The Maturation of Oocyte Follicular Epithelium of Podarcis s. sicula Is Promoted by d-Aspartic Acid

We investigated whether the maturation of oocyte follicular epithelium of lizard is affected by d-aspartic acid (d-Asp). Our results demonstrated that d-Asp is endogenously present in the oocytes, and its distribution varies during the reproductive cycle and following intraperitoneal administration. At previtellogenesis, it is observed in the cytoplasm and nucleus of pyriform cells, in intermediate cells, in some small cells of the granulosa, in the ooplasm, and in some thecal elements. At vitellogenesis, d-Asp is localized in the proximity of the zona pellucida, in the theca, and in the ooplasm. Injected d-Asp is mainly captured by pyriform cells and ooplasm of previtellogenic oocytes, but a moderate accumulation is evident in the cytoplasm of some small granulosa cells and in the theca. d-Asp also increases the ovarian and plasmatic levels of 17β-estradiol and decreases those of testosterone. As a direct and/or indirect consequence of d-Asp, previtellogenic oocytes grow up and mature, resulting in a higher accumulation of carbohydrates in the granulosa, zona pellucida, and ooplasm, but also a reduction in the thickness of the granulosa layer and an increase of the theca stratum. Taken together, our results show that d-Asp may be related to the synchrony of reproduction, either enhancing the growth and maturation of follicular epithelium or influencing its endocrine functions. (J Histochem Cytochem 58:157–171, 2010)     

Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.     

Differences in Anionic Dependence of the Synaptic Efflux of d-Aspartic Acid and γ-Aminobutyric Acid

The synaptosomal effluxes of D-aspartate and gamma-aminobutyric acid (GABA) induced by a substitution of the Cl- ions with propionate in the incubation medium were measured in preparations of hippocampal tissue homogenates. The efflux of aspartate was significantly higher than spontaneous efflux at 125 mM Cl- (normal = 144 mM) and was increased with decreasing Cl- concentration. GABA efflux was much less sensitive to a reduction in Cl- concentration than D-aspartate. The efflux was Ca2+-dependent in both cases.     

Determination Method for l-Ascorbic Acid in Foods with Immobilized Ascorbate Oxidase

Pumpkin (Cucurbita moschata) ascorbate oxidase was entrapped within 6% (w/v) Ca-alginate gel beads, and then the beads were treated with 1% (w/v) glutaraldehyde for 20 hr at 4°C. The immobilized ascorbate oxidase was much more stable than the free form. Under the optimum conditions, the immobilized enzyme remained fully active for 3 months and after 50 assays. A linear relationship was found between immobilized ascorbate oxidase activity and l-ascorbic acid concentration in the range of 2 ~ 20 μg/ml. The immobilized preparation could be employed for the simple and rapid determination of l-ascorbic acid in foods.     

Sterilization by Means of Hydrochloric Acid Vapour

As heat sterilization of glass bottles often results in breakage and of most plastic containers in deformation, we investigated a rapid low-temperature sterilization method as an alternative. Vapour evolving from hydrochloric acid was chosen because it does not leave toxic residues which might contaminate food packed in treated containers. Vapour evolving from 0.25 ml of 31% (w/w) hydrochloric acid reduced the number of viable spores of aerobic and anaerobic bacteria in bottles (300 ml) by a factor of at least 2500 and that from 0.01 ml of 37% (w/w) hydrochloric acid reduced the number of mould spores by a factor of over 40 000 within 5 min at 20°C.     

D- 型氨基酸氧化酶抑制剂的发展

D-型氨基酸氧化酶(D-Amino acid oxidase,DAAO)抑制剂可以阻止D-型氨基酸(主要是D-型丝氨酸)的降解和过氧化氢的生成,在治疗精神分裂症阴性症状和认知障碍与镇痛等方面均表现出较好的疗效。从第一个DAAO抑制剂芳香羧酸类的苯甲酸到经过烯醇互变的α-羟基酮喹类抑制剂喹诺林-2,3-二酮,DAAO抑制剂结构上总共经历了3代变化,抑制剂与酶之间的相互作用模式逐渐加强,其抑制活性升高了数万倍,脂溶性增加,酸性减弱,理化性质逐渐优化。本文就近10年DAAO抑制剂的结构发展与生物活性之间的关系进行综述。     

测定芦荟素和芦荟大黄素的硼砂光度法

利用硼砂与芦荟中两种主要有效成分的不同作用将可见分光光度法和荧光分光光度法结合提出了测定芦荟素和芦荟大黄素含量的新方法.芦荟素的测定下限为0.00004g/L;芦荟大黄素的测定下限为0.0005g/L.此方法重复性好,样品分析结果相对标准偏差小于7%,加标回收率92～110%.     

