Ultrastructure of basidiospores and mycelium of Lenzites saepiaria

Hyde, James M. (University of Mississippi Medical School, Jackson), and Charles H. Walkinshaw. Ultrastructure of basidiospores and mycelium of Lenzites saepiaria. J. Bacteriol. 92:1218-1227. 1966.-Ungerminated and germinated basidiospores and 2-day-old mycelial cultures of Lenzites saepiaria were similar in their fine structure. Fixation with glutaraldehyde, followed by osmium tetroxide, was far superior to permanganate. Cell organelles were seen in cytoplasm of spores and hyphae, and clamp connections were abundant in hyphal elements. Numerous lomasomes, vesicular bodies, and complex concentric membranes occurred in the cytoplasm and were often associated with the cell membrane or the dolipore membrane (parenthesome) of the septum. Endoplasmic reticulum was not found, but numerous ribosomes were seen; polyribosome groupings were frequently noted. The nucleus was bounded by a double membrane which contained few pores. Germinating spores exhibited one or more large osmiophilic bodies in association with a vacuole and membranous elements. Other than possessing a thin wall, the emerging germ tube was similar in structure to the parent spore.     

Ultrastructure of protoplasts from mycelium and microconidia of Trichophyton mentagrophytes

Protoplast formation from mycelium and microconidia of Trichophyton mentagrophytes was achieved with Novozym 234. Pretreatment procedures with dithiothreitol or urea mercaptoethanol sodium lauryl sulphate before digestion with Novozym 234 greatly reduced protoplast yield from mycelium. Snail gut enzyme did not protoplasts in good yield. Scanning electron microscopy of mycelium protoplasts showed the acquired spherical shape. The plasma membrane appeared finely granular although remnants of cell wall could sometimes be observed. Transmission electron microscopy showed the cell interior of these protoplasts was plasmolysed. Microconidia treated with Novozym 234 displayed a range of cell wall digestion, with intact protoplasts showing distinct cytoplasmic organelles.     

Ultrastructure of the mycelium and spores of Actinomadura fastidiosa sp. nov]

A new species of the Actinomadura genus, A. fastidiosa sp. nov., is described. The ultrastructure of the vegetative mycelium and spores of this organism was studied. The vegetative cells have a multilayered cell wall, often consisting of five layers with different thickness and electron density. The spores are similar to the vegetative cells by their inner structure but have a thicker wall.     

Stabilization studies of Fomes sclerodermeus laccases

Stability of laccase isoenzymes from a crude extract obtained from Fomes sclerodermeus grown on wheat bran medium was studied. The variables assessed were temperature, pH and additives. As revealed by PAGE, three bands of laccase, each with different thermal inactivation pattern, were detected in the crude extract: after 6h at 50 degrees C and pH 8, Lc2 was the most resistant, while the Lc1 and Lc3 bands were almost completely inactivated. This pattern of inactivation was observed at all temperatures and pH tested. Laccase activity was more stable in the 5-10 pH range when incubated at 40 and 50 degrees C; at 30 degrees C and 24h the enzyme remained fully active in the 3-11 pH range. The effect of additives (veratryl alcohol, trehalose, glycerol, mannitol, glutaraldehyde, CuSO(4) and 1-HBT) on laccase stability was tested. The stability was enhanced with CuSO(4) (1.25 mM), glycerol (0.2%) and mannitol (1%). The presence of both CuSO(4) and glycerol caused a 3-fold increase in the half-life values.     

The anti-influenza virus effect of Phellinus igniarius extract

Herbal medicine has been used in the orient for thousands of years to treat large and small ailments, including microbial infections. Although there are treatments for influenza virus infection, there is no treatment for drug-resistant viruses. It is time that we explored and exploited the multi-component nature of herbal extracts as multi-drug combination therapies. Here, we present data on the anti-influenza virus effect of a medicinal mushroom, Phellinus igniarius. The P. igniarius water extract was effective against influenza A and B viruses, including 2009 pandemic H1N1, human H3N2, avian H9N2, and oseltamivir-resistant H1N1 viruses. Virological assays revealed that the extract may interfere with one or more early events in the influenza virus replication cycle, including viral attachment to the target cell. Therefore, our results provide new insights into the use of P. igniarius as an anti-influenza medicine.     

Function of laccase in the white-rot fungus Fomes annosus

1. Thioglycolic acid, a Cu-chelating agent, totally inhibited extracellular laccase activity without affecting growth and morphology of Fomes annosus.
2. In the presence of thioglycolic acid Fomes annosus cleaved high molecular weight lignosulfonate with a molecular weight range of 2×10⁶ to 1000. In the absence of thioglycolic acid the polymerizing activity of laccase prevented the detection of lignosulfonate breakdown products.
3. Oxidative polymerization of a lignin monomer, coniferyl alcohol, occurred in the presence but not in the absence of laccase activity.
4. Catechol and guaiacol added to the medium at a concentration of 2 mmol, are normally oxidized by fungal laccase and strongly inhibit growth. Presence of thioglycolic acid prevented the oxidation of these phenols and simultaneously permitted normal growth.

