木犀草素通过抑制p65 NF-κB、促进p85 PI3K调节微血管内皮细胞VCAM-1表达

通过抑制微血管内皮细胞血管细胞黏附分子(VCAM)-1的表达,木犀草素可阻遏中性粒细胞与微血管内皮细胞的黏附,起到抗炎作用。木犀草素调节VCAM-1表达与三条信号通路有关：丝裂原活化蛋白激酶(MAPK)、核因子kappa B (NF-κB)/IκB和磷脂酰肌醇3激酶(PI3K)/Akt通路。其中,MAPK和NF-κB/IκB通路参与VCAM-1正向调节,PI3K/Akt通路参与VCAM-1负向调节。本文研究了木犀草素对微血管内皮细胞该三条通路中的关键蛋白p38 MAPK、p65 NF-κB、p85 PI3K磷酸化。结果表明：木犀草素在反应的30s和1min促进p38 MAPK磷酸化,在30 s、1 min和5 min促进p85 PI3K磷酸化,而在30 s、1 min、5 min和30 min抑制p65 NF-κB磷酸化。阻抑p38 MAPK通路导致VCAM-1表达下调,而p38 MAPK抑制剂SB203580可通过抑制p38 MAPK磷酸化也下调VCAM-1,提示木犀草素对微血管内皮细胞VCAM-1的调节作用独立于p38 MAPK磷酸化。由此可知,木犀草素通过抑制p65 NF-κB磷酸化或促进p85 PI3K磷酸化调节微血管内皮细胞VCAM-1表达。本文为木犀草素抗炎作用的分子机制研究提供了新的线索。     

木犀草素通过抑制p65 NF-κB、促进p85 PI3K调节微血管内皮细胞VCAM-1表达（英文）

通过抑制微血管内皮细胞血管细胞黏附分子(VCAM)-1的表达,木犀草素可阻遏中性粒细胞与微血管内皮细胞的黏附,起到抗炎作用.木犀草素调节VCAM-1表达与三条信号通路有关:丝裂原活化蛋白激酶(MAPK)、核因子kappa B(NF-κB)/IκB和磷脂酰肌醇3激酶(PI3K)/Akt通路.其中,MAPK和NF-κB/IκB通路参与VCAM-1正向调节,PI3K/Akt通路参与VCAM-1负向调节.本文研究了木犀草素对微血管内皮细胞该三条通路中的关键蛋白p38 MAPK、p65 NF-κB、p85 PI3K磷酸化.结果表明:木犀草素在反应的30 s和1 min促进p38 MAPK磷酸化,在30 s、1 min和5 min促进p85 PI3K磷酸化,而在30 s、1 min、5 min和30 min抑制p65 NF-κB磷酸化.阻抑p38 MAPK通路导致VCAM-1表达下调,而p38 MAPK抑制剂SB203580可通过抑制p38 MAPK磷酸化也下调VCAM-1,提示木犀草素对微血管内皮细胞VCAM-1的调节作用独立于p38 MAPK磷酸化.由此可知,木犀草素通过抑制p65 NF-κB磷酸化或促进p85 PI3K磷酸化调节微血管内皮细胞VCAM-1表达.本文为木犀草素抗炎作用的分子机制研究提供了新的线索.     

