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College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea
Sang Mong Lee
Department of Sericultural and Entomological Biology, Miryang National University, Miryang 627-130, Korea
Eunju Park
Division of Life Sciences, Kyungnam University, Masan 631-260, Korea
Iksoo Kim
Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea
Yeon Ho Je
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
Byung Rae Jin 1
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea
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ABSTRACT |
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We describe the molecular characterization of the Cu,Zn superoxide dismutase (SOD1) gene of Cordyceps militaris, which is one of the entomopathogenic fungi called a vegetable wasp and plant worm. The SOD1 gene of C. militaris spans 922 bp and consisted of three introns and four exons coding for 154 amino acid residues. The deduced amino acid sequence of the C. militaris SOD1 cDNA showed 88% identity to Claviceps purpurea SOD1, 82% to Neurospora crassa SOD1, and 75–64% to SOD1 sequences from other fungi. The C. militaris SOD1 possesses the typical metal binding ligands of six histidines and one aspartic acid common to fungal SOD1s. The cDNA encoding C. militaris SOD1 was expressed as a 17-kDa polypeptide in the baculovirus-infected insect Sf9 cells. The enzyme activity of the purified recombinant C. militaris SOD1 was approximately 568 U per mg–1. Southern blot analysis of the genomic DNA suggested the C. militaris SOD1 was a single gene. Northern and Western blot analysis and enzyme activity assays indicated SOD1 was expressed constitutively. This is the first report of an SOD1 gene from any entomopathogenic fungus.
Key words: Cordyceps militaris, Cu, Zn superoxide dismutase (SOD1), entomopathogenic fungus, SOD gene
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Dalton et al 1999).
The first line of defense against ROS includes the enzymatic activity of superoxide dismutase (SOD) that catalyzes the disproportion of superoxide to hydrogen peroxide and water (McCord and Fridovich 1969, Fridovich 1986). SOD mainly removes highly toxic O2– and also prevents O2– mediated reduction of iron and subsequent OH– generation. The SOD enzyme is classified into three forms based on metal requirements of the active site namely Cu,Zn SOD (SOD1), Mn-SOD (SOD2) and Fe-SOD. Cu,Zn SOD is found primarily in the cytosol of eukaryotes (Crapo et al 1992). Mn-SOD is present in the mitochondria of both prokaryotes and eukaryotes, and Fe-SOD is found in both eubacteria and archaebacteria (Fridovich 1995).
In the case of SOD1, this enzyme binds on one copper and one zinc ion and displays the Greek Key ß-barrel fold (Tainer 1982). SOD1 was found in green algae and in all higher eukaryote species (Fridovich 1995). Also SOD1 genes have been identified in numerous species of fungi, plants, insects and mammals. Molecular characterization of SOD1 has been investigated in various fungal species (Chary et al 1990, Oberegger et al 2000, Chaturvedi et al 2001, Moore et al 2002). Mutants of the yeast Saccharomyces cerevisiae and the filamentous fungus Neurospora crassa lacking SOD1 are sensitive to oxygen and superoxide generating agents (Chary et al 1994, Jamieson et al 1994). SOD1 of Aspergillus fumigatus has been shown to be a valuable immunodiagnostic marker for Aspergillus infections (Holdom et al 2000). Characterization of an SOD1 gene knock-out mutant of Cryptococcus neoformans var. gattii revealed that SOD1 is directly involved in the virulence of this fungal pathogen (Narasipura et al 2003).
A great variety of entomopathogenic fungi have been described and several of them are being investigated for their pharmaceutical use (Yamada 1984, Samson et al 1988, Montefiori et al 1989, Carlile and Watkinsom 1996, Shimizu 1997). Of these entomopathogenic fungi, "vegetable wasp and plant worm" as a common name is an interesting subject for pharmaceutical use. In general "vegetable wasp and plant worm" refers to the fruiting body growing on the larval, pupal or adult integument of the insect host. One such insect-born mushroom is produced from the larvae or pupae attacked by entomopathogenic fungi, mainly Cordyceps species, a member of the Hhypocreales order of the Ascomycetes. They infect the larva, pupa or imago of insects, kill them and then form a fruiting body on the insect. In the Orient, some Cordyceps species are one of the most potent herbs in traditional medicine and are used widely as a tonic for longevity, endurance and vitality. It has shown that Cordyceps species can produce many kinds of bioactive compounds, and the medicinal benefits of Cordyceps species were reported (Kneifel et al 1977; Furuya et al 1983; Xu et al 1992; Kuo et al 1996; Zhu et al 1998a, b; Nakamura et al 1999; Zhao et al 2000). Because of its importance in traditional medicine, C. militaris is mass-produced in the silkworm, Bombyx mori, pupae (Lee et al 2001).
