A new fungal immunomodulatory protein (Fip) has been purified from the edible mushroom, Volvariella volvacea, and designated Fip-vvo. Analysis of the purified protein by SDS/PAGE followed by Coomassie Blue staining demonstrated that Fip-vvo is a single polypeptide with an apparent molecular mass of 15 kDa. Periodic acid/Schiff staining showed that this single polypeptide lacks carbohydrates. Using an in vitro bioassay measuring blast-formation stimulatory activity, Fip-vvo was shown to stimulate the maximum proliferation of human peripheral blood lymphocytes at a concentration of 5 microg/ml. Fip-vvo was capable of agglutinating rat red blood cells. Neither haemagglutination nor mitogenic activities were inhibited by mono- or dimeric sugars. In vivo, repeat administration of Fip-vvo greatly reduced the production of BSA-induced Arthus reaction in mice, whereas little effect was observed on the prevention of systemic anaphylaxis reactions. The selectively enhanced transcriptional expression of interleukin (IL)-2, IL-4, interferon-gamma, tumour necrosis factor-alpha, lymphotoxin and IL-2 receptor by Fip-vvo was also demonstrated by reverse transcriptase-PCR. This finding suggests that Fip-vvo exerts its immunomodulatory effects via cytokine regulation. In addition, the complete amino acid sequence of Fip-vvo was obtained by direct protein sequencing. This protein consists of 112 amino acid residues with a blocked N-terminal end and has a calculated molecular mass of 12667 Da not including the N-terminal blocking group. By gel filtration analysis, Fip-vvo exhibited a molecular mass of 26 kDa for the native molecules in PBS. This result indicates that native Fip-vvo is most likely a non-covalently associated homodimeric molecule.

Nicotinamide N-methyltransferase (NNMT) was recently identified as one clear cell renal cell carcinoma (ccRCC)-associated gene by analyzing full-length complementary DNA-enriched libraries of ccRCC tissues. The aim of this study is to investigate the potential role of NNMT in cellular invasion. A strong NNMT expression is accompanied with a high invasive activity in ccRCC cell lines, and small interfering RNA-mediated NNMT knockdown effectively suppressed the invasive capacity of ccRCC cells, whereas NNMT overexpression markedly enhanced that of human embryonic kidney 293 (HEK293) cells. A positive correlation between the expression of NNMT and matrix metallopeptidase (MMP)-2 was found in ccRCC cell lines and clinical tissues. The treatment of blocking antibody or inhibitor specific to MMP-2 significantly suppressed NNMT-dependent cellular invasion in HEK293 cells. Furthermore, SP-1-binding region of MMP-2 promoter was found to be essential in NNMT-induced MMP-2 expression. The specific inhibitors of PI3K/Akt signaling markedly decreased the binding of SP1 to MMP-2 promoter as shown by chromatin immunoprecipitation assay. We also demonstrated that PI3K/Akt pathway plays a role in NNMT-dependent cellular invasion and MMP-2 activation. Moreover, short hairpin RNA-mediated knockdown of NNMT expression efficiently inhibited the growth and metastasis of ccRCC cells in non-obese diabetic severe combined immunodeficiency mice. Taken together, the present study suggests that NNMT has a crucial role in cellular invasion via activating PI3K/Akt/SP1/MMP-2 pathway in ccRCC.

