NAD~+对小麦叶片线粒体甘氨酸氧化和三羧酸代谢的调节

外源NAD~+对小麦叶片线粒体内甘氨酸、苹果酸及α—酮戊二酸氧化都有促进作用。当几种呼吸底物同时存在时,其中甘氨酸的氧化抑制了其他底物的同时氧化,因为催化这两类废物氧化的酶对NAD~+的亲和力和对NADH/NAD~+比值的敏感程度有差异,催化甘氨酸氧化的甘氨酸脱羧酶对线粒体基质内可利用的NAD~+的亲和力分别比苹果酸脱氢酶和α—酮戊二酸脱氢酶的亲和力大约1或2倍。另外,甘氨酸亦可通过保持线粒体基质内高NADH/NAD~+比值来影响三羧酸环的正常代谢。     

草酰乙酸—苹果酸穿梭系统对甘氨酸和α-酮戊二酸氧化的调节

草酰乙酸—苹果酸穿梭系统通过改变线粒体基质内NAD~+的可利用量或NADH/NAD~+比率对甘氨酸和α-酮戊二酸氧化进行调节。该穿梭系统利用不同来源的NADH的能力相近。     

氧化磷酸化作用和有关同题的研究 VI.可溶性因子在鼠肝线粒体碎片琥珀酸氧化偶联NAD~+需能还原中的作用

鼠肝线粒体经超声波处理得到的碎片在用适当量水洗一次后很不稳定,催化琥珀酸氧化偶联NAD~+需能还原的活力很低,此时加入线粒体经超声波处理后47,000×g离心的清液后线粒体碎片催化NAD~+需能还原的活力即显著地增大。由上清液所促进的NAD~+需能还原也同样地被Amytal与DNP所抑制。实验结果表明在上清液中合有线粒体催化琥珀酸氧化偶联NAD~+需能还原所必需的可溶性因子,上清液中可溶性因子甚不稳定,在25℃放置50分钟活力丧失60%,用pH沉淀及DEAE离子交换纤维素柱层析分离等方法可以将可溶性因子提纯约100倍。提纯后的可溶性因子ATP酶活力甚低。     

川芎Ⅲ号碱对鼠肝线粒体氧化磷酸化作用的研究

 川芎Ⅲ号碱是中药川芎中的一种生物碱。本文实验观察到:川芎Ⅲ号碱有降低鼠肝线粒体氧耗量,氧化磷酸化解偶联和减少ATP的生成作用。并发现:该化合物在低浓度(<10μmol/L)可阻断NAD~+链的电子传递,其作用点在NADH脱氢酶系统鱼籐酮敏感部位。此外,该化合物通过激活线粒体ATP酶水解活性,加速ATP的分解。作者对上述结果进行了简要的讨论。     

花生幼苗下胚轴质膜氧化还原系统

花生幼苗下胚轴细胞膜制剂具有氧化NAD(P)H,还原Fe(CN)_6~(3-)与EDTA-Fe~(3 )的能力,当Fe(CN)_6~(3-)浓度为1mmol/L时,膜制剂氧化NADH和NAD(P)H的K_m分别为100和110μmol/L;V_(max)为1400和710nmol mg~(-1) proteinmin~(-1),最适pH为8.0。NADH 0.25 mmol/L浓度下,膜制剂还原Fe(CN)_6~(3-)的K_m为40,V_(max)为1300;还原EDTA-Fe~(3 )的K_m为125,V_(max)为180,最适pH分别为8.0与7.0。氧为该系统天然受体,鱼藤酮、抗霉素A与CN~-对其活性无影响,10μmol/L的DCCD抑制其活性约20％,10μmol/L的SHAM抑制近50％活性。     

