Reusable System for Phenol Detection in an Aqueous Medium Based on Nanodiamonds and Extracellular Oxidase from Basidiomycete Neonothopanus nambi

Doklady Biochemistry and Biophysics - A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto...     

Stimulation of luminescence of mycelium of luminous fungus Neonothopanus nambi by ionizing radiation

The luminescent system of higher luminous fungi is not fully understood and the enzyme/substrate pair of the light emission reaction has not been isolated. It was suggested that luminescence of fungi involves oxidase‐type enzymes, and reactive oxygen species are important for fungal light production. Generation of reactive oxygen species can be stimulated by ionizing irradiation, which has not been studied for luminous fungi. We report the effect of X‐irradiation on the luminescence of fungus Neonothopanus nambi. Experiments were performed with mycelium on a home‐built setup based on an X‐ray tube and monochromator/photomultiplier tube. Application of X‐rays does not change the emission spectrum, but after approximately 20 min of continuous irradiation, light production from unsupported mycelium starts growing and increases up to approximately five times. After peaking, its level decreases irrespective of the presence of X‐irradiation. After staying at a certain level, light production collapses to zero, which is not related to the drying of the mycelium or thermal impact of radiation. The observed shape of kinetics is characteristic of a multistage and/or chain reaction. The time profile of light production must reflect the current levels of radicals present in the system and/or the activity of enzyme complexes involved in light production. Copyright © 2014 John Wiley & Sons, Ltd.     

Extracellular Oxidase from the Neonothopanus nambi Fungus as a Promising Enzyme for Analytical Applications

The Protein Journal - The extracellular enzyme with oxidase function was extracted from the Neonothopanus nambi luminescent fungus by using mild processing of mycelium with β-glucosidase and...     

中国野生发光真菌新记录种Neonothopanus nambi的分离、鉴定及其形态观察

【目的】在中国福建地区采集分离一株野生发光真菌,菌种编号为MRC-lf3,对其进行形态观察及分子鉴定以确定其分类地位,并观察其整个生活史的形态特征。【方法】采用组织分离技术获得野生发光真菌纯培养物,结合形态学及ITS进化分析确定该菌的分类地位,应用单反相机记录子实体不同生长时期的形态特征及发光情况。【结果】该发光真菌的菌丝、子实体、孢子的形态均与Neonothopanus nambi最为相似,ITS序列的进化分析结果也支持该发光真菌为Neonothopanus nambi。其子实体颜色深浅和发光强度均受到光照强度的影响,光照可使子实体颜色变暗、发光强度减弱。【结论】从福建省福州市采集分离获得一株大型野生发光真菌,经鉴定该菌为Neonothopanus nambi,是中国首次发现的Neonothopanus属真菌,该发现也将中国大陆的野生发光真菌总数增加至8种。     

Nanodiamonds as an effective adsorbent for immobilization of extracellular peroxidases from luminous fungus Neonothopanus nambi to construct a phenol detection system

Modified nanodiamonds (MNDs) produced by detonation synthesis can be used as an effective adsorbent to immobilize extracellular peroxidases of the luminous basidiomycete Neonothopanus nambi. The enzymes are firmly immobilized on MND particles and exhibit catalytic activity. The indicator system (the MND–enzyme complex) reused many times retains its ability to catalyze reaction of co-oxidation of phenol and 4-aminoantipirine in the presence of hydrogen peroxide and remains functionally active during long-term storage (for 1?month or longer) in aqueous suspensions at 4?°C. MNDs and enzymes of higher fungi can be effectively used to construct new reusable indicator systems for analytical applications such as monitoring contamination of aquatic environments by phenolic compounds.     

