Optimization of Se- and Zn-Enriched Mycelium of Lentinula edodes (Berk.) Pegler as a Dietary Supplement with Immunostimulatory Activity.

Mycelial cultures of Lentinula edodes, an edible and medicinal mushroom, have been used in our previous research to obtain selenium-containing immunomodulatory preparations. Our current attempts to obtain a new preparation containing both selenium and zinc, two micronutrients necessary for the functioning of the immune system, extended our interest in the simultaneous accumulation of these elements by mycelia growing in media enriched with selenite and zinc(II) ions. Subsequently, we have studied the effects of new L. edodes mycelium water extracts with different concentrations of selenium and zinc on the activation of T cell fraction in human peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis was used to measure the expression of activation markers on human CD4+ and CD8+ T cells stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs). It was demonstrated that statistically significant changes were observed for PD-1 and CD25 antigens on CD8+ T cells. The selenium and zinc content in the examined preparations modified the immunomodulatory activity of mycelial polysaccharides; however, the mechanisms of action of various active ingredients in the mycelial extracts seem to be different.

The Effect of Novel Selenopolysaccharide Isolated from Lentinula edodes Mycelium on Human T Lymphocytes Activation, Proliferation, and Cytokines Synthesis.

Polysaccharides isolated from Lentinula edodes are bioactive compounds with immunomodulatory properties. In our previous studies from L. edodes mycelium, we have isolated a selenium(Se)-enriched fraction (named Se-Le-30), a mixture of linear 1,4-α-glucan and linear 1,3-ß- and 1,6-ß-glucans. In this study, we analyzed the effects of Se-Le-30 on the activation and proliferation of human T lymphocytes stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs) and on the production of cytokines by peripheral blood mononuclear cells (PBMCs). Se-Le-30 had effects on T cell proliferation induced by Abs against CD3 and CD28. It significantly inhibited the proliferation of CD3-stimulated CD4+ and CD8+ T cells and enhanced the proliferation of CD4+ T cells stimulated with anti-CD3/CD28 Ab. Moreover, Se-Le-30 downregulated the number of CD3-stimulated CD4+CD69+ cells, CD4+CD25+ cells, as well as CD8+CD25+ cells, and upregulated the expression of CD25 marker on CD4+ and CD8+ T cells activated with anti-CD3/CD28 Abs. Furthermore, Se-Le-30 enhanced the synthesis of IFN-γ by the unstimulated and anti-CD3/CD28-stimulated PBMCs, inhibited synthesis of IL-2 and IL-4 by CD3-stimulated cells, and augmented the synthesis of IL-6 and IL-10 by unstimulated, CD3-stimulated, and CD3/CD28-stimulated PBMCs. Together, we demonstrated that Se-Le-30 exerts immunomodulatory effects on human T lymphocytes. These observations are of importance for the prospective use of Se-Le-30 in research or as a therapeutic compound.

Selenium-Containing Exopolysaccharides Isolated from the Culture Medium of Lentinula edodes: Structure and Biological Activity.

In continuation of our research on the influence of selenium incorporation on the biosynthesis, structure, and immunomodulatory and antioxidant activities of polysaccharides of fungal origin, we have isolated from a post-culture medium of Lentinula edodes a selenium (Se)-containing exopolysaccharide fraction composed mainly of a highly branched 1-6-α-mannoprotein of molecular weight 4.5 × 106 Da, with 15% protein component. The structure of this fraction resembled mannoproteins isolated from yeast and other mushroom cultures, but it was characterized by a significantly higher molecular weight. X-ray absorption fine structure spectral analysis in the near edge region (XANES) suggested that selenium in the Se-exopolysaccharide structure was present mainly at the IV oxidation state. The simulation analysis in the EXAFS region suggested the presence of two oxygen atoms in the region surrounding the selenium. On the grounds of our previous studies, we hypothesized that selenium-enriched exopolysaccharides would possess higher biological activity than the non-Se-enriched reference fraction. To perform structure-activity studies, we conducted the same tests of biological activity as for previously obtained mycelial Se-polyglucans. The Se-enriched exopolysaccharide fraction significantly enhanced cell viability when incubated with normal (human umbilical vein endothelial cells (HUVEC)) cells (but this effect was absent for malignant human cervical HeLa cells) and this fraction also protected the cells from oxidative stress conditions. The results of tests on the proliferation of human peripheral blood mononuclear cells suggested a selective immunosuppressive activity, like previously tested Se-polyglucans isolated from L. edodes mycelium. The Se-exopolysaccharide fraction, in concentrations of 10-100 µg/mL, inhibited human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. As with previously obtained Se-polyglucans, in the currently tested Se-polymannans, the selenium content increased the biological activity. However, the activity of selenium exopolysaccharides in all tests was significantly lower than that of previously tested mycelial isolates, most likely due to a different mode of selenium binding and its higher degree of oxidation.

