Hypoxia adaptation and hemoglobin mutation in Tibetan chick embryo

Tibetan chick, as an indigenous chick breed at high altitudes, is well known for its ability to survive under conditions of reduced oxygen supply. The hypoxia toleration of Tibetan chick is stronger than that of other breeds in low land and stands out in the embryo. The hatchability of low land breeds approximates to zero under hypoxia at simulated altitudes of above 4000 m[1] while the natural hatchability reaches 70%―80% in Tibetan chick. An incubator which can automatically control oxygen…     

Hypoxia adaptation and hemoglobin mutation in Tibetan chick embryo

Tibetan chick lives at high altitudes between 2600 and 4200 m with a high hatchability and low land breeds survive rarely with a hatchability of 3.0% under hypoxia of simulated 4200 m. Under hypoxia of whole 21 d, the hatchability of Tibetan chick and Recessive White Feather broiler differed with a greatest disparity from day 4 to 11 and also significantly in other stages except from day 1 to 3. Hypoxia in each stage did not reduce significantly survival rate of this stage except hatchability. These two results indicated that the hypoxia in the early stage had an adverse effect on the later stage. All exons encoding chick hemoglobins were sequenced to analyze gene polymorphism. The functional mutation Met-32(B13)-Leu, related with hypoxia, was found in α^D globin chain and the mutation frequency increased with increased altitude. In addition, under hypoxic conditions, the population with higher mutation frequency had a higher hatchability. The automated homology model building was carried out using crystal structure coordinates of chick HbD. The results indicated that the substitution Met-32(B13)-Leu provides a more hydrophobic environment which leads to higher stability of heme and oxygen affinity of hemoglobin. The occurrence of the mutation Met-32(B13)-Leu is related to the origin of Tibetan chick.     

Hypoxia regulates avian cardiac Arnt and HIF-1alpha mRNA expression

The aryl hydrocarbon receptor nuclear translocator (Arnt) and hypoxia-inducible factor (HIF)-1alpha mediate cellular responses to hypoxia. We investigated the ability of hypoxia to regulate Arnt and HIF-1alpha mRNA in the heart in vivo. We cloned avian Arnt, developed an in vivo model of chronic cardiac hypoxia, and measured expression of cardiac Arnt and HIF-1alpha mRNA by quantitative RT-PCR. Chronic hypoxic exposure (24 h to 15% O(2)) of day 9 chick embryos resulted in a 30-fold increase in covalent binding of (3)H-misonidazole, a hypoxic tissue marker, to cardiac tissue, and a 2-fold induction of cardiac inducible nitric oxide synthase mRNA, compared to normoxic controls. In this same model, cardiac Arnt mRNA expression decreased by 35%, while HIF-1alpha mRNA expression increased 400%. These data suggest that regulation of Arnt and HIF-1alpha mRNA expression may contribute to the physiological responses of the heart during prolonged hypoxia.     

鸡缺氧诱导因子-1[[alpha]]基因的差异表达与低氧适应性

低氧诱导因子-1(HIF-1)是在低氧的癌细胞中发现的一种转录激活因子, 在生物体氧平衡调节中起关键作用。藏鸡是对高原低氧、低温环境有着极强适应能力的高原土著品种, 相对而言, 白来航鸡和寿光鸡为两个低地鸡种。在常氧环境下对这3个鸡品种进行全期模拟低氧孵化, 结果显示, 藏鸡的孵化率显著高于两个低地鸡品种, 表现出了高度的耐受低氧环境的能力, 而对于低地鸡, 一定程度的低氧环境对其孵化是致命的。利用Taqman探针法FQRT-PCR技术检测了藏鸡、白来航鸡、寿光鸡HIF-1[[alpha]] 的组织特异性表达。结果表明, HIF-1[[alpha]] mRNA在3个鸡品种的大脑和骨骼肌组织均有表达, 并有明显的组织差异性, 脑的表达量最大; 并且发现常氧条件下孵化时, 藏鸡胚胎的大脑组织内HIF-1[[alpha]] 基因的表达量与低氧孵化的低地鸡胚胎相接近.     

缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。     

藏羚羊低氧诱导因子1α基因的克隆与组织表达

为探讨低氧诱导因子1α (hypoxia inducible factor l alpha,HIF-1α)在藏羚羊(Pantholops hodgsonii)高原低氧适应机制中的作用,采用RT-PCR和RACE技术克隆藏羚羊HIF-1α基因的cDNA序列,同时采用real-time PCR和Western blot方法...     