大肠杆菌NAD+合成关键酶的克隆表达及发酵优化

【目的】烟酰胺腺嘌呤二核苷酸(NAD~+)在细胞基因表达、氧化还原反应、能量代谢以及调控细胞生命周期中具有重要的作用,其细胞内含量是能量效率的关键因素。强化辅因子合成策略,获得高产NAD~+菌株,对于NAD~+依赖型氧化还原反应的速率和调节相关生化合成途径的代谢流具有重要意义。【方法】首先通过内源性调节,对代谢途径中的关键酶基因进行强化,过量表达和共表达NAD~+合成途径中的关键酶基因pncB、nadD和nadE;其次,通过外源调节增加NAD~+前体物,优化诱导条件提高发酵过程中关键酶的表达量,增加NAD~+的合成量;最后在单因素优化试验的基础上,以NAD~+含量为响应值,采用Box-Bohnken试验设计方法,研究3个显著性影响因素相互作用对NAD~+积累量的影响,确定最佳的优化条件。【结果】根据关键酶基因强化策略,构建了7株重组菌,其中重组菌E.coli BL21/p ET-21a-nad E-pncB胞内NAD~+含量相比初始菌株E.coli BL21/pET-21a提高了405.2%。通过对该菌株诱导条件和NAD~+合成前体的优化,使用Design Expert 8.0分析实验数据,得出该重组菌株的最佳发酵条件为:诱导温度控制在15–20 oC,OD_(600)为0.6–0.8时添加IPTG 0.63 mmol/L、烟酸15.8 mg/L、诱导时长控制在24 h。NAD~+含量在最优条件下实验验证值可达43.16μmol/g DCW,与优化前相比提高了123.6%,与初始菌株相比提高了1029.8%。【结论】在大肠杆菌中共表达关键酶基因pncB和nadE,胞内NAD~+合成量明显增加,前体物以及诱导条件的外源调节使NAD~+积累量达到最佳优化值。实现了提高NAD~+含量的目标,胞内辅因子浓度的增加为提高生物催化效率奠定了可行性基础。     