Selective Biological Effects of Selenium-Enriched Polysaccharide (Se-Le-30) Isolated from Lentinula edodes Mycelium on Human Immune Cells.

A common edible mushroom Lentinula edodes, is an important source of numerous biologically active substances, including polysaccharides, with immunomodulatory and antitumor properties. In the present work, the biological activity of the crude, homogenous (Se)-enriched fraction (named Se-Le-30), which has been isolated from L. edodes mycelium by a modified Chihara method towards human peripheral blood mononuclear cells (PBMCs) and peripheral granulocytes, was investigated. The Se-Le-30 fraction, an analog of lentinan, significantly inhibited the proliferation of human PBMCs stimulated with anti-CD3 antibodies or allostimulated, and down-regulated the production of tumor necrosis factor (TNF)-α by CD3+ T cells. Moreover, it was found that Se-Le-30 significantly reduced the cytotoxic activity of human natural killer (NK) cells. The results suggested the selective immunosuppressive activity of this fraction, which is non-typical for mushroom derived polysaccharides.

Identification of the Primary Structure of Selenium-Containing Polysaccharides Selectively Inhibiting T-Cell Proliferation.

We previously described the biosynthesis, isolation, and immunosuppressive activity of the selenium-containing polysaccharide fraction isolated from the mycelial culture of Lentinula edodes. Structural studies have shown that the fraction was a protein-containing mixture of high molar mass polysaccharides α- and ß-glucans. However, which of the components of the complex fraction is responsible for the immunosuppressive activity non-typical for polysaccharides of fungal origin has not been explained. In the current study, we defined four-polysaccharide components of the Se-containing polysaccharide fraction determined their primary structure and examined the effect on T- and B-cell proliferation. The isolated Se-polysaccharides, α-1,4-glucan (Mw 2.25 × 106 g/mol), unbranched ß-1,6-d-glucan, unbranched ß-1,3-d-glucan and ß-1,3-branched ß-1,6-d-glucan (Mw 1.10 × 105 g/mol), are not typical as components of the cell wall of L. edodes. All are biologically active, but the inhibitory effect of the isolated polysaccharides on lymphocyte proliferation was weaker, though more selective than that of the crude fraction.

Selenium-containing polysaccharides from Lentinula edodes-Biological activity.

We hypothesized that selenium(Se)-enriched polysaccharides would possess superior biological activity when compared to those non-enriched. To verify this hypothesis, we obtained by biotechnological methods a Se-enriched analog of Japanese anticancer drug lentinan and, as a reference, the non-Se-enriched fraction. We tested the effects of the obtained fractions on the proliferation of human peripheral blood mononuclear cells. The results suggested a selective immunosuppressive activity, non-typical for mushroom derived polysaccharides. Both fractions caused significant inhibition of human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. The inhibitory effect was not due to the toxicity of the examined polysaccharides. In normal (HUVEC) or malignant (HeLa) cells tested fractions significantly enhanced cell viability and protected the cells from oxidative stress conditions. However, we observed no effect of the polysaccharide fractions on the production of reactive oxygen species by granulocytes in vitro. The selenium content increased the biological activity of the tested polysaccharide fractions.

Selenized polysaccharides - Biosynthesis and structural analysis.

The main objective of our research was to analyze the structure of the Se-containing polysaccharides and to examine how the selenium is bound to the polysaccharide molecule. During investigation of the biosynthesis of new immunomodulators, we isolated a selenium (Se)-containing polysaccharide-protein fraction containing proteoglycans of molecular weights of 3.9 × 106 Da and 2.6 × 105 Da, composed of glucose or mannose, nearly 8% of protein and 190 µg Se/g dry weight. X-ray absorption spectroscopy (XAS) data analysis in the near edge region (XANES) confirmed that selenium in the Se-polysaccharides structure is present at the -II oxidation state and that Se is organically bound. The simulation analysis in the EXAFS (extended X-ray absorption fine structure) region suggested that selenium is most likely bound by a glycosidic-link in a ß-1,3 or α-1,4-glycosidic bond or substituted for oxygen in a pyranosidic ring. Calculations performed with Gaussian 03 software predicted deformations in the polysaccharide structure caused by the incorporation of the selenium atom including change in bond lengths and torsion angles and, as a result, disappearance of hydrogen bonds in the vicinity of the selenium atoms.