担子菌漆酶的分离纯化及其性质研究

采用硫酸铵盐析、DEAE纤维素柱层析、Phenyl SepharoseTM6 Fast Flow疏水柱层析等方法,得到电泳纯的漆酶同工酶Lac1,纯化倍数为318.4,活力回收率为18.6％。用SDSPAGE测得该酶分子量为60.3kD,而经质谱分析为55.94kD。最适反应温度为65℃,最适反应pH值为2.2～2.8,酶的等电点pI（室温）为4.02,其N末端序列为AIGPVTDL,用硫酸酚法测得其含糖量为49.2％。25℃条件下,以ABTS(2,2'azinobis(3ethylbenzthiazoline6sulphonate)为底物的Km为17.5μmol/L。该酶在45℃,pH3.0～9.5较稳定。Cu^2＋对酶活有明显的促进作用,Fe^2＋完全抑制酶的活性,Mn^2＋和Ag^＋对酶活无明显影响。DTT（Dithiothreitol,二硫苏糖醇）和NaN3完全抑制酶的活性。Koshland试剂对漆酶的活力影响比较大,色氨酸可能是酶活力的必需基团。     

Light and Temperature Requirements during Fruit-Body Development of a Basidiomycete Mushroom, Coprinus congregates

Light initiated fruit body primordia of Coprinus congregatus Bull, ex Fr. grown at relatively high temperature (25°C) require a single dark period or low-temperature induction for completion of fruit-body development. The dark period requirement varied with the temperature regime during the inductive dark period A minimum requirement of 2.5 h was found al 15–20°C. Darkness always promotes development of fruit-body primordial, but cannot, be regarded as an absolute necessity until temperature exceeds about 17.5°C. Normal development of me primordia without darkness was obtained by lowering the temperature to 10°C for 6 h. It appeared that at high temperatures two successive stimuli were required for basidiocarp maturation, a light-off and a subsequent signal light-on signal. On the contrary, at 10°C a single low-temperature signal seemed to be involved. Thus, induction of fruit-body development could be produced by alternative pathways. These developmental features have been extended to other fungi and compared with the flowering processes of some short-day plants.     

Fruiting at High Temperature and Its Genetic Control in the Basidiomycete Flammulina velutipes

Of 10 geographic strains of Flammulina velutipes, 4 were found capable of fruiting at 22°C (Fr_H) rather than at the typical 15°C (Fr_L). Crosses made between Fr_H and Fr_L monokaryons were never observed to fruit at 22°C. However, some hybrids did fruit at the intermediate temperature of 18°C when grown on appropriate substrates, indicating incomplete dominance of the low-temperature requirement. Analysis of progeny of five Fr_H × Fr_L crosses indicated that a minimum of two genes appears to control the requirement for fruiting at ≤15°C. The genes are not closely linked to either incompatibility locus.     

Blue Light Overrides Repression of Asexual Sporulation by Mating Type Genes in the Basidiomycete Coprinus cinereus

Monokaryotic mycelia of the homobasidiomycete Coprinus cinereus form asexual spores (oidia) constitutively in abundant numbers. Mycelia with mutations in both mating type loci (Amut Bmut homokaryons) also produce copious oidia but only when exposed to blue light. We used such an Amut Bmut homokaryon to define environmental and inherent factors that influence the light-induced oidiation process. We show that the Amut function causes repression of oidiation in the dark and that light overrides this effect. Similarly, compatible genes from different haplotypes of the A mating type locus repress sporulation in the dark and not in the light. Compatible products of the B mating type locus reduce the outcome of light on A-mediated repression but the mutated B function present in the Amut Bmut homokaryons is not effective. In dikaryons, the coordinated regulation of asexual sporulation by compatible A and B mating type genes results in moderate oidia production in light. Copyright 1998 Academic Press.     

Thermodynamics of Light Emission and Free-Energy Storage in Photosynthesis

A Planck law relationship between absorption and emission spectra is used to compute the fluorescence spectra of some photosynthetic systems from their absorption spectra. Calculated luminescence spectra of purple bacteria agree well but not perfectly with published experimental spectra. Application of the Planck law relation to published activation spectra for Systems I and II of spinach chloroplasts permits independent calculation of the luminescence spectra of the two systems; if the luminescence yield of System I is taken to be one-third the yield of System II, then the combined luminescence spectrum closely fits published experimental measurement.

