曾　宽，黄　芳，包　建，李梦珠，王小川(华中科技大学同济医学院病理生理学系，湖北武汉430000)

银杏叶提取物对阿尔茨海默病的预防与治疗作用及其机制

曾宽，黄芳，包建，李梦珠，王小川△
(华中科技大学同济医学院病理生理学系，湖北武汉430000)

目的:探讨银杏叶提取物(GBE)对阿尔茨海默病(AD)的预防与治疗作用及其机制。方法:大鼠尾静脉注射高半胱氨酸(Hcy) 2周构建AD模型。实验分为对照组(生理盐水)、模型组(Hcy)和预防组(Hcy + GBE)，治疗组(Hcy注射2周后给予GBE 1周)。水迷宫检测大鼠学习记忆。Western blot检测大鼠海马和皮质中tau及其磷酸化、GSK-3β及其磷酸化、PP2A及其甲基化、PSD95、synapsin I的变化。结果: (1)水迷宫提示模型组大鼠潜伏期显著高于其它3组。(2)大鼠海马和皮质中tau、GSK-3β和PP2A表达无变化，tau磷酸化和PP2A甲基化模型组显著高于其它3组，GSK-3β磷酸化和PSD95、synapsin I表达模型组显著性低于其它3组。结论: GBE能通过调节GSK-3β和PP2A活性预防和治疗Hcy导致的大鼠学习记忆障碍和tau过度磷酸化。

The investigation of unique functional and structural aspects of peroxisomes (POs) requires the preparation of highly purified frac-tions of this organelle.In this report，differential centrifugation separation of POs from of tissues and cells and purification by density gradient ultracentrifugation were described.POs were purified by different ways: (1) Metrizamide gradient centrifugation.Layer the light or the heavy enriched peroxisomal fraction on the top of thawed gradient (adjusting densities to 1.12，1.155，1.19，1.225 and 1.26 g/mL，respectively).Centrifuge gradients in a vertical-type rotor at an integrated force of 1.256×106g/min (gmax= 39 000)，highly purified heavy peroxisomes band at 1.23～1.24 g/mL and light peroxisomes band at 1.20～1.21 g/mL.(2) Isotonic Percoll gradient.Layer the crude POs on the 1.135 g/mL cold isotonic Percoll，centrifuged for 1 h at 35 000×g in a fixed angle rotor，PO band near the top of the gradient.(3) Nycodenz gradient.The crude PO fraction was layered on to the 1.30，1.24 and 1.15 g/mL step Nycoden gradient，centrifuged for 1 h at 35 000×g in a fixed angle rotor.(4) Iodixanol gradient.Centrifugation of light mitochondrial fractions in 20% to 40% (w/v) iodixanol gradients，made iso-osmotic by the addition of sucrose，resulted in an excellent separation of peroxisomes from the remaining organelles.Peroxisomes banded around 30% (w/v) iodixanol (at the density of 1.175 g/mL).These procedures allowed for the rapid isolation of highly pure POs from tissue and cutured cells.Morphological examination of the peroxisomal fractions confirmed the near absence of other organelles and revealed.

ATP is used by protein kinases to phosphorylate targeted substrates.Recent findings underscore its additional key roles in regulation.Here we observe that ATP binding protects the phosphorylated T308 (pT308) by stabilizing the closed conformation of the Akt1 kinase domain and identify hot spot residues in the allosteric propagation pathway from ATP to pT308.These data highlight a new model of kinase regulation and offer a potential avenue to allosteric drug therapy.

In this study，we created a hypoxic environment in a sealed bottle as oxygen was gradually consumed by the mouse inside.Then we recorded the survival time of the mouse and calculated 2 indexes，standard hypoxic tolerance time (STT) and adjusted standard hypoxic tolerance time (ATT) for evaluating hypoxic tolerance of the mouse.Using methazolamide as the positive control and saline the negative control，we were able to evaluate hypoxic tolerance of mice with this simple method.As the mice were grouped according to body weight or bottle volume，we found a strongly negative correlation between STT with body weight，but not bottle volume，suggesting that different body weight may lead to false positive or negative errors to the results expressed as STT.On the other hand，both false positive and negative errors were rectified when we used ATT as the hypoxic tolerant index.With this method，we will be able to preliminarily screen anti-hypoxic medicines using mice as the experiment animals.When the body weight is strictly matched in all groups in the experiment，STT can be used as the index of hypoxic tolerance.Otherwise，ATT can be a better index for evaluation of hypoxic tolerant ability as the impact of body weight o n the results can be practically eliminated.

*［Foundation item］Supported by National Natural Science Foundation of China (No.81071610; No.81471814)

△Corresponding author E-mail: winneygaocn@aliyun.com

Our current study aims to investigate the alleviative effect exerted by oxygen intake in population at an altitude of 3 658 m.The symptomatic remission，brain function，physiological parameters were all recorded before，15 d after and 30 d after oxygen intake，and 15 d after termination.We found that oxygen intake improved the body function both physiologically and metabolically，and 30 min daily oxygen intake at least 2 times a week was beneficial for population living at 3 658 m.Subgroup analysis on the population at 3 658 m (n =263) was also conducted based on the diagnosis of CMS high risk (n =161) and healthy controls (n =102).It was generally showed that oxygen inhalation could alleviate the symptoms like cyanosis，palpitation and headache in this cohort，but exerted no significant effect on other related clinical findings including sleep quality，brain function，liver and renal functions in different subgroups.Therefore，oxygen inhalation should be closely monitored and the best efficacy can be achieved through individualized treatment.

△Corresponding author

▲These authors contribute equally to this work

Isolation of peroxisomes from tissues and cells by differential and density gradient centrifugation

LIN Yuan-xi，SONG Xin-ai，WANG Ya-wen
(Department of Physiology and Pathophysiology，Xi’an Jiaotong University School of Medicine，Xi’an 710061，China)

Mechanism of ATP-dependent allosteric protection of Akt kinase phosphorylation

LU Shao-yong，ZHANG Jian
(School of Medicine，Shanghai Jiaotong University，Shanghai 200240，China.E-mail: jian.zhang@sjtu.edu.cn)

A simple method for evaluating hypoxic tolerance in mice*

XU Gang1，2，GAO Yu-qi1，2，GAO Yi-xing4，GAO Wen-xiang2，3△
(1Institute of Medicine and Hygienic Equipment for High Altitude Region，2Ministry of Education High Altitude Medicine Key Laboratory，3Department of Pathophysiology and High Altitude Pathology，College of High Altitude Military Medicine，
4Southwest Hospital Institute of Pathology and Southwest Cancer Center，Third Military Medical University，Chongqing 400038，China)

Optimal personalized oxygen inhalation significantly alleviates symptoms of chronic mountain sickness patients through improving overall health conditions

FENG Bin1▲，ZHANG Gang2，4，5▲，LIU Fu-yu3，4，5，LI Peng2，4，5，ZHENG Shan-jun2，4，5，GAI Lu-yue1，GAO Yu-qi3，4，5△
(1Department of Cardiology，Chinese PLA General Hospital，Beijing 100853，China;2Department of High Altitude Military Hygiene，3Institute of Medicine and Hygienic Equipment for High Altitude Region，4Key Laboratory of High Altitude Medicine，Ministry of Education，College of High Altitude Military Medicine，Third Military Medical University，Chongqing 400038，China;5The Key Laboratory of High Altitude Medicine，People's Liberation Army，Chongqing 400038，China)

△E-mail: 773847352@qq.com