Ni2+胁迫下嗜酸氧化亚铁硫杆菌亚铁氧化应激效应

【目的】嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)作为湿法冶金的主要功能菌,其在重金属Ni²⁺胁迫下的亚铁氧化应激机制还不清楚。【方法】在两步法培养体系中,设计由低到高Ni²⁺胁迫浓度,利用分光光度法、电化学法和实时荧光定量PCR法等从宏观到微观探究Ni²⁺胁迫下A.ferrooxidans菌亚铁氧化应激效应。【结果】两步培养法体系下A.ferrooxidans菌能够耐受较高浓度的Ni²⁺毒性(≤30 g/L Ni²⁺)。当Ni²⁺胁迫浓度增加至40 g/L时,与对照组(不添加Ni²⁺胁迫)相比,A.ferrooxidans亚铁氧化率和速率显著降低(24 h的亚铁氧化抑制率约为59.4%),亚铁氧化代谢相关的rus操纵子各功能基因的表达量也显著下调,尤其是基因cyc1(最高下调了16倍)和coxBACD(4个不同亚基最高下调2.7–7.4倍);同时Fe²⁺氧化相关的胞外电子传递能力也降低,对照组(0 g/L Ni²⁺,29.0µA)最大还原峰电流值高于实验组(40 g/L Ni²⁺,24.5µA)。【结论】超高浓度Ni²⁺胁迫环境对A.ferrooxidans菌有毒性,导致该菌rus操纵子上各功能基因表达量下调,同时造成胞外电子传递速率降低,最终致使A.ferrooxidans菌亚铁氧化能力降低。研究结果将为含Ni固废高效生物浸出提供理论支持。     

人肌型特异烯醇化酶放免分析法及初步应用

建立了人肌型特异烯醇化酶(hMSE)的放免分析法,抗血清的亲和常数为5.1×10⁹L/mol,采用改良的BHR法制备了 ¹²⁵I-hMSE,后者非特异结合率为3%,与抗血清(1∶10³)结合率达50.16%;批内和批间CV分别为8.6%和13%.回收率为95%-105%.标准曲线范围为5-320μg/L.最小检出率为5μg/L.最佳反应条件:0.1mol/LpH7.4PBS(含5mmol/L MgSO₄,0.1%吐温-20,0.1%NaN₃);反应温度和时间:37℃反应0.5h和4℃,0.5h,作为快速测定;或选用4℃反应24h.65例健康人血清hMSE浓度为23.9±10.9μg/L(x±s).hMSE超过57μg/L(x+3s).为阳性值.测定了AMI和肌病患者,血清hMSE明显升高.     

牛小脑肌醇磷脂激酶PI(4)K高产率纯化与特征

对牛小脑膜区肌醇磷脂激酶进行了11 500倍纯化,过程包括：TritonX-100抽提,硫酸铵沉淀,阳离子交换层析(phosphocellulose),亲和层析(Heparin Sepharose CL-6B)和阴离子交换层析(DEAE10,FPLC)等.纯化程度可达95％以上,对SDS-PAGE电泳结果进行扫描分析测其分子质量为56 ku.纯化的肌醇磷脂激酶的特异活性为450 nmol/mg·min, 动力学性质表现为ATP的表观K_m值为7.9×10^-7 mol/L,PI的表观K_m值为6.6×10^-7 mol/L. 腺嘌呤核苷是该酶的有效抑制剂,3.5×10^-7 mol/L腺嘌呤核苷可使该酶活力降低约50％,而TritonX-100对该酶活力具有刺激作用,0.5% TritonX-100可使该酶表现为最高活力.     