Preserved placental oxygenation and development during severe systemic hypoxia

Local tissue oxygenation profoundly influences placental development. To elucidate the impact of hypoxia on cellular and molecular adaptation in vivo, pregnant mice at embryonic days 7.5-11.5 were exposed to reduced environmental oxygen (6-7% O2) for various periods of time. Hypoxia-inducible factor (HIF)-1alpha mRNA was highly expressed in the placenta, whereas HIF-2alpha was predominantly found in the decidua, indicating that HIF-1 is a relevant oxygen-dependent factor involved in placental development. During severe hypoxia, HIF-1alpha protein was strongly induced in the periphery but, however, not in the labyrinth layer of the placenta. Accordingly, no indication for tissue hypoxia in this central area was detected with 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide staining and VEGF expression as hypoxic markers. The absence of significant tissue hypoxia was reflected by preserved placental architecture and trophoblast differentiation. In the search for mechanisms preventing local hypoxia, we found upregulation of endothelial nitric oxide synthase (NOS) expression in the labyrinth layer. Inhibition of NOS activity by N(omega)-nitro-L-arginine methyl ester application resulted in ubiquitous placental tissue hypoxia. Our results show that placental oxygenation is preserved even during severe systemic hypoxia and imply that NOS-mediated mechanisms are involved to protect the placenta from maternal hypoxia.     

HIF-1alpha, HIF-2alpha and VHL mRNA expression in different cell lines during hypoxia

Hypoxia inducible factor-1alpha (HIF-1alpha) mRNA expression is significantly decreased under hypoxia in different cell lines exposed directly to hypoxia or treated with dimethyloxalylglycine which mimics hypoxic effects under normoxic conditions. However, the decreased expression of HIF-1alpha mRNA is accompanied by an increase of HIF-1alpha protein (pHIF-1alpha) level as well as by overexpression of known HIF-dependent genes (VEGF, Glut1, PFKFB-3 and PFKFB-4) under hypoxic conditions or with the use of dimethyloxalylglycine. Expression of HIF-1alpha mRNA also depends on iron because desferrioxamine and cobalt chloride produce similar to hypoxia effects on the levels of this mRNA. It was shown that HIF-1alpha mRNA expression did not change significantly in some cell lines (SKBR3, MDA-MB468 and BT549) under hypoxia. However, in these cell lines hypoxia decreases expression of HIF-2alpha mRNA, another member of HIF-alpha gene family, as a result of cell specific regulation of HIF-alpha genes under hypoxia. Moreover, hypoxia slightly induces expression of PFKFB-4 mRNA in SKBR3, MDA-MB468 and BT549 as compared to other cell lines where this effect of hypoxia was much stronger and adaptation to hypoxia is controlled by HIF-1alpha. Hypoxia slightly reduces expression of tumor suppressor VHL which targets HIF-1alpha for ubiquitination. Thus, our results clearly demonstrated down regulation of HIF-1alpha or HIF-2alpha in different cell lines by hypoxia.     

Stabilization of hypoxia-inducible factor-1alpha is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells

It has been suggested that blood vessel formation is an important event coupled to bone formation. The expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been shown to be greatly stimulated in osteoblasts by hypoxic stimuli such as deprivation of oxygen and treatment with cobalt. In other cell types, hypoxia-inducible factor-1 (HIF-1) that binds hypoxia-response element (HRE) has been shown to mediate gene expression induced by hypoxic stimuli. In this study, we investigated the effects of hypoxic stimuli on HIF-1, HRE, and VEGF in osteoblastic cell lines. Exposure of these cells to hypoxia or cobalt resulted in a great increase in the protein level of HIF-1alpha and the gene expression of VEGF. Transforming growth factor-beta1, prostaglandin E2, dexamethasone, and 1,25-dihydroxyvitamin D3 that have been shown to regulate VEGF gene expression in osteoblasts had no effect on HIF-1alpha induction. Blocking the enzymatic activity of phosphatidylinositol 3-kinase, p38, MEK-1 did not have any effect on the cobalt-stimulated increase of HIF-1alpha in these cells. In contrast, N-acetylcysteine (NAC), a scavenger of reactive oxygen species, abolished the cobalt induction of HIF-1alpha and that of the VEGF and a HRE-driven reporter genes. However, the hypoxia responses were not affected by NAC. These findings suggest that hypoxia and cobalt can induce VEGF gene expression in osteoblasts by increasing the level of HIF-1alpha protein through different mechanisms.     

Chick embryo culture using duck egg shell--first successful hatch

The suitability of duck egg shell (DES) for chick embryo culture was investigated. Chick embryos were transferred into DESs with all egg contents after 3 days of normal incubation and cultured. The vessels made of polyethylene cling film were used for shell-less control. Among 35 embryos cultured in DESs, 21 survived until 16 days of incubation (13 days after transfer) and finally 3 newly hatched chicks were obtained at 22 days of incubation. One of them died 4 days later, but remaining two became full-grown cocks showing normal body weight and production of fertile sperms. Among 37 embryos cultured in polyethylene vessels, none survived over the period of 19 days of incubation. It is suggested that DES culture system may be useful for the various experiments using chick embryos.     

Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.