Mikiro Tada

Mushroom Tyrosinase Inhibitory Activity of Esculetin Isolated from Seeds of Euphorbia lathyris L.

A tyrosinase inhibitor was isolated from the seeds of Euphorbia lathyris L. by bioassay-guided fractionation and purification, using silica gel column chromatography. It was identified as esculetin by comparing its physical properties and spectral data with those of an authentic sample. The IC50 value of esculetin in the mushroom tyrosinase activity test was 43 μM. The kinetic study indicates that esculetin exhibited competitive inhibition against the oxidation of 3-(3,4-dihydroxyphenyl)-alanine by mushroom tyrosinase. The structure-activity relationships among five esculetin analogs suggest that hydroxyl groups at the C6 and C7 positions of the coumarin skeleton played an important role in the expression of tyrosinase inhibitory activity.

Effects of exogenous application of proline and betaine on the growth of tobacco cultured cells under saline conditions

Abstract The effects of the application of exogenous proline and betaine on the growth of tobacco cultured cells subjected to salt stress were investigated. Both proline and betaine mitigated the inhibition of growth of tobacco cells under saline conditions, but the harmful effect of salinity was less reduced by betaine than by proline. The amount of intracellular betaine in tobacco cells cultured in the NaCI medium supplemented with 20 mm betaine was larger than that of intracellular proline in the NaCI medium with 20 mm proline. The 1,1-diphenyl2-picrylhydrazyl (DPPH) experiments showed that proline displayed an antioxidant activity and that the antioxidant activity of betaine was not detectable. The malondialdehyde (MDA) assay demonstrated that exogenous proline but not betaine decreased the amount of MDA in salt-stressed tobacco cells. These results suggest that the difference in the mitigation effects between proline and betaine may be responsible for the difference in the antioxidant activity.

Exogenous proline mitigates the inhibition of growth of Nicotiana tabacum cultured cells under saline conditions

Abstract The addition of exogenous proline (10 mm) to Na100-saline culture medium, modified LS medium (Linsmaier and Skoog 1965: Physiol. Plant., 18, 100–127) with 100 mm NaCl promoted the growth of tobacco (Nicotiana tabacum L., cv. Bright Yellow-2) suspension cells unadapted to salt stress without maintaining a high ratio of K+ to Na+ ions under salinity conditions. The addition of exogenous glutamic acid or alanine were not comparable to that of exogenous proline. The proline contents of the NaCl-unadapted cells became much higher when the cells were grown in Na100-saline culture medium with 10 mm proline than when the cells were cultured without proline. The accumulation of K+, Na+, counter ions was sufficient to compensate for the increase of the water potential of the cells caused by salinity. These results suggest that exogenous proline does not act as a nitrogen store and that proline may act as a protectant for enzymes and membranes against salt inactivation rather than as a compatible solute in tobacco suspension cells.

Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein

Because sea cucumbers lack a well-developed immune system and can ingest pathogenic bacteria together with food, some form of active antibacterial substances must be present in the body for defense. In this study, the cDNA of an i-type lysozyme from the sea cucumber Stichopus japonicus (designated SjLys) was cloned by RT-PCR and RACE PCR techniques. The full length cDNA of SjLys was 713 bp with an open reading frame of 438 bp coding for 145 amino acids. Two catalytic residues (Glu34 and Asp47), conserved in i-type lysozymes, and a highly conserved region near the active site, MDVGSLSCG(P\Y)(Y\F)QIK, were detected in SjLys. In addition, the domain structure analysis of SjLys showed that it is highly similar to the medicinal leech destabilase, which belongs to a new phylogenetic family of invertebrate lysozymes possessing both glycosidase and isopeptidase activities. To gain insight into the in vitro antimicrobial activities of SjLys, the mature peptide coding region was heterologously expressed in Escherichia coli. The recombinant SjLys protein displayed an inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. A remarkable finding is that the recombinant SjLys exhibited more potent activities against all tested bacterial strains after heat-treating at 100 degrees C for 50 min. These results indicated that the S. japonicus lysozyme is an enzyme with combined enzymatic (glycosidase) and nonenzymatic antibacterial action.