Fungal SOD1 genes have been studied from various species but not from any entomopathogenic fungi. The antioxidant activities of the SOD1 in fruiting bodies of C. sinensis have been reported without isolating the corresponding gene (Yamaguchi et al 2000). This paper describes the complete nucleotide sequence and the exon/intron structure of the SOD1 gene from C. militaris, which is one of the most valued Cordyceps spp. The C. militaris SOD1 cDNA was expressed in baculovirus-infected insect cells, and the activity of the purified recombinant enzyme was determined. Furthermore, SOD1 expression in C. militaris is analyzed. This is the first report of molecular cloning, expression and characterization of an SOD1 gene from an entomopathogenic fungus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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.
cDNA library screening, nucleotide sequencing and data analysis.— –
A cDNA library was constructed using poly(A)+ mRNA isolated from the fruiting bodies of C. militaris in the vector Uni-ZAP XR (Stratagene, La Jolla, California). Sequencing of randomly selected clones harboring cDNA inserts was performed to generate a library of expressed sequence tags (ESTs). For DNA sequencing, plasmid DNA was extracted using the Wizard minipreparation kit (Promega, Madison, Wisconsin). Sequence of each cDNA clone was determined using an automatic sequencer (model 310 Genetic Analyzer; Perkin-Elmer Applied Biosystems, Foster City, California). The sequences were compared using the DNASIS and BLAST programs provided by the NCBI. GenBank, EMBL and SwissProt databases were searched for sequence homology using a BLAST algorithm program. MacVector (version 6.5, Oxford Molecular Ltd.) was used to align the amino acid sequences of SOD1. Phylogenetic analysis of fungal SOD1 amino acid sequences was performed using PAUP (Phylogenetic Analysis Using Parsimony) version 4.0 (Swofford 2000).
Genomic DNA isolation, PCR of the SOD1 gene and Southern blot analysis.— – Genomic DNA was extracted from the fruiting body of C. militaris using a WizardTM Genomic DNA Purification Kit, according to the manufacturer’s instructions (Promega). The primers used for amplification of a genomic DNA encoding the SOD1 were 5'-GACAAAAT-CATCCAAAATGGTCAAAGCAG-3' for the translational start sequence region and 5'-GCCTCTTAGTTGGCGA-CGCCAATGACAC-3' for the 3' coding region. The genomic DNA was then used as a template for PCR. After a 35-cycle amplification (94 C for 30 s, 50 C for 40 s, 72 C for 2 min), PCR products were analyzed with 1.0% agarose gel electrophoresis. The PCR product then was purified with a PCR Purification Kit (QIAGEN) and cloned into the plasmid vector pGem-T (Promega).
Genomic DNA extracted from the fruiting body of C. militaris was digested with Sal I, Eco RV, Hind III, Sma I or Xba I and electrophoresed in 1.0% agarose gel. The DNA from the gel was transferred onto a nylon blotting membrane (Schleicher & Schuell, Dassel, Germany) and hybridized at 42 C with a probe in a hybridization buffer containing 5 x SSC, 5 x Denhardt’s solution, 0.5% SDS, and 100 µg/mL denatured salmon sperm DNA. The 652 bp SOD1 cDNA clone was labeled with [
-32P] dCTP (Amersham, Arlington Heights, Illinois) using the Prime-It II Random Primer Labeling Kit (Stratagene) for use as a probe for hybridization. After hybridization, the membrane filter was washed three times for 30 min each in 0.1% SDS and 0.2 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate) at 65 C and exposed to autoradiography film.
