藏羚羊骨骼肌肌红蛋白含量及乳酸脱氢酶、苹果酸脱氢酶活力的研究

目的:探讨藏羚羊骨骼肌对低氧环境的适应机制。方法:以生活在同海拔高度(4 300 m)的藏绵羊和低海拔绵羊(1 800 m)为对照,用分光光度法测定三种动物骨骼肌中肌红蛋白(Mb)含量、乳酸(LA)含量,酶活力法测定三种动物骨骼肌中乳酸脱氢酶(LDH)和苹果酸脱氢酶(MDH)活力。结果:藏羚羊骨骼肌中Mb含量明显高于藏绵羊和低海拔绵羊(P<0.05),而藏绵羊和低海拔绵羊间无明显差异。LA含量和LDH活力明显低于藏绵羊和低海拔绵羊(P<0.05),而MDH活力及MDH/LDH比值显著高于藏绵羊和低海拔绵羊(P<0.05),藏绵羊和低海拔绵羊间无明显差异。结论:藏羚羊可能通过增加骨骼肌中Mb的含量,提高其在低氧环境获取氧的能力,且藏羚羊骨骼肌组织中有氧代谢比例高,这可能与肌肉中Mb含量较高有关,推测藏羚羊较高的Mb含量可能是其适应高原缺氧条件的分子基础之一。     

藏羚羊和藏系绵羊血液内分泌激素水平的比较研究

为了探讨藏羚羊适应高原环境的内分泌系统的功能特点,我们对藏羚羊(9只)和藏系绵羊(10只)进行了内分泌激素水平的比较。在海拔4300m地区捕获到动物后,随即运至海拔2800m实验基地,次日早晨抽取右侧颈静脉血,采用放射免疫法(radioimmunoassay,RIA)或酶联免疫吸附实验法(enzyme-linked immunosorbent assay,ELISA)测定下丘脑-腺垂体-外周腺轴20项内分泌激素水平,用导管插入术记录动物心率(heart rate,HR)、收缩压(systolic blood pressure,SBP)、舒张压(diastolic bloodpressure,DBP)和平均肺动脉压(mean pulmonary arterial pressure,mPAP)。使用血细胞分析仪测定血红蛋白(hemoglobin,Hb)含量。结果显示,藏羚羊血液中FT3、FT4、AngⅡ水平显著低于藏系绵羊,而藏羚羊血液中TRH、CRH、F、GHRH、E2、Ald、ACTH、CGRP水平则显著高于藏系绵羊。相对于藏系绵羊,藏羚羊HR、mPAP、SBP、DBP和Hb含量明显较低。藏羚羊和藏系绵羊的H...     

肌酸对游泳大鼠乳酸、糖原含量和乳酸脱氢酶活性的影响

为探讨肌酸对提高大鼠运动能力的作用 ,观察了肌酸对游泳大鼠血清、心肌和骨骼肌乳酸、糖原含量和乳酸脱氢酶 (LDH)活性的影响。实验用雄性wistar大鼠 2 4只 ,随机分为正常组、游泳对照组和游泳补充肌酸组。两个游泳组每天游泳训练 1h,9天后 ,游泳 4h ,测定血清、心肌和骨骼肌乳酸水平 ,测定血清和骨骼肌乳酸脱氢酶活性以及心肌与骨骼肌糖原含量。结果显示 :肌酸可抑制游泳运动后大鼠血清、心肌和骨骼肌乳酸浓度以及血清LDH活性的升高幅度 ,抑制心肌和骨骼肌糖原含量及骨骼肌LDH活力的下降。以上结果表明 ,肌酸可改善运动后机体乳酸和糖原的代谢 ,降低运动性疲劳 ,提高大鼠的运动能力     

