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Cloning and expression of hypoxia-inducible factor 1 alpha from the hibernating ground squirrel, Spermophilus tridecemlineatus

机译:冬眠地松鼠Spermophilus tridecemlineatus的缺氧诱导因子1α的克隆与表达

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Mammalian hibernation is associated with apnoic breathing patterns and a hypoxia-hypothermia connection has been suggested as part of the mechanism by which body temperature is reduced as animals enter torpor. Hence, we hypothesized that changes in the expression of the hypoxia inducible factor (HIF-1) may potentially be involved in regulating hibernation-responsive gene targets. The expression of the alpha subunit of HIF-1 was quantified at both gene and protein levels in four organs of the thirteen-lined ground squirrel, Spermophilus tridecemlineatus. Reverse transcription-PCR showed no change in hif-1 alpha transcript levels in the liver, lung, skeletal muscle or brown adipose tissue of euthermic versus hibernating animals but HIF-1 alpha protein levels were elevated by 60-70% in the two organs responsive for thermogenesis (brown adipose and skeletal muscle). Furthermore, assessment of DNA binding by HIF-1 in nuclear extracts from brown adipose revealed 6-fold higher levels in hibernator tissue than in euthermic controls suggesting increased expression of HIF-1 responsive genes during hibernation. The complete nucleotide sequence of hif-1 alpha from ground squirrels, the first hif-1 alpha sequence amplified from a hibernating mammal, was obtained using PCR amplification and 3' and 5' RACE. Amino acid sequence analysis revealed 90-95% identity with the HIF-1 alpha protein from other mammals. Several unique amino acid sequence substitutions were identified that may affect protein conformation and could possibly function to counteract low temperature effects on HIF-1 alpha conformation at near 0 degrees C body temperatures during torpor. (c) 2005 Elsevier B.V. All rights reserved.
机译:哺乳动物的冬眠与呼吸暂停模式有关,并且低氧-体温过低的连接已被建议作为动物进入r锅时降低体温的机制的一部分。因此,我们假设缺氧诱导因子(HIF-1)的表达变化可能潜在地参与调节休眠响应基因靶标。 HIF-1的α亚基的表达在基因和蛋白质水平上都在13个内衬的地松鼠Spermophilus tridecemlineatus的四个器官中进行了定量。逆转录-PCR结果显示,与冬眠动物相比,正常动物的肝,肺,骨骼肌或棕色脂肪组织中的hif-1 alpha转录水平没有变化,但在两个有反应的器官中,HIF-1 alpha蛋白水平却提高了60-70%用于生热(棕色脂肪和骨骼肌)。此外,HIF-1在棕色脂肪核提取物中对DNA结合的评估显示,冬眠者组织中HIF-1响应基因的表达增加,而冬眠者组织中冬眠者组织中的HIF-1水平高6倍。使用PCR扩增以及3'和5'RACE获得了来自地松鼠的hif-1α的完整核苷酸序列,这是从冬眠的哺乳动物中扩增出的第一个hif-1α序列。氨基酸序列分析显示与其他哺乳动物的HIF-1α蛋白具有90-95%的同一性。鉴定了几个独特的氨基酸序列取代,这些取代可能会影响蛋白质构象,并可能在抵消0℃体温下在低温下对HIF-1α构象的低温影响。 (c)2005 Elsevier B.V.保留所有权利。

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