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.

Growth inhibition of capsaicin on HeLa cells is not mediated by intracellular calcium mobilization.

The effects of capsaicin on cellular growth and intracellular calcium mobilization were examined in human cervical carcinoma derivation, HeLa cells. Capsaicin inhibited cellular growth and increased intracellular calcium level in HeLa cells. This capsaicin-induced intracellular calcium concentration rise was blocked by capsazepin, vanilloid (capsaicin) receptor antagonist. But, an intracellular calcium chelator BAPTA/AM did not block the inhibitory effect of capsaicin on cellular growth. These observations suggest that intracellular calcium mobilization is not required for the capsaicin-induced inhibition of cellular growth.

Autophagy plays a potential role in the process of sea cucumber body wall “melting” induced by UV irradiation

The changes of tissue appearances and structures in the process of UV-induced “melting” for sea cucumber (Stichopus japonicus) body wall were studied. And the localization and determination of acid phosphatase (ACP), Cathepsin B and Cathepsin L activities were also investigated. The results show that the connective tissue was damaged with many hollows emerging and the regular collagen bundles were broken apart into irregular fragments. Margination of condensed chromatin at the nuclear membrane was observed. Both Golgi’s body and endoplasmic reticulum swelled, curled, and eventually double-or multi-lamellar vesicles were formed. A number of autophagic vesicles distributed in all through the whole cytoplasm. ACP becomes more active after UV irradiation. The activities of cathepsin B and cathepsin L increased in UV-treated sea cucumbers and both achieved their maximum under certain conditions. It indicates that autophagy plays a potential role in the “melting” process for sea cucumber body wall induced by UV irradiation.

Negative correlation between the ratio of K+ to Na+ and proline accumulation in tobacco suspension cells

Abstract The concentrations of K+, Na+, and proline and the ratio of K+ to Na+ (K+ / Na+) were analyzed in NaCl-unadapted and NaCl-adapted tobacco (Nicotiana tabacum) cells in suspension culture. At 3 to 5 d after inoculation, the NaCl-unadapted cells cultured in 100 mmol L−1 NaCl saline culture medium (Na100 medium) accumulated 28.7 mmol L−1 proline with a low ratio of K+ to Na+ (= 2.8) and the NaCl-adapted cells cultured in the Na100 medium contained 6.28 mmol L−1 proline with a high K+ / Na+ ratio (≧ 7.5). The contents of amino acids for the NaCl-adapted cells in the Na100 medium were similar to those for the NaCl-unadapted cells in a modified LS medium (standard medium). At 14 d after inoculation, the NaCl-unadapted cells in the Na100 medium contained 4.77 mmol L−1 proline and restored the K+ / Na+ ratio from 2.8 to 6.2. These results indicate the presence of a negative correlation between the K+ / Na+ ratio and proline accumulation and suggest that a balance between the K+ / Na+ ratio and proline accumulation may be the factor involved in determining the salt tolerance of plant cells.

Antifungal activity of the fermentation product of herbs by lactic acid bacteria against tinea.

The fermentation product of herbs by lactic acid bacteria (FHL), in which Enterococcus faecalis TH10 predominated, was assayed for antifungal activity against tinea. The antifungal activity of FHL was as high as that of a synthetic fungicide. Autoclaving FHL did not reduce its antifungal activity, whereas neutralizing it did. The results suggested that nonproteinaceous compounds or organic acids in FHL could inhibit the growth of the dermatophyte tinea under low-pH conditions, and that malonic acid and acetic acid could have especially high antifungal activity against tinea.

Docosahexaenoic Acid Induces Apoptosis via the Bax-Independent Pathway in HL-60 Cells

We attempted to determine whether docosahexaenoic acid (DHA)-induced apoptosis is mediated via the Bax-mediated pathway in human myeloid leukemia HL-60 cells. DHA-induced apoptosis was confirmed by morphological analysis and caspase-3 activation. But, cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition (MPT), did not inhibit DHA-induced Bax translocation to mitochondria or caspase-3 activation. These data suggest that DHA can induce apoptosis via the Bax-independent pathway.