Vincent Eng Choo Ooi

Free radical scavenging activities of mushroom polysaccharide extracts

The superoxide and hydroxyl radical scavenging activities of eight mushroom antitumor polysaccharide extracts were investigated using phenazin methosulphate-NADH-nitroblue tetrazolium system and ascorbic acid-Cu(2+)-cytochrome C system respectively. The results showed that six of eight mushroom polysaccharide extracts had superoxide and hydroxyl radical scavenging activities. The protein content of the polysaccharide extracts appeared to contribute a direct effect on free radical scavenging activity. However, none of the mushroom polysaccharide extracts had antioxidative activity as measured by detecting malondialdehyde (MDA) contents of liver microsomes.

The immunomodulatory and antitumor activities of lectins from the mushroom Tricholoma mongolicum

TML-1 and TML-2 were two lectins isolated from the mushroom Tricholoma mongolicum. They did not differ appreciably in their pH stability and cationic requirement for hemagglutinating activity. They both stimulated the production of nitrite ions and activated the macrophages in mice. The two lectins were able to inhibit the growth of implanted sarcoma 180 cells by 68.84% and 92.39% respectively. The growth of tumor cells in the mouse peritoneal cavity was also inhibited by the two lectins with TML-2 expressing a greater potency.

Immunomodulatory and antitumor activities of a polysaccharide-peptide complex from a mycelial culture of Tricholoma sp., a local edible mushroom

A polysaccharide-peptide complex (PSPC) with immunomodulatory and antitumor activities was obtained from a submerged mycelial culture of Tricholoma sp., a local edible mushroom. The polysaccharide-peptide complex exhibited a molecular weight of 17 K in gel filtration and a single band after SDS-polyacrylamide gel electrophoresis. It was characterized by non-adsorption on both DEAE-Sepharose CL-6B and CM-cellulose. It could activate the macrophages, stimulate the proliferation of T-cells, and inhibit the growth of sarcoma 180 in mice. It possessed more potent immunomodulatory and antitumor activities than Coriolus versicolor polysaccharopeptide (PSP) and deserves to be studied as a potential agent for immunomodulation and cancer therapy.

ANALYSIS OF IMMUNOMODULATING CYTOKINE mRNAs IN THE MOUSE INDUCED BY MUSHROOM POLYSACCHARIDES

The immunomodulating action of two mushroom antitumor polysaccharides, polysaccharide-protein complex (PSPC) and lentinan, was elucidated through analysing the expression profile of cytokines during a time course (0 h to 48 h) after their administration. Among the 5 cytokine genes, the induction of a marked increase in the mRNA levels of IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma and M-CSF by PSPC and lentinan was observed in the peritoneal exudate cells and splenocytes. However, the time point of their increased production was different after PSPC and lentinan administration.

Polysaccharide—peptide complexes from the cultured mycelia of the mushroom coriolus versicolor and their culture medium activate mouse lymphocytes and macrophages

The aim of this study was to investigate the effects of the mushroom Coriolus versicolor on cells of the immune system. The cultured mycelia of the mushroom Coriolus versicolor and their culture medium were separately extracted with boiling water. The resulting polysaccharopeptide preparations were designated intramycelial (IM) and extramycelial materials (EM), and were separated by gel filtration before determining their effects on lymphocytes and macrophages in vitro and in vivo. After gel filtration on Sepharose 6B, only a single peak with a molecular weight of 13-19 KDa was obtained. Gel filtration of IM and EM on Sephadex G-50 revealed the presence of a larger peak of 28 KDa (from IM) and 15 KDa (from EM) and a smaller peak of 3.5 KDa. IM, EM and their large molecular peaks enhanced the mitogenic response of T-cells from BALB/c mice in vitro. Splenocytes from C57BL/6 mice pre-treated by force-feeding with IM and EM demonstrated an augmented mitogenic response to Con A. The macrophages of C57BL/6 mice that had been pre-treated with IM or EM showed an enhanced production of nitrite ions. The results indicate that both mouse lymphocytes and macrophages were activated by preparations of polysaccharopeptide from cultured mycelia and culture medium of C. versicolor. However, no direct cytotoxic activity against fibroblasts, hepatoma cells and choriocarcinoma cells could be demonstrated.