用基因芯片检测DPYD等位基因在受试人群中的发生频率

二氢嘧啶脱氢酶基因（DPYD基因）所编码的二氢嘧啶脱氢酶（DPD酶）是氟化嘧啶类抗肿瘤药物代谢的主要限速酶,其活性存在显著的个体差异,并因此影响药物的疗效和毒副作用.大部分编码低/无活性酶的突变型等位基因是由于基因中的单核苷酸多态性（single nucleotide polymorphism,SNP）造成的,检测这些SNPs是预测患者对药物的反应和实现个体化给药方案的基础.制备并优化了用于检测DPYD基因中6个已知SNPs所编码的等位基因（DPYD^*2,^*3,^*4,^*5,^*9,^*12）的基因芯片,建立了该芯片的基因分型标准.并利用该芯片检测了肿瘤患者（112例）、肾病患者（83例）和健康者（45例）中DPYD突变型等位基因的发生频率.在受试人群中,突变型等位基因DPYD^*5和DPYD^*9平均发生率分别为32.08%和11.25%,未发现DPYD^*2,^*3,^*4,^*12突变型等位基因.而且以上单碱基突变的发生率在肿瘤患者、肾病患者和健康者间以及男性、女性肿瘤患者间无显著性差异,表明其与疾病的发生或性别无显著性关联.对20例标本的基因分型结果采用直接测序法进行验证,19例基因芯片分型结果与直接测序法结果相一致.DPYD^*5、DPYD^*9突变型等位基因在受试人群中具有较高的发生率.利用基因芯片能够对其实现快速准确的检测.     

重组刺桐胰蛋白酶抑制剂a的制备及其在tPA突变体纯化中的应用

将基因工程菌株E.coliBL21(DE3) pET22b-mETIa高密度发酵,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导,重组刺桐胰蛋白酶抑制剂a(rETIa)蛋白在E.coli中得到较高水平表达,表达量占菌体总蛋白的40%以上.经菌体破碎、包涵体变性、复性,二步柱层析纯化得到电泳纯的rETIa蛋白.测得rETIa对t-PA突变体(NTA)的抑制平衡常数K_i为8.72×10^－8 mol/L.据此利用纯化的rETIa蛋白制备rETIa-Sepharose 4B亲和层析柱.直接一步纯化NTA复性液,纯化的NTA纯度达90 %以上,收率为96.2 %,纯化倍数为13.2,比活为(565.7±71.3) U/μg.     

酸性区3融合重链促进基于蛋白质内含子的双载体B域缺失型凝血因子Ⅷ转基因表达

最近的研究表明,蛋白质内含子(intein)介导的B区缺失型凝血因子Ⅷ (BDD-FⅧ)的轻链和重链剪接可顺式促进后者的分泌,而且剪接反应在细胞内、外均可发生．为进一步提高基于蛋白质内含子的双载体转BDD-FⅧ基因的功效,将具有促进重链分泌作用的位于Pro¹⁶⁴⁰～Ser¹⁶⁹⁰的酸性区3(acidic region-3,AR-3)引入重链,检验对蛋白质内含子剪接的BDD-FⅧ蛋白分泌和活性的影响．用融合蛋白内含子的附加ar-3重链(HCAR3IntN)基因和轻链(IntCLC)基因共转染培养的HEK293细胞,分别用ELISA和Coatest法定量分析分泌至培养上清中剪接BDD-FⅧ蛋白量和生物活性,并用免疫印迹观察了细胞内的BDD-FⅧ剪接．结果显示,共转HCAR3IntN和IntCLC基因细胞,分泌至上清的剪接BDD-FⅧ蛋白量和活性分别为(173±26) μg/L和(1.31±0.15) U/ml,明显高于未添加ar-3的蛋白质内含子融合重链(HCIntN)与轻链(IntCLC)基因共转染细胞[(102±12) μg/L和(0.79±0.09) U/ml],提示AR-3对蛋白质内含子剪接的BDD-FⅧ蛋白分泌和活性有明显改善作用．而且,分别转HCAR3IntN和IntCLC基因细胞混合培养后的上清中,亦检测到剪接的BDD-FⅧ蛋白和活性[(35±7) μg/L和(0.28±0.08) U/ml],表明蛋白质内含子可进行不依赖细胞机制的蛋白质剪接．另外,转基因细胞总蛋白呈现明显的可与FⅧ多克隆抗体进行反应的剪接BDD-FⅧ蛋白条带,直观地反映细胞内BDD-FⅧ的剪接．为动物模体内运用蛋白质反式剪接技术的双腺相关病毒载体(AAV)转BDD-FⅧ基因实验提供了依据．     

L-丝氨酸生产菌的选育及不同碳源对发酵的影响

以一株黄假单胞属(Pseudomonas flava)菌株A3为出发菌株,经过紫外(UV)诱变和硫酸二乙酯(DES)逐级诱变处理和选育,选育出一株能够以甲醇为唯一碳源的兼性甲基营养型菌JW-01(Mth^R、Gly^R)。在含甘氨酸30g/L、甲醇1%的发酵培养基中发酵3d后L-丝氨酸产量为6．2g/L,较出发菌株提高了67．6%。该菌具有较好的传代稳定性。     

