A New Protocol for the Detection of Sterigmatocystin-producing Aspergillus Section Versicolores Using a High Discrimination Polymerase.

Aspergillus section Versicolores species, except Aspergillus sydowii, produce a carcinogenic mycotoxin sterigmatocystin (STC). Since these fungi are found in varied environmental milieu including indoor dust and food products, our aim was to develop a sensitive and convenient assay to detect STC producing fungal strains. We made use of a high discrimination DNA polymerase (HiDi DNA polymerase), for single nucleotide polymorphism (SNP)-based PCR amplification. Using specific primer pairs based on the SNPs between A. sydowii and other strains of Aspergillus section Versicolores, we succeeded in amplifying the genomic DNA all target strains except A. sydowii. These results confirm that the SNP-based PCR amplification technique, using a high discrimination DNA polymerase, was a reliable and robust screening method for target fungal strains.

New Insight on Fumigation Action of Essential Oil, Commercial Fungicide and Low Oxygen Microenvironment on Museum Mold, Alternaria alternata.

Fumigation has been the most convenient method in the field of pest control in museums. In this study, as fumigants, ethanol 70%, deltamethrin (commercial pesticide (CP) ) , essential oil (EO) from Pinus regida, and low oxygen microenvironment (0.1%, (LOM) ) were tested individually and jointly against museum fungal strain Alternaria alternata. Three concentrations of each CP and EO were chosen for evaluating the individual effect. In the joint action fumigation process, three lower concentrations of CP and EO were tested in LOM. The rate of mycelial growth inhibition at each fumigation process was determined by two steps: 1) directly after the fumigation process and 2) after 7 d of the inoculation of the fumigated spores in new medium and incubating it in normal condition. The results demonstrated that applying of each chemical (CP or EO) in LOM enhanced its fungicidal activity and that effect of EO improved from fungistatic to fungicidal by jointing with LOM.

Fungicidal Effects of Ultraviolet Light (254 nm) Irradiation on Contaminated Museum Packing and Storing Materials.

In storage of modern museums, collections are packed and stored with acid-free paper-based materials for keeping safe and stable conditions. Direct contact of fungal contaminated packing and storing materials with the collections is concerned about expanding of infection in storage facilities. In this study, fungicidal effects of UV light irradiation on the materials such as archival board and Japanese tissue paper contaminated with Penicilliun commune and Chaetomium globosum were tested. The analyzed materials were divided into two groups; Group 1 was examined with 20 µl of spore suspensions of fungi (106 cfu/ml) ; and Group 2 was tested on Czapek- Dox agar medium modified without sugar and inoculated with 100 µl of the spore suspensions of fungi (106 cfu/ml) . Six doses of UV irradiation were examined on Group 1 and five doses on Group 2 in addition to control. The assessment was done by using 1) adenosine triphosphate (ATP) bioluminescence assay and double staining to determine the cell viability; 2) observation under light microscope to evaluate morphophysiological change of tested fungi (spores and hyphae) . Because of the thinness and high transparency of tissue paper, UV irradiations were highly efficient to fungicide its fungal contamination compared with archival board. In spite of the high resistance of C. globosum spores, the rate of growth was slow, and with a little amount of perithecia or fruiting bodies and a high amount of ycelium (which damaged rapidly through UV irradiation) . This may be due to a low relative humidity of the incubation environment. Minimum dosage of UV irradiation with fungicidal effectiveness against all fungal contamination was estimated as 118 J/cm2.

Hypoxia induces expression of a GPI-anchorless splice variant of the prion protein.

The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI-anchorless PrP (GPI(-) PrPSV), was unglycosylated and soluble in non-ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non-neuronal tissues. When long-term passaged T98G cells were placed in a low-oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI(-) PrPSV expression in T98G cells.

The activities of mycotoxins derived from Fusarium and related substances in a short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells).