RNA isolation and Northern blot analysis.— –
Total RNA was isolated from mycelium (1 wk postinoculation) and fruiting body (2 or 3 wk p.i.) of C. militaris by using the Total RNA Extraction Kit (Promega). Total RNA (10 µg/lane) from C. militaris was denatured by glyoxalation (McMaster and Carmichael 1977), transferred onto a nylon blotting membrane (Schleicher & Schuell) and hybridized at 42 C with a probe in a hybridization buffer. Hybridization condition, fragment labeling and filter washing were as described for the Southern blot analysis.
Construction of baculovirus transfer vector.— – The 652 bp C. militaris SOD1 cDNA from pBlueScript-CmSOD1 was sub-cloned between the Eco RI and Xho I sites of pBacPAK9 (Clontech, Palo Alto, California) to produce the transfer vector pBacPAK9-CmSOD1. In the this vector, the C. militaris SOD1 cDNA is under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV).
Cell culture and construction of recombinant virus.— –
The Spodoptera frugiperda IPLB Sf21-AE (Vaughn et al 1977) clone 9 (Sf9) cells were maintained at 27 C in TC100 medium (GIBCO BRL LIFE Technologies, Gaithersburg, Maryland), supplemented with 10% fetal bovine serum (FBS; GIBCO BRL LIFE Technologies) as described by standard methods (O’Reilly et al 1992). Wild-type AcNPV and recombinant AcNPV were propagated in Sf9 cells. The titer was expressed as plaque-forming units (PFU) per mL (O’Reilly et al 1992).
For the construction of recombinant virus, 35 mm cell culture dishes were seeded with 1.0–1.5 x 106 cells and incubated at 27 C for 1 h to allow cell attachment. One microgram of BacPAK6 viral DNA (Clontech), 5 µg of p-BacPAK9-CmSOD1 in 20 mM HEPES buffer in a final volume of 50 µL were mixed in a polystyrene tube. Fifty microliters of 100 µg/mL LipofectinTM (GIBCO BRL LIFE Technologies) were mixed gently with the DNA solution, and the mixture was incubated at room temperature for 30 min. The cells were washed twice with 2 mL serum-free TC100 medium and refed with 1.5 mL serum-free TC100 medium. The Lipofectin-DNA complexes were added drop-wise to the medium covering the cells while the dish was gently swirled. After incubation at 27 C for 5 h, TC100 medium containing antibiotics and 10% FBS was added to each dish and incubation at 27 C was continued. At 5 d p.i., the supernatant was harvested, clarified by centrifugation at 2000 rpm for 5 min, and stored at 4 C. Recombinant AcNPV was plaque purified on six well plates seeded with 1.5 x 106 Sf9 cells as described by O’Reilly et al (1992).
SDS-polyacrylamide gel electrophoresis (PAGE).— –
Insect Sf9 cells were mock-infected or infected with the wild-type AcNPV and recombinant AcNPV in a 35 mm diam dish (1 x 106 cells) at a multiplicity of infection (MOI) of 5 PFU per cell. After incubation at 27 C, cells were harvested at 1, 2 and 3 d p.i. The harvested cells were washed twice with PBS and mixed with protein sample buffer and boiled. Total cellular lysates of C. militaris were prepared from mycelium (1 wk p.i.) and fruiting body (2 or 3 wk p.i.) and mixed with protein sample buffer and boiled. All samples were subjected to 12% SDS-PAGE (Laemmli 1970).
Purification of recombinant C. militaris SOD1.— – Insect Sf9 cells were infected with the recombinant AcNPV expressing C. militaris SOD1 at a MOI of 5 PFU per cell. The cells were harvested at 3 d p.i., sonicated, and clarified by centrifugation (10 000 g) at 4 C for 10 min. The supernatant was adjusted to 1 M ammonium sulphate and applied to a HiTrap desalting column (Pharmacia LKB) equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM PMSF with a flow rate of 0.5 mL/min. The recombinant C. militaris SOD1-enriched fractions, identified by SDS-PAGE, were subjected to gel permeation chromatography on a Superdex 200 HR 10/30 column (Pharmacia LKB) in 20 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM PMSF with a flow rate of 0.5 mL/min. Fractions containing purified recombinant C. militaris SOD1 were identified by SDS-PAGE and enzyme activity.