研究报告: 电化学适配体传感器用于乳酸快速检测的研究

目的 构建用于检测L-乳酸的新型电化学适配体传感器。方法 基于金钯-掺氮多壁碳纳米管纳米复合材料（Au/Pd-N-MWCNTs）修饰的玻碳电极，通过三螺旋分子开关（triple-helix molecular switch，THMS）触发具有RNA剪切活性的Pb²⁺辅助的脱氧核酶（DNAzyme）对电极表面固定化信号探针的循环剪切效应，实现L-乳酸的超灵敏电化学检测。采用差分脉冲伏安法（DPV）记录电流信号变化。结果 信号探针浓度4 μmol/L、Pb²⁺浓度4 μmol/L、DNAzyme剪切孵育时间60 min为传感器最优测试条件。在最优实验条件下，该L-乳酸传感器线性范围为1~20 mmol/L，检出限为0.51 mmol/L。此外，该适配体传感器具有优异的稳定性（RSD=4.56%）、重现性（RSD=2.80%）和选择性。在人血清样本中检测L-乳酸时回收率为105.60%~110.80%，RSD为2.35%~4.56%，与传统方法具有较好的一致性。结论 该适配体传感器能实现L-乳酸的超灵敏检测，在生物医学诊断、食品工业和环境监测等领域具有广泛的应用前景。     

悬浮培养HEK-293 N3S细胞生产重组腺病毒Ad-GFP的实验研究

利用5L生物反应器悬浮培养HEK-293 N3S细胞生产携带绿色荧光蛋白基因的重组腺病毒(recombinant adenovirus-green fluorescent protein,Ad-GFP),为规模化生产腺病毒基因药物建立一种稳定可行的生产工艺。复苏的种子细胞进行逐级放大最后接入5L搅拌式生物反应器中,采用含5％胎牛血清（FBS）的DMEM/F12培基灌流培养293 N3S细胞,当细胞密度达到(2～4)×10⁶个/mL时感染Ad-GFP,48h后收获细胞,经两步氯化铯超速离心获得纯化的Ad-GFP。采用紫外分光光度计比色法和高压液相色谱法（HPLC）测定病毒颗粒数和纯度,采用组织培养半数感染剂量（TCID₅₀）法检测腺病毒的感染滴度。连续培养10～12d,细胞密度可达到(2～4)×10⁶6个/mL左右,纯化的Ad-GFP感染滴度和颗粒数分别为1.0×10¹¹IU/mL和1.68×10¹²VP/mL,比活性为6.0%,A₂₆₀A₂₈₀比值为1.33,产品纯度达到99.2%。建立了5L生物反应器悬浮培养293 N3S细胞生产重组腺病毒Ad-GFP的生产工艺,对携带其他基因的重组腺病毒药物生产具有一定的指导意义。     

代谢工程强化脱氮副球菌DYTN-1去除氮素污染物

【目的】脱氮副球菌(Paracoccus denitrificans)是一种环境友好的α-变形菌纲菌株,在有氧条件下也可进行反硝化过程,具有较好的脱氮能力。本研究以脱氮副球菌DYTN-1为底盘细胞,筛选氮素诱导型启动子用于强化硝化和反硝化途径,进而达到代谢工程强化脱氮副球菌DYTN-1去除氮素污染物的目的。【方法】通过接合转移的方法分别将过表达amoA、amoB、hao和nirS基因的重组质粒导入脱氮副球菌DYTN-1细胞中。经过荧光定量检测和氮素定量检测对脱氮副球菌DYTN-1的基因元件和氮去除能力进行表征。【结果】从基因组中挖掘了6个受NO₂^‒、NO₃^‒和NH₄⁺诱导的启动子,诱导差异为2‒26倍;且过表达nirS的菌株用2 g/L KNO₃处理24 h后培养基中NO₃^‒的残余量为野生型菌株的67%。同时过表达hao和nirS基因的菌株在用1 g/L NH₄Cl和2 g/L KNO₃处理12 h后,其NO₃^‒的剩余量仅为野生型菌株的50%,且最终总氮的降解效率达79.5%,剩余总氮仅为野生型菌株的一半。【结论】上述研究表明,利用筛选获得的启动子工具在P. denitrificans DYTN-1中进行代谢工程改造强化氮素污染物的去除具有可行性。     

