Hironori Umetsu

Purification, crystallisation and characterisation of carboxypeptidase from wheat bran

Abstract A carboxypeptidase was purified and crystallised from wheat bran. Disc gel electrophoresis at pH 4·0 and ultracentrifugal analysis revealed that the enzyme was essentially homogeneous. The sedimentation constant and isoelectric point were determined to be 6·3 S and 6·0, respectively. The molecular weight of the enzyme was estimated to be 118,000 by a gel filtration method. The enzyme liberated carboxyl terminal amino acid residues from a wide range of N -substituted dipeptides and tripeptides which contain l -proline. It had a pH optimum at pH 4·0 for Z -Glu-Tyr ( Z -benzyloxycarbonyl). The K m and k cat values for Z -Glu-Tyr at pH 4·0 and 30°C were 0·19 m m and 20 s −1 , respectively. The enzyme hydrolysed Z -Gly-Pro-Leu-Gly-Pro and bradykinin sequentially at pH 4·0 from their carboxyl terminal amino acid residues. The enzyme activity was completely inhibited by DFP.

Two detection methods of genetically modified maize and the state of its import into Japan

Abstract We detected genetically modified maize (GM-maize) with two different methods and examined the state of its import into Japan. A method using a cetyltrimethylammonium bromide buffer efficiently extracted DNA. Measurement of the extracted DNA at 260/230 nm and 260/280 nm indicated that the DNA was pure enough for polymerase chain reaction (PCR) amplification. Agarose gel electrophoresis of the PCR amplified products resolved a band at 329 bp for the zein gene and 437 and 522 bp for the introduced genes in Bt11, 619 bp in Event176, 194 bp in MON810 and 231 bp in LIBERTY maize. GM-maize was also detected by an enzyme-linked immunosorbent assay (ELISA) and there was no conflict in the results between the ELISA and the PCR method. However, the PCR method had advantages in distinguishing the lines of GM-maize. GM-maize was contained in the maize seeds imported into Japan between January and March in 2000 and MON810 counted for the largest percentage of the GM-maize.

Homology modeling and structural analysis of human P-glycoprotein.

Homology modeling and structural analysis of human P-glycoprotein (hP-gp) were performed with a software package the Molecular Operating Environment (MOE). A mouse P-gp (mP-gp; PDB code: 3G5U) was selected as a template for the 3D structure modeling of hP-gp. The modeled hP-gp showed significant 3D similarities at the drug biding site (DBS) to the mP-gp structure. The contact energy profiles of the hP-gp model were in good agreement with those of the mP-gp structure. Ramachandran plots revealed that only 3.5% of the amino acid residues were in the disfavored region for hP-gp. Further, docking simulations between 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and the P-gp models revealed the similarity of the ligand-receptor bound location between the hP-gp and mP-gp models. These results indicate that the hP-gp model was successfully modeled and analyzed. To the best of our knowledge, this is the first report of a hP-gp model with a naturally occurring isothiocyanate, and our data verify that the model can be utilized for application to target hP-gp for the development of antitumor drugs. Abbreviations ABC - ATP-binding cassette, ASE-Dock - alpha sphere and excluded volume-based ligand-protein docking, DBS - drug biding site, MDR - multidrug resistance, MOE - Molecular Operating Environment, ITC - isothiocyanate, P-gp - P-glycoprotein.

Structural insight into the ligand-receptor interaction between glycyrrhetinic acid (GA) and the high-mobility group protein B1 (HMGB1)-DNA complex

Structural analysis of the high-mobility group protein B1 (HMGB1)-DNA complex and a docking simulation between glycyrrhetinic acid (GA) and the HMGB1-DNA complex were performed with a software package the Molecular Operating Environment (MOE). An HMGB1-DNA (PDB code: 2GZK) was selected for the 3D structure modeling of the HMGB1-DNA complex. The Site Finder module of the MOE identified 16 possible ligand-binding sites in the modeled HMGB1-DNA complex. The docking simulation revealed that GA possibly inhibits functions of HMGB1 interfering with Lys90, Arg91, Ser101, Tyr149, C230 and C231 in the HMGB1-DNA complex. To the best of our knowledge, this is the first report of an HMGB1-DNA complex with GA, and our data verify that the GA-HMGB1-DNA model can be utilized for application to target HMGB1 for the development of antitumor drugs. Abbreviations ASE-Dock - alpha sphere and excluded volume-based ligand-protein docking, CNS - central nervous system, GA - glycyrrhetinic acid, GL - glycyrrhizin, HMGB1 - high-mobility group protein B1, LBS - ligand-biding site, MOE - Molecular Operating Environment, SRY - sex-determining region on the Y chromosome.

Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus

Abstract. Seventeen strains of the genus Paecilomyces were examined for their ability to produce serine carboxypeptidase. Paecilomyces carneus IFO 7012 exhibited the highest potency for serine carboxypeptidase production. A maximum yield of serine carboxypeptidase was obtained by koji culture of the strain at 22°C for 7 days. The serine carboxypeptidase was purified to homogeneity from an extract of the koji culture. The molecular weight of the enzyme was estimated to be 47,000 by HPLC. The isoelectric point of the enzyme was determined to be 4.0, and the optimum pH was 4.0 toward benzyloxycarbonyl-L-glutamyl-L-tyrosine (Z-Glu-Tyr) and benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala), respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and p-chloromercurybenzoate. Relative hydrolysis rates of N-acylpeptides and kinetic studies indicated that the enzyme preferred substrates having bulky amino acids in the penultimate position from their carboxy-termini.

