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The teaching team of medical functional sciences in Fudan University carried out exper-imental courses targeting problem-based designing and reformed the present teaching system. These experi-mental courses gave up the traditional teacher-based teaching method and guided students to learn knowledge and understand theories independently. At the same time, students could discuss the problems thoroughly, design the experimental plans and verify their hypothesis. This teaching method combines PBL, flipped classes, experimental designs and statistics analysis, which could provide experience for the education of medical functional sciences in medical colleges.
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Brain iron deposition has been proposed to play an important role in the pathophysiology of Alzheimer disease (AD). The aim of this study was to investigate the correlation of brain iron accumulation with the severity of cognitive impairment in patients with AD by using quantitative MR relaxation rate R2' measurements. Fifteen patients with AD, 15 age- and sex-matched healthy controls, and 30 healthy volunteers underwent 1.5T MR multi-echo T2 mapping and T2* mapping for the measurement of transverse relaxation rate R2' (R2'=R2*-R2). We statistically analyzed the R2' and iron concentrations of bilateral hippocampus (HP), parietal cortex (PC), frontal white matter (FWM), putamen (PU), caudate nucleus (CN), thalamus (TH), red nucleus (RN), substantia nigra (SN), and dentate nucleus (DN) of the cerebellum for the correlation with the severity of dementia. Two-tailed t-test, Student-Newman-Keuls test (ANOVA) and linear correlation test were used for statistical analysis. In 30 healthy volunteers, the R2' values of bilateral SN, RN, PU, CN, globus pallidus (GP), TH, and FWM were measured. The correlation with the postmortem iron concentration in normal adults was analyzed in order to establish a formula on the relationship between regional R2' and brain iron concentration. The iron concentration of regions of interest (ROI) in AD patients and controls was calculated by this formula and its correlation with the severity of AD was analyzed. Regional R2' was positively correlated with regional brain iron concentration in normal adults (r=0.977, P<0.01). Iron concentrations in bilateral HP, PC, PU, CN, and DN of patients with AD were significantly higher than those of the controls (P<0.05); Moreover, the brain iron concentrations, especially in parietal cortex and hippocampus at the early stage of AD, were positively correlated with the severity of patients' cognitive impairment (P<0.05). The higher the R2' and iron concentrations were, the more severe the cognitive impairment was. Regional R2' and iron concentration in parietal cortex and hippocampus were positively correlated with the severity of AD patients' cognitive impairment, indicating that it may be used as a biomarker to evaluate the progression of AD.
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The changes of glycogen synthase kinase-3(GSK-3)and protein phosphatase-2A(PP-2A),and the role of them in the regulation of the abnormally hyperphosphorylated some sites of tau in the cortex of diabetic rat were investigated.The diabetic rat model was induced by streptozotocin.The activities of GSK-3,PP-2A were measured by liquid scintillation for incorporated radioactivity in control,DM,DM + Li2CO3 groups.The level of hyperphosphorylated tau and the expression of 2PP-2A was measured respectively by Western blot.It is suggested that GSK-3 activity increases,PP-2A activity and expression decrease,and hyperphosphorylated tau be produced at Ser198/Ser199/Ser202 and Ser396/Ser404 in DM rats cortex.After the DM rat were treated with Li2CO3,the inhibition of GSK-3 activity and the improvement of PP-2A activity were found,and hyperphosphorylation of tau at Ser198/Ser199/Ser202 and Ser396/Ser404 were deduced.These studies firstly suggested that an increase of GSK-3 activity might inhibit PP-2A activity,and which produce hyperphosphorylated of tau at Ser198/Ser199/Ser202 and Ser396/Ser404 in DM rat cortex in common.
