鸭梨多酚氧化酶基因反义表达载体的构建及农杆菌介导的遗传转化

根据鸭梨多酚氧化酶基因序列设计引物,PCR扩增该基因3′端450bp的片段,并将该片段反向插入真核表达载体pB I121的CaMV 35S启动子和NOS终止子之间,首次构建了鸭梨PPO基因的反义表达载体;其后,在农杆菌EHA105的介导下,成功实现了PPO反义基因对鸭梨组培苗的遗传转化。经Northern杂交和酶活检测证实,转基因鸭梨植株体内的多酚氧化酶基因转录和翻译水平均得到明显抑制,从而为耐褐化梨新品种的培育奠定了基础。     

蕨菜多酚氧化酶的酶学性质

研究了蕨菜[Pteridium aquilinum (L．) Kuhn var．latiusculum (Desv．) Underw．]多酚氧化酶的动力学性质,结果表明：以邻苯二酚为底物,该酶最适pH为7．4,最适温度为25℃,60℃以上使酶迅速失活,动力学方程v=619．08[S]/(0．031 [S]),Vc、异Vc钠、NaHSO3、L-Cys可完全抑制酶活性,饱和NaCl能显著抑制酶活性,蔗糖、SDS对酶有激活作用。该酶能催化邻苯二酚、焦性没食子酸氧化,但对焦性没食子酸亲和力更强。     

Cross-linked agarose for separation of low molecular weight natural products in hydrophilic interaction liquid chromatography

Following its market introduction in 1982, the cross-linked 12% agarose gel media Superose 12 has become widely known as a tool for size exclusion chromatography of proteins and other biological macromolecules. In this review it is shown that, when appropriate mobile phases are used, Superose possesses adsorption properties similar to that of traditional media for hydrophilic interaction liquid chromatography (HILIC). This is illustrated by the separation and purification of low molecular weight compounds such as polyphenols including active components of traditional Chinese medicinal herbs and green tea. Structural features of the cross-linked agarose that likely cause the observed adsorption effects are discussed aswell. These are identified as being primarily ether bonds acting as strong hydrogen bond acceptors as well as hydrophobic residues originating from the cross-linking reagents.     

Identification of a potent xanthine oxidase inhibitor from oxidation of caffeic acid

Inhibitory activity of Fe-ion-catalyzed radical oxidation products from 22 types of phenolic compounds toward xanthine oxidase (XO) was investigated. Phenols are readily oxidizable compounds in nature and, thus, showed potent antioxidant activities. Among the phenols screened in this study, noticeable activity was observed in the oxidation product of caffeic acid, whereas almost no XO-inhibitory activity of caffeic acid was observed. Assay-guided purification of the oxidation product of caffeic acid afforded a highly potent XO inhibitor, with an IC₅₀ value that was calculated to be 60 nmol L⁻¹, which indicated XO-inhibitory activity much stronger than that of allopurinol (IC₅₀ = 1 μmol L⁻¹), a potent XO inhibitor and excellent medicine for the treatment of gout. The chemical structure of this new XO inhibitor was investigated by one- and two-dimensional NMR and HR–ESI–MS analyses, and the unique tetracyclic structure was confirmed by synthesis starting from commercially available 1,2,4-trimethoxybenzene and 3,4-dimethoxylbenzoyl chloride.     

香蕉芒果果皮中酶活性与炭疽病菌潜伏侵染的关系

在树上正生长发育的香蕉果实,随着果实长大,果皮中过氧化物酶(POD)和多酚氧化酶(PPO)活性不断升高。采后的香蕉和芒果果实的果皮中,POD和PPO活性变化与呼吸代谢相关。苯丙氨酸解氨酶(PAL)主要分布在果皮中,并随着果实成熟度的提高,其活性呈极显著下降趋势。感病果实的果皮中三种酶活性均比健康果皮高。说明果皮中三种酶活性的变化与炭疽菌的潜伏侵染有关。     

采后荔枝果皮色素、总酚及有关酶活性的变化

成熟度约八成的“淮枝”在采收后2天内,色素的合成代谢仍相当活跃,类胡萝卜素,花色素苷的含量增高,多酚氧化酶和苯丙氨酸解氨酶的活性增大,总酚保持原有水平,叶绿素降解,反映了果实达到完全成熟的特征,随后,上述各项参数因果实的衰老而下降,与采收当天相比,7天后花色素苷为90％,类黄酮为59％,总酚为71％,苯丙氨酸解氨酶活性为46％,讨论了酚类,花色素苷及两种酶活性之间的关系及对荔枝果实在成熟,衰老褐变中的作用。     

Potentiation of dietary restriction-induced lifespan extension by polyphenols

Dietary restriction (DR) extends lifespan across multiple species including mouse. Antioxidant plant extracts rich in polyphenols have also been shown to increase lifespan. We hypothesized that polyphenols might potentiate DR-induced lifespan extension. Twenty week old C57BL/6 mice were placed on one of three diets: continuous feeding (control), alternate day chow (Intermittent fed, IF), or IF supplemented with polyphenol antioxidants (PAO) from blueberry, pomegranate, and green tea extracts (IF + PAO). Both IF and IF + PAO groups outlived the control group and the IF + PAO group outlived the IF group (all p < 0.001). In the brain, IF induced the expression of inflammatory genes and p38 MAPK phosphorylation, while the addition of PAO reduced brain inflammatory gene expression and p38 MAPK phosphorylation. Our data indicate that while IF overall promotes longevity, some aspects of IF-induced stress may paradoxically lessen this effect. Polyphenol compounds, in turn, may potentiate IF-induced longevity by minimizing specific components of IF-induced cell stress.     

