酵母细胞渗透压调节与甘油代谢

酵母甘油代谢与调控的信息主要来自于酿酒酵母和酿酒酵母细胞对高渗应答的研究。本文综述了酵母细胞非胁迫条件下的甘油合成与分解代谢特征;甘油在酵母细胞渗透压调节过程中的作用与酵母耐高渗机理;增强甘油合成的外环境及其甘油合成的途径工程;以及酵母感受上高渗信息及控制在高渗协迫条件下甘油合成的高渗甘油应答途径。     

酵母细胞对高渗环境的适应与胞内甘油累积

甘油是包括酿酒酵母在内的许多种酵母细胞中的主要相容性溶质。为适应在高渗环境下的生存,酵母细胞将在胞内累积甘油。胞内甘油累积的增加可由甘油合成的增强,甘油利用的减弱,细胞膜通透性下降导致的胞内甘油流失的减少以及从环境中吸取更多的甘油而产生。本文综述了酵母细胞对环境渗透压变化的信号传导,高渗诱导的基因表达,环境渗透压升高时酵母细胞内甘油的累积以及甘油合成的限速步骤。     

渗透压敏感和耐高渗酵母菌在海藻酸钠-壳聚糖-海藻酸钠微囊中的生长和代谢

为了研究微囊微环境中渗透压对微囊内不同渗透压敏感性细胞生长、代谢的影响, 分别以渗透压敏感型酿酒酵母Y02724与耐高渗酵母Hansel为细胞模型, 考察了有氧条件下这两种细胞在海藻酸钠-壳聚糖-海藻酸(Alginate-chitosan-alginate, ACA)微胶囊中的生长、代谢状态。主要检测了细胞比生长速率、最大产物生成量以及代谢物乙醇、甘油分泌量等的变化。实验结果分析表明, 渗透压胁迫可能是导致不同渗透压敏感性细胞在微囊微环境中生长代谢特征变化的因素之一, 即微囊微环境内可能存在渗透压胁迫。     

酵母细胞渗透压调节与甘油代谢

酵母甘油代谢与调控的信息主要来自于酿酒酵母和酿酒酵母细胞对高渗应答的研究。本文综述了酵母细胞非协迫条件下的甘油合成与分解代谢特征;甘油在酵母细胞渗透压调节过程中的作用与酵母耐高渗机理;增强甘油合成的外环境及其甘油合成的途径工程;以及酵母感受胞外高渗信息及控制在高渗协迫条件下甘油合成的高渗甘油应答途径。     

蛋白纳米颗粒与细胞力电平衡

人体细胞的生命活动依赖于膜电位极化和胞内外离子不对称动态平衡(也称生物渗透压平衡)。膜电位改变及离子含量组成变化均参与了细胞对环境理化刺激的应激反应,调控其对环境改变的适应。最近的研究发现：人体血浆及细胞内的蛋白纳米颗粒变化参与了细胞膜电位的调控,与细胞内外离子的重新分布及跨膜渗透压的改变密切相关。电压依赖离子通道的激活及开放程度,是诱导细胞离子重新分布的重要调控机制,其活性与蛋白纳米颗粒调控的膜电位改变密切相关,且离子组成也参与了蛋白纳米颗粒吸附离子诱导膜电位变化的调节。因而,蛋白纳米颗粒是调控细胞膜电位平衡及生物渗透压平衡的重要物理机制,这一协同调控的力电活动与多种与蛋白纳米颗粒相关疾病的发生及治疗密切相关,该机制的阐明能为解析当前多种临床疑难疾病的发病机制提出新的研究方向。     

粗糙脉孢菌转录因子LAH-3参与渗透压调控

【目的】通过构建转录因子lah-3基因缺失突变体,研究lah-3基因缺失突变体菌株的渗透压表型,进而探究lah-3基因在渗透压调控中的作用。【方法】采用同源基因重组敲除技术构建lah-3基因缺失突变体。用4%NaCl和1 mol/L Sorbitol进行渗透压处理。利用Northern blot检测渗透压应答基因的表达。利用Westhern blot检测LAH-3蛋白磷酸化修饰水平,OS-2蛋白的表达水平及其磷酸化修饰水平。【结果】在转录因子lah-3基因缺失突变体中,渗透压应答基因gcy-1、stl-1以及pck-1的表达水平都明显降低,而且在渗透压刺激下,LAH-3蛋白磷酸化修饰水平升高。LAH-3的磷酸化修饰不受OS-2调控。lah-3基因的缺失既不影响OS-2蛋白的表达水平,也不影响其在渗透压刺激后的磷酸化修饰。【结论】粗糙脉孢菌中转录因子LAH-3参与调控渗透压应答基因的转录,但其响应过程不依赖于OS-2 MAPK信号通路。     

颈动脉注射高渗氯化钠液和牵拉左心房对猫下丘脑前部单位电活动的影响

在氨基甲酸乙酯-氯醛糖麻醉下,经咽部暴露下丘脑。实验在三碘季铵酚制动并人工呼吸条件下进行。用玻璃微电极在细胞外记录下丘脑前部(AH)单位电活动,观察其对颈总动脉注射高渗 NaGl 液及牵拉左心房的反应。共记录到32个单位,其自发放电频率1—13次/s。对照条件下所记录的视上核(SO)单位中,多数对高渗刺激和牵拉左心房分别表现为兴奋和抑制,少数则被前者抑制,而被后者所兴奋。11个下丘脑前核-视上核(Ha-SO)区域的单位,接受高渗刺激后5s 内放电频率增加61.6±37.0%(平均值±标准误),持续时间约1min;偏内侧近 Ha 的单位,在牵拉左心房时其放电频率减少23.5±10.5%;偏外侧近 SO 单位对高渗刺激呈特异的单相反应,此可能即为 Verney 渗透压感受器。AH 偏内侧的 Ha-SO 单位对高渗刺激和牵拉左心房呈会聚反应。对两种刺激引起 AH 单位反应的特征及其整合作用的机理作了简要的讨论。     