抑制p38MAPK信号通路对Bpiv3复制的影响

由牛副流感病毒3型(Bovine parainfluenza virus type 3,Bpiv3)感染引起的牛副流感病已成为各国牛场最重要的传染病之一,每年都会给世界养牛业造成巨大的经济损失,但关于该病致病的分子机制研究较少。本研究通过观察Bpiv3感染对MDBK细胞中丝裂原活化蛋白激酶(MKK3)及其下游分子p38丝裂酶原活化的蛋白激酶(p38MAPK)的表达的影响,探讨相关的信号转导机制,对p38 MAPK通路在Bpiv3感染过程中的作用进行了初步研究。Bpiv3感染细胞后,采用Western Blot检测MKK3,p38 MAPK在蛋白水平的表达变化,并采用ELISA法检测细胞上清中IL-6,IL-8,IL-13和TNF-α的水平变化,采用SPSS 12软件进行统计学分析。结果表明,Bpiv3在感染后能够诱导MKK3的激活以及p38的磷酸化,激活了p38 MAPK信号通路。而且p38 MAPK信号通路参与了Bpiv3的复制过程。ELISA检测Bpiv3感染后以及使用抑制剂SB202190处理后的细胞上清中IL-6、IL-8、IL-13和TNF-α的水平发现,p38 MAPK信号通路参与了Bpiv3诱导的炎症反应。研究证实Bpiv3感染能够激活p38 MAPK通路,显著上调MKK3的表达并诱导p38发生磷酸化,进一步激活下游分子发挥生物学活性,促进Bpiv3的复制及诱导促炎细胞因子的产生。p38 MAPK信号通路的激活可能是Bpiv3感染诱发炎症反应的机制之一。     

miR-196b-5p促进成肌细胞增殖分化

MicroRNA (miRNA)是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子，在动植物中参与转录后基因表达调控。大量研究表明，miRNA调控骨骼肌的发育，主要表现在肌卫星细胞的激活及增殖、分化、肌管的形成等生物学过程。本实验室前期对大白猪背最长肌(longissimus dorsi,LD)和比目鱼肌(soleus muscle,Sol)进行miRNA测序，筛选鉴定到一个在不同骨骼肌中差异表达并且序列高度保守的miR-196b-5p，目前miR-196b-5p在骨骼肌方面的研究尚未见报道。本研究进一步设计合成miR-196b-5p mimics和inhibitor对C2C12细胞进行miR-196b-5p过表达及干扰表达，利用蛋白免疫印迹、实时荧光定量PCR检测、流式细胞术、免疫荧光染色等方法探究miR-196b-5p对成肌细胞增殖分化的影响，并利用生物信息学预测和双荧光素酶报告系统鉴定了miR-196b-5p的靶基因。结果显示，过表达miR-196b-5p显著增加细胞周期基因Cyclin B、Cyclin D和Cyclin E的mRNA和蛋白表达水平(P<0....     

聚合度7-15的壳寡糖抑制脂多糖刺激的单核细胞产生TNF-α和IL-8的作用研究

本文通过建立脂多糖刺激的单核细胞炎症损伤模型,观察聚合度7-15的壳寡糖对炎性单核细胞白细胞介素-8(IL-8)和肿瘤坏死因子-α(TNF-α)表达的影响,及对p38丝裂原激活的蛋白激酶(p38 mitogen-activated protein kinases,p38MAPK)信号通路磷酸化的影响。采用p38信号通路抑制剂(SB203580)验证抑制p38信号通路对脂多糖诱导的单核细胞表达IL-8和TNF-α的作用,从而探索壳寡糖抑制单核细胞炎性损伤的分子机制。结果表明壳寡糖可抑制脂多糖诱导的单核细胞表达IL-8和TNF-α,并且抑制p38信号蛋白的磷酸化水平。因此,初步认为壳寡糖可能通过抑制炎性U937细胞中p38MAPK信号通路抑制IL-8和TNF-α的表达。     

细胞衰老过程中JNK、p38和Akt的活性变化

研究发现衰老成纤维细胞抗凋亡. 为探明其机制,检测与凋亡相关的信号传导通路JNK、p38和Akt在细胞衰老过程中是否发生改变.在本研究中,UV被用作JNK和p38传导通路的诱导剂,胎牛血清用作Akt通路的诱导剂.结果表明: p38和Akt在年轻和衰老细胞中均能被相应的诱导剂活化;相反, UV照射却不能有效激活衰老细胞中JNK的活性.结果提示:衰老细胞对凋亡诱导不敏感可能与JNK不能被有效活化有关.     