Cell transformation assays using BALB/3T3 cells can mimic the two-stage process of chemical carcinogenesis in experimental animals. A short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells), which was developed by Ohmori et al. and modified by Asada et al., has been reported to detect both tumor initiators and promoters as transformation initiators and promoters, respectively, with their differences based on their protocols. In this new short-term assay, we examined mycotoxins derived from Fusarium and related substances for the initiation and promotion activities of the transformation. The tested substances included deoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, fumonisin B(1), fumonisin B(2), zearalenone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol and beta-zearalenol. Fumonisin B(1) and T-2 toxin were positive for promoting activity in the assay. Especially, T-2 toxin was active at concentrations as low as 0.001-0.002microg/mL in the culture medium. From a comparison between the results of this study and published carcinogenicity assay data, it was expected that the Bhas 42 cell transformation assay had a good correlation with the two-stage carcinogenicity tests using experimental animals for estimation of the tumor-promoting activity.

In vitro bioassay of endotoxin using fluorescein as a pH indicator in a macrophage cell culture system.

Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH-indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS-detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.

Propagation of a protease-resistant form of prion protein in long-term cultured human glioblastoma cell line T98G.

Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), a lethal, neurodegenerative condition, occur in sporadic, genetic and transmitted forms. CJD is associated with the conversion of normal cellular prion protein (PrP(C)) into a protease-resistant isoform (PrP(res)). The mechanism of the conversion has not been studied in human cell cultures, due to the lack of a model system. In this study, such a system has been developed by culturing cell lines. Human glioblastoma cell line T98G had no coding-region mutations of the prion protein gene, which was of the 129 M/V genotype, and expressed endogenous PrP(C) constitutively. T98G cells produced a form of proteinase K (PK)-resistant prion protein fragment following long-term culture and high passage number; its deglycosylated form was approximately 18 kDa. The PK-treated PrP(res) was detected by immunoblotting with the mAb 6H4, which recognizes residues 144-152, and a polyclonal anti-C-terminal antibody, but not by the mAb 3F4, which recognizes residues 109-112, or the anti-N-terminal mAb HUC2-13. These results suggest that PrP(C) was converted into a proteinase-resistant form of PrP(res) in T98G cells.

Byssochlamysol, a new antitumor steroid against IGF-1-dependent cells from Byssochlamys nivea. I. Taxonomy, fermentation, isolation and biological activity.

A new antitumor steroid, byssochlamysol, was isolated from the mycelium of Byssochlamys nivea M#5187. Byssochlamysol inhibited IGF-1-dependent growth of MCF-7 human breast cancer cells with an IC50 of 20 ng/ml, whereas serum-dependent cell growth was not inhibited by less than 10 microg/ml of byssochlamysol. This substance induced apoptosis in IGF-1-dependent Colo320DM human colon cancer cells.

Byssochlamysol, a new antitumor steroid against IGF-1-dependent cells from Byssochlamys nivea. II. Physico-chemical properties and structure elucidation.

The structure of byssochlamysol, a new antitumor metabolite against IGF-1-dependent cancer cells from Byssochlamys nivea M#5187, was determined to be a highly oxidized ergostane steroid as shown in Fig. 1 by NMR studies.

G1-dependent prion protein expression in human glioblastoma cell line T98G.

Human glioblastoma cell line T98G produced a cellular form of prion protein (PrP(C)), and we confirmed expression of PrP mRNA by RT-PCR. Immunoblot analysis of whole cell lysate revealed one major (35 kDa) and two faint bands (31, 25 kDa) that reacted with monoclonal anti-human PrP antibody 3F4. Cells treated with tunicamycin produced only a 25 kDa band, representing a deglycosylated form of PrP. Similarly, peptide: N-glycosidase F treatment of whole cell lysate altered the Asn-linked form to the deglycosylated form. When T98G cells were cultured for a longer period, the amount of PrP(C) per cell increased on Day 4 to 16 in a time-dependent manner. When the cells were cultured at high cell-density, the cells on Day 4 produced the same amount of PrP(C) as those on Day 16 of the usual culture. Moreover, in a serum-free medium, cells cultured at a low cell-density produced the same amount of PrP(C) as those cultured at the high cell-density. These results demonstrate that PrP(C) production in T98G cells was dependent on the phase of the cell cycle, probably the G1 phase.