Subcomponents in humic acid structure contribute to the differential responses of Aspergillus oryzae strains to humic acid.

Humic acid (HA) is a complex natural organic macromolecule, can be decomposed to low-molecular compounds by some soil fungi and then influences the growth of fungi. Aspergillus oryzae is a fungus domesticated from its ancestor, which was supposed to live in soil. Group 3 strains of A. oryzae hold fewer aflatoxin-biosynthetic genes than group 1 strains and may differently response to HA because of the deletion of some genes along with the domestication. However, effect of HA on growth of A. oryzae group 1 and group 3 strains remains unclear. In this study, four strains of A. oryzae in group 1 and four in group 3 were point inoculated on equivalent medium (pH 7.3) with two commercially available HAs. The growth of RIB40 was the most stimulated among group 1 strains and that of RIB143 was the most inhibited among group 3 strains. To identify the basis of these differences, we examined the possible effects of HA subcomponents including polyphenol and minerals on the growth of RIB40 and RIB143. Polyphenol represented by gallic acid (GA), a partial structure common with model HA, and mineral ions including Al 3+ , Ca 2+ , Ti 4+ , Mn 2+ , Sr 2+ , and Ba2+ contributed to stimulating the growth of RIB40, whereas these components generally did not affect the growth of RIB143. Thus, our findings indicate that the sub-compositions of HAs, including GA and several minerals, were the main factors driving the different responses of RIB40 and RIB143 to HAs.

Characterization of a (D)-stereoselective aminopeptidase (DamA) exhibiting aminolytic activity and halophilicity from Aspergillus oryzae.

ß-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the ß-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [ß-Ala-para-nitroanilide (pNA)], the enzyme preferred D-Leu-pNA and D-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward D-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0-11.0. DamA also exhibited aminolytic activity, producing D-Leu-D-Leu-NH2 from D-Leu-NH2 as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from D-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a D-amino acid at the N-terminus as well as physiologically active peptides.

Humoral immune responses to CTL epitope peptides from tumor-associated antigens are widely detectable in humans: a new biomarker for overall survival of patients with malignant diseases.

Both cellular and humoral immune responses are crucial to induce potent anti-tumor immunity, but most of currently conducted peptide-based cancer vaccines paid attention to cellular responses alone, and none of them are yet approved as a therapeutic modality against cancer patients. We investigated humoral immune responses to CTL epitope peptides derived from tumor-associated antigens in healthy donors and patients with various diseases to facilitate better understanding of their distribution patterns and potential roles. Bead-based multiplex assay, ELISA, and Western blotting were used to measure immunoglobulins reactive to each of 31 different CTL epitope peptides. Importantly, the sums of anti-peptide IgG levels specific to 31 CTL epitope peptides were well correlated with better overall survival (OS) in patients with malignant diseases. Our results suggested that humoral immune responses to CTL epitope peptides were widely detectable in humans. Measurement of immunoglobulins specific to CTL epitope peptides may provide a new biomarker for OS of patients with malignant diseases, although it still remains to be determined whether the correlations between humoral immune responses to epitope peptides and OS are observed only for the CTL epitopes used, or also for other panels of peptides. Quantity of circulating IgG reactive to these peptides was also discussed.

Characterization of an Aspergillus oryzae cysteinyl dipeptidase expressed in Escherichia coli.

Cysteinyl dipeptidase from Aspergillus oryzae (CdpA) was produced in Escherichia coli and purified. The enzyme showed activity specific toward cysteine-containing dipeptides, but its substrate specificity was distinct from those of other cysteinyl dipeptidases of the M20 family. It was optimally active at pH 7-8 and stable at pH 6-9 and at up to 40 °C.

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.

A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae.

Aspergillus oryzae is resistant to many kinds of antibiotics, which hampers their use to select transformants. In fact, the fungus is resistant to over 200microg/ml of bleomycin (Bm). By enhancing the susceptibility of A. oryzae to Bm using Triton X-100 as a detergent and an ATP-binding cassette (ABC) pump inhibitor, chlorpromazine, to the growing medium, we established a novel transformation system by Bm selection for A. oryzae. In a medium containing these reagents, A. oryzae showed little growth even in the presence of 30microg Bm/ml. Based on these findings, we constructed a Bm-resistance expression cassette (BmR), in which blmB encoding Bm N-acetyltransferase from Bm-producing Streptomyces verticillus was expressed under the control of a fungal promoter. We obtained a gene knockout mutant efficiently by Bm selection, i.e., the chromosomal ligD coding region was successfully replaced by BmR using ligD disruption cassette consisted of ligD flanking sequence and BmR through homologous recombination.

Mitogen limitation and bone morphogenetic protein-4 promote neurogenesis in SFME cells, an EGF-dependent neural stem cell line.

