Functional characterization and FTIR-based 3D modeling of full length and truncated forms of Scorpio maurus venom phospholipase A2

Background

Heterodimeric phospholipase A₂ from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity.

Growth media in anaerobic fermentative processes: The underestimated potential of thermophilic fermentation and anaerobic digestion

Fermentation and anaerobic digestion of organic waste and wastewater is broadly studied and applied. Despite widely available results and data for these processes, comparison of the generated results in literature is difficult. Not only due to the used variety of process conditions, but also because of the many different growth media that are used. Composition of growth media can influence biogas production (rates) and lead to process instability during anaerobic digestion. To be able to compare results of the different studies reported, and to ensure nutrient limitation is not influencing observations ascribed to process dynamics and/or reaction kinetics, a standard protocol for creating a defined growth medium for anaerobic digestion and mixed culture fermentation is proposed. This paper explains the role(s) of the different macro- and micronutrients, as well as the choices for a growth medium formulation strategy. In addition, the differences in nutrient requirements between mesophilic and thermophilic systems are discussed as well as the importance of specific trace metals regarding specific conversion routes and the possible supplementary requirement of vitamins. The paper will also give some insight into the bio-availability and toxicity of trace metals. A remarkable finding is that mesophilic and thermophilic enzymes are quite comparable at their optimum temperatures. This has consequences for the trace metal requirements of thermophiles under certain conditions. Under non-limiting conditions, the trace metal requirement of thermophilic systems is about 3 times higher than for mesophilic systems.     

Hemin-incorporated nanoflowers as enzyme mimics for colorimetric detection of foodborne pathogenic bacteria

Rapid, sensitive and point-of-care detection of foodborne pathogenic bacteria is essential for food safety. In this study, we found that hemin-concanavalin A hybrid nanoflowers (HCH nanoflowers), as solid mimic peroxidase, could catalyze oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of H₂O₂ to a green-colored product. HCH nanoflowers, integrating the essential functions of both biological recognition and signal amplification, meet the requirements of signal labels for colorimetric immunoassay of bacteria. In view of the excellent peroxidase mimetic catalytic activity of HCH nanoflowers, a colorimetric biosensing platform was newly constructed and applied for sensitive detection of foodborne Escherichia coli O157:H7 (E. coli O157:H7). The corresponding detection limits was as low as 4.1?CFU/mL with wide linear ranges (10¹–10⁶?CFU/mL).     

Optimized allosteric inhibition of engineered protein tyrosine phosphatases with an expanded palette of biarsenical small molecules

Protein tyrosine phosphatases (PTPs), which catalyze the dephosphorylation of phosphotyrosine in protein substrates, are important cell-signaling regulators, as well as potential drug targets for a range of human diseases. Chemical tools for selectively targeting the activities of individual PTPs would help to elucidate PTP signaling roles and potentially expedite the validation of PTPs as therapeutic targets. We have recently reported a novel strategy for the design of non-natural allosteric-inhibition sites in PTPs, in which a tricysteine moiety is engineered within the PTP catalytic domain at a conserved location outside of the active site. Introduction of the tricysteine motif, which does not exist in any wild-type PTP, serves to sensitize target PTPs to inhibition by a biarsenical compound, providing a generalizable strategy for the generation of allosterically sensitized (as) PTPs. Here we show that the potency, selectivity, and kinetics of asPTP inhibition can be significantly improved by exploring the inhibitory action of a range of biarsenical compounds that differ in interarsenical distance, steric bulk, and electronic structure. By investigating the inhibitor sensitivities of five asPTPs from four different subfamilies, we have found that asPTP catalytic domains can be broadly divided into two groups: one that is most potently inhibited by biarsenical compounds with large interarsenical distances, such as AsCy3-EDT₂, and one that is most potently inhibited by compounds with relatively small interarsenical distances, such as FlAsH-EDT₂. Moreover, we show that a tetrachlorinated derivative of FlAsH-EDT₂, Cl4FlAsH-EDT₂, targets asPTPs significantly more potently than the parent compound, both in vitro and in asPTP-expressing cells. Our results show that biarsenicals with altered interarsenical distances and electronic properties are important tools for optimizing the control of asPTP activity and, more broadly, suggest that diversification of biarsenical libraries can serve to increase the efficacy of these compounds in targeted control of protein function.     