Immunomodulation and antitumor activity of polysaccharide-protein complex from the culture filtrates of a local edible mushroom, Tricholoma lobayense

1. The polysaccharide-protein complex (PSPC) isolated from the culture filtrate of Tricholoma lobayense showed antitumor activity in mice. 2. PSPC had the ability to restore the phagocytic function of peritoneal exudate cells (PEC) and T-cell mitogenic activity of tumor-bearing mice. 3. An increase in the production of reactive nitrogen intermediates and tumor necrosis factor (TNF) was determined in PEC of mice that had received PSPC. 4. The present study showed that PSPC both induced the various immune responses in vivo and exhibited cytotoxicity against tumor cell lines in the presence of PSPC at doses of 30 and 60 micrograms/ml in vitro.

Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide-protein complexes

Two antitumor polysaccharide-protein complexes, PSPC and PSK from mushrooms, were compared for their modulating effect on cytokine and cytokine receptor gene expression. RNA samples were isolated from the splenocytes and peritoneal exudate cells of the untreated or treated mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the cytokine gene expression. Nine out of 17 cytokine mRNAs and 5 out of 6 cytokine receptor mRNAs were detected in the splenocytes and peritoneal exudate cells from both untreated and treated mice. However, IL-4 was only detected in the splenocytes while IL-7 and IL-1R(typeI) were only detected in the peritoneal exudate cells. Among the 9 cytokine genes, the expression level of M-CSF was up-regulated in splenocytes and peritoneal exudate cells of the mice by PSPC and PSK. The expression level of TNF-alpha was only up-regulated in the peritoneal exudate cells by PSK, but not by PSPC.

Lectin activity in fruiting bodies of the edible mushroom Tricholoma mongolicum

This study was undertaken to ascertain the existence and some of the characteristics of lectin in the fruiting bodies of the edible mushroom Tricholoma mongolicum. The fractionation procedure utilized entailed aqueous extraction, (NH4)2SO4 precipitation, dialysis, ion exchange chromatography on DEAE‐Sepharose and gel filtration on Sephadex G‐100. A similar procedure has been successfully employed for isolating lectins from cultured mycelia of the same species. It was found that the fruiting body lectin could not be purified with the same facility as the mycelial lectins. Although lectins from both fruiting bodies and cultured mycelia were adsorbed on DEAE‐Sepharose and possessed similar carbohydrate specificities, the fruiting body lectin was adsorbed on ConA‐Sepharose and hence probably glycoprotein in nature whereas the mycelial lectins are unglycosylated proteins.

STUDIES ON PURIFICATION OF A LECTIN FROM FRUITING BODIES OF THE EDIBLE SHIITAKE MUSHROOM LENTINUS EDODES

Abstract A lectin, with a specificity for N-acetylgalactosamine and N-acetylglucosamine and a molecular weight of 43 kDa, was isolated from fruiting bodies of the edible shiitake mushroom Lentinus edodes. The purification procedure entailed extraction with aqueous buffer, ammonium sulfate precipitation, gel filtration on Sephadex G-100 and affinity chromatography on N-acetylgalactosamine-agarose. The lectin was unique in that it was tenaciously bound on anion exchangers including DEAE-cellulose, DEAE-Sepharose, DEAE-Sephadex, Q-Sepharose, Dowex and PEI-cellulose and also on hydroxyapatite and phenyl Sepharose. It was largely unadsorbed on cation exchangers including CM-cellulose, CM-Sepharose, SP-Sepharose and Amberlite, and also on protein G-Sepharose, Red Sepharose and Affi-gel Blue gel, wheat germ lectin-Sepharose and p-aminophenyl- d -glucopyranoside agarose.

A protein with inhibitory activity on cell-free translation from cultured mycelia of the edible mushroom Tricholoma lobayense

Cultured mycelia of the edible mushroom Tricholoma lobayense were extracted with cold saline. Proteins were precipitated from the extract by addition of (NH4)2SO4. The precipitate was dissolved and dialyzed before ion exchange chromatography on DEAE-cellulose. Ability to inhibit translation in a rabbit reticulocyte lysate was located in the unadsorbed fraction which was then subjected to affinity chromatography on Affi-gel Blue gel. The strongest activity was again retained by the unadsorbed fraction. Ion exchange chromatography on CM-cellulose resulted in fractionation of this fraction into an unadsorbed and two adsorbed peaks. Cell-free translation inhibitory activity was concentrated in the fraction eluted with 100 mM NaCl in 10 mM NH4OAc (pH 5.4). The translation-inhibitory protein possessed a molecular weight of 30 kDa as estimated by gel filtration using a fast protein liquid chromatography system and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.