Monthly Archives: September 2010

Medical Studies

Function of the iron-binding chelator produced by Coriolus versicolor in lignin biodegradation.

Wang L, Yan W, Chen J, Huang F, Gao P.

State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China.

Abstract

An ultrafiltered low-molecular-weight preparation of chelating compounds was isolated from a wood-containing culture of the white-rot basidiomycete Coriolus versicolor. This preparation could chelate Fe3+ and reduce Fe3+ to Fe2+, demonstrating that the substance may serve as a ferric chelator, oxygen-reducing agent, and redox-cycling molecule, which would include functioning as the electron transport carrier in Fenton reaction. Lignin was treated with the iron-binding chelator and the changes in structure were investigated by 1H-NMR, 13C-NMR, difference spectrum caused by ionization under alkaline conditions and nitrobenzene oxidation. The results indicated that the iron-binding chelator could destroy the beta-O-4 bonds in etherified lignin units and insert phenolic hydroxyl groups. The low-molecular-weight chelator secreted by C. versicolor resulted in new phenolic substructures in the lignin polymer, making it susceptible to attack by laccase or manganese peroxidase. Thus, the synergic action of the iron-binding chelator and the lignocellulolytic enzymes made the substrate more accessible to degradation.

PMID: 18246309 [PubMed – indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/pubmed/18246309

Medical Studies

Production of polygalacturonase from Coriolus versicolor grown on tomato pomace and its chromatographic behaviour on immobilized metal chelates.

do Rosário Freixo M, Karmali A, Arteiro JM.

Department of Chemistry, Universidade de Evora, Rua Ramalho Ortigão N 59, Evora, Portugal.

Abstract

Tomato pomace and pectin were used as the sole carbon sources for the production of polygalacturonase from a strain of Coriolus versicolor in submerged culture. The culture of C. versicolor grown on tomato pomace exhibited a peak of polygalacturonase activity (1,427 U/l) on the third day of culture with a specific activity of 14.5 U/mg protein. The production of polygalacturonase by C. versicolor grown on pectin as a sole carbon source increased with the time of cultivation, reaching a maximum activity of 3,207 U/l of fermentation broth with a specific activity of 248 U/mg protein. The levels of different isoenzymes of polygalacturonase produced during the culture growth were analysed by native PAGE. Differential chromatographic behaviour of lignocellulosic enzymes produced by C. versicolor (i.e. polygalacturonase, xylanase and laccase) was studied on immobilized metal chelates. The effect of ligand concentration, pH, the length of spacer arm and the nature of metal ion were studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). The adsorption of these lignocellulosic enzymes onto immobilized metal chelates was pH-dependent since an increase in protein adsorption was observed as the pH was increased from 6.0 to 8.0. The adsorption of polygalacturonase as well as other enzymes to immobilized metal chelates was due to coordination of histidine residues which are available at the protein surface since the presence of imidazole in the equilibration buffer abolished the adsorption of the enzyme to immobilized metal chelates. A one-step purification of polygalacturonase from C. versicolor was devised by using a column of Sepharose 6B-EPI 30-IDA-Cu(II) and purified enzyme exhibited a specific activity of about 150 U/mg protein, final recovery of enzyme activity of 100% and a purification factor of about 10. The use of short spacer arm and the presence of imidazole in equilibration buffer exhibited a higher selectivity for purification of polygalacturonase on this column with a high purification factor. The purified enzyme preparation was analysed by SDS-PAGE as well as by “in situ” detection of enzyme activity.

PMID: 18253772 [PubMed – indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/pubmed/18253772

Medical Studies

Selection of white-rot basidiomycetes for bioconversion of mustard (Brassica compestris) straw under solid-state fermentation into energy substrate for rumen micro-organism.

Tripathi MK, Mishra AS, Misra AK, Vaithiyanathan S, Prasad R, Jakhmola RC.

Division of Animal Nutrition, Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India. mktripathi@gmail.com

Abstract

AIMS: Selection of white-rot fungi of bio-conversion of mustard straw (MS) into feed for ruminants.

