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Ganoderma lucidum polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function

Previous studies demonstrated Ganoderma lucidum polysaccharides (GL-PS), a form of bioactive β-glucan can stimulate the maturation of monocyte-derived dendritic cells (DC). The question of how leukemic cells especially in monocytic lineage respond to GL-PS stimuli remains unclear.

In this study, we used in vitro culture model with leukemic monocytic cell-lines THP-1 and U937 as monocytic effectors cells for proliferation responses and DCs induction. We treated the THP-1 and U937 cells with purified GL-PS (100 μg/mL) or GL-PS with GM-CSF/IL-4. GL-PS alone induced proliferative response on both THP-1 and U937 cells but only THP-1 transformed into typical DC morphology when stimulated with GL-PS plus GM-CSF/IL-4. The transformed THP-1 DCs had significant increase expression of HLA-DR, CD40, CD80 and CD86 though not as high as the extent of normal monocyte-derived DCs. They had similar antigen-uptake ability as the normal monocyte-derived DCs positive control. However, their potency in inducing allogeneic T cell proliferation was also less than that of normal monocyte-derived DCs.

Our findings suggested that GL-PS could induce selected monocytic leukemic cell differentiation into DCs with immuno-stimulatory function. The possible clinical impact of using this commonly used medicinal mushroom in patients with monocytic leukemia (AML-M4 and M5) deserved further investigation.

Extracellular Expression of a Functional Recombinant Ganoderma lucidium Immunomodulatory Protein by Bacillus subtilis and Lactococcus lactis

Bacillus subtilis and Lactococcus lactis are ideal hosts for the production of extracellular heterologous proteins of major commercial importance. A recombinant gene for the novel Ganoderma lucidium immunomodulatory protein LZ-8, recombinant LZ-8, was designed encoding the same amino acid sequence but using the preferred codons for both strains and was synthesized by overlapping extension PCR. Using the signal peptide (SP) from subtilisin YaB (SPYaB), recombinant LZ-8 was expressed extracellularly in Bacillus subtilis and Lactococcus lactis. In the absence of SPYaB, recombinant LZ-8 was expressed extracellularly in B. subtilis, but not in L. lactis. The three expressed recombinant LZ-8s showed different capacities for modulating the production of Th1 and Th2 cytokines by peripheral blood mononuclear cells and of tumor necrosis factor alpha by a macrophage cell line.

Cholesterol-lowering properties of Ganoderma lucidum in vitro, ex vivo, and in hamsters and minipigs

There has been renewed interest in mushroom medicinal properties. We studied cholesterol lowering properties of Ganoderma lucidum (Gl), a renowned medicinal species.

Organic fractions containing oxygenated lanosterol derivatives inhibited cholesterol synthesis in T9A4 hepatocytes. In hamsters, 5% Gl did not effect LDL; but decreased total cholesterol (TC) 9.8%, and HDL 11.2%. Gl (2.5 and 5%) had effects on several fecal neutral sterols and bile acids. Both Gl doses reduced hepatic microsomal ex-vivo HMG-CoA reductase activity. In minipigs, 2.5 Gl decreased TC, LDL- and HDL cholesterol 20, 27, and 18%, respectively (P < 0.05); increased fecal cholestanol and coprostanol; and decreased cholate.

Overall, Gl has potential to reduce LDL cholesterol in vivo through various mechanisms. Next steps are to: fully characterize bioactive components in lipid soluble/insoluble fractions; evaluate bioactivity of isolated fractions; and examine human cholesterol lowering properties. Innovative new cholesterol-lowering foods and medicines containing Gl are envisioned.

Comparative Studies of Delignification Caused by Ganoderma Species

Isolates of six species of Ganoderma in the G. lucidum complex were evaluated for their ability to decay wood of Quercus hypoleucoides A. Camus and Abies concolor (Gord. and Glend.) Lindl. ex. Hildebr. by using in vitro agar block decay tests. Morphological, ultrastructural, and chemical studies of decayed wood were used to determine the extent of delignification or simultaneous decay caused by each species of Ganoderma. All species decayed both white fir and oak wood; however, less percent weight loss (%WL) occurred in white fir than oak. In white fir, isolates of two undescribed Ganoderma species (RLG16161, RLG16162, JEA615, and JEA625) caused significantly higher%WL (21 to 26%) than that in G. colossum, G. oregonense, G. meredithiae, and G. zonatum (10 to 16%). Only Ganoderma sp. isolates JEA615 and JEA625 caused delignification, with JEA615 causing a lignin-to-glucose gram loss ratio of 1.6:1. Morphological and ultrastructural studies confirmed delignification by this fungus and showed that some delignification had occurred by all of the species, although areas of delignification were limited to small regions adjacent to simultaneously decayed cells. In oak, G. colossum caused significantly less%WL (22 to 35%) than the other species (38 to 52%). All of the species, except G. meredithiae, caused delignification with lignin-to-glucose gram loss ratios ranging from 1.4 to 4.9:1. Extensive delignification by isolates of G. colossum and G. oregonense was observed; moderate delignification was caused by the other species. Ganoderma meredithiae caused a simultaneous decay, with only small localized regions of cells delignified, while delignification by G. zonatum was irregular, with specific zones within the cell wall delignified. The thermophilic and chlamydosporic G. colossum has the capacity to cause extensive delignification and appears ideally suited for use in lignin degradation studies and biotechnological applications of lignin-degrading fungi.

Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction

Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-α. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells.

Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach.

Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.

Signaling mechanisms of enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified from Ganoderma lucidum

The polysaccharide from Ganoderma lucidum (PS-G) has been reported to enhance immune responses and to elicit antitumor effects. In our previous study, we found that PS-G efficiently inhibited spontaneously and Fas-enhanced neutrophil apoptosis when cultured in vitro. Since phagocytosis and chemotaxis play essential roles in host defense mediated by neutrophils, it is of great interest to know the effect of PS-G on these two cell functions, and the molecular events leading to these actions.

Effect of 26-Oxygenosterols from Ganoderma lucidum and Their Activity as Cholesterol Synthesis Inhibitors

Ganoderma lucidum is a medicinal fungus belonging to the Polyporaceae family which has long been known in Japan as Reishi and has been used extensively in traditional Chinese medicine. We report the isolation and identification of the 26-oxygenosterols ganoderol A, ganoderol B, ganoderal A, and ganoderic acid Y and their biological effects on cholesterol synthesis in a human hepatic cell line in vitro. We also investigated the site of inhibition in the cholesterol synthesis pathway. We found that these oxygenated sterols from G. lucidum inhibited cholesterol biosynthesis via conversion of acetate or mevalonate as a precursor of cholesterol. By incorporation of 24,25-dihydro-[24,25-3H2]lanosterol and [3-3H]lathosterol in the presence of ganoderol A, we determined that the point of inhibition of cholesterol synthesis is between lanosterol and lathosterol. These results demonstrate that the lanosterol 14α-demethylase, which converts 24,25-dihydrolanosterol to cholesterol, can be inhibited by the 26-oxygenosterols from G. lucidum. These 26-oxygenosterols could lead to novel therapeutic agents that lower blood cholesterol.

Lignin-Modifying Enzymes of the White Rot Basidiomycete Ganoderma lucidum

Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (Mr of 40,000 and 66,000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity (~100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.

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