Ligand-receptor Interaction between Triterpenoids and the 11β-Hydroxysteroid dehydrogenase type 2 (11βHSD2) Enzyme Predicts Their Toxic Effects against Tumorigenic r/m HM-SFME-1 Cells

The present study deals with in silico prediction and in vitro evaluation of the selective cytotoxic effects of triterpenoids on tumorigenic human c-Ha-ras and mouse c-myc cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells. Ligand fitting of five different triterpenoids to 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) was analyzed with a molecular modeling method, and glycyrrhetinic acid (GA) was the best-fitted triterpenoid to the ligand binding site in 11βHSD2. Analysis of antiproliferative effects revealed that GA, oleanolic acid, and ursolic acid had selective toxicity against the tumor cells and that GA was the most potent triterpenoid in its selectivity. The toxic activity of the tested triterpenoids against the tumor cells showed good correlations with the partition coefficient (logP) and polar surface area values. Time-lapse microscopy, fluorescence staining, and confocal laser scanning microscopic observation revealed that GA induced morphologic changes typical of apoptosis such as cell shrinkage and blebbing and also disrupted the cytoskeletal proteins. Furthermore, GA exhibited a strong inhibitory effect on 11βHSD2 activity in the tumor cells. Our current results suggest that analysis of the ligand-receptor interaction between triterpenoids and 11βHSD2 can be utilized to predict their antitumor effects and that GA can be used as a possible chemopreventive and therapeutic antitumor agent. To the best of our knowledge, this is the first report on in silico prediction of the toxic effects of triterpenoids on tumor cells by 11βHSD2 inhibition.

Effects of phytosulfokine-α on growth and tropane alkaloid production in transformed roots of Atropa belladonna

Phytosulfokine (PSK)-α is a sulphated pentapeptide, isolated fromthe medium of cultured Asparagus officinalis mesophyllcells, that promotes cell proliferation. It is a putative key factor inconditioned medium required for the growth of low-density plant cell cultures.The present study investigates the effect of PSK-α on growth and tropanealkaloid production in Atropa belladonna hairy rootstransformed with Agrobacteriumrhizogenes (MAFF 03-01724). Although the growth rates ofhairy roots cultured in medium with orwithout PSK-α for 4 weeks did not show any differences, the productivityof tropane alkaloids, especially of hyoscyamine, was enhanced by10−7 or 10−8 M PSK-α. Inaddition, the content of tropane alkaloids in transformed roots treated withPSK-α was 1.4 times higher than that of untreated roots after 4 weeks ofculture. The time course of growth and tropane alkaloid production inAtropa belladonna transformed roots suggested thatPSK-α influenced the growth of transformed roots during the activegrowingphase, but not tropane alkaloid production.

Substrate Specificity of Aminopeptidase from the Mid-gut Gland of the Scallop (Patinopecten yessoensis)

An action for various peptides and a kinetic study for amino acid p-nitroanilides (pNAs) and 4-methylcoumaryl-7-amides (MCAs) were performed with purified aminopeptidase from the mid-gut of the scallop. The enzyme preferred dipeptides having Ala, Met, and Phe in the amino-terminal or the penultimate position from the amino-termini. The catalytic efficiencies, k cat⁄K m values for Ala-pNA and MCA were the highest in the tested substrates, and those for pNA and MCA substrates having Met or Phe were the next highest. The enzyme was found to be a new alanine-specific aminopeptidase.

Characteristics of Cold-Preserved Embryogenic Suspension Cells in Fennel, Foeniculum vulgare Miller

Summary An embryogenic suspension cell line of fennel, Foeniculum vulgare Miller, directly induced from hypocotyl, was developed. The suspension cells were cold-preserved at 4 °C for up to 12 weeks and then cultured at 25 °C for 2 weeks. The packed cell volume of the cultured cells decreased as the cold-preservation period became longer. The cells, with a size of 32-82μm and having embryogenic potential, diminished suddenly with the passage of the cold-preservation period. The cells cold-preserved for 2 to 6 weeks were capable of forming normal somatic embryos that were identical to those obtained from the cells without cold-preservation. The resulting somatic embryos regenerated to develop into normal plantlets when cultured in hormone-free Murashige and Skoog medium under illumination. Esterase isozyme analysis on polyacrylamide gel electrophoresis revealed the appearance of different bands in the embryos treated with 4-week and 6-week cold-preservation, in contrast to the embryos from the cells without cold-preservation. Anethole was detected in the methanol extracts of embryos induced from the cold-preserved cells at the same level as that of embryos induced from the cells without cold-preservation.

Mouse 11β-Hydroxysteroid Dehydrogenase Type 2 for Human Application: Homology Modeling, Structural Analysis and Ligand-Receptor Interaction:

Mouse (m) 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) was homology-modeled, and its structure and ligand-receptor interaction were analyzed. The modeled m11βHSD2 showed significant 3D similarities to the human (h) 11βHSD1 and 2 structures. The contact energy profiles of the m11βHSD2 model were in good agreement with those of the h11βHSD1 and 2 structures. The secondary structure of the m11βHSD2 model exhibited a central 6-stranded all-parallel β-sheet sandwich-like structure, flanked on both sides by 3-helices. Ramachandran plots revealed that only 1.1% of the amino acid residues were in the disfavored region for m11βHSD2. Further, the molecular surfaces and electrostatic analyses of the m11βHSD2 model at the ligand-binding site exhibited that the model was almost identical to the h11βHSD2 model. Furthermore, docking simulation and ligand-receptor interaction analyses revealed the similarity of the ligand-receptor bound conformation between the m11βHSD2 and h11βHSD2 models. These results indicate that the m11βHSD2 model was successfully evaluated and analyzed. To the best of our knowledge, this is the first report of a m11βHSD2 model with detailed analyses, and our data verify that the mouse model can be utilized for application to the human model to target 11βHSD2 for the development of anticancer drugs.