METHODS AND RESULTS: Mustard straw was cultured with Ganoderma applanatum, Coriolus versicolor and Phanerochaete chrysosporium for solid-state fermentation at 35 degrees C from 7 to 63 days for delignification and for 21 days to study dry matter digestibility and protein enrichment. Lignin loss in fungus cultured straw varied between 100 and 470 g kg(-1) lignin. Delignification was higher between 7 and 28 days fermentation with C. versicolor. Among the three fungi P. chrysosporium was the most effective in degrading lignin for longer fermentation. In-vitro dry matter digestibility (IVDMD) and crude protein content was higher in C. versicolor cultured straw. Large quantity of straw was cultured by C. versicolor for 21 days, for in vivo evaluation. Mean pH and metabolites of rumen fermentation were not different while, pH and volatile fatty acid increased at 6 h postfermentation on cultured straw feeding. Cultured straw fermentation increased (P = 0.001) small holotricks and reduced (P = 0.005) large holotricks population. Fungus cultures straw did not improve microbial enzyme concentration.

CONCLUSIONS: Coriolus versicolor and P. chrysosporium were the promising fungus for MS bio-delignification.

SIGNIFICANCE AND IMPACT OF THE STUDY: Coriolus versicolor treated MS improved dry matter digestibility and protein content.

PMID: 18266643 [PubMed – indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/pubmed/18266643

Autoimmune Disease, Breast Cancer, Cancer, Leukemia, Medical Studies, PSP

Polysaccharopeptide enhances the anticancer activity of doxorubicin and etoposide on human breast cancer cells ZR-75-30.

Wan JM, Sit WH, Louie JC.

Food and Nutritional Science Division, School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, PR China. jmfwan@hkusua.hku.hk

Abstract

In search of natural bioactive microbial compounds with adjuvant properties, we have previously showed that the polysaccharopeptide (PSP), isolated from Chinese medicinal mushroom Coriolus versicolor, was able to enhance the cytotoxicity of certain S-phase targeted-drugs on human leukemic HL-60 cells via some cell-cycle and apoptotic-dependent pathways. The present study aimed to investigate whether the synergism of mechanisms of PSP with certain chemotherapeutic drugs also applies to human breast cancer. PSP treatment enhanced the cytotoxicity of doxorubicin (Doxo), etoposide (VP-16) but not cytarabine (Ara-C). Bivariate bromodeoxyuridine (BrdUrd)/DNA flow cytometry analysis estimated a longer DNA synthesis time (Ts) for the PSP treated cancerous cells suggesting that PSP enhanced the apoptotic effect of Doxo and VP-16 via creating an S-phase trap in the human breast cancer cell line ZR-75-30. The participation of PSP in the apoptotic machinery of the chemotherapeutic agents was further supported by a reduced ratio of protein expression of Bcl-xL/Bax of the cancer cells. This study provides further insight into the synergistic mechanisms of PSP and supports the hypothesis that the anticancer potentials of PSP is not limited to leukemia but may also be used as an adjuvant therapy for breast cancers.

PMID: 18292947 [PubMed – indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/pubmed/18292947

in vitro, Medical Studies, Melanoma, Tumor

Anti-tumor effect of Coriolus versicolor methanol extract against mouse B16 melanoma cells: in vitro and in vivo study.

Harhaji Lj, Mijatovi? S, Maksimovi?-Ivani? D, Stojanovi? I, Momcilovi? M, Maksimovi? V, Tufegdzi? S, Marjanovi? Z, Mostarica-Stojkovi? M, Vucini? Z, Stosi?-Grujici? S.

Department of Immunology, Institute for Biological Research Sinisa Stankovi?, Belgrade University, Belgrade, Serbia.

Abstract

Numerous studies have shown immunostimulatory and anti-tumor effects of water and standardized aqueous ethanol extracts derived from the medicinal mushroom, Coriolus versicolor, but the biological activity of methanol extracts has not been examined so far. In the present study we investigated the anti-tumor effect of C. versicolor methanol extract (which contains terpenoids and polyphenols) on B16 mouse melanoma cells both in vitro and in vivo. In vitro treatment of the cells with the methanol extract (25-1600 microg/ml) reduced melanoma cell viability in a dose-dependent manner. Furthermore, in the presence of the methanol extract (200 microg/ml, concentration IC(50)) the proliferation of B16 cells was arrested in the G(0)/G(1) phase of the cell cycle, followed by both apoptotic and secondary necrotic cell death. In vivo methanol extract treatment (i.p. 50 mg/kg, for 14 days) inhibited tumor growth in C57BL/6 mice inoculated with syngeneic B16 tumor cells. Moreover, peritoneal macrophages collected 21 days after tumor implantation from methanol extract-treated animals exerted stronger tumoristatic activity ex vivo than macrophages from control melanoma-bearing mice. Taken together, our results demonstrate that C. versicolor methanol extract exerts pronounced anti-melanoma activity, both directly through antiproliferative and cytotoxic effects on tumor cells and indirectly through promotion of macrophage anti-tumor activity.

