高原鼠兔组织中精子特异性乳酸脱氢酶的作用机理

高原鼠兔Ochotona curzoniae具有很强的高原低氧适应能力。前期研究发现,精子特异性乳酸脱氢酶(LDH-C4)在高原鼠兔体细胞中表达。为阐明LDH-C4在高原鼠兔组织中的作用机理,应用RNA干扰技术沉默高原鼠兔心肌、肝脏和脑组织中的Ldh-c基因;应用荧光定量PCR和Western Blot方法,测定Ldh-c基因在心肌、肝脏和脑组织中的表达水平;应用生物化学方法测定沉默Ldh-c基因后,心肌、肝脏和脑组织中LDH比活力、乳酸和ATP的含量。结果表明,腹腔注射腺病毒p MultiRNAi-Ldhc能极显著降低高原鼠兔组织中Ldh-c基因的表达水平,在mRNA和蛋白水平,心肌中Ldh-c基因的表达分别下降48.11%和19.27%;肝脏中Ldh-c基因的表达分别下降70.16%和25.82%;脑中Ldh-c基因的表达分别下降49.08%和25.36%。沉默Ldh-c基因表达后,高原鼠兔心肌、肝脏和脑组织中LDH比活力、乳酸和ATP的含量也显著降低,分别下降25.58%、41.94%和21.23%,28.16%、15.90%和24.66%以及16.65%、12.78%和18.50%。这些结果说明,高原鼠兔在低氧环境中,其心肌、肝脏和脑组织通过LDH-C4催化无氧糖酵解获得部分ATP,增强对低氧环境的适应能力。     

高原鼠兔脑组织中精子特异性乳酸脱氢酶的作用

高原鼠兔对高原低氧环境有很强的适应性。研究发现,精子特异性乳酸脱氢酶(LDH-C4)基因在高原鼠兔脑组织中表达,为阐明LDH-C4在高原鼠兔脑组织中的作用,应用荧光定量PCR和Western blot方法,测定了Ldh-c基因在高原鼠兔脑组织中的表达水平;应用对精子特异性乳酸脱氢酶(LDH-C4)特异性的抑制剂(N-isopropyl oxamate),通过肌肉注射后,研究抑制剂对高原鼠兔脑组织中LDH比活力、乳酸和ATP含量的影响。结果表明,在mRNA和蛋白水平,Ldh-c基因在高原鼠兔脑组织中均有表达,相对表达水平分别为0.38±0.05和0.74±0.13;当肌肉注射1 m L 1 mol/L的抑制剂30 min后,血液中抑制剂浓度为0.08 mmol/L;与对照组相比,抑制剂组脑组织中LDH比活力、乳酸和ATP含量显著下降,抑制剂对LDH、乳酸和ATP的抑制率分别为30.78%、46.47%和21.04%。结果表明,精子特异性乳酸脱氢酶基因在高原鼠兔脑组织中表达。LDH-C4通过催化无氧糖酵解过程,为其脑组织生命活动提供至少20%的ATP,可能使高原鼠兔减小在低氧环境中对氧气的依赖,增强对低氧环境的适应能力。     

低氧暴露下高原鼠兔肺组织间隙连接蛋白40表达分析

低氧性肺血管收缩反应钝化是高原鼠兔适应低氧环境的重要策略,但参与该生理代偿反应的功能基因尚不明确。间隙连接蛋白40 (Connexin40, Cx40) 在哺乳动物肺血管内皮表达。本研究对生活在海拔3 200 m的高原鼠兔进行28 d模拟海拔5 000 m低氧处理,以Sprague Dawley (SD) 大鼠为对照,采用免疫组化法分析模拟低氧处理后高原鼠兔和SD大鼠肺组织形态结构,qPCR和蛋白印记法检测Cx40基因和蛋白表达量变化,探究Cx40在高原鼠兔低氧性肺血管收缩反应钝化中的潜在作用。结果显示,低氧处理后,高原鼠兔肺泡呈空泡囊状,Cx40蛋白在支气管和肺血管中均表达,Cx40 基因mRNA水平随着低氧暴露而升高,但其蛋白质水平呈下降趋势,肺支气管Cx40蛋白无明显变化。SD大鼠肺血管和肺支气管表达的Cx40蛋白均无明显变化。暗示生活在高海拔低氧环境中的高原鼠兔,Cx40蛋白下调可抑制血管收缩信号,减弱低氧性肺血管收缩反应,使低氧性肺血管收缩反应钝化,以适应高原缺氧环境。研究结果可为高原土著动物适应高寒缺氧环境提供基础理论数据。     

