Ca 2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) is a member of a family of Ca 2+/calmodulin-regulated protein kinases which also includes Ca 2+/calmodulin-dependent protein kinases Ⅰ and Ⅲ, myosin light chain kinases and phosphorylase kinase. Unlike the other members of this family, CaMKⅡ is multifunctional protein kinase and distributes in a variety of tissues. It is especially abundant in neuronal system. In hippocampus, CaMKⅡ is about 2% of the total protein. Studies have shown that CaMKⅡ plays an important role in a variety of biological processes, such as regulation of gene transcription, synthesis of neurotransmitter, phosphorylation of cytoskeletonal protein, hippocampal learning and memory formation.

Bradykinin (BK) is a calcium/calmodulin dependent protein kinase Ⅱ (CaMKⅡ) specific activator, and Cyclosporin A (CSA) is reported to suppress protein phosphotase (PP)-2B activity. In vitro studies have shown that CaMKⅡ and PP-2B play an important role in Alzheimer-like phosphorylation of microtube-associated protein tau. To reconstitute an animal model based on the imbalance of protein kinase (s) and protein phosphatase (s) seen in Alzheimer brain, we injected BK and/or CSA into rat hippocampus. The results from behavioral study showed that an obvious disturbance in learning and memory was seen with BK or BK plus CSA injected rats. Moreover, the behavior abnormality appeared earlier in aged rats than young adults of the same kind after the injection. On the other hand, no obvious dysfunction in living and behavior was observed with CSA alone injected rats. The results obtained by immunohistochemical assay indicated that the staining for M4\, 12E8\, PHF-1 and CaMKⅡ was stronger, and for Tau-1 was weaker in BK injected rats compared with Control group. It was also found that the binding of M4 and PHF-1 but not 12E8 to tau was significantly increased in CSA injected rats. As the same as BK injection, binding of Tau-1 to tau was decreased after CSA injection. The immunostaining for 12E8\,PHF-1 and CaMKⅡ was increased, whereas for Tau-1\, M4\, and GSK-3 was decreased after combination injection of BK and CSA. In addition, the staining of PP-2B decreased in all the three models. To our knowledge, this is the first data shown in vivo that the activation of CaMKⅡ induces both Alzheimer-like tau phosphorylation and behavioral disturbance.

Objective To reconstitute an animal model based on the imbalance of protein kinase(s) and protein phosphatase(s) seen in Alzheimer brain. Methods The injection of bradykinin (BK) into hipocampus was performed, and their behaviors were observed by electronic attack-jump experiment and phosphorylation of tau using immunohistochemical assay. Results The results of behavior studying showed that an obvious disturbance in learning and memory was seen in BK injected rats. The results obtained by immunohistochemical assay indicated that the staining for M4?12E8?PHF-1 and CaMK-Ⅱ was stronger, and for tau-1 was weaker in BK injected rats as compared with the control group. The BK injected rats showed obvious deficit in behavior [mistakes made by model and control rats during electronic attack-jump experiment:8.3?2.5 and 6.9?3.1, P

Objective To investigate the in vivo effect of melatonin(Mel) on calyculin A(CA)-induced tau hyperphosphorylation in neuroblastoma cells(N2awt).Methods We treated N2awt cells with CA or CA and 50 ?mol/L Mel,detected the level of tau phosphorylation with immunofluorescence,and assayed the activities of GSK3 and the ratio of GSK-3? phosphorylated at Ser9 site to total GSK-3?.Results CA treatment led to tau hyperphosphorylation accompanied with the increased activity of GSK-3 and the decreased ratio of GSK-3? phosphorylated at Ser9 site to total GSK-3?.When the cells were incubated simultaneously with CA and 50 ?mol/L Mel,the CA-induced tau hyperphosphorylation,GSK-3 activation and the ratio of GSK-3? phosphorylated at Ser9 site to total GSK-3? decrease were attenuated.Conclusion Melatonin protects neuroblastoma cells from CA-induced tau hyperphosphorylation.Its protection may be related to the regulation of GSK-3 activity and the ratio of GSK-3? phosphorylated at Ser9 site to total GSK-3? increase.

This paper explores the existing gene doping problems of athletes in bioethical aspect,describes the development of gene doping,and points out that strengthening the anti-doping education,further improving legal system and strengthening an effective supervision and anti-doping research are main focus of anti-doping work currently.