Preparation of polyclonal antiserum and Western blot analysis.— – The purified recombinant C. militaris SOD1 (about 10 µg) was mixed with equal volume of Freund’s complete adjuvant (a total of 200 µL, Sigma) and injected into Balb/c mice. Three successive injections were performed with 1 wk interval beginning a week after the first injection with antigens mixed with equal volume of Freund’s incomplete adjuvant (a total of 200 µL, Sigma). Blood was collected 3 d after the last injection and centrifuged at 13 000 rpm for 5 min. The supernatant antibodies were stored at –70 C until use.
Western blot analysis was carried out using ECL Western blotting analysis system, according to the manufacturer’s instructions (Amersham Pharmacia Biotech). SDS-PAGE was carried out as described above. Proteins were blotted to a sheet of nitrocellulose transfer membrane (Schleicher & Schuell) (Towbin et al 1979). The blotting was performed in transfer buffer consisting of 25 mM Tris and 192 mM glycine in 20% methanol at 30 volts overnight at 4 C. After blotting the membrane was blocked by incubation in 1% BSA solution 2 h at room temperature. The blocked membrane was incubated with antiserum solution (1:1000 v/v) 1 h at room temperature and washed in TBST (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.05% Tween 20). The membrane then was incubated with antimouse IgG horseradish peroxidase (HRP) conjugate and HRP-streptavidin complex. After repeated washing, the membrane was incubated with ECL detection reagents (Amersham Pharmacia Bio-tech) and exposed to autoradiography film.
Determination of enzyme activity.— – The activity of the purified recombinant C. militaris SOD1 and extracts from mycelium (1 wk p.i.) or fruiting body (2 or 3 wk p.i.) was determined using a commercial assay kit (SOD-test Wako; Wako Pure Chemicals, Osaka, Japan) according to the manufacturers’ instructions. The SOD1 assay system is formatted for measuring the percentage of nitroblue tetrazolium reduced by superoxide anion generated by xanthine plus xanthine oxidase. The units of SOD1 activity were derived from a standard curve constructed using authentic SOD1 (Sigma). The SOD1 activity is expressed as units mg–1 protein. The protein concentration was determined by using BCA protein assay reagent kit (Pierce Co.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. The C. militaris SOD1 cDNA contains an open reading frame of 462 bp encoding 154 amino acid residues.
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. Comparison of the genomic sequence with the sequence of the SOD1 cDNA revealed the presence of four exons and three introns (FIG. 1B). The sequences at the exon-intron boundaries confirmed to the consensus sequences (Roberts et al 1988, Chary et al 1990), including an invariant GT at the intron 5' boundary and an invariant AG at its 3' boundary.
Comparison of the C. militaris SOD1 deduced amino acid sequence with other fungal SOD1 sequences is shown in FIG. 2. The residues maintaining the active site geometry, Gly45, Gly62, Pro75, Gly83, Gly139 and Gly142, and the metal binding sites, His47, 49, 64, 121 for copper and His64, 72, 81 and Asp84 for zinc, were conserved in the C. militaris SOD1 (Chary et al 1990). Arg144, considered as an important residue for SOD1 enzyme activity (Malinowski and Fridovich 1979, Borders et al 1985), and two cysteine residues at positions 58 and 147, which form an intrachain disulfide bridge, all were conserved in the C. militaris SOD1 sequence.
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). Within the Ascomycetes fungal clade, the C. militaris, C. purpurea and N. crassa SOD1s formed a subgroup. The C. militaris SOD1 amino acid sequence was 88% and 82% identical to those of C. purpurea and N. crassa respectively. The protein sequence identity of C. militaris SOD1 to the other fungal SOD1s ranged from 75% to 64%.
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To examine the expression of C. militaris SOD1 by recombinant virus in insect cells, SDS-PAGE and Western blot analysis were performed to analyze the protein synthesis in Sf9 cells infected with the recombinant virus (FIG. 4). The recombinant C. militaris SOD1 was present as a single band of about 17 kDa polypeptide in the cells infected with the recombinant virus, but not in the cells infected with the wild-type AcNPV or mock-infected cells.
