A berberine-aniline blue fluorescent staining procedure for suberin,lignin, and callose in plant tissue

Summary A fluorescent staining procedure to detect suberin, lignin and callose in plants has been developed. This procedure greatly improves on previous methods for visualizing Casparian bands in root exodermal and endodermal cells, and performs equally well on a variety of other plant tissues. Berberine was selected as the most suitable replacement forChelidonium majus root extract after comparing the staining properties of the extract with those of four of its constituent alkaloids. Aniline blue counterstaining efficiently quenched unwanted background fluorescence and nonspecific berberine staining, while providing a fluorochrome for callose. When used with multichambered holders which allow simultaneous processing of freehand sections, this efficient staining procedure facilitates morphological studies involving large numbers of samples.Abbreviations ISCC-NBS Inter-Society Color Council-National Bureau of Standards - UV ultraviolet light     

Cometabolic turnover of aniline,phenol and some of their monochlorinated derivatives by the Rhodococcus mutant strain AM 144

One-step conversion of aniline, phenol and some of their monochlorinated derivatives into the corresponding catechols by resting pre-adapted cells of the Rhodococcus mutant strain AM 144 (defective in synthesis of catechol 1,2-dioxygenase) was shown to depend on the availability of an additional metabolizable carbon substrate, e.g. glucose or acetate. A stoichiometric relation existed between the amount of the latter compounds added and the amount of aniline (or phenol, respectively) converted into catechol suggesting that the primary function of the cosubstrates was to provide reducing power to the oxygenative transformation reaction. The observed cosubstrate-dependence generally parallels that seen in previous studies on turnover of different monochloroaromatic non-growth substrates by aromatics-utilizing Rhodococcus wildtype-strains. Cell cultures of strain AM 144 growing at the expense of acetate also proved able to convert aniline into catechol. Typically, growth of the cells was retarded during the phase of aniline transformation as compared to the respective control cultures. Based on the results of these model experiments, it was concluded that (i) in natural microbial communities cometabolically active bacteria would hardly enrich under cometabolic conditions over fast-growing non-cometabolizing bacteria if the latter organisms will tolerate the particular non-growth substrate, and (ii) cometabolizing bacteria would have a selective advantage only if the non-growth substrate to be transformed is a toxic one or if it can serve as a potential nutrient source (e.g., of nitrogen or sulfur).Abbreviations MCA monochloroaniline - MCP monochlorophenol - MCC monochlorocatechol - TLC thin-layer chromatography - MS mass spectrometry - GLC gas-liquid chromatography - UV ultraviolet (range of the spectrum)     

Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol-aniline coupling hydrogen donor

The hydrogen donor couples pyrocatechol-aniline and phenol-aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV-Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol-aniline was converted to a pink-colored reagent with a lambda(max) of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol-aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol-aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of K(m) (12.5mM) and V(max) (12.2 mM min(-1)mg(-1)) were calculated for pyrocatechol-aniline. Regarding the superiority of pyrocatechol-aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay.     

Anaerobic treatment of highly concentrated aniline wastewater using packed-bed biofilm reactor

The performance of packed-bed biofilm reactor (PBBR) with self-floating bio-carriers was investigated to treat highly concentrated organic nitrogenous aniline wastewater with a COD value as high as 24,000 mg/L. With 45 vol% of carrier charge inside the reactor, the aniline wastewater can be effectively treated with 94% of COD removal efficiency at a low organic loading rate (OLR) of 0.9 kg COD/(m³ d). The removal efficiency decreased gradually down to 75% when OLR increased to 12.27 kg COD/(m³ d) that corresponded to 1 day of HRT. Separate tests with biofilm alone showed that the conversion contribution of the biofilm was about half of the overall COD conversion by the biofilm plus sludge system at the same OLRs of 3–4 kg COD/(m³ d), and that the biofilm had higher activity than suspended sludge. Ammonium released from decomposed aniline was increased gradually from 500 to 1700 mg/L with the OLR increase from 0.9 to 12.27 kg COD/(m³ d), which resulted in inhibitory effect to the microorganism due to the toxicity of free ammonia. Batch anaerobic toxicity tests showed that the biofilm was less sensitive to toxic compounds than suspended sludge and could tolerate higher concentration of free ammonia.     