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A fungal immunomodulatory protein (Fip-gts) was purified from Ganoderma tsugae. The DNA encoding Fipgts was isolated from a cDNA library of G. tsugae by reverse transcriptase-polymerase chain reaction. The complete amino acid sequence of Fip-gts, deduced from the nucleotide sequence of the cDNA, was the same as LZ-8 isolated from Ganodermn lucidum. Recombinant Fip-gts was expressed as a glutathione S-transferase fusion protein in Escherichia coli with a yield of 20 mg/liter of culture. Recombinant Fip-gts, purified to homogeneity, had the same blast formation stimulatory activity to human peripheral blood lymphocytes as native Fip-gts.The yeast two-hybrid system and site-directed mutagenesis were used to determine whether dimerization of Fip-gts occurred. Deletion analysis of the N-terminal amphipathic ␣-helix domain of Fip-gts identified a sequence of about 10 amino acids responsible for inducing immunomodulatory activity. Non-functional Fip-gts deletion mutants did not form dimers, whereas wild type Fip-gts did as determined by gel filtration. A mutant with deletions at Leu-5, Phe-7, and Leu-9 lost the amphipathic characteristics of the N-terminal domain and the ability to form dimers as well as its immunomodulatory activity.Fusion of Fip-gts with the DNA binding and the transactivation domains of GAL4 resulted in the activation of the lacZ activator gene, indicating the interaction of Fip-gts with it itself. The dimerization domain was further defined by analyzing the ability of the N-terminal 13 amino acids or Leu-5, Phe-7, and Leu-9 deletion mutants of Fip-gts to interact with the wild type Fip-gts. These experiments confirmed the N-terminal amphipathic ␣-helix as the dimerization domain and suggest that the dimerization of Fip-gts may play an important role in Fip-gts immunomodulatory activity.A new family of fungal immunomodulatory proteins (Fips) 1 has recently been established. Four Fips have been isolated and purified from Ganodermn lucidum, Flammulina veltipes, Volvariella volvacea, and Ganoderma tsugae and designated as LZ-8, Fip-fve, Fip-vvo, and Fip-gts, respectively (1-3).Fips are mitogenic in vitro for human peripheral blood lymphocytes (hPBLs) and mouse splenocytes, and induce a bellshaped dose-response curve similar to that for lectin mitogens. Activation of hPBLs with Fips results in the increased production of IL-2, IFN-␥, and tumor necrosis factor-␣ molecules associated with ICAM-1 expression (2, 3). Fips can also act as immunosuppressive agents; in vivo these proteins can prevent systemic anaphylactic reactions and significantly decrease footpad edema during the Arthus reaction (1, 2). LZ-8 can also suppress autoimmune diabetes in young female non-obese diabetes mice (4). Furthermore, LZ-8 has a significant effect on cellular immunity, as shown by the increase of graft survival in transplanted allogenic mouse skin and allogenic pancreatic rats (5) without producing the severe toxic effects on pancreatic islets associated with prednisolone and cyclosporin A treatment (6, 7).The Fips identifi...

A new fungal immunomodulatory protein (FIP-fie) has been isolated and purified from the edible golden needle mushroom (Flammulina velutipes). The apparent molecular mass of FIP-fie determined by SDS/PAGE agrees well with the value of 12704 Da calculated from its amino acid composition and sequence. The complete amino acid sequence of FIP-fie was elucidated by protein sequencing techniques. FIP-fie consists of 114 amino acid residues with an acetylated amino end, and lacks methionine, halfcystine and histidine residues. FIP-fie was able to hemagglutinate human red blood cells. The immunomodulatory activity of FIP-fie was demonstrated by its stimulatory activity toward human peripheral blood lymphocytes, and its suppression of systemic anaphylaxis reactions and local swelling of mouse footpads. FIP-fie was found to enhance the transcriptional expression of interleukin-2 and interferon-y. Keywords. Flarnmulina velutipesGolden needle mushrooms (Flammulina velutipes) are a popular edible mushroom in Asia. We previously reported the isolation of a cardiotoxin, flammutoxin, from this mushroom [l]. Many polysaccharide biological response modifiers with antitumor activities, such as lentinan [2, 31 Schizophyllan [4] and OK-432 [5], have been isolated from fungi or bacteria. An anticarcinogenic and immunomodulatory protein has also been isolated from the edible grass mushroom, Volvariella volvacea [6]. Another fungal immunomodulatory protein (FIP), Ling Zhi-8, was isolated from the mycelia of Ganoderma. lucidum [7]. In this study, we describe the isolation and characterization of FIP-fie from the edible golden needle mushroom, E velutipes. The immunomodulatory and hemagglutinating activities of this FIP were studied, and the mechanism of immunornodulatory activity was examined. The complete amino acid sequence was determined by protein sequencing techniques. MATERIALS AND METHODSIsolation of FIP-fie. The fruit bodies of E velutipes (400 g) were homogenized with 1 1 ice-cold 5% acetic acid in the presence of 0.05 M 2-mercaptoethanol [6], and soluble proteins in the supernatant were precipitated by addition of ammonium sul-Correspondence to J. Y. Lin, Abbreviations. FIP-fie, fungal immunomodulatory protein isolated from Flammulina velutipes; IL-2, interleukin-2 ; IFN-y, interferon-y ; RT, reverse transcriptase.