齐墩果酸通过PPARγ调控人脐静脉内皮细胞抗氧化损伤作用

目的探讨齐墩果酸(OA)对ox-LDL诱导的内皮细胞氧化损伤的作用及其机制。方法实验分组:对照组,ox-LDL模型组,ox-LDL+OA(10μmol/L,20μmol/L,40μmol/L)组,ox-LDL+OA(10μmol/L,20μmol/L,40μmol/L)+GW9662,GW9662单独处理组。采用MTT法检测HUVECs细胞活力;酶联免疫吸附试验法检测HUVECs细胞SOD活力、GSH活力以及MDA含量;活性氧检测试剂盒检测HUVECs细胞ROS水平;Western blot检测HUVECs细胞PPARγ蛋白表达水平,所有指标的检测都进行生物学重复。采用方差分析和两样本t检验进行统计学分析。结果 MTT结果显示,ox-LDL组的细胞存活率为(49.17±0.62)%,OA(10μmol/L、20μmol/L、40μmol/L)预处理后存活率分别为(68.51±1.16)%、(82.64±0.73)%、(92.37±0.13)%,可减弱ox-LDL对HUVECs细胞存活率的降低且呈剂量依赖性关系,差异具有统计学意义(t=24.35,26.18,35.17;P=0.034,0.027,0.008)。本研究还发现,OA对ox-LDL诱导的HUVECs细胞SOD、GSH活性的降低和MDA、ROS水平的增加具有抑制作用且呈剂量依赖关系。ox-LDL组的细胞SOD、GSH、ROS和MDA水平分别为(16.12±0.06)μmol/g、(132.16±2.11)μmol/g、(2.63±0.02)k U/g、(158.12±0.39)%,ox-LDL+OA(10μmol/L)组的细胞SOD、GSH、ROS和MDA水平分别为(10.60±0.14)μmol/g、(108.36±2.05)μmol/g、(2.41±0.21)k U/g、(136.18±1.24)%,ox-LDL+OA(20μmol/L)组的细胞SOD、GSH、ROS和MDA水平分别为(13.28±0.09)μmol/g、(129.58±0.09)μmol/g、(2.26±0.15)k U/g、(126.43±1.51)%,ox-LDL+OA(40μmol/L)组的细胞SOD、GSH、ROS和MDA水平分别为(14.86±0.16)μmol/g、(131.47±0.76)μmol/g、(2.14±0.08)k U/g、(112.39±1.07)%(F=26.38,31.27,56.82,41.16;P=0.005,0.004,0.002,0.003)。Western blot结果显示,OA有效促进HUVECs细胞PPARγ蛋白水平提高。与ox-LDL+OA(20μmol/L)组(65.37±0.15)%比较,ox-LDL+OA(20μmol/L)+GW9662组的细胞活力为(52.89±0.16)%,差异有统计学意义(t=16.47,P=0.035)。进一步发现ox-LDL+OA(10μmol/L)组SOD、GSH、MDA、ROS水平为(10.58±0.13)μmol/g、(102.46±0.06)μmol/g、(2.42±0.08)k U/g、(144.38±2.02)%,oxLDL+OA(10μmol/L)+GW9662组的SOD、GSH、MDA、ROS水平分别为(8.42±0.05)μmol/g、(88.38±0.48)μmol/g、(2.83±0.01)k U/g、(154.41±1.04)%,两组比较差异有统计学意义(t=38.47,39.25,43.69,41.27;P=0.008,0.008,0.006,0.006);ox-LDL+OA(20μmol/L)组SOD、GSH、MDA、ROS水平(13.25±0.05)μmol/g、(122.59±0.33)μmol/g、(2.23±0.16)k U/g、(123.94±0.15)%,ox-LDL+OA(20μmol/L)+GW9662组的SOD、GSH、MDA、RO水平分别为(10.59±0.12)μmol/g、(106.42±0.15)μmol/g、(2.61±0.07)k U/g、(138.12±1.15)%,两组比较差异有统计学意义(t=46.08,38.11,49.35,35.59;P=0.005,0.008,0.004,0.009);oxLDL+OA(40μmol/L)组SOD、GSH、MDA、ROS水平分别为(15.88±0.14)μmol/g、(140.26±1.05)μmol/g、(2.02±0.13)k U/g、(187.52±0.68)%,ox-LDL+OA(40μmol/L)+GW9662组的SOD、GSH、MDA、RO水平(13.65±0.03)μmol/g、(124.61±1.27)μmol/g、(2.49±0.04)k U/g、(126.51±0.73)%,两组比较差异有统计学意义(t=48.04,38.62,45.14,50.13;P=0.004,0.008,0.005,0.002),此外,本研究还发现,抑制PPARγ后,OA抑制ox-LDL诱导的HUVECs细胞的氧化损伤仍存在剂量效应。结论 OA可以通过PPARγ抑制x-LDL诱导HUVECs细胞氧化损伤。     

重金属镉通过DNA氧化损伤途径诱导细胞中心体扩增

目的:研究氯化镉(CdCl_2)对细胞中心体扩增的影响,及活性氧(ROS)和DNA损伤在CdCl_2诱导细胞中心体扩增中的作用。方法:用不同浓度(0、10、20、40μmol/L)CdCl_2处理HCT116细胞48 h,MTT法检测细胞活性;用低浓度无毒性的CdCl_2处理细胞48 h,免疫荧光实验观察细胞内中心体的扩增;用CdCl_2(20μmol/L)、CdCl_2+N-乙酰半胱氨酸(NAC)处理细胞2 h,活性氧检测试剂盒检测细胞内ROS水平的变化;用CdCl_2(20μmol/L)、CdCl_2+NAC处理细胞6 h,彗星电泳试剂盒检测细胞内DNA损伤水平的变化;用CdCl_2(20μmol/L)、CdCl_2+NAC处理细胞48 h,免疫荧光观察细胞内中心体的扩增。结果:20μmol/L或以下CdCl_2处理细胞48 h不影响细胞活性;CdCl_2 20μmol/L或以下无毒性剂量CdCl_2诱导细胞中心体发生扩增(P0.01),并呈剂量依赖效应;在20μmol/L CdCl_2处理下,细胞内ROS和DNA损伤水平均明显升高(P0.01),当有抗氧化剂NAC存在时,细胞内升高的ROS和DNA损伤水平均被明显抑制(P0.01);抗氧化剂NAC对CdCl_2诱导的中心体扩增也有明显的抑制效果(P0.01)。结论:氯化镉通过DNA氧化损伤途径诱导细胞中心体扩增。     

