Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

Optimum modification for the highest cytotoxicity of cationized ribonuclease

Cationization of a protein is considered to be a powerful strategy for internalizing a functional protein into cells. Cationized proteins appear to adsorb to the cell surface by electrostatic interactions, then enter the cell in a receptor- and transporter-independent fashion. Thus, in principle, all cell types appear to take up cationized proteins. Since ribonucleases (RNases) have a latent cytotoxic potential, cationized RNases could be useful cancer chemotherapeutics. In this study, we investigated the effect of the degree of cationization on the cytotoxicity of RNase A by modifying carboxyl groups with ethylenediamine. We found that there is an optimum degree of modification for cytotoxicity, in which 5 to 7 out of 11 carboxyl groups in RNase A are modified, toward MCF-7 and 3T3-SV-40 cells. More interestingly, the cytotoxicity of cationized RNase As correlates well with the value of [RNase activity] x [estimated concentration of RNase free from RNase inhibitor], mimicking the practical enzymatic activity of cationized RNase As in cytosol. The results indicate that cationization of a protein to an optimum level is important for maintaining protein function in the cytosol. Sophisticated protein cationization techniques will help to advance protein transduction technology.     

Laminin‐421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells

Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC‐CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat‐treatment. The chemotactic activity of LEC‐CM was abolished by immunodepletion with anti‐laminin‐1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC‐CM contained laminin‐421. When melanoma C8161 cells were treated with function‐blocking antibodies to integrin α3 or α6, their chemotactic responses to LEC‐CM were markedly reduced. Furthermore, the knock‐down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC‐CM. These data suggest that laminin‐421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.     

Porphyromonas gingivalis Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells

The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, Porphyromonas gingivalis, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that P. gingivalis increased IL-33 expression in the cytoplasm of human gingival epithelial cells in vitro. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from P. gingivalis did not increase IL-33 expression. Specific inhibitors of P. gingivalis proteases (gingipains) suppressed IL-33 mRNA induction by P. gingivalis and the P. gingivalis gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by P. gingivalis. These results indicate that the PAR-2/IL-33 axis is promoted by P. gingivalis infection in human gingival epithelial cells through a gingipain-dependent mechanism.     

Distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, in pulmonate snails

The distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, were examined in the nervous system of pulmonate snails. We chemically identified the authentic NdWFamide from a land snail (Euhadra congenita) and a freshwater snail (Lymnaea stagnalis). NdWFamide potentiated the heartbeat of those snails. Immunohistochemistry using anti-NdWFamide antibody demonstrated the distribution of NdWFamide-containing neurons and fibers in the central nervous system, as well as peripheral tissues, such as the cardiovascular region and accessory sex organs. These results suggest that NdWFamide is a neuropeptide mediating the neural regulation of the activity of the cardiovascular and reproductive systems of snails.     

Bacterial Contribution to Dissolved Organic Matter in Eutrophic Lake Kasumigaura,Japan

Incubation experiments using filtered waters from Lake Kasumigaura were conducted to examine bacterial contribution to a dissolved organic carbon (DOC) pool. Bacterial abundance, bacterial production, concentrations of DOC, total dissolved amino acids (TDAA), and total dissolved neutral sugars (TDNS) were monitored during the experiments. Bacterial production during the first few days was very high (20 to 35 μg C liter⁻¹ day⁻¹), accounting for 40 to 70% of primary production. The total bacterial production accounted for 34 to 55% of the DOC loss during the experiment, indicating high bacterial activities in Lake Kasumigaura. The DOC degradation was only 12 to 15%, whereas the degradation of TDAA and TDNS ranged from 30 to 50%, suggesting the preferential usage of TDAA and TDNS. The contribution of bacterially derived carbon to a DOC pool in Lake Kasumigaura was estimated using d-amino acids as bacterial biomarkers and accounted for 30 to 50% of the lake DOC. These values were much higher than those estimated for the open ocean (20 to 30%). The ratio of bacterially derived carbon to bulk carbon increased slightly with time, suggesting that the bacterially derived carbon is more resistant to microbial degradation than bulk carbon. This is the first study to estimate the bacterial contribution to a DOC pool in freshwater environments. These results indicate that bacteria play even more important roles in carbon cycles in freshwater environments than in open oceans and also suggests that recent increases in recalcitrant DOC in various lakes could be attributed to bacterially derived carbon. The potential differences in bacterial contributions to dissolved organic matter (DOM) between freshwater and marine environments are discussed.     

Mitochondrial superoxide production contributes to vancomycin-induced renal tubular cell apoptosis

Vancomycin chloride (VCM), a glycopeptide antibiotic, is widely used for the therapy of infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is a major adverse effect in VCM therapy. In this study, we investigated the cellular mechanisms underlying VCM-induced renal tubular cell injury in cultured LLC-PK1 cells. VCM induced a concentration- and time-dependent cell injury in LLC-PK1 cells. VCM caused increases in the numbers of annexin V-positive/PI-negative cells and TUNEL-positive cells, indicating the involvement of apoptotic cell death in VCM-induced renal cell injury. The VCM-induced apoptosis was accompanied by the activation of caspase-9 and caspase-3/7 and reversed by inhibitors of these caspases. Moreover, VCM caused an increase in intracellular reactive oxygen species production and mitochondrial membrane depolarization, which were reversed by vitamin E. In addition, mitochondrial complex I activity was inhibited by VCM as well as by the complex I inhibitor rotenone, and rotenone mimicked the VCM-induced LLC-PK1 cell injury. These findings suggest that VCM causes apoptotic cell death in LLC-PK1 cells by enhancing mitochondrial superoxide production leading to mitochondrial membrane depolarization followed by the caspase activities. Moreover, mitochondrial complex I may play an important role in superoxide production and renal tubular cell apoptosis induced by VCM.     

Presence of oxidized protein hydrolase in human cell lines, rat tissues, and human/rat plasma

Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.     

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.