过表达磷脂酶D3影响成肌细胞中Akt磷酸化水平

磷脂酶D(PLD)催化卵磷脂(Phosphatidylc holine,PC)水解产生胆碱(Choline)和磷脂酸(Phosphatidic acid,PA),其代谢产物参与调控细胞内许多生理和生化过程。在过表达磷脂酶D3(PLD3)的成肌细胞(C2C12细胞)中,研究了PLD3对胰岛素刺激后Akt通路激活的影响。研究结果表明,PLD3过表达细胞的Akt磷酸化水平比对照组低,并且不受胰岛素浓度变化的调控。虽然PLD3过表达细胞中Akt磷酸化水平随胰岛素刺激时间的延长而有所增加,但磷酸化总水平比对照组低。磷脂酶D抑制剂丁-1醇能够抑制对照组胰岛素刺激下Akt磷酸化,却不能抑制PLD3过表达细胞的Akt磷酸化,并且PLD3过表达细胞Akt磷酸化水平比对照组高6倍。用磷脂酸(PA)做刺激时,对照组的Akt磷酸化明显增加,而PLD3过表达细胞株的Akt磷酸化没有显著变化;用PA和胰岛素同时刺激时,PLD3过表达株和对照组的Akt磷酸化均比PA单独刺激时降低。这说明PLD3的过表达抑制成肌细胞内胰岛素信号的传导。     

PI3K和受体型酪氨酸激酶介导家蚕麻痹肽免疫诱导信号的传递

【目的】普遍存在于鳞翅目昆虫中的ENF肽具有非常相似的结构和生理功能, 对昆虫的发育、 免疫、 应激反应等方面都起着重要的调控作用。有研究报道, 作为ENF肽家族成员之一的家蚕Bombyx mori麻痹肽（paralytic peptide, PP）通过激活MAPK来调控家蚕的免疫应答, 但其完整的分子作用机制尚不明确。本研究旨在解析传递家蚕PP免疫诱导信号的分子通路。【方法】首先通过荧光定量PCR检测了多种昆虫细胞系在PP诱导下抗菌肽基因的转录水平, 并利用Western blot检测p38 MAPK的磷酸化水平, 然后利用不同信号传递分子的抑制剂处理BmE细胞筛选参与传递PP免疫诱导信号的分子。【结果】PI3K抑制剂LY294002和受体型酪酸激酶抑制剂Genistein抑制了PP对BmE细胞抗菌肽基因表达的诱导作用; 且BmE细胞和家蚕血细胞在PP的诱导下, Akt和大小约为70 kDa的细胞膜蛋白均出现磷酸化水平的动态变化。【结论】PI3K/Akt信号通路和Genistein敏感性的受体型酪氨酸激酶介导了PP免疫诱导信号的传递。     

低氧对骨骼肌成肌细胞增殖分化的影响及相关机制研究进展

骨骼肌中成肌细胞作为肌源性祖细胞具有自我更新和生成新的肌纤维的能力,而氧气水平对调节成肌细胞的肌生成能力至关重要。这篇综述中描述了低氧状态下细胞的分子调控,氧水平对成肌细胞在肌生成中的影响和骨骼肌成肌细胞增殖和分化的转录调节。本文还概述了多种信号通路在分子调控中的机制,其中Notch,Wnt信号通路是依赖于低氧诱导因子HIF-1α介导的机制;PI3K-Akt-mTOR、p38 MAPK、p53信号传导则是独立于HIF-1α在低氧下调节成肌分化的途径。因此,深入研究和进一步揭示机制中涉及的信号通路为治疗低氧对骨骼肌损伤性的疾病提供证据和参考。     

举重运动通过激活PI3K/Akt信号途径并促进肌肉卫星细胞活化来改善肌肉功能

为了考察举重运动对卫星细胞活化和PI3K/Akt信号通路的影响,本研究将60只SD大鼠随机分为对照组(未进行运动)、耐力运动组和举重运动组。耐力运动组和举重运动组大鼠分别进行8周相应的运动。采用双重免疫荧光染色检测大鼠骨骼肌组织中活化的卫星细胞数(Pax7+/MyoD+)。采用Western blotting检测大鼠骨骼肌组织中Akt、mTOR、p70S6K、4E-BP1、IL-1β和IL-6的表达。采用苏木精和曙红(HE)染色评价骨骼肌形态。采用日立7060全自动生化分析仪检测大鼠血清肌酸激酶(CK)水平。采用邻苯三酚比色法检测血清超氧化物歧化酶(SOD)活性。研究发现,运动后,举重运动组活化的卫星细胞数显著高于耐力运动组。运动后,举重运动组的Akt、mTOR、p70S6K和4E-BP1的磷酸化水平显著高于耐力运动组,而IL-1β和IL-6蛋白表达水平显著低于耐力运动组。运动后,举重运动组的血清CK水平显著低于耐力运动组,而SOD水平显著高于耐力运动组。本研究表明举重运动可促进肌肉卫星细胞的活化并且激活PI3K/Akt信号通路及下游分子。此外,举重运动还可提高机体抗氧化能力并预防炎症损伤。     

