Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.

BIOMIMITICS OF OXIDASE AND OXYGENASE THE ELECTRONIC SPECTRA AND THE OXYGEN UPTAKE KINTICS FOR MPc AND MTPP (M = Fe, Co, Cu, Pc AND TFP - ARE DIANIONS OF PHTHALOCYANIN AND TETRAPHENYLPORPHYRINE, RESPECTIVELY)

The reaction rates of MTPP with oxygen in air are Inas than that with pure oxygen, the ratio is roughly the same as to the partial presence of imygen in air, The influences of S-ligand etbanethiol and O- litand Vc on the above Systems have also been investigated, the former makes the MP hands having more changes and the reaction rate constants becoming greater, the latter has less influence.

果实成熟衰老与抗病性的调控机制研究

　　果实为开花植物所特有的发育器官，在种子的成熟和传播过程中发挥着重要作用。同时，肉质果实中含有丰富的营养物质，包括纤维素、维生素、抗氧化剂等，成为人们饮食的重要组成部分。由于果实的成熟衰老和抗病性直接影响果品的质量和市场价值，因此，研究果实成熟衰老和抗病性的调控机制具有重要的理论意义和应用前景。本文主要利用蛋白质组学的方法，探讨外源化学物质抑制果实成熟衰老和诱导抗病性的调控机制。 1. 硅对果实的抗病性诱导：用硅酸钠（1%）处理采后的甜樱桃果实，再接种褐腐病原菌（Molinilia fracticola），置于20C下，观测贮藏期间果实的发病率，并分析硅处理后诱导的主要蛋白质及调控机制。研究结果表明：硅酸钠处理可显著抑制贮藏期间褐腐病的发生，其抑病机理与硅诱导PR-蛋白的表达，提高果实的抗氧化水平，减轻由病原菌侵染造成的氧化胁迫相关。同时，硅处理还能保护细胞骨架结构，有利于增强果实对病原菌入侵的抵抗力。 2. 水杨酸对果实的抗病性诱导：用水杨酸（SA，2mM）在果园处理三种成熟度的甜樱桃果实，然后接种青霉病原菌（Penicillium expansum）观察其发病情况，并取样分析参与抗病性应答的主要蛋白质及调控机制。试验结果表明：SA处理能显著降低青霉病的发病率和抑制病斑扩展，而且SA对低成熟度甜樱桃果实的抗性诱导效果更好。在八成熟的果实中，有5个热激蛋白和4个脱氢酶蛋白被SA诱导，这些蛋白参与了糖酵解和三羧酸循环。抗氧化蛋白和PR蛋白主要参与较低成熟度果实的抗性应答，而热激蛋白和脱氢酶在较高成熟度果实的抗性应答中更明显，SA诱导的抗性与代谢途径相关。 　　3. 草酸对果实的抗性诱导：用5mM的草酸处理冬枣果实后，接种青霉菌（P. expansum），观察果实发病情况，测定果实相关的生理指标，分析参与果实抗性应答的主要蛋白质及调控机制。结果表明：草酸能明显延缓冬枣果实的衰老，提高果实对青霉菌的抗性。草酸处理能抑制果实乙烯的释放量和呼吸强度，延缓叶绿素的降解，减少乙醇积累。利用蛋白质组学的研究方法证实了在25个参与了草酸处理应答的蛋白中，胱硫醚-β-合酶结构域包含蛋白（CBB domain-containing protein）和3个与光合作用相关蛋白[二磷酸核酮糖羧化酶/加氧酶（Ribulose bisphosphate carboxylase/oxygenase activase, chloroplast precursor），二磷酸核酮糖羧化酶/加氧酶大亚基结合蛋白（RuBisCO large subunit-binding protein subunit beta, chloroplast precursor），植物光系统Ⅱ放氧复合蛋白2（PSII oxygen-evolving complex protein 2）]的表达量上调，乙醇脱氢酶的表达量出现下调。草酸处理还提高了与乙烯合成前体相关蛋白的表达，抑制了ACC合成酶的活性。草酸提高果实抗病的机制与延缓果实成熟衰老和保持果实抗性有关。 4. 果实衰老的调控机制：采用高氧（100%）和低氧（2-3%）处理苹果果实，观察果实衰老的进程，并基于蛋白质组学的研究方法，探讨苹果果实衰老与线粒体蛋白质组的关系。结果表明，在苹果衰老过程中有22个蛋白的表达量发生变化，这些蛋白主要参与了三羧酸循环，电子传递，碳代谢和胁迫应答。高氧处理能诱导氧化胁迫，加速了果实的衰老。质谱鉴定结果证明：在高氧胁迫下，超氧化物歧化酶（manganese superoxide dismutase，MnSOD）和线粒体外膜通道蛋白（porin) 的表达量降低，MnSOD的活性受到抑制，由此提高了线粒体中超氧阴离子的含量，增加了蛋白质的氧化损伤。 此外，高氧处理改变了porin的功能，导致了线粒体膜的透势发生变化，从而引起外膜损伤。由此阐明了活性氧在果实的成熟衰老调控中的重要作用。

