OBJECTIVETo reveal the quality variation of polysaccharides, triterpenoids and proteins in spores and fruiting bodies of Ganoderma lucidum from producing areas, different varieties, harvesting parts and periods, and wall-breaking treatments.

METHODSpores and fruiting bodies from varieties of Longzhi No. 1 and Hunong No. 1 were collected as test samples, together with wall-broken spores sold in domestic main producing areas. The anthrone-sulfuric acid colorimetric method was used to determine the content of total polysaccharides. The vanillin-glacial acetic acid-perchloric acid colorimetric method was used to determine the content of total triterpenoids. The Lowry method was used to determine the content of total proteins.

RESULTThe content ranges of total polysaccharides, total triterpenoids, and total proteins from 6 domestic main producing areas were 0.40% - 2.25%, 1.36%-3.15% and 0.74% -1.91% respectively. The content ranges of total polysaccharides, triterpenoids, and proteins in the fruiting bodies from 2 varieties cultured in Zhejiang were 0.25% -1.42%, 0.44% -1.42% and 1.82% -3.67% respectively. In addition, the ranges of samples from wall-unbroken spores were 0.41% - 0.91%, 0.09% - 0.12%, 0.78% - 0.90% respectively and wall-broken spores are 1.03% - 2.25%, 1.89% - 3.15%, 0.96% - 1.04% respectively.

CONCLUSIONThere are significant differences in the contents of main chemical ingredients of wall-broken G. lucidum spores saled in the markets. The samples from Zhejiang contain high content of total polysaccharides and triterpenoids, and samples from Fujian contains more proteins. Between the 2 major varieties cultured in Zhejiang, Longzhi No. 1 contains higher content of triterpenoids, but Hunong No. 1 has more polysaccharides. Contents of triterpenoids and polysaccharides from wall-broken spores are much higher than those of fruiting bodies. The stipes from fruiting bodies contains more polysaccharides than those of the pileus, while the triterpenoids contents are higher in the pileus than stipes. The pileus and stipes collected in the second year contain higher content of polysaccharides than the first year's samples, but the contents of triterpenoids are lower. Wall-breaking treatment would significantly improve the extraction and dissolution rate of total triterpenoids and polysaccharides.

Fungal Proteins ; analysis ; Polysaccharides ; analysis ; Reishi ; chemistry ; Spores, Fungal ; chemistry ; Triterpenes ; analysis

OBJECTIVETo analysis the nutrient and effective ingredients of in Cordyceps militaris and make the best use of its medical value.

METHODAdenosine, cordycepin, polysaccharides, cordyceps acid, protein and fat in different parts of C. militaris were extracted, they are quantified by HPLC and other colorimetric analysis.

RESULTThe contents of polysaccharide was found to be 86.49 mg x g(-1) in C. militaris, 6.82 mg x g(-1) of adenosine in stroma, 13.28 mg x g(-1) of cordycepin and 44.07 mg x g(-1) of cordyceps acid in sclerolium.

CONCLUSIONIn different parts of C. militaris, the biosynthesis of effective ingredients is different. The total amount of effective ingredients is highest in C. militaris, the production of cordycepin and cordyceps acid is highest in sclerotium in comparison with other parts. Growth of C. militaris largely relies on its capability to utilize fat and protein from silkworm.

Adenosine ; analysis ; Animals ; Bombyx ; chemistry ; microbiology ; Cordyceps ; chemistry ; Deoxyadenosines ; analysis ; Fungal Proteins ; analysis ; Polysaccharides ; analysis

OBJECTIVETo compare the difference in protome expression between yeast form and mould form of Penicillium marneffei.

METHODSSurface enhanced laser desorption/ionization (SELDI) time-of-flight mass spectra were performed to compare the expressed proteins between yeast form and mould form of Penicillium marneffei. Protein profiling was read by PBSIIC ProteinChip Reader and the proteome database was analyzed by Proteinchip Software 3.2.0.

RESULTSSeventy-five distinct proteins were found in the yeast form and mould form of Penicillium marneffei, in which 10 proteins were up-regulated in yeast form and 3 in mould form. The proteins 2900 and 3151 were only expressed in the yeast form and the proteins 13,151 and 13,285 only in mould form.

CONCLUSIONSELDI technique can identify the difference of the expressed low-molecular-mass proteins between the mould form and yeast form of Penicillium marneffei.