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). The recombinant C. militaris SOD1 was purified as a single band of 17 kDa by SDS-PAGE with an enzymatic activity of approximately 568 U mg-1 protein (FIG. 5, TABLE I).
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).
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). SOD1 transcripts and protein was detected at all three growth stages. Enzyme activity also was analyzed and was 14.7, 15.7 and 18.4 U mg–1 protein at 1, 2 and 3 wk p.i. respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Bordo et al 1994, Chaturvedi et al 2001), representing that the regions are involved in the metal binding and catalysis. His47, 49, 64 and 121 theoretically function as copper binding sites, whereas His64, 72, 81 and Asp84 theoretically function as zinc binding sites. Cys58 and 147 are believed to be involved in disulfide bond formation, while Arg144 is believed to be necessary to guide the superoxide radical to the active site (Malinowski and Fridovich 1979, Borders et al 1985, Parker et al 1986, Chaturvedi et al 2001). Among the known SOD1 sequences, C. militaris SOD1 was closest to C. purpurea (88% protein sequence identity), which causes ergot disease on a wide range of host grasses and belongs to the hypocreals of the ascomycetes, and showed high protein sequence identity to fungal SOD1 sequences ranging from 82% to 64%. The amino acid sequence identity further reflected on the phylogenetic relationships. Fungal SOD1 is divided into two separate subclades, ascomycetes and basidiomycetes (Chaturvedi et al 2001). The C. militaris SOD1 clustered with fungal SOD1 and formed a subgroup with C. purpurea SOD1 and N. crassa SOD1 within the ascomycetes subclade.
The C. militaris SOD1 genomic DNA consisted of three introns and four exons coding for 154 amino acid residues. This size of 154 amino acid residues is conserved in SOD1 genes known from the ascomycetes and basidiomycetes. Although C. militaris and N. crassa (Chary et al 1990) SOD1 genes are consisted of four exons, C. purpurea (Moore et al 2002) and three varieties of C. neoformans (Chaturvedi et al 2001) are composed of six exons. In terms of intron boundaries, furthermore, the consensus sequences were well conserved in these four species.
The genomic organization of the SOD1 gene in C. militaris analyzed by Southern blot hybridization clearly showed a single band under high-stringency conditions when five restriction enzymes, which have no internal digesting site within C. militaris SOD1 gene, were employed. This single hybridization signal suggests that the SOD1 gene exists as a single copy in C. militaris genome, as is true for N. crassa (Chary et al 1990) and C. neoformans (Chaturvedi et al 2001).
The C. militaris SOD1 cDNA was expressed in the insect Sf9 cells and the recombinant C. militaris SOD1 is detected as a band with approximately 17 kDa polypeptide in SDS-PAGE. Furthermore, the purified recombinant C. militaris SOD1 showed activity of approximately 568 U mg–1 protein.
The expression of C. militaris SOD1 with growth stage performed by Northern blot analysis revealed that the C. militaris SOD1 mRNA was expressed from mycelium to fruiting body and its expression was not strong after forming of fruiting body. Similarly, enzyme assay and Western blot analysis confirmed an increase of SOD1 activity with growth stage and the presence of about 17 kDa SOD1 polypeptide in both mycelium and fruiting body. It is likely that the C. militaris SOD1 is synthesized for the entire growth stage, suggesting that C. militaris SOD1 plays an important role in protection against oxidative damage during the entire growth period. Although experiments have not been performed to assay the SOD1 enzyme activity in the infection and/or growth stage of Cordyceps species, previous studies reported that SOD1 of A. fumigatus has been shown to represent a valuable immunodiagnostic marker for Aspergillus infections by proving the expression of this enzyme during pathogenic growth (Holdom et al 2000) and the major SOD1 of the phytopathogen C. purpurea is not essential for pathogenicity and is expressed during the entire infection period (Moore et al 2002).