常氧下高原鼠兔HIF-1αmRNA的表达

低氧诱导因子—1(HIF—1)是一种由低氧诱导因子—1α(HIF—1α)和芳香烃核受体转录因子(ARNT)组成的二聚体转录因子,在生物体氧平衡调节中起关键作用。高原鼠兔(Dchotorua curzoniae)是生活在青藏高原海拔3000m以上区域,有着极强的低氧、低温耐受能力的特有自然种群。它通过高的基础代谢率和高的氧利用率来适应高原低温、缺氧环境。将高原鼠兔带到北京,常氧室温条件下适应7d。RT—PCR扩增高原鼠兔HIF—1α cDNA片断,^32P标记作为特异性探针,Northern杂交检测了常氧条件下高原鼠兔HIF—1α的组织特异性表达。结果显示,HIF—1α mRNA在高原鼠兔多种组织中均有表达,并且表现出明显的组织差异性。其中脑的表达量最大,肾其次,心、脾和肝的表达相对较少。     

大鼠脑红蛋白(NGB)的原核表达、抗体制备及其细胞分布

脑红蛋白 (NGB)是新发现的与脑内氧供应密切相关的分子 .为了检测细胞内脑红蛋白的表达、亚细胞分布从而对该分子进行深入的功能研究 ,成功地将大鼠脑红蛋白基因编码区构建于原核表达载体pGEX 4T 2 ,转化大肠杆菌BL2 1(DE3) ,获得融合表达产物 .对含有融合蛋白的包含体进行溶解和复性 ,用谷胱甘肽S 转移酶 (GST)亲和层析柱纯化 ,通过免疫家兔获得了兔源性抗NGB多克隆抗体 .采用Western印迹分析技术 ,用该抗体检测NGB基因的真核表达产物 ,证明该抗体有较好的针对NGB蛋白的专一性 ,可用于对NGB的结构和功能研究 .同时 ,用该抗体进行免疫组化分析发现 ,正常成年大鼠神经系统中有较多的NGB免疫反应阳性细胞分布 ,提示NGB是与神经系统功能密切相关的重要分子     

高原鼢鼠的学习记忆能力与其脑组织中foxP2基因的高表达有关(英文)

高原鼢鼠(Myospalax baileyi)终生营地下洞道生活,具有较强的学习记忆能力和定位洞道中障碍物的能力。叉头框蛋白P2(forkhead box p2,FOXP2)是一种与口面部协调、运动协调功能有关,并且与学习记忆、以及定位功能有密切联系的转录因子。为了研究和探讨foxP2基因与高原鼢鼠发达的学习记忆能力之间的关系,我们克隆了高原鼢鼠foxP2基因的编码区序列;以地面动物高原鼠兔为对照,应用real-time PCR和Western blot分别测定高原鼢鼠脑组织中foxP2 mRNA和FOXP2蛋白的表达水平;应用免疫组织化学法检测FOXP2蛋白在高原鼢鼠脑组织不同区域中的表达。结果显示:(1)与其它哺乳类动物相比,高原鼢鼠foxP2编码区序列相对保守,但其编码的氨基酸序列显示存在两个特殊的氨基酸残基突变;(2)高原鼢鼠foxP2mRNA的表达量极显著高于高原鼠兔(P0.01);(3)高原鼢鼠FOXP2蛋白的表达量极显著高于高原鼠兔(P0.01);(4)FOXP2蛋白在高原鼢鼠脑组织不同区域均有表达,其中以大脑皮层、丘脑和纹状体区域表达量较高。本研究结果提示,高原鼢鼠发达的学习记忆能力与foxp2基因在脑组织中的高表达有着密切的联系。     