Serum-free mouse embryo (SFME) cells are an epidermal growth factor (EGF)-dependent established line derived from brains of 16-d-old Balb/c mouse embryos. SFME cells grow indefinitely in serum-free medium without replicative senescence, chromosomal abnormalities, or malignant transformation. SFME cells express nestin, a neural stem cell marker, under serum-free conditions. Exposure to serum or transforming growth factor beta (TGF-beta) leads to a marked increase in differentiation toward the astrocytic lineage with expression of glial fibrillary acidic protein and other astrocyte markers. In this study, we show that treatment of SFME cells with bone morphogenetic protein-4 (BMP-4), another member of the TGF-beta family, led to differentiation toward a neuronal lineage under conditions of low mitogenic stimulation (0.5 ng/mL) by EGF and fibroblast growth factor. Maximum mitogenic stimulation with 50 ng/mL EGF blocked the BMP-4 effect on neuronal differentiation, but did not block TGF-beta-induced expression of markers of the astrocytic lineage. BMP-4 treatment also enhanced the activity of the neuron-specific enolase (NSE) promoter in SFME-NSE-lacZ cells that carry the gene for bacterial beta-galactosidase under the control of the NSE promoter. Extended BMP-4 treatment caused SFME cells to express a neuronal phenotype synthesizing gamma-aminobutyric acid. These results indicate that SFME cells have the capacity to generate both neurons and astrocytes in vitro, which resemble the behavior of EGF-dependent multipotential stem cells in the central nervous system, and establish a relationship between effects of BMP-4 and degree of mitogenic stimulation by other peptide growth factors.

Identification of a novel cytotoxic protein, Cry45Aa, from Bacillus thuringiensis A1470 and its selective cytotoxic activity against various mammalian cell lines.

Parasporal inclusion proteins produced by Bacillus thuringiensis strain A1470 exhibit strong cytotoxicity against human leukemic T cells when activated by protease treatment. One of the cytotoxic proteins was separated by anion exchange chromatography and gel filtration chromatography and designated Cry45Aa. Its gene was then expressed in recombinant Escherichia coli, in which the Cry45Aa precursor was accumulated in an inclusion body. It was solubilized in sodium carbonate buffer and processed with proteinase K, and cytotoxic activities of the protein against various mammalian cell lines were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay. The protein exhibited high cytotoxic activity against CACO-2, Sawano, MOLT-4, TCS, and HL60 cells and moderate activity against U-937 DE-4, PC12, and HepG2 cells. On the other hand, the EC50 values against Jurkat, K562, HeLa, A549, Vero, COS-7, NIH3T3, CHO, and four normal tissue cells (human primary hepatocyte cells, UtSMC, MRC-5, and normal T cells) were >2 microg/mL.

Pluripotent differentiation in vitro of murine ES-D3 embryonic stem cells.

Although the ES-D3 murine embryonic stem cell line was one of the first derived, little information exists on the in vitro differentiation potential of these cells. We have used immunocytochemical and flow cytometric methods to monitor ES-D3 embryoid body differentiation in vitro during a 21-d period. Spontaneous differentiation of embryoid body cells was induced by leukemia inhibitory factor withdrawal in the absence of feeder cells. The pluripotent stem cell markers Oct-3/4, SSEA-1, and EMA-1 were found to persist for at least 7 d, whereas the primitive endoderm marker cytokeratin endo-A was expressed at increasing levels from day 6. The localization of these antigens within the embryoid bodies suggested that embryonic ectoderm- and primitive endoderm-derived tissues were segregated. Localized expression of class III beta-tubulin and sarcomeric myosin also was detected, indicating that representatives of all three embryonic germ layers were present after induction of differentiation in vitro.

Suppression of UVC-induced cell damage and enhancement of DNA repair by the fermented milk, Kefir.

An aqueous extract of Kefir, fermented milk originally produced in the Caucasus mountains, suppressed morphological changes of human melanoma HMV-1 and SK-MEL cells and human normal fibroblastTIG-1 cells caused by UVC-irradiation, suggesting that UV damage can be suppressed by the Kefir extract. The addition of the Kefir extract after UVC-irradiation of HVM-1 cells resulted in a remarkable decrease in intracellular reactive oxygen species (ROS) which had been increased by UVC irradiation. The Kefir extract also stimulated unscheduled DNA synthesis and suppressed UVC-induced apoptosis of HMV-1 cells. A colony formation assay revealed that the Kefir extract rescued HMV-1 cells from cell death caused by UVC irradiation. The Kefir extract, as well as methyl methanethiosulfonate which is known to enhance the nucleotide excision repair (NER) activity, exhibited strong thymine dimer repair-enhancing activity. Epigalocatechin exhibited a weak NER activity but vitamins A, C, and E and catechin showed no NER activity. The thymine dimer repair-enhancing factors in the Kefir extract were heat-stable and assumed to be molecules with a molecular weight of less than 5000. The treatment of HMV-1 cells with the Kefir extract during or before UVC- irradiation also prevented the generation of ROS and thymine dimmer, and suppressed the apoptosis of HMV-1 cells, suggesting that application of Kefir can prevent UV damage.