鲢鱼解偶联蛋白2全长cDNA序列的克隆及其组织表达

从淡水食毒藻鱼类鲢鱼(Hypophthalmichthysmolitrix)肝脏,通过简并引物克隆解偶联蛋白2(un-couplingprotein2,UCP2)cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏UCP2cDNA全序列。序列分析结果表明,鲢鱼肝脏UCP2cDNA全长1452bp,其中5′-UTR长337bp,3′-UTR长182bp,编码区933bp,编码310个氨基酸,推测的氨基酸序列包含线粒体内膜载体蛋白3个特征结构及解偶联蛋白(UCPs)的特征序列。对鲢鱼不同组织UCP2的表达调控研究发现,鲢鱼组织UCP2基因在肠道、肝脏、肌肉、脂肪组织均大量表达,而在脑组织表达量较低,这与鲢鱼体内微囊藻毒素在这几个组织的分布完全一致,表明UCP2的功能可能与抑制微囊藻毒素引发过量活性氧(ROS)生成有关。     

解偶联蛋白2对活性氧的抑制作用

线粒体在能量代谢和自由基代谢中占据十分重要的地位。电子传递过程中形成的活性氧(reactive oxygen species,ROS)履行着众多生理功能,但过多或持续存在的ROS可能与癌症、衰老、糖尿病、动脉硬化、局部缺血或再灌注损伤等的发生有关。解偶联蛋白2(uncoupling protein 2,UCP2)作为线粒体内膜质子转运家族中的一个新成员,通过解偶联作用能降低线粒体内膜电势,使活性氢产生减少。UCP2抑制ROS产生的作用日益受到关注。     

线粒体解偶联蛋白UCP2的研究进展

本文综述了线粒体解偶联蛋白2（uncoupling protein2,UCP2）研究方面的进展。UCP2定位于线粒体内膜上,通过消散线粒体内膜的质子梯度调节线粒体的功能,包括线粒体内膜电位、ATP合成、呼吸链ROS产生、线粒体钙库的存储和释放等。目前,UCP2的质子漏机理并不清楚,但体内实验表明UCP2活性可被过氧化物激活。特别是近年来UCP2调控胰岛素分泌方面的研究取得了重要进展。     

鲢鱼解偶联蛋白2全长cDNA序列的克隆及其组织表达

从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏，通过简并引物克隆解偶联蛋白2(uncoupling protein 2，UCP2) cDNA核心序列，应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列，最后通过序列拼接获得鲢鱼肝脏UCP2 cDNA全序列。序列分析结果表明，鲢鱼肝脏UCP2 cDNA全长1 452 bp，其中5′-UTR长337 bp，3′-UTR长182 bp，编码区933 bp，编码310个氨基酸，推测的氨基酸序列包含线粒体内膜载体蛋白3个特征结构及解偶联蛋白(UCPs)的特征序列。对鲢鱼不同组织UCP2的表达调控研究发现，鲢鱼组织UCP2基因在肠道、肝脏、肌肉、脂肪组织均大量表达，而在脑组织表达量较低，这与鲢鱼体内微囊藻毒素在这几个组织的分布完全一致，表明UCP2的功能可能与抑制微囊藻毒素引发过量活性氧(ROS)生成有关。     

真鲷肝脏解偶联蛋白2(UCP2)基因及其功能的探讨

从真鲷(Pagrus major)肝脏通过简并引物PCR克隆解偶联蛋白2(UCP2)cDNA部分序列。该片段长674bp,编码224个氨基酸残基。推测的此部分氨基酸序列包含线粒体载体蛋白的特征结构,并与其它脊椎动物UCP2氨基酸序列同源性在72．8％以上。对变温动物色类UCP2组织表达调控研究表明：与哺乳类UCP2基因不同,真鲷UCP2基因在肝脏大量表达,而在腹腔肠系膜脂肪组织则仅有痕迹量表达,两者表达水平相差20倍以上。饲料中添加10％绿鳕油或48h饥饿对真鲷肝脏UCP2基因的表达水平均无显著影响,表明UCP2基因在脂肪含量高的鱼类肝脏表达十分稳定,为维持其基本功能所必需。真鲷肝脏和腹腔肠系膜脂肪组织UCP2基因表达水平的强烈反差,与鱼类这两种贮脂器官完全不同的氧化活性相一致[动物学报49(1)：110—117,2003]。     