Objective To investigate the relationship between protein phosphatase inhibition, tau hyperphosphorylation and neuronal death seen in Alzheimer disease (AD). Methods Co culture of protein phosphatase inhibitor okadaic acid (OA) and neuroblastoma cells (SH SY5Y), by agarose gel electrophoresis to detect DNA fragmentation, and in situ hybridization by TdT mediated biotin labeled dNTP nick end labeling (TUNEL) to further detect the cell apoptosis. Results Incubation of SY5Y cells with 10 nmol/L OA for 24 or 48 hours led to the appearance of DNA fragmentation and a remarkable increase of positive cells from 2 16%?0 94% to 18 05%?3 57% ( P

Objective To investigate the in vivo effect of melatonin on calyculin A-induced neurofilament(NF) hyperphosphorylation in neuroblastma cells(N2awt).Methods N2awt cells were treated with CA or CA and different concentration melatonin or CA and vitamin E,the levels of neurofilament phosphorylation and the level of PP-2A were detected,and the activities of PP-2A were assayed.Results Calyculin A treatment led to neurofilament hyperphosphorylation by decreasing the level and activity of PP-2A.Both melatonin and vitamin E had protective effect on calyculin A-induced neurofilament hyperphosphorylation,although melatonin increased the activity of PP-2A while vitamin E did not.Morever,melatonin partially attenuated the decreasing of PP-2A level. Conclusion Melatonin protects neuroblastma cells from CA-induced neurofilament hyperphosphorylation through the regulation of PP-2A level and the increase of PP-2A activity.

Objective To observe and explore the effectiveness of irbesartan hydrochlorothiazide(IH) combine with Bisoprol‐ol fumarate(BIS) on youth hypertension .Methods Randomly divided 96 patients in our hospital from September 2012 to February 2015 into observation group and control group(48 cases in each/group) .the IH treatment was given to the control group based on regular drug ,while the BIS was given to the observation group on the basis of the control group .systolic pressure(SBP) ,diastic pressure(DBP) and heart rate(HR) ,left ventricular end diastolic diameter(LVEDD) ,E peak and left ventricular ejection fraction (LVEF) in the two groups before and after treatment were detected ,and comprehensive efficacy were evaluated based on blood pressure improvement .Results Before treatment ,the difference of SBP ,DBP ,HR ,LVEDD ,E peak and LVEF between the two groups was not statistically significant (P>0 .05);after treatment ,SBP ,DBP and HR were (116 .4 ± 11 .8)mm Hg ,(85 .3 ± 6 .7) mm Hg and (65 .2 ± 7 .1)times/min in the observation group ,respectively ,while in the control group ,SBP ,DBP and HR were (132 .8 ± 14 .6)mm Hg ,(96 .3 ± 6 .2)mm Hg and (75 .2 ± 8 .1)times/min ,respectively ,the difference was statistically significant(P<0 .05);after treatment ,LVEDD in observation group was significantly lower than the control group ,while LVEF ,E peak were significantly higher(P< 0 .05);the total effective rate in observation group was 95 .8% (46/48) ,while total effective rate was 75 .0% (36/48) in the control group ,the difference was statistically significant(P<0 .05) .Conclusion IH combined with BIS can significantly improve blood pressure in patients with juvenile hypertension and has significant effect ,thus it is a safe and effective therapy for juvenile hypertension .

BACKGROUND:It can be theoretical y speculated that the combination of titanium, micro-arc oxidation coating and arginine-glycin-aspartic acid (RGD) polypeptide wil show better mechanical and biological properties. OBJECTIVE:To observe the microstructure and cellproliferation of the titanium and micro-arc oxidation coatings modified with RGD polypeptide by different modification methods. METHODS:Ninety specimens of pure titanium and micro-arc oxidation coatings were divided into three groups, with 30 specimens in each group. The first 30 specimens were pure titanium physical y decorated with RGD polypeptide only. The other two groups of specimens were physical and chemical coupling adsorption RGD polypeptide micro-arc oxidation samples, respectively. The fluorescence microscope was used to observe the effects and amounts of grafting RGD polypeptide on each sample. Content of the RGD polypeptide on the surface of the specimens were measured by X-ray photoelectron spectroscopy. The mouse bone marrow stromal cells were cultured on the surfaces of three groups samples, and the adhesion and proliferation of the cells cultured for 3 hours, 12 hours, 24 hours and 3 days were observed by optical microscope respectively. RESULTS AND CONCLUSION:There were green fluorescence spots, with varying size and amount, on the surface of specimens in three groups. In the unit field of view, the fluorescence was the strongest in chemical coupling adsorption RGD polypeptide micro-arc oxidation samples, indicating that these specimens were grafted with many polypeptides. In physical coupling adsorption RGD polypeptide micro-arc oxidation samples, smal amount of polypeptides were found on the surface, and the content of the RGD polypeptide was the highest in chemical coupling adsorption RGD polypeptide micro-arc oxidation samples. No apparent cytotoxicity was observed in three groups. The cellproliferation was the best in chemical coupling adsorption RGD polypeptide micro-arc oxidation samples. Experimental findings suggest that, chemical coupling method can wel fix the RGD polypeptides on the surface of pure titanium samples containing micro-arc oxidation, without any cytotoxicity, which contribute to promote the cellgrowth and proliferation.

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.