scholarly journals Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell

1998 ◽  
Vol 333 (1) ◽  
pp. 129-137 ◽  
Author(s):  
Toshio TOMITA ◽  
Dai ISHIKAWA ◽  
Takayasu NOGUCHI ◽  
Eisaku KATAYAMA ◽  
Yohichi HASHIMOTO
Keyword(s):  
Sucrose Gradient ◽  
Edible Mushroom ◽  
Human Erythrocytes ◽  
Flammulina Velutipes ◽  
Target Cells ◽  
Potassium Ions ◽  
Fruiting Bodies ◽  
Sucrose Gradient Ultracentrifugation ◽  
Ring Shaped Structure ◽  
Cytolytic Action

Flammutoxin has been previously isolated as a cardiotoxic and cytolytic polypeptide of 22 or 32 kDa from the fruiting bodies of the edible mushroom Flammulina velutipes. In the present study, we purified flammutoxin as a single haemolytic protein of 31 kDa and studied the mode of its cytolytic action. (1) Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. (2) Flammutoxin did not lyse human erythrocytes in the presence of non-electrolytes with hydrodynamic diameters of > 5.0 nm, although it caused leakage of potassium ions and swelling of the cells under the same conditions. (3) Experiments including solubilization of cell-bound toxin with 2% (w/v) SDS at 20 °C and subsequent Western immunoblots showed that flammutoxin formed a band corresponding to 180 kDa under the conditions where it lysed erythrocytes. (4) Electron microscopy of flammutoxin-treated human erythrocytes revealed the presence of a ring-shaped structure with outer and inner diameters of 10 and 5 nm, respectively, on the cells. (5) A ring-shaped toxin oligomer of the same dimensions was solubilized from the toxin-treated human erythrocytes with 2% (w/v) SDS at 20 °C and isolated by a sucrose-gradient ultracentrifugation. These data indicated that flammutoxin assembles into a ring-shaped oligomer possessing a hydrophilic pore of 4–5 nm on target cells.

scholarly journals Development of a Transgenic Flammulina velutipes Oral Vaccine for Hepatitis B

2019 ◽  
Vol 57 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Li-Hsin Huang ◽  
Hao-Yeh Lin ◽  
Ying-Tzu Lyu ◽  
Chiau-Ling Gung ◽  
Ching-Tsan Huang
Keyword(s):  
Hepatitis B ◽  
Oral Vaccine ◽  
Expression Level ◽  
Flammulina Velutipes ◽  
Total Soluble Protein ◽  
Control Group ◽  
Fruiting Bodies ◽  
Oral Vaccines ◽  
Virus Surface ◽  
Low Expression

Orally administered fungal vaccines show promise for the prevention of infectious diseases. Edible mushrooms are deemed appropriate hosts to produce oral vaccines due to their low production cost and low risk of gene contamination. However, their low expression level of antigens has limited the potential development of oral vaccines using mushrooms. The low expression level might result from impurity of the transgenic mycelia since dikaryotic mycelia are commonly used as transformation materials. In this study, stable transgenic hepatitis B virus surface antigen (HBsAg) in Flammulina velutipes transformants was obtained by Agrobacterium-mediated transformation, followed by fruiting and basidiospore mating. The formation of HBsAg was detected by western blot analysis. The expression levels of HBsAg in transgenic F. velutipes fruiting bodies were (129.3±15.1), (110.9±1.7) and (161.1±8.5) ng/g total soluble protein. However, the values may be underestimated due to incomplete protein extraction. Two of the four pigs in the experimental group produced positive anti-HBsAg-specific IgG after being fed the HBsAg transgenic F. velutipes fruiting bodies for 20 weeks, while no anti-HBsAg antibody was detected in the control group. One of the positive pigs had HBsAg titres of 5.36 and 14.9 mIU/mL in weeks 10 and 14, respectively, but expression faded thereafter. The other positive pig displayed HBsAg titres of 9.75, 17.86 and 39.87 mIU/mL in weeks 14, 18 and 20, respectively. The successful immunogenicity in pigs fed transgenic F. velutipes fruiting bodies demonstrated the potential of using the fungus as an oral vaccine.

Abstract 488: Sinoatrial Nodal Pacemaker Cells (SANC) Exhibit High Basal CA Activated Adenylyl Cyclase (AC) Activity and Express Multiple Distinctly Localized AC Types

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
David R Graham ◽  
Antoine Younes ◽  
Alexey Lyashkov ◽  
Anna Sheydina ◽  
Maria Volkova ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Sucrose Gradient ◽  
Soluble Fraction ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lipid Domains ◽  
Rt Pcr ◽  
Pacemaker Cells ◽  
Pacemaker Function ◽  
Sucrose Gradient Ultracentrifugation