重组高原鼠兔瘦素原核表达载体的构建、蛋白表达及纯化

瘦素(leptin)由ob 基因编码,对调节能量代谢起着重要作用。本研究建立了高原鼠兔瘦素的原核表达系统,并对其进行了原核表达。研究中作者从高原鼠兔瘦素基因的cDNA 文库中,扩增编码高原鼠兔瘦素的核酸序列,并利用DNA 基因重组技术将其克隆到原核表达载体pET30a(+ )中,构建了高原鼠兔瘦素原核表达载体pET30a(+ )/ ppleptin。对目的片段进行测序确认后,将其转化到大肠杆菌BL21 中,并利用IPTG 诱导外源性目的蛋白表达。表达的包涵体蛋白经溶解及变性后上柱纯化。重组质粒经测序检测后,表明原核载体构建正确。同时,SDS - PAGE 凝胶电泳结果显示,重组菌在16KD 处有明显新增条带,纯化后的目的蛋白条带纯度较高。该结果为高原鼠兔瘦素的后续基础研究提供了基础资料。     

免疫组织化学方法检测脑红蛋白在大鼠中枢神经系统的分布

目的 探讨脑红蛋白（NGB）基因在中枢神经系统中的分布。方法 用免疫组织化学ABC法研究了NGB蛋白在成年大鼠脑内的分布和定位。结果 NGB蛋白在成年大鼠脑中有非常广泛的表达。其分布区域包括大脑皮质,海马,丘脑和下丘脑的部分核团,脑桥及小脑,NGB免疫反应阳性物质定位于神经元的细胞质。结论 NGB蛋白在大鼠脑中有非常广泛的表达,提示NGB基因在中枢神经系统的功能活动中可能起重要作用。     

青藏高原高原鼠兔Na+,K+-ATP酶β2亚基基因编码区的克隆与分析

目的:克隆青藏高原高原鼠兔Na ,K -ATP酶β2亚基(ATP1B2)的基因编码区,并分析其序列特征,以揭示高原鼠兔低氧适应的分子基础。方法:采用RT-PCR技术从高原鼠兔脑组织中扩增出ATP1B2基因编码区cDNA序列并进行序列测定,采用生物信息学技术对其进行分析。结果:ATP1B2基因编码区由873bp组成,编码290个氨基酸残基。序列分析结果显示,高原鼠兔ATP1B2编码区的核酸序列与兔、人、牛、大鼠、小鼠及狗分别有99%、93%、91%、91%、90%和90%的同源性。结论:克隆出青藏高原高原鼠兔ATP1B2基因编码区,为进一步了解高原鼠兔低氧适应的分子机制提供了基础。     

用pLNCX2携带人MCPH1基因shRNA重组逆转录病毒载体的构建及鉴定

目的建立逆转录病毒介导的MCPH1基因RNA干扰表达体系并观察其在宫颈癌Caski细胞中对MCPH1表达的影响。方法将人MCPH1基因RNA干扰双链DNA片段重组到逆转录病毒质粒pLNCX2中,构建携带人MCPHI基因RNA干扰的逆转录病毒载体pLNCX2-shRNA—MCPH1,经PT67细胞包装后,产生的重组逆转录病毒感染宫颈癌细胞株Caski细胞,并用G418筛选产生稳定的细胞克隆,用RT—PCR和Western印迹检测细胞中MCPH1mRNA和蛋白表达的变化。结果重组逆转录病毒质粒经测序鉴定正确,逆转录病毒感染Caski细胞后用G418筛选出稳定的细胞克隆,RT—PCR和Western印迹检测人MCPH1mRNA和蛋白表达水平明显低于阴性对照组和未干扰组。结论携带人MCPHI基因RNA干扰双链DNA片段的逆转录病毒感染Caski细胞后能明显抑制MCPHImRNA和蛋白表达,为进一步研究MCPH1在宫颈癌中的作用奠定了基础。     