In conclusion the gene cloning, expression and molecular characterization of a novel SOD1 in C. militaris are reported in this study. The results might provide a framework for future studies on the SOD1 in entomopathogenic fungi.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: brjin{at}daunet.donga.ac.kr
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LITERATURE CITED |
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Bordo D, Djinovic K, Bolognedi M. 1994. Conserved patterns in the Cu,Zn superoxide dismutase family. J Mol Biol 238:366–386.[CrossRef][Medline]
Carlile MJ, Watkinsom SC. 1996. The fungi. New York: Academic Press. p 1–152.
Chary P, Dillon D, Schroeder AL, Natvig DO. 1994. Superoxide dismutase (sod-1) null mutants of Neurospora crassa: oxidative stress sensitivity, spontaneous mutation rate and response to mutagens. Genetics 137:723–730.[Abstract]
———, Hallewell RA, Natvig DO. 1990. Structure, exon pattern, and chromosome mapping of the gene for cytosolic copper-zinc superoxide dismutase (sod-1) from Neurospora crassa. J Biol Chem 265:18961–18967.
Chaturvedi S, Hamilton AJ, Hobby P, Zhu G, Lowry CV, Chaturvedi V. 2001. Molecular cloning, phylogenetic analysis and three-dimensional modeling of Cu,Zn superoxide dismutase (CnSOD1) from three varieties of Cryptococcus neoformans. Gene 268:41–51.[CrossRef][Medline]
Crapo JD, Oury T, Rabouille C, Slot JW, Chang LY. 1992. Copper, zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc Natl Acad Sci USA 89:10405–10409.
Dalton TP, Shertzer HG, Puga A. 1999. Regulation of gene expression by reactive oxygen. Annu Rev Pharmacol Toxicol 39:67–101.[CrossRef][Medline]
Fridovich I. 1986. Superoxide dismutases. Adv Enzymol Rel Areas Mol Biol 58:61–97[Medline]
———. 1995. Superoxide radical and superoxide dismutase. Ann Rev Biochem 64:97–112.[CrossRef][Medline]
Furuya T, Hirotani M, Matsuzawa M. 1983. N6-(2-hydroxyethyl)adenosine, a biologically active compound from cultured mycelia of Cordyceps and Isaria species. Phytochemistry 22:2509–2512.[CrossRef]
Holdom MD, Lechenne B, Hay RJ, Hamilton AJ, Monod M. 2000. Production and characterization of recombinant Aspergillus fumigatus Cu,Zn superoxide dismutase and its recognition by immune human sera. J Clin Microbial 38:558–562.
Jamieson DJ, Rivers SL, Stephen DW. 1994. Analysis of Saccharomyces cerevisiae proteins induced by peroxide and superoxide stress. Microbiology 140:3277–3283.[Abstract]
Kneifel H, Konig WA, Loeffler W, Muller R. 1977. Ophio-cordin, an antifungal antibiotics of Cordyceps ophioglossoides. Arch Microbiol 113:121–130.[CrossRef][Medline]
Kuo YC, Tsai WJ, Shiao MS, Chen CF, Lin CY. 1996. Cordyceps sinensis as an immunomodulatory agent. Am J Chin Med 24:111–125.[CrossRef][Medline]
Laemmli UK. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685.[CrossRef][Medline]
Lee SM, Park NS, Cho SY, Hwang JS, Jin BR. 2001. Production of the wild entomopathogenic fungi, Cordyceps militaris, in the silkworm, Bombyx mori. Int J Indust Entomol 3:105–108.
Malinowski DP, Fridovich I. 1979. Chemical modification of arginine at the active site of the bovine erythrocyte superoxide dismutase. Biochemistry 18:5909–5917.[CrossRef][Medline]
McCord JM, Fridovich I. 1969. The utility of superoxide dismutase in studying free radical reactions. I. Radicals generated by the interaction of sulfite, dimethyl sulfoxide, and oxygen. J Biol Chem 244:6056–6063.
McMaster GK, Carmichael GG. 1977. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74:4835–4838.