Experimental and computational approaches to quantitative proteomics: status quo and outlook

Proteomics has come a long way from the initial qualitative analysis of proteins present in a given sample at a given time (“cataloguing”) to large-scale characterization of proteomes, their interactions and dynamic behavior. Originally enabled by breakthroughs in protein separation and visualization (by two-dimensional gels) and protein identification (by mass spectrometry), the discipline now encompasses a large body of protein and peptide separation, labeling, detection and sequencing tools supported by computational data processing. The decisive mass spectrometric developments and most recent instrumentation news are briefly mentioned accompanied by a short review of gel and chromatographic techniques for protein/peptide separation, depletion and enrichment. Special emphasis is placed on quantification techniques: gel-based, and label-free techniques are briefly discussed whereas stable-isotope coding and internal peptide standards are extensively reviewed. Another special chapter is dedicated to software and computing tools for proteomic data processing and validation. A short assessment of the status quo and recommendations for future developments round up this journey through quantitative proteomics.     

Biotransformation and toxicity of aniline and aniline derivatives in cyanobacteria

Agmenellum quadruplicatum strain PR-6 and Oscillatoria sp. strain JCM grown photoautotrophically in the presence of aniline metabolized the aromatic amine to formanilide, acetanilide and p-aminophenol. The metabolites were isolated by either thin-layer, gas-liquid or high pressure liquid chromatography and identified by comparison of their chromatographic, ultraviolet absorbance and mass spectral properties with those of authentic compounds. The toxicity of aniline derivatives towards Agmenellum quadruplicatum strain PR-6 indicated that the cyanobacterium was extremely sensitive to o-, m- and p-aminophenols, and phenylhydroxylamine.Abbreviations TLC thin layer chromatography - HPLC high pressure liquid chromatography - GC/MS gas chromatography/mass spectrometry - m/e mass to charge ratio     

Directed evolution of aniline dioxygenase for enhanced bioremediation of aromatic amines

The objective of this study was to enhance the activity of aniline dioxygenase (AtdA), a multi-component Rieske non-heme iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA, so as to create an enhanced biocatalyst for the bioremediation of aromatic amines. Previously, the mutation V205A was found to widen the substrate specificity of AtdA to accept 2-isopropylaniline (2IPA) for which the wild-type enzyme has no activity (Ang EL, Obbard JP, Zhao HM, FEBS J, 274:928–939, 2007). Using mutant V205A as the parent and applying one round of saturation mutagenesis followed by a round of random mutagenesis, the activity of the final mutant, 3-R21, was increased by 8.9-, 98.0-, and 2.0-fold for aniline, 2,4-dimethylaniline (24DMA), and 2-isopropylaniline (2IPA), respectively, over the mutant V205A. In particular, the activity of the mutant 3-R21 for 24DMA, which is a carcinogenic aromatic amine pollutant, was increased by 3.5-fold over the wild-type AtdA, while the AN activity was restored to the wild-type level, thus yielding a mutant aniline dioxygenase with enhanced activity and capable of hydroxylating a wider range of aromatic amines than the wild type. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

生精细胞及精子赖氨酸含量的研究

本实验取１０只Ｗｉｓｔａｒ大鼠的睾丸和附睾,睾丸石蜡切片,附睾精子涂片后用苯胺蓝染色显示赖氨酸含量。结果是睾丸生精小管中精原细胞和精母细胞染色较深即赖氨酸含量较高,精子细胞和精子染色渐淡即赖氨酸含量降低,而附睾精子显示,在附睾头部的精子染色较深,附睾尾部的精子几乎不着色,应用显微分光光度计测定附睾精子,计算出头部的精子赖氨酸含量在１左右,尾部的精子赖氨酸含量接近于零。本实验还检测了１０例正常人及１０例不育者精子的赖氨酸,结果为正常人精子的赖氨酸含量较低,不育者精子赖氨酸含量高且畸形率也高。提示精子赖氨酸含量高是核蛋白转型异常的征象,可能是男性不育的一个重要原因。     

The aniline blue fluorochrome specifically stains the septum of both live and fixed Schizosaccharomyces pombe cells

Abstract A novel method is described which uses aniline blue for the specific fluorescent staining of the septa of dividing cells of the fission yeast, Schizosaccharomvces pombe . It gives the same results with live and fixed cells. In fixed or, more generally, dead cells there is no staining of the cytoplasm: this renders aniline blue superior to other dyes previously used to stain the septum of S. pombe . This feature allows quantitative analysis of the septum index for fixed samples and, therefore, makes aniline blue the stain of choice for cell cycle kinetic studies.