Towards catalyst compartimentation in combined chemo- and biocatalytic processes: Immobilization of alcohol dehydrogenases for the diastereoselective reduction of a β-hydroxy ketone obtained from an organocatalytic aldol reaction

The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turned out to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized with their cofactors on a commercially available superabsorber based on a literature known protocol. The use of the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to high conversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the second reaction cycle. A comparable result was obtained when no cofactor was added although a water rich reaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastereoselective reduction of an organocatalytically prepared enantiomerically enriched aldol adduct, leading to high conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at a lower catalyst and cofactor amount being still sufficient for biotransformations with “free” enzymes the immobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strong decrease of conversion was observed already in the second reaction cycle, thus indicating a significant leaching effect of catalyst and/or cofactor.     

NADP-Dependent enzymes. I: Conserved stereochemistry of cofactor binding

The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2′-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moeties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design. © 1997 Wiley-Liss Inc.     

NADP-Dependent enzymes. II: Evolution of the mono- and dinucleotide binding domains

Nicotinamide adenine dinucleotides [NAD and NADP with both referred to as NAD(P)] are among the more diffuse redox cofactors. Despite their stereochemical similarity where the only difference is a phosphomonoester on the ribose near the adenine of NADP, they show different biochemical reactivities with NAD behaving as an oxidant and NADP as a reductant. NAD(P)-dependent enzymes generally share a common open α/β fold with few exceptions only recently structurally characterized. This study of the molecular evolution of the NAD(P) binding domains, possible given the large number of known molecular structures, addresses two main questions: 1) can a common fold exist in different biological systems (divergent evolution) and 2) does a relationship exist among similar biological systems that display different folds (convergent evolution)? Both the structures of mono- and dinucleotide binding domains have been classified by cluster analysis based on the similarity evaluated by their main chain Cα superposition. Moreover, the cofactor conformations and the stereochemical characteristics of their pockets have also been classified by analogous methods on the basis of the published tertiary structures. Two primary results appear: 1) the classification of the mononucleotide binding domains is different from that of the dinucleotide binding folds and 2) both divergent and convergent evolutionary pathways can be hypothesized, the latter less frequently observed and less pronounced but nevertheless evident. The generally accepted hypothesis that dinucleotide binding domains have evolved by gene duplication of primordial genes coding for the smaller mononucleotide binding domains is acceptable but the two halves of the resulting dinucleotide binding domains are evolutionarly uncorrelated. The NH₂-terminal mononucleotide binding domain is less variable than the COOH-terminal half, probably because it involves the binding of the ADP moiety of NAD(P) invariant in all examined systems. There is evidence to postulate that evolutionary pathways for NAD(P)-dependent enzymes are both divergent and convergent. In fact, nearly all combinations of similarity/dissimilarity in overall fold, cofactor conformation, and cofactor binding pocket structural characteristics for each enzyme pair examined are possible. The NAD(P)-dependent enzymes apparently provide a canonical example of an evolutionary principle that “anything goes.” © 1997 Wiley-Liss Inc.     

Arabidopsis thaliana nicotinate/nicotinamide mononucleotide adenyltransferase (AtNMNAT) is required for pollen tube growth

While mammals and fungi possess nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) isoforms, Arabidopsis thaliana only contains a single NMNAT gene, AtNMNAT (At5g55810). We analyzed the enzymatic activity of the AtNMNAT-encoded protein to determine the role of AtNMNAT in plant development. AtNMNAT catalyzed the synthesis of nicotinate adenine dinucleotide (NaAD) from nicotinate mononucleotide (NaMN) in the Preiss-Handler-dependent pathway, and of nicotinamide adenine dinucleotide (NAD) from nicotiamide mononucleotide (NMN) in the Preiss-Handler-independent pathway. Prominent AtNMNAT expression was detected in the male gametophyte. Moreover, AtNMNAT expression was spatio-temporally regulated during microspore development and pollen tube growth. Disruption of the AtNMNAT gene (atnmnat mutant) was characterized by a decrease in NAD content in pollen. Cytological examinations revealed that the atnmnat mutant was gametophytically impaired in in vivo and in vitro pollen tube growth. Our results suggest that metabolic fulfillment via the NAD pathway is indispensable for normal pollen growth and subsequent normal seed production.     

