Kyoya Takahata

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.

Cross-linking of a synthetic partial-length (1-28) peptide of the Alzheimer β/A4 amyloid protein by transglutaminase

Cerebral deposits of β/A4 amyloid protein is a pathologic sign of Alzheimers disease. A synthetic partial‐length (1–28) peptide of this protein contains one glutamine and two lysine residues. Here we show that this peptide can be a substrate of transglutaminase, which catalyzes cross‐linking between glutamine and lysine residues in peptides, by demonstrating the formation of multimeric peptides due to the action of this enzyme. A modified (LyS28 to l‐norleucine) version of the synthetic peptide was also cross‐linked, but another modified version (Lys16 to l‐norleucine) was very poorly cross‐linked, indicating that Lys16 is involved exclusively in the cross‐linking of the partial‐length peptide catalyzed by transglutaminase.

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.

The inhibitory effects of okadaic acid on platelet function

Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin‐induced platelet aggregation (IC50 = 0.8 μM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50‐kDa protein and 20‐kDa myosin light chain (MLC20). The 50‐kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20, OA decreased the thrombin‐induced phosphorylation of 47‐kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5−10 μM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin‐induced platelet activation.

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.

Purification and partial characterization of CD9 antigen of human platelets

CD9 antigen (p24) was purified from human platelets and partially characterized. The yield was 75 μg from 10 units of platelet concentrates. p24 (38 000 copies/platelet) has intramolecular disulfide bond(s) and, in SDS‐PAGE, consists of major 24‐kDa molecule and minor 26‐ to 31‐kDa molecules. The N‐terminal sequence of p24, PVKGOTKXIKYLLFGFNFIF, indicates that the protein has not previously been characterized and amino terminus (position 12–20) is hydrophobic.

PROTECTION FROM CHEMOTHERAPY-INDUCED ALOPECIA BY DOCOSAHEXAENOIC ACID

Chemotherapy-induced alopecia is a common side effect of many chemotherapeutic drugs used for the treatment of cancer. Several methods currently utilized to prevent chemotherapy-induced alopecia are unsatisfactory. Research of new treatments has been hampered, in part, by the lack of a suitable and reproducible experimental animal model to determine the cellular target of anticancer drugs in the hair follicle and how the interaction of these drugs with that target leads to hair loss. Recently made observations in the young rat model (1) have provided a new insight into this problem and opened new avenues for further investigation. Using this model, Cece et al. (2) reported that chemotherapy treatment induced apoptosis in a hair follicle cell. Thus we examined whether docosahexaenoic acid (DHA), which had been found to have a modulator effect on neural apoptosis, could prevent chemotherapy-induced alopecia in this model. Lactating Sprague-Dawley rats with 10 newborns/mother were purchased. In these experiments, 8-d-old rats were used for cytosine arabinoside (Ara-C)-induced alopecia, and 11 -dold fats were used for etoposide (VP-16)-induced alopecia. All chemotherapy was given i.p., and DHA was given p.o. Degree of alopecia was assessed on the 17-d-old (Ara-C) or 20-d-old (VP-16) animals. The results are as follows: (i) all rats in the chemotherapytreated group became totally alopecia; (ii) in these alopeciainduced groups, apoptosis was detected by TUNEL staining method in skin sections; (iii) in contrast, most rats in the DHA-treated group were protected from chemotherapyinduced alopecia; (iv) recombinant human interleukinlc~ (rhlL lc0 or intrinsic IL 1 inducer, lipopolysaccharide, also could strongly protect alopecia; and (v) the effect of DHA could not be neutralized by ILl-receptor antagonist. In conclusion, it is suggested that DHA protection against chemotherapy-induced alopecia could be mediated either directly or indirectly through apoptosis modulation without involving ILl systems.

Effects of Oxygenated Carotenoid β-Cryptoxanthin on Morphological Differentiation and Apoptosis in Neuro2a Neuroblastoma Cells

Beta-Cryptoxanthin (βCx) was investigated for cell functions in neuroblastoma Neuro2a cells. The following results were obtained. 1. Beta-Cx induced neurite outgrowth. 2. Beta-Cx inhibited the etoposide-induced activation of caspase-3 activity in a dose-dependent manner. These data suggest a bioregulatry function of βCx in the control of differentiation and apoptosis in Neuro2a cells.

SUPPRESSIVE EFFECTS OF ASCORBATE DERIVATIVES ON ULTRAVIOLET-B-INDUCED INJURY IN HACAT HUMAN KERATINOCYTES

Abstract The aging of skin, including sunburning, is caused by ultraviolet (UV) irradiation. Here, we examined the inhibitory effect of ascorbic acid (AsA) and its derivatives AsA 2-phosphate (AA-2P) and AsA 2-glucoside (AA-2G) on UV-B– induced cytotoxicity in HaCaT keratinocytes. Results show that cell viability significantly decreased when exposed to UV-B at 0.1–0.4 J/cm2 in a dose-dependent manner. In this study, AsA could not inhibit cytotoxicity, but AA-2P and AA-2G was able to cancel the harmful effect of UV-B when treated at high levels of 0.5–5 mM. These results indicate that the masking of the C-2 OH group may be an effective modification for AsA to inhibit UV-B–induced cytotoxicity in human keratinocytes.

ENZYMATIC PREPARATION OF SPHINGOSINE 1-PHOSPHATE

Sphingosine 1-phosphate was prepared from sphingosine by two steps involving conversion of sphingosine to phosphatidylsphingosine catalyzed by phospholipase D and enzymatic hydrolysis of the intermediate to a diacyglycerol and sphingosine 1-phosphate with phospholipase C.