渗透压突然改变对大肠埃希菌水通道蛋白GlpF基因表达的影响

目的 探讨水-甘油通道蛋白(glycerol protein ficilitator,GlpF)的生理功能及对细菌生长繁殖的影响.方法 将大肠埃希菌接种于等渗透压的液体培养基(1 IM)培养,18h后,突然改变培养液的渗透压,在30、60、120 min时间点检测细菌的A600nm值,RT-PCR分析其GlpF的表达.结果 突然改变渗透压后,大肠埃希菌数量均有不同程度的减少.在1/2 IM组、1/4 IM组细菌的A600nm值与其各自对照组相比,变化并不明显,但其细菌GlpF表达量明显降低.在高渗组,2 IM组的A600nm值与等渗组相比变化不大,而其GlpF表达量明显高于等渗组.结论 在环境渗透压突然改变时,细菌可以通过调控GlpF的表达来实现对细菌细胞内外水份的调节,以维持胞内环境稳定.     

表达耐辐射异常球菌IrrE蛋白对产丁二酸大肠杆菌耐渗透压的影响

高渗透压胁迫是降低生物法制备丁二酸生产效率的关键因素之一。为提高丁二酸生产菌株对高渗透压胁迫的耐受性能,本研究考察了外源引入全局调控蛋白IrrE提高大肠杆菌耐高渗透压胁迫性能的可行性。试验结果表明,在不同浓度Na+胁迫下,重组菌生长和发酵性能明显提升。在5 L罐发酵中,重组菌最大细胞干重、糖耗和丁二酸产量比对照菌分别提高了15.6%、22%和23%,表明引入IrrE蛋白可提高菌株对高渗透压胁迫的耐受能力。进一步比较重组菌和对照菌胞内相容性物质海藻糖和甘油的浓度后发现,重组菌胞内海藻糖和甘油浓度明显提高,其最大积累量分别是对照菌的1.3和3.8倍,推测IrrE可通过增加胞内相容性物质的积累提高菌株对高渗透压胁迫的耐受性。     

高渗应激对肾髓细胞的影响

肾髓细胞长期暴露于高渗环境并能正常发挥功能。在过去多年中,研究者在探索肾髓细胞是如何在高渗的环境中得以生存并发挥功能的过程中发现,细胞能产生一系列变化来适应高渗应激,例如有机渗透物的聚积以及热休克蛋白的表达增高等。然而,无论是在体内还是体外,即便是细胞适应了高渗环境之后,高盐引起DNA断裂仍持续存在,但细胞却仍能快速增殖并发挥功能。当盐浓度降低时,DNA断裂被迅速修复。本文对肾髓细胞在高渗应激下发生的损伤性改变及其保护性反应等方面的新近研究进展予以综述。     

Osmotic response element-binding protein (OREBP) is an essential regulator of the urine concentrating mechanism

OREBP (osmotic response element-binding protein), also called TonEBP or NFAT5, is thought to induce the expression of genes that increase the accumulation of organic osmolytes to protect cells against a hypertonic environment. To investigate the consequences of lacking OREBP activity, transgenic (Tg) mice that overexpress OREBPdn (dominant negative form of OREBP) specifically in the epithelial cells of the renal collecting tubules were generated. These mice showed impairment in their urine concentrating mechanism, most likely due to reduced expression of the aquaporin AQP2 and the urea transporter UT-A1 and UT-A2 mRNAs. When deprived of water or after the administration of a vasopressin analogue, urine osmolality of the Tg mice was significantly increased but not to the same extent as that of the wild type mice. The expression of AQP2 and UT-A1, but not UT-A2 mRNAs, was increased to the same level as that of the wild type mice in the water deprivation state, indicating that the vasopressin regulatory mechanism was not affected by OREBPdn. These data indicate that in addition to vasopressin, OREBP is another essential regulator of the urine concentrating mechanism. Furthermore, the OREBPdn Tg mice developed progressive hydronephrosis soon after weaning, confirming the osmoprotective function of OREBP implicated by the in vitro experiments.     

Regulation of nucleocytoplasmic trafficking of transcription factor OREBP/TonEBP/NFAT5

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity.     

Transgenic mice expressing dominant-negative osmotic-response element-binding protein (OREBP) in lens exhibit fiber cell elongation defect associated with increased DNA breaks

Osmotic-response element-binding protein (OREBP), also known as TonEBP or NFAT5, is thought to be responsible for the induction of osmolyte-accumulating genes when cells are under hypertonic stress. Recent studies suggest that OREBP also plays a role in water reabsorption in the kidney, T-cell proliferation, and embryonic development. We developed transgenic mice that express the dominant-negative OREBP (OREBPdn) specifically in the lens because our earlier studies showed that it is particularly sensitive to osmotic stress. The transgenic mice developed nuclear cataract soon after birth, suggesting defects in lens development. The developing transgenic lenses showed incomplete elongation of fiber cells and formation of vacuoles. This is accompanied by evidence of DNA strand breaks, activation of p53, and induction of checkpoint kinase, suggesting that the developing fiber cells lacking OREBP are in a similar physiological state as cells experiencing hypertonic stress. These results indicate that OREBP-mediated accumulation of osmolytes is essential during elongation of the lens fiber cells.