PMID: 18313195 [PubMed – indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/pubmed/18313195

Medical Studies

Development of a bioreactor system for the decolorization of dyes by Coriolus versicolor f. antarcticus.

Diorio LA, Mercuri AA, Nahabedian DE, Forchiassin F.

Department of Biodiversity and Experimental Biology, University of Buenos Aires, Argentina. luisdiorio@bg.fcern.uba.ar

Abstract

Decolorization of 100 microM malachite green (MG) by Coriolus versicolor f. antarcticus using a two-phase bioreactor, was investigated. In the first phase the decolorization ability of this fungus, growing under conditions of solid-state fermentation (SSF), was proved; in the second phase the capacity of the enzymes present in extracts from the solid residues was exploited. During the first phase using the same culture in the bioreactor, five consecutive charges were made, each with 75 ml of 100 microM MG solution, at 28 degrees C. Each cycle ended when MG solution reached a decolorization of 50%, at this time the bioreactor was discharged to a stainless steel coil at 50 degrees C, initiating the second phase of decolorization. Time required in order to reach 50% decolorization during the first phase varied between 25 and 65 min, with an average retention time of 48 min. The second stage had a retention time of 120 min. Residual MG after this phase varied from 0% to 6.3%. The role of laccase and Mn-peroxidase in MG decolorization is discussed. Toxicity of MG solutions before and after decolorization treatments was assayed using Lumbriculus variegatus as test organism.

PMID: 18359061 [PubMed – indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/pubmed/18359061

Cancer, Immunotherapy, Medical Studies, PSK

The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines via arrest of cell cycle and induction of apoptosis.

Jiménez-Medina E, Berruguilla E, Romero I, Algarra I, Collado A, Garrido F, Garcia-Lora A.

Servicio de Análisis Clínicos e Inmunologia, Hospital Universitario Virgen de las Nieves, Universidad de Granada, Av, de las Fuerzas Armadas 2, 18014 Granada, Spain. evajimenez@fundacionhvn.org

Abstract

BACKGROUND: Protein-bound polysaccharide (PSK) is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated.

METHODS: The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells.

RESULTS: PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G0/G1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation.

CONCLUSION: These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells.

PMID: 18366723 [PubMed – indexed for MEDLINE]PMCID: PMC2291471Free PMC Article

http://www.ncbi.nlm.nih.gov/pubmed/18366723

Medical Studies

Enhanced production of laccase from Coriolus versicolor NCIM 996 by nutrient optimization using response surface methodology.

Arockiasamy S, Krishnan IP, Anandakrishnan N, Seenivasan S, Sambath A, Venkatasubramani JP.

Department of Biotechnology, Kalasalingam University, Krishnankoil, Tamil Nadu, India. asanthiagu@rediffmail.com

Abstract

Plackett and Burman design criterion and central composite design were applied successfully for enhanced production of laccase by Coriolus versicolor NCIM 996 for the first time. Plackett and Burman design criterion was applied to screen the significance of ten nutrients on laccase production by C. versicolor NCIM 996. Out of the ten nutrients tested, starch, yeast extract, MnSO(4), MgSO(4) x 7H(2)O, and phenol were found to have significant effect on laccase production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Plackett-Burman design. The optimized medium composition for production of laccase was (g/l): starch, 30.0; yeast extract, 4.53; MnSO(4), 0.002; MgSO(4) x 7H(2)O, 0.755; and phenol, 0.026, and the optimum laccase production was 6,590.26 (U/l), which was 7.6 times greater than the control.

PMID: 18459071 [PubMed – indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Krishnan%20IP%22[Author]

Medical Studies

Biomimetic synthesis and characterisation of protein capped silver nanoparticles.

Sanghi R, Verma P.