Selective Cytotoxic Activity of Se-Methyl-Seleno-L-Cysteine- and Se-Polysaccharide-Containing Extracts from Shiitake Medicinal Mushroom, Lentinus edodes (Agaricomycetes).

Numerous formulations derived from the shiitake medicinal mushroom, Lentinus edodes, demonstrate anticancer activities. We hypothesized that isolates from selenium (Se)-enriched mycelia of L. edodes would possess stronger cancer-preventive properties than current preparations. The aim of this study was to investigate whether the presence of Se-methyl-seleno-L-cysteine in mycelial extracts of L. edodes affects their cytotoxic activity (makes them stronger) or whether they are as effective as Se-containing polysaccharides. Extracts were prepared from Se-containing mycelia under various conditions and assayed for cytotoxic activity in cancer (PC3 and HeLa) and normal (HMEC-1) cell lines. The chemical composition of the extracts was examined; specifically, the amounts of potentially cytotoxic Se compounds (methylselenocysteine, selenomethionine, and Se-containing polysaccharides) were measured. The relationship between extract composition and biological activity was characterized. Mycelial cultures were cultivated in a 10-L bioreactor in medium enriched with sodium selenite. Mycelial extracts were prepared either at 100°C or at 4°C in acidic solution. Total Se content was determined using the atomic absorption spectrometry method, and methylselenocysteine and selenomethionine contents were measured using reverse-phase high-performance liquid chromatography. Protein, carbohydrate, and polyphenolic contents were determined with spectrophotometric methods, and Se-containing polysaccharides were measured with the use of precipitation. Anticancer activity of mycelial extracts was examined using the MTT cell viability assay. Extracts containing Se-methyl-seleno-L-cysteine or Se-polysaccharides prepared at 4°C and 100°C, respectively, display moderate, time-dependent, specific cytotoxic activity in HeLa and PC3 cell lines. The effect in HeLa cells is more pronounced in the extract prepared at 4°C than at 100°C. The effect is almost equal for the PC3 cell line. However, both extracts have no effect or only slightly stimulate normal (HMEC-1) cell viability. The selective cytotoxic activity of L. edodes extracts in cancer (PC3 and HeLa) cells is due to the presence of both Se-methyl-seleno-L-cysteine and selenated polysaccharides, perhaps in combination with other active ingredients.

A Search for the Optimum Selenium Source to Obtain Mushroom-Derived Chemopreventive Preparations.

The objective of this research was to test whether selenium-yeast (Se-yeast) is a better source of selenium than sodium selenite for accumulation in mycelia and immunoactive cell wall polysaccharides. Culture media were enriched in selenium to a concentration of 20 µg/mL. Selenium was added to the medium either in the form of sodium selenite or in form of Se-yeast (Sel-Plex; Alltech Inc., Lexington, KY). The total selenium concentrations in the mycelium biomass and in the isolated crude polysaccharides were determined using atomic absorption spectroscopy. We found that selenium accumulated more efficiently in cultures enriched with Se-yeast. A higher concentration of selenium was also found in the crude polysaccharide fractions isolated from the mycelium grown in Se-yeast-enriched media. With the use of the needle trap gas chromatography-mass spectrometry method, we found that there are significant differences in the composition of the volatile aroma and flavor compounds secreted by the mycelia cultivated in different media.

Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis.

In this report the optimization of biosynthesis of tacrolimus, the immunosupressant widely used in transplantology and dermatology was described. The enhancement of the productivity of Streptomyces tsukubaensis strain was achieved by development of new precursors of tacrolimus biosynthesis, which should allow to reduce the costs of the process. The enrichment of the fermentation medium in pyridine-2-carboxylic acid (picolinic acid), piperidine-2-carboxylic acid (pipecolic acid), pyridine-3-carboxylic acid (nicotinic acid) or pyridine-3-carboxylic acid amide (nicotinamide) caused significant growth of the productivity of tacrolimus: 7-fold, 6-fold, 3-fold and 5-fold, respectively. The optimum concentration of the precursors in medium was 0.0025-0.005%. The investigation of the kinetics of tacrolimus biosynthesis together with the analysis of the impact of tested compounds on the culture growth and NAD (nicotinamide adenine dinucleotide) concentration in S. tsukubaensis cells enables to put forward a hypothesis concerning the mechanism of action of tested culture medium additives. The compounds active as tacrolimus precursors (pipecolic and picolinic acids) are more effective than these active mainly as the growth promoters (nicotinamide and nicotinic acid). Nicotinamide and nicotinic acid--vitamin B3 components--promote S. tsukubaensis growth most probably due to the stimulation of NAD/NADP biosynthesis.