鸳鸯茉莉花色变化过程中的生理生化特性研究

以3年生鸳鸯茉莉(Brunfelsia acuminata Benth.)5个不同花期(花蕾期、初开期、全开期、过渡期、末花期)的花瓣为试材,研究其开花过程中花瓣的色度值、类黄酮、类胡萝卜素、花色苷含量、细胞p H值以及相关酶活性的变化,并分析各指标之间的相关性。结果显示,除全开期外,其它4个时期各色度值变化不明显,初开期、全开期和过渡期,明亮参数L值逐渐增大,红绿参数a值、黄蓝参数b值和彩度C值急剧降低;类黄酮含量在过渡期最低,其它阶段无显著差异;类胡萝卜素含量变化呈上升-下降-稳定的趋势,全开期达到最高;花色苷含量则随开花进程不断下降,花蕾期最高;花蕾期、初开期、全开期的SOD活性变化不显著,过渡期时显著升高,末花期又迅速回落到最低值;POD活性不断上升,末花期为花蕾期的28.73倍;CAT活性前期变化不显著,末花期时有一定幅度的升高;花瓣色度各指标、花色苷含量、上述酶的活性、p H值等几个参数间均存在极显著相关性,但SOD活性、类胡萝卜素、类黄酮与所有参数的相关性均不显著。研究结果表明花色苷是影响鸳鸯茉莉花色变化的决定性代谢产物,POD和CAT活性是调节花色苷降解速率的主要抗氧化酶。     

Immunocytochemical localization of enzymes involved in the C3 and C4 pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara

Eleocharis vivipara link, an amphibious leafless sedge, develops traits of C₄ photosynthesis and Kranz anatomy in the terrestrial form but develops C₃-like traits with non-Kranz anatomy when submerged. The cellular localization of C₃ and C₄ enzymes in the photosynthetic cells of the two forms was investigated by immunogold labeling and electron microscopy. The terrestrial form has mesophyll cells and three kinds of bundle sheath cell, namely, parenchyma sheath cells, non-chlorophyllous mestome sheath cells, and Kranz cells. Phosphoenol-pyruvate carboxylase (PEPCase) was present in the cytosol of both the mesophyll cells and the parenchyma sheath cells, with higher-density labeling in the latter, but not in the Kranz cells. Pyruvate, Pi dikinase (PPDK) was found at high levels in the chloroplasts of both the mesophyll cells and the parenchyma sheath cells with some-what stronger labeling in the latter. This enzyme was also absent from the Kranz cells. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found in the chloroplasts of all types of photosynthetic cell, but labeling was significantly less intense in the parenchyma sheath cells than in other types of cell. The submerged form also has three types of photosynthetic cell, as well as non-chlorophyllous mestome sheath cells, but it lacks the traits of Kranz anatomy as a consequence of modification of the cells. Rubisco was densely distributed in the chloroplasts of all the photosynthetic cells. However, PEPCase and PPDK were found in both the mesophyll cells and the parenchyma sheath cells but at lower levels than in the terrestrial form. These data reveal that the terrestrial form has a unique pattern of cellular localization of C₃ and C₄ enzymes, and they suggest that this pattern and the changes in the extent of accumulation of the various enzymes are the main factors responsible for the difference in photosynthetic traits between the two forms.Abbreviations CAM crassulacean acid metabolism - MC meso phyll cell - PSC parenchyma sheath cell - KC Kranz cell - PEP-Case phosphoenolpyruvate carboxylase - PPDK pyruvate, Pi dikinase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - LS large subunit - RuBP ribulose-1,5-bisphosphate This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology). The author is grateful to Drs M. Matsuoka and S. Muto for providing the antisera and Dr. M. Samejima for his advice at the early stages of this study.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Alteration of Phospholipase C-δ Protein Level and Specific Activity in Alzheimer's Disease

Abstract: Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. We have previously demonstrated that an antibody to an isozyme of PLC, PLC-δ, produced intense staining of neurofibrillary tangles in the brains of patients with Alzheimer's disease. In the present study, we investigated the protein level and activity of this enzyme in control and Alzheimer brains. Western blot analysis using a specific antibody for PLC-δ showed that the concentration of PLC-δ protein was significantly higher in the cytosolic fraction of Alzheimer's disease cortical tissue than in control brains. The activity of PLC-δ, which hydrolyzes phosphatidylinositol, was also investigated, and we found that PLC-δ activity was not significantly different in the Alzheimer and control cytosolic fractions. These results indicate that the specific activity of PLC-δ is decreased in Alzheimer brains and suggest that inactivation of PLC-δ might be related to the pathophysiology of this disease.     

凤眼莲、水花生若干光合作用参数与酶类的研究

在正常的生长条件下,凤眼莲有较高的叶面系数、叶绿素含量和光合强度。叶片可溶性糖含量也高。在低温胁迫下,凤眼莲和水花生根系活力和过氧化物酶、过氧化氢酶、细胞色素氧化酶、淀粉酶等酶活性均明显下降。试验结果表明,凤眼莲对低温胁迫较水花生更为敏感。