Advantages of 2'-O-methyl oligoribonucleotide probes for detecting RNA targets.

We have compared various kinetic and melting properties of oligoribonucleotide probes containing 2'-O-methylnucleotides or 2'-deoxynucleotides with regard to their use in assays for the detection of nucleic acid targets. 2'-O-Methyl oligoribonucleotide probes bound to RNA targets faster and with much higher melting temperatures (Tm values) than corresponding 2'-deoxy oligoribonucleotide probes at all lengths tested (8-26 bases). Tm values of both probes increased with length up to approximately 19 bases, with maximal differences in Tm between 2'-O-methyl and 2'-deoxy oligoribonucleotide probes observed at lengths of 16 bases or less. In contrast to RNA targets, 2'-O-methyl oligoribonucleotide probes bound more slowly and with the same Tm to DNA targets as corresponding 2'-deoxy oligoribonucleotide probes. Because of their greatly enhanced Tm when bound to RNA, 2'-O-methyl oligoribonucleotide probes can efficiently bind to double-stranded regions of structured RNA molecules. A 17 base 2'-O-methyl oligoribonucleotide probe was able to bind a double-stranded region of rRNA whereas the same 17 base 2'- deoxy oligoribonucleotide probe did not. Due to their enhanced Tm when bound to RNA targets, shorter 2'-O-methyl oligoribonucleotide probes can be used in assays in place of longer 2'-deoxy oligoribonucleotide probes, resulting in enhanced discrimination between matched and mismatched RNA targets. A 12 base 2'-O-methyl oligoribonucleotide probe had the same Tm as a 19 base 2'-deoxy oligoribonucleotide probe when bound to a matched RNA target but exhibited a much larger decrease in Tm than the 2'-deoxy oligoribonucleotide probe when bound to an RNA target containing either 1 or 2 mismatched bases. The increased Tm, faster kinetics of hybridization, ability to bind to structured targets and increased specificity of 2'-O-methyl oligoribonucleotide probes render them superior to corresponding 2'-deoxy oligoribonucleotides for use in assays that detect RNA targets.     

Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization.

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain two mismatches between the two heat-stable enterotoxins. Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled. After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C. Results were consistent with suckling-mouse tests for heat-stable enterotoxins. A stronger signal was obtained on paper filters than on nitrocellulose filters. Enterotoxigenic E. coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E. coli cells. This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.     

Analysis of local order in the spatial distribution of cell surface molecular assemblies.

Electron micrographs obtained from platinum-carbon replicas of freeze-cleaved or freeze-dried cells and exhibiting intramembranous or externally-disposed particles were analysed statistically for degree of non-random particle association. Analysis was based on use of an analogy to the radial distribution function, g(r), which provided the average density of neighbor particles around a particle in the micrograph at 30 Å increments from particle center. The ratio of average density of neighbors around a particle to overall particle density in the micrograph was then determined as a function of distance from particle center. For a random plot of 90 Å particles, the computed ratio deviated from 1 by less than 10% for a distance up to 900 Å from particle center. In a field of similarly-sized intramembranous particles within the freeze-cleaved surface membrane of a mouse fibroblast, there were indications of slight non-random particle associations at distances of 120–240 Å and 300–330 Å from particle center, while a significant association of particles comprising an intercellular gap junction was obtained at 90–120 Å. Virus-related molecular assemblies on the surfaces of fibroblasts infected with a temperature-sensitive mutant of murine luekemia virus and grown under conditions that did not permit virus assembly to occur were analysed similarly. Significant deviation from randomness was found at several distances from particle center, both on areas of membrane that were populated by large numbers of particles and on low-density areas.     

Unique oxidation of imidazolidine nitroxides by potassium ferricyanide: strategy for designing paramagnetic probes with enhanced sensitivity to oxidative stress

Potassium ferricyanide (PF), routinely employed for the oxidation of sterically-hindered hydroxylamines to nitroxides, is considered to be chemically inert towards the latter. In the present study, we report on an unexpected oxidative fragmentation of the imidazolidine nitroxides containing hydrogen atom in the 4-position of the heterocycle (HIMD) by PF resulting in the loss of the EPR signal. The mechanistic EPR, spectrophotometric, electrochemical and HPLC-MS studies support the assumption that the HIMD fragmentation is facilitated by the proton abstraction from the 4-position of the oxoammonium cation formed as a result of the initial one-electron HIMD oxidation. Increase in steric hindrance around the radical fragment by introducing ethyl substituents decreased the rate of ascorbate-induced HIMD reduction by more than 20 times, but did not affect the rate of ferricyanide-induced HIMD oxidation. This preferential sensitivity of HIMDs to oxidative processes has been used to detect peroxyl radicals in the presence of high concentration of the reducing agent, ascorbate. HIMD-based EPR probes capable to discriminate oxidative and reductive processes might find application in biomedicine and related fields for monitoring the oxidative stress and reactive radical species in biological systems.     

