Transformation of the cultivated mushroom,Agaricus bisporus, to hygromycin B resistance

Application of biotechnology to the cultivated mushroom,Agaricus bisporus, has been hampered thus far by the lack of a transformation system. Here, transformation of both a homo- and a heterokaryotic strain ofA. bisporus to hygromycin B resistance is described. Transforming DNA was integrated into theA. bisporus genome and stably maintained throughout vegetative growth. Transformants of the heterokaryotic strain formed transgenic fruiting bodies. Promoters derived from the unrelated ascomyceteAspergillus nidulans and fromA. bisporus itself, were able to drive expression of the hygromycin B resistance gene. Expression controlled by a fragment of 265 bp from theA. bisporus GPD promoter was sufficient to generate transformants. However, transformation efficiency was not enhanced by using this homologous promoter.     

In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes

Lentinula edodes, known as “shiitake” is one of the widely used medicinal mushrooms in the Orient. Antitumour activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC₅₀ values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to zymosan showed SI_comit/SI_mit ratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.     

Lampteromyces bioluminescence : 3. Structure of lampteroflavin, the light emitter in the luminous mushroom, L. japonicus

The structure of lampteroflavin, the light emitter in the luminous mushroom (Lampteromyces japonicus), was determined to be a new riboflavin α- -riboside, its structure being elucidated on the basis of fast atom bombardment tandem mass spectrometry, NMR, and CD studies.     

Occurrence of a unique amino acid racemase in a basidiomycetous mushroom, Lentinus edodes

Free -alanine was detected in a cell extract of the fruit-body of an edible basidiomycetous mushroom, Lentinus edodes (Shiitake), by means of reverse-phase high performance liquid chromatography. We also found an amino acid racemase activity in L. edodes fruit-body, and purified the enzyme. The enzyme has a molecular weight of approximately 86,000, and consists of two subunits of identical molecular weight (44,000). The optimal pH of the enzyme activity is around pH 9.5 for both -to- and -to- alanine racemization. The enzyme requires pyridoxal 5′-phosphate as a cofactor. K_m and V_max values for -alanine were 37.3 mM and 520 nmol/min/mg, respectively; for -alanine, they were 9.21 mM and 141 nmol/min/mg, respectively. The equilibrium constant was calculated to be 1.10, which is consistent with the theoretical value for the racemase reaction. The ability of the enzyme to catalyze the racemization of various -amino acids was investigated. The enzyme catalyzes the racemization of -serine (relative reaction rate, 144% of rate for -alanine), -alanine (100%), -homoserine (17.1%), -2-aminobutyrate (5.6%), -glutamate (4.5%), and -asparagine (3.2%). To the best of our knowledge, this is the first report of an amino acid racemase produced by a basidiomycetous mushroom.     

Substrate dependent larval development and emergence of the mushroom pests Lycoriella auripila and Megaselia halterata

Pasteurized, spawned, full-grown and immediately-cased full-grown compost were simultaneously exposed to natural populations of the mushroom pests Lycoriella auripila (Winnertz) (Diptera: Sciaridae) and Megaselia halterata (Wood) (Diptera: Phoridae). Different numbers of adults emerged from each of these composts. Highest numbers of L. auripila emerged from spawned and pasteurized compost whereas lowest numbers of L. auripila emerged from full-grown compost. the emergence from full-grown compost was delayed, which could be explained by the delayed development of the larvae in this type of compost. High numbers of M. halterata emerged from compost that was completely colonized by the mycelia of the edible white button mushroom Agaricus bisporus (Lange) Imbach. The immediate covering of the compost with a casing layer significantly lowered the numbers of emerging M. halterata flies. Compared with the emergence pattern from full-grown and immediately-cased full-grown compost, adult M. halterata showed a delayed pattern of emergence in spawned compost. Adult M. halterata did not emerge from pasteurized compost. The results of these experiments enabled us to improve the timing of the application of insect pathogenic nematodes in the control of the larvae of both insect pests.     

Inter- and intraspecific variation in infectivity ofSteinernema spp. to larvae of the mushroom flyLycoriella solani

Laboratory bioassays were done to assess the susceptibility of larvae of the mushroom sciarid flyLycoriella solani Winnertz (Diptera; Sciaridae) to 16 isolates comprising five species of the entomopathogenic nematode,Steinernema (Nematoda; Steinernematidae). Each isolate was tested by challenging sciarid larvae with single nematodes. Six isolates were also tested in dose-response experiments. Variations in nematode infectivity occurred (p<0.001) at both inter- and intraspecific levels.Steinernema feltiae (Filipjev) was the most virulent species: two isolates, Nemasys (LD₅₀=1.5–3.9) and Sus 94 (1.8–3.9) were superior to the rest.Steinernema kraussei (Steiner) was the least infectious nematode tested as its infection probability never exceeded 0.10. In addition, lethal dose₅₀ values for this species ranged from 9.0–62.6 and two isolates failed consistently to infect any hosts.     

银杏外种皮提取物对酪氨酸酶的抑制作用

以银杏外种皮为材料,采用水、乙醇和乙醇-乙醚三种不同的方法分离提取其中的活性物质(分别命名为1#,2#,3#),研究它们对蘑菇酪氨酸酶催化L-多巴(L-DOPA)氧化活力的影响。结果表明,这3种提取物均对蘑菇酪氨酸酶有抑制作用,1#,2#和3#对酶抑制作用的IC50分别为2.25、1.75和0.32mg/mL。抑制作用动力学结果表明:三种提取物对酶的抑制作用均表现为混合型,相应的抑制常数KI依次为2.11、1.62和0.29mg/mL;KIS依次为2.80、2.33和0.45mg/mL。结果显示,采用乙醇-乙醚提取的银杏外种皮提取物对酪氨酸酶抑制作用最强。     

The A mating-type genes of the mushroom Coprinus cinereus are not differentially transcribed in monokaryons and dikaryons

Summary The A mating type factor of Coprinus cinereus regulates part of a developmental sequence that leads to the conversion of the asexual monokaryon into the fertile dikaryon. The A42 factor is a complex of seven genes, at least four of which are involved in determining the specificity of mating interactions. In this report we show that the A42 genes are constitutively expressed in both monokaryons and dikaryons. This has important implications with respect to intracellular recognition of a compatible mating, which requires an interaction between proteins already present within the cells of the mating partners, and for the subsequent maintenance of dikaryotic growth.     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

香菇C91-3转录本Unigene 24277基因克隆表达及其生物学活性的研究

通过对香菇C_91-3转录本Unigene 24277基因的生物信息学分析,克隆表达含RCC1结构域的Unigene 24277基因,并研究其抗肿瘤活性。从香菇C_91-3菌丝体中提取总RNA,反转录合成cDNA,并利用Rapid Amplification of cDNA Ends（RACE）技术获得基因全长。NCBI数据库分析提示其含有RCC1结构域。PCR扩增RCC1结构域,将其克隆产物与pET-32a（+）载体连接,热转化至E.coil Rosetta-gami（DE3）中诱导表达,纯化、复性后,通过MTT法研究其抗肿瘤活性。结果显示原核表达载体构建成功,重组蛋白成功诱导表达,并初步证明了重组蛋白具有抑制肿瘤细胞增殖活性的功能,为后续抗肿瘤机制的探究奠定了基础。