不同海拔牦牛骨骼肌中乳酸脱氢酶三种亚基基因表达的比较

为了阐明高原低氧对牦牛（Bos mutus）骨骼肌中乳酸脱氢酶（LDH）三种亚基基因（LDHA、LDHB和LDHC）表达的影响,本实验分别选取高海拔（4 200 m）、中海拔（3 200 m）和低海拔（1 900 m）三个海拔位置养殖的临床健康成年雄性牦牛各5头,采用实时荧光定量PCR(qRT-PCR)和蛋白质印迹法检测牦牛骨骼肌中LDH三种亚基基因的m RNA表达和蛋白表达水平。结果表明,随海拔的升高,牦牛骨骼肌中LDHA m RNA的表达逐渐下降;LDHB m RNA先降低后升高,在高海拔组牦牛中表达最高,相对表达量为2.82±0.12,与低海拔组（1.01±0.07）、中海拔组（0.73±0.06）牦牛LDHB mRNA表达量差异显著（P <0.05）;LDHC mRNA的表达量随海拔的升高呈下降趋势,且低海拔组（1.10±0.16）、中海拔组（0.86±0.16）、高海拔组（0.69±0.12）组间两两相比均差异显著（P <0.05）。LDHA和LDHC蛋白表达量随海拔的升高呈下降趋势,且LDHA蛋白表达量在低海拔组（1.00±0.00）、中海拔组（0.88±0.0...     

藏羚羊肌红蛋白(MGB)基因编码区的克隆与分析

为了探讨藏羚羊低氧适应的分子生物学机制,从藏羚羊(Pantholops hodgsonii)骨骼肌组织中提取总RNA,通过逆转录聚合酶联反应(RT-PCR)技术扩增出MGB基因编码区cDNA片段,并与pGEM-T Easy载体连接构建重组质粒,转化DH5α菌。扩增培养后鉴定阳性质粒并进行序列测定,结果与NCBI数据库进行比较。结果显示,MGB基因编码区由465bp组成,编码154个氨基酸。其序列与绵羊、牛、猪、人、小鼠、大鼠的同源性分别是98%、97%、90%、86%、82%、81%。推测出的氨基酸序列与绵羊、牛有98%同源性,而与猪、人、小鼠及大鼠分别有89%、85%、80%、79%的同源性。与NCBI数据库登录的绵羊的MGB cDNA序列比较发现,藏羚羊MGB基因的21位和78位密码子分别发生了突变(GGT→GAT和GAA→AAG),其对应的氨基酸分别由甘氨酸变为天冬氨酸及谷氨酸变为赖氨酸。至此,成功克隆出藏羚羊MGB基因编码区,为进一步揭示藏羚羊低氧适应的分子机制打下基础。     

藏羚羊和藏野驴粪便真菌多样性比较研究

为探究西藏地区反刍动物(藏羚羊)和单胃草食动物(藏野驴)粪便中真菌群落结构,本研究采用ITS1区高通量测序方法分析西藏羌塘国家自然保护区藏羚羊和藏野驴新鲜粪便中真菌的多样性.结果表明:从5头藏羚羊新鲜粪便中共鉴定出5个门、15个纲、32个目、45个科和56个属的真菌;从5头藏野驴新鲜粪便中共鉴定出3个门、10个纲、18...     