酒精性心肌病大鼠心肌解偶联蛋白2表达及能量代谢变化

目的:观察心肌解偶联蛋白2(uncoupling protein 2,UCP2)在酒精性心肌病(alcoholic cardiomyopathy,ACM)时表达变化及对心肌能量代谢的影响.方法:将Wistar大鼠分为三组,酒精组(A组,10只)、少量饮酒组(B组,7只)和对照组(C组,7只),三组给予相同饮食,酒精组通过采取逐渐增加饮用酒精浓度并长期定量摄入的方法建立ACM模型,少量饮酒组长期饮用少量低浓度酒精,对照组以水代酒.6个月后心脏彩超测定心功能;计算左室/体重指数;RT-PCR法测定心肌组织UCP2 mRNA表达;Western blot法测定心肌UCP2蛋白表达;高效液相色谱分析法测定心肌三磷酸腺苷酸(ATP)、二磷酸腺苷酸(ADP)、单磷酸腺苷酸(AMP)和磷酸肌酸(PCr)含量.结果:酒精组左心室射血分数和左心室短轴缩短率均低于对照组和少量饮酒组(P均＜0.01),左心室舒张末期内径则高于对照组和少量饮酒组(P＜0.01),左室/体重指数明显增加(P＜0.01);酒精组UCP2 mRNA及蛋白表达高于对照组和少量饮酒组(P均＜0.01);酒精组ATP、ADP、AMP和PCr较对照组和少量饮酒组明显减少(P均＜0.01),相关性分析显示心肌组织ATP水平与UCP2蛋白表达呈显著负相关(r=0.896,P＜0.01).结论:ACM时心肌UCP2表达明显增加,导致心肌能量代谢障碍,恶化心脏功能.     

解偶联蛋白2与衰老

解偶联蛋白(uncoupling protein,UCP)属于线粒体内膜上的一类载体蛋白,其生理作用是消除线粒体膜电位,使氧化磷酸化解偶联,从而抑制酸腺苷(adenosine triphosphate,ATP)合成,能量以热能形式散发.研究发现UCP2具有一种质子漏功能,表现对线粒体活性氧(reactive oxygen species,ROS)产生的调控和降低ROS的功能.在不同组织器官,不同代谢状态下UCP2的生理功能对细胞的影响不完全相同.特别是近年来的研究发现,UCP2参与了能量代谢、ROS的产生、子宫内膜退化、衰老等过程,并且与非酒精性脂肪肝、抗肥胖、动脉粥样硬化、局部缺血以及缺血再灌注损伤和2型糖尿病等有一定的相关性,倍受人们的关注.     

解偶联蛋白2与衰老

解偶联蛋白（uncoupling protein,UCP）属于内膜上的一类载体蛋白,其生理作用是消除线粒体膜电位,使氧化磷酸化解偶联,从而抑制酸腺苷（adenosine triphosphate,ATP）合成,能量以热能形式散发。研究发现UCP2具有一种质子漏功能,表现对线粒体活性氧（reactive oxygen species,ROS）产生的调控和降低ROS的功能：在不同组织器官,不同代谢状态下UCP2的生理功能对细胞的影响不完全相同。特别是近年来的研究发现,UCP2参与了能量代谢、ROS的产生、子宫内膜退化、衰老等过程,并且与非酒精性脂肪肝、抗肥胖、动脉粥样硬化、局部缺血以及缺血再灌注损伤和2型糖尿病等有一定的相关性,倍受人们的关注。     

解偶联蛋白及功能研究进展

解偶联蛋白(ucP,uncoupling protein)是一类线粒体内膜上的载体,属于线粒体载体超家族,可以将H^ 从线粒体内膜渗漏到线粒体基质中,减少ATP的合成并产生热能。已知UCPl在小鼠中有维持体温和能量稳态的重要作用。而UCP2和UCP3可控制活性氧(reactive oxygen species,ROS)产生、调节脂肪酸氧化,并且在肥胖和糖尿病发生中有重要作用。     