     

Resin and the Resistance of Conifers to Fomes annosus

Roots of young trees of different conifer species, Scots pine,Corsican pine, Sitka spruce, and Douglas fir were inoculatedwith Fomes annosus. In the pines, infection was negligible butin the other genera the central wood was extensively invaded.The process of infection was studied further in Sitka spruceby means of a root-severing technique in which an inoculum blockwas forced between the cut ends of a root. In most trees a centralrot developed which in some extended up into the butt: the meanextent of growth recorded was 30 cm in 14 months. In the severedroot portions infection also occurred but F. annosus was subsequentlyreplaced to a considerable extent by other micro-organisms,particularly in trees growing on a peat soil. A similar centralrot developed after inoculations of Norway spruce. The resistance of pine sapwood and the outer wood of spruceand Douglas fir can be explained at least partially by the structureand viability of the resin duct system. Larch, Douglas fir,spruce, and pine form a series showing increasing complexityand activity of the resin system. In addition, studies carriedout on the resin yields of pines growing under different conditionsshowed that the resistance of a tree to F. annosus is correlatedwith its ability to mobilize resin. Site factors, forest management,host age vigour, genetic constitution, and past history mayall influence resin mobilization.     

桑黄粗多糖除蛋白及抗肿瘤活性

旨在研究Sevag法除桑黄粗多糖中蛋白的最佳工艺,并通过噻唑蓝(MTT)细胞比色法测定除蛋白前后桑黄多糖的抗肿瘤活性。在单因素试验Sevag用量、氯仿与正丁醇体积比、萃取次数、震荡时间的基础上,设计正交试验,经过方差分析,确定Sevag法除桑黄粗多糖蛋白的最佳方案;并且对除蛋白后多糖与粗多糖进行体外抗肿瘤实验。Sevag法除桑黄粗多糖蛋白的最佳工艺:Sevag用量50 mL、V(氯仿)/V(正丁醇)=3、最佳萃取次数6次、最佳振荡时间20 min,在此条件下,多糖的得率为64.8%。体外细胞试验表明除蛋白后多糖与粗多糖对肿瘤细胞EC109和SiHa的抑制率没有明显差异。     

Darstellung und Eigenschaften einer Peroxidase aus Phellinus igniarius

Zusammenfassung Mit Hilfe verschiedener Präparationsmethoden wurde aus dem Mycel und der Nährlösung des Basidiomyceten Phellinus igniarius eine Peroxidase (EC 1.11.1.7) gewonnen.Das Enzym ist gegenüber äußeren Einflüssen (pH, Salzkonzentration, Temperatur) bemerkenswert stabil. Sein pH-Optimum liegt im sauren Bereich. Zwei Isoenzyme wurden gefunden. Die Molmasse, der isoelektrische Punkt, die Michaelis-Menten-Konstante, die Indolessigsäure-Oxidase-Aktivität sowie verschiedene spektrale und analytische Eigenschaften dieser Peroxidase wurden bestimmt. Die Aktivität des Enzyms läßt sich durch Effektoren sowohl hemmen als auch verstärken. Es ist anzunehmen, daß das Enzym einen intra- und einen extracellulären Wirkbereich hat.

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Purification and characterization of peroxidase from Phellinus igniarius

A Peroxidase (EC 1.11.1.7) of the basidiomycet Phellinus igniarius was derived from mycel and a medium containing glucose and extract of yeast by using various methods of preparation.The enzyme resists extreme conditions (pH, temperature, salt concentration). Its optimum pH for activities is in the acid range. Two isoenzymes were found. The molecular weight, isoelectric point, Michaelis-Menten constant, indolacetic acid oxidase activity and spectral and analytical properties of this peroxidase were determined. It is assumed that the enzyme has an intracellular as well as an extracellular field of activity.

     

Metal-binding capacity of arbuscular mycorrhizal mycelium

Experiments with excised mycelium of several Glomus spp. with different histories of exposure to heavy metals were carried out to measure their capacities to bind Cd and Zn. Cd sorption was followed for up to 6 h of incubation to determine its time course relationships. Controls treated with a metabolic inhibitor were included to evaluate whether sorption was due to active uptake or passive adsorption. The effect of ion competition (effects of Ca or Zn on Cd sorption) and general measurements of cation exchange capacity (CEC) of roots and hyphae were also performed. The results showed that AM mycelium has a high metal sorption capacity relative to other microorganisms, and a CEC comparable to other fungi. Metal sorption was rapid (<30 min) and appeared mainly to be due to passive adsorption. Adsorption was highest in a metal-tolerant G. mosseae isolate and intermediate for a fungus isolated from a soil treated with metal-contaminated sludge. The former adsorbed up to 0.5 mg Cd per mg dry biomass, which was three times the binding capacity of non-tolerant fungi, and more than 10 times higher than reported values for, e.g., the commonly used biosorption organism Rhizopus arrhizus. The implications of these results for AM involvement in plant protection against excess heavy metal uptake are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