高原鼢鼠、鼠兔及大鼠心肌和骨骼肌乳酸脱氢酶活力及同工酶谱

为了探讨高原鼢鼠和鼠兔的低氧适应机理,用紫外分光光度法测定了高原鼢鼠、鼠兔及大鼠心肌和骨骼肌乳酸脱氢酶(Lactate Dehydrogenase,LDH)的活力,通过聚丙烯酰胺凝胶电泳分离了LDH同工酶.结果显示:鼢鼠、鼠兔、大鼠心肌LDH活力分别为16.90±2.00 U/mg pro,20.55±2.46 U/mg pro,38.26±6.78 U/mg pm,鼢鼠和鼠兔差异不显著(P>0.05),大鼠与鼠兔差异显著(P<0.05),大鼠与鼢鼠差异极显著(P<0.01);骨骼肌LDH活力,鼢鼠、鼠兔、大鼠分别为39.34±3.74 U/mg pro,78.33±9.54 U/mg pro,67.80±10.89 u/mg pr0,大鼠和鼠兔差异不显著(P>0.05),二者均极显著高于鼢鼠(P<0.01).高原鼠兔与鼢鼠LDH同工酶M亚基的迁移率较为相近,而H亚基差别较大;鼢鼠和大鼠H亚基的迁移率较为相近,而M亚基差别较大.鼢鼠和鼠兔心肌LDH同工酶亚基组成以H亚基为主,M亚基含量较低,而骨骼肌LDH同工酶亚基组成以M业基为主,H业基含量较低.大鼠心肌和骨骼肌LDH同工酶H亚基和M亚基含量均很丰富.说明高原鼢鼠和鼠兔虽然生活在极其缺氧的环境中,但它们的组织并不缺氧;高原鼢鼠和鼠兔以不同的策略适应高原缺氧.     

麦洼牦牛乳酸脱氢酶-B基因多态性的PCR-RFLP分析

为了从分子水平上检测麦洼牦牛Bos grunniens乳酸脱氢酶-1(LDH1)的H亚基编码基因Ldhb的多态性,实验提取79头麦洼牦牛基因组DNA,采用PCR-RFLP技术分析Ldhb基因多态性,并测定牦牛背最长肌中肌红蛋白含量。实验建立的牦牛Ldhb的G896A突变位点的PCR-RFLP检测方法,在79头麦洼牦牛中检测到Ldhb-AA和Ldhb-AG两种基因型,其中AG基因型频率为16.46%,并且这些样品的乳酸脱氢酶-1的电泳迁移率快于Ldhb-AA样品。本实验未检测到Ldhb-GG基因型;麦洼牦牛Ldhb基因多态性与背最长肌中的肌红蛋白含量之间未见相关性。     

甘肃鼢鼠与SD大鼠骨骼肌低氧适应的比较

运用组织学和紫外分光光度法,对常氧、低氧2周和4周的甘肃鼢鼠(Myospalax cansus)及SD大鼠(Rattus norvegicus)骨骼肌形态结构、乳酸脱氢酶(LDH)、琥珀酸脱氢酶(SDH)活力及肌红蛋白(Mb)浓度进行测定。结果显示,甘肃鼢鼠骨骼肌形态结构在常氧、低氧2周及4周后变化不明显;SD大鼠随低氧时间延长发生显著变化,低氧2周后,肌纤维明显萎缩,间隙增大,结构较紊乱;低氧4周后,肌纤维破裂,呈细丝状,不规则,大小不一致,肌节紊乱。甘肃鼢鼠LDH活性随低氧时间增长逐渐降低,但无显著性差异(P0.05);在不同时间低氧处理后,甘肃鼢鼠前后肢的LDH活性均极显著低于SD大鼠(P0.01);常氧条件下,甘肃鼢鼠前后肢SDH活性低于SD大鼠,但无显著差异(P0.05),低氧2周及4周后,与SD大鼠呈极显著差异(P0.01);常氧条件下,甘肃鼢鼠前后肢Mb浓度均显著高于SD大鼠(P0.05),低氧2周及4周后,极显著高于SD大鼠(P0.01)。结果表明,甘肃鼢鼠骨骼肌较SD大鼠有着更强的低氧耐受力。     

藏羚寄生蠕虫卵粪检调查初报

2004年12月25日~2005年1月5日对位于青藏公路楚玛尔河大桥附近集群的藏羚(Pantholopshodgsoni),随机拣取其新鲜粪便共计36份带回实验室。利用漂浮法、沉淀法对粪样进行寄生虫蠕虫卵检查,结果显示,粪样中存在有细颈属(Nematodirus)、马歇尔属(Marshallagia)线虫卵和莫尼茨属(Moniezia)绦虫卵,其感染率在19.4%~94.4%之间,感染强度(EPG)分别为5.58、5.11和2.86。结果表明,冬季藏羚的寄生蠕虫感染率高,但感染种类少和感染强度低。     

Lactate Dehydrogenase Activity is Inhibited by Methylmalonate in vitro

Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K _i=3.02±0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.S. R. Mirandola and E. N. Maciel contributed equally to this work.     

