Katsuyoshi Mitsunaga

Estrogen receptor binding assay of chemicals with a surface plasmon resonance biosensor.

We have developed a simple assay method for the evaluation of estrogen receptor (ER) binding capacity of chemicals without the use of radio- or fluorescence-labeled compounds. We used the solution competition assay by the BIACORE biosensor, a surface plasmon resonance biosensor, with estradiol as a ligand, human recombinant ER(alpha) (hrER(alpha)) as a high molecular weight (hmw) interactant and test chemicals as analytes. For the ligand, aminated estradiol with a spacer molecule (E2-17PeNH) was synthesized and immobilized on a carboxymethyl dextran-coated sensor chip by the amine coupling method. The injection of the hmw interactant hrER(alpha) to the biosensor raised the sensorgram, indicating its binding to the ligand E2-17PeNH. The binding of test chemicals to hrERalpha was determined as a reduction in the hrER(alpha) binding to E2-17PeNH. The dissociation constant for the binding to hrER(alpha) was calculated for estrone (4.29 x 10(-9)M), estradiol (4.04 x 10(-10)M), estriol (8.35 x 10(-10)M), tamoxifen (2.16 x 10(-8)M), diethylstilbestrol (1.46 x 10(-10)M), bisphenol A (1.35 x 10(-6)M) and 4-nonylphenol (7.49 x 10(-6)M), by plotting the data according to an equation based on mass action law. This method can also be used as a high throughput screening method.

Structural mechanism and photoprotective function of water-soluble chlorophyll-binding protein.

A water-soluble chlorophyll-binding protein (WSCP) is the single known instance of a putative chlorophyll (Chl) carrier in green plants. Recently the photoprotective function of WSCP has been demonstrated by EPR measurements; the light-induced singlet-oxygen formation of Chl in the WSCP tetramer is about four times lower than that of unbound Chl. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function. The WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl molecules. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl molecules are tightly packed and isolated from bulk solvent. With reference to the novel Chl-binding mode, we propose that the photoprotection mechanism may be based on the inhibition of physical contact between the Chl molecules and molecular oxygen.

Canthin-6-one alkaloids from Eurycoma longifolia

Five new canthin-6-one alkaloids, 9,10-dimethoxycanthin-6-one, 10-hydroxy-9-methoxycanthin-6-one, 11-hydroxy-10-methoxycanthin-6-one, 5,9-dimethoxycanthin-6-one and 9-methoxy-3-methylcanthin-5,6-dione were isolated from the bark and wood of Eurycoma longifolia, along with six known canthin-6-one alkaloids and two known β-carboline alkaloids. Their structures were determined from spectroscopic data and other chemical evidence.

Chromones from Harrisonia perforata

Five new chromones, perforatins C-G, together with 10 known compounds were isolated from the wood of Harrisonia perforata.

Proteomic analysis of indium embryotoxicity in cultured postimplantation rat embryos

Indium embryotoxicity was investigated by proteomic analysis with two-dimensional electrophoresis of rat embryos cultured from day 10.5 of gestation for 24h in the presence of 50 microM indium trichloride. In the embryo proper, indium increased quantity of several protein spots including those identified as serum albumin, phosphorylated cofilin 1, phosphorylated destrin and tyrosyl-tRNA synthetase. The increased serum albumin, derived from the culture medium composed of rat serum, may decrease the toxicity of indium. The increase of phosphorylated cofilin 1 might be involved in dysmorphogenicity of indium through perturbation of actin functions. In the yolk sac membrane, indium induced quantitative and qualitative changes in the protein spots. Proteins from appeared spots included stress proteins, and those from decreased or disappeared spots included serum proteins, glycolytic pathway enzymes and cytoskeletal proteins, indicating yolk sac dysfunction. Thus, several candidate proteins that might be involved in indium embryotoxicity were identified.

Proteomic analysis of selenium embryotoxicity in cultured postimplantation rat embryos.