Unique oxidation of imidazolidine nitroxides by potassium ferricyanide: Strategy for designing paramagnetic probes with enhanced sensitivity to oxidative stress

Abstract

Potassium ferricyanide (PF), routinely employed for the oxidation of sterically-hindered hydroxylamines to nitroxides, is considered to be chemically inert towards the latter. In the present study, we report on an unexpected oxidative fragmentation of the imidazolidine nitroxides containing hydrogen atom in the 4-position of the heterocycle (HIMD) by PF resulting in the loss of the EPR signal. The mechanistic EPR, spectrophotometric, electrochemical and HPLC–MS studies support the assumption that the HIMD fragmentation is facilitated by the proton abstraction from the 4-position of the oxoammonium cation formed as a result of the initial one-electron HIMD oxidation. Increase in steric hindrance around the radical fragment by introducing ethyl substituents decreased the rate of ascorbate-induced HIMD reduction by more than 20 times, but did not affect the rate of ferricyanide-induced HIMD oxidation. This preferential sensitivity of HIMDs to oxidative processes has been used to detect peroxyl radicals in the presence of high concentration of the reducing agent, ascorbate. HIMD-based EPR probes capable to discriminate oxidative and reductive processes might find application in biomedicine and related fields for monitoring the oxidative stress and reactive radical species in biological systems.     

The reduction of trimethylarsine oxide to trimethylarsine by thiols: a mechanistic model for the biological reduction of arsenicals

Trimethylarsine oxide is reduced to trimethylarsine in aqueous solution by a variety of thiols and dithiols including cysteine, glutathione, and lipoic acid. Kinetic results and other observations suggest that the rate-determining step is the production of [Me₃AsSR]⁺ from an initially formed Me₃As(SR)OH species, and that the reduction occurs via a two-electron transfer from Me₃As(SR)₂ affording Me₃As and RS-SR. A simple model for the biological methylation of arsenic is proposed based on oxidative methylation of arsenic(III) by S-adenosylmethionine and reduction by a thiol such as lipoic acid.     

玉米秸秆和异Vc钠生产废液固态发酵生产高蛋白饲料研究

以玉米秸秆为原料,以麸皮和异Vc钠生产废液(WEP)为辅料进行生物蛋白饲料固态发酵研究.通过菌种配伍试验,确定了混菌发酵菌种为白地霉、产朊假丝酵母和枯草芽孢杆菌.在此基础上通过单因素优化试验确定了秸秆蛋白饲料的最优发酵条件:以玉米秸秆(5 g)和麸皮(1 g)为基料,4%WEP营养液,固液比1:4(g/mL),初始pH值4.5;以麸皮浸汁作种子培养液,种龄24 h,各菌接种比例为产朊假丝酵母∶白地霉∶枯草芽孢杆菌=3:1:1,接种量2 mL;28℃、静置发酵2 d,在此条件下,秸秆饲料中真蛋白含量为6.21%,比对照提高了23.95%.该研究为秸秆和异Vc钠生产废液的高质化利用提供了新的思路和途径.     

Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes.

A rapid method for the identification of bacterial cells using 16S rRNA-directed, fluorescently tagged oligonucleotide probes has been developed. The parameters evaluated for their effect on labeling intensity included storage time, type of fixative, time of fixation, treatment time with methanol:formaldehyde and treatment time with borohydride. The results of tests using a variety of microorganisms, both Gram-positive and Gram-negative, are presented. Using this method, cells are spotted onto slides and stored desiccated until hybridized. This method may be especially applicable to environmental samples, which comprise diverse cell types and frequently require storage prior to examination.     