桑黄栽培子实体成分分析及其显微形态观察

采用扫描电子显微镜观察人工栽培桑黄子实体的显微形态及其结构,如菌管、子实层、担孢子、锥形刚毛及离散菌丝结构等。分析结果表明:桑黄幼嫩子实体中全氨基酸含量可达11.22%,是成熟子实体(4.28%)的2.62倍,其多糖含量(1.18%)也较成熟子实体(0.64%)高许多。其多糖组分主要为甘露糖、半乳糖、葡萄糖,摩尔比分别为3.820:0.314:1.000(幼嫩子实体)和3.260:0.112:1.000(成熟子实体)。     

桑黄菌丝体粗多糖的提取方法研究

采用正交试验设计,以桑黄菌丝体粗多糖含量为考察指标,用苯酚—硫酸法,分别确定了热水浸提法、微波辅助提取法和超声提取法的最佳工艺。通过极差分析得出:热水浸提法的最优工艺为浸提时间3 h、浸提3次、液料质量比50∶1、浸提温度90℃,粗多糖提取率为2.10%;微波提取法的最优工艺为微波处理15 min、液料质量比50∶1、提取3次,提取率为4.18%;超声提取法的最优工艺为超声30 min、提取2次、液料质量比60∶1、温度60℃、频率60 Hz,提取率为3.02%。微波辅助法与热水浸提法相比,时间缩短,且提取率提高近1倍;与超声提取法相比,时间缩短1/2,但提取率提高40%。因此,微波辅助提取法速度更快、提取效率更高、操作更简便,优于其他2种方法。     

Investigation of the electrostatic charge of basidiospores of the Phellinus igniarius group

The polarity and magnitude of primary electric charges carried by basidiospores in the airborne state were investigated in living fungal fruiting bodies under natural forest conditions using a portable experimental device designed by the author. The operating principle was the falling of spores in the homogeneous horizontal electric field. The vertical and horizontal components of the trajectories of the spores were determined according to their deposition sites on electrodes (vertical metal plates). Altogether 33 samples of spores were examined for polarity, 10 of these samples (with 10⁴–10⁶ spores per sample) also were used to calculate the mean spore charge-to-mass quotient and the mean spore charge. The detection limits of spore charge-to-mass quotient varied in the range from (4.9±2.3)×10^?5 to (1.36±0.33)×10^?4 C kg^?1. Basidiospores (subglobose, smooth, diameter of 4–6 μm) of the closely related (sibling) species Phellinus alni, P. nigricans, P. populicola and P. tremulae (Hymenochaetales, Basidiomycota) carried positive electrical charges that have mean values from 48 to 305 elementary charges. The intraspecies variation of the spore charge could depend on the natural variation in spore size.     

Effect of Spruce Root Constituents on Extracellular Enzymes of Fomes annosus

Fractions of acetone extracts from heartwood, sapwood and wood from the reaction zone in roots of Norway spruce, attacked by Fomes annosus, were tested concerning their effect on the activity of cellulase, polygalacturonase, aryl-β-glucosidase and laccase, obtained from culture filtrates of the same fungus. A specific inhibition of polygalacturonase was recorded with the resinous fractions. The hydrolysing enzymes were more inhibited by the lignans of the reaction zone in the presence of fungal laccase than in the absence of laccase. The pH dependence of the inhibiting effect of phenols varied among the enzymes. The four enzymes were inhibited by partly different lignan fractions, and synergistic effects between minor compounds were indicated. The phenolic fractions inhibited weakly the biosynthesis of the enzymes. It was obvious that results from enzyme inhibition studies in vitro do not reflect the complicated mutual interrelationships between production and activity of the extracellular fungal enzymes and the chemical constituents of the host in vivo.     

木蹄层孔菌子实体化学成分及对肿瘤细胞的抑制作用的研究

运用硅胶、sephadex LH-20等柱色谱,从木蹄层孔菌子实体的乙醇提取物分离得到12个化合物,通过单体的理化性质、NMR和MS技术鉴定单体的结构为3-十六碳酸酯-7,22-二烯麦角甾醇（1）,十八烷酸（2）,棕榈酸（3）,7,22-二烯麦角甾-3-酮（4）,麦角甾-7,22-二烯-3-醇（5）,5,8-过氧麦角甾-6,22-二烯-3-醇（6）,3,3-二甲氧基-7,22-二烯麦角烷（7）,28-乙酰白桦脂醇（8）,白桦脂醇（9）,β-羟基十八烷酸（10）,9,10-二羟基十八烷酸（11）,瑞香素（12）。采用Alamar Blue法检测单体化合物对人肺癌细胞NCI-H 460和人胃癌细胞SGC-7901的抑制活性。结果表明,化合物4对NCI-H 460细胞株的抑制活性最高,化合物9对SGC-7901的抑制活性最高。