BACKGROUND Developmental toxicity of selenium (Se) is a nutritional, environmental and medicinal concern. Here, we investigated Se embryotoxicity by proteomic analysis of cultured rat embryos. METHODS Rat embryos at day 9.5 or 10.5 of gestation were cultured for 48 or 24 h, respectively, in the presence of sodium selenate (100 or 150 microM) or sodium selenite (20 or 30 microM). Proteins from the embryo proper and yolk sac membrane were analyzed by two-dimensional electrophoresis for quantitative changes from those in control embryos. Proteins with quantitative changes were identified by mass spectrometric analysis. RESULTS Growth inhibition and morphological abnormalities of cultured embryos were observed in all the Se treatment groups. By the analysis of the embryo proper, actin-binding proteins were identified as proteins with quantitative changes by selenate: increased phosphorylated-cofilin 1, increased phosphorylated-destrin, decreased drebrin E, and decreased myosin light polypeptide 3. Many proteins showed similar changes between selenate and selenite, including increased ATP-synthase, decreased acidic ribosomal phosphoprotein P0, and decreased pyrroline-5-carboxylate reductase-like. In the yolk sac membrane, antioxidant proteins were identified for protein spots with quantitative changes by selenite: increased peroxiredoxin 1 and increased glutathione S-transferase. CONCLUSION The identified proteins with quantitative changes by selenate or selenite were considered to be candidate proteins involved in Se embryotoxicity: the actin-binding proteins for selenate embryotoxicity, proteins with the similar changes for the common Se embryotoxicity and antioxidant proteins for modification of Se embryotoxicity by redox-related treatments. These proteins may also be used as biomarkers in developmental toxicity studies.

In vivo/in vitro study in rat embryos on indium-caused tail malformations.

Pathogenesis of indium-caused tail malformations was investigated by in vivo and in vitro experiments. In the in vivo experiment, pregnant Wistar rats received single intravenous administration of indium trichloride at 0.4 mg/kg on day 10 of gestation, and their embryos were examined on days 11, 12 and 13. Embryos in the indium group showed caudal hypoplasia from day 11. Increased apoptosis was observed in their tailbud on day 11. Similar effects were observed in the in vitro experiment, when day 10 rat embryos were cultured in the presence of indium trichloride at 50 microM for 24 h and for further 24 h in the absence of indium. It was considered from these results that caudal hypoplasia probably due to excessive cell loss by increased apoptosis in the tailbud accounted for indium-caused tail malformations in rat fetuses, and that indium-caused embryotoxic effects were direct effects on the conceptus.

Alkaloids and quassinoids of Brucea mollis var. tonkinensis

Abstract Two new canthin-6-one alkaloids, bruceollines C and G, were isolated from the root bark and root wood of Brucea mollis var. tonkinensis , together with three known canthin-6-one alkaloids, 11-hydroxycanthin-6-one, 1-hydroxy-11-methoxycanthin-6-one and 11-hydroxy-1-methoxycanthin-6-one, and four known β-carboline alkaloids, 1-hydroxymethyl-β-carboline, 1-ethyl-β-carboline, 1-(2′-hydroxyethyl)-β-carboline and β-carboline-1-propionic acid, and six known quassinoids, brucein B, brucein D, brusatol, bruceoside B, yadanziolide A and soulameanone. Their structures were elucidated based on the spectral and chemical evidence.

Complement component C3 functions as an embryotrophic factor in early postimplantation rat embryos

A presumed embryotrophic factor for early postimplantation rat embryos, partially purified from rat serum, was identified as complement component C3 (C3), the central component of the complement system, by sequence analysis of its N-terminal. Purified rat C3 showed embryotrophic activity for rat embryos cultured from day 9.5 of gestation for 48 h in the culture medium composed of rabbit serum. The maximum embryotrophic activity of C3 was observed around 0.5 mg/ml, a level which is lower than rat serum C3 levels. In the culture medium composed of rat serum, cultured rat embryos selectively consumed C3, and C3-depletion by cobra venom factor affected embryonic growth. Inactivation of the internal thiolester bond of C3, the critical functional site for its activity in the complement system, by methylamine had no effects on its embryotrophic activity. Purified rabbit C3 had only weak embryotrophic activity for cultured rat embryos, suggesting species specificity of the embryotrophic activity of C3. Immunochemical analyses showed the specific presence of C3 on the visceral yolk sac, but not on the embryo proper of day 9.5 or 10.5 rat embryos both in utero and in vitro. In analysis using fluorescein-labeled rat C3, unfragmented C3s bound to the visceral yolk sac stronger than C3b, the primary active fragment of C3 in the complement system. These results indicate that C3, which has always been considered to be detrimental to embryos, functions as an embryotrophic factor by novel mechanisms probably through the visceral yolk sac. The present study thus provides new insights into functions of C3 and postimplantation embryonic growth.

Rearranged limonoids from Harrisonia brownii

Abstract A new limonoid, brownin C, was isolated from the bark of Harrisonia brownii, and a new limonoid, brownin G, from the wood of the same plant. Their structures were determined by spectral techniques and X-ray crystal structure analysis.