Fungal Proteins ; analysis ; Penicillium ; growth & development ; metabolism ; Protein Array Analysis ; methods ; Proteome ; analysis ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods ; Spores, Fungal ; metabolism ; Yeasts ; metabolism

BACKGROUNDCandida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.

METHODSCandida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSA total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1), genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.

CONCLUSIONSThe up-regulation of the gene encoding the multidrug resistance efflux pump CDR1 may contribute to the terbinafine resistance in Candida albicans. However, the precise roles of other affected genes remain unclear, further studies of these genes and their respective products that play roles in the context of antifungal resistance are warranted.

Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; genetics ; Ergosterol ; biosynthesis ; Fungal Proteins ; genetics ; Gene Expression Profiling ; Genome, Fungal ; Membrane Transport Proteins ; genetics ; Naphthalenes ; pharmacology ; Oligonucleotide Array Sequence Analysis

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.
Cordyceps ; chemistry ; Desiccation ; methods ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; analysis ; Molecular Weight

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.
Actins/analysis ; Animals ; Cytoskeletal Proteins/*analysis ; Fungal Proteins/*analysis ; Histocytochemistry ; Microscopy, Immunoelectron ; Pneumocystis/*chemistry/cytology ; Rats ; Rats, Wistar ; Support, Non-U.S. Gov't ; Tropomyosin/analysis ; Tubulin/analysis

In this study, we analyzed the physical interactions of the dominant negative isoform of MoYpt7. Our results show that MoYpt7 interacts with MoGdi1. The dominant negative isoform of MoYpt7 (dominant negative isoform, N125I) is essential for colony morphology, conidiation, and pathogenicity in the rice blast fungus. These results further demonstrate the biological functions of MoYpt7 in Magnaporthe oryzae.
DNA Mutational Analysis ; Fungal Proteins/metabolism* ; Gene Expression Regulation, Fungal ; Genes, Fungal ; Green Fluorescent Proteins/metabolism* ; Magnaporthe/genetics* ; Microscopy, Fluorescence ; Mutation ; Oryza/microbiology* ; Phenotype ; Plant Diseases/microbiology* ; Protein Isoforms

Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles of appressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST (expressed sequence tag) database of M. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTH11, beta subunit of G protein and SGT1 involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results.
Cell Proliferation ; Fungal Proteins ; metabolism ; Fungal Structures ; metabolism ; Gene Expression Profiling ; methods ; Magnaporthe ; metabolism ; Oligonucleotide Array Sequence Analysis ; methods ; Proteome ; metabolism

In order to investigate the effects of fungal elicitor and macroporous adsorption resin on shikonin accumulation in hairy roots of arnebia euchroma (Royle) Johnst, we used spectrophotometry to determine the total naphthoquinone content of the hairy roots, by adding different volume ratio of Aspergillus niger elicitor, Aspergillus oryzae elicitor, and the macroporous resin into the M-9 liquid medium at different culture time. The results show that the total naphthoquinone content was 2.28 times higher than the control when we added mixed elicitors of Aspergillus niger and Aspergillus oryzae at the ratio of 2.5:50 in the 10th day of hairy roots cultivating. The total naphthoquinone content was 3.71 times higher than that of the control, when we added macroporous adsorption resin NKA-9. Aspergillus niger elicitor exhibited synergistic effect with Aspergillus oryzae elicitor to enhance the naphthoquinone. Also, the total naphthoquinone level was 4.17 times higher than that of the control by adding mixed fungal elicitor and macroporous adsorption resin NKA-9 in the bioreactor. Aspergillus oryzae and mixed elicitor could promote the hairy roots proliferation, and macroporous adsorption resin NKA-9 and mixed elicitor increased the total naphthoquinone content. In summary, the measure developed for Arnebia euchroma (Royle) Johnst hairy roots cultivating in bioreactors may potential for large-scale production of naphthoquinone.
Aspergillus niger ; metabolism ; Boraginaceae ; metabolism ; Fungal Proteins ; pharmacology ; Naphthoquinones ; analysis ; metabolism ; Plant Roots ; chemistry ; metabolism ; Porosity ; Resins, Synthetic ; pharmacology

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.
Bacterial Proteins ; biosynthesis ; genetics ; Fermentation ; Fungal Proteins ; biosynthesis ; genetics ; Lipase ; biosynthesis ; genetics ; Oxygen ; analysis ; pharmacology ; Pichia ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; Truncated Hemoglobins ; biosynthesis ; genetics