蚯蚓纤溶酶的分离纯化及部分序列的测定

以新鲜蚯蚓为原料,经过保温抽提、乙醇沉淀、DEAE-SepharoseFastFlow离子交换层析、Lysine-Sepharose4B亲和层析以及SDS-PAGE制备电泳等纯化步聚,得到一种纯度达95％以上的蚯蚓纤溶酶．该酶具有强烈的溶解纤维蛋白的作用及蛋白酶活性,平板法测得其比活性为90OUK单位／毫克蛋白,TAME法测得其比活性为2500O单位／毫克蛋白．酶学性质研究表明其最适反应温度为65℃,最适反应PH值为8．5．该酶的分子量为33kD,等电点为pH3．5．还对该酶进行了氨基酸组成分析,并测定了其N端部分序列．     

纤维素衍生物在固定酶和药物中的应用

本文综述了纤维素衍生物在固定酶和药物中的应用。     

Degradation of agarophytic red algal cell wall components by new crude enzyme preparations

Several new crude enzyme preparations were isolated from a marine association of the agarolytic bacterium Cytophaga diffluens and the infusorium Uronema marinum, an axenic culture of Cytophaga diffluens, some species of land micro- and macromycetes adapted to assimilate red algal biomass and from the marine mollusc Littorina littorea. Fungal and mollusc enzyme preparations were shown to have cellulase, xylanase, protease and agarase activities. Fungal agarase activity was revealed only after 3–4 passages of the culture on the medium containing algal biomass. Enzyme preparations from the association and the pure bacterial culture growing on the medium with bactoagar as the sole carbon source contained only agarase activity. The maximum specific agarase activity was found in a preparation from the marine association. The preparations obtained can be used for isolating protoplasts and single cells from red seaweed thalli. This revised version was published online in June 2006 with corrections to the Cover Date.     

利用mtCOI PCR-RFLP技术鉴定中国境内九个烟粉虱隐种

烟粉虱Bemisia tabaci (Gennadius)是一个物种复合体, 包括31个以上形态上无法区分的隐种, 其中少数隐种是世界性入侵害虫。目前, 在中国境内分布有2个入侵隐种和13个土著隐种。快速、 高效的鉴别方法对掌握烟粉虱田间发生规律及制定相关防控策略具有重要意义。然而到目前为止, 除了mtCOI基因测序比对外, 尚未有一种简便的方法可以有效地区分多个烟粉虱隐种。本研究采用mtCOI PCR-RFLP技术, 单独或组合使用TaqI, VspI, Van91I, NcoI和FokI这5种限制性内切酶, 酶切烟粉虱mtCOI的PCR扩增片段, 鉴别分布在中国境内的9个烟粉虱隐种。结果表明, 单独使用TaqI酶切, 可鉴别出MEAM1和China 1两个隐种, 使用TaqI+NcoI分步酶切可鉴定出MED和Asia 1两个隐种, 使用TaqI+Van91I分步酶切可鉴定出Asia II 3和Asia II 9两个隐种, 使用TaqI+VspI+FokI分步酶切可鉴定出Asia II 1, Asia II 6和Asia II 7 3个隐种。本研究为高效鉴别中国境内的多个烟粉虱隐种提供了方法。     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Effect of ammonium-N on malic enzyme and lipid production in Rhodotorula glutinis grown on monosodium glutamate wastewater

Rhodotorula glutinis, an oil producing strain, can utilize monosodium glutamate (MSG) wastewater as a raw material for lipid production. The effects of ammonium-N in the MSG wastewater (ammonium 15,000–25,000?mg/L, COD 30,000–50,000?mg/L) on cell growth, lipid accumulation and malic enzyme activity of R. glutinis have been studied. Four initial ammonium sulfate concentrations in the medium were set, which were 20, 60, 100, and 140?g/L. With an increase in the ammonium sulfate concentration, the uptake of ammonia nitrogen and lipid accumulation increased while the biomass decreased at 72?h. The maximum value of ammonia nitrogen consumption reached 5.77?g/L for an initial ammonium sulfate concentration of 140?g/L at 72?h. In addition, 60?g/L ammonium sulfate concentration may be an appropriate concentration for R. glutinis cultivation. The activity of the malic enzyme was measured and the results showed that there was a linear relationship between the intracellular lipid content and the total malic enzyme activity.     

Activation of Nrf2/Keap1 signaling and autophagy induction against oxidative stress in heart in iron deficiency

We investigated the effects of dietary iron deficiency on the redox system in the heart. Dietary iron deficiency increased heart weight and accumulation of carbonylated proteins. However, expression levels of heme oxygenase-1 and LC3-II, an antioxidant enzyme and an autophagic marker, respectively, in iron-deficient mice were upregulated compared to the control group, resulting in a surrogate phenomenon against oxidative stress.     

Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.