高粱NADP苹果酸脱氢酶的纯化及其分子特性

用Sephadex G-100柱和制备性等电聚焦电泳纯化了高梁叶片NADP苹果酸脱氢酶,得到电泳均一的酶制剂,酶比活力提高350倍。天然酶含有两个分子量为4万D的亚基。动力学研究表明Km(OAA)=31μmol/L:K_m(NADPH)=48 μmol/L;K_m(Mal)=11m mol/L:K_m(NADP)=45μmol/L。NADP为NADPH的竞争性抑制剂,抑制常数K_i=190 μmol/L。 在体外DTT为NADP苹果酸脱氢酶的激活所必需。DTT对该酶的激活受一小分子蛋白、NADP及离子强度所调节。     

海水亚硝酸氧化细菌初始富集过程中硝酸盐的定性检测

本文对海水亚硝酸氧化细菌初始富集过程中硝酸盐的定性检测方法及其适用范围进行了研究。结果表明, 在NaNO2起始浓度为100mg/L的亚硝酸氧化细菌初始富集培养系统中,1mL培养液中残余的NaNO2,可先用 1．0mol/L盐酸溶液20μL和50g/L氨基磺酸铵溶液10-20μL将其去除,然后再用二苯胺试剂对培养液中经亚硝酸 氯化细菌转化来的NaNO3进行定性检测,可检测出的NaNO3浓度下限在20mg/L左右。在NaNO2起始浓度不同的 富集培养系统中,去除NaNO2所需盐酸溶液、氨基磺酸铵溶液的量可根据其起始浓度按比例相应增减,但NaNO2的 起始浓度不宜超过200mg/L。该方法亦适用于淡水亚硝酸氧化细菌初始富集培养过程中硝酸盐的定性检测。       

Chromium(III) Oxidation Coupled with Microbially Mediated Mn(II) Oxidation

Abstract

Reductive immobilization of Cr(VI) has been widely explored as a cost-effective approach for Cr-contaminated site remediation. In soils containing manganese oxides, however, the immobilized form of chromium, i.e., Cr(III), could potentially be reoxidized. In this study, batch experiments were conducted to assess whether there were any microbial processes that could accelerate Cr(III) oxidation in aerobic, manganese-containing systems. The results showed that in the presence of at least one species of manganese oxidizers, Pseudomonas putida, Cr(III) oxidation took place at low concentrations of Cr(III). About 30–50% of added Cr(III) (10–200 μ M) was oxidized to Cr(VI) within five days in the systems with P. putida and biogenic Mn oxides. The rate of Cr(III) oxidation was approximately proportional to the initial concentration of Cr(III) up to 100 μ M, but the growth of P. putida was partially inhibited by Cr(III) at 200 μ M and totally stopped when it reached 500 μ M. Cr(III) oxidation was dependent upon the biogenic formation of Mn oxides, though the oxidation rate was not directly proportional to the amount of Mn oxides formed. Chromium(III) oxidation took place through a catalytic pathway, in which the microbes mediated Mn(II) oxidation to form Mn-oxides, and Cr(III) was subsequently oxidized by the biogenic Mn-oxides.     