Consideration of the entropy associated with the excited state of the absorbing molecules is used to compute the oxidation-reduction potentials and maximum free-energy storage resulting from light absorption. Spinach chloroplasts under an illumination of 1 klux of white light can produce at most a potential difference of 1.32 ev for System I, and 1.36 ev for System II. In the absence of nonradiative losses, the maximum amount of free energy stored is 1.19 ev and 1.23 ev per photon absorbed for Systems I and II, respectively. The bacterium Chromatium under an illumination of 1 mw/cm² of Na D radiation can produce at most a potential difference of 0.90 ev; the maximum amount of free energy stored is 0.79 ev per photon absorbed.

The combined effect of partial thermodynamic reversibility and a finite trapping rate on the amount of luminescence is considered briefly.

     

Methods of Studying Ultraweak Photon Emission from Biological Objects: I. History,Types and Properties,Fundamental and Application Significance

Biophysics - This review is devoted to the methods for studying the ultraweak luminescence of biological objects; in addition to modern methods aimed at studies in the visible, near-infrared, and...     

Kinetics of Light Emission and Oxygen Consumption by Bioluminescent Bacteria

Oxygen plays a key role in bacterial bioluminescence. The simultaneous and continuous kinetics of oxygen consumption and light emission during a complete exhaustion of the exogenous oxygen present in a closed system has been investigated. The kinetics are performed with Vibrio fischeri, V. harveyi, and Photobacterium phosphoreum incubated on respiratory substrates chosen for their different reducing power. The general patterns of the luminescence time courses are different among species but not among substrates. During steady-state conditions, substrates, which are less reduced than glycerol, have, paradoxally, a better luminescence efficiency. Oxygen consumption by luciferase has been evaluated to be 17% of the total respiration. Luciferase is a regulatory enzyme presenting a positive cooperative effect with oxygen and its affinity for this final electron acceptor is about 4–5 times higher than the one of cytochrome oxidase. The apparent Michaelis constant for luciferase has been evaluated to be in the range of 20 to 65 nM O₂. When O₂ concentrations are as low as 10 nM, luminescence can still be detected; this means that above this concentration, strict anaerobiosis does not exist. By n-butyl malonate titration, it was clearly shown that electrons enter the luciferase pathway only when the cytochrome pathway is saturated. It is suggested that, in bioluminescent bacteria, luciferase acts as a free-energy dissipating valve when anabolic processes (biomass production) are impaired.     

rhsBLyS及其突变体对小鼠血清免疫球蛋白分泌及B细胞增殖分化的影响

为了进一步研究所构建的人可溶性B淋巴细胞刺激因子 (hsBLyS)及其突变体hsBY A(Cys14 6→Ala14 6 )和hsBY V(Cys14 6→Val14 6 )在体内的生物活性 ,将 3种重组蛋白注射入BALb C小鼠体内 ,进而分析重组蛋白对小鼠血清免疫球蛋白分泌和脾脏B细胞增殖分化的影响 ,并对 3种蛋白的体内生物活性进行了比较 .结果表明 ,3种蛋白均能促进小鼠血清总IgG和IgM的分泌明显增加 ,统计学检验显示突变体rhsBY V诱导这 2种免疫球蛋白分泌的能力最强 ;3种蛋白还诱导小鼠脾脏增大、脾脏成熟B细胞的相对数量增加 .该高活性突变体hsBY V的获得提示 ,Cys14 6并非BLyS发挥正常功能所必需 ,将Cys14 6突变成Val14 6有可能提高BLyS的生物活性 .     