Effect of fiber type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat muscle

AMP-activated protein kinase (AMPK) may mediate the stimulatory effect of contraction and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on glucose transport in skeletal muscle. In muscles with different fiber type composition from fasted rats, AICAR increased 2-deoxyglucose transport and total AMPK activity approximately twofold in epitrochlearis (EPI), less in flexor digitorum brevis, and not at all in soleus muscles. Contraction increased both transport and AMPK activity more than AICAR did. In EPI muscles, the effects of AICAR and contractions on glucose transport were partially additive despite a lower AMPK activity with AICAR compared with contraction alone. In EPI from fed rats, glucose transport responses were smaller than what was seen in fasted rats, and AICAR did not increase transport despite an increase in AMPK activity. AICAR and contraction activated both alpha(1)- and alpha(2)-isoforms of AMPK. Expression of both isoforms varied with fiber types, and alpha(2) was highly expressed in nuclei. In conclusion, AICAR-stimulated glucose transport varies with muscle fiber type and nutritional state. AMPK is unlikely to be the sole mediator of contraction-stimulated glucose transport.     

A microcomputer-based analyser for evaluation of contractile properties of a cardiac muscle

A computer controlled equipment is described for computer analysis of isometric contractions of cardiac muscle by different modes of excitation. Both the method and the programme are presented. The following parameters are analysed: half and full contraction time, half relaxation time, maximum force developed during contraction, maximum rate of force development. The same computer is simultaneously used for controlling the stimulation.     

Protein metabolism during endurance exercise

After reviewing all the available results from our laboratory and numerous reports in the literature concerning changes that have occurred in various aspects of protein metabolism during exercise, a number of conclusions can be drawn with some degree of confidence. During exercise, protein synthesis is depressed and this change leaves amino acids available for catabolic processes. The rate of leucine oxidation appears to be increased during exercise, and there is a movement of amino acids, mostly in the form of alanine, from muscle to liver where the rate of gluconeogenesis is increased as a result of exercise. These changes in protein metabolism are probably physiologically significant in at least three ways: amino acid conversion to citric acid cycle intermediates enhances the rate of oxidation of acetyl-CoA generated from glucose and fatty acid oxidation; increased conversion of amino acids to glucose helps to prevent hypoglycemia; oxidation of some amino acids may provide energy for muscular contraction.     

Shared strategies in gene organization among prokaryotes and eukaryotes

Although genes in prokaryotes and eukaryotes are transcribed and translated by very different mechanisms, they may be organized in their respective chromosomes in surprisingly similar ways. Here, I examine common modes of maintaining nonrandom gene organization in both prokaryotes and eukaryotes, the different ways these organizations have likely arisen, and classes of organization that may be unique to one group or the other.     

Different types of variant muscle nociception after intermittent and continuous neuromuscular stimulation in rats

Summary Critical assessment of experimental muscle-pain models resulting from maximal muscle contraction may provide a means of assessing hypersensitivity and the central nociceptive mechanisms involved in diffused muscle pain. The aim of the present study was, therefore, to investigate the patterns of nociceptive behavior and neuronal changes in the rat spinal cord after two modes of maximal muscle contraction. The gastrocnemius muscle of adult male Sprague-Dawley rats was subjected to continuous (10 min) or intermittent (60 min, 10/50 s on/off ratio) premodulated electrical stimulation of median frequency. Similar peak forces but different patterns of contraction output were generated by these two stimulation modes. Nociceptive behavioral scores and hind-leg oedema were significantly greater in the continuous group compared to the controls; however, significant difference was not demonstrated for either parameter comparing the intermittent and control groups. The sensory threshold was slightly reduced after the intermittent stimulation, and elevated after the continuous modality. The elevation of sensory threshold could be reversed by naloxone administration. More Fos-labeled nuclei were noted for both of the stimulation groups relative to the controls. The Fos-labeled nuclei were larger for the intermittent group than for the continuous and control analogs. The results of the present study suggest that prolonged contraction from continuous stimulation results in specific nociceptive neuron activation, muscle lesion and endogenous opioid release causing exaggerated nociception.     