Degradation of procyanidins by Aspergillus fumigatus: identification of a novel aromatic ring cleavage product

Aspergillus fumigatus was able to grow on apple-purified procyanidins (PCs). PCs concentration decreased 30% over the first 60 h of liquid fermentation. The mean degree of polymerization (DPn) of apple-purified PCs increased from 8 to 15 during the fermentation. A fungal enzyme extract from the liquid fermentation was used to study procyanidin B2 [(-)-epicatechin-(4beta-8)-(-)-epicatechin] degradation. The major degradation product (PB2-X) had a retention time of 10.5 min and a molecular mass at m/z 609. High-performance liquid chromatography/multiple fragment mass spectrometry (HPLC/MS(n)) was used for the structural characterization of PB2-X as well as that of thiolysis-treated PB2-X. Twelve fragment ions at m/z 565, 547, 457, 439 (two fragment ions), 421, 413, 377, 395, 351, 287 and 277 were completely identified. It was therefore deduced that the terminal unit of procyanidin B2 dimer was modified by an oxygenase from A. fumigatus leaving the extension unit intact. In addition, FT-IR analysis confirmed a lactone formation in (-)-epicatechin moiety involved in oxidative degradation. Two reaction schemes were postulated for the interpretation of the results.     

Epinephrine quantification in pharmaceutical formulations utilizing plant tissue biosensors

A plant tissue biosensor associated with flow injection analysis is proposed to determine epinephrine in pharmaceutical samples. The polyphenol oxidase enzymes present in the fibers of a palm tree fruits (Livistona chinensis), catalyses the oxidation of epinephrine to epinephrinequinone as a primary product. This product is then electrochemically reduced (at −0.10 V versus Ag/AgCl_sat) on the biosensor surface and the resulting current is used for the quantification of epinephrine. The biosensor provides a linear response for epinephrine in the concentration range from 5.0 × 10⁻⁵ to 3.5 × 10⁻⁴ mol l⁻¹. The limit of detection estimated for this interval was 1.5 × 10⁻⁵ mol l⁻¹ and the correlation coefficient of 0.998, working under a flow rate of 2.0 ml min⁻¹ and using a sample loop of 100 μl. The repeatability (R.S.D. for 10 consecutive determinations of a 3.0 × 10⁻⁴ mol l⁻¹ epinephrine solution) was 3.1%. The results obtained by the method here proposed were compared with the official UV spectrophotometric procedure and also using a plant tissue reactor. The responses obtained with the proposed strategies were in good agreement with both ways of analyses, whereas the values obtained by the official spectrophotometric method was strongly affected by benzoic acid, present in the formulation of pharmaceutical product utilized for inhalation. Such favorable results obtained with the carbon paste biosensor or utilizing the bioreactor, joined with the simplicity of its preparation turns these procedures very attractive for epinephrine quantification in pharmaceutical products.     

Changes in gas exchange, proline accumulation and antioxidative enzyme activities in three olive cultivars under contrasting water availability regimes

Changes in photosynthetic performance, osmolyte accumulation and the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and polyphenol oxidase (PPO) were investigated in one-year-old olive cultivars (Chemlali, Meski and Picholine) subjected to contrasting water availability regimes under arid climatic conditions in Tunisia. Shoot elongation rates (SER) and photosynthetic performance were markedly reduced by the water deficit regime (WD) in all cultivars except for Chemlali, which proved to be superior to the other two cultivars with respect to drought tolerance. Higher photosynthetic performance (net photosynthesis (P_n), stomatal conductance (g_s) and transpiration rates (E)) in the Chemlali and Meski cvs. compared to Picholine olive allowed them to maintain better plant water status and shoot elongation rates. Under WD conditions, Chemlali showed a greater capability for proline accumulation. Leaves grown under WD conditions showed signs of oxidative stress such as reduced chlorophyll and carotenoid concentrations. Nevertheless, different cultivars developed certain antioxidative defense mechanisms, including elevated SOD, APX and CAT activities. In contrast, PPO activity decreased under WD circumstances. Comparatively, Chemlali olive displayed better antioxidative enzyme activity, and thus better protection against oxidative stress. These results show that the ability of olive trees to up-regulate the enzymatic antioxidative system might be an important attribute linked to drought tolerance. These findings demonstrate that the association of higher P_n, proline accumulation and antioxidative defenses could be effective in a water-limited environment and may be useful selection criteria in breeding programs with the objective of improving drought tolerance and growth of olive trees, at least under the described environmental conditions.