MONITORING OF INTRACELLULAR OXYGEN -CONCENTRATION DURING HYPOXIA INDUCIBLE FACTOR ACTIVATION USING OXYGEN PHOSPHORECSENCE QUENCHING

哺乳动物细胞的生存非常依赖氧气。尽管几位学者报道了信号转导途径的发现，但细胞感知和响应氧自由基减少的分子机制尚不清楚。一个典型的例子是，低氧刺激了许多基因的转换，这些基因对低氧状态下的存活至关重要，同时也刺激了细胞凋亡。低氧对某些基因转录的促进作用主要由低氧诱导因子-1（HIF-1）介导。然而，目前尚不清楚这种转移因子是由低氧诱导的能量代谢变化激活的，还是由直接通过氧通道的氧不足激活的。为了回答这一问题，我们试图从基因表达和细胞内氧合状态两个方面进行研究。 ...更多信息 38氨基酸。随着GFP cDNA的克隆，一些生物学家对GFP和GFP融合蛋白的表达产生了极大的兴趣，将其作为一种独特的荧光团来监测一系列细胞内病变，特别是蛋白质的定位。缺氧诱导因子-1（HIF- 1）是一种转录调节蛋白，在缺氧时从细胞质向细胞核转移。我们观察了用钴模拟低氧处理的GFP标记的HIF-1的核翻译。氧依赖性的磷光猝灭是磷光体和分子氧之间碰撞频率和这些碰撞过程中能量转移效率的函数。我们研制了一种用共聚焦显微镜测定钯卟啉磷光寿命的系统。利用以往报道的磷光衰减常数，估算出培养基中的氧浓度，在正常氧下为194微摩尔，在缺氧下为29微摩尔。我们尝试测定胞质溶胶和培养基之间的氧梯度。少

MAMMALIAN CELLS ARE CRITICALLY DEPENDENT ON OXYGEN FOR SURVIVAL. THE MOLECULAR MECHANISMS BY WHICH CELLS SENSE AND RESPOND TO A REDUCTION IN OXYGEN SUPPLY ARE NOT KNOWN ALTHOUGH FINDINGS ON THE SIGNAL TRANSDUCTION PATHWAY HAVE BEEN REPORTED BY SEVERAL AUTHORS. A TYPICAL EXAMPLE IS THAT HYPOXIA STIMULATES TRANSCRIPTION OF A NUMBER OF GENES CRUCIAL TO SURVIVAL IN THE HYPOXIC STATE, AS WELL AS APOPTOSIS. THE ENHANCEMENT OF TRANSCRIPTION OF SOME GENES BY HYPOXIA IS MAINLY MEDIATED BY HYPOXIA-INDUCIBLE FACTOR-1 (HIF-1). HOWEVER, IT IS NOT CLEAR WHETHER THIS TRANSCRIPTION FACTOR ARE ACTIVATED BY HYPOXIA-INDUCED CHANGES IN ENERGY METABOLISM OR BY OXYGEN SHORTAGE DIRECTLY THROUGH AN OXYGEN SENSOR. TO ANSWER THIS, WE ATTEMPTED TO CORRELATE THE GENE-EXPRESSION AND INTRACELLULAR OXYGENATION STATE.GREEN FLUORESCENT PROTEIN (GFP) FROM AEQUOREA VICTORIA IS A POWERFUL FLUOROPHORE FORMED AUTOCATALYTICALLY BY INTERNAL CYCLIZATION AND OXIDATION OF THE SER-TYR-GLY SEQUENCE AT POSITIONS 65-67 WITHIN THE 2 … More 38 AMINO ACID. FOLLOWING THE CLONING OF THE CDNA FOR GFP, SEVERAL INVESTIGATORS TOOK A GREAT INTEREST IN THE EXPRESSION OF GFP AND GFP-FUSION PROTEINS AS A UNIQUE FLUOROPHORE TO MONITOR A RANGE OF INTRACELLULAR PROCESSES, ESPECIALLY THE LOCALIZATION OF PROTEINS. HIF- 1 IS THE TRANSCRIPTION REGULATORY PROTEIN AND TRANSLOCATE FROM THE CYTOPLASM TO THE NUCLEUS DURING HYPOXIA. WE OBSERVED THE NUCLEAR TRANSLAOCATION OF GFP LABELED HIF-1 BY COBALT TREATMENT WHICH MIMICS HYPOXIA.OXYGEN-DEPENDENT QUENCHING OF PHOSPHORESCENCE IS A FUNCTION OF THE FREQUENCY OF COLLISION BETWEEN THE PHOSPHOR AND MOLECULAR OXYGEN AND OF THE EFFICIENCY OF ENERGY TRANSFER DURING THESE COLLISMNS. WE DEVELOPED A DETECTION SYSYTE FOR PALLADIUM PORPHYRIN PHOSPHORESCENCE LIFETIME USING CONFOCAL MICROSCOPE. USING PHOSPHORESCENCE DECAY CONSTANT PREVIOUS REPORTED, THE OXYGEN CONCENTRATIONS IN THE MEDIUM WERE ESTIMATED AS 194 MICROMOLAR UNDER NORMOXIA AND 29 MICROMOLAR UNDER ANOXIA, RESPECTIVELY. WE PLAN TO DETERMINE OXYGEN GRADIENT BETWEEN CYTOSOL AND MEDIUM. Less