藏羚羊成纤维细胞系的建立及其核型分析

藏羚羊(Pantholops hodgsonii)是我国特有的国家Ⅰ级重点保护野生动物,生存在高海拔低氧环境下,具有高原生物的细胞遗传特性。实验采用组织块贴壁培养法进行原代培养,后经差异贴壁法联合消化排除法纯化,成功构建了藏羚羊成纤维细胞系,并对培养不同代次细胞的形态、生长动力学、染色体核型等进行了研究。结果表明,培养得到的藏羚羊细胞为典型的成纤维细胞,第7、10、15代细胞群体倍增时间分别为26、48、50 h。细胞的生长均为"S"型。染色体中二倍体染色体(2n=60)占主体,所占比例为65%～91%,包括性染色体X、Y在内均为端着丝粒类型,染色体组的总相对长度为193.45,平均相对长度为3.22,未发现随体、次缢痕等特殊标志性特征。     

Activity of Lactate Dehydrogenase in Serum and Cerebral Cortex of Immature and Mature Rats after Hypobaric Hypoxia

In our previous studies we have found both an increase of lipid peroxidation damage (expressed as levels of thiobarbituric acid-reactive substances) in brain and plasma lactate concentration in 21-day-old rats after a 30-min exposure to hypobaric hypoxia. Pretreatment of rats with l-carnitine decreased both parameters. The aim of our present study was to determine if the l-carnitine-dependent decrease of plasma lactate could be due to a modification of lactate dehydrogenase (LDH) activity. We followed brain and blood serum LDH activity of 14-, 21- and 90-day-old Wistar rats. We found an increase of brain LDH activity with age. However, we did not observe any significant differences in LDH activity after exposure to hypobaric hypoxia or l-carnitine pretreatment. In contrast to brain, serum LDH activity did not show any clear age-dependence. The hypoxia exposure increased LDH activity of 21-day-old rats only. Pretreatment of rats with l-carnitine decreased serum LDH activity of 21- and 90-day-old rats probably due to membrane stabilizing role of l-carnitine. In conclusions, acute hypobaric hypoxia and/or l-carnitine pretreatment modified serum but not brain LDH activity.     

Regulation of Glycerol Phosphate Dehydrogenase and Lactate Dehydrogenase Activity by Forskolin and Dibutyryl Cyclic AMP in the C6 Glial Cells

We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP.     

中国美利奴羊H-FABP基因多态性及其与部分肉质性状的相关性

利用聚合酶链式反应-单链构象多态（PCR-SSCP）技术检测中国美利奴羊（Ovis aries var. Merino）心型脂肪酸结合蛋白基因（H-FABP）外显子2的单核苷酸多态性（SNPs）和遗传多态性,分析其与肌内脂肪（IMF）含量、肌纤维直径和肌纤维密度的相互关系,为该品种绵羊的分子标记辅助选择提供理论依据。结果显示,H-FABP基因外显子2有AA、AB和BB 3种基因型,AA型和BB型在778位均发生了C缺失,939位均发生了A→G转换,BB型还在789位发生了T→C转换,该突变导致所编码氨基酸发生了缬氨酸→丙氨酸的替换;BB型为IMF的优势基因型,与AB型相比差异显著（P<0.05）,与AA型相比差异极显著（P<0.01）;BB型对肌纤维直径存在负相关。结果提示,中国美利奴羊H-FABP基因外显子2具有多态性,该基因可能是中国美利奴羊肉质性状的主效基因,或者与控制肉质性状的主效基因相连锁。     