Relationship between COPD and polymorphisms of HOX-1 and mEPH in a Chinese population

Recent studies have proposed that susceptibility to chronic obstructive pulmonary disease (COPD) might be related with the polymorphisms of some genes encoding antioxidant enzymes, such as heme oxygenase-1 (HOX-1) and microsomal epoxide hydrolase (mEPH).

Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalysis in Escherichia coli

APC (allophycocyanin) is widely used for fluorescence tagging and may be a promising antioxidant agent for use within the food and pharmaceutical industries. Chromophore attachment to apo-ApcA (apo-APC alpha-subunit without chromophore) can be auto-catalysed both in vitro and in vivo. In the present study, a plasmid containing genes of apo-ApcA and chromophore synthetases (HOI (ferredoxin-dependent haem oxygenase) and PcyA (phycocyanobilin:ferredoxin oxidoreductase)] was constructed and expressed in Escherichia coli. The results show that holo-ApcA (APC alpha-subunit with chromophore) can be synthesized by autocatalysis in E. coli. Recombinant holo-ApcA showed the same spectral and fluorescent properties as PC (phycocyanin) and could serve as a good substitute for native PC for fluorescent tagging. Moreover, recombinant ApcA can inhibit hydroxyl and peroxyl radicals more strongly than holo-ApcA and native APC. The EC50 values were 296.4 +/- 22.4 mu g/ml against hydroxyl radicals and 38.5 +/- 2.6 mu g/ml against peroxyl radicals.

雨生红球藻Rubisco在虾青素积累过程中的表达分析

虾青素因其具有极强的抗氧化性及优越的着色作用，被广泛应用于营养保健和水产养殖中，备受国内外研究者的关注。红球藻是目前虾青素的生物来源中较有优势的一条途径。 我们选取雨生红球藻作为研究材料，收集其生长过程中四个不同的阶段，分别为绿色游动细胞阶段，绿色不动细胞阶段，绿褐色不动细胞阶段（开始积累虾青素），以及红色不动孢子阶段。本研究中提取了核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）的粗提液，并测定了酶活。其编码基因rbcL的mRNA表达量也被测定。另外，光合速率与呼吸速率的比值（P/R）通过测定与计算得出，各个阶段藻细胞中虾青素的含量由分析得出。本研究还应用了叶绿素荧光测定方法，确定了光系统II潜在最大活性（Fv / Fm），光系统II实际活性（ΦPSII），电子传递速率（ETR）和非光化学淬灭参数（NPQ）。 结果表明，绿色游动细胞的生长状态最佳，其P/R、Fv / Fm、ΦPSII均为最大，NPQ为最小。这说明在此状态的细胞中，光系统II的活性最强；但是其Rubisco活性与rbcL表达量均为最小。相比之下，在绿褐色不动细胞中，P/R和NPQ的值较低，Fv / Fm、ΦPSII和ETR值都最小，但Rubisco活性与rbcL表达量均为最高。 结合工业生产虾青素的方法，我们认为，Rubisco或许参与了虾青素的合成，而非Calvin循环为色素合成提供前体和能量。因此，在生产过程中适当加入碳源，比如CO2，可以有效增大虾青素的产量。