Oxidation of cysteine and homocysteine by bovine albumin

The autooxidation of cysteine and homocysteine to their disulfide forms was determined by measuring the time course of thiol groups disappearance. We found the oxidative chemistry of cysteine and homocysteine to be quite different. In the absence of added Cu(II), cysteine autooxidized at a slower rate than homocysteine, though in its presence cysteine oxidation was much faster, homocysteine being found to be a poor responder to copper catalysis. Albumin speeded up the spontaneous oxidation of both aminothiols, the reaction being faster with cysteine than with homocysteine. The copper content of different albumins was found to be highly variable, ranging from 12.75 to 0.64 microg Cu(II)/g albumin. We propose that copper bound to albumin possesses redox cycling activity to perform cysteine oxidation since: (i) copper elimination by copper chelators markedly reduces oxidation; and (ii) a positive correlation exists between the albumin copper content and the oxidation reaction rate.     

厌氧氨氧化菌混培物生长及代谢动力学研究

研究了厌氧氨氧化菌混培物的动力学特性.测得细胞产率系数1.573mgVS(mmolNH4+)-1;细胞衰减常数0.052mgVS(g@VS@d)-1.厌氧氨氧化菌混培物的最大氨氧化速率1.320～2.761mmol(gVS@d)-1,最大亚硝酸盐转化(反硝化)速率14.497mmol(gVS@d)-1.厌氧氨氧化菌混培物利用氨的Km值1.801～4.215mmol@L-1,利用亚硝酸盐的Km值0.468mmol@L-1.氨自身的抑制常数38.018～98.465mmol@L,实际最大氨氧化速率的氨浓度16.656mmol@L-1.亚硝酸盐对厌氧氨氧化的抑制常数5.401～11.995mmol@L-1.厌氧氨氧化的最适pH7.605.厌氧氨氧化的最适温度30℃.Vmaxa、Kma、Kia和Kin的活化能依次为37.316、30.239、33.695和30.473kJ@mol-1.     

齐墩果酸氧化产物的成分研究

以齐墩果酸为原料,分别用高锰酸钾和SeO2/H2O2(30%)进行氧化。从产物中分离得到3个化合物,经1H NMR、13C NMR、2D-NMR、MS等波谱分析,分别鉴定为3,11-二羰基-12,17-二烯-28-去甲基齐墩果烷(1)、3β-羟基-11-烯-13,28-内酯-齐墩果烷(2)和3α,12β,13α-三羟基-28-羧基齐墩果烷(3),收率依次是4.5%、6.4%、2%,其中化合物1和3为新化合物。     

Iron (II) Ions Induced Oxidation of Ascorbic Acid and Glucose

Lipid peroxidation (LPO) of polyunsaturated fatty acids (PUFAs) is suspected to be involved in the generation of chronic diseases. A model reaction for LPO is the air oxidation of PUFAs initiated by Fe²⁺ and ascorbic acid. In the course of such model reactions glycolaldehyde (GLA) was detected as main aldehydic product. Since it is difficult to explain the generation of GLA by oxidation of PUFAs, it was suspected that GLA might be derived by oxidation of ascorbic acid. This assumption was verified by treatment of ascorbic acid with Fe²⁺.

Produced aldehydic compounds were trapped by addition of pentafluorobenzylhydroxylamine hydrochloride (PFBHA-HCl), trimethylsilylated and finally identified by gas chromatography/mass spectrometry (GC/MS). Oxidation of ascorbic acid with O₂ in presence of iron ions produced not only glycolaldehyde (GLA), but also glyceraldehyde (GA), dihydroxyacetone (DA) and formaldehyde. Glyoxal (GO) and malondialdehyde (MDA) were detected as trace compounds.

The yield of the aldehydic compounds was increased by addition of lipid hydroperoxides (LOOH) or H₂O₂. The buffer influenced the reaction considerably: Iron ions react with Tris buffer by producing dihydroxyace-tone (DA). Since ascorbic acid is present in biological systems and Fe²⁺ ions are obviously generated by cell damaging processes, the production of GLA and other aldehydic components might add to the damaging effects of LPO.

Glucose suffers also oxidation to short-chain aldehydic compounds in aqueous solution, but this reaction requires addition of equimolar amounts of Fe²⁺ together with equimolar amounts of H₂O₂ or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HPODE). Therefore this reaction, also influenced by the buffer system, seems to be not of biological relevance.     