脑红蛋白与Na^＋，K^＋-ATP酶β2亚基的相互作用及作用位点的鉴定

为研究神经系统特异性携氧蛋白———脑红蛋白 (NGB)保护神经元耐受缺氧损伤的分子机制 ,利用酵母双杂交系统从人胎脑cDNA文库中筛选与其有相互作用的蛋白质。序列分析表明 ,其中一个克隆的编码产物与Na ,K ATP酶 β2亚基 (NKA1b2 )序列一致。随后采用PCR方法从人胎脑cDNA文库中扩增获得NKA1b2全长cDNA。蛋白质结合实验表明 ,原核表达的NGB与体外转录翻译得到的NKA1b2在细胞外有结合作用。免疫共沉淀实验证明二者在生理条件下能够以复合物的形式存在。利用NGB系列短截体研究相互作用的位点发现 ,NGB蛋白N末端 1～ 75位氨基酸可与NKA1b2结合 ,但结合力很弱 ,而其C末端 75个氨基酸则与NKA1b2无结合作用 ,由此推测NGB蛋白整体的三维结构是结合所必需的。     

Functional properties of neuroglobin and cytoglobin. Insights into the ancestral physiological roles of globins

Neuroglobin and cytoglobin are two recently discovered vertebrate globins, which are expressed at low levels in neuronal tissues and in all tissues investigated so far, respectively. Based on their amino acid sequences, these globins appear to be phylogenetically ancient and to have mutated less during evolution in comparison to the other vertebrate globins, myoglobin and hemoglobin. As with some plant and bacterial globins, neuroglobin and cytoglobin hemes are hexacoordinate in the absence of external ligands, in that the heme iron atom coordinates both a proximal and a distal His residue. While the physiological role of hexacoordinate globins is still largely unclear, neuroglobin appears to participate in the cellular defence against hypoxia. We present the current knowledge on the functional properties of neuroglobin and cytoglobin, and describe a mathematical model to evaluate the role of mammalian retinal neuroglobin in supplying O2 supply to the mitochondria. As shown, the model argues against a significant such role for neuroglobin, that more likely plays a role to scavenge reactive oxygen and nitrogen species that are generated following brain hypoxia. The O2 binding properties of cytoglobin, which is upregulated upon hypoxia, are consistent with a role for this protein in O2-requiring reactions, such as those catalysed by hydroxylases.     

Globins and hypoxia adaptation in the goldfish, Carassius auratus

Goldfish (Carassius auratus) may survive in aquatic environments with low oxygen partial pressures. We investigated the contribution of respiratory proteins to hypoxia tolerance in C. auratus. We determined the complete coding sequence of hemoglobin alpha and beta and myoglobin, as well as partial cDNAs from neuroglobin and cytoglobin. Like the common carp (Cyprinus carpio), C. auratus possesses two paralogous myoglobin genes that duplicated within the cyprinid lineage. Myoglobin is also expressed in nonmuscle tissues. By means of quantitative real-time RT-PCR, we determined the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and cytoglobin in goldfish exposed to prolonged hypoxia (48 h at Po(2) ~ 6.7 kPa, 8 h at Po(2) ~ 1.7 kPa, 16 h at Po(2) ~ 6.7 kPa) at 20 degrees C. We observed small variations in the mRNA levels of hemoglobin, neuroglobin and cytoglobin, as well as putative hypoxia-responsive genes like lactate dehydrogenase or superoxide dismutase. Hypoxia significantly enhanced only the expression of myoglobin. However, we observed about fivefold higher neuroglobin protein levels in goldfish brain compared with zebrafish, although there was no significant difference in intrinsic myoglobin levels. These observations suggest that both myoglobin and neuroglobin may contribute to the tolerance of goldfish to low oxygen levels, but may reflect divergent adaptive strategies of hypoxia preadaptation (neuroglobin) and hypoxia response (myoglobin).     

