Stepwise sRNA targeting of structured bacterial mRNAs leads to abortive annealing

1. Download : Download high-res image (137KB)
2. Download : Download full-size image

     

Desferrioxamine as an Electron Donor. Inhibition of Membranal Lipid Peroxidation Initiated by H202 –Activated Metmyoglobin and Other Peroxidizing Systems

Desferoxamine (DFO) involvement in several peroxidative systems was studied. These sytems included: a) membranal lipid peroxidation initiated by H₂O₂-activated metmyoglobin (or methemoglobin); b) phenol-red oxidation by activated metmyoglobin or horseradish peroxidase (HRP): c) β-carotene-linoleate couple oxidation stimulated by lipoxygenase or hemin. Desferrioxamine was found to inhibit all these systems but not ferrioxamine (FO). Phenol-red oxidation by H₂0₂-horseradish peroxidase was inhibited competitively with DFO. Kinetic studies using the spectra changes in the Soret region of metmyoglobin suggest a mechanism by which H₂0₂ reacts with the iron-heme to form an intermediate of oxy-ferryl myoglobin that subsequently reacts with DFO to return the activated compound to the resting state. These activities of DFO resemble the reaction of other electron donors.     

Determination of the pH dependence,substrate specificity,and turnovers of alternative substrates for human ornithine aminotransferase

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

Interaction between cadmium, zinc and silver-substituted plastocyanin and cytochrome b 6 f complex — heavy metals toxicity towards photosynthetic apparatus

This paper reports the results of research on the interaction between the cytochrome f of the active cytochrome b ₆ f complex (incubated with Cd-, Zn-, and Ag-substituted plastocyanins) and Cu-plastocyanin. The presented studies show, that the metal derivatives of plastocyanin can have an influence on the photosynthetic electron transfer path: cytochrome b ₆ f complex — photosystem I. The metal-substituted plastocyanins occupy the plastocyanin electron transfer site of the cytochrome f. The stopped-flow measurements show, that although the metal derivatives of plastocyanin do not influence the rate of cyt f- Pc electron transfer, creation of the non-electron-transfer complexes characterised by a strong binding between the cyt f and substituted plastocyanins and their slow release, dependent on the redox state of the substituted metal, results in the decrease of a turnover of the cytochrome complex. The research was done in the Department of Plant Biochemistry, Freiburg University, Sch?nzlestrasse 1, 79 104 Freiburg, Germany     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Monitoring Migration and Measuring Biomass in Benthic Biofilms: The Effects of Dark/far-red Adaptation and Vertical Migration on Fluorescence Measurements

Pulse modulated fluorescence has increasingly been used as an ecological tool to examine changes in the vertical distribution of microphytobenthic cells within the upper layers of estuarine sediments (most often using the minimum fluorescence yield F(o)) as well as to indicate the health of the community (using the maximum PS II quantum efficiency F(v)/F(m)). However, the practicalities of in situ measurements, often dictates that short dark adaptation periods must be used ( approximately 15 min). The use of far-red light as an alternative to dark adaptation was investigated in natural migratory microphytobenthic biofilms and artificial non-migratory biofilms. Prolonged periods of darkness ( approximately 24 h) were not adequate to achieve 'true' measurements of F(o) and F(v)/F(m), which require complete oxidation of Q(A) and full reversal of non-photochemical quenching (NPQ). In some instances, stable values were only achieved using far-red light. Prolonged exposure to dark/far-red light led to a downwards migration of cells in natural assemblages, as seen by a reduction in both F(o) and the maximum fluorescence yield (F(m)). In non-migratory biofilms, F(m) increased in the dark and far-red treatments, indicating a reversal of NPQ, whereas F(o) decreased in far-red light but increased in the dark. It is suggested that far-red light and darkness differentially affected the balance between NPQ reversal and Q(A) oxidation that lead to the measured F(o) yield. The use of far-red light as an alternative to dark adaptation is discussed and the implications of short (e.g., 15 min) dark adaptation times used in situ are discussed with reference to the vertical migration of cells within sediment biofilms.     

Modified α-amylase activity among insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais

Fitness cost is usually associated with insecticide resistance and may be mitigated by increased energy accumulation and mobilization. Preliminary evidence in the maize weevil (Coleoptera: Curculionidae) suggested possible involvement of amylases in such phenomenon. Therefore, α-amylases were purified from an insecticide-susceptible and two insecticide-resistant strains (one with fitness cost [resistant cost strain], and the other without it [resistant no-cost strain]). The main α-amylase of each strain was purified by glycogen precipitation and ion-exchange chromatography (≥70-fold purification, ≤19% yield). Single α-amylase bands with the same molecular mass (53.7 kDa) were revealed for each insect strain. Higher activity was obtained at 35-40 °C and at pH 5.0-7.0 for all of the strains. The α-amylase from the resistant no-cost strain exhibited higher activity towards starch and lower inhibition by acarbose and wheat amylase inhibitors. Opposite results were observed for the α-amylase from the resistant cost strain. Although the α-amylase from the resistant cost strain exhibited higher affinity to starch (i.e., lower K_m), its V_max-value was the lowest among the strains, particularly the resistant no-cost strain. Such results provide support for the hypothesis that enhanced α-amylase activity may be playing a major role in mitigating fitness costs associated with insecticide resistance.     

生物膜法强化净化氨氮污染水体及其微生物群落解析

【目的】比较不同营养条件及挂膜方式下生物膜法对氨氮污染水体的净化效果及其功能微生物群落结构。【方法】设置空白(Blank)、自然成膜(Raw)、预附脱氮菌强化挂膜(PCC)3组生物膜反应器,利用末端限制性片段长度多态性(T-RFLP)技术和非度量多维标度(NMDS)分析方法对生物膜反应器转化氨氮过程中微生物群落结构及其演替过程进行动态解析。【结果】在C/N=1:1时,除PCC在起始阶段短暂具有较高的氨氮脱除效率外,Blank、Raw和PCC最终均表现出较低的氨氮转化效率(10%-20%)。改变C/N=2:1后,Raw和PCC对人工合成污水中NH4+-N的转化率均提高至95%以上,而且Raw与PCC的群落结构在C/N=2:1时具有较高的相似性,优势菌群主要为γ-变形菌纲(Gammaproteobacteria)、放线菌纲(Actinobacteria)和硝化螺菌纲(Nitrospira)。【结论】C/N是影响生物膜反应器氨氮去除效果及驱动生物膜反应器中细菌群落结构发生改变的重要因子。