Structure of alanine dehydrogenase from Archaeoglobus: active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin

The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases. The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.     

Crystal structure of the flavoprotein ArsH from Sinorhizobium meliloti

Purified ArsH from Sinorhizobium meliloti exhibits NADPH:FMN-dependent reduction of molecular O2 to hydrogen peroxide and catalyzes reduction of azo dyes. The structure of ArsH was determined at 1.8A resolution. ArsH crystallizes with eight molecules in the asymmetric unit forming two tetramers. Each monomer has a core domain with a central five-stranded parallel beta-sheet and two monomers interact to form a classical flavodoxin-like dimer. The N- and C-terminal extensions of ArsH are involved in interactions between subunits and tetramer formation. The structure may provide insight in how ArsH participates in arsenic detoxification.     

Pulse fluorimetry study of energy transfers between tryptophan residues and NADPH in beef liver glutamate dehydrogenase complexes

A method is proposed to determine the rates of singlet energy transfers in an array of chromophores containing a finite number of donors and fluorescent acceptors. This method is based on measurements of transfer efficiency coupled with pulse fluorimetry. Three classes of donors can be distinguished which differ in their energy transfer rate. The rates of the first, the second and the third class are respectively greater than, of the order of, and smaller than the emission rate. The method is applied to the study of the energy transfers from tryptophan residues to NADPH, in ternary and quaternary glutamate dehydrogenase complexes. Practically, all these tryptophan residues belong to the first class. They can be divided into two subclasses having different transfer rate values. The distances between these residues and the NADPH site are of the order of 2.5 nm. In addition, the ligand binding induces a protein conformational change, leading to a fluorescence quenching of the tryptophanyl emission.     

Biofuel anode based on d-glucose dehydrogenase,nicotinamide adenine dinucleotide and a modified electrode

A biofuel cell anode has been made from a modified graphite electrode and immobilized d-glucose dehydrogenase [β-d-glucose:NAD(P)⁺ 1-oxidoreductase, EC 1.1.1.4 7] so that energy could be drawn from the conversion of d-glucose to d-gluconic acid. An equivalent amount of dihydronicotinamide adenine dinucleotide (NADH) was formed from NAD⁺ and reduced the surface groups of the modified electrode. Reoxidationn of the latter produced the electrons necessary for a power output from the cell. Electrode modification was made by adsorption of N,N-dimethyl-7-amino 1,2-benzophenoxazinium onto the graphite. A current density of 0.2 mA cm^?2 at a cell voltage of ～0.8 V was obtained for more than 8 h with a simulated oxygen cathode. The internal resistance in the cell, in particular in the separator, appeared to be the main current-limiting factor.     

Variation in the sequence of a mitochondrial nadh dehydrogenase i gene fragment among six natural populations of Schistosoma japonicum from China

The present study investigated the level of genetic variation among Schistosoma japonicum populations of different geographical origins from mainland China. Polymerase chain reaction-based methods were employed to determine the sequence for a subunit of the mitochondrial NADH dehydrogenase I gene for populations from Zhejiang, Anhui, Jiangxi, Hunan, Hubei and Sichuan. No variation was detectable in the NADH dehydrogenase I sequence within populations from Zhejiang and Hubei, whereas sequence variation of 0.2% was detected within populations from Anhui, Jiangxi, Hunan and Sichuan. Pairwise comparison of the sequences representing the six different populations revealed genetic differences ranging from 0 to 0.6%.     

NAD^+与细胞衰老

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD^+)作为糖酵解、三羧酸循环和氧化磷酸化中关键酶的辅助因子,参与了细胞的物质代谢、能量合成、损伤DNA的修复等多种生理病理过程。近年来越来越多的研究发现,细胞内NAD^+水平在机体或细胞衰老过程中呈明显下降趋势,而补充NAD^+能延缓细胞/机体的衰老,使NAD^+及其前体物质在细胞衰老中的作用受到广泛关注。该文就NAD^+及其前体物质与细胞代谢、衰老的关系及相关分子机制研究的最新进展进行综述,以期深入认识NAD^+与细胞衰老的内在联系,为细胞衰老相关的基础及应用研究提供理论参考。