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AIM: To probe into tau hyperphosphorylation at PHF- 1 sites induced by glycogen synthase kinase - 3β(GSK- 3β) in vivo. METHODS: Twenty - one rats were randomly allocated to three groups as follows: GSK - 3β transfection group, vector group and control group; 0.1 μg/3μL GSK- 3β- HA plasmid or vector was injected bilaterally into cerebrum of the rats respectively, rats without injection were controls. Western blotting and immunohistochemical staining of cortex were carried out to detect the expression of GSK- 3β- HA plasmid and tau phosphorylation using phosphorylation- dependent tau antibody PHF- 1. RESULTS: After transfection with GSK- 3β- HA for 48 h, GSK - 3β - HA was expressed in GSK- 3β transfection group; and hyperphosphorylated tau at PHF- 1 sites accumulated in neurons in the transfected areas. The hyperphosphorylated tau colocalized largely with GSK- 3β expressed by the transfected GSK- 3β plasmid. CONCLUSIONS: Transfection with GSK- 3βin vivo can induce tau hyperphosphorylation involving the pathogenesis of neurodegenerative disorders. These data further prove that GSK- 3β is a key kinase to induce tau hyperphosphorylation and may be a therapeutic target for tauopathy- related neurodegenerative diseases.
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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.
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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.
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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.
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<p><b>OBJECTIVE</b>To reconstitute an Alzheimer's disease model by administering bradykinin (BK) or cyclosporine A (CSA) to the rat hippocampus.</p><p><b>METHODS</b>BK or CSA was administered to the rat hippocampus using a stereotaxic apparatus. The behavior of the rats was observed with an electronic attack jump platform. The phosphorylation of Tau protein was examined through immunohistochemical assay.</p><p><b>RESULTS</b>Behavior studies showed that an obvious disturbance in learning and memory was seen in BK injected rats.No obvious dysfunction was observed in CSA injected rats. The results obtained by immunohistochemical assay indicated that the staining of M4, 12E8, paired helical filament-1 (PHF-1) and calcium/calmodulin-dependent protein kinase II (CaMKII) was stronger, and that of Tau-1 was weaker in BK injected rats compared with the control group. We also found that the binding of M4 and PHF-1 but not 12E8 to Tau was significantly increased in CSA injected rats. As for BK injection, binding of Tau-1 to Tau was decreased after CSA injection.</p><p><b>CONCLUSION</b>To our knowledge, this is the first data showing in vivo that the activation of CaMKII induces both Alzheimer-like Tau phosphorylation and behavioral disturbances.</p>
Subject(s)
Animals , Rats , Alzheimer Disease , Bradykinin , Toxicity , Calcium , Metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases , Metabolism , Cyclosporine , Toxicity , Disease Models, Animal , Hippocampus , Metabolism , Immunohistochemistry , Phosphorylation , Rats, Sprague-Dawley , tau Proteins , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore the association between the abnormal phosphorylation sites found in Alzheimer disease (AD) tau and the inhibition of its biological activity.</p><p><b>METHODS</b>Ultracentrifugation, chromatography, manual Edman degradation and autosequence techniques were used to prepare and phosphorylate human recombinant tau, isolate and purify 32P tau peptides and determine phosphorylation sites.</p><p><b>RESULTS</b>Phosphorylation of tau by casein kinase 1 (CK-1), cyclic AMP-dependent protein kinase (PKA) and glycogen synthetase kinase 3 (GSK-3) separately inhibited its biological activity and the inhibition of this activity by (CSK-3 was significantly increased if tau was prephosphorylated by CK-1 or PKA. The most potent inhibition was seen by a combined phosphorylation of tau with PKA and GSK-3. The treatment of tau by PKA and GSK-3 combination induced phosphorylation of tau at Ser-195, Ser-198, Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-262, Ser-356, Ser-404, whereas Thr-181, Ser-184, Ser-262, Ser-356 and Ser-400 were phosphorylated by GSK-3 alone under the same condition.</p><p><b>CONCLUSION</b>Phosphorylation of tau by PKA plus GSK-3 at Thr-205 might play a key role in tau pathology in AD.</p>
Subject(s)
Humans , Alzheimer Disease , Metabolism , Binding Sites , Casein Kinases , Cyclic AMP-Dependent Protein Kinases , Metabolism , Glycogen Synthase Kinase 3 , Metabolism , In Vitro Techniques , Microtubules , Metabolism , Phosphorylation , Protein Kinases , Metabolism , tau Proteins , MetabolismABSTRACT
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.