Atmospheric Oxidation of Hydrocarbons

Hydrocarbon oxidation in the atmosphere proceeds generally by the following sequence of reactions: hydrocarbon + OH → alkyl radical + H₂O, alkyl radical + O,(³I) → alkylperoxy radical, alkylperoxy radical + NO → alkoxy radical + NO₂, alkoxy radical + O₂(³X) -→ aldehyde + HO,. The atmospheric lifetimes of hydrocarbons are determined by their reactivity towards OH as well as by the average OH concentration level. They are compound specific and vary from several hours to several years. Hydrocarbon oxidation chains couple with other trace gases (O_v, HO_x, and NO_v). For the conditions of the average continental atmosphere an increase of the oxidative potential (HO_v, O_x) is predicted through hydrocarbon oxidation.     

Oxidation and metal-ion affinities of a novel cyclic tetrasaccharide

The cyclic tetrasaccharide, cyclo-(-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was oxidized in high yield to a dicarboxylic acid, cyclo-(-->6)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA-(1-->). The parent and oxidized compound were then screened for the ability to form stable complexes with 20 metal cations. Ion-exchange thin-layer chromatography was utilized to survey binding in aqueous and 50% methanolic solutions. The screening identified Pb2+, Fe2+ and Fe3+ as forming strong metal chelates with the oxidized cyclic tetrasaccharide. The stoichiometry of the oxidized cyclic tetrasaccharide and Pb2+ complex was determined to be 1:1 using aqueous gel-permeation chromatography. Perturbations between the free and complexed structure were examined using NMR spectroscopy. Molecular simulations were used to identify a probable structure of oxidized cyclic tetrasaccharide complexed with Pb2+.     

Cysteine and Crocin Oxidation Catalyzed by Horseradish Peroxidase

The amino acid cysteine is oxidized by horseradish peroxidase, and the water-soluble carotenoid crocin is bleached by cooxidation. The rnonophenol p-hydroxyacetophenone stimulates oxygen uptake, cysteine oxidation and crocin bleaching, whereas its concentration does not change. Superoxide dismutase significantly enhances all these oxidative reactions. Addition of H₂O₂ is not required for these peroxidase-catalyzed oxidations.     

Cysteine and Crocin Oxidation Catalyzed by Horseradish Peroxidase

The amino acid cysteine is oxidized by horseradish peroxidase, and the water-soluble carotenoid crocin is bleached by cooxidation. The rnonophenol p-hydroxyacetophenone stimulates oxygen uptake, cysteine oxidation and crocin bleaching, whereas its concentration does not change. Superoxide dismutase significantly enhances all these oxidative reactions. Addition of H₂O₂ is not required for these peroxidase-catalyzed oxidations.     

嗜热革节孢多糖单加氧酶氧化方式

多糖单加氧酶(polysaccharidemonooxygenase,PMO)是一种铜离子依赖的氧化酶,属于辅助活性酶类第九家族(auxiliary activity 9,AA9),在存在电子供体维生素C(vitamin C,Vc)的情况下,可以氧化裂解纤维素的多糖链,显著提高纤维素的酶解效率。本文克隆了嗜热革节孢Scytalidium thermophilumAA9家族的一个编码基因pmo7651,并在毕赤酵母GS115进行诱导表达,通过His标签获得了重组蛋白PMO7651-His。以磷酸膨胀纤维素(PASC)为底物进行酶促反应,薄层层析法(TLC)结果显示PMO7651酶解产物主要为纤维二糖至纤维五糖;飞行时间质谱法(MALDI-TOF-MS)和溴氧化法确定PMO7651具有C1、C4、C6位的氧化活性;底物结合平面的3个芳香族氨基酸位点突变对酶的活性具有不同程度的影响;在PMO7651帮助下,纤维素酶的降解效率均具有不同程度的提高。