The Effects of Nano-anatase TiO2 on the Activation of Lactate Dehydrogenase from Rat Heart

Lactate dehydrogenase (LDH, EC1.1.1.27), widely expressed in the heart, liver, and other tissues, plays an important role in glycolysis and glyconeogenesis. The activity of LDH is often altered upon inflammatory responses in animals. Nano-TiO₂ was shown to provoke various inflammatory responses both in rats and mice; however, the molecular mechanism by which TiO₂ exerts its toxicity has not been completely understood. In this report, we investigated the mechanisms of nano-anatase TiO₂ (5 nm) on LDH activity in vitro. Our results showed that LDH activity was greatly increased by low concentration of nano-anatase TiO₂, while it was decreased by high concentration of nano-anatase TiO₂. The spectroscopic assays revealed that the nano-anatase TiO₂ particles were directly bound to LDH with mole ratio of [nano-anatase TiO₂] to [LDH] was 0.12, indicating that each Ti atom was coordinated with five oxygen/nitrogen atoms and a sulfur atoms of amino acid residues with the Ti–O(N) and Ti–S bond lengths of 1.79 and 2.41 Å. We postulated that the bound nano-anatase TiO₂ altered the secondary structure of LDH, created a new metal ion-active site for LDH, and thereby enhanced LDH activity.     

藏鹀的自然历史、威胁和保护

藏鹀(Emberiza koslowi)是我国青藏高原东部的特有珍稀鸟种,目前关于它的资料非常匮乏.为了解藏鹀的分布、数量和基本生活史特征,促进对于该物种的有效保护,自2005年起以青海省果洛州久治县白玉乡为中心对藏鹀进行了持续6年的观察,并针对其面临的威胁采取了相应的保护措施.结果显示,藏鹀主要分布在青海的玉树、果洛和四川的阿坝一带海拔3 500～4 700 m范围内的适宜栖息地,该分布区比原有认知更靠东北,更为狭小且海拔更高.用样线法开展的藏鹀数量监测,在7.4 km2的调查范围内记录到一个18 ～33只的稳定种群.此外,还对藏鹀的筑巢、育雏和争斗等行为进行了详细描述.食肉动物的捕食、冬季食物缺乏和牲畜踩踏鸟卵是藏鹀面临的最直接威胁.通过持续监测、与当地牧民协商、建立保护小区并开展有针对性的保护,藏鹀种群趋于稳定.     

中国林蛙乳酸脱氢酶多基因系统及基因间连锁关系的研究

（１）用聚丙烯酰胺凝胶电泳对山西产中国林蛙４个地理群的３３３只林蛙进行了分析,结果表明：中国林蛙的ＬＤＨ由ＬＤＨ－Ａ,ＬＤＨ－Ｂ和ＬＤＨ－Ｃ３个基因决定。ＬＤＨ－Ａ是单态座位,ＬＤＨ－Ｂ和ＬＤＨ－Ｃ均为多态座位,每个多态座位均有两个等位基因。ＬＤＨ－Ｂ与ＬＤＨ－Ｃ呈紧密连锁关系。认为ＬＤＨ－Ｃ是ＬＤＨ－Ｂ的重复产物。（２）热稳定性、尿素处理稳定性及组织特异性研究表明：ＬＤＨ对温度和尿素处理稳定性顺序为Ａ４＞Ｂ’４＞Ｂ４＞Ｃ４。Ａ４在骨骼肌和肝脏等组织中活力最大,Ｂ４在心肌和卵巢中活力最强,ＬＤＨ－Ｃ主要在眼球和卵巢中表达。（３）Ｌｄｈ－ｂ和Ｌｄｈ－ｂ＇在不同地理群间呈差异分布,随着纬度的增高,Ｌｄｈ－ｂ在种群中的频率增大。