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The neurofilament proteins (NFPs) from different neuronal tissues including Alzheimer and Huntington disease gray matter, rat brain gray, white matter and spinal cord were separated biochemically into two major fractions. A systematic investigation on the distribution, expression and phosphorylation of NFPs in those fractions was undertaken in the present study. It was found that only non-phosphorylated NF-H and NF-M, but not NF-L subunit were detected in Alzheimer brain gray matter high speed supernatant, whereas all neurofilament subunits including non-phosphorylated and phosphorylated were measured in high speed pellet fraction of the same tissue. The hyperphosphorylation of NF-H and NF-M in Alzheimer brain was shown by phosphorylation dependent monoclonal antibodies SMI31 and SMI34. This hyperphosphorylation was confirmed by non-phosphorylation dependent antibody SMI32 with dephosphosphorylation of the samples. Furthermore, an increased amount of NF-H, NH-M and NF-L, detected by SMI33 and NR4 respectively, was also observed in Alzheimer samples, in which the elevation in NF-L was significant. A significantly different immunoblot patterns in distribution, expression and phosphorylation were determined in various position of the neural system and alternative fractions. To our best knowledge, this is the first data shown definite abnormality of NFPs in Alzheimer disease. The information obtained in the present study will be extremely valuable in further study of the proteins both in physiological and pathological conditions.
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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
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AIM: To explore the effect of ovariectomization on Alzheimer-like phosphorylation of Tau in hippocampus of Sorague-Dawlery rats. METHODS: An animal model was developed using ovariectimized (OVX) rats, and the phosphorylation of Tau protein was measured by Western blot. RESULTS: The levels of phosphorylated Tau at PHF-1epitope were elevated in ovariectimized rat brain hippocampus 4 weeks and 8 weeks after ovariectomization, when compared with sham-OVX rats (P0.05). CONCLUSION: Ovariectomization may induce Alzheimer-like hyperphosphorylation of Tau protein in brain hippocampus of rats. [
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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
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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
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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.
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Objective To investigate the reduction of amyloid beta peptide (A?) production induced by insulin in the cortex of diabetic rat. Methods 15 male SD rats were randomly divided into control group, diabetes group, and diabetes plus insulin group. The diabetic rats were induced by streptozotocin. The activity of glycogen synthase kinase-3 (GSK-3) was measured by 32P liquid scintillography for incorporated radioactivity in control group, DM group, DM+insulin group, the production of A? was determined by sandwich ELISA in each group, and the expression of APP was determined by Western-blot. Results In control group, the activity of GSK-3 (1.04?0.11), the production of A? (A?_ 40 (40.92?5.34) pg/?l, A?_ 42 (29.64?3.19) pg/?l)and the levels of full-length APP(1.05?0.08) was low, but in DM group, the activity of GSK-3 (2.02?0.12) and the production of A?(A?_ 40 (67.53?11.69) pg/?l, A?_ 42 (45.02?4.10) pg/?l) increased (P
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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.
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Rat models with common peroneal nerve injury were treated with modified Decoction for Invigorating Yang orally to observe its effect on the nervous functional recovery after peripheral nerve injury. The result showed that the motor nerve conduction velocity of the drug group during the whole observation period was higher than that of the control group (p
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AIM: To explore the effect of nuclear factor-?B (NF-?B) on the anti-apoptosis induced by brain ischemia preconditioning (IP). METHODS: Temporary middle cerebral artery occlusion for 20 min followed three days reperfusion before 6 hours middle cerebral artery occlusion (MCAO) trancranially was used as preconditioning in Wistar rats. The protective role was evaluated by analyzing the infarct volume. The status of neuronal apoptosis was observed by TUNEL. The expression of NF?B p65 protein, the assay of SOD activity and MDA concentration were analyzed by using the methods of immunohistochemistry and cytochemistry. RESULTS: Compared to the control group, 20 min ischemic preconditioning, which did not produce neuronal damage obviously, reduced the infarct volume significantly after MCAO 6 h and obviously decreased the number of neural cell apoptosis in penumbra (P