Expression of Cu,Zn-superoxide dismutase gene from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its resistance to oxidative stress

The gene for the Cu, Zn-superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of the yeast P. pastoris. The SOD was purified from the cultured yeast by ammonium sulfate precipitation and DEAE-cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overexpressed SOD protein was shown to have immunologically biologic activity and to be enzymatically active. The yeast overexpressing Cu, Zn-SOD appeared to be more resistant to oxidative stress such as paraquat, menadione, and heat shock.     

荧光/化学发光探针成像检测超氧阴离子自由基的研究进展

超氧阴离子自由基(O·-2)是细胞内氧气单电子还原后最先产生的一类含氧的高活性物种(活性氧,ROS),与生命过程息息相关.正常稳态浓度的O·-2起重要的信号调控作用,包括细胞的增殖、分化、自噬等.但O·-2浓度的异常,又与癌症、神经退行性疾病、糖尿病等多种疾病的发生发展密切相关.因此,监测O·-2浓度的变化对揭示相关疾病的机理具有至关重要作用.由于荧光成像检测方法具有诸多优势,发展高灵敏、高选择性检测O·-2的荧光探针成为揭示相关疾病发生发展分子机制的关键切入点.近年来,随着荧光显微技术的发展,研究者开发了多种荧光/化学发光探针,实现了对细胞及活体内O·-2水平的可视化监测.本文综述了近五年用于检测O·-2的分子探针、纳米探针、蛋白探针以及化学发光探针的研究进展,并对其发展前景进行了展望.     

荧光光谱分析超氧化物歧化酶的构象变化及其功能

通过荧光光谱研究了枯草芽孢杆菌超氧化物歧化酶(SOD)的分子构象在不同浓度盐酸胍、尿素以及不同pH条件下的变化及其与功能的关系.结果表明,SOD构象与功能的变化与其荧光光谱的变化有明显的对应关系.随着盐酸胍浓度的增加,SOD发射峰红移,荧光强度不断下降,且这两种变化同步发生;酶则出现快失活慢变构现象.而利用尿素变性SO...     

DNA interaction, superoxide scavenging and cytotoxicity studies on new copper(II) complexes derived from a tridentate ligand

The copper complexes [Cu(Pyimpy)(H₂O)](ClO₄)₂ (1), [Cu(Pyimpy)₂](ClO₄)₂ (2), [Cu(Pyimpy)(Cl)₂]·2H₂O (3·2H₂O), [Cu(Pyimpy)(N₃)(ClO₄)]₂ (4) and [Cu(Pyimpy)(SCN)(ClO₄)]₂ (5) were synthesized and characterized by spectroscopic techniques, crystal structures and electrochemical studies (Pyimpy: (2-((2-phenyl-2-(pyridin-2-l)hydrazono)methyl)pyridine)). The superoxide scavenging activity of the two water soluble complexes 1 and 3 was examined. DNA interaction studies by UV-Vis absorption spectral changes during a titration experiment indicated the generation of new species. These small molecule SOD mimics exhibited excellent DNA cleavage activity in the presence of H₂O₂ as well as 2-mercaptoethanol. Complexes 1-5 exhibited better cytotoxicity compared to CuCl₂·2H₂O and the ligand Pyimpy, and showed more potency than cisplatin for MCF-7, PC-3 and HEK-293 cells. Complex 3 exhibited the highest potency for MCF-7, PC-3 and HEK-293 cells compared to the other complexes.     

Synthesis,characterization, crystal structures,and superoxide dismutase activity of copper(II) octahedral complexes containing tri- and monodentate ligands

Three new copper(II) complexes [Cu(PSBP)₂](NO₃)(BF₄) (1), [Cu(DAPBMA)₂](BF₄)₂ (2), and [Cu(ImH)₄(NO₃)₂] (3), where PSBP = 4-phenylsemicarbazide-2-benzoylpyridine, DAPBMA = 2,6-diacetylpyridine-bis-4-methoxyaniline, and ImH = Imidazole, have been synthesized and characterized by elemental analysis, FAB mass spectrometry, magnetic susceptibility, X-band electron paramagnetic resonance (EPR), electronic spectroscopy, and cyclic voltammetry. Frozen solution EPR spectra of the complexes have axial features with g _∥ > g _⊥ > 2.003 suggesting the presence of a d _{x ²? y ²} ground state. Single crystal X-ray analyses of 1–3 reveal the presence of distorted octahedral geometry. All complexes exhibit significant superoxide dismutase activity.     

Over-expression of extracellular superoxide dismutase in mouse synovial tissue attenuates the inflammatory arthritis

Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular- superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC- SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1β, TNFα, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.     

铅染毒家兔脂质过氧化水平和神经传导速度的变化及关系研究

为观察铅对脂质过氧化水平和神经传导速度(NCV)的影响,并探讨两者关系,1 6只家兔随机分为2组,每组8只,染铅组饮用0 1 %醋酸铅水,对照组予以去离子水,持续8周,测定了两组家兔股神经NCV及血浆和脑组织丙二醛(MDA)、总超氧化物歧化酶(T -SOD)水平,分析了MDA、T -SOD与NCV之间的关系。结果表明,染铅组NCV显著低于对照组(P <0 0 1 ) ,NCV与脑组织MDA之间存在相关关系(r=-0 5 2 1 ,P <0 0 5 ) ,但染铅组MDA、T -SOD与对照组比较未达到显著性差别(P >0 0 5 )。提示铅中毒能引起NCV减慢,这与脂质过氧化水平有关,脂质过氧化损伤可能是铅致中毒性周围神经病的机制之一。