Ken-Ichi Kusumoto

Directed deletions in the aflatoxin biosynthesis gene homolog cluster of Aspergillus oryzae.

To investigate the structure of the aflatoxin gene cluster in Aspergillus oryzae, 39 strains belonging to this species were examined for the existence of pksA, fas-1A, aflR and vbs, and the results compared with those for ver-1 obtained previously. These five genes are involved in aflatoxin biosynthesis in Aspergillus parasiticus. The strains examined were categorized into three groups; group 1, having the five homologs; 2, having ver-1 and vbs; and 3, having vbs homologs. Long-PCR analysis of the regions between the five homologs in A. oryzae IFO 4135, coupled with Southern-hybridization analysis, shows that those homologs are clustered with the same arrangement as in A. parasiticus. These results suggest that directed deletions of the cluster occur in A. oryzae strains. The possible breakpoints of the deletions in the strains of group 2 and 3 were estimated.

Didodecyldimethylammonium bromide (DDAB) induces caspase-mediated apoptosis in human leukemia HL-60 cells.

Didodecyldimethylammonium bromide (DDAB) is widely used for an efficient delivery system into mammalian cells. However, the biological activities of DDAB nanoparticles in mammalian cells are insufficiently understood. The purpose of this study was to establish a critical role of DDAB in cellular response. Here, we demonstrate that DDAB is a potent inducer of cell death in a wide range of tumor cell lines, wherein leukemia cells (HL-60 and U937) and neuroblastoma cells (Neuro2a) were more sensitive to DDAB than carcinoma cells such as HepG2 and Caco-2 cells. Moreover, in HL-60 cells, treatment with DDAB led to increased numbers of apoptotic cells with fragmented DNA (99.6%) and high levels of caspase-3 activation in comparison to that with actinomycin D. Cotreatment with a caspase-8 inhibitor (Z-IETD-FMK) or a polyethylene glycol (Mr 2000) (PEG 2000) effectively prevented the activation of caspase-3 induced by DDAB. These results suggest that DDAB can trigger caspase-3-mediated apoptosis through the extrinsic caspase-8 pathway and cytotoxic pore formation in cell membrane. Therefore, the present findings provide new insight into the biological activity of DDAB to induce caspase-mediated apoptosis.

Aspergillus oryzae with and without a homolog of aflatoxin biosynthetic gene ver-1

Abstract Part of the nucleotide sequence of the ver-1 homolog in each of two strains of Aspergillus oryzae, Aspergillus sojae, and Aspergillus flavus were compared with two homologs in Aspergillus parasiticus. The homologs in A. oryzae and A. sojae (non-aflatoxin-producers) exhibited an extremely high degree (93.8–99.8% for A. oryzae, and 96.0–99.5% for A. sojae) of sequence identity with that of A. flavus and A. parasiticus. No two strains within the same species, except A. sojae, were identical. No sequence fingerprint was found to distinguish between A. oryzae and A. flavus, or between A. sojae and A. parasiticus. The predicted partial amino acid sequences (181 amino acids) of the ver-1 homologs had at most two amino acid changes relative to A. parasiticus SYS-4 ver-1. Transcripts of ver-1 homologs in the strains of A. oryzae and A. sojae examined were not detected by the polymerase chain reaction coupled with reverse transcription. By Southern analysis, a total of 46 strains of A. oryzae were examined for the presence of the ver-1 homolog. The homolog was detected in 38 strains, but not in 8 strains. Morphologically, strains with and without the ver-1 homolog were indistinguishable. Thus, A. oryzae contains strains with and without a homolog of the aflatoxin biosynthetic gene ver-1.

Inhibitors of platelet lipoxygenase from Ponkan fruit

An activity-guided separation for inhibitors of rat platelet 12-lipoxygenase led to the isolation of two compounds, 4-O-feruloyl-5-O-caffeoylquinic acid (IC50; 5.5 microM) and methyl 4-O-feruloyl-5-O-caffeoylquinate (IC50; 1.9 microM) from the peel of Ponkan fruit (Citrus reticulata). The complete structure of each phenolic ester was determined by NMR spectroscopy [1H and 13C NMR spectra, 1H-1H correlation spectroscopy (COSY), 1H-detected heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC) spectroscopies] and other spectral methods.

Changes in molecular weight and carbohydrate composition of cell wall polyuronide and hemicellulose during ripening in strawberry fruit

Abstract Polyuronides and hemicelluloses from the cell wall of developing strawberry fruit ( Fragaria ananasa , Duch. cv. Toyonoka) were examined for molecular weight distribution, and carbohydrate composition prior to and following gel filtration chromatography. In non-fractionated polyuronides, the galacturonic acid and arabinose concentrations decreased during development. The concentration of rhamnose markedly increased, whereas that of arabinose declined in low-molecular-weight polyuronides ( M r kDa ). A significant reduction of hemicelluloses was detected in the high-molecular-weight region ( M r >170 kDa ), whereas little change was observed in the sugar composition of these polymers. Polygalacturonase activity was consistently reduced during ripening.

Putrescine accumulation in banana fruit with ripening during storage.

Abstract Considerable accumulation of the free polyamine, putrescine (Put), was found in both pulp and peel tissue of unchilled and rewarmed bananas (Musa AAA group) during normal fruit ripening with climacteric ethylene production, while free Put remained at the original level in continuously chilled fruit. The bound Put content in both tissues of the unchilled and rewarmed fruits increased ca 3–10 fold. No competitive relationship was observed between Put and ethylene production in the banana fruit.

