Katsuyoshi Kamiie

Molecular cloning and characterization of a novel carboxylesterase-like protein that is physiologically present at high concentrations in the urine of domestic cats (Felis catus)

Normal mammals generally excrete only small amounts of protein in the urine, thus avoiding major leakage of proteins from the body. Proteinuria is the most commonly recognized abnormality in renal disease. However, healthy domestic cats ( Felis catus ) excrete proteins at high concentrations (about 0.5 mg/ml) in their urine. We investigated the possible cause of proteinuria in healthy cats, and discovered a 70 kDa glycoprotein, which was excreted as a major urinary protein in cat urine, irrespective of gender. To elucidate the biochemical functions and the excretion mechanism of this protein, we cloned the cDNA for this protein from a cat kidney cDNA library. The deduced amino acid sequence shared 47% identity with the rat liver carboxylesterase (EC 3.1.1.1), and both the serine hydrolase active site and the carboxylesterase-specific sequence were conserved. Therefore we named this protein cauxin (carboxylesterase-like urinary excreted protein). In contrast to the mammalian carboxylesterases, most of which are localized within the cells of various organs, cauxin was expressed specifically in the epithelial cells of the distal tubules, and was secreted efficiently into the urine, probably because it lacked the endoplasmic reticulum retention sequence (HDEL). Based on our finding that cauxin is not expressed in the immature cat kidney, we conclude that cauxin is involved in physiological functions that are specific for mature cats. Recently, cauxin-like cDNAs were found from human brain and teratocarcinoma cells. These data suggest that cauxin and cauxin-like human proteins are categorized as a novel group of carboxylesterase multigene family.

Novel effects of glycyrrhetinic acid on the central nervous system tumorigenic progenitor cells: induction of actin disruption and tumor cell-selective toxicity.

Licorice extracts are used worldwide in foods and medicines, and glycyrrhetinic acid (GA) is a licorice component that has been reported to induce various important biological activities. In the present study, we show that GA induces actin disruption and has tumor cell-selective toxic properties, and that its selectivity is superior to those of all the clinically available antitumor agents tested. The cytotoxic activity of GA and the tested antitumor agents showed better correlation with the partition coefficient (log P) values rather than the polar surface area (PSA) values. For selective toxicity against tumor cells, GA was most effective at 10 microM that was the same concentration as the previously reported maximum plasma GA level reached in humans ingesting licorice. These results suggest that GA could be utilized as a promising chemopreventive and therapeutic antitumor agent. The underlying mechanisms involved in the selective toxicity to tumor cells by GA are also preliminarily discussed.

Cloning and Expression of Bombyx mori Silk Gland Elongation Factor 1γ in Escherichia coli

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of α-, β-, γ-, and δ-subunits. EF-1α•GTP catalyzes the binding of aminoacyl-tRNA to ribosomes concomitant with the hydrolysis of GTP. EF-1βγδ catalyzes the exchange of EF-1α-bound GDP for exogenous GTP and stimulates the EF-1α-dependent binding of aminoacyl-tRNA to ribosomes. EF-1γ cDNA, which contains an open reading frame (ORF) encoding a polypeptide of 423 amino acid residues, was amplified and cloned by PCR from a silk gland cDNA library. The calculated molecular mass and predicted pI of the product were 48,388 Da and 5.84, respectively. The silk gland EF-1γ shares 67.3% amino acid identity with Artemia salina EF-1γ. The N-terminal domain (amino acid residues 1–211) of silk gland EF-1γ is 29.3% identical to maize glutathione S-transferase. We demonstrated that silk gland EF-1γ bound to glutathione Sepharose, suggesting that the N-terminal domain of EF-1γ may have the capacity to bind to glutathione.

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.

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.

Molecular docking and structural analysis of cofactor-protein interaction between NAD+ and 11β-hydroxysteroid dehydrogenase type 2

Molecular docking and structural analysis of the cofactor-protein interaction between NAD+ and human (h) or mouse (m) 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) were performed with the molecular operating environment (MOE). 11βHSD1 (PDB code: 3HFG) was selected as a template for the 3D structure modeling of 11βHSD2. The MOE docking (MOE-dock) and the alpha sphere and excluded volume-based ligand-protein docking (ASE-dock) showed that both NAD+-h11βHSD2 and NAD+-m11βHSD2 models have a similar binding orientation to the template cofactor-protein model. Our present study also revealed that Asp91, Phe94, Tyr232 and Thr267 could be of importance in the interaction between NAD+ and 11βHSD2. NADP+ was incapable of entering into the cofactor-binding site of the 11βHSD2 models. The present study proposes the latest models for 11βHSD2 and its cofactor NAD+, and to the best of our knowledge, this is the first report of a m11βHSD2 model with NAD+.

Interaction between Elongation Factors 1β and 1γ from Bombyx mori Silk Gland

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: α (51 kDa), β (26 kDa), γ (49 kDa), and δ (33 kDa). The EF-1α subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1α-bound GDP is then exchanged for GTP by the EF-1βγδ complex. To facilitate analysis of the roles of the individual EF-1β, γ, and δ subunits in GDP/GTP exchange on EF-1α, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1β, EF-1γ, and the carboxyl-terminal half of EF-1δ were expressed, purified, and examined for protein:protein interactions by gel filtration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1β and γ subunits in a 1:1 molar ratio was detected by gel filtration. A higher molecular weight species containing an excess of EF-1γ relative to EF-1β was also detected. The amino-terminal region of EF-1β (amino acid residues 1-129) was sufficient for binding to EF-1γ. The carboxyl-terminal half of EF-1δ did not appear to form a complex with EF-1γ.

Interaction between elongation factors 1beta and 1gamma from Bombyx mori silk gland.

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: α (51 kDa), β (26 kDa), γ (49 kDa), and δ (33 kDa). The EF-1α subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1α-bound GDP is then exchanged for GTP by the EF-1βγδ complex. To facilitate analysis of the roles of the individual EF-1β, γ, and δ subunits in GDP/GTP exchange on EF-1α, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1β, EF-1γ, and the carboxyl-terminal half of EF-1δ were expressed, purified, and examined for protein:protein interactions by gel filtration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1β and γ subunits in a 1:1 molar ratio was detected by gel filtration. A higher molecular weight species containing an excess of EF-1γ relative to EF-1β was also detected. The amino-terminal region of EF-1β (amino acid residues 1-129) was sufficient for binding to EF-1γ. The carboxyl-terminal half of EF-1δ did not appear to form a complex with EF-1γ.

Intracellular accumulation of structurally varied isothiocyanates correlates with inhibition of nitric oxide production in proinflammatory stimuli-activated tumorigenic macrophage-like cells.

In the present study, we analyzed the intracellular accumulation of 6-(methylsulfinyl)hexyl isothiocyanate (6MITC) and its analogs in proinflammatory stimuli-activated J774.1 cells to predict the biological potencies of the ITCs. Our present analyses exhibited that the intracellular accumulation was in the order of 6MITC>2b>2e≈2c>2g>2d>2f>2h. Investigation of reactivity of the ITCs with glutathione (GSH) in the tumor cells revealed partial inhibition of GSH by the ITCs. Furthermore, the inhibition of nitric oxide (NO) production in the tumor cells was ascribed to the intracellularly accumulated ITCs. The NO suppression was correlated with the inhibition of tumor cell growth. Our present results suggest that the intracellular accumulation of the ITCs can be used to predict their biological potencies, such as inhibition of NO production that was correlated with suppression of tumor cell growth. To the best of our knowledge, this is the first report to predict the biological potency of 6MITC and its analogs with their intracellular accumulation.