Montefiori DC, Sobol Jr RW, Li SW, Reichenbach NL, Suhadolnik RJ, Charubala R, Pfleiderer W, Modliszewski A, Robinson Jr WE, Mitchill WM. 1989. Phosphorothioate and cordycepin analogues of 2',5'-oligoadenylate: Inhibition of human immunodeficiency type I reverse transcriptase and infection in vitro. Proc Natl Acad Sci USA 86:7191–7194.
Moore S, de Vries OMH, Tudzynski P. 2002. The major Cu,Zn SOD of the phytopathogen Claviceps purpurea is not essential for pathogenicity. Mol Plant Pathol 3: 9–22.
Mount DW. 1996. Reprogramming transcription. Nature 383:763–764.[CrossRef][Medline]
Nakamura K, Yamaguchi Y, Kagota S, Kwon YM, Shinozuka K, Kunitomo M. 1999. Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis on Lewis lung carcinoma and B16 melanoma cells in syngeneic mice. Jpn J Pharmacol 79:335–341.[CrossRef][Medline]
Narasipura SD, Ault JG, Behr MJ, Chaturvedi V, Chaurvedi S. 2003. Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence. Mol Microbiol 47:1681–1694.[CrossRef][Medline]
Oberegger H, Zadra I, Schoeser M, Haas H. 2000. Iron starvation leads to increased expression of Cu/Zn-superoxide dismutase in Aspergillus. FEBS Lett 485:113–116.[CrossRef][Medline]
O’Reilly DR, Miller LK, Luckow VA. 1992. Baculovirus Expression Vectors: A Laboratory Manual, New York: WH Freeman & Co.
Parker MW, Bossa F, Barra D, Bannister WH, Bannister JV. 1986. In: Rotilio G, ed. Superoxide and superoxide dismutase in chemistry, biology and medicine. Amsterdam: Elsevier Sci Publ. p 237–245.
Roberts AN, Berlin V, Hager KM, Yanofsky C. 1988. Molecular analysis of a Neurospora crassa gene expressed during conidiation. Mol Cell Biol 8:2411–2418.
Samson R, Evans C, Large JP. 1988. Atlas of entomopathogenic fungi. Berlin: Springer-Verlag.
Shimizu D. 1997. Illustrated vegetable wasps and plant worms. Tokyo: Seibundo Shinko-sha.
Steinman HM. 1980. The amino acid sequence of copper-zinc superoxide dismutase from Bakers’ yeast. J Biol Chem 255:6758–6765.
Swofford DL. 2000. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. MA: Sinauer Sunderland.
Tainer JA, Getzoff ED, Beem KM, Richardson JS, Richardson DC. 1982. Determination and analysis of the 2 A ° structure of copper, zinc superoxide dismutase. J Mol Biol 160:181–217.[CrossRef][Medline]
Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitro-cellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354.
Vaughn JL, Goodwin RH, Thompkins GJ, McCawley P. 1977. The establishment of two insect cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13:213–217.[Medline]
Xu RH, Peng XE, Chen GZ, Chen GL. 1992. Effects of Cordyceps sinensis on natural killer activity and colony formation of B16 melanoma. Chin Med J 105:97–101.[Medline]
Yamada H. 1984. Structure and antitumor activity of an alkali-soluble polysaccharides from Cordyceps ophiglossoides. Carbohydr Res 125:107–115.[CrossRef][Medline]
Yamaguchi Y, Kagota S, Nakamura K, Shinozuka K, Kunitomo M. 2000. Antioxidant activity of the extracts from fruiting bodies of cultured Cordyceps sinensis. Phytother Res 14:647–649.[CrossRef][Medline]
Zhao LW, Xiao XW, Wei YC. 2000. Inhibitory effect of Cordyceps sinensis and Cordyceps militaris on human glomerular mesangial cell proliferation induced by native LDL. Cell Biochem Funct 18:93–97.[CrossRef][Medline]
Zhu JS, Halpern GM, Jones K. 1998a. The scientific rediscovery of a precious ancient Chinese herbal regimen: Cordyceps sinensis: Part I. J Altern Complement Med 4: 289–303.[Medline]
———, ———, ———. 1998b. The scientific rediscovery of a precious ancient Chinese herbal regimen: Cordyceps sinensis: Part II. J Altern Complement Med 4:429–457.[Medline]
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