Gene transfer effects on various cationic amphiphiles in CHO cells.

Gene transfer is an important tool to explore genomic, cell biologic, or gene therapeutic research. In this paper we report that several cationic amphiphiles have the potential to efficiently deliver DNA into CHO cells, which is one of the cell lines considered to be important for production of proteins including therapeutic proteins. We have found that O,O′-ditetradecanoyl-N-(trimethylammonio acetyl) diethanolamine chloride (14Dea2), among 29 types of cationic amphiphiles tested, shows a transfection efficiency of more than 40% in CHO cells. In addition, the results from a series of hydrocarbon chains of varying lengths bound to a connector have shown that an optimal chain length is important for the efficient delivery of DNA into cells. Moreover, flow cytometer analysis has shown that 14Dea2 transfection leads to high levels of expression of the reporter gene (green fluorescent protein) in individual cells. These findings have suggested that 14Dea2 is able to effectively deliver a number of plasmids into a cell nucleus. Thus, our system might be a powerful tool for high efficiency gene transfer and production of high levels of recombinant protein.

Homologs of the aflatoxin biosynthetic gene ver-1 in strains of Aspergillus oryzae and related species☆

Abstract Partial nucleotide sequences of two ver-1 homologs in Aspergillus parasiticus SYS-4 were analyzed. One of the homologs appeared to have a termination codon at amino acid position 87 and seems to be a non-functional pseudogene. Comparison of the nucleotide sequence between the another ver-1 homolog of SYS-4 and ver-1 of A. parasiticus NRRL 5862 led to identification of a frameshift within an exon; thus a part of amino acid sequences for the ver-1 homolog of SYS-4 and ver-1 of NRRL 5862 seems to differ greatly. With the ver-1 region of SYS-4 as a probe, DNA homologous to ver-1 was detected in some strains of Aspergillus oryzae and Aspergillus sojae , which are non-aflatoxin-producers, in addition to strains of Aspergillus flavus and A. parasiticus .

Anti-Melanogenic Activity of Yacon Leaves in Mouse Melanoma Cells

Understanding of natural compounds for the biological functions, including skin-whitening, anti-obese, anti-cancer activity, is essential for the effective design and selection of appropriate compounds as potential applications in basic and therapeutic research. The purpose of this study was to identify the novel biological function of yacon harvesting in Kurume city (Fukuoka, Japan). Yacon (Smallanthus sonchifolius, Asteraceae), originating from the Andean of South America, is a rich source of beta-1, 2-oligofructans and polyphenols. The plant has become popular in Japan, New Zealand, and Central Europe. Recently, it was reported that aqueous leaf extracts in yacon have a hypoglycemic activity in normal and diabetic rats. However, the biological functions of yacon have been controversial. Here, we found that yacon leaf extracts have an anti-melanogenic activity to suppress melanin synthesis in mouse B16 melanoma cells, which has been attracting a lot of attention as beauty aid. First, we estimated the extraction effects of yacon leaves with various solvents on suppression of melanogenesis. Subsequently, the leaf extracts treated with ethanol were isolated by fractionation on a reverse-phase high-pressure liquid chromatography C-18 column following a silica-gel column. The purified fraction was homogeneously detected by thin-layer chromatography (TLC) analysis. The identification and characterization of this compound as a melanogenic inhibitor will be presented in the future. Our results suggest that this approach can propose a novel biological function of traditional food yacon for suppressing melanin synthesis.

Thin-Film Assembly of Totally Designed Lipidic Materials as Gene Carrier in Mouse Embryonic Neural Stem Cells: Thin-Film Assembly of Lipidic Material as Potential Gene Carrier

Neural stem cells present in developing embryonic and adult nervous systems are immature and uncommitted cells that possess self-renewal potential and produce a vast array of highly specialized neural cells belonging to the central nervous system. Several studies have reported the development of gene delivery system for these neural cells as a potential application in gene therapy for neurodegenerative diseases and for understanding the genetic basis of brain development and function. Gene delivery system based on a variety of materials such as lipids, polypeptides, amino-dendrimers, and polyethylenimine have been used in mammalian cells, and currently, these systems are known to possess therapeutic applications. However, the transfer efficiencies of most lipidic materials with regard to neural cells, including neural stem cells, progenitor cells, neurons, and glia, are less than 5% compared to those of virus vectors. Understanding of lipidic materials used for gene delivery system is essential for the effective design and development of potential applications in basic and therapeutic research. This study aimed to evaluate the biological activity of totally synthesized ditetradecylacetyldiethanolaminetrimethylammonium (TMA-C2-DEA-C14) as gene carriers for neural stem cells. The transfer abilities were estimated by expressing green fluorescent protein (GFP) in mouse embryonic neural stem cells. Here, we demonstrate that lipidic assembly of TMA-C2-DEA-C14, which was self-organized by incubation in water for a month at 25°C, can provide an efficient gene delivery with low cytotoxicity (approximately 40% of GFP-expressed neural stem cells). Moreover, electron microscopic analysis showed that TMA-C2-DEA-C14 assembly is characterized by thin-film structures with polygonal shapes (approximately 2.7 μm), and after association with DNA, their structures dramatically changes to form liposome complexes that can effectively deliver DNA into the cellular cytoplasm of neural stem cells. Our findings suggest that this approach can serve as a novel model for the development of lipidic materials on nonviral gene delivery system.