Dual fluorophore-nitroxide probes for analysis of vitamin C in biological liquids

A new method for quantitative analysis of vitamin C in biological and chemical liquids was proposed. The method is based on the use of dual molecule consisting of a fluorescent chromophore and a nitroxide radical. In the dual molecule, the nitroxide acts as a quencher of the fluorescence of the chromophore fragment. Reduction of the nitroxide fragment by ascorbic acid results in decay of ESR signal and enhancement of the fluorescence. By performing the series of pseudo-first-order reactions between the dual molecule and ascorbic acid and consequent plotting rate constants versus ascorbic acid concentrations the calibration curves for the vitamin C analysis were obtained. Variations of chemical structure of fluorophore and nitroxide fragments allow to regulate fluorescent properties and redox potentials of the dual molecules. The proposed fluorophore-nitroxide hybrids retain all features of the spin labels and fluorescence probes gaining new advantages for monitoring redox reactions and radical processes by two independent techniques: ESR and steady-state fluorescent spectroscopy. The method was applied to the vitamin C analysis in commercial fruit juices.     

Lymphocyte transformation in vitro : V. Thymidine incorporation into unstimulated lymphocytes

In vitro conditions and kinetics of ¹⁴C-thymidine incorporation into unstimulated lymphocytes were studied. Lymphocytes cultured during 3 to 9 days displayed a progressive increase of thymidine uptake with time. The addition of varying numbers of autologous mitomycin-treated lymphocytes to cultures containing a fixed number of untreated lymphocytes markedly enhanced thymidine uptake per non-mitomycin-treated lymphocytes. In the latter cultures a sharp rise of thymidine uptake between the 7th day of culture was seen. Supernatants of non-stimulated lymphocytes, whether mitomycin-treated or not, showed no stimulating effect on autologous normal cells in these experiments. Although the mechanism of the enhancing effect of mitomycin-treated autologous lymphocytes remains unclear, it obviously may interfere in culture experiments in which the antigen specific stimulatory effect of mitomycin-treated cells is measured, which can only be detected by including the appropriate controls.     

The convenience of the use of allosteric probes for the study of lipid--protein interactions in biological membranes: thermodynamic considerations.

One of the most commonly used methods to study enzyme (protein)-structure interactions at a more specific level is the use of the Arrhenius plots to detect the influence of the “environment” on the enzyme. We want to point out here that the use of a suitable “allosteric enzyme” would be a more sensitive method to detect influences of the environment than the study of Arrhenius plots. According to simple thermodynamic considerations, a change of more than 2.8 kcal/mol in the interaction between enzyme and membrane would be needed to give a noticeable change in the position of the break (T_i) of the corresponding Arrhenius plot. On the other hand, feeble changes in the membrane-enzyme interaction, of the order of 0·7–0·8 kcal/mol, would be enough to give a significant change in the values of n (Hill coefficient).     

流域尺度水土保持生物措施减沙效益研究

1　引　　言黄土高原位于黄河中游 ,流经黄土高原的 9大支流进入河口镇至花园口的中游河段 ,总径流量为 187× 10 8ｍ3 ,输沙量占黄河流域总沙量的 90 % .建国以来以修建梯田坝地、造林种草、建谷坊、调整种植结构为主的水土保持工程、生物治理长年不懈 ,其目的在于减水减沙 ,改善流域生态环境 .但是 ,除部分中小尺度流域进入生态、经济效益良性循环的轨道外 ,大部分地区生态环境仍然十分脆弱 ,部分地区有恶化之势 .因此 ,定量评价水土保持工程、生物措施的治理效益是广为关注的研究课题 ,虽相关的研究成果不少 ,但是绝大部分工作是评价水土…     

Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects

Chemical probes for free radicals in biology are important tools; fluorescence and chemiluminescence offer high detection sensitivity. This article reviews progress in the development of probes for "reactive oxygen and nitrogen" species, emphasizing the caution needed in their use. Reactive species include hydrogen peroxide; hydroxyl, superoxide, and thiyl radicals; carbonate radical-anion; and nitric oxide, nitrogen dioxide, and peroxynitrite. Probes based on reduced dyes lack selectivity and may require a catalyst for reaction: despite these drawbacks, dichlorodihydrofluorescein and dihydrorhodamine have been used in well over 2,000 studies. Use in cellular systems requires loading into cells, and minimizing leakage. Reactive species can compete with intracellular antioxidants, changes in fluorescence or luminescence possibly reflecting changes in competing antioxidants rather than free radical generation rate. Products being measured can react further with radicals, and intermediate probe radicals are often reactive toward antioxidants and especially oxygen, to generate superoxide. Common probes for superoxide and nitric oxide require activation to a reactive intermediate; activation is not achieved by the radical of interest and the response is thus additionally sensitive to this first step. Rational use of probes requires understanding and quantitation of the mechanistic pathways involved, and of environmental factors such as oxygen and pH. We can build on this framework of knowledge in evaluating new probes.