In SANC constituitive AC generates high basal cAMP, inducing PKA-dependent phosphorylation that regulates Ca2+ cycling, that is essential for normal pacemaker function. Our goals were to identify, in rabbit SANC, the types of AC expressed, and their Ca2+ sensitivity and location. Radioimmunoassay (with total phosphodiasterase inhibition) showed a high Ca2+ activated basal AC activity. AC activity increased 5-fold from Ca2+ free (EGTA) to 1 uM free Ca2+. RT PCR (using specifically designed rabbit primers) showed that AC types II and V, and Ca2+ activated types, I and VIII, are expressed in SANC. The organization of these distinct AC types within calveolar or non-calveolar membrane microdomains was determined in pooled SANC isolated from 5 hearts, using triton x100, and sucrose gradient ultracentrifugation. Lipid domains segregated into caveolin containing and non-caveolin containing membrane microdomains, where AC activity was concentrated (fig , AC activity). Immunoblots demonstrated localization of different AC types between these two membrane domains, with AC I, II, V/VI localizing to caveolin containing lipid rafts, and AC VIII present in both caveolin and GM1 lipid domains, and also in the soluble fraction (fig ). In summary, multiple ACs, both Ca2+ activated and non-CA2+ activated types, are expressed in SANC, and these reside in distinct calveolar and non-calveolar lipid domains. We conclude that constituitive basal AC activity is, generated, in part, at least, by a Ca2+ activated AC. type.

scholarly journals Combining transcriptomics and metabolomics to reveal the underlying molecular mechanism of ergosterol biosynthesis during the fruiting process of Flammulina velutipes

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Ruihong Wang ◽  
Pengda Ma ◽  
Chen Li ◽  
Lingang Xiao ◽  
Zongsuo Liang ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Differentially Expressed ◽  
Flammulina Velutipes ◽  
Cdna Libraries ◽  
Ergosterol Biosynthesis ◽  
Biosynthesis Pathway ◽  
Fruiting Bodies ◽  
Regulatory Relationship ◽  
Illumina Hiseq ◽  
Hiv Drugs

Abstract Background Flammulina velutipes has been recognized as a useful basidiomycete with nutritional and medicinal values. Ergosterol, one of the main sterols of F. velutipes is an important precursor of novel anticancer and anti-HIV drugs. Therefore, many studies have focused on the biosynthesis of ergosterol and have attempted to upregulate its content in multiple organisms. Great progress has been made in understanding the regulation of ergosterol biosynthesis in Saccharomyces cerevisiae. However, this molecular mechanism in F. velutipes remains largely uncharacterized. Results In this study, nine cDNA libraries, prepared from mycelia, young fruiting bodies and mature fruiting bodies of F. velutipes (three replicate sets for each stage), were sequenced using the Illumina HiSeq™ 4000 platform, resulting in at least 6.63 Gb of clean reads from each library. We studied the changes in genes and metabolites in the ergosterol biosynthesis pathway of F. velutipes during the development of fruiting bodies. A total of 13 genes (6 upregulated and 7 downregulated) were differentially expressed during the development from mycelia to young fruiting bodies (T1), while only 1 gene (1 downregulated) was differentially expressed during the development from young fruiting bodies to mature fruiting bodies (T2). A total of 7 metabolites (3 increased and 4 reduced) were found to have changed in content during T1, and 4 metabolites (4 increased) were found to be different during T2. A conjoint analysis of the genome-wide connection network revealed that the metabolites that were more likely to be regulated were primarily in the post-squalene pathway. Conclusions This study provides useful information for understanding the regulation of ergosterol biosynthesis and the regulatory relationship between metabolites and genes in the ergosterol biosynthesis pathway during the development of fruiting bodies in F. velutipes.

scholarly journals In vitro Cultivation of Newly Reported Wild Edible Mushroom Volvariella bomybycina from Nepal

2017 ◽  
Vol 5 (1) ◽  
pp. 27-31
Author(s):  
Pradip Kumar Chaudhary ◽  
Mitesh Shrestha ◽  
Bal Hari Poudel ◽  
Mahesh Kumar Adhikari
Keyword(s):  
Natural Habitat ◽  
Culture Method ◽  
Edible Mushroom ◽  
Edible Mushrooms ◽  
In Vitro Cultivation ◽  
Fruiting Bodies ◽  
Wild Edible Mushrooms ◽  
Tissue Culture Method ◽  
Wild Edible Mushroom

Wild edible mushrooms are becoming endangered all over the world. Very few wild edible mushrooms are found in natural habitat. Volvariella bombycina is an edible and medicinal mushroom. The mushroom was collected in natural habitat growing on Populus tree. Mycelium of the mushroom was developed in PDA slant tubes by tissue culture method, incubated at 25°C for 1-2 weeks. Spawn was developed in wheat grains after incubation at 25°C for 2-3 weeks. Substrates were formulated for the development of fruiting bodies by combination of paddy straw, saw dust and rice husk. Fruiting bodies of V. bombycina was cultivated in these substrates after incubation at 28 ± 2°C for 2-4 weeks. The work describes the optimized process for in vitro culture of wild edible mushroom Volvariella bomybycina.Nepal Journal of Biotechnology. Dec. 2017 Vol. 5, No. 1: 27-31

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