Studying intracellular transport using high-pressure freezing and Correlative Light Electron Microscopy

Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopy in one experiment. Light microscopy (LM) is especially suited for providing a general overview with data from lots of different cells and by using live cell imaging it can show the history or sequence of events between or inside cells. Electron microscopy (EM) on the other hand can provide a much higher resolution image of a particular event and provide additional spatial information, the so-called reference space. CLEM thus has certain strengths over the application of both LM and EM techniques separately. But combining both modalities however generally also means making compromises in one or both of the techniques. Most often the preservation of ultrastructure for the electron microscopy part is sacrificed. Ideally samples should be visualized in its most native state both in the light microscope as well as the electron microscope. For electron microscopy this currently means that the sample will have to be cryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for using cryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressure freezing in combination with live cell imaging. In addition we examine some of the EM analysis tools that may be useful in combination with CLEM techniques.     

The Relation between Photophosphorylation and Delayed Light Emission in Chloroplasts

One millisecond delayed light emission has been studied in isolated coupled lettuce (Lactuca sativa var. romaine) chloroplasts. Delayed light emission was increased upon addition of ferricyanide or 2,3,5,6-tetramethyl-p-phenylene diamine. In the presence of ferricyanide, the magnitude of the signal was increased by the addition of ADP (in the absence of orthophosphate), ATP, DI0-9, or phlorizin. The signal was also increased by the addition of NaCl and by the addition of NH₄Cl in the presence of a high NaCl concentration. The signal of delayed light emission was decreased by the addition of gramicidin, valinomycin, and by the addition of NH₄Cl in the presence of a low NaCl concentration.     

Induction of Dimorphism in the Basidiomycete Lenzites saepiaria

Sugars can serve as the germinant for basidiospores of the wood-rotting fungus Lenzites saepiaria. Hexoses sterilized by autoclaving were better germinants than hexoses that were sterilized by filtration. The degradation products in heated hexose which were responsible for the stimulation of germination were levulinic and formic acid. Another product of hexose degradation, hydroxymethyl furfural, had a marked effect on outgrowth of L. saepiaria basidiospores and on the development of mycelia. Basidiospores that germinated in the presence of hydroxymethyl furfural yielded large rounded bodies that, in some cases, developed as a chain of yeastlike cells. Addition of hydroxymethyl furfural to developing mycelia resulted in the production of chains of round yeastlike structures. Similar results were obtained by treating basidiospores or mycelia with phenethyl alcohol.     

流行性出血热患者尿液中融合细胞的检测及其意义

我们观察到流行性出血热患者尿液中的“多核巨细胞”并非是上皮细胞脱落后的偶然堆积,而是细胞之间发生了融合。经免疫组化染色发现所有融合细胞内均有特异性EHF病毒抗原颗粒。提示流行性出血热病毒的直接作用与尿液中融合细胞的形成有关。     

Delayed Fluorescence and Ultraweak Light Emission from Isolated Chloroplasts (Comparison of Emission Spectra and Concentration Dependence)

The ultraweak light emission of isolated chloroplasts (Hidegand Inaba (1991) Photochem. Photobiol. 52: 137) was investigatedin comparison to delayed light emission. We compared the concentrationdependence and the spectral distribution of the light emittedfrom isolated chloroplasts stored in the dark for 10 s, 2 min(delayed light emission), 4 and 10 h (ultraweak light emission),respectively. In samples with low chlorophyll concentration, spectra of allemission phenomena were maximal at 685–695 nm, but spectraof ultraweak light, especially that of long term (10 h) emission,were broader in the 700–800 nm region than spectra ofdelayed light, indicating emission from a bigger variety ofchlorophyll molecules. The intensity of delayed light and short term (4 h) ultraweaklight exhibited a simple, saturating exponential dependenceon chlorophyll concentration, while long term (10 h) ultraweaklight emission was best described as a saturating exponentialcontaining a quadratic function of the concentration. This differencesuggests that long term ultraweak light emission is broughtabout by reactions distinct from the earlier described mechanismof electron transport related dark photoemission. (Received November 15, 1991; Accepted May 18, 1992)