解偶联蛋白与肥胖及2型糖尿病发病的关系

解偶联蛋白（UCPs）是线粒体内膜上的一种转运蛋白,它能够降低线粒体内膜上的质子梯度,使底物氧化和ADP磷酸化解偶联,减少ATP的产生。基于其功能,解偶联蛋白基因被视为肥胖病及2型糖尿病的重要候选基因。UCP同系物过表达的遗传工程小鼠表现出对饮食导致的肥胖具有耐受性,同时UCP2基因3＇非翻译区的45bp插入／缺失以及UCP3基因C-55T多态与肥胖表型的相关性等研究结论支持这一假说。本文对UCP基因与多基因控制的肥胖病及2型糖尿病发病的相关研究进行综述和讨论。 Abstract:Uncoupling proteins (UCPs) are mitochondria carrier proteins,which are able to dissipate the proton gradient of the inner mitochondria membrane.The uncoupling procedure reduces the amount of ATP generated through an oxidation of fuels.Therefore,UCPs are suggested as candidate genes for human obesity or type II diabetes mellitus.Experimental evidences,that genetically engineered mice over expressing different UCP homologues were resistant to diet-induced obesity and 45bp insertion polymorphism in the UCP2 3＇untranslated region and C-55T in UCP3 promoter region were associated with obesity related phenotype,supported the hypothesis.The roles of UCP genes in polygenic obesity and type II diabetes are evaluated and discussed in this paper.     

Structure and composition of teleost scales from snakehead Channa argus (Cantor) (Perciformes: Channidae)

The structure of teleost scales from snakehead Channa argus was investigated using thermogravimetric analysis (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive analysis of X-rays (EDAX), Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction (XRD). Thermal treatment of fish scales indicates that the fibrillary plate is partially calcified. SEM shows two kinds of scale denticles, arranged along the circuli in the anterior field and the lateral fields, respectively. TEM indicates the stratum laxum with abundant fibrils, chromatophores and capillary blood vessels within the scale covering, and shows the fibrillary plate as an 'orthogonal plywood structure' of stratified lamellae, consisting of 80–100 nm diameter collagen fibres co-aligned in individual lamellae and alternated by c. 90° of the fibre alignment between adjacent lamellae. EDAX, FTIR and XRD show that the mineral phase of the scales is a carbonated hydroxyapatite with a Ca:P molar ratio of 1·85.     

Association of the ins/del polymorphisms of uncoupling protein 2 (UCP2) with BMI in a Korean population

To elucidate the potential impact of the variants of the UCP2 gene on obesity phenotypes, we have genotyped four polymorphisms in UCP2 among 988 Korean subjects using TaqMan^® methods, and three common haplotypes with frequency greater than 0.1 were constructed in the Korean population. No significant associations were detected with the risk of metabolic syndrome by logistic regression analyses. However, the 45 base-pair ins/del polymorphism (+3474 ins/del) in the 3′ untranslated region (3′ UTR) showed significant association with body mass index (P = 0.007, P^corr = 0.02) and waist circumference (P = 0.005). Further subgroup analysis revealed that the genetic effects were more apparent among female subjects. In addition, a summary of the controversial genetic effects on obesity mediated by UCP2 polymorphisms from previous studies is also given. Our results suggest that subjects with a 45 bp insertion allele of UCP2+3474 ins/del might have a higher risk of developing obesity, although the biological effects of this variant are not completely known.     

Effects of rosiglitazone on global ischemia-induced hippocampal injury and expression of mitochondrial uncoupling protein 2

We investigate the effect of rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPARgamma) with anti-inflammatory and anti-oxidative actions, on hippocampal injury and its roles in mitochondrial uncoupling protein 2 (UCP2) expression caused by transient global ischemia (TGI) in rats. Increased UCP2 expression was observed in mitochondria of hippocampal CA1 2-24h after TGI/reperfusion, with maximal expression levels at 6-18h. Administration of rosiglitazone to hippocampus 30min prior to the onset of TGI further enhanced mitochondrial UCP2 expression 2-6h following TGI/reperfusion. Rats subjected to TGI/reperfusion displayed a significant increase in lipid peroxidation, based on increased malondialdehyde (MDA) levels, in hippocampal CA1 mitochondria 2-6 h after reperfusion. Rosiglitazone significantly attenuated TGI/reperfusion-induced lipid peroxidation and suppressed hippocampal CA1 neuronal death based on the surviving neuronal counts. In conclusion, our results provide correlative evidence for the "PPARgamma-->UCP2-->neuroprotection" cascade in ischemic brain injury.     

The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis

A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.     

人重组磷脂酶D_2变构体cDNA和蛋白质序列分析

采用VectorNTI、DNATools等计算机分析软件及信息库,研究人重组磷脂酶D2(rhPLD2)变构体的生物学特征。rhPLD2具有多种基因结构和功能调控元件,其编码的蛋白质具有发挥功能所必需的活性保守基序和一定的空间结构,表明rhPLD2应是一种具有一定生物学功能的异质性蛋白质。     

Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.