HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF MANNITOL-1-PHOSPHATE DEHYDROGENASE FROM THE BASIODIOMYCETE PHOLIOTA NAMEKO

ABSTRACT The gene encoding mannitol-1-phosphate dehydrogenase, mpd, which has similar function with alcohol dehydrogenase, has been sequenced and characterized from the basiodiomycete Pholiota nameko. The coding region of mpd was composed of 2992 bp and found to encode a polypeptide of 359 amino acids that has similarity with Laccaria bicolor. To evaluate the expression level of mannitol-1-phosphate from P. nameko, mpd cDNA was inserted into pCold shock vector and expressed in host BL21 (DE3) by 24 h induction with 1 mM isopropyl 1-thio-β-Dgalactopyranoside at 15 O C after 2 h growth at 37 O C. The purified protein was detected by SDS-PAGE and western blot

Mycelial Growth-promoting Potential of Extracellular Metabolites of Paraburkholderia spp. Isolated from Rhizopogon roseolus Sporocarp

This study aimed to investigate the effect of potential metabolite(s) produced by Paraburkholderia spp. isolated from the Rhizopogon roseolus (shouro mushroom) sporocarp on the mycelial growth of R. roseolus. For this purpose, we selected two molecularly identified bacteria: P. fungorum GIB024 and P. caledonica KN1. Direct confrontation assay at three different distances, a pour plate method that sampled bacterial spent broth either with and without agitation at 25 °C, and an indirect confrontation assay was carried out in order to assess the R. roseolus growth-promoting ability of Paraburkholderia spp. These assessments were carried out in a 1:5 diluted Melin-Norkran-modified medium with glucose (hs-dMMN) and without glucose (ls-dMMN). GIB024 promoted the growth of R. roseolus in ls-dMMN in short distance, whereas KN1 inhibited the growth of the fungus in that condition. In hs-dMMN, both bacteria have neutral or slightly promotion effect toward R. roseolus. We determined from the spent broth analysis that Paraburkholderia spp. that grew axenically under static conditions had a more pronounced mycelial growth-promoting effect on R. roseolus than under agitation conditions. We also found that high concentration of spent broth resulted in a decrease in mycelial growth-promoting ability. Volatile metabolite(s) produced by both bacteria did not promote the mycelial growth of R. roseolus. In conclusion, Paraburkholderia spp. exhibited a species- and nutrient (sugar)-dependent ability to promote the mycelial growth of R. roseolus, and the bacterial soluble metabolite(s) play a crucial role in their growth-promoting ability

Vaccination with liposome-coupled glypican-3-derived epitope peptide stimulates cytotoxic T lymphocytes and inhibits GPC3-expressing tumor growth in mice

AbstractBecause therapeutic manipulation of immunity can induce tumor regression, anti-cancer immunotherapy is considered a promising treatment modality. We previously reported that glypican-3 (GPC3), an oncofetal antigen overexpressed in hepatocellular carcinoma (HCC), is a useful target for cytotoxic T lymphocyte (CTL)-mediated cancer immunotherapy, and we have performed clinical trials using the GPC3-derived peptide vaccine. Although vaccine-induced GPC3-peptide-specific CTLs were often tumor reactive in vitro and were correlated with overall survival, no complete response was observed. In the current study, we synthesized liposome-coupled GPC3-derived CTL epitope peptide (pGPC3-lipsome) and investigated its antitumor potential. Vaccination with pGPC3-liposome induced peptide-specific CTLs at a lower dose than conventional vaccine emulsified in incomplete Freund's adjuvant. Coupling of pGPC3 to liposomes was essential for effective priming of GPC3-specific CTLs. In addition, immunization with pGPC3-liposome inhibited GPC3-expressing tumor growth. Thus, vaccination with tumor-associated antigen-derived epitope peptides coupled to the surfaces of liposomes may be a novel therapeutic strategy for cancer

Enhancement of antitumor effect by peptide vaccine therapy in combination with anti-CD4 antibody: Study in a murine model

Purpose: The clinical efficacy of cancer peptide vaccine therapy is insufficient. To enhance the anti-tumor effect of peptide vaccine therapy, we combined this therapy with an anti-CD4 mAb (GK1.5), which is known to deplete CD4+ cells, including regulatory T cells (Tregs). Methods: To determine the treatment schedule, the number of lymphocyte subsets in the peripheral blood of mice was traced by flow cytometry after administration of anti-CD4 mAb. The ovalbumin (OVA)257–264 peptide vaccine was injected intradermally and anti-CD4 mAb was administered intraperitoneally into C57BL/6 mice at different schedules. We evaluated the enhancement of OVA peptide-specific cytotoxic T lymphocyte (CTL) induction in the combination therapy using the ELISPOT assay, CD107a assay, and cytokine assay. We then examined the in vivo metastasis inhibitory effect by OVA peptide vaccine therapy in combination with anti-CD4 mAb against OVA-expressing thymoma (EG7) in a murine liver metastatic model. Results: We showed that peptide-specific CTL induction was enhanced by the peptide vaccine in combination with anti-CD4 mAb and that the optimized treatment schedule had the strongest induction effect of peptide-specific CTLs using an IFN-γ ELISPOT assay. We also confirmed that the CD107a+ cells secreted perforin and granzyme B and the amount of IL-2 and TNF produced by these CTLs increased when the peptide vaccine was combined with anti-CD4 mAb. Furthermore, metastasis was inhibited by peptide vaccines in combination with anti-CD4 mAb compared to peptide vaccine alone in a murine liver metastatic model. Conclusion: The use of anti-CD4 mAb in combination with the OVA peptide vaccine therapy increased the number of peptide-specific CTLs and showed a higher therapeutic effect against OVA-expressing tumors. The combination with anti-CD4 mAb may provide a new cancer vaccine strategy

Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR▿

Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h