higher than that of healthy controls (1.7±0.5) ( F=21.27, P<0.001). In the osteoporosis group, the reduced bone mass group, and the normal bone mass group, age (66±8), (62±10), (50±13), disease course (15±10), (9±8, (7±7), BMI (20±4) kg/m 2, (22±3) kg/m 2, (24±3) kg/m 2, NLR (3.9±2.3, 2.7±1.2, 2.6±1.0), CRP (41±43) mg/L, (28±34) mg/L, (18±26) mg/L, ESR (46±30) mm/1 h, (36±26) mm/1 h, (26±20) mm/1 h were significantly different among the three groups ( χ2=32.92, P<0.001; H=17.41, P<0.001; F=12.04, P<0.001; H=11.62, P=0.030; H=13.78, P=0.001; F=7.18, P=0.001). Correlation analysis showed that NLR was correlated with CRP, ESR, anti-CCP antibody, femoral neck bone mineral density, DAS28 score and age. The correlation coefficients were 0.49 ( P<0.001), 0.39 ( P<0.001), 0.30( P<0.001), -0.18( P=0.042), 0.50( P<0.001), 0.17( P=0.046), respectively. Femoral neck was correlated with age, BMI, course of disease, CRP, ESR. The correlation coefficients were -0.46( P<0.001), 0.38 ( P<0.001),-0.39 ( P<0.001), -0.34 ( P<0.001), the correlation coefficients of L1-4 with age, BMI, CRP and ESR were -3.41( P<0.001), 0.39( P<0.001), -0.22( P=0.010), -2.42( P=0.005), respectively. There was no correlation between bone mineral density and DAS28 and anti-CCP antibody. Ordered multi-classification Logistic regression analysis showed that: age, course of disease, NLR and ESR were risk factors for osteoporosis, and their OR values were 1.12 ( P<0.001), 1.05 ( P=0.025), 1.29 ( P=0.031), 1.02 ( P=0.039), 0.28 ( P=0.008), respectively. Body mass index ( OR=0.76, P<0.001) were protective factors. ROC curve showed that the AUC area of NLR was 0.68, the AUC area of NLR, BMI, age, sex and course of disease was 0.90, the cut-off value was 0.20, sensitivity was 0.95, and specificity was 0.73[95% CI(0.84, 0.95)]. Conclusion:In osteoporosis, NLR is related to bone mass and disease activity of patients with rheumatoid arthritis. Combined with other related indexes, NLR can be used as a predictive diagnostic index and has a guiding role in clinical practice.