光和底物对乙醇酸氧化酶的基因表达的诱导

从菠菜中提纯了乙醇酸氧化酶并制备其抗体,经免疫双扩散、Ｗｅｓｔｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ证实水稻和豌豆黄化苗中不存在乙醇酸氧化酶。在黑暗中,底物可促进该酶基因的表达,而在黄化苗光照初期,推测光可能是不经过底物促进该酶基因的表达。     

D-氨基酸氧化酶研究进展

D-氨基酸氧化酶(D-amino acid oxidase:oxidoreductase, DAAO, EC 1.4.3.3)是一种以黄素腺嘌呤(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸的氨基生成相应的酮酸和氨。在体内D-氨基酸的代谢中起着重要作用。主要介绍了D-氨基酸氧化酶的生理功能和应用、表达条件优化及通过定点突变对酶学性质的研究。     

水杨酸、乙酰水杨酸对番茄幼苗叶片中PPO和POD的诱导作用

以番茄品种改良美国908和合作906为试验材料,研究了水杨酸(SA)和乙酰水杨酸(ASA)喷雾处理6-7叶期幼苗后,叶片内多酚氧化酶(PPO)和过氧化物酶(POD)活性在120 h内的变化.结果显示:SA和ASA对2个番茄品种的适宜诱导浓度分别为1 mmol/L和1.39 mmol/L;SA和ASA对PPO活性的诱导效果无显著差异,但对POD活性的诱导效果SA极显著强于ASA;合作906的PPO活性增幅显著大于改良美国908,而POD活性增幅却显著小于改良美国908,且合作906对诱导处理反应更敏感.研究表明,SA和ASA能显著提高番茄幼苗叶片的PPO、POD活性,而酶活性变化在品种和诱导剂间有显著差异.     

Modification of Tobacco Leaf Glycolate Oxidase Activity by Chlorogenic Acid and Other Polyphenols

Glycolate oxidase extracted from tobacco leaves (Nicotiana tabacum L. cv. NC-95) and assayed by the 2,6-dichlorophe-nolindophenol reduction method was stimulated by chlorogenic acid and other o-diphenols but not by p-diphenols such as hydroquinone. Chlorogenic acid also protected the enzyme against certain enzyme antagonists. A novel assay utilizing horseradish peroxidase with the chromogen o-dianisidine was developed for detecting glycolate oxidase in conjunction with disc electrophoresis. Dissociation of glycolate oxidase into an active monomer during ammonium sulfate fractiona-tion was confirmed electrophoretically. After electrophoresis, flavin mononucleotide was required for monomer activity whereas chlorogenic acid was inhibitory to enzyme band development.     

Ascorbic Acid Oxidase: An Enzyme in Search of a Role

Ascorbic acid oxidase (AAO) has been fully characterized at molecular level, yet its functional role is unclear. The properties of the enzyme and the main hypotheses on its function are discussed. Recent data and reappraisal of previous observations suggest that AAO could be part of a dynamic mechanism operating whenever plant cells have to control oxygen availability. This revised version was published online in July 2006 with corrections to the Cover Date.     

Determination of Copper Content in o-Diphenol Oxidase by Neutron Activation Analysis

A polyphenol oxidase (o-diphenol oxidase) [o-diphenol: O₂ oxidoreductase E. C. 1.10. 3.1] from sweet potato named component II_b was highly purified. The copper content of this enzyme was measured by neutron activation analysis. Samples were analyzed with or without chemical separation after neutron irradiation. The copper content of the enzyme was determined to be 0.27%, and the minimum molecular weight of this enzyme was caluculated to be 23,500.     

The Determination of Phaseic Acid by Monoclonal Antibody-Based Enzyme immunoassay

A monoclonal antibody PA3-2-B3, IgG₁ (Λ) is described which specifically recognizes phaseic acid and shows very little cross-reactivity (0.14%) with abscisic acid or dihydrophaseic acid (0.88%). Based on this antibody, an enzyme immunoassay was developed which displays a linearity range from 15 pg to 3 ng of phaseic acid. Results obtained with this assay agree with those obtained by gas chromatography-electron capture detection. Using the novel enzyme immunoassay, as well as an established immunoassay for abscisic acid, levels of these two compounds in leaves of Phaseolus vulgaris were determined as a function of plant age, water stress, recovery from stress, and feeding of abscisic acid through the transpiration stream. The production of phaseic acid in a microsomal system from bean leaves was demonstrated. The results show a regulation of the plant's capacity to metabolize abscisic acid to phaseic acid as a function of water stress.