A globin gene of ancient evolutionary origin in lower vertebrates: evidence for two distinct globin families in animals

Hemoglobin, myoglobin, neuroglobin, and cytoglobin are four types of vertebrate globins with distinct tissue distributions and functions. Here, we report the identification of a fifth and novel globin gene from fish and amphibians, which has apparently been lost in the evolution of higher vertebrates (Amniota). Because its function is presently unknown, we tentatively call it globin X (GbX). Globin X sequences were obtained from three fish species, the zebrafish Danio rerio, the goldfish Carassius auratus, and the pufferfish Tetraodon nigroviridis, and the clawed frog Silurana tropicalis. Globin X sequences are distinct from vertebrate hemoglobins, myoglobins, neuroglobins, and cytoglobins. Globin X displays the highest identity scores with neuroglobin (approximately 26% to 35%), although it is not a neuronal protein, as revealed by RT-PCR experiments on goldfish RNA from various tissues. The distal ligand-binding and the proximal heme-binding histidines (E7 and F8), as well as the conserved phenylalanine CD1 are present in the globin X sequences, but because of extensions at the N-terminal and C-terminal, the globin X proteins are longer than the typical eight alpha-helical globins and comprise about 200 amino acids. In addition to the conserved globin introns at helix positions B12.2 and G7.0, the globin X genes contain two introns in E10.2 and H10.0. The intron in E10.2 is shifted by 1 bp in respect to the vertebrate neuroglobin gene (E11.0), providing possible evidence for an intron sliding event. Phylogenetic analyses confirm an ancient evolutionary relationship of globin X with neuroglobin and suggest the existence of two distinct globin types in the last common ancestor of Protostomia and Deuterostomia.     

Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions

Recent evidence suggests that the reaction of nitrite with deoxygenated hemoglobin and myoglobin contributes to the generation of nitric oxide and S-nitrosothiols in vivo under conditions of low oxygen availability. We have investigated whether ferrous neuroglobin and cytoglobin, the two hexacoordinate globins from vertebrates expressed in brain and in a variety of tissues, respectively, also react with nitrite under anaerobic conditions. Using absorption spectroscopy, we find that ferrous neuroglobin and nitrite react with a second-order rate constant similar to that of myoglobin, whereas the ferrous heme of cytoglobin does not react with nitrite. Deconvolution of absorbance spectra shows that, in the course of the reaction of neuroglobin with nitrite, ferric Fe(III) heme is generated in excess of nitrosyl Fe(II)-NO heme as due to the low affinity of ferrous neuroglobin for nitric oxide. By using ferrous myoglobin as scavenger for nitric oxide, we find that nitric oxide dissociates from ferrous neuroglobin much faster than previously appreciated, consistently with the decay of the Fe(II)-NO product during the reaction. Both neuroglobin and cytoglobin are S-nitrosated when reacting with nitrite, with neuroglobin showing higher levels of S-nitrosation. The possible biological significance of the reaction between nitrite and neuroglobin in vivo under brain hypoxia is discussed.     

Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure, expression pattern, and ligand binding

Neuroglobin has been identified as a respiratory protein that is primarily expressed in the mammalian nervous system. Here we present the first detailed analysis of neuroglobin from a non-mammalian vertebrate, the zebrafish Danio rerio. The zebrafish neuroglobin gene reveals a mammalian-type exon-intron pattern in the coding region (B12.2, E11.0, and G7.0), plus an additional 5'-non-coding exon. Similar to the mammalian neuroglobin, the zebrafish protein displays a hexacoordinate deoxy-binding scheme. Flash photolysis kinetics show the competitive binding on the millisecond timescale of external ligands and the distal histidine, resulting in an oxygen affinity of 1 torr. Western blotting, immune staining, and mRNA in situ hybridization demonstrate neuroglobin expression in the fish central nervous system and the retina but also in the gills. Neurons containing neuroglobin have a widespread distribution in the brain but are also present in the olfactory system. In the fish retina, neuroglobin is mainly present in the inner segments of the photoreceptor cells. In the gills, the chloride cells were identified to express neuroglobin. Neuroglobin appears to be associated with mitochondria-rich cell types and thus oxygen consumption rates, suggesting a myoglobin-like function of this protein in facilitated oxygen diffusion.