The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation

Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3‐kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin‐activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho‐aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho‐aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle. Copyright © 2014 John Wiley & Sons, Ltd.     

Propranolol prevents epinephrine from limiting insulin-stimulated muscle glucose uptake during contraction.

Beta-blockade results in rapid glucose clearance and premature fatigue during exercise. To investigate the cause of this increased glucose clearance, we studied the acute effects of propranolol on insulin-stimulated muscle glucose uptake during contraction in the presence of epinephrine with an isolated rat muscle preparation. Glucose uptake increased in both fast- (epitrochlearis) and slow-twitch (soleus) muscle during insulin or contraction stimulation. In the presence of 24 nM epinephrine, glucose uptake during contraction was completely suppressed when insulin was present. This suppression of glucose uptake by epinephrine was accompanied by a decrease in insulin receptor substrate (IRS)-1-phosphatidylinositol 3 (PI3)-kinase activity. Propranolol had no direct effect on insulin-stimulated glucose uptake during contraction. However, epinephrine was ineffective in attenuating insulin-stimulated glucose uptake during contraction in the presence of propranolol. This ineffectiveness of epinephrine to suppress insulin-stimulated glucose uptake during contraction occurred in conjunction with its inability to completely suppress IRS-1-PI3-kinase activity. Results of this study indicate that the effectiveness of epinephrine to inhibit insulin-stimulated glucose uptake during contraction is severely diminished in muscle exposed to propranolol. Thus the increase in glucose clearance and premature fatigue associated with beta-blockade could result from the inability of epinephrine to attenuate insulin-stimulated muscle glucose uptake.     

WATER RELATIONS IN THE CELL : I. THE CHLOROPLASTS OF NITELLA AND OF SPIROGYRA

Chloroplasts may contract under natural conditions and give up water to the rest of the cell, thus indicating changes in metabolism or constitution. Such contractions may be produced experimentally. In Nitella the chloroplasts are ellipsoid bodies which, under natural conditions, may contract to spheres with a loss of volume. This may be brought about by lead acetate, ferric chloride, and digitonin: the contraction may occur while the cell is alive. The contraction in lead acetate is reversible (in lead nitrate little or no contraction occurs). In Spirogyra the chloroplast is a long, spirally coiled ribbon which may contract under natural conditions to a short nearly straight rod with a loss of volume. This can be brought about by inorganic salts and in other ways while the cell is still alive.     

Adrenoceptor and local modulator control of cutaneous blood flow in thermal stress

Blood flow to the skin is controlled by body temperatures in two ways: core and mean skin temperature combine in the central nervous system to form a reflex mechanism that controls the frequency of activity in sympathetic nerves to the cutaneous blood vessels; and local mechanisms independent of reflex effects control contractile response to the sympathetic transmitter norepinephrine (NE) at different temperatures. Cutaneous vessels differ in responsiveness to NE across temperatures: in limbs and tails, the superficial vessels constrict more strongly to NE when cooled, while the deep vessels show weaker responses to NE when cooled. This allows the limb to dissipate heat when warm and to conserve heat when cool. The mechanism for this difference in thermal response of deep and superficial vessels is not completely known, but may relate to differences in the adrenoceptors on which NE acts, and/or to the actions of locally produced substances that modulate the responses to NE in different ways at different temperatures. This paper discusses the alpha1- and alpha2-adrenoceptors involved in contraction of deep and superficial cutaneous vessels and also describes the roles of the local modulator nitric oxide, which interacts with adrenoceptors to affect cutaneous blood flow.     

The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.