Facility for Ecological and Analytical Testing, Indian Institute of Technology Kanpur, Kanpur, India. rsanghi@iitk.ac.in

Abstract

A controlled and up-scalable route for the biosynthesis of silver nanopartilces (NPs) mediated by fungal proteins of Coriolus versicolor has been undertaken for the first time. The fungus when challenged with silver nitrate solution accumulated silver NPs on its surface in 72h which could be reduced to 1h by tailoring the reaction conditions. Under alkaline conditions, the reaction was much faster and could easily proceed at room temperature even without stirring. The resulting Ag NPs displayed controllable structural and optical properties depending on the experimental parameters such as pH and reaction temperatures. The average size, morphology, and structure of particles were determined by AFM, TEM, XRD and UV/Visible absorption spectrophotometry. Fourier transform infrared study disclosed that the amino groups were bound to the particles, which was accountable for the stability of NPs. It further confirmed the presence of protein as the stabilizing and capping agent surrounding the silver NPs. Experiments were conducted both with, media in which fungus was initially harvested and that of pristine fungal mycelium alone. Under normal conditions, in the case of media extracellular synthesis took place whereby other than the fungal proteins, glucose was also responsible for the reduction. In the case of fungal mycelium, the intracellular formation of Ag NPs, could be tailored to give both intracellular and extracellular Ag NPs under alkaline conditions whereby the surface S-H groups of the fungus played a major role.

http://www.ncbi.nlm.nih.gov/pubmed/18625550

Clinical Trials, Immunotherapy, Medical Studies, Melanoma, PSK, PSP

Evaluation of widely consumed botanicals as immunological adjuvants.

1 Comment

Ragupathi G, Yeung KS, Leung PC, Lee M, Lau CB, Vickers A, Hood C, Deng G, Cheung NK, Cassileth B, Livingston P.

Laboratory of Tumor Vaccinology, Melanoma and Sarcoma Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, United States. ragupatg@mskcc.org

Abstract

BACKGROUND: Many widely used botanical medicines are claimed to be immune enhancers. Clear evidence of augmentation of immune responses in vivo is lacking in most cases. To select botanicals for further study based on immune enhancing activity, we study them here mixed with antigen and injected subcutaneously (s.c.). Globo H and GD3 are cell surface carbohydrates expressed on glycolipids or glycoproteins on the cell surface of many cancers. When conjugated to keyhole limpet hemocyanin (KLH), mixed with an immunological adjuvant and administered s.c. the magnitude of the antibody responses against globo H, GD3 and KLH depend largely on the potency of the adjuvant. We describe here the results obtained using this s.c. immunization model with seven botanicals purported to have immune stimulant effects.

METHODS: Groups of 5-10 mice were immunized with globo H-KLH or GD3-KLH mixed with botanical, saline or positive control immunological adjuvant, s.c. three times at 1 week intervals. Antibody responses were measured 1 and 2 weeks after the 3rd immunization. The following seven botanicals and fractions were tested: (1) H-48 (Honso USA Co.), (2) Coriolus versicolor raw water extract, purified polysaccharide-K (PSK) or purified polysaccharide-peptide (PSP) (Institute of Chinese Medicine (ICM)), (3) Maitake extract (Yukiguni Maitake Co. Ltd. and Tradeworks Group), (4) Echinacea lipophilic, neutral and acidic extracts (Gaia Herbs), (5) Astragalus water, 50% or 95% ethanol extracts (ICM), (6) Turmeric supercritical (SC) or hydro-ethanolic (HE) extracts (New Chapter) or 60% ethanol extract (ICM) and (7) yeast beta-glucan (Biotec Pharmacon). Purified saponin extract QS-21 (Antigenics) and semisynthetic saponin GPI-0100 (Advanced BioTherapies) were used as positive control adjuvants. Sera were analyzed by ELISA against synthetic globo H ceramide or GD3 and KLH.

RESULTS: Consistent significant adjuvant activity was observed after s.c. vaccination with the Coriolus extracts (especially PSK), a 95% ethanol extract of Astragalus and yeast beta-glucan, and (to a lesser extent) Maitake. Antibodies against KLH in all cases and against globo H in most cases were induced by these botanicals. Little or no adjuvant activity was demonstrated with H-48 or Echinacea extracts or the Astragalus water extract. Experiments with GD3-KLH as immunogen confirmed the adjuvant activity of the Coriolus, yeast beta-glucan and Astragalus extracts. While extraction with ethanol concentrated the active ingredients in Astragalus, it had no impact on Coriolus where the 90% ethanol precipitate and solute were equally active.

CONCLUSIONS: Some, but not all, botanicals purported to be immune stimulants had adjuvant activity in our model. PSK and Astragalus were surprisingly active and are being further fractionated to identify the most active adjuvant components.

PMID: 18640165 [PubMed – indexed for MEDLINE]PMCID: PMC2565